Molecular identification of hyaluronate lyase, not hyaluronidase, as an intrinsic hyaluronan-degrading enzyme in Clostridium perfringens

Molecular identification of hyaluronate lyase, not hyaluronidase, as an intrinsic hyaluronan-degrading enzyme in Clostridium perfringens | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Molecular identification of hyaluronate lyase, not hyaluronidase, as an intrinsic hyaluronan-degrading enzyme in Clostridium perfringens Wataru Hashimoto, Tomoya Kumon, Sayoko Oiki This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4310962/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 22 Oct, 2024 Read the published version in Scientific Reports → Version 1 posted 12 You are reading this latest preprint version Abstract Clostridium perfringens , an opportunistic pathogen in the human intestine, causes gas gangrene by producing various toxins. A clostridial enzyme degrading the host extracellular matrix hyaluronan (HA) has been considered a critical virulence factor as mu-toxin hyaluronidases including endo-β- N -acetylglucosaminidases (Nags). Here we show that, distinct from Nags, hyaluronate lyase (HysA) is an intrinsic HA-degrading enzyme. C. perfringens (ATCC 13124) was found to assimilate host-derived extracellular mucosubstances, HA and mucin, which induced expression of the HA-related genetic cluster, including hysA , but repressed nag genes. The recombinant C. perfringens HysA showed an HA-degrading activity toward HA through β-elimination reaction. The HA-degrading enzyme in the culture supernatant of C. perfringens exhibited the lyase activity and was identical to the recombinant HysA on the native-PAGE gel, followed by activity straining. These results demonstrated that the intrinsic HA-degrading enzyme of C. perfringens is hyaluronate lyase HysA, but not hyaluronidases NagH, NagJ, and NagK. Biological sciences/Microbiology/Bacteriology Biological sciences/Microbiology/Pathogens Biological sciences/Biochemistry/Enzymes Biological sciences/Biochemistry/Glycobiology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Approximately 38 trillion bacteria, more than all human cells, are estimated to be indigenous to the human gut 1 . Intestinal bacteria establish competitive and symbiotic relationships through interactions with the host and other bacteria in the gut, where various nutrients are intermittently supplied by the host's diet 2–4 . The gut microbiota is suggested to be correlated with host health, such as regulation of the immune system, allergy, inflammatory bowel disease, obesity, diabetes, and cancer 5–11 . While some beneficial bacteria, e.g. , genera of Bifidobacterium and Lactobacillus , are indigenous to the gut, opportunistic pathogens, including genera of Clostridium and Streptococcus , can also exist. However, the mechanism of bacterial colonization in the gut remains to be clarified. All animal cells, including the gut, are covered with a noncellular component called extracellular matrices. Extracellular matrices, composed of water, proteins, and polysaccharides, provide a physical scaffold for cells and promote cell differentiation and homeostasis 12 . Proteoglycans, a part of extracellular matrices, comprise glycosaminoglycans (GAGs) linked to core proteins. GAGs are heteropolysaccharides with a disaccharide-repeating unit of uronic acid (or galactose) and amino sugar 13 . Chondroitin sulfate C (CSC) and heparin (HP) are representative sulfated GAGs that constitute proteoglycans, while hyaluronan (HA), a representative nonsulfated GAG, exists independently of proteoglycans. HA is the most abundant GAG in the gut epithelium 14 . In Streptococcus species, polysaccharide lyase and phosphotransferase system (PTS) are involved in the degradation and import of HA, respectively 15,16 . Our previous studies have demonstrated the streptococcal action on HA as follows 17–21 : Hyaluronate lyase is inducibly expressed in the presence of HA and essential for the assimilation of nutrient HA, followed by pathogenic damage to host tissues. Unsaturated HA disaccharide generated from HA through the lyase reaction is imported into the cytoplasm in the specific PTS, followed by degradation by cytoplasmic unsaturated glucuronyl hydrolase (UGL). The resultant unsaturated uronic acid is metabolized into glycolysis by isomerase, dehydrogenase, and other enzymes (Supplementary Fig. 1) 18–21 . These enzymes and transporter are encoded as a GAG genetic cluster in the streptococcal genome 19 . Some pathogenic bacteria, such as Streptococcus pneumoniae and Staphylococcus aureus , degrade GAGs and infect host cells 16,22 . Intestinal epithelial cells are covered with a mucin layer outside the extracellular matrices. Mucin is a macromolecule composed of core proteins linked to sugar chains, including sialic acid, fucose, galactose, and amino sugar. The mucin layer, formed of mucins secreted from goblet cells within the intestinal epithelium, contains a low-density outer mucus layer and a high-density inner mucus layer. Intestinal bacteria are present in the outer mucus layer and utilize the mucin 3,23 . In contrast, the inner mucus layer functions as a barrier to block interactions between bacteria and host, thus leaving the bacteria undetectable 23,24 . However, it remains unclear whether pathogens that infect and invade host epithelial cells degrade the mucin layer. Clostridium perfringens is a Gram-positive, anaerobic, and spore-forming bacterium that belongs to the Bacillota phylum, one of the most abundant phyla among intestinal bacteria. C. perfringens is a major component of the adult human gut microbiota, increasing in abundance from the ileum, the cecum, and the rectum 25 . However, this bacterium can cause life-threatening gas gangrene and food poisoning in humans and animals 26 . The clostridial virulence is largely attributed to the production of approximately twenty different toxins. For example, alpha-toxins, such as phospholipase, degrade phosphatidylcholine and sphingomyelin in the plasma membrane and cause endocytosis and cell death through the formation of diacylglycerol and ceramide 27 . C. perfringens also produce degrading enzymes such as hyaluronidase (mu-toxin), collagenase (kappa-toxin), proteases, and sialidase, to decompose connective tissue, leading to colonization and infection of the surrounding tissues 28 . Furthermore, mu-toxin, while itself is non-lethal, facilitates the spread of the major alpha-toxin. An endo-β- N -acetylglucosaminidase, NagH, has been identified as the hyaluronidase by screening recombinant lambda phages for glucuronidase activity 29 . Although NagH has long been considered the HA-degrading enzyme and a virulence factor, the intrinsic enzyme crucial for HA degradation has not yet been elucidated. Two complete genome sequences of C. perfringens strains 13 and ATCC 13124 were described by Shimizu et al. and Garry et al. , respectively 30,31 . Based on these studies, five candidate genes, nagH (CPE0191), nagI (CPE0881), nagJ (CPE1234), nagK (CPE1279), and nagL (CPE1523), coding for hyaluronidases and nanJ (CPE0553) coding for sialidase, have been thought to be virulence factors in C. perfringens strain 13 30 . The nagH gene (CPF_0184) coding for hyaluronoglucosaminidase, nagJ (CPF_0875) coding for putative O -linked N -acetylglucosaminidase annotated as nagI in C. perfringens strain 13, nagJ (CPF_1442) and nagK (CPF_1487) were conserved in the C. perfringens strain ATCC 13124 genome. However, the nagL gene was not conserved. VirS and VirR, a two-component system involved in regulating the expression of virulence factors, have also been identified 32 . VirS, a membrane sensor protein, receives environmental and intracellular signals via Agr, an accessory gene regulator, causing autophosphorylation. VirR is activated in the cytoplasm by receiving a phosphate group from VirS, resulting in control of transcriptional expression. The VirS/VirR regulon positively regulates major toxins, such as phospholipase C and collagenase, suggesting an important role during host infection. At the same time, the system mainly acts as a global repressor that negatively regulates general genes 33 . In contrast, RevR, a virulence regulator, is a global activator 34 . Transcriptome analysis by RNA sequence (RNA-seq) and DNA microarray of these three regulators of virulence factors indicated that the hyaluronidase candidate genes ( nagH , nagI , and nagJ ) and sporulation protein ( sigF , ftsN , spoIIIAG , sigG , and spoIVA ) as virulence factors are positively regulated by RevR, contrary to the negative regulation by VirR 28,34–36 . Based on this observation, NagH also potentially degrades HA in C. perfringens . This study identifies the intrinsic HA-degrading enzyme in C. perfringens by transcriptome analysis and enzyme characterization. Results and Discussion Degradation and assimilation of HA by C. perfringens We previously found the GAG genetic cluster, in the C. perfringens strain ATCC 13124 genome as well as in the genomes of Streptococcus agalactiae and S. pneumoniae (Fig. 1 ) 21 . While hysA (CPF_0394), coding for hyaluronate lyase, and hepC (CPF_0406), coding for heparin lyase II/III-like protein, were found in the cluster, the HA-degrading enzyme in C. perfringens remained to be identified. nagHIJK , coding for hyaluronidases, which have been considered as candidate genes for HA degradation, were not found in the GAG genetic cluster. On the other hand, the cluster contains homologous genes coding for transporter and enzymes involved in the degradation and metabolism of HA. Therefore, hysA possibly plays a role in HA degradation as well as in virulence factors. In addition, it has been suggested that C. perfringens degrades and assimilates mucin by a sialidase, NanI, indicating that degradation of host mucosubstances such as HA and mucin is important for C. perfringens to colonize the human intestine 37,38 . To clarify the clostridial molecular mechanism of HA degradation, we first examined the GAG degradation ability of C. perfringens and other related species, such as Clostridium butyncum and Clostridioides difficile , through the halo plate assay method (Fig. 2 a, b). After clostridial cells were grown on halo plates containing GAGs and bovine serum albumin (BSA) in a nutrient-poor medium (Fig. 2 a) or a nutrient-rich medium (Fig. 2 b), acetic acid was added to confirm the presence or absence of a halo in a white precipitate. C. perfringens formed a halo in the presence of HA, while no halo was detected in the presence of CSC or HP, indicating HA degradation by C. perfringens . However, other species tested showed no halo, suggesting no GAG degradation. HA assimilation by C. perfringens was also examined by culturing in a nutrient-poor medium, excluding BSA and agar from the halo plate (Fig. 2 c). C. perfringens cells showed significantly higher growth in the presence of HA than in the absence of HA. Moreover, C. perfringens also grew in the nutrient-poor medium containing mucin, indicating that the bacterial cells assimilate mucin and HA. Therefore, C. perfringens grown on the nutrient-poor medium in the presence of HA was expected to express genes involved in the degradation, import, and assimilation of GAGs in response to HA. Transcriptome analysis of C. perfringens in the presence of HA or mucin To investigate the gene expression in the presence of HA or mucin, we performed an RNA-seq analysis of C. perfringens cells grown in the nutrient-poor medium in the presence and absence of HA or mucin. The data are shown in Supplementary Fig. 2. In the case of C. perfringens cells grown in the nutrient-poor medium, the dataset contained 49,915,882 reads with a length of 101 nucleotides. The reads obtained from the cells grown in the nutrient-poor medium containing HA and mucin were 49,913,574 and 68,868,196, respectively. The trimming data by sliding window method results were as follows: the nutrient-poor medium, 44,776,526; the nutrient-poor medium containing HA, 44,736,304; and the nutrient-poor medium containing mucin, 60,852,264. The mapping data occupied 90.70%, 90.53%, and 89.33%, respectively. Normalization was performed using RPKM (Reads Per Kilobase per Million mapped reads). Of the 2,921 genes, 2,735 genes for which RPKM could be calculated were analyzed. Figure 3 a shows the number of differentially expressed genes by 10-fold or more in the nutrient-poor medium containing HA compared with the nutrient-poor medium. In the nutrient-poor medium in the presence of HA, 65 genes were highly expressed, while the expression level of 92 genes was significantly decreased. No genes decreased in the nutrient-poor medium containing mucin, while seven genes showed a high expression level. In response to both HA and mucin, the expression of bioB (CPF_1795), coding for biotin synthase, the product of which is an essential vitamin for animals, was elevated 49.3-fold and 12.5-fold in the nutrient-poor medium containing HA and mucin, respectively, compared with the nutrient-poor medium. This suggests the possibility that the gut-predominant C. perfringens provides the host with essential biotin through the assimilation of HA and mucin. In the nutrient-poor medium containing HA, the following genes involved in cell proliferation were highly expressed: synthesis and transport of amino acid [CPF_0170 coding for cysteine synthase, CPF_0756 for the proton/sodium glutamate symporter, and minC (CPF_2394) for the probable septum site determining protein], purine-pyrimidine synthase [CPF_1001 for hypothetical protein and xpt1 (CPF_0319) for xanthine phosphoribosyltransferase], and choline expression enzyme (CPF_0608 for MarR family/choline/ethanolamine kinase). In contrast, genes involved in the degradation of amino acids (CPF_1245 for L-serine dehydratase), transport of sugar (CPF_2652 for maltose/maltodextrin-binding protein and CPF_1113 for sugar-binding protein), and utilization of mucin [ gngC (CPF_1119) for endo-β-galactosidase, nanA (CPF_0178) for N -acetylneuraminate lyase, and fucP (CPF_1052) for L-fucose: H + symporter permease] had low expression levels. The expression of operon genes (CPF_0890–CPF_0903) responsible for the utilization of ethanolamine abundantly in the intestinal tract and available for various pathogenic bacteria were also decreased: eutA (CPF_0890) for ethanolamine utilization protein, CPF_0891 for ethanolamine ammonia-lyase large subunit, CPF_0892 for ethanolamine ammonia-lyase small subunit, eutL (CPF_0893) for ethanolamine utilization, CPF_0894 for ethanolamine utilization, CPF_0895 for ethanolamine utilization, eutM (CPF_0896) for ethanolamine utilization, CPF_0897 for ethanolamine utilization cobalamin adenosyltransferase, CPF_0898 for ethanolamine utilization, CPF_0899 for ethanolamine utilization protein, eutN (CPF_0900) for ethanolamine utilization, CPF_0901 for ethanolamine utilization, eutH (CPF_0902) for ethanolamine utilization, and eutQ (CPF_0903) for ethanolamine utilization 39 . These downregulated genes did not seem essential to assimilate HA as a carbon source. In the nutrient-poor medium containing mucin, genes coding for endo-β-galactosidase (CPF_1119) involved in the utilization of mucin and constituents of GAG genetic cluster (CPF_0397 for 2-dehydro-3-deoxyphosphogluconate aldolase, CPF_0398 for 2-dehydro-3-deoxygluconokinase, and CPF_0406 for heparin lyase II/III-like protein) were up-regulated. Furthermore, changes in the expression of genes involved in C. perfringens strain 13 virulence are shown in Table 1 . As a result, expression of nagH (CPF_0184), two nagJ (CPF_0875 and CPF_1442), and nagK (CPF_1487), which have been thought to encode major toxins as hyaluronidases, were unexpectedly lowly expressed in the nutrient-poor medium containing HA. On the other hand, virS (CPF_1751), a regulator of virulence factors, was highly expressed. A gene cluster homologous to the GAG genetic cluster of S. pneumoniae was found to be up-regulated in both nutrient-poor media containing each of HA and mucin. Table 2 shows changes in the expression level of each gene constituting the GAG genetic cluster in the nutrient-poor medium containing HA or mucin compared with the nutrient-poor medium. Table 1 Expression change of virulence genes of C. perfringens C. perfringens strain ATCC 13124 Name C. perfringens strain 13 Name Nutrient-poor + HA /nutrient-poor Nutrient-poor + mucin /nutrient-poor CPF_0184 nagH CPE_0191 nagH -4.45 1.08 CPF_1442 nagJ CPE_1234 nagI -1.92 1.29 CPF_0875 nagJ CPE_0881 nagJ -5.67 1.09 CPF_1487 nagK CPE_1279 nagK -3.34 1.34 CPF_1751 virS CPE_1500 virS 2.24 1.26 Table 2 Expression change of constituent genes of GAG genetic cluster name ATCC13124 Nutrient-poor + HA /nutrient-poor Nutrient-poor + mucin /nutrient-poor hysA CPF_0394 1.82 4.80 kduI CPF_0395 9.62 10.99 kduD CPF_0396 7.83 9.41 kdgA CPF_0397 9.63 11.97 kdgK CPF_0398 9.57 11.88 kduI CPF_0399 2.28 6.87 ugl CPF_0400 2.16 8.36 PTS-EIIB CPF_0401 2.76 8.67 PTS-EIIC CPF_0402 2.43 7.96 PTS-EIID CPF_0403 2.41 8.03 PTS-EIIA CPF_0404 6.59 9.27 yajC CPF_0405 8.51 9.89 hepC CPF_0406 10.02 13.64 HysA is the primary enzyme that degrades HA in S. pneumoniae . In C. perfringens , nagH , two nagJ , and nagK genes, which have been thought to encode degraders of HA, had repressed expression levels in the presence of HA, while expression of the hysA homologous gene (CPF_0394) increased. A comparison of RPKM obtained by RNA-seq suggested that the HA-degrading enzyme in C. perfringens may be a product of hysA but not the previous candidates (Fig. 3 b). The GAG genetic cluster, including hysA , enables unsaturated uronic acid generated from GAG degradation to join glycolysis 21 . The expression of genes involved in the utilization of amino sugars was also investigated (Fig. 3 c). The expression level of nagB (CPF_2744) for glucosamine-6-phosphate deaminase and CPF_1718 for phosphofructokinase family protein responsible for metabolism of amino sugar into glycolysis, were up-regulated, whereas glmS (CPF_2636) for fructose-6-phosphate aminotransferase for reverse pathway had decreased expression. Based on the above results, HysA, encoded in the GAG genetic cluster, likely is the enzyme in C. perfringens responsible for the degradation of HA. Moreover, unsaturated uronic acid and amino sugar, the resultant monosaccharides derived through HA degradation, were metabolized by glycolysis, unsaturated uronic acid were metabolized by GAG genetic cluster, and amino sugar by the Nag pathway. Characterization of recombinant CPF_0394, CpeHysA To characterize HysA, the gene for CPF_0394 was cloned into the pET21b vector and expressed in Escherichia coli BL21-Gold(DE3)pLysS system. CPF_0394 is composed of 1,003 amino acid residues. The HA degradation ability was examined by the halo plate assay method using the cell extract obtained from the recombinant E. coli cells, resulting in the formation of a halo (Fig. 4 a). Therefore, the recombinant enzyme was purified by metal affinity, anion exchange, and gel filtration chromatographies to homogeneity. As a result of SDS-PAGE followed by staining with coomassie brilliant blue (CBB), the molecular size of CPF_0394 indicated by a single band was approximately 113 kDa, consistent with the estimated value (Fig. 4 b). The absorbance at 235 nm increased in the CPF_0394 and HA reaction mixture. This indicates that CPF_0394 is a hyaluronate lyase (CpeHysA) directly acting on HA and producing unsaturated HA disaccharide with an absorbency at 235 nm, corresponding to C = C double bonds in unsaturated uronic acid residues. CpeHysA was further characterized by monitoring the absorbance increase at 235 nm (Fig. 4 c-f). To determine the optimal pH, the enzyme activity was assayed in the presence of HA in different pH buffers at 30°C. CpeHysA showed maximum activity at pH 5.5 and no activity below pH 4.5. The enzyme maintained more than 50% of activity in a pH range of 5.0–8.0. CpeHysA showed the highest activity at 60°C, and almost all activity was lost at 70°C. To assess the thermostability, CpeHysA was preincubated at different temperatures in Tris(hydroxymethyl) aminomethane-hydrochloride (Tris-HCl) for 10 min, followed by monitoring the enzyme activity in Tris-HCl at 30°C. The activity was maintained at 55°C, while the enzyme lost most activity at 60°C. As shown in Fig. 4 f, CpeHysA exhibited little degradation activity toward CSC and HP, indicating that the enzyme was specific for HA. Structural model of CpeHysA To investigate the structural conservation in HysA, we modeled the structure of CpeHysA using AlphaFold2 40 . The model structure was superimposed with the crystal structure of S. pneumoniae hyaluronate lyase (SpnHysA) in a complex with unsaturated HA disaccharide (PDB ID, 1C82) (Fig. 5 a) because both CpeHysA and SpnHysA are categorized to polysaccharide lyase family 8 on the CAZy database 41 . While the sequence identity between CpeHysA and SpnHysA is 31%, the root-mean-square deviation is 1.5 Å, indicating that both structures are very similar. The length of amino acid residues of CpeHysA and SpnHysA are 1,003 and 1,066, respectively. However, the three-dimensional structure of SpnHysA consists of Ala168—Ala893, fully functional truncated length. Similar to SpnHysA, the CpeHysA model is divided into two domains (N- and C-terminal domains) linked by a short loop. Unsaturated HA disaccharide is bound to the cleft between N- and C-terminal domains. At the disaccharide-binding site of SpnHysA, Asn349 attracts electrons on the carboxylate group of uronic acid residue to make the C5 proton more acidic 42,43 . His399 removes a relatively acidic C5 proton by imidazole side chain, forming an unsaturated C = C (C4 and C5) double bond. Tyr408 provides a proton to the glycosidic bond oxygen to break the linkage. All three important residues (Asn349, His399, and Tyr408) are structurally conserved in the CpeHysA model (Asn223, His273, and Tyr282) (Fig. 5 b), suggesting the physiological function of the enzyme for the degradation of HA through β-elimination reaction. Overall structure and three catalytically important residues of CpeHysA are also well conserved in our family 8 xanthan lyase (Supplementary Fig. 3) 44 . Identification of HA-degrading enzyme in C. perfringens To identify the intrinsic HA-degrading enzyme in C. perfringens , the culture supernatant and cell extract of C. perfringens in addition to the purified recombinant CpeHysA were subjected to native-PAGE, followed by activity staining with halo plate assay method using HA as a substrate (Fig. 6 a, b). Only one clear halo in the clostridial culture supernatant and cell extract was detected at the same position corresponding to CpeHysA on the native-PAGE gel, suggesting that the HA-degrading enzyme in C. perfringens is CpeHysA. Furthermore, the absorbance at 235 nm increased in the reaction mixtures of HA in the presence of the culture supernatant and cell extract, indicating that the HA-degrading enzyme was a hyaluronate lyase catalyzing a β-elimination reaction through production of unsaturated saccharides with C = C double bonds showing the absorbance at 235 nm. The specific activity of HA-degrading enzyme in the culture supernatant (3.6 units/mg) was remarkably higher than that in the cell extract (0.0041 units/mg), demonstrating that C. perfringens secreted the enzyme extracellularly. Thin-layer chromatography (TLC) revealed that the enzyme released unsaturated HA disaccharide (Fig. 6 c). According to the JSPS KAKENHI report (Grant number: 24590538, https://kaken.nii.ac.jp/ja/file/KAKENHI-PROJECT-24590538/24590538seika.pdf ), the recombinant NagH, NagI, NagJ, NagK, and NagL of C. perfringens strain 13 genes have not shown HA-degrading activity and the recombinant HysA homolog of C. perfringens strain NCTC8237 (ATCC 13124) has exhibited HA-degrading activity. This report is consistent with our native-PAGE, followed by activity staining, enzyme assay, and TLC results. Therefore, CpeHysA was identified as the intrinsic HA-degrading enzyme in C. perfringens . In summary, we identified hyaluronate lyase (CpeHysA), not hyaluronidases, as an HA-degrading enzyme in C. perfringens . Transcriptome analysis showed that HA increased the expression level of hysA and other constituent genes in the GAG genetic cluster but decreased nagHJK . A sole HA-degrading enzyme in the clostridial culture supernatant corresponded to CpeHysA, indicating that CpeHysA degrades HA as a virulence factor in C. perfringens (Fig. 7 ). Since HA induced the GAG genetic cluster, the resultant unsaturated HA disaccharide is likely degraded and metabolized as follows: PTS (CPF_0401—0404) imports unsaturated HA disaccharide into the cytoplasm through phosphorylation of the substrate. In the cytoplasm, UGL (CPF_0400) degrades unsaturated HA disaccharides to unsaturated uronic acid and phosphorylated amino sugar. 4-Deoxy-L- threo -5-hexosulose-uronic acid (DHU) is nonenzymatically converted from unsaturated uronic acid and is metabolized by KduI (CPF_0395 and CPF_0399), the complex structure of which with substrate analogs has recently reported in Lactocaseibacillus (45), and KduD (CPF_0396). The resultant metabolites finally flow into glycolysis. This study is a significant finding that newly identified hyaluronate lyase as an HA-degrading enzyme distinct from conventional hyaluronidases in C. perfringens . Methods Materials Sodium hyaluronate derived from Streptococcus equi was purchased from Sigma-Aldrich. Sodium chondroitin sulfate C and sodium heparin were obtained from Nacalai Tesque. Mucin from the porcine stomach purchased from Fujifilm Wako Pure Chemical Co. was subjected to purification 46 . All other reagents of special grade were commercially available. Microorganisms and culture conditions C. perfringens strain ATCC 13124 (JCM 1290), C. butyricum ATCC 25752 (JCM 1390), and C. difficile ATCC 9689 (JCM 1296) used for experiments were purchased from the RIKEN BioResource Center Microbial Materials Development Office (JCM). Gifu Anaerobic Medium (GAM) medium [1% peptone, 0.3% soy peptone, 1% protease peptone, 1.35% digested serum powder, 0.5% yeast extract, 0.22% meat extract, 0.12% liver extract, 0.3% glucose, 0.25% potassium dihydrogen phosphate, 0.3% sodium chloride, 0.5% soluble starch, 0.03% L-cysteine hydrochloride, and 0.03% sodium thioglycolate (pH 7.1)] was used as a standard medium under anaerobic conditions at 37°C. The medium for assimilation assay and RNA-seq analysis of C. perfringens was prepared as follows: the nutrient-poor medium [0.1% potassium dihydrogen phosphate, 0.1% disodium hydrogen phosphate, 0.01% magnesium sulfate heptahydrate including 20-fold diluted GAM (0.05% peptone, 0.015% soy peptone, 0.05% protease peptone, 0.0675% digested serum powder, 0.025% yeast extract, 0.011% meat extract, 0.006% liver extract, 0.015% glucose, 0.015% sodium chloride, 0.025% soluble starch, 0.0015% L-cysteine hydrochloride, and 0.0015% sodium thioglycolate)] in the presence or absence of 0.2% HA or mucin. Briefly, C. perfringens cells grown in GAM medium overnight were harvested, washed, and suspended with saline to an optical density of 1 at 600 nm (OD 600 = 1). The cell suspensions were inoculated into the nutrient-poor medium in the presence or absence of HA or mucin and cultured for a few days with periodical monitoring of OD 600 . For the degradation assay, 1% BSA and 1% agar were added to the nutrient-poor medium in the presence of HA. For the nutrient-rich halo plates, 0.2% CSC or HP, 1% BSA, and 1% agar were added to GAM medium. Because no white precipitate was detected in the nutrient-rich HA plate after the addition of acetic acid, a glucose-rich medium [0.1% potassium dihydrogenphosphate, 0.1% disodium hydrogenphosphate, 0.01% magnesium sulfate heptahydrate, 0.1% yeast extract, 5% glucose] containing 0.2% HA, 1% BSA, and 1% agar was alternatively used to detect HA degradation. Luria-Bertani (LB) medium [1% tryptone, 0.5% yeast extract, and 1% sodium chloride (pH 7.2)] was used as a medium for E. coli . E. coli BL21-Gold(DE3)pLysS cells harboring the pET21b-CPF_0394 plasmid were cultured at 30°C in LB medium containing 0.1 mg/ml sodium ampicillin to an OD 600 = 0.3–0.7. Then, 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added and incubated at 16°C for 2 days. Halo plate assay for HA degradation The C. perfringens cell suspension was inoculated in the degradation assay medium and anaerobically cultured at 37°C for 7 days 47 . After the addition of 2 M acetic acid 2 mL, the HA-degraded position formed a clear halo. RNA-seq analysis RNA extraction from C. perfringens cells was subjected to the hot phenol method as follows. C. perfringens cells in the logarithmic growth phase were collected by centrifugation at 2,600 × g at 4°C for 5 min and washed with saline. ISOGEN 300 µL (Nippon Gene) was added to the cell suspension, and the mixture was stirred with glass beads by vortexing for 4 min. After further addition of ISOGEN 700 µL, the mixture was incubated at 65°C for 30 min. The supernatant was collected by centrifugation at 12,000 × g at 4°C for 15 min, followed by the addition of chloroform 200 µL. After vortexing for 15 seconds, the mixture was incubated at room temperature for 3 min and centrifuged at 12,000 × g at 4°C for 15 min. Isopropanol (500 µL) was added to the obtained aqueous layer and mixed by inversion, followed by incubation at room temperature for 5 min and centrifugation at 12,000 × g at 4°C for 10 min. Ethanol was added to the resulting precipitate and was centrifuged at 7,500 × g at 4°C for 5 min to obtain a precipitate. The resultant precipitate was dried in a desiccator and dissolved with 100 µL of RNase-free water. Of the obtained RNA extract, 80 µL was used for RNA-seq, and 20 µL was used for purity and quality confirmation. The RNA extract was immediately frozen with liquid nitrogen and stored at − 80°C. The purity and quality of the RNA extract was confirmed by 0.9% agarose gel electrophoresis at 100 mV for 30 min. A quality check of RNA and RNA-seq analysis were performed by Macrogen Co. Japan. DNase treatment was added to the RNA extract to eliminate DNA contamination. To prepare the sequencing libraries, Ribo-Zero rRNA Removal Kit (Bacteria) and TruSeq Stranded Total RNA Sample Prep Kit (Illumina) were used. Paired-end (101 bp) RNA-seq analysis was performed on the Illumina NovaSeq 6000 sequencer. Trimmed reads were mapped to the reference genome ( C. perfringens strain ATCC 13124) with Bowtie. After the read mapping, HTseq was used for expression profiling. The analysis of differentially expressed genes was performed on 3 comparison pairs using RPKM. Construction of plasmid for expression of CPF_0394, CpeHysA To construct the protein expression system in E. coli , the CPF_0394 (CpeHysA) gene was amplified via polymerase chain reaction (PCR) using C. perfringens genomic DNA as a template and oligonucleotides as In-Fusion primers (Supplementary Table 1). The reaction mixture contained PCR buffer for KOD FX Neo (TOYOBO), 4 nmol of dNTPs, 0.8 U of KOD FX Neo, 3 pmol of forward and reverse primers, and genomic DNA. The PCR reaction conditions were as follows: 94°C for 2 min followed by 30 cycles of 98°C for 10 sec, 45.4°C for 30 sec, and 68°C for 2 min. The CPF_0394 gene fragment and pET21b vector were digested with NdeI and XhoI mixed with In-Fusion HD Enzyme Premix Kit (Takara Bio), followed by incubation at 50°C for 15 min. After the transformation of E. coli BL21-Gold(DE3)pLysS cells with the resultant plasmid, the nucleotide sequence of the objective gene was confirmed by DNA sequencing. DNA manipulations were carried out as described elsewhere 48 . Protein purification E. coli BL21-Gold(DE3)pLysS cells harboring pET21b-CPF_0394 were cultured in LB containing ampicillin and IPTG, and harvested by centrifugation at 6,800 × g at 4°C for 10 min. The obtained cells were suspended in 20 mM Tris-HCl (pH 7.5) and disrupted by an ultrasonic generator (Insonator Model 201M, Kubota) at 9 kHz, 0°C for 20 min. The disrupted cells were centrifuged at 20,000 × g at 4°C for 20 min to obtain supernatant as the cell extract. The cell extract was subjected to metal affinity chromatography using TALON resin (Clontech). The cell extract mixed with TALON resin was washed with equilibration buffer [20 mM Tris-HCl (pH 7.5), 500 mM NaCl, and 10 mM imidazole (pH 8.0)] and eluted with elution buffer [20 mM Tris-HCl (pH 7.5), 500 mM NaCl, and 500 mM imidazole (pH 8.0)]. Every eluted fraction was collected, and the purity of the protein in the fractions was assessed by SDS-PAGE 49 . Fractions containing partially purified protein were collected and applied to anion exchange chromatography using Resource Q (GE Healthcare). After washing with equilibration buffer [20 mM Tris-HCl (pH 7.5)], the proteins were eluted with gradient elution buffer [20 mM Tris-HCl (pH 7.5) and 0–1 M NaCl]. Fractions were selected by SDS-PAGE and applied to a gel filtration chromatography using HiLoad 16/60 Superdex 200 pg (GE Healthcare). The proteins were separated by molecular size with elution buffer [20 mM Tris-HCl (pH 7.5) and 0.15 M NaCl] and were subjected to SDS-PAGE, followed by protein staining with CBB. Collected fractions containing the purified protein were combined and dialyzed against 20 mM Tris-HCl (pH 7.5). Enzyme assay The activity of hyaluronate lyase (CPF_0394, CpeHysA) was assayed by monitoring the increase in absorbance at 235 nm derived from the C = C double bonds in unsaturated HA disaccharide. The reaction mixture comprised 50 mM Tris-HCl (pH 7.5), 0.1% HA, and purified CpeHysA. To determine the optimal pH, the enzyme activity was measured in 50 mM buffer of sodium acetate (pH 4.0–5.5), potassium phosphate (pH 6.0–7.5), Tris-HCl (pH 7.5–8.5), and glycine-NaOH (pH 7.5–10.0) at 30°C. The optimal temperature was determined by measuring the enzyme activity in 50 mM Tris-HCl (pH 7.5) at 30°C–70°C. To examine the thermostability, the enzyme was preincubated for 10 min at 30°C–70°C, followed by measuring the activity in 50 mM Tris-HCl (pH 7.5) at 30°C. To measure the intracellular and extracellular activity of HysA in C. perfringens , the bacterial cells grown in 100 mL of the nutrient-poor medium containing HA were harvested to obtain cell pellet and culture supernatant. The resultant cell pellet was subjected to ultrasonication and centrifugation to obtain the cell extract. The culture supernatant and cell extract were subjected to concentration by ammonium sulfate precipitation, dialysis against 20 mM Tris-HCl (pH 7.5), and enzyme assay. TLC The reaction mixture of HA in the presence of the culture supernatant or cell extract of C. perfringens was boiled and centrifuged. The resultant supernatant was subjected to TLC using a developing solvent of 1-butanol: acetic acid: water = 3:2:2. After spraying with 10% sulfuric acid in ethanol, the TLC plate was heated to visualize the HA degradation products. Native-PAGE C. perfringens cells were grown in the nutrient-poor medium containing HA in the logarithmic growth phase and centrifuged to obtain the culture supernatant and bacterial cells. The resultant cells were washed and suspended in 20 mM Tris-HCl (pH 7.5). The cells were ultrasonically disrupted as described above, and the cell extract was obtained by centrifugation of the disrupted cells at 20,000 × g at 4°C for 20 min. The culture supernatant, cell extract, and the recombinant purified CpeHysA were subjected to native-PAGE with 6% separation gel at 0°C in a running buffer consisting of 43 mM imidazole and 35 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH 7.4). After electrophoresis, the gel was placed on the halo assay plate and incubated at 37°C for 24 h. The gel was removed, and acetic acid was added to confirm the degradation of HA. Statistics Significant differences were statistically determined using Student’s t -test (** p < 0.01, * p < 0.05). Declarations Competing interests The authors declare no competing interest. Author Contribution W. H. designed the study, T. K. and S. O. performed the experiments, T. K., S. O., and W. H. analyzed the data, and T. K., S. O., and W. H. wrote the manuscript. Acknowledgments This work was supported in part by JSPS KAKENHI Grant Numbers 15H04629, 18H02166, and 21H02156 (W.H.). The authors would like to thank Enago ( www.enago.com ) for the English language review. Data Availability RNA-seq data have been deposited to the GEO database under accession GSE240236. References Sender, R., Fuchs, S. & Milo, R. Revised estimates for the number of human and bacteria cells in the body. 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A., Anderson, C. J. & Kendall, M. M. Ethanolamine influences human commensal Escherichia coli growth, gene expression, and competition with enterohemorrhagic E. coli O157:H7. mBio 9, e01429-18 (2018). Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583–589 (2021). Drula, E. et al. The carbohydrate-active enzyme database: functions and literature. Nucleic Acids Res 50, D571-D577 (2022). Li, S. et al. Structural basis of hyaluronan degradation by Streptococcus pneumoniae hyaluronate lyase. EMBO J 19, 1228–1240 (2000). Ponnuraj, K. & Jedrzejas, M. J. Mechanism of hyaluronan binding and degradation: Structure of Streptococcus pneumoniae hyaluronate lyase in complex with hyaluronic acid disaccharide at 1.7 Å resolution. J Mol Biol 299, 885–895 (2000). Maruyama, Y., Hashimoto, W., Mikami, B. & Murata, K. Crystal structure of Bacillus sp. GL1 xanthan lyase complexed with a substrate: insights into the enzyme reaction mechanism. 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Supplementary Files Supplementary.docx Cite Share Download PDF Status: Published Journal Publication published 22 Oct, 2024 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 17 Jun, 2024 Reviews received at journal 16 Jun, 2024 Reviewers agreed at journal 06 Jun, 2024 Reviews received at journal 06 Jun, 2024 Reviews received at journal 22 May, 2024 Reviewers agreed at journal 14 May, 2024 Reviewers agreed at journal 13 May, 2024 Reviewers invited by journal 09 May, 2024 Editor assigned by journal 09 May, 2024 Editor invited by journal 28 Apr, 2024 Submission checks completed at journal 25 Apr, 2024 First submitted to journal 23 Apr, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4310962","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":296568073,"identity":"57398d52-d32e-4b18-9527-ce0795fc0214","order_by":0,"name":"Wataru Hashimoto","email":"data:image/png;base64,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","orcid":"","institution":"Kyoto University","correspondingAuthor":true,"prefix":"","firstName":"Wataru","middleName":"","lastName":"Hashimoto","suffix":""},{"id":296568075,"identity":"ef115e9b-b281-46a3-b5e2-93caf291ff23","order_by":1,"name":"Tomoya Kumon","email":"","orcid":"","institution":"Kyoto University","correspondingAuthor":false,"prefix":"","firstName":"Tomoya","middleName":"","lastName":"Kumon","suffix":""},{"id":296568077,"identity":"c52afead-58db-48c7-8e30-19fd19744fe2","order_by":2,"name":"Sayoko Oiki","email":"","orcid":"","institution":"Kyoto University","correspondingAuthor":false,"prefix":"","firstName":"Sayoko","middleName":"","lastName":"Oiki","suffix":""}],"badges":[],"createdAt":"2024-04-23 09:38:44","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4310962/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4310962/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-024-73955-y","type":"published","date":"2024-10-22T15:57:56+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":55730295,"identity":"d1906abe-478e-4177-a9b5-8cefe84780e3","added_by":"auto","created_at":"2024-05-02 11:03:29","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":63294,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGAG genetic clusters. a\u003c/strong\u003e \u003cem\u003eStreptococcus agalactiae\u003c/em\u003e, gbs_XXXX; \u003cem\u003eStreptococcus pneumoniae\u003c/em\u003e, spr_XXXX. \u003cstrong\u003eb\u003c/strong\u003e \u003cem\u003eClostridium perfringens\u003c/em\u003e, CPF_XXXX. Genes coding for proteins and enzymes for import, degradation, and metabolism of GAGs. XXXX, ID no. of each gene.\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4310962/v1/7a62612a850572e36cd592d8.jpg"},{"id":55730297,"identity":"e712d45b-0c8d-466e-9357-e9c1e947b040","added_by":"auto","created_at":"2024-05-02 11:03:29","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":75302,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDegradation and assimilation of GAGs and mucin by \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC. perfringens\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. a, b\u003c/strong\u003e halo assay for the degradation of GAGs (HA, CSC, and HP). Plates before (left) and after (right) addition of acetic acid in nutrient-poor (\u003cstrong\u003ea\u003c/strong\u003e) and nutrient-rich medium (\u003cstrong\u003eb\u003c/strong\u003e). \u003cstrong\u003ec\u003c/strong\u003e growth of \u003cem\u003eC. perfringens\u003c/em\u003e cells in the presence of HA or mucin. Circle, nutrient-poor medium; triangle, nutrient-poor medium containing HA; and square, nutrient-poor medium containing mucin. Each measurement represents the mean of three individual experiments. Significant differences from the control (nutrient-poor medium) were determined using Student’s \u003cem\u003et\u003c/em\u003e-test (\u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4310962/v1/66a988280aea5b110b65208b.jpg"},{"id":55731122,"identity":"599e193e-cbd4-466c-9f8a-89e5e08cecb6","added_by":"auto","created_at":"2024-05-02 11:11:29","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":48869,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscriptome analysis of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC. perfringens\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e in the presence of HA or mucin. a\u003c/strong\u003e the number of differentially expressed genes in nutrient-poor medium containing HA (white) or mucin (black). \u003cstrong\u003eb\u003c/strong\u003e RPKM of candidate genes for HA degradation. Nutrient-poor medium (gray) containing HA (white) or mucin (black). \u003cstrong\u003ec\u003c/strong\u003e the expression of genes involved in the utilization of amino sugar.\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4310962/v1/78ef72cc1782210d9536056b.jpg"},{"id":55730299,"identity":"f0c77393-a038-493b-91a2-22e081c7d882","added_by":"auto","created_at":"2024-05-02 11:03:29","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":61981,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression, purification, and characterization of CPF_0394 (CpeHysA). a\u003c/strong\u003e halo assay for HA degradation using the cell extract obtained from the CpeHysA-expressing recombinant \u003cem\u003eE. coli\u003c/em\u003e. Plates before (left) and after (right) addition of acetic acid. +, BL21-Gold(DE3)pLysS harboring pET21b-CPF_0394; and \u003cem\u003e—\u003c/em\u003e, BL21-Gold(DE3)pLysS. \u003cstrong\u003eb\u003c/strong\u003e SDS-PAGE followed by CBB staining of the purified CpeHysA. Lane M, unstained marker of protein standard; lane P, purified CpeHysA. \u003cstrong\u003ec\u003c/strong\u003e Optimal pH. Buffers were sodium acetate (circle), potassium phosphate (triangle), Tris-HCl (square), and glycine-NaOH (rhombus). The activity measured in sodium acetate (pH 5.5) at 30°C was taken as 100%. \u003cstrong\u003ed\u003c/strong\u003e Optimal temperature. The activity measured in Tris-HCl (pH 7.5) at 60°C was taken as 100%. \u003cstrong\u003ee\u003c/strong\u003e Thermostability. The activity measured in Tris-HCl (pH 7.5) at 30°C using CpeHysA preincubated at 45°C was taken as 100%. \u003cstrong\u003ef\u003c/strong\u003e Substrate specificity. The activity measured in Tris-HCl (pH 7.5) at 30°C using HA was taken as 100%. Each measurement represents the mean of three individual experiments.\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4310962/v1/d19c86e130d0017e8e1967ac.jpg"},{"id":55731123,"identity":"ee3ddb6b-8030-40a1-8bae-f6711ab08058","added_by":"auto","created_at":"2024-05-02 11:11:29","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":95567,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStructural model of CpeHysA. a\u003c/strong\u003esuperimposition of CpeHysA and SpnHysA. Blue, the AlphaFold2 model of CpeHysA; pink, crystal structure of SpnHysA (PDB ID, 1C82). Ball models show unsaturated HA disaccharides. \u003cstrong\u003eb\u003c/strong\u003e the disaccharide-binding site of SpnHysA. Blue, the AlphaFold2 model of CpeHysA; pink, crystal structure of SpnHysA. Stick models indicate the conserved amino acid residues.\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4310962/v1/267da978439ee2af38a445e1.jpg"},{"id":55730301,"identity":"52f5420e-da14-4923-a84b-7e336c4fb9a1","added_by":"auto","created_at":"2024-05-02 11:03:29","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":57420,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHA-degrading activity of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC. perfringens\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. \u003c/strong\u003eNative-PAGE followed by CBB staining (\u003cstrong\u003ea\u003c/strong\u003e) and halo-forming activity staining (Plates before (left) and after (right) addition of acetic acid. The yellow frame indicates the native-PAGE gel.) (\u003cstrong\u003eb\u003c/strong\u003e), and TLC (\u003cstrong\u003ec\u003c/strong\u003e). CpeHysA, the purified recombinant CpeHysA; \u003cem\u003eC. perfringens\u003c/em\u003e S, the culture supernatant of \u003cem\u003eC. perfringens\u003c/em\u003e; \u003cem\u003eC. perfringens\u003c/em\u003e C, the cell extract of \u003cem\u003eC. perfringens\u003c/em\u003e; \u003cem\u003eC. perfringens\u003c/em\u003e C (x40), 40-fold concentrated \u003cem\u003eC. perfringens\u003c/em\u003e C. HA, hyaluronan; ∆HA, unsaturated HA disaccharide.\u003c/p\u003e","description":"","filename":"Picture6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4310962/v1/6b2d10e2a9b3d5559bbb1dec.jpg"},{"id":55730302,"identity":"815929c5-1ad0-4346-8a71-8bf9d823d7a9","added_by":"auto","created_at":"2024-05-02 11:03:30","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":69479,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eC. perfringens\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e model for degradation, import, and metabolism of HA. \u003c/strong\u003eHysA (CPF_0394) degrades HA to unsaturated HA disaccharides on the cell surface. PTS (CPF_0401—0404) in the cytoplasmic membrane imports unsaturated HA disaccharide into the cytoplasm through phosphorylation. In the cytoplasm, UGL (CPF_0400) degrades unsaturated HA disaccharides to unsaturated uronic acid and phosphorylated amino sugar. DHU, nonenzymatically converted from unsaturated uronic acid, is metabolized by KduI (CPF_0395 and CPF_0399) and KduD (CPF_0396). The resultant is finally metabolized into glycolysis.\u003c/p\u003e","description":"","filename":"Picture7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4310962/v1/18762f8bdbe46a913efcabd7.jpg"},{"id":67684466,"identity":"cbe589a3-05db-41bb-a0a4-009d6012e38b","added_by":"auto","created_at":"2024-10-28 16:23:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1256277,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4310962/v1/38603a58-4dc1-4705-86fe-cd1f74080adc.pdf"},{"id":55730300,"identity":"8a21eb43-6ebf-4277-9070-af9308312f77","added_by":"auto","created_at":"2024-05-02 11:03:29","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":7542305,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-4310962/v1/1d68ca4b4f88e775028dbb62.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Molecular identification of hyaluronate lyase, not hyaluronidase, as an intrinsic hyaluronan-degrading enzyme in Clostridium perfringens","fulltext":[{"header":"Introduction","content":"\u003cp\u003eApproximately 38 trillion bacteria, more than all human cells, are estimated to be indigenous to the human gut\u003csup\u003e1\u003c/sup\u003e. Intestinal bacteria establish competitive and symbiotic relationships through interactions with the host and other bacteria in the gut, where various nutrients are intermittently supplied by the host's diet\u003csup\u003e2\u0026ndash;4\u003c/sup\u003e. The gut microbiota is suggested to be correlated with host health, such as regulation of the immune system, allergy, inflammatory bowel disease, obesity, diabetes, and cancer\u003csup\u003e5\u0026ndash;11\u003c/sup\u003e. While some beneficial bacteria, \u003cem\u003ee.g.\u003c/em\u003e, genera of \u003cem\u003eBifidobacterium\u003c/em\u003e and \u003cem\u003eLactobacillus\u003c/em\u003e, are indigenous to the gut, opportunistic pathogens, including genera of \u003cem\u003eClostridium\u003c/em\u003e and \u003cem\u003eStreptococcus\u003c/em\u003e, can also exist. However, the mechanism of bacterial colonization in the gut remains to be clarified.\u003c/p\u003e \u003cp\u003eAll animal cells, including the gut, are covered with a noncellular component called extracellular matrices. Extracellular matrices, composed of water, proteins, and polysaccharides, provide a physical scaffold for cells and promote cell differentiation and homeostasis\u003csup\u003e12\u003c/sup\u003e. Proteoglycans, a part of extracellular matrices, comprise glycosaminoglycans (GAGs) linked to core proteins. GAGs are heteropolysaccharides with a disaccharide-repeating unit of uronic acid (or galactose) and amino sugar\u003csup\u003e13\u003c/sup\u003e. Chondroitin sulfate C (CSC) and heparin (HP) are representative sulfated GAGs that constitute proteoglycans, while hyaluronan (HA), a representative nonsulfated GAG, exists independently of proteoglycans. HA is the most abundant GAG in the gut epithelium\u003csup\u003e14\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn \u003cem\u003eStreptococcus\u003c/em\u003e species, polysaccharide lyase and phosphotransferase system (PTS) are involved in the degradation and import of HA, respectively\u003csup\u003e15,16\u003c/sup\u003e. Our previous studies have demonstrated the streptococcal action on HA as follows\u003csup\u003e17\u0026ndash;21\u003c/sup\u003e: Hyaluronate lyase is inducibly expressed in the presence of HA and essential for the assimilation of nutrient HA, followed by pathogenic damage to host tissues. Unsaturated HA disaccharide generated from HA through the lyase reaction is imported into the cytoplasm in the specific PTS, followed by degradation by cytoplasmic unsaturated glucuronyl hydrolase (UGL). The resultant unsaturated uronic acid is metabolized into glycolysis by isomerase, dehydrogenase, and other enzymes (Supplementary Fig.\u0026nbsp;1)\u003csup\u003e18\u0026ndash;21\u003c/sup\u003e. These enzymes and transporter are encoded as a GAG genetic cluster in the streptococcal genome\u003csup\u003e19\u003c/sup\u003e. Some pathogenic bacteria, such as \u003cem\u003eStreptococcus pneumoniae\u003c/em\u003e and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, degrade GAGs and infect host cells\u003csup\u003e16,22\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIntestinal epithelial cells are covered with a mucin layer outside the extracellular matrices. Mucin is a macromolecule composed of core proteins linked to sugar chains, including sialic acid, fucose, galactose, and amino sugar. The mucin layer, formed of mucins secreted from goblet cells within the intestinal epithelium, contains a low-density outer mucus layer and a high-density inner mucus layer. Intestinal bacteria are present in the outer mucus layer and utilize the mucin\u003csup\u003e3,23\u003c/sup\u003e. In contrast, the inner mucus layer functions as a barrier to block interactions between bacteria and host, thus leaving the bacteria undetectable\u003csup\u003e23,24\u003c/sup\u003e. However, it remains unclear whether pathogens that infect and invade host epithelial cells degrade the mucin layer.\u003c/p\u003e \u003cp\u003e \u003cem\u003eClostridium perfringens\u003c/em\u003e is a Gram-positive, anaerobic, and spore-forming bacterium that belongs to the Bacillota phylum, one of the most abundant phyla among intestinal bacteria. \u003cem\u003eC. perfringens\u003c/em\u003e is a major component of the adult human gut microbiota, increasing in abundance from the ileum, the cecum, and the rectum\u003csup\u003e25\u003c/sup\u003e. However, this bacterium can cause life-threatening gas gangrene and food poisoning in humans and animals\u003csup\u003e26\u003c/sup\u003e. The clostridial virulence is largely attributed to the production of approximately twenty different toxins. For example, alpha-toxins, such as phospholipase, degrade phosphatidylcholine and sphingomyelin in the plasma membrane and cause endocytosis and cell death through the formation of diacylglycerol and ceramide\u003csup\u003e27\u003c/sup\u003e. \u003cem\u003eC. perfringens\u003c/em\u003e also produce degrading enzymes such as hyaluronidase (mu-toxin), collagenase (kappa-toxin), proteases, and sialidase, to decompose connective tissue, leading to colonization and infection of the surrounding tissues\u003csup\u003e28\u003c/sup\u003e. Furthermore, mu-toxin, while itself is non-lethal, facilitates the spread of the major alpha-toxin. An endo-β-\u003cem\u003eN\u003c/em\u003e-acetylglucosaminidase, NagH, has been identified as the hyaluronidase by screening recombinant lambda phages for glucuronidase activity\u003csup\u003e29\u003c/sup\u003e. Although NagH has long been considered the HA-degrading enzyme and a virulence factor, the intrinsic enzyme crucial for HA degradation has not yet been elucidated. Two complete genome sequences of \u003cem\u003eC. perfringens\u003c/em\u003e strains 13 and ATCC 13124 were described by Shimizu \u003cem\u003eet al.\u003c/em\u003e and Garry \u003cem\u003eet al.\u003c/em\u003e, respectively\u003csup\u003e30,31\u003c/sup\u003e. Based on these studies, five candidate genes, \u003cem\u003enagH\u003c/em\u003e (CPE0191), \u003cem\u003enagI\u003c/em\u003e (CPE0881), \u003cem\u003enagJ\u003c/em\u003e (CPE1234), \u003cem\u003enagK\u003c/em\u003e (CPE1279), and \u003cem\u003enagL\u003c/em\u003e (CPE1523), coding for hyaluronidases and \u003cem\u003enanJ\u003c/em\u003e (CPE0553) coding for sialidase, have been thought to be virulence factors in \u003cem\u003eC. perfringens\u003c/em\u003e strain 13\u003csup\u003e30\u003c/sup\u003e. The \u003cem\u003enagH\u003c/em\u003e gene (CPF_0184) coding for hyaluronoglucosaminidase, \u003cem\u003enagJ\u003c/em\u003e (CPF_0875) coding for putative \u003cem\u003eO\u003c/em\u003e-linked \u003cem\u003eN\u003c/em\u003e-acetylglucosaminidase annotated as \u003cem\u003enagI\u003c/em\u003e in \u003cem\u003eC. perfringens\u003c/em\u003e strain 13, \u003cem\u003enagJ\u003c/em\u003e (CPF_1442) and \u003cem\u003enagK\u003c/em\u003e (CPF_1487) were conserved in the \u003cem\u003eC. perfringens\u003c/em\u003e strain ATCC 13124 genome. However, the \u003cem\u003enagL\u003c/em\u003e gene was not conserved.\u003c/p\u003e \u003cp\u003eVirS and VirR, a two-component system involved in regulating the expression of virulence factors, have also been identified\u003csup\u003e32\u003c/sup\u003e. VirS, a membrane sensor protein, receives environmental and intracellular signals via Agr, an accessory gene regulator, causing autophosphorylation. VirR is activated in the cytoplasm by receiving a phosphate group from VirS, resulting in control of transcriptional expression. The VirS/VirR regulon positively regulates major toxins, such as phospholipase C and collagenase, suggesting an important role during host infection. At the same time, the system mainly acts as a global repressor that negatively regulates general genes\u003csup\u003e33\u003c/sup\u003e. In contrast, RevR, a virulence regulator, is a global activator\u003csup\u003e34\u003c/sup\u003e. Transcriptome analysis by RNA sequence (RNA-seq) and DNA microarray of these three regulators of virulence factors indicated that the hyaluronidase candidate genes (\u003cem\u003enagH\u003c/em\u003e, \u003cem\u003enagI\u003c/em\u003e, and \u003cem\u003enagJ\u003c/em\u003e) and sporulation protein (\u003cem\u003esigF\u003c/em\u003e, \u003cem\u003eftsN\u003c/em\u003e, \u003cem\u003espoIIIAG\u003c/em\u003e, \u003cem\u003esigG\u003c/em\u003e, and \u003cem\u003espoIVA\u003c/em\u003e) as virulence factors are positively regulated by RevR, contrary to the negative regulation by VirR\u003csup\u003e28,34\u0026ndash;36\u003c/sup\u003e. Based on this observation, NagH also potentially degrades HA in \u003cem\u003eC. perfringens\u003c/em\u003e. This study identifies the intrinsic HA-degrading enzyme in \u003cem\u003eC. perfringens\u003c/em\u003e by transcriptome analysis and enzyme characterization.\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003e \u003cb\u003eDegradation and assimilation of HA by\u003c/b\u003e \u003cb\u003eC. perfringens\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe previously found the GAG genetic cluster, in the \u003cem\u003eC. perfringens\u003c/em\u003e strain ATCC 13124 genome as well as in the genomes of \u003cem\u003eStreptococcus agalactiae\u003c/em\u003e and \u003cem\u003eS. pneumoniae\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e1\u003c/span\u003e)\u003csup\u003e21\u003c/sup\u003e. While \u003cem\u003ehysA\u003c/em\u003e (CPF_0394), coding for hyaluronate lyase, and \u003cem\u003ehepC\u003c/em\u003e (CPF_0406), coding for heparin lyase II/III-like protein, were found in the cluster, the HA-degrading enzyme in \u003cem\u003eC. perfringens\u003c/em\u003e remained to be identified. \u003cem\u003enagHIJK\u003c/em\u003e, coding for hyaluronidases, which have been considered as candidate genes for HA degradation, were not found in the GAG genetic cluster. On the other hand, the cluster contains homologous genes coding for transporter and enzymes involved in the degradation and metabolism of HA. Therefore, \u003cem\u003ehysA\u003c/em\u003e possibly plays a role in HA degradation as well as in virulence factors. In addition, it has been suggested that \u003cem\u003eC. perfringens\u003c/em\u003e degrades and assimilates mucin by a sialidase, NanI, indicating that degradation of host mucosubstances such as HA and mucin is important for \u003cem\u003eC. perfringens\u003c/em\u003e to colonize the human intestine\u003csup\u003e37,38\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo clarify the clostridial molecular mechanism of HA degradation, we first examined the GAG degradation ability of \u003cem\u003eC. perfringens\u003c/em\u003e and other related species, such as \u003cem\u003eClostridium butyncum\u003c/em\u003e and \u003cem\u003eClostridioides difficile\u003c/em\u003e, through the halo plate assay method (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, b). After clostridial cells were grown on halo plates containing GAGs and bovine serum albumin (BSA) in a nutrient-poor medium (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e2\u003c/span\u003ea) or a nutrient-rich medium (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e2\u003c/span\u003eb), acetic acid was added to confirm the presence or absence of a halo in a white precipitate. \u003cem\u003eC. perfringens\u003c/em\u003e formed a halo in the presence of HA, while no halo was detected in the presence of CSC or HP, indicating HA degradation by \u003cem\u003eC. perfringens\u003c/em\u003e. However, other species tested showed no halo, suggesting no GAG degradation. HA assimilation by \u003cem\u003eC. perfringens\u003c/em\u003e was also examined by culturing in a nutrient-poor medium, excluding BSA and agar from the halo plate (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). \u003cem\u003eC. perfringens\u003c/em\u003e cells showed significantly higher growth in the presence of HA than in the absence of HA. Moreover, \u003cem\u003eC. perfringens\u003c/em\u003e also grew in the nutrient-poor medium containing mucin, indicating that the bacterial cells assimilate mucin and HA. Therefore, \u003cem\u003eC. perfringens\u003c/em\u003e grown on the nutrient-poor medium in the presence of HA was expected to express genes involved in the degradation, import, and assimilation of GAGs in response to HA.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTranscriptome analysis of\u003c/b\u003e \u003cb\u003eC. perfringens\u003c/b\u003e \u003cb\u003ein the presence of HA or mucin\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate the gene expression in the presence of HA or mucin, we performed an RNA-seq analysis of \u003cem\u003eC. perfringens\u003c/em\u003e cells grown in the nutrient-poor medium in the presence and absence of HA or mucin. The data are shown in Supplementary Fig.\u0026nbsp;2. In the case of \u003cem\u003eC. perfringens\u003c/em\u003e cells grown in the nutrient-poor medium, the dataset contained 49,915,882 reads with a length of 101 nucleotides. The reads obtained from the cells grown in the nutrient-poor medium containing HA and mucin were 49,913,574 and 68,868,196, respectively. The trimming data by sliding window method results were as follows: the nutrient-poor medium, 44,776,526; the nutrient-poor medium containing HA, 44,736,304; and the nutrient-poor medium containing mucin, 60,852,264. The mapping data occupied 90.70%, 90.53%, and 89.33%, respectively. Normalization was performed using RPKM (Reads Per Kilobase per Million mapped reads). Of the 2,921 genes, 2,735 genes for which RPKM could be calculated were analyzed. Figure\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e3\u003c/span\u003ea shows the number of differentially expressed genes by 10-fold or more in the nutrient-poor medium containing HA compared with the nutrient-poor medium. In the nutrient-poor medium in the presence of HA, 65 genes were highly expressed, while the expression level of 92 genes was significantly decreased. No genes decreased in the nutrient-poor medium containing mucin, while seven genes showed a high expression level.\u003c/p\u003e \u003cp\u003eIn response to both HA and mucin, the expression of \u003cem\u003ebioB\u003c/em\u003e (CPF_1795), coding for biotin synthase, the product of which is an essential vitamin for animals, was elevated 49.3-fold and 12.5-fold in the nutrient-poor medium containing HA and mucin, respectively, compared with the nutrient-poor medium. This suggests the possibility that the gut-predominant \u003cem\u003eC. perfringens\u003c/em\u003e provides the host with essential biotin through the assimilation of HA and mucin.\u003c/p\u003e \u003cp\u003eIn the nutrient-poor medium containing HA, the following genes involved in cell proliferation were highly expressed: synthesis and transport of amino acid [CPF_0170 coding for cysteine synthase, CPF_0756 for the proton/sodium glutamate symporter, and \u003cem\u003eminC\u003c/em\u003e (CPF_2394) for the probable septum site determining protein], purine-pyrimidine synthase [CPF_1001 for hypothetical protein and \u003cem\u003expt1\u003c/em\u003e (CPF_0319) for xanthine phosphoribosyltransferase], and choline expression enzyme (CPF_0608 for MarR family/choline/ethanolamine kinase). In contrast, genes involved in the degradation of amino acids (CPF_1245 for L-serine dehydratase), transport of sugar (CPF_2652 for maltose/maltodextrin-binding protein and CPF_1113 for sugar-binding protein), and utilization of mucin [\u003cem\u003egngC\u003c/em\u003e (CPF_1119) for endo-β-galactosidase, \u003cem\u003enanA\u003c/em\u003e (CPF_0178) for \u003cem\u003eN\u003c/em\u003e-acetylneuraminate lyase, and \u003cem\u003efucP\u003c/em\u003e (CPF_1052) for L-fucose: H\u003csup\u003e+\u003c/sup\u003e symporter permease] had low expression levels. The expression of operon genes (CPF_0890\u0026ndash;CPF_0903) responsible for the utilization of ethanolamine abundantly in the intestinal tract and available for various pathogenic bacteria were also decreased: \u003cem\u003eeutA\u003c/em\u003e (CPF_0890) for ethanolamine utilization protein, CPF_0891 for ethanolamine ammonia-lyase large subunit, CPF_0892 for ethanolamine ammonia-lyase small subunit, \u003cem\u003eeutL\u003c/em\u003e (CPF_0893) for ethanolamine utilization, CPF_0894 for ethanolamine utilization, CPF_0895 for ethanolamine utilization, \u003cem\u003eeutM\u003c/em\u003e (CPF_0896) for ethanolamine utilization, CPF_0897 for ethanolamine utilization cobalamin adenosyltransferase, CPF_0898 for ethanolamine utilization, CPF_0899 for ethanolamine utilization protein, \u003cem\u003eeutN\u003c/em\u003e (CPF_0900) for ethanolamine utilization, CPF_0901 for ethanolamine utilization, \u003cem\u003eeutH\u003c/em\u003e (CPF_0902) for ethanolamine utilization, and \u003cem\u003eeutQ\u003c/em\u003e (CPF_0903) for ethanolamine utilization\u003csup\u003e39\u003c/sup\u003e. These downregulated genes did not seem essential to assimilate HA as a carbon source.\u003c/p\u003e \u003cp\u003eIn the nutrient-poor medium containing mucin, genes coding for endo-β-galactosidase (CPF_1119) involved in the utilization of mucin and constituents of GAG genetic cluster (CPF_0397 for 2-dehydro-3-deoxyphosphogluconate aldolase, CPF_0398 for 2-dehydro-3-deoxygluconokinase, and CPF_0406 for heparin lyase II/III-like protein) were up-regulated. Furthermore, changes in the expression of genes involved in \u003cem\u003eC. perfringens\u003c/em\u003e strain 13 virulence are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. As a result, expression of \u003cem\u003enagH\u003c/em\u003e (CPF_0184), two \u003cem\u003enagJ\u003c/em\u003e (CPF_0875 and CPF_1442), and \u003cem\u003enagK\u003c/em\u003e (CPF_1487), which have been thought to encode major toxins as hyaluronidases, were unexpectedly lowly expressed in the nutrient-poor medium containing HA. On the other hand, \u003cem\u003evirS\u003c/em\u003e (CPF_1751), a regulator of virulence factors, was highly expressed. A gene cluster homologous to the GAG genetic cluster of \u003cem\u003eS. pneumoniae\u003c/em\u003e was found to be up-regulated in both nutrient-poor media containing each of HA and mucin. Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows changes in the expression level of each gene constituting the GAG genetic cluster in the nutrient-poor medium containing HA or mucin compared with the nutrient-poor medium.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eExpression change of virulence genes of \u003cem\u003eC. perfringens\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eC. perfringens\u003c/em\u003e strain ATCC 13124\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eName\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eC. perfringens\u003c/em\u003e\u003c/p\u003e \u003cp\u003estrain 13\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eName\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNutrient-poor\u0026thinsp;+\u0026thinsp;HA\u003c/p\u003e \u003cp\u003e/nutrient-poor\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNutrient-poor\u0026thinsp;+\u0026thinsp;mucin\u003c/p\u003e \u003cp\u003e/nutrient-poor\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCPF_0184\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003enagH\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCPE_0191\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003enagH\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e-4.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.08\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCPF_1442\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003enagJ\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCPE_1234\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003enagI\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e-1.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.29\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCPF_0875\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003enagJ\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCPE_0881\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003enagJ\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e-5.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.09\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCPF_1487\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003enagK\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCPE_1279\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003enagK\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e-3.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.34\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCPF_1751\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003evirS\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCPE_1500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003evirS\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.26\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eExpression change of constituent genes of GAG genetic cluster\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ename\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eATCC13124\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNutrient-poor\u0026thinsp;+\u0026thinsp;HA\u003c/p\u003e \u003cp\u003e/nutrient-poor\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNutrient-poor\u0026thinsp;+\u0026thinsp;mucin\u003c/p\u003e \u003cp\u003e/nutrient-poor\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ehysA\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCPF_0394\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4.80\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ekduI\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCPF_0395\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e10.99\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ekduD\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCPF_0396\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e9.41\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ekdgA\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCPF_0397\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e11.97\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ekdgK\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCPF_0398\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e11.88\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ekduI\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCPF_0399\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e6.87\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eugl\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCPF_0400\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e8.36\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePTS-EIIB\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCPF_0401\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e8.67\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePTS-EIIC\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCPF_0402\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e7.96\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePTS-EIID\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCPF_0403\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e8.03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePTS-EIIA\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCPF_0404\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e9.27\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eyajC\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCPF_0405\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e9.89\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ehepC\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCPF_0406\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e13.64\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eHysA is the primary enzyme that degrades HA in \u003cem\u003eS. pneumoniae\u003c/em\u003e. In \u003cem\u003eC. perfringens\u003c/em\u003e, \u003cem\u003enagH\u003c/em\u003e, two \u003cem\u003enagJ\u003c/em\u003e, and \u003cem\u003enagK\u003c/em\u003e genes, which have been thought to encode degraders of HA, had repressed expression levels in the presence of HA, while expression of the \u003cem\u003ehysA\u003c/em\u003e homologous gene (CPF_0394) increased. A comparison of RPKM obtained by RNA-seq suggested that the HA-degrading enzyme in \u003cem\u003eC. perfringens\u003c/em\u003e may be a product of \u003cem\u003ehysA\u003c/em\u003e but not the previous candidates (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). The GAG genetic cluster, including \u003cem\u003ehysA\u003c/em\u003e, enables unsaturated uronic acid generated from GAG degradation to join glycolysis\u003csup\u003e21\u003c/sup\u003e. The expression of genes involved in the utilization of amino sugars was also investigated (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). The expression level of \u003cem\u003enagB\u003c/em\u003e (CPF_2744) for glucosamine-6-phosphate deaminase and CPF_1718 for phosphofructokinase family protein responsible for metabolism of amino sugar into glycolysis, were up-regulated, whereas \u003cem\u003eglmS\u003c/em\u003e (CPF_2636) for fructose-6-phosphate aminotransferase for reverse pathway had decreased expression. Based on the above results, HysA, encoded in the GAG genetic cluster, likely is the enzyme in \u003cem\u003eC. perfringens\u003c/em\u003e responsible for the degradation of HA. Moreover, unsaturated uronic acid and amino sugar, the resultant monosaccharides derived through HA degradation, were metabolized by glycolysis, unsaturated uronic acid were metabolized by GAG genetic cluster, and amino sugar by the Nag pathway.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCharacterization of recombinant CPF_0394, CpeHysA\u003c/h2\u003e \u003cp\u003eTo characterize HysA, the gene for CPF_0394 was cloned into the pET21b vector and expressed in \u003cem\u003eEscherichia coli\u003c/em\u003e BL21-Gold(DE3)pLysS system. CPF_0394 is composed of 1,003 amino acid residues. The HA degradation ability was examined by the halo plate assay method using the cell extract obtained from the recombinant \u003cem\u003eE. coli\u003c/em\u003e cells, resulting in the formation of a halo (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Therefore, the recombinant enzyme was purified by metal affinity, anion exchange, and gel filtration chromatographies to homogeneity. As a result of SDS-PAGE followed by staining with coomassie brilliant blue (CBB), the molecular size of CPF_0394 indicated by a single band was approximately 113 kDa, consistent with the estimated value (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e4\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eThe absorbance at 235 nm increased in the CPF_0394 and HA reaction mixture. This indicates that CPF_0394 is a hyaluronate lyase (CpeHysA) directly acting on HA and producing unsaturated HA disaccharide with an absorbency at 235 nm, corresponding to C\u0026thinsp;=\u0026thinsp;C double bonds in unsaturated uronic acid residues. CpeHysA was further characterized by monitoring the absorbance increase at 235 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e4\u003c/span\u003ec-f). To determine the optimal pH, the enzyme activity was assayed in the presence of HA in different pH buffers at 30\u0026deg;C. CpeHysA showed maximum activity at pH 5.5 and no activity below pH 4.5. The enzyme maintained more than 50% of activity in a pH range of 5.0\u0026ndash;8.0. CpeHysA showed the highest activity at 60\u0026deg;C, and almost all activity was lost at 70\u0026deg;C. To assess the thermostability, CpeHysA was preincubated at different temperatures in Tris(hydroxymethyl) aminomethane-hydrochloride (Tris-HCl) for 10 min, followed by monitoring the enzyme activity in Tris-HCl at 30\u0026deg;C. The activity was maintained at 55\u0026deg;C, while the enzyme lost most activity at 60\u0026deg;C. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e4\u003c/span\u003ef, CpeHysA exhibited little degradation activity toward CSC and HP, indicating that the enzyme was specific for HA.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eStructural model of CpeHysA\u003c/h2\u003e \u003cp\u003eTo investigate the structural conservation in HysA, we modeled the structure of CpeHysA using AlphaFold2\u003csup\u003e40\u003c/sup\u003e. The model structure was superimposed with the crystal structure of \u003cem\u003eS. pneumoniae\u003c/em\u003e hyaluronate lyase (SpnHysA) in a complex with unsaturated HA disaccharide (PDB ID, 1C82) (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e5\u003c/span\u003ea) because both CpeHysA and SpnHysA are categorized to polysaccharide lyase family 8 on the CAZy database\u003csup\u003e41\u003c/sup\u003e. While the sequence identity between CpeHysA and SpnHysA is 31%, the root-mean-square deviation is 1.5 \u0026Aring;, indicating that both structures are very similar. The length of amino acid residues of CpeHysA and SpnHysA are 1,003 and 1,066, respectively. However, the three-dimensional structure of SpnHysA consists of Ala168\u0026mdash;Ala893, fully functional truncated length. Similar to SpnHysA, the CpeHysA model is divided into two domains (N- and C-terminal domains) linked by a short loop. Unsaturated HA disaccharide is bound to the cleft between N- and C-terminal domains. At the disaccharide-binding site of SpnHysA, Asn349 attracts electrons on the carboxylate group of uronic acid residue to make the C5 proton more acidic\u003csup\u003e42,43\u003c/sup\u003e. His399 removes a relatively acidic C5 proton by imidazole side chain, forming an unsaturated C\u0026thinsp;=\u0026thinsp;C (C4 and C5) double bond. Tyr408 provides a proton to the glycosidic bond oxygen to break the linkage. All three important residues (Asn349, His399, and Tyr408) are structurally conserved in the CpeHysA model (Asn223, His273, and Tyr282) (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e5\u003c/span\u003eb), suggesting the physiological function of the enzyme for the degradation of HA through β-elimination reaction. Overall structure and three catalytically important residues of CpeHysA are also well conserved in our family 8 xanthan lyase (Supplementary Fig.\u0026nbsp;3)\u003csup\u003e44\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIdentification of HA-degrading enzyme in\u003c/b\u003e \u003cb\u003eC. perfringens\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo identify the intrinsic HA-degrading enzyme in \u003cem\u003eC. perfringens\u003c/em\u003e, the culture supernatant and cell extract of \u003cem\u003eC. perfringens\u003c/em\u003e in addition to the purified recombinant CpeHysA were subjected to native-PAGE, followed by activity staining with halo plate assay method using HA as a substrate (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, b). Only one clear halo in the clostridial culture supernatant and cell extract was detected at the same position corresponding to CpeHysA on the native-PAGE gel, suggesting that the HA-degrading enzyme in \u003cem\u003eC. perfringens\u003c/em\u003e is CpeHysA. Furthermore, the absorbance at 235 nm increased in the reaction mixtures of HA in the presence of the culture supernatant and cell extract, indicating that the HA-degrading enzyme was a hyaluronate lyase catalyzing a β-elimination reaction through production of unsaturated saccharides with C\u0026thinsp;=\u0026thinsp;C double bonds showing the absorbance at 235 nm. The specific activity of HA-degrading enzyme in the culture supernatant (3.6 units/mg) was remarkably higher than that in the cell extract (0.0041 units/mg), demonstrating that \u003cem\u003eC. perfringens\u003c/em\u003e secreted the enzyme extracellularly. Thin-layer chromatography (TLC) revealed that the enzyme released unsaturated HA disaccharide (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). According to the JSPS KAKENHI report (Grant number: 24590538, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://kaken.nii.ac.jp/ja/file/KAKENHI-PROJECT-24590538/24590538seika.pdf\u003c/span\u003e\u003cspan address=\"https://kaken.nii.ac.jp/ja/file/KAKENHI-PROJECT-24590538/24590538seika.pdf\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), the recombinant NagH, NagI, NagJ, NagK, and NagL of \u003cem\u003eC. perfringens\u003c/em\u003e strain 13 genes have not shown HA-degrading activity and the recombinant HysA homolog of \u003cem\u003eC. perfringens\u003c/em\u003e strain NCTC8237 (ATCC 13124) has exhibited HA-degrading activity. This report is consistent with our native-PAGE, followed by activity staining, enzyme assay, and TLC results. Therefore, CpeHysA was identified as the intrinsic HA-degrading enzyme in \u003cem\u003eC. perfringens\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eIn summary, we identified hyaluronate lyase (CpeHysA), not hyaluronidases, as an HA-degrading enzyme in \u003cem\u003eC. perfringens\u003c/em\u003e. Transcriptome analysis showed that HA increased the expression level of \u003cem\u003ehysA\u003c/em\u003e and other constituent genes in the GAG genetic cluster but decreased \u003cem\u003enagHJK\u003c/em\u003e. A sole HA-degrading enzyme in the clostridial culture supernatant corresponded to CpeHysA, indicating that CpeHysA degrades HA as a virulence factor in \u003cem\u003eC. perfringens\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Since HA induced the GAG genetic cluster, the resultant unsaturated HA disaccharide is likely degraded and metabolized as follows: PTS (CPF_0401\u0026mdash;0404) imports unsaturated HA disaccharide into the cytoplasm through phosphorylation of the substrate. In the cytoplasm, UGL (CPF_0400) degrades unsaturated HA disaccharides to unsaturated uronic acid and phosphorylated amino sugar. 4-Deoxy-L-\u003cem\u003ethreo\u003c/em\u003e-5-hexosulose-uronic acid (DHU) is nonenzymatically converted from unsaturated uronic acid and is metabolized by KduI (CPF_0395 and CPF_0399), the complex structure of which with substrate analogs has recently reported in \u003cem\u003eLactocaseibacillus\u003c/em\u003e (45), and KduD (CPF_0396). The resultant metabolites finally flow into glycolysis. This study is a significant finding that newly identified hyaluronate lyase as an HA-degrading enzyme distinct from conventional hyaluronidases in \u003cem\u003eC. perfringens\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e "},{"header":"Methods","content":"\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003eSodium hyaluronate derived from \u003cem\u003eStreptococcus equi\u003c/em\u003e was purchased from Sigma-Aldrich. Sodium chondroitin sulfate C and sodium heparin were obtained from Nacalai Tesque. Mucin from the porcine stomach purchased from Fujifilm Wako Pure Chemical Co. was subjected to purification\u003csup\u003e46\u003c/sup\u003e. All other reagents of special grade were commercially available.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eMicroorganisms and culture conditions\u003c/h2\u003e \u003cp\u003e \u003cem\u003eC. perfringens\u003c/em\u003e strain ATCC 13124 (JCM 1290), \u003cem\u003eC. butyricum\u003c/em\u003e ATCC 25752 (JCM 1390), and \u003cem\u003eC. difficile\u003c/em\u003e ATCC 9689 (JCM 1296) used for experiments were purchased from the RIKEN BioResource Center Microbial Materials Development Office (JCM). Gifu Anaerobic Medium (GAM) medium [1% peptone, 0.3% soy peptone, 1% protease peptone, 1.35% digested serum powder, 0.5% yeast extract, 0.22% meat extract, 0.12% liver extract, 0.3% glucose, 0.25% potassium dihydrogen phosphate, 0.3% sodium chloride, 0.5% soluble starch, 0.03% L-cysteine hydrochloride, and 0.03% sodium thioglycolate (pH 7.1)] was used as a standard medium under anaerobic conditions at 37\u0026deg;C. The medium for assimilation assay and RNA-seq analysis of \u003cem\u003eC. perfringens\u003c/em\u003e was prepared as follows: the nutrient-poor medium [0.1% potassium dihydrogen phosphate, 0.1% disodium hydrogen phosphate, 0.01% magnesium sulfate heptahydrate including 20-fold diluted GAM (0.05% peptone, 0.015% soy peptone, 0.05% protease peptone, 0.0675% digested serum powder, 0.025% yeast extract, 0.011% meat extract, 0.006% liver extract, 0.015% glucose, 0.015% sodium chloride, 0.025% soluble starch, 0.0015% L-cysteine hydrochloride, and 0.0015% sodium thioglycolate)] in the presence or absence of 0.2% HA or mucin. Briefly, \u003cem\u003eC. perfringens\u003c/em\u003e cells grown in GAM medium overnight were harvested, washed, and suspended with saline to an optical density of 1 at 600 nm (OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1). The cell suspensions were inoculated into the nutrient-poor medium in the presence or absence of HA or mucin and cultured for a few days with periodical monitoring of OD\u003csub\u003e600\u003c/sub\u003e. For the degradation assay, 1% BSA and 1% agar were added to the nutrient-poor medium in the presence of HA. For the nutrient-rich halo plates, 0.2% CSC or HP, 1% BSA, and 1% agar were added to GAM medium. Because no white precipitate was detected in the nutrient-rich HA plate after the addition of acetic acid, a glucose-rich medium [0.1% potassium dihydrogenphosphate, 0.1% disodium hydrogenphosphate, 0.01% magnesium sulfate heptahydrate, 0.1% yeast extract, 5% glucose] containing 0.2% HA, 1% BSA, and 1% agar was alternatively used to detect HA degradation.\u003c/p\u003e \u003cp\u003eLuria-Bertani (LB) medium [1% tryptone, 0.5% yeast extract, and 1% sodium chloride (pH 7.2)] was used as a medium for \u003cem\u003eE. coli\u003c/em\u003e. \u003cem\u003eE. coli\u003c/em\u003e BL21-Gold(DE3)pLysS cells harboring the pET21b-CPF_0394 plasmid were cultured at 30\u0026deg;C in LB medium containing 0.1 mg/ml sodium ampicillin to an OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.3\u0026ndash;0.7. Then, 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added and incubated at 16\u0026deg;C for 2 days.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eHalo plate assay for HA degradation\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eC. perfringens\u003c/em\u003e cell suspension was inoculated in the degradation assay medium and anaerobically cultured at 37\u0026deg;C for 7 days\u003csup\u003e47\u003c/sup\u003e. After the addition of 2 M acetic acid 2 mL, the HA-degraded position formed a clear halo.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eRNA-seq analysis\u003c/h2\u003e \u003cp\u003eRNA extraction from \u003cem\u003eC. perfringens\u003c/em\u003e cells was subjected to the hot phenol method as follows. \u003cem\u003eC. perfringens\u003c/em\u003e cells in the logarithmic growth phase were collected by centrifugation at 2,600 \u0026times; \u003cem\u003eg\u003c/em\u003e at 4\u0026deg;C for 5 min and washed with saline. ISOGEN 300 \u0026micro;L (Nippon Gene) was added to the cell suspension, and the mixture was stirred with glass beads by vortexing for 4 min. After further addition of ISOGEN 700 \u0026micro;L, the mixture was incubated at 65\u0026deg;C for 30 min. The supernatant was collected by centrifugation at 12,000 \u0026times; \u003cem\u003eg\u003c/em\u003e at 4\u0026deg;C for 15 min, followed by the addition of chloroform 200 \u0026micro;L. After vortexing for 15 seconds, the mixture was incubated at room temperature for 3 min and centrifuged at 12,000 \u0026times; \u003cem\u003eg\u003c/em\u003e at 4\u0026deg;C for 15 min. Isopropanol (500 \u0026micro;L) was added to the obtained aqueous layer and mixed by inversion, followed by incubation at room temperature for 5 min and centrifugation at 12,000 \u0026times; \u003cem\u003eg\u003c/em\u003e at 4\u0026deg;C for 10 min. Ethanol was added to the resulting precipitate and was centrifuged at 7,500 \u0026times; \u003cem\u003eg\u003c/em\u003e at 4\u0026deg;C for 5 min to obtain a precipitate. The resultant precipitate was dried in a desiccator and dissolved with 100 \u0026micro;L of RNase-free water. Of the obtained RNA extract, 80 \u0026micro;L was used for RNA-seq, and 20 \u0026micro;L was used for purity and quality confirmation. The RNA extract was immediately frozen with liquid nitrogen and stored at \u0026minus;\u0026thinsp;80\u0026deg;C.\u003c/p\u003e \u003cp\u003eThe purity and quality of the RNA extract was confirmed by 0.9% agarose gel electrophoresis at 100 mV for 30 min. A quality check of RNA and RNA-seq analysis were performed by Macrogen Co. Japan. DNase treatment was added to the RNA extract to eliminate DNA contamination. To prepare the sequencing libraries, Ribo-Zero rRNA Removal Kit (Bacteria) and TruSeq Stranded Total RNA Sample Prep Kit (Illumina) were used. Paired-end (101 bp) RNA-seq analysis was performed on the Illumina NovaSeq 6000 sequencer. Trimmed reads were mapped to the reference genome (\u003cem\u003eC. perfringens\u003c/em\u003e strain ATCC 13124) with Bowtie. After the read mapping, HTseq was used for expression profiling. The analysis of differentially expressed genes was performed on 3 comparison pairs using RPKM.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eConstruction of plasmid for expression of CPF_0394, CpeHysA\u003c/h2\u003e \u003cp\u003eTo construct the protein expression system in \u003cem\u003eE. coli\u003c/em\u003e, the CPF_0394 (CpeHysA) gene was amplified via polymerase chain reaction (PCR) using \u003cem\u003eC. perfringens\u003c/em\u003e genomic DNA as a template and oligonucleotides as In-Fusion primers (Supplementary Table\u0026nbsp;1). The reaction mixture contained PCR buffer for KOD FX Neo (TOYOBO), 4 nmol of dNTPs, 0.8 U of KOD FX Neo, 3 pmol of forward and reverse primers, and genomic DNA. The PCR reaction conditions were as follows: 94\u0026deg;C for 2 min followed by 30 cycles of 98\u0026deg;C for 10 sec, 45.4\u0026deg;C for 30 sec, and 68\u0026deg;C for 2 min. The CPF_0394 gene fragment and pET21b vector were digested with NdeI and XhoI mixed with In-Fusion HD Enzyme Premix Kit (Takara Bio), followed by incubation at 50\u0026deg;C for 15 min. After the transformation of \u003cem\u003eE. coli\u003c/em\u003e BL21-Gold(DE3)pLysS cells with the resultant plasmid, the nucleotide sequence of the objective gene was confirmed by DNA sequencing. DNA manipulations were carried out as described elsewhere\u003csup\u003e48\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eProtein purification\u003c/h2\u003e \u003cp\u003e \u003cem\u003eE. coli\u003c/em\u003e BL21-Gold(DE3)pLysS cells harboring pET21b-CPF_0394 were cultured in LB containing ampicillin and IPTG, and harvested by centrifugation at 6,800 \u0026times; \u003cem\u003eg\u003c/em\u003e at 4\u0026deg;C for 10 min. The obtained cells were suspended in 20 mM Tris-HCl (pH 7.5) and disrupted by an ultrasonic generator (Insonator Model 201M, Kubota) at 9 kHz, 0\u0026deg;C for 20 min. The disrupted cells were centrifuged at 20,000 \u0026times; \u003cem\u003eg\u003c/em\u003e at 4\u0026deg;C for 20 min to obtain supernatant as the cell extract. The cell extract was subjected to metal affinity chromatography using TALON resin (Clontech). The cell extract mixed with TALON resin was washed with equilibration buffer [20 mM Tris-HCl (pH 7.5), 500 mM NaCl, and 10 mM imidazole (pH 8.0)] and eluted with elution buffer [20 mM Tris-HCl (pH 7.5), 500 mM NaCl, and 500 mM imidazole (pH 8.0)]. Every eluted fraction was collected, and the purity of the protein in the fractions was assessed by SDS-PAGE\u003csup\u003e49\u003c/sup\u003e. Fractions containing partially purified protein were collected and applied to anion exchange chromatography using Resource Q (GE Healthcare). After washing with equilibration buffer [20 mM Tris-HCl (pH 7.5)], the proteins were eluted with gradient elution buffer [20 mM Tris-HCl (pH 7.5) and 0\u0026ndash;1 M NaCl]. Fractions were selected by SDS-PAGE and applied to a gel filtration chromatography using HiLoad 16/60 Superdex 200 pg (GE Healthcare). The proteins were separated by molecular size with elution buffer [20 mM Tris-HCl (pH 7.5) and 0.15 M NaCl] and were subjected to SDS-PAGE, followed by protein staining with CBB. Collected fractions containing the purified protein were combined and dialyzed against 20 mM Tris-HCl (pH 7.5).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eEnzyme assay\u003c/h2\u003e \u003cp\u003eThe activity of hyaluronate lyase (CPF_0394, CpeHysA) was assayed by monitoring the increase in absorbance at 235 nm derived from the C\u0026thinsp;=\u0026thinsp;C double bonds in unsaturated HA disaccharide. The reaction mixture comprised 50 mM Tris-HCl (pH 7.5), 0.1% HA, and purified CpeHysA. To determine the optimal pH, the enzyme activity was measured in 50 mM buffer of sodium acetate (pH 4.0\u0026ndash;5.5), potassium phosphate (pH 6.0\u0026ndash;7.5), Tris-HCl (pH 7.5\u0026ndash;8.5), and glycine-NaOH (pH 7.5\u0026ndash;10.0) at 30\u0026deg;C. The optimal temperature was determined by measuring the enzyme activity in 50 mM Tris-HCl (pH 7.5) at 30\u0026deg;C\u0026ndash;70\u0026deg;C. To examine the thermostability, the enzyme was preincubated for 10 min at 30\u0026deg;C\u0026ndash;70\u0026deg;C, followed by measuring the activity in 50 mM Tris-HCl (pH 7.5) at 30\u0026deg;C.\u003c/p\u003e \u003cp\u003eTo measure the intracellular and extracellular activity of HysA in \u003cem\u003eC. perfringens\u003c/em\u003e, the bacterial cells grown in 100 mL of the nutrient-poor medium containing HA were harvested to obtain cell pellet and culture supernatant. The resultant cell pellet was subjected to ultrasonication and centrifugation to obtain the cell extract. The culture supernatant and cell extract were subjected to concentration by ammonium sulfate precipitation, dialysis against 20 mM Tris-HCl (pH 7.5), and enzyme assay.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eTLC\u003c/h2\u003e \u003cp\u003eThe reaction mixture of HA in the presence of the culture supernatant or cell extract of \u003cem\u003eC. perfringens\u003c/em\u003e was boiled and centrifuged. The resultant supernatant was subjected to TLC using a developing solvent of 1-butanol: acetic acid: water\u0026thinsp;=\u0026thinsp;3:2:2. After spraying with 10% sulfuric acid in ethanol, the TLC plate was heated to visualize the HA degradation products.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eNative-PAGE\u003c/h2\u003e \u003cp\u003e \u003cem\u003eC. perfringens\u003c/em\u003e cells were grown in the nutrient-poor medium containing HA in the logarithmic growth phase and centrifuged to obtain the culture supernatant and bacterial cells. The resultant cells were washed and suspended in 20 mM Tris-HCl (pH 7.5). The cells were ultrasonically disrupted as described above, and the cell extract was obtained by centrifugation of the disrupted cells at 20,000 \u0026times; \u003cem\u003eg\u003c/em\u003e at 4\u0026deg;C for 20 min. The culture supernatant, cell extract, and the recombinant purified CpeHysA were subjected to native-PAGE with 6% separation gel at 0\u0026deg;C in a running buffer consisting of 43 mM imidazole and 35 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH 7.4). After electrophoresis, the gel was placed on the halo assay plate and incubated at 37\u0026deg;C for 24 h. The gel was removed, and acetic acid was added to confirm the degradation of HA.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eStatistics\u003c/h2\u003e \u003cp\u003eSignificant differences were statistically determined using Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test (**\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, *\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e "},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eW. H. designed the study, T. K. and S. O. performed the experiments, T. K., S. O., and W. H. analyzed the data, and T. K., S. O., and W. H. wrote the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eThis work was supported in part by JSPS KAKENHI Grant Numbers 15H04629, 18H02166, and 21H02156 (W.H.). The authors would like to thank Enago (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.enago.com\u003c/a\u003e\u003c/span\u003e\u003cspan address=\"http://www.enago.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) for the English language review.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eRNA-seq data have been deposited to the GEO database under accession GSE240236.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSender, R., Fuchs, S. \u0026amp; Milo, R. Revised estimates for the number of human and bacteria cells in the body. PLoS Biol 14, e1002533 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFerraris, P. et al. 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Nature 227, 680\u0026ndash;685 (1970).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4310962/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4310962/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eClostridium perfringens\u003c/em\u003e, an opportunistic pathogen in the human intestine, causes gas gangrene by producing various toxins. A clostridial enzyme degrading the host extracellular matrix hyaluronan (HA) has been considered a critical virulence factor as mu-toxin hyaluronidases including endo-β-\u003cem\u003eN\u003c/em\u003e-acetylglucosaminidases (Nags). Here we show that, distinct from Nags, hyaluronate lyase (HysA) is an intrinsic HA-degrading enzyme. \u003cem\u003eC. perfringens\u003c/em\u003e (ATCC 13124) was found to assimilate host-derived extracellular mucosubstances, HA and mucin, which induced expression of the HA-related genetic cluster, including \u003cem\u003ehysA\u003c/em\u003e, but repressed \u003cem\u003enag\u003c/em\u003e genes. The recombinant \u003cem\u003eC. perfringens\u003c/em\u003e HysA showed an HA-degrading activity toward HA through β-elimination reaction. The HA-degrading enzyme in the culture supernatant of \u003cem\u003eC. perfringens\u003c/em\u003e exhibited the lyase activity and was identical to the recombinant HysA on the native-PAGE gel, followed by activity straining. These results demonstrated that the intrinsic HA-degrading enzyme of \u003cem\u003eC. perfringens\u003c/em\u003e is hyaluronate lyase HysA, but not hyaluronidases NagH, NagJ, and NagK.\u003c/p\u003e","manuscriptTitle":"Molecular identification of hyaluronate lyase, not hyaluronidase, as an intrinsic hyaluronan-degrading enzyme in Clostridium perfringens","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-02 11:03:24","doi":"10.21203/rs.3.rs-4310962/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-06-17T17:07:44+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-16T08:11:34+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"81012386759504049303286576190016701152","date":"2024-06-06T19:16:01+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-06T13:06:49+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-22T11:28:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"167375218366113452128880186534364607935","date":"2024-05-14T12:31:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"29425485895316005931772353132355871921","date":"2024-05-14T01:27:40+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-05-09T12:27:34+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-05-09T11:27:11+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-04-29T01:16:27+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-04-25T06:29:55+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-04-23T09:17:51+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"75231a47-6bfe-4d3c-8dfd-9b7fec6c5d1a","owner":[],"postedDate":"May 2nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":31276267,"name":"Biological sciences/Microbiology/Bacteriology"},{"id":31276268,"name":"Biological sciences/Microbiology/Pathogens"},{"id":31276269,"name":"Biological sciences/Biochemistry/Enzymes"},{"id":31276270,"name":"Biological sciences/Biochemistry/Glycobiology"}],"tags":[],"updatedAt":"2024-10-28T16:14:08+00:00","versionOfRecord":{"articleIdentity":"rs-4310962","link":"https://doi.org/10.1038/s41598-024-73955-y","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2024-10-22 15:57:56","publishedOnDateReadable":"October 22nd, 2024"},"versionCreatedAt":"2024-05-02 11:03:24","video":"","vorDoi":"10.1038/s41598-024-73955-y","vorDoiUrl":"https://doi.org/10.1038/s41598-024-73955-y","workflowStages":[]},"version":"v1","identity":"rs-4310962","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4310962","identity":"rs-4310962","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}