Biological function and mechanism of flpL gene in regulating pathogenicity of Aeromonas hydrophila

Biological function and mechanism of flpL gene in regulating pathogenicity of Aeromonas hydrophila | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (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];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Biological function and mechanism of flpL gene in regulating pathogenicity of Aeromonas hydrophila Hanyang Jiao , Kemei Liu , Ziyi Liao , View ORCID Profile Adeeba Naseer , Hua Ye , Hao Xu , Yun Li , Yongyao Yu , HuiQing Mei , View ORCID Profile Ronghua Wu doi: https://doi.org/10.1101/2025.02.17.638635 Hanyang Jiao a Integrative Science Center of Germplasm Creation in Western China (CHONGQING) Science City & Aquaculture Engineering Technology Research Center, College of Fisheries, Southwest University , Chongqing 400715, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Kemei Liu a Integrative Science Center of Germplasm Creation in Western China (CHONGQING) Science City & Aquaculture Engineering Technology Research Center, College of Fisheries, Southwest University , Chongqing 400715, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ziyi Liao a Integrative Science Center of Germplasm Creation in Western China (CHONGQING) Science City & Aquaculture Engineering Technology Research Center, College of Fisheries, Southwest University , Chongqing 400715, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Adeeba Naseer a Integrative Science Center of Germplasm Creation in Western China (CHONGQING) Science City & Aquaculture Engineering Technology Research Center, College of Fisheries, Southwest University , Chongqing 400715, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Adeeba Naseer Hua Ye a Integrative Science Center of Germplasm Creation in Western China (CHONGQING) Science City & Aquaculture Engineering Technology Research Center, College of Fisheries, Southwest University , Chongqing 400715, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Hao Xu a Integrative Science Center of Germplasm Creation in Western China (CHONGQING) Science City & Aquaculture Engineering Technology Research Center, College of Fisheries, Southwest University , Chongqing 400715, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Yun Li a Integrative Science Center of Germplasm Creation in Western China (CHONGQING) Science City & Aquaculture Engineering Technology Research Center, College of Fisheries, Southwest University , Chongqing 400715, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Yongyao Yu b Department of Aquatic Animal Medicine, College of Fisheries, Huazhong Agricultural University , Wuhan, Hubei 430070, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site HuiQing Mei c Chongqing Fisheries Technology Extension Center , Chongqing 401121, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: starwu061{at}126.com 275621593{at}qq.com Ronghua Wu a Integrative Science Center of Germplasm Creation in Western China (CHONGQING) Science City & Aquaculture Engineering Technology Research Center, College of Fisheries, Southwest University , Chongqing 400715, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Ronghua Wu For correspondence: starwu061{at}126.com 275621593{at}qq.com Abstract Full Text Info/History Metrics Preview PDF ABSTRACT Aeromonas hydrophila is human-animal commensal bacterium that seriously threatens the development of aquaculture and human health. Type IV pili (T4P) are essential for bacterial physiological function. The relationship between the molecular mechanism of flpL gene affiliated to T4P and the pathogenicity is still unclear. In the research, A. hydrophila with a genetically stable deletion of flpL gene (Δ flpL ) was constructed. The median lethal dose of Δ flpL for Crucian carp was 4.87 times higher compared to wild-type strain, suggesting that Δ flpL significantly reduced pathogenicity. The attenuation may be attributed to a reduced capacity for biofilm formation and downregulation of the expression levels of virulence-related genes in Δ flpL . Meanwhile, the significant increase in swimming capacity and adhesion can be related to the upregulation of flpC, tapM, tapB and tapP genes. In conclusion, flpL gene plays essential role in the pathogenicity, adhesion, motility, and capacity for biofilm formation in A. hydrophila . The research aims to clarify the pathogenesis of A. hydrophila , and lay the foundation for developing live attenuated vaccines through the targeted modification flpL gene. 1. Introduction Aeromonas hydrophila is a gram-negative bacterium frequently present in aquatic habitats ( Ji et al , 2015 ). It can infect fish, domesticated animals and mammals, causing a critical threat to the aquaculture industry and public security ( Fang et al , 2004 ). A. hydrophila is capable of causing significant disease outbreaks in various economically essential fish species, including Crucian carp ( Carassius auratus ) ( Feng et al , 2022 ), Micropterus salmoides ( Yang et al , 2023 ) and Oreochromis niloticus ( Aly et al , 2023 ). The fish infected by A. hydrophila usually appear focal symptoms, including skin ulcers, tissue swelling and surface hyperemia, leading to hemorrhagic septicemia and ulcerative syndrome ( Fernandez-Bravo & Figueras, 2020 ). In recent years, there was rising incidence of aquatic animal deaths due to A. hydrophila infections ( Suresh & Pillai, 2023 ; Li et al , 2023b ). These occurrences have resulted in significant economic losses and have severely hindered the advancement of aquaculture. Recently, the prevention and treatment of A. hydrophila infections frequently involved the antibiotics ( Khalil et al , 2021 ). However, large and frequent usage of antibiotics in aquaculture, not only caused serious pollution to the aquatic environment, but also raised the problem of drug-resistance and residues in fish ( Liang et al , 2024 ). Currently, various types of vaccines have emerged to combat A. hydrophila infections, including inactivated vaccines ( Zhang et al , 2020 ), DNA vaccines ( Liu et al , 2022 ), subunit vaccines ( Zhang et al , 2023 ) and live attenuated vaccines ( Li et al , 2021 ). Among these, the live attenuated vaccines closely resemble the natural growth state and are characterized by low production costs, minimal toxicity, and robust immune induction. ( Mohd-Aris et al , 2019 ). Deleted virulence genes responsible for pathogenicity were used to develop live attenuated vaccines ( Yi et al , 2024 ; Chi et al , 2023 ). Virulence factors could increase the possibility of pathogenicity to the host ( Sarkar et al , 2021 ). The pathogenic factors of A. hydrophila are multifactorial, including pili, flagella, adhesins, cytotoxins, hemolysins and lipases ( Rasmussen-Ivey et al , 2016 ). Among them, pili are surface structures of bacterium that consist of repeated subunits which interact either covalently or non-covalently. Pili are essential virulence factors of proteobacteria that significantly contribute to bacterial physiology, including adhesion, biofilm formation and cell motility ( Lukaszczyk et al , 2019 ). The classification of A. hydrophila pili is based on their assembly pathways, which include type I pili and type IV pili. The type IV pili can be further categorized into three distinct types: Flp pili, Tap pili, and MSHA pili. The tight adherence macromolecular transport system represents an essential type II secretion mechanism. The tad gene functions to encode the components required for the assembly of adhesive Flp pili ( Tomich et al , 2007 ). The Flp pili were first reported in Actinobacillus pleuropneumoniae ( Li et al , 2019 ), the causative agent of locally invasive periodontitis. These pili are essential for colony morphology and biofilm formation, which are important virulence factors. The flpL gene is located in the flpABCDEFGHIJKL gene cluster, contributing to product Flp pili pseudopilin ( Boyd et al , 2008 ). Study have revealed that flp mutant strain of A. pleuropneumoniae significantly reduced capacity of biofilm formation, cellular adherence, resistance against pathogenicity, and survival in swine blood compared with the wide strain ( Li et al , 2019 ). The function and molecular mechanism of the flpL gene, which belongs to the Flp pili, remain unclear in A. hydrophila . Therefore, we hypothesized that the flpL gene might be crucial for the pathogenic mechanisms of A. hydrophila . In the study, the flpL gene deletion strain of A. hydrophila (Δ flpL ) was generated through homologous recombination. We compared various parameters between the wild-type strain (WT) and Δ flpL . These findings elucidated the effects and underlying mechanisms of the flpL gene on the pathogenicity and biological properties of A. hydrophila . The study will provide theoretical evidence supporting the potential development of Δ flpL as a live attenuated vaccine against A. hydrophila infections. 2. Materials and methods 2.1 Bacteria, plasmid, and cultivation Table 1 summarizes bacteria and plasmids utilized in this research. The WT was obtained from our laboratory’s previous collection ( Wu et al , 2020 ). In addition, the WT was cultivated in BHI medium at a temperature of 28℃. The host Escherichia coli SM10λpir bacterium was cultured in LB medium at a temperature of 37℃. In order to attain positive selected strains, the concentrations of antibiotics are ampicillin chloramphenicol. 15% sucrose was used to screen for gene deletion strains. View this table: View inline View popup Download powerpoint Table 1. Bacteria and plasmids used in the study 2.2 Establishment of A. hydrophila mutant strain (Δ flpL ) The flpL gene deletion strain was established by referencing our previously constructed method ( Xiong et al , 2024 ; Lu et al , 2024a ; Long et al , 2024 ; Lu et al , 2024b ). Firstly, the primers were designed using the comprehensive genome sequence of A. hydrophila (NCBI Reference Sequence: NC_021290.1). Secondly, upstream and downstream homology arms were amplified separately. Thirdly, the recombinant fragment was constructed by joining the fragments obtained above using overlap PCR. The fragment was ligated to the enzymatic site of pRE112 plasmid to acquire the pRE112-Δ flpL . Afterwards, the pRE112-Δ flpL was transformed into E. coli SM10λpir, and confirmed successful recombination. Next, the SM10λpir with pRE112-Δ flpL recombinant plasmid was reorganized into the WT for conjugation. Finally, by Amp and Cm sensitivity and sucrose resistance, the Δ flpL mutant confirmed through PCR with flpL -a/ flpL -d primers and direct DNA sequencing of the mutation sites, were selected. The successfully screen colony was inoculated into BHI medium at a ratio of 1:10 per hour, and samples were taken 12 hours later and validated using flpL -a/ flpL -d primers. All primers used for establishing Δ flpL are detailed in Table 2 . View this table: View inline View popup Download powerpoint Table 2. Primers used for establishment of Δ flpL . 2.3 Median lethal dose (LD 50 ) Healthy Crucian carp, with an average weight of 20 ± 1 g, were sourced from a farm in Chongqing, China. The water temperature was maintained at 26 ± 1℃. During the two-week acclimation period, the fish were fed daily at 1% of their body weight. After confirming the absence of A. hydrophila infection through bacteriological testing of eight randomly selected individuals, the experiment proceeded. To examine the LD 50 of WT and Δ flpL , bacterial suspensions were prepared by diluting the cultures in fresh BHI broth to achieve four different concentrations: 3 × 10 7 CFU/mL, 3 × 10 6 CFU/mL, 3 × 10 5 CFU/mL and 3 × 10 4 CFU/mL. Crucian carp (n = 12 per group) were randomly assigned to 9 groups. Each group received an intraperitoneal injection of 200 µL from above the four concentration gradients of WT and Δ flpL , individually. The control group received an equivalent injection of sterile BHI broth. The fish were monitored for a period of two weeks, during which mortality was recorded following infection until them stabilized. The LD 50 values of WT and Δ flpL for Crucian carp were analyzed according to previous method ( Finney, 1985 ). 2.4 Growth curve analyses To determine the growth capacity, the same concentrations of WT and Δ flpL bacterial fluids were respectively inoculated into BHI both. The WT and Δ flpL medium were taken every hour, and the optical density (OD) at 600 nm (OD 600 ) was monitored until the culture reached a stationary phase. The experiment was replicated at least three times to construct growth curves for both the WT and Δ flpL strains. 2.5 Hemolytic activity To measure the haemolytic activity, the same volume of WT and Δ flpL was taken during the logarithmic growth phase of bacteria (OD 600 = 0.5), and then inoculated into Columbia blood agar plates. Afterwards, plates were subsequently cultured at 28℃ for 24 hours to investigate the difference between WT and Δ flpL . 2.6 Motility assay The bacterial liquids of WT and Δ flpL were respectively aspirated 2 µL to the swimming and swarming nutrient medium ( Chi et al , 2023 ), using the same experimental manipulation of hemolytic activity test. This experiment was replicated a minimum of three times to verify swimming and swarming capacity. 2.7 Biofilm formation test The assessment of biofilm formation capacity was based on the methodology that we previously published ( Chi et al , 2023 ; Xiong et al , 2024 ). In short, bacterial suspensions of WT and Δ flpL , both adjusted to an OD 600 of 0.5, were inoculated into 96-well plates and incubated for 24 hours. Subsequently, the bacteria in each well were fixed using 99% methanol and stained with 0.1% crystal violet. The dye was then dissolved with 33% glacial acetic acid, and the capacity of biofilm formation between WT and Δ flpL was quantified by examining the OD 575 ( Stepanović et al , 2000 ). 2.8 Adhesion capacity Absolute fluorescence quantitative PCR was conducted to determine difference in adherence ( Gao et al , 2016 ; Mansour et al , 2019 ). The healthy Crucian carp were randomly allocated into three groups. Afterwards, the fish were subjected to immersion in WT (3 × 10 6 CFU/mL), Δ flpL (3 × 10 6 CFU/mL) and sterile PBS for a period of 2 hours. Gill tissue samples weighing 30 mg were collected from WT and Δ flpL for DNA extraction. Next, the two types of DNA were normalized to the same concentration for qPCR analysis, and Ct values were recorded. The Ct values were brought to the standard curve constructed by our previous study to acquire the A. hydrophila of adhered bacteria ( Xiong et al , 2024 ). Meanwhile, the gills were uniformly ground and coated on Rimler-shotts medium to observe the difference between WT and Δ flpL more clearly. 2.9 Expression of virulence-related genes To reveal the molecular mechanisms with the impact of flpL deletion on virulence and biological characteristics, real-time quantitative PCR (RT-qPCR) was conducted to compare the expression levels of virulence-associated genes in WT and Δ flpL. Total RNA was extracted from WT and Δ flpL strains, followed by the elimination of genomic DNA contamination and synthesis of complementary DNA (cDNA), all performed according to the manufacturer’s protocols using the respective kits. Expression of some essential virulence genes were determined by RT-PCR test, including some type IV pili related genes, lip , aha , aerA and hly gene, and analyzed relative expression levels between WT and Δ flpL . The dates were analyzed using the 2 -ΔΔCt method ( Ganger et al , 2020 ). The 16S rRNA is the internal gene as control. The fluorescence intensities of each relative gene were analyzed in triplicate. Aiming to exclude non-specific amplification problems, and the specificity was validated according to the melting curve of the RT-qPCR products. The experiments were independently performed four times. The primers for RT-qPCR are provided in Table 3 . View this table: View inline View popup Table 3. Sequences of the primers for RT-qPCR. 2.10 Statistical analysis Statistical analysis was conducted using SPSS v.26.0 and Origin2021 software. All results are expressed as the mean ± SD (standard deviation), and one-way analysis of variance (ANOVA) was employed for comparisons among groups. 3. Results 3.1 Establishment of Δ flpL strain The lengths of the homology arms located upstream and downstream of the flpL gene were 546 bp and 428 bp, respectively ( Fig. 1A ). The fusion fragment obtained by overlap PCR, measuring 974 bp ( Fig. 1B ). The pRE112-Δ flpL was successfully transferred into E. coli SM10λpir by detecting 1314 bp ( Fig. 1C ). The first successfully homologous recombination resulted in a double band at 1514 bp and 974 bp ( Fig. 1D ). The second successfully homologous recombination resulted in a single band at 974 bp ( Fig. 1E ). Ultimately, the genetic stability assay WT as control was a 1514 bp band and the Δ flpL had a 974 bp band. This finding indicated that the Δ flpL exhibited stable inheritance and maintain genetic stability ( Fig. 1F ). Download figure Open in new tab Fig. 1. Establishment of Δ flpL mutant strain. (A): PCR results for the upstream and downstream homologous arms of flpL gene. M is DL2000 DNA Marker, 1-3 are the upstream homologous arms, 4-6 are the downstream homologous arms. (B): Results of upstream and downstream homologous arms overlap PCR product gel recovery. M is DL2000 DNA Marker, 7-9 are the flpL gene deletion fragment. (C): Transformation of pRE112-Δ flpL recombinant plasmid into SM10λpir verified by colony PCR. M: DL2000 DNA Marker, 10-13 and 15-16 are PCR results of pRE112-Δ flpL recombinant plasmid colonies, 14 is SM10λpir harboring the pRE112 plasmid. (D): The results of the first successfully homologous recombinant PCR. M: DL2000 DNA Marker, 17-20 are strains with double bands, 21 is the WT served as the control. (E): The results of the second successfully homologous recombinant PCR. M: DL2000 DNA Marker, 22-23 are strains with targeted bands, 24 is WT served as the control. (F): Results of the genetic stability assessment for the mutant strain. M: DL2000 DNA Marker, 25-33 are randomly sampled Δ flpL bacterial samples within 50 generations, 34 is WT served as the control. 3.2 LD 50 values The LD 50 values for WT and Δ flpL were 5.33 × 10 5 CFU/tail and 2.60 × 10 6 CFU/tail, respectively. Following infection with WT, Crucian carp exhibited superficial bleeding and abdominal swelling ( Fig. 2A ). In contrast, Crucian carp infected with Δ flpL showed only superficial hemorrhaging with milder symptoms, with no notable abdominal distension ( Fig. 2B ). Bacteria obtained from deceased fish were identified by 16S rDNA universal primers. Subsequent PCR product sequencing confirmed that the isolates were A. hydrophila . The results demonstrated that the LD 50 of Δ flpL was 4.87-fold higher than that of WT, suggesting a significant reduction the virulence of A. hydrophila ( P < 0.01). Download figure Open in new tab Fig. 2. The influence of WT and Δ flpL on virulence (**means P < 0.01). (A): Symptoms observed in Crucian carp infected with WT. (B): Symptoms obsessed in Crucian carp infected with Δ flpL . (C): LD 50 of WT and Δ flpL for Crucian carp. Download figure Open in new tab Fig. 3. Growth curves of Δ flpL and WT. 3.3 Influence of the flpL gene on the growth capacity The OD 595 values for Δ flpL and WT did not show significant difference after 15 hours of incubation at 28°C, indicating that deletion of the flpL gene did not have a significant influence on the growth capacity. 3.4 Effect of flpL gene on motility The diameter of colonies on swimming capacity of Δ flpL and WT were 24.59 ± 0.65 mm and 8.98 ± 0.35 mm, respectively ( Fig. 4A-B ). The results showed that the mutation of the flpL gene have a significant enhance of the swimming capacity of A. hydrophila ( P < 0.01). On the swarming plates, the travel distance of Δ flpL and WT were 14.09 ± 0.65 mm and 14.91 ± 1.20 mm, respectively. The results demonstrated that there was no significant change on the swarming capacity of Δ flpL and WT ( P > 0.05, Fig. 4C-D ). Download figure Open in new tab Fig. 4. Influence of the flpL gene on the motility of A. hydrophila (ns represents no significant difference ( P > 0.05), **means P < 0.01). (A, B): Diameter of colonies on swimming capacity of WT and Δ flpL . (C, D) Diameter of colonies on swarming capacity of WT and Δ flpL . 3.5 Hemolytic activity The result of haemolytic activity demonstrated that both Δ flpL and WT exhibited β-hemolysis ( Fig. 5A ). No significant change in hemolytic activity was observed between Δ flpL and WT ( P > 0.05). However, the morphology of two kinds of colonies was different on Columbia blood agar plates. Δ flpL had smooth colony edges, but WT colony had untidy edges with fissures. Download figure Open in new tab Fig. 5. Comparison of haemolytic activity of Δ flpL and WT (ns represents not significant ( P > 0.05), *means P < 0.05, **means P < 0.01). (A): Hemolytic activity assay of Δ flpL and WT. (B): Comparison of colony diameters between WT and Δ flpL bacterial strains. 3.6 Biofilm formation test Through the 96-well plates method, the microplate reader detected OD 575 values of 0.432 ± 0.005 for Δ flpL and 0.632 ± 0.011 for WT ( Fig. 6 ). These results indicated that the flpL gene deletion reduced the biofilm formation capacity of A. hydrophila . Download figure Open in new tab Fig. 6. Biofilm formation of WT and Δ flpL strains (**means P < 0.01). Download figure Open in new tab Fig. 7. The adhesion levels between WT and Δ flpL (**means P < 0.01). (A): The quantity of bacteria attaching to the gills at 2 hours. (B): WT was coated following a 500-fold dilution. (C): Δ flpL was coated following a 500-fold dilution. Download figure Open in new tab Fig. 8. RT-qPCR result (ns represents no significance ( P > 0.05), *means P < 0.05, **means P < 0.01). 3.7 Adhesion capacity In our previous study, we constructed the standard curve that delineates the relationship between the quantity of A. hydrophila and the Ct value ( Xiong et al , 2024 ). Based on the standard curve and equation ( y = − 3.501 x + 38.31), the adhesion levels of WT and Δ flpL were determined to be 1.32 × 10 7 CFU/g and 1.66 × 10 8 CFU/g, respectively. The adhesion capacity of the flpL gene was significantly enhanced, increasing 12.6-fold relative to WT. The Δ flpL group exhibited a notably higher number of bacterial colonies on RS solid medium compared to the WT group. 3.8 virulence gene expression The expression levels of several virulence genes were analyzed in WT and Δ flpL . As illustrated in Fig. 9, flpC related Flp type IV pili and Tap type IV pili genes including tapM , tapB , tapP , showed significantly up-regulated ( P < 0.05). Conversely, the genes associated with MSHA type IV pili, lip , aha , aerA and hly , exhibited notable down-regulation in Δ flpL . 3. Discussion Flp pili, similar to other two types of T4P pili, greatly contribute to bacterial adherence, virulence, and capacity of biofilm formation ( Long et al , 2024 ; Lu et al , 2024a ). The flpL gene is a part of the T4P pili gene family. However, its function and the molecular mechanism in the virulence of A. hydrophila still unclear. This study makes the first attempt to elucidate the function of the flpL gene on virulence. The result demonstrated that the LD 50 of Δ flpL was 4.87-fold higher than that of WT in Crucian carp, indicating the flpL gene is important for virulence of A. hydrophila . This finding aligns with study in Actinobacillus pleuropneumoniae ( Li et al , 2019 ), Haemophilus ducreyi ( Spinola et al , 2003 ), Pasteurella multocida ( Fuller et al , 2000 ). Several studies have reported that unlike MSHA and Tap pili, the Flp pili appear to have a limited contribution to pathogenicity ( Hadi et al , 2012 ; Ellison et al , 2022 ). However, in the study, the decrease in pathogenicity of the flpL gene was more significant than the deletion of genes related MSHA and Tap pili ( Long et al , 2024 ; Lu et al , 2024a ). To investigated the mechanisms for the reduced pathogenicity in Δ flpL , biological characteristics associated with bacterial virulence were analyzed, including growth ability, motility, hemolytic activity, biofilm formation ability and cell adhesion. The results demonstrated that detection the flpL gene did not influence the growth capacity. The influence of the flpL gene on growth capacity was in accordance with previous studies in other type IV pili genes, such as flp gene in Actinobacillus pleuropneumoniae ( Li et al , 2019 ) and mshQ gene in A. hydrophila ( Qin et al , 2014 ). There is no significant difference observed in swarming capacity. The enhanced of swimming capacity in Δ flpL indicated that the flpL gene could affect A. hydrophila motility, which was consistent with some finding in previous studies that Flp pili gene deletions would affect bacterial motility ( Carvia-Hermoso et al , 2024 ; Zatakia et al , 2014 ). Hemolysin is a one of the most critical virulence factors that plays critical role in bacterial pathogenicity ( Zhao et al , 2024 ). The hemolytic activity of Δ flpL did not show a significant difference compared to that of WT, and both were β-hemolytic. However, the morphology of Δ flpL and WT strains had obvious difference. The edges of Δ flpL colonies are smooth, while the edges of WT colonies show a jagged shape. Previous studies have shown that the formation of folded and jagged colonies is linked to the biofilm formation capacity ( Weissman et al , 2020 ). Therefore, we analyzed the difference of biofilm formation capacity between WT and Δ flpL. The findings demonstrated that a substantial reduction in biofilm formation occurred in Δ flpL compared with WT, indicating that the smooth edges of Δ flpL colonies on Columbia blood agar plates might be linked to the reduced biofilm formation capacity. Consistently, a previous study in Xylella fastidiosa also found that the type IV pili are essential for both motility and biofilm formation ( Li et al , 2007 ). In addition, type IV pili play a key role in the adhesion processes of pathogenic gram-negative bacteria ( Piepenbrink et al , 2014 ). Adhesion to host cells, which is the initial step of infection, heavily relies on the presence of pili. Studies have demonstrated that pili influence the adhesion capacity of Aeromonas schubertii ( Piepenbrink et al , 2014 ), Pseudomonas aeruginosa ( Beaussart et al , 2014 ), Sulfolobus acidocaldarius ( Charles-Orszag et al , 2023 ). The present study demonstrated a significant enhance in the adhesion capacity of Δ flpL . The result was different from other previous studies on the deletion of genes related to the type IV pili ( Qin et al , 2014 ; Li et al , 2023a ). To sum up, Δ flpL exhibited a significant decrease in virulence for Crucian carp through reducing biofilm formation capacity, though swimming capacity and adhesion were enhanced. To investigate the molecular mechanism of Δ flpL on pathogenicity, the expression levels of several critical virulence genes were analyzed, and cross-talks between flpL and other type virulence genes were found. The type IV pili is widely distributed fibers on bacterial surfaces that participate in diverse physiological behavior, including virulence, biofilm formation, motility and protein secretion ( Ellison et al , 2022 ). The Δ flpL may affect the expression levels of other genes belonging to the same type IV pili genes. It is possible that the existence of overlapping ORF regions between different virulence factors in the bacterial genome, the deletion of one virulence factor using homologous recombination also impacts on the expression levels of other virulence genes in the same gene cluster. The current study found that the expression levels of flpC , tapM , tapB and tapP genes showed significant increase. The appearances of up-regulated expression of these genes might contribute to the enhanced swimming ability and increased adhesion capacity. The MSHA type IV pili primarily contribute significantly to biofilm formation ( Hadi et al , 2012 ). The genes of MSHA type IV pili were down-regulated, which may explain the decrease in biofilm formation. Lipoprotein servers as a critical bacterial secretion system that plays a significant role in pathogenesis ( Allaoui et al , 1992 ). The aha gene is critical for adhesion and pathogenicity ( Song et al , 2019 ). Aerolysin, an exotoxin encoded by aerA gene, is highly cytotoxic, hemolytic, and enterotoxicity ( Howard et al , 1987 ), playing an critical role in the virulence of A. hydrophila ( Singh et al , 2008 ). The hly gene related to bacterial virulence, encodes pore-forming cytolysin listeriolysin. In the research, we speculate that the reduced expression of lip , aha , aerA and hly genes might be essential factors contributing to the decreased pathogenicity of the Δ flpL strain. However, the specific signaling pathway underlying this requires further investigation. 4. Conclusion In conclusion, the first genetically stable flpL gene of A. hydrophila knockout strain was successfully constructed in this study. Our findings demonstrated that the Δ flpL exhibits significantly decreased pathogenicity for Crucian carp. This may be result from the decreased biofilm formation capacity and decreased expression levels of specific virulence-associated genes. Moreover, the significant increases in swimming capacity and adhesion may be related to the up-regulation of flpC, tapM, tapB and tapP genes. Motility and adhesion are strongly associated with the rate of bacterial infections. These results underscore the pivotal role of the flpL gene in pathogenicity, adhesion, motility, and biofilm formation capacity. This study will provide theoretical evidence supporting the potential development of Δ flpL as a live attenuated vaccine against A. hydrophila infections. 5. CRediT authorship contribution statement Hanyang Jiao : Conceptualization, Methodology, Writing-Original Draft, Preparation, Writing-Review & Editing. Kemei Liu : Investigation, Methodology, Software. ZiYi Liao : Methodology, Software. Adeeba Naseer : Resources, Software. Hua Ye : Writing-Review & Editing. Hao Xu : Writing-Review & Editing. Yun Li : Writing-Review & Editing. Yongyao Yu : Writing-Review & Editing. HuiQing Mei : Writing-Review & Editing. Ronghua Wu : Conceptualization, Methodology, Validation, Writing-Review & Editing. 6. Declaration of interest statement The authors declare no conflicts of interest. 7. Data availability The data that has been used is confidential. 8. Acknowledgements This study was funded by National Natural Science Foundation of China (32102831). Footnotes Hanyang Jiao Email: joehey.cqps{at}foxmail.com ; Kemei Liu Email: 1369134328{at}qq.com ; Ziyi Liao Email: 18684555387{at}163.com ; Adeeba Naseer Email: adeebanaserr44{at}gmail.com ; Hao Xu: xuhao{at}email.swu.edu.cn ; Hua Ye Email: yhlh2000{at}126.com ; Yun Li Email: aquatics{at}swu.edu.cn ; Yongyao Yu Email: yuyy{at}mail.hzau.edu.cn References 1. ↵ Allaoui A , Sansonetti P & Parsot C ( 1992 ) Mxij, a Lipoprotein Involved in Secretion of Shigella Ipa Invasins, Is Homologous to Yscj, a Secretion Factor of the Yersinia Yop Proteins . 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Share Biological function and mechanism of flpL gene in regulating pathogenicity of Aeromonas hydrophila Hanyang Jiao , Kemei Liu , Ziyi Liao , Adeeba Naseer , Hua Ye , Hao Xu , Yun Li , Yongyao Yu , HuiQing Mei , Ronghua Wu bioRxiv 2025.02.17.638635; doi: https://doi.org/10.1101/2025.02.17.638635 Share This Article: Copy Citation Tools Biological function and mechanism of flpL gene in regulating pathogenicity of Aeromonas hydrophila Hanyang Jiao , Kemei Liu , Ziyi Liao , Adeeba Naseer , Hua Ye , Hao Xu , Yun Li , Yongyao Yu , HuiQing Mei , Ronghua Wu bioRxiv 2025.02.17.638635; doi: https://doi.org/10.1101/2025.02.17.638635 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Microbiology Subject Areas All Articles Animal Behavior and Cognition (7625) Biochemistry (17652) Bioengineering (13874) Bioinformatics (41890) Biophysics (21429) Cancer Biology (18567) Cell Biology (25467) Clinical Trials (138) Developmental Biology (13365) Ecology (19874) Epidemiology (2067) Evolutionary Biology (24294) Genetics (15591) Genomics (22478) Immunology (17717) Microbiology (40331) Molecular Biology (17153) Neuroscience (88496) Paleontology (666) Pathology (2828) Pharmacology and Toxicology (4817) Physiology (7635) Plant Biology (15114) Scientific Communication and Education (2044) Synthetic Biology (4286) Systems Biology (9817) Zoology (2268)