NorK, a novel norfloxacin efflux pump in Staphylococcus aureus

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Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results NorK, a novel norfloxacin efflux pump in Staphylococcus aureus Paul Briaud , Jessica Baude , Sylvère Bastien , Laura Camus , View ORCID Profile François Vandenesch , View ORCID Profile Karen Moreau doi: https://doi.org/10.1101/850768 Paul Briaud a CIRI, Centre International de Recherche en Infectiologie, Inserm U1111, Université Lyon1, Ecole Normale Supérieure de Lyon , CNRS UMR5308, Lyon, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jessica Baude a CIRI, Centre International de Recherche en Infectiologie, Inserm U1111, Université Lyon1, Ecole Normale Supérieure de Lyon , CNRS UMR5308, Lyon, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Sylvère Bastien a CIRI, Centre International de Recherche en Infectiologie, Inserm U1111, Université Lyon1, Ecole Normale Supérieure de Lyon , CNRS UMR5308, Lyon, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Laura Camus a CIRI, Centre International de Recherche en Infectiologie, Inserm U1111, Université Lyon1, Ecole Normale Supérieure de Lyon , CNRS UMR5308, Lyon, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site François Vandenesch a CIRI, Centre International de Recherche en Infectiologie, Inserm U1111, Université Lyon1, Ecole Normale Supérieure de Lyon , CNRS UMR5308, Lyon, France b Institut des agents infectieux, Hospices Civils de Lyon , Lyon, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for François Vandenesch Karen Moreau a CIRI, Centre International de Recherche en Infectiologie, Inserm U1111, Université Lyon1, Ecole Normale Supérieure de Lyon , CNRS UMR5308, Lyon, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Karen Moreau For correspondence: karen.moreau{at}univ-lyon1.fr Abstract Full Text Info/History Metrics Preview PDF Abstract A novel efflux pump similar to Nor efflux pumps and designated as NorK was identified in Staphylococcus aureus . It contributes to norfloxacin resistance and presents a high level of expression in different strains. Its expression is not regulated by MgrA, unlike other genes of the nor gene family, suggesting that NorK could be important in the absence of expression of other nor genes. Staphylococcus aureus is a major opportunistic bacterial pathogen responsible for a variety of diseases ( 1 ). Its success in causing life-threatening infections is due to its considerable production of toxins and ability to develop resistance to a wide spectrum of antibacterial compounds ( 2 , 3 ). Various systems have been developed by S. aureus to make it resistant to these substances, such as the alteration of drug binding sites, drug inactivation and the involvement of efflux pumps. The term multidrug resistance (MDR) pumps refers to numerous efflux pumps that are classified into two major groups: (i) ABC-type transporters, which utilize ATP hydrolysis to extrude target drugs, and (ii) secondary multidrug transporters, which exploit proton and sodium gradients as energy sources. Among this latter group, the major facilitator superfamily (MFS) has been studied extensively in staphylococcal species. In S. aureus , the Nor protein family belongs to the MFS and can extrude a broad range of antibiotic substances such as the fluroquinolones (FQ), norfloxacin and ciprofloxacin. Four different pumps involved in FQ resistance have been described previously ( 4 – 7 ). NorA and NorB are responsible for a relatively high-level of resistance to hydrophobic quinolones, whereas NorC seems to be involved in a low-level of resistance ( 6 ). The NorD substrates remain to be discovered. Here, we report a new chromosomally encoded Nor protein called NorK, which is highly expressed independently of MgrA regulation and involved in norfloxacin resistance. Identification of a new putative efflux pump In a previous study (Briaud et al., 2019), we identified 4 different nor related genes from RNA sequencing data, using S. aureus NCTC8325 as a reference genome (GenBank CP000253 ). Three of them have already been described: norB (SAOUHSC_01448), norC (SAOUHSC_00058) and norD (SAOUHSC_02762). The last gene SAOUHSC_02740 was encoded for an unknown protein and annotated as a putative drug transporter. The so-called norK gene is 1.401 nucleotides long (Fig. S1) and encodes for a hypothetical protein of 466 amino acids. This protein shares relatively high homology with NorB (57% identity and 76% similarity) and NorC (59% identity and 74% similarity) but low homology with the NorA and NorD pumps (16% identity and 34% similarity with NorA, 16% identity and 39% similarity with NorD). A phylogenetic analysis was conducted on Nor proteins and major chromosomically encoded MFS efflux pumps from S. aureus NCTC8325 using ClustalW alignment and the Maximum Likelihood method. As depicted by the cluster formed by both proteins ( Fig.1 ), NorK is closely related to NorB and NorC. ClustalW alignment of NorK with NorB and NorC proteins revealed that highly conserved domains were shared ( Fig.2 ). Moreover, the NorK protein presented 14 transmembrane domains predicted by the Phyre2 web portal ( 8 ), similar to those observed in the NorB and NorC pumps. Together these data suggest that NorK belongs to the same family as the Nor proteins previously described. To assess if this pump was widely distributed among S. aureus , BLAST analysis on a large collection of genotypically diverse bacteremia isolates ( 9 ) was performed. The results showed that 98% (123/126) harbor norK genes (identity and coverage >90%). Download figure Open in new tab FIG. 1. Phylogenetic tree of S. aureus chromosomally encoded major MFS efflux pumps. Accession numbers from the Staphylococcus aureus NCTC 8325 strain are indicated in parentheses. Proteic sequences were aligned by the ClustalW method and the tree was built using the Maximum Likelihood method based on the Le Gascuel 2008 model ( 14 ) with a bootstrap of 10000. The tree with the highest log likelihood (−7115.58) is shown. The percentage of trees (>70) in which the associated taxa clustered together is shown next to the branches. The Multi-Antimicrobial Extrusion Protein (MATE) MdtK sequences from E. coli (KAB2830459) was used to root the tree. Download figure Open in new tab FIG. 2. Amino acid sequence alignments of Staphylococcus aureus NCTC 8325 NorB (YP 499970), NorC (YP 498663) and NorK (YP 501210) proteins. Alignment was done using ClustalW. Identical amino acids are shown in red, similar amino acids in blue. Grey boxes represent predicted transmembrane domains by Phyre2 web portal ( 8 ). The expression of norK genes was explored by RT-qPCR (protocol in supplementary data) on 5 different laboratory strains of S. aureus and compared to norA, B, C and D gene expressions. The norK transcript was consistently detected in all the strains tested at a level similar to norA and norD genes ( Fig. 3 and S2), supporting functionality. Download figure Open in new tab FIG. 3. nor genes expression. S. aureus strains RN6390• ( 14 ), HG001▾ (15), SH1000▪ (16), Newman♦ (16), SF8300▴ (17), were cultivated in BHI for 8 hours at 37°C with shaking (200rpm). At 4 hours of culture, RNAs were extracted and nor gene expression was monitored by RT-qPCR. The results are represented as the mean +/- standard error. Quantification after 2, 6 and 8 hours of culture are presented in figure S2 MgrA can act as a repressor or an activator of norA, norB and norC genes ( 5 – 7 ) depending on the RsbU strain background and as a result of its phosphorylation state (22). We thus explored norK gene expression in the Newman wild-type (WT, rsbU+) and the Newman Δ mgrA strain ( 11 ). mgrA deletion had no impact on norK expression while increased norA and norC and decreased norB expression were observed as expected for a RsbU positive background ( Fig. 4 ) ( 10 , 12 ). Download figure Open in new tab FIG. 4. MgrA regulation of nor gene expression. Newman wild-type (black) and Newman Δ mgrA (grey) strains were cultivated in BHI at 37°C for 8 hours. At 4 hours of culture, RNAs were extracted and nor gene expression was monitored by RT-qPCR. The results are depicted as mean + standard error from three independent experiments. Statistical analyses were performed by using an unpaired t-test (* P<0,05, ** P<0,001, *** P<0,0001). NorK efflux activity and involvement in antibiotic resistance To assess the functionality of NorK, thus its efflux activity, an overexpressing S. aureus strain was built (protocol in supplementary data) and an ethidium bromide efflux assay was performed as previously described ( 13 ). The rate of fluorescent loss in norK -overexpressing strains was significantly higher compared to the WT strain ( Fig. 5 ). Antibiotic efflux was tested by time-kill assay in the presence of a representative anti-staphylococcal fluoroquinolone, norfloxacin, as previously described ( 14 ). Wild-type and norK -overexpressing strains were cultivated for 8 hours in Mueller-Hinton broth supplemented with norfloxacin at ½MIC (0.19µg/mL), MIC (0.38µg/mL) and 2MIC (0.76µg/mL). At 2, 4, 6 and 8 hours, bacteria were enumerated by plating on TSA. The survival rates of the norK -overexpressing strain were significantly higher (between 0.5 log and 1 log CFU/ml) than those for the WT strain. Download figure Open in new tab FIG. 5. Quantification of ethidium bromide efflux over time by S. aureus RN6390 WT (•) and norK overexpressing (▪) strains. Bacteria were resuspended in efflux buffer with EtBr (2µg/mL) and reserpine (25µg/mL), to prevent efflux. After 30min, bacteria were washed to remove reserpine and EtBr efflux was monitored by measuring the decrease of fluorescence. One experiment representative of three independent experiments is depicted. Download figure Open in new tab FIG. 6. Time–kill curves of S. aureus RN6390 WT (•) and norK overexpressing (▪) strains. Bacteria were grown in Mueller-Hinton broth (MHB) for 8 hours. Bacteria were resuspended at 1.10 6 CFU/mL in MHB supplemented with norfloxacin at ½ MIC (0.19µg/mL), MIC (0.38µg/mL) and 2MIC (0.76µg/mL). Bacteria were plated on MHA every two hours to count living cells. The results are indicated as the percentage of survival rate calculated by dividing the number of bacteria at a time point by the number of bacteria at T0. One experiment representative of three independent experiments is depicted. In conclusion, NorK is a new member of the nor gene family and contributes to S. aureus norfloxacin resistance. NorK is widely distributed within S. aureus strains and its high level of expression may point to an important role in antibiotic resistance and fitness during the colonization of infectious niches. Moreover, the MgrA-independent regulation of norK indicates that its expression could be induced even though other nor -regulated genes were not expressed. Further studies will be necessary to explore the expression profiles of nor genes in different conditions (e.g., antibiotic stresses, infectious niches). Authors’ contributions PB, JB, LC, FV and KM contributed to the design of the study. PB and JB conducted the experiments. SB conducted and analyzed all the bioinformatics works. PB and KM collected the data and wrote the first draft of the manuscript. All the authors contributed to manuscript revision and approved the submitted version. None of the authors have any conflict of interest to report. Acknowledgments This work was funded by Inserm. P. Briaud was funded by the French Ministry of Education and Research, and L. Camus was funded by the Fondation pour la Recherche Médicale (grant number ECO20170637499). References 1. ↵ Tong SYC , Davis JS , Eichenberger E , Holland TL , Fowler VG. 2015 . Staphylococcus aureus Infections: Epidemiology, Pathophysiology, Clinical Manifestations, and Management . Clin Microbiol Rev 28 : 603 – 661 . OpenUrl Abstract / FREE Full Text 2. ↵ Jenkins A , Diep BA , Mai TT , Vo NH , Warrener P , Suzich J , Stover CK , Sellman BR. 2015 . Differential expression and roles of Staphylococcus aureus virulence determinants during colonization and disease . mBio 6 : e02272 – 02214 . 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