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Put a Bow on It: Knotted Antibiotics Take Center Stage
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Abstract
Ribosomally-synthesized and post-translationally modified peptides (RiPPs) are a large class of natural products produced across all domains of life. The lasso peptides, a subclass of RiPPs with a lasso-like structure, are structurally and functionally unique compared to other known peptide antibiotics in that the linear peptide is literally “tied in a knot” during its post-translational maturation. This underexplored class of peptides brings chemical diversity and unique modes of action to the antibiotic space. To date, eight different lasso peptides have been shown to target three known molecular machines: RNA polymerase, the lipid II precursor in peptidoglycan biosynthesis, and the ClpC 1 subunit of the Clp protease involved in protein homeostasis. Here, we discuss the current knowledge on lasso peptide biosynthesis as well as their antibiotic activity, molecular targets, and mechanisms of action.
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Mechanistic Understanding of Lanthipeptide Biosynthetic Enzymes
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M. Repka, Jonathan R. Chekan, Satish K Nair … (2017)
Lanthipeptides are ribosomally synthesized and post-translationally modified peptides (RiPPs) that display a wide variety of biological activities, from antimicrobial to antiallodynic. Lanthipeptides that display antimicrobial activity are called lantibiotics. The post-translational modification reactions of lanthipeptides include dehydration of Ser and Thr residues to dehydroalanine and dehydrobutyrine, a transformation that is carried out in three unique ways in different classes of lanthipeptides. In a cyclization process, Cys residues then attack the dehydrated residues to generate the lanthionine and methyllanthionine thioether cross-linked amino acids from which lanthipeptides derive their name. The resulting polycyclic peptides have constrained conformations that confer their biological activities. After installation of the characteristic thioether cross-links, tailoring enzymes introduce additional post-translational modifications that are unique to each lanthipeptide and that fine-tune their activities and/or stability. This review focuses on studies published over the past decade that have provided much insight into the mechanisms of the enzymes that carry out the post-translational modifications.
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Evolution of Multidrug Resistance during Staphylococcus aureus Infection Involves Mutation of the Essential Two Component Regulator WalKR
Introduction In hospitals worldwide infections with methicillin-resistant S. aureus (MRSA) remain a significant cause of morbidity and mortality, with a small number of clones accounting for a large number of hospital acquired infections. In Australasia, multi-locus sequence type (MLST) 239 (ST239) is the major hospital acquired clone of MRSA, and has been present in the region for over 30 years. This clone is resistant to almost all antibiotic classes; therefore the mainstay of therapy for serious MRSA infections has been the glycopeptide antibiotic vancomycin. However, resistant strains have recently emerged [1], and although the level of resistance is low there is an impact on treatment outcome [2]. These vancomycin-intermediate S. aureus (VISA, vancomycin MIC 4–8 µg/ml) and heterogenous-VISA (hVISA, vancomycin MIC ≤2 µg/ml with a “resistant subpopulation”) strains are increasingly common, however the genetics of resistance are incompletely defined [3]. While the emergence of VISA in Australia has been in strains of the ST239 clone [4], the first VISA strain Mu50 was reported from Japan in 1997 [5], and a number of other reported VISA strains belong to the same clonal complex as Mu50 (CC5) [6]–[8]. In many cases VISA emerge from fully-vancomycin susceptible S. aureus (VSSA) parental strains during persistent infection [6], [8], [9], and in some cases this has been associated with the evolution of daptomycin non-susceptibility despite the absence of exposure to daptomycin [10]. VISA strains appear to arise via sequential point mutations in key staphylococcal regulatory genes [11]–[13], however the breadth of mutations that can contribute to resistance are poorly defined. In addition, it is not clear if there are differences in resistance mechanisms and pathways to VISA in different clones of S. aureus. Commonly described phenotypic changes in VISA compared to VSSA include increased cell wall thickness and reduced autolytic activity [7], [14], [15], in addition to other significant phenotypic changes that are predicted to impact the virulence of the organism. These include a reduction in biofilm formation, reduced activity of the agr quorum sensing system, and enhanced capsule production [3], [4], [15], [16]. The link(s) between development of antimicrobial resistance and the regulation of these virulence factors is unknown. A number of studies have used comparative genomics of paired S. aureus isolates to detect mutations that occur in the resistant strain compared to the parent strain, including a landmark study by Mwangi et al where increasing vancomycin resistance in sequential clinical isolates of S. aureus were linked to accumulated mutations in the increasingly resistant strain [11]. However, the genetic loci where mutations in clinical S. aureus strains have been experimentally confirmed using allelic replacement experiments to contribute to VISA are limited to vraSR, graRS, and more recently rpoB [13], [17], [18]. We have previously used functional genomics to show that a point mutation in graS can lead to reduced vancomycin susceptibility in one clinical pair of ST239 VSSA (JKD6009) and VISA (JKD6008) [18]. However this mutation, while leading to a reduction in vancomycin susceptibility, did not restore the full VISA resistance profile. It is worth noting that all these studies have focussed on a total of three clinical and laboratory induced VISA isolates, and screening of additional ST239 VISA strains has failed to demonstrate that mutations in these loci are common to other VISA [4], suggesting that there are mutations in as yet undefined loci contributing to VISA in other clinical isolates. Daptomycin is an antibiotic that exerts its effect at the cell membrane, and while a link between VISA and daptomycin non-susceptibility has been demonstrated [10], [19], the genetics of this relationship are undefined. While mutations in mprF, rpoB and rpoC are thought to be the genetic basis for daptomycin non-susceptibility in S. aureus, mutations have also been detected in walK [20]. The contribution of the walK mutations to daptomycin non-susceptibility has not been defined as these strains also harboured mprF mutations. To expand understanding of the mechanisms of VISA and identify other loci contributing to vancomycin resistance we fully sequenced four additional clinical pairs of VSSA and VISA, and then compared them to the fully assembled and annotated genome of our previously described VISA strain JKD6008 [21]. We also re-analyzed our original sequenced pair (JKD6008 and JKD6009) after fully assembling the genome sequence of the VISA strain JKD6008. Using this approach, followed by allelic replacement experiments, we show that WalKR (also known as YycGF and VicKR) plays a major role during the in vivo evolution of extensive drug resistance in clinical S. aureus. Our findings highlight an unexpected degree of plasticity within this essential two-component regulator and are particularly pertinent, with inhibitors of WalK recently proposed as novel anti-staphylococcal agents [22]–[24]. Results Isolate Selection and Characteristics To identify the genetic mechanisms leading to VISA five clinical pairs of VSSA and VISA were selected, where the resistant strain evolved from the susceptible parent strain during failed vancomycin therapy [15], [18]. In addition, we also examined eight global non-paired hVISA and VISA (Table 1). The clinical pairs were selected from patients that had been treated with vancomycin, and not daptomycin. This meant that any changes in daptomycin susceptibility linked to vancomycin exposure and increasing vancomycin resistance could be specifically assessed. The duration of in vivo vancomycin exposure ranged from 8 to 42 days in these clinical isolate pairs and the majority of isolates fulfilled the criteria for VISA (vancomycin broth MIC 4–8 µg per ml). For all VISA strains in the isolate pairs an increase in daptomycin MIC was seen compared to the parental VSSA strain, and five of the 13 hVISA/VISA isolates were daptomycin non-susceptible (Table 1). All of the clinical strains were typed using the StaphyType96 DNA Array (CLONDIAG, Jena, Germany) to predict the sequence type, mec type, and agr type (Table 1). All of the clinical pairs were from Australia and New Zealand, and were ST239 MRSA, the dominant hospital clone of MRSA in the region, while the additional isolates represented a range of staphylococcal sequence types within clonal complexes 5 and 8. 10.1371/journal.ppat.1002359.t001 Table 1 Methicillin-resistant Staphylococcus aureus clinical strains used in study. Strain Origin Clonal Complex Typing ST- mec agr type Vanco BMD (µg/mL) Dapto Etest MIC (µg/mL) Etest GRD vanco 48hs (µg/mL) Etest GRD teico 48hs (µg/mL) Resistance Phenotype Drug Exposurea Reference or Source Isolate Pairs Pair 1 JKD6000 Australia 8 ST239-III [3A] agr_I 1 0.25 1.5 4 VSSA, DS [4], [15] JKD6001 8 ST239-III [3A] agr_I 4 1.5 4 12 VISA, DNS Vanc 13 d [4], [15] Pair 2 JKD6004 Australia 8 ST239-III [3A] agr_I 2 0.5 1.5 2 VSSA, DS [4], [15] JKD6005 8 ST239-III [3A] agr_I 4 2 3 32 VISA, DNS Vanc 8 d [4], [15] Pair 3 JKD6009 New Zealand 8 ST239-III [3A] agr_I 2 0.19 1.5 3 VSSA, DS [4], [15] JKD6008 8 ST239-III [3A] agr_I 4 0.5 3 32 VISA, DS Vanc 42 d [4], [15] Pair 4 JKD6021 Australia 8 ST239-III [3A] agr_I 1 0.19 1.5 4 VSSA, DS [4], [15] JKD6023 8 ST239-III [3A] agr_I 4 1 3 12 VISA, DS Vanc 15 d [4], [15] Pair 5 JKD6052 Australia 8 ST239-III [3A] agr_I 1 0.25 1 2 VSSA, DS [4], [15] JKD6051 8 ST239-III [3A] agr_I 4 0.5 4 >32 VISA, DS Vanc 32 d [4], [15] Additional Isolates BPH0191 Australia 8 ST239-III [3A] agr_I 2 1 3 24 hVISA, DS This study PC3 USA 5 ST5 – II [2A] agr_II 8 3 8 >32 VISA, DNS [6] Sweden 307 Sweden 5 ST5 – II [2A] agr_II 8 1.5 6 32 VISA, DNS M. Wootton VISA 3759 Scotland 8 ST247 – I [1B] agr_I 4 0.5 4 >32 VISA, DS [62] BPH0062 Sth. Africa 8 ST247 – I [1B] agr_I 2 1 3 16 hVISA, DS Jan Bell BPH0065 Hong Kong 8 ST239-III [3A] agr_I 8 3 16 >32 VISA, DNS Jan Bell BPH0073 Taiwan 8 ST239-III [3A] agr_I 4 1 6 16 VISA, DS Jan Bell BPH0088 Japan 5 ST5 – II [2A] agr_II 2 0.38 3 8 hVISA, DS Jan Bell NB. aThe number of days of in vivo vancomycin exposure between the first and last isolate in the pair. BMD, broth microdilution; Etest GRD, Etest for glycopeptide resistance detection; VSSA, vancomycin-susceptible S. aureus; VISA, vancomycin-intermediate S. aureus; hVISA, heterogenous-VISA; DNS, daptomycin non-susceptible; dapto, daptomycin; vanco, vancomycin; teico, teicoplanin. Genome Comparisons Highlight Mutations in walKR Associated with Reduced Vancomycin and Daptomycin Susceptibility in S. aureus In an earlier study of the VSSA/VISA pair JKD6009/JKD6008 we compared partially assembled 454 GS20 sequences and found six nucleotide substitutions in JKD6008 [18]. We then showed that a mutation occurring in the sensor region of the graS gene partly explained the reduced vancomycin susceptibility of this strain. To now comprehensively address the question of mutations that contribute to VISA we used our recently completed JKD6008 reference genome [21] which we have shown is closely related to other Australasian ST239 strains (unpublished data), and is therefore an appropriate reference genome for analysis, and used our read-mapping technique to re-examine the genetic differences between JKD6008 and JKD6009 as well as comparing four other clinical VSSA/VISA pairs (Table 1, strain pairs 1, 2, 4 and 5). Using either SOLiD or Illumina technologies, high coverage short-read sequences were obtained for the clinical pairs with high mean fold coverage (JKD6009 [SOLiD] mean fold coverage 338.7x; other strains [Illumina] mean fold coverage 85.5 to 230.6x). The list of differences between JKD6009 and JKD6008 increased from six to 10, but each pair presented a limited list of mutations in the VISA strain compared to its VSSA parent (Table 2). The most interesting observation was the presence of a previously undetected SNP in the walK gene of JKD6008. Strikingly, three of the four other clinical pairs also had single mutations within the walKR locus (Table 2). In one pair (JKD6004/JKD6005), the only mutation identified was a single SNP in walR of VISA strain JKD6005. JKD6051 was the only VISA strain among the five sequenced pairs without a walKR mutation. Mutations of potential interest in this strain included a mutation in the regulator SarR (A68T), and a mutation in RpoB (H481Y). 10.1371/journal.ppat.1002359.t002 Table 2 Results of whole genome sequence comparison of five pairs of VSSA and VISA. Isolate Pair and Mutation no. Mutation in VISA (Sa_JKD6008 coordinate) Locus (Sa_JKD6008) Gene Function Effect of Mutation Pair 1 - JKD6000/JKD6001 1 G to A (24673) SAA6008_00018 walR Response regulator, essential A96T 2 G to A (1217720) Intergenic 3 T to A (2006385) SAA6008_01867 Putative phage protein Q22H 4 G to A (2142758) SAA6008_02029 dUTPase Silent 5 T to G (2142774) SAA6008_02029 dUTPase N2T SAA6008_02030 acetyltransferase E136D 6 T to C (2150689) Intergenic 7 C to T (2151557) Intergenic Pair 2 - JKD6004/JKD6005 1 A to G (25010) SAA6008_00018 walR Response regulator, essential K208R Pair 3 - JKD6009/JKD6008 1 G to A (25769) SAA6008_00019 walK Sensor kinase, essential G223D 2 A to G (360905) SAA6008_00313 glpT Glycerol-3-phosphate transporter Silent 3a C to T (734466) SAA6008_00676 graS T136I 4a G to A (976097) SAA6008_00920 addA ATP-dependent nuclease subunit A Silent 5a T to A (1721456) SAA6008_01608 tgt Queuine tRNA-ribosyltransferase F365Y 6 C to T (2391832) Intergenic 7a C to T (2470905) SAA6008_02357 Sodium/bile acid symporter family protein P128S. 8a G to A (2754296) SAA6008_02622 Pyridine nucleotide-disulphide reductase G268D 9a C to T (2846492) Intergenic 10 G to A (2892543) SAA6008_02743 ABC transporter ATP binding protein Silent Pair 4 - JKD6021/JKD6023 1 G to T (25903) SAA6008_00019 walK Sensor kinase, essential V268F 2 G to A (57470) SAA6008_00049 Cadmium-transporting ATPase A163T 3 C to T (454602) Intergenic 4 C to A (637292) Intergenic 5 A to T (727814) SAA6008_00669 Hypothetical Protein Premature stop 6 T to C (916693) Intergenic 7 G to A (2022797) Intergenic 8 G to A (2403501) SAA6008_02288 rpsJ 30S ribosomal protein S10 Silent Pair 5 - JKD6052/JKD6051 1 C to A (46858) SAA6008_00039 mecA PBP2a A97S 2 C to T (600465) SAA6008_00548 rpoB DNA directed RNA polymerase beta subunit H481Y 3 G to A (849972) SAA6008_00779 trxB Thioredoxin-disulfide reductase A311T 4 T insertion (1015671) SAA6008_00955 CHP Premature stop 5 T to C (1062271) SAA6008_01000 menB Naphthoate synthase Silent 6 C to T (2443041) SAA6008_02331 sarR Staphylococcal accessory regulator R A68T 7 G to A (2892543) SAA6008_02743 ABC transporter ATP binding protein Silent NB. aThese mutations were previously detected and described prior to genome closure and re-analysis. VSSA, vancomycin-susceptible S. aureus; VISA, vancomycin-intermediate S. aureus. Frequency and Role of walKR Mutations in Reduced Vancomycin and Daptomycin Susceptibility in S. aureus Given the previous reports of diverse genetic pathways involved in VISA we were surprised to find a single locus that was mutated in four of our five pairs of sequenced strains. To extend this analysis the walKR locus was sequenced from 8 additional, unpaired global isolates of hVISA/VISA that were available for study (Table 3). This demonstrated that, in addition to the four VISA strains in the genome sequencing analysis, 6 of the 8 additional resistant strains also had a mutation in the walKR locus. The downstream genes yycHIJ are involved in repression of walR in B. subtilis [25], therefore we sequenced yycHIJ for the two strains where a mutation in walKR was not detected but no mutations were found (Table 3). 10.1371/journal.ppat.1002359.t003 Table 3 Screening for walKR mutations in unique (non-paired) strains. Isolate Molecular Type Phenotype WalK WalR YycHIJ BPH0191 ST239-III [3A] hVISA, DS del 469-470 ND - PC3 ST5 – II [2A] VISA, DNS A567D ND - Sweden 307 ST5 – II [2A] VISA, DNS del 337-340 ND - VISA 3759 ST247 – I [1B] VISA, DS ND ND ND BPH0062 ST247 – I [1B] hVISA, DS ND ND ND BPH0065 ST239-III [3A] VISA, DNS T595I ND - BPH0073 ST239-III [3A] VISA, DS N382S ND - BPH0088 ST5 – II [2A] hVISA, DS L14F ND - Note: DS, daptomycin susceptible; DNS, daptomycin non-susceptible; -, yycHIJ amplification and sequencing not performed in these strains. Analysis of the positions of the mutations within the walKR genes in this study indicates that they are not limited to a specific domain or region (Figure 1A ). Indeed, the mutations occur across the spectrum of the domains that contribute to two-component regulator function. Each change therefore presumably exerts its effect via distinct mechanisms. For example the WalR A96T mutation occurs in a conserved region that is important for phosphorylation-mediated protein conformational changes [26] (Figure 1B ), while the K208R mutation occurs in a highly conserved α3 DNA recognition helix region (Figure 1B ). Recently, the crystal structure of DNA-binding domain of S. aureus WalR protein has been solved, and the interaction of the protein with target DNA examined [27]. Modelling the WalR DNA-binding domain with the K208R mutation from the VISA strain JKD6005 highlights its proximal location to the critical α3–β5 DNA binding loop (Figure 1C ). 10.1371/journal.ppat.1002359.g001 Figure 1 Analysis of location of walKR mutations detected in study isolates. (A) Schematic figure of the walKR operon showing protein domains and positions of identified mutations. Protein domains were defined according to Dubrac et al [25]. White regions of walK represent membrane spanning regions. (B) Amino acid sequences of the walR and walK genes. Domains are highlighted in color according to figure 1A . Identified mutations are indicated in bold type with an asterix. Membrane spanning regions of walK are italicized. The walR aspartic acid that is phosphorylated by walK and the walK histidine in the HisKA domain that is autophosphorylated are shown by arrows. Conserved regions are underlined and referred to in the text. (C) Ribbon diagram of the DNA binding domain of a WalR monomer from VISA strain JKD6005 (pink) modelled against the 1.87 Å structure of the WalR DNA-binding domain (PDB: 2ZXJ) from VSSA Staphylococcus aureus (blue). Shown is the position of the substituted amino acid (K->R) near the critical α3–β5 DNA binding loop (indicated by the arrow). Within WalK, amino acid substitutions were detected in a range of functional domains (Figure 1), including the G223D mutation in JKD6008 at a highly conserved residue required for a sharp reverse turn between the α1 helix and the connector region of the HAMP domain [28], the 3-amino acid deletion Δ337–340 in BPH0191 in the β scaffold of the PAS domain, which is also known to be involved in its structural integrity [29], and the N382S mutation in WalK from BPH0073 in the phosphor-acceptor domain, only 3 amino acids from the conserved histidine that undergoes autophosphorylation and is essential for autokinase activity (Figure 1B ). Generation and Whole Genome Sequencing of walKR Mutants To measure the impact of these single nucleotide changes in walKR, bi-directional allelic replacement experiments were performed using two of the clinical pairs (Table 2, pairs 2 and 3). The walK mutation from VISA strain JKD6008 was introduced into the parent VSSA JKD6009, generating TPS3130 (Table 4). We have previously generated a GraS T136I mutation in JKD6009 (JKD6208) [18], therefore to measure the impact of the walK/graS double mutation, the walK mutation from VISA JKD6008 was also introduced into the previously produced graS mutant (JKD6208), generating TPS3128 (Table 4). The walR allele from the VISA strain JKD6005 was used to replace the walR allele in the VSSA parent JKD6004 generating TPS3190, and the walR allele from VSSA parent (JKD6004) was used to replace the walR allele from VISA strain (JKD6005) generating the vancomycin-susceptible strain TPS3124 (Table 4). 10.1371/journal.ppat.1002359.t004 Table 4 Impact of mutations in WalKR on vancomycin and daptomycin susceptibility. Isolate Description Standard vanco Etest (µg/mL) Standard dapto Etest (µg/mL) Macro Etest vanco 48hs (µg/mL) Macro Etest teico 48hs (µg/mL) Mutant set 1 JKD6009 Parent VSSA 1.5 0.19 4 6 JKD6008 Clinical VISA 4 0.5 12 16 JKD6208 JKD6009, GraS T136I 2 0.25 6 8 TPS3130 JKD6009, WalK G223D 3 0.25 8 16 TPS3128 JKD6009, GraS T136I and WalK G223D 4 0.75 12 16 Mutant set 2 JKD6004 Parent VSSA 1.5 0.5 4 6 JKD6005 Clinical VISA 4 2 8 12 TPS3124 JKD6005, WalR R208K (wildtype) from JKD6004 1.5 0.75 4 6 TPS3190 JKD6004, WalR K208R 4 2 8 12 Note: vanco, vancomycin; dapto, daptomycin; teico, teicoplanin; macro Etest uses a 2 McFarland inoculum. We next sequenced the genomes of S. aureus mutants TPS3130 and TPS3190 to determine if unintended mutations had been introduced during the pKOR1-mediated allelic exchange process, particularly in other regulatory loci such as agr. Coverage of the reference strain JKD6008 was 99.7% and 96.9% for TPS3130 and TPS3190, respectively. For strain TPS3190, Ion Torrent sequencing confirmed the expected walR mutation at position 25010 and the presence of a single silent mutation in walK at position 26026, but no other changes. The unintended change at nucleotide 26026 was a PCR-induced error, introduced during cloning in pKOR1. The situation with strain TPS3130 was more complex. TPS3130, which is VSSA strain JKD6009 modified by replacing its walK gene with the allele from VISA strain JKD6008 (conferring the G223D amino acid change) had the predicted SNP at position 25769 (Figure S1) but also carried an additional four SNPs not present in JKD6009 (Table 5). Two of these four SNPs were the same as changes found in VISA strains JKD6008 and we propose that these might be compensatory mutations linked to the walK mutation. The probability of these changes occurring by chance at exactly these positions in JKD6008 and TPS3190 is small (p 1 µg per ml) [52]. Clinical isolates underwent molecular characterisation using the DNA microarray StaphyType96 (CLONDIAG, Jena, Germany). DNA extraction was performed using the DNeasy Tissue Kit (Qiagen, Hilden, Germany), and the microarray and data analysis were performed as previously described [53]. The DNA microarray assigns strains to clonal complexes (CC) using reference strains previously defined by multilocus sequence typing (MLST) and spa typing. Pulsed-field gel electrophoresis using SmaI enzyme restriction was performed using the CHEF DR III system (BioRad, Berkeley, California) to confirm clonal group if necessary [54]. DNA Methods, Molecular Techniques and Construction of Mutants Standard procedures were used for DNA manipulation, molecular techniques, PCR, sequencing and plasmid extraction. To generate walK and walR mutants, allelic replacement experiments were performed using the vector pKOR1, as described previously [18]. The locus containing the walKR mutation was amplified from JKD6008, JKD6004, and JKD6005 for exchange into the respective parental strains using primers 1901 and 1908 (Table S2). The amplified product was cloned into the attB sites of pKOR1 and then transformed into E. coli DH5alpha. After transformation into E. coli, pKOR1 with the integrated walKR locus was extracted and sequenced to confirm the correct sequence, prior to performing allelic exchange in RN4220 intermediate and the clinical S. aureus strain. To confirm that no other significant mutations were introduced during the homologous recombination, the whole walKR locus was sequenced from the mutant strains using oligonucleotides covering the whole replaced sequence and its flanking ends (Table S2). High Throughput DNA Sequencing Genome sequences for four ST239 VSSA and VISA clinical pairs (VSSA: JKD6000, JKD6004, JKD6021 and JKD6052; VISA: JKD6001, JKD6005, JKD6023, and JKD6051) were obtained from an Illumina Genome Analyzer II using 36-cycle paired-end chemistry. SOLiDv2 26 bp mate-pair sequencing was also performed on the clinical VSSA strain JKD6009, for which previous 454 GS20 shotgun sequence data was available. Single-end genome sequencing of the two laboratory-induced mutants TPS3130 and TPS3190 was performed using Ion Torrent sequencing as described [55]. TPS3130 yielded 88.7 Mbp from four 314 chips and TPS3190 yielded 56.9 Mbp from two 314 chips. Comparative Genomics A read mapping approach was used to compare the sequences from the four VSSA/VISA clinical pairs described above, the previously described clinical pair JKD6009/JKD6008 (NCBI Genbank accession numbers NZ_ABSA00000000 and CP002120) and the Ion Torrent sequences from TPS3130 and TPS3190. The reads from all genomes were aligned to the JKD6008 reference using SHRiMP 2.0 [56]. SNPs were identified using Nesoni v0.52, which uses the aligned reads of each genome to the reference to construct a tally of putative differences at each position, including substitutions, insertions, and deletions [www.bioinformatics.net.au]. This tally was input to a Bayesian model to decide whether a base (or deletion) could be called for the position, and if so, whether it differed from the reference. Each position is treated as possibly containing a mixture of bases. A particular base is called if the likelihood that it makes up more than 50% of the mixture exceeds a threshold, set to 0.99 by default. This likelihood is calculated by updating a prior distribution over all possible mixtures, in the form of a Dirichlet distribution, as bases are observed. A similar procedure is used to call the presence or absence of insertions between positions in the reference. Using the whole genome sequence of JKD6008 as a reference a global SNP analysis was performed, and allelic variability at any nucleotide position was tallied to generate a global SNP analysis for every genome compared to JKD6008. Protein modelling was performed using the crystal structure of the WalR DNA-binding domain of S. aureus (PDB structure: 2ZXJ) using ModBase [27], [57]. Phenotypic Assays Biofilm and autolytic assays were performed as described previously [15]. Cell wall thickness was measured by taking 100 readings of wall thickness per strain from multiple cells after preparation of electron microscopic images as described previously [15]. Galleria mellonella Virulence Assay This invertebrate S. aureus infection model was used to study the virulence of clinical and mutant strains as previously described [44]. Briefly, a HPLC syringe was used to inject 10 µL of bacterial suspension (approx 1.0×106 CFU) into the last left proleg of each caterpillar. Bacterial colony counts were performed to confirm consistency of inoculum, and caterpillars monitored daily for 6 days. Multiple biological replicates were performed for each strain. Microarray Transcriptional Analysis Microarray transcriptional analysis was performed with TIGR version 9 S. aureus arrays, as previously described [4]. For preparation of total RNA shaking flasks (50 mL BHI broth in 250 mL flasks) were inoculated with 500 µL overnight BHI broth culture and incubated on a 225rpm shaker at 37°C. Optical density was closely monitored, and one millilitre of sample was collected at exponential growth phase (optical density at 600 nm of 0.5) and 0.5 mL RNA stabilization reagent (RNA later, Qiagen) was added and mixed immediately. The mixture was allowed to stand in room temperature for 10 minutes before total RNA was extracted using the RNeasy micro kit (Qiagen). RNA extractions and hybridisations were performed on four different occasions, and the dye swapped with each biological replicate. The images were combined and quantified and then imported into BASE and analyzed using Bioconductor and Limma [58], [59]. The fold ratio of gene expression for the mutant strains TPS3130 and TPS3190 relative to the parental MRSA strain JKD6009 and JKD6004, respectively, were calculated. Using a modified t-test p values were calculated and adjusted for multiple testing using false discovery rate (FDR) correction. A ≥ 1.5-fold change with p<0.05 was considered significant. Quantitative RT-PCR To investigate activity of the agr locus (RNAIII) and confirm the microarray transcriptional results qRT-PCR was performed for RNAIII, argH, purF, pyrA, ureA, atl, and gltB, using oligos in Table S2. RNA was prepared from exponential phase cultures as previously described [15]. Two on-column DNase I digestion steps were performed and cDNA synthesis using SuperScript II RNase H reverse transcriptase (Invitrogen) included a SuperScript II negative control to confirm the absence of genomic DNA. Relative expression was determined as previously described [15], and was normalised against gyrB as previously described [60]. Results were obtained from 3 biological replicates each performed in triplicate. Statistical Analysis Statistical analyses of mutant strains were performed using the two-tailed Mann Whitney U test, with a p<0.05 set for statistical significance. Kaplan Meier plots of G. mellonella killing results were analyzed using the log rank test. All analyses were performed using Prism for Macintosh ver 5.0 (GraphPad Software Inc., CA, USA) Accession Numbers For the sequenced clinical strains JKD6000, JKD6001, JKD6004, JKD6005, JKD6009, JKD6021, JKD6023, JKD6051, JKD6052 the reads have been deposited in the NCBI Sequence Read Archive under study accession number SRA027352. The Ion Torrent reads for the mutant strains TPS3130 and TPS3190 have been deposited in the NCBI Sequence Read Archive under study accession number SRA044879.2. Microarray data has been submitted to GEO with accession number GSE29157. Protein modelling was performed using the crystal structure of the WalR DNA-binding domain of S. aureus (PDB structure: 2ZXJ). Supporting Information Figure S1 Results of Ion Torrent sequencing of mutant strain TPS3130. (A) Non-ambiguous read mapping of Ion Torrent sequences for TPS3130 against the whole genome sequence of reference strain JKD6008, demonstrating genome coverage and depth (repeat regions excluded). (B) Detailed analysis of the read coverage results for the walKR operon confirms the presence of the G to A mutation at position 25769 in the reference strain JKD6008 and the mutant TPS3130. (TIF) Click here for additional data file. Figure S2 Quantitative real-time PCR confirmation of microarray results. Using qRT-PCR the fold ratio of gene expression for the mutant strain (TPS3130 or TPS3190) was compared to the parent strain (JKD6009 or JKD6004) for six genes (argH, purF, pyrA, ureA, atl and gltB). qRT-PCR results are presented as mean ± SEM for at least 3 biological replicates. Microarray expression results for the same genes also shown. (TIF) Click here for additional data file. Table S1 List of differentially regulated genes common to the WalK (TPS3130) and WalR (TPS3190) mutants generated in this study compared to parental strains, based on global microarray transcriptional analysis. (PDF) Click here for additional data file. Table S2 List of primers used in this study. (DOCX) Click here for additional data file.
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New insights into the WalK/WalR (YycG/YycF) essential signal transduction pathway reveal a major role in controlling cell wall metabolism and biofilm formation in Staphylococcus aureus.
Sarah Dubrac, Ivo Boneca, Olivier Poupel … (2007)
The highly conserved WalK/WalR (also known as YycG/YycF) two-component system is specific to low-G+C gram-positive bacteria. While this system is essential for cell viability, both the nature of its regulon and its physiological role have remained mostly uncharacterized. We observed that, unexpectedly, Staphylococcus aureus cell death induced by WalKR depletion was not followed by lysis. We show that WalKR positively controls autolytic activity, in particular that of the two major S. aureus autolysins, AtlA and LytM. By using our previously characterized consensus WalR binding site and carefully reexamining the genome annotations, we identified nine genes potentially belonging to the WalKR regulon that appeared to be involved in S. aureus cell wall degradation. Expression of all of these genes was positively controlled by WalKR levels in the cell, leading to high resistance to Triton X-100-induced lysis when the cells were starved for WalKR. Cells lacking WalKR were also more resistant to lysostaphin-induced lysis, suggesting modifications in cell wall structure. Indeed, lowered levels of WalKR led to a significant decrease in peptidoglycan biosynthesis and turnover and to cell wall modifications, which included increased peptidoglycan cross-linking and glycan chain length. We also demonstrated a direct relationship between WalKR levels and the ability to form biofilms. This is the first example in S. aureus of a regulatory system positively controlling autolysin synthesis and biofilm formation. Taken together, our results now define this signal transduction pathway as a master regulatory system for cell wall metabolism, which we have accordingly renamed WalK/WalR to reflect its true function.
Author and article information
Journal
Journal ID (nlm-ta): Antibiotics (Basel)
Journal ID (iso-abbrev): Antibiotics (Basel)
Journal ID (publisher-id): antibiotics
Title:
Antibiotics
Publisher:
MDPI
ISSN
(Electronic):
2079-6382
Publication date
(Electronic):
11
August
2019
Publication date Collection:
September
2019
Volume: 8
Issue: 3
Electronic Location Identifier: 117
Affiliations
Author notes
Author information
Stephanie Tan
https://orcid.org/0000-0001-7829-2064
Justin Nodwell
https://orcid.org/0000-0001-7909-1803
Article
Publisher ID: antibiotics-08-00117DOI: 10.3390/antibiotics8030117
PMC ID: 6784204
PubMed ID: 31405236
SO-VID: 47bed28a-6014-47c3-9a90-ed2f5a043baf
Copyright © © 2019 by the authors.
License:
Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).
History
Date
received
: 18
July
2019
Date
accepted
: 09
August
2019
Categories
Subject:
Review
Data availability: