Identification of an antivirulence agent targeting the master regulator of virulence genes in Staphylococcus aureus.

The emergence of bactericidal antibiotic-resistant strains has increased the demand for alternative therapeutic agents, such as antivirulence agents targeting the virulence regulators of pathogens. Staphylococcus aureus exoprotein expression (sae) locus, the master regulator of virulence gene expression in multiple drug-resistant S. aureus, is a promising therapeutic target. In this study, we screened a small-molecule library using a SaeRS green fluorescent protein (GFP)-reporter that responded to transcription controlled by the sae locus. We identified the compound, N-(2-methylcyclohexyl)-11-oxo-10,11-dihydrodibenzo[b,f][1,4]thiazepine-8-carboxamide (SKKUCS), as an efficient repressor of sae-regulated GFP activity. SKKUCS inhibited hemolysin production and reduced α-hemolysin-mediated cell lysis. Moreover, SKKUCS substantially reduced the expression levels of various virulence genes controlled by the master regulators, sae, and the accessory gene regulator (agr), demonstrating its potential as an antivirulence reagent targeting the key virulence regulators. Furthermore, autokinase inhibition assay and molecular docking suggest that SKKUCS inhibits the kinase activity of SaeS and potentially targets the active site of SaeS kinase, possibly inhibiting ATP binding. Next, we evaluated the efficacy and toxicity of SKKUCS in vivo using murine models of staphylococcal intraperitoneal and skin infections. Treatment with SKKUCS markedly increased animal survival and significantly decreased the bacterial burden in organs and skin lesion sizes. These findings highlight SKKUCS as a potential antivirulence drug for drug-resistant staphylococcal infections.

Diabetes mellitus promotes the nasal colonization of high virulent Staphylococcus aureus through the regulation of SaeRS two-component system.

Diabetic foot infections are a common complication of diabetes. Staphylococcus aureus is frequently isolated from diabetic foot infections and commonly colonizes human nares. According to the study, the nasal microbiome analysis revealed that diabetic patients had a significantly altered nasal microbial composition and diversity. Typically, the fasting blood glucose (FBG) level had an impact on the abundance and sequence type (ST) of S. aureus in diabetic patients. We observed that highly virulent S. aureus ST7 strains were more frequently colonized in diabetic patients, especially those with poorly controlled FBG, while ST59 was dominant in healthy individuals. S. aureus ST7 strains were more resistant to human antimicrobial peptides and formed stronger biofilms than ST59 strains. Critically, S. aureus ST7 strains displayed higher virulence compared to ST59 strains in vivo. The dominance of S. aureus ST7 strains in hyperglycemic environment is due to the higher activity of the SaeRS two-component system (TCS). S. aureus ST7 strains outcompeted ST59 both in vitro, and in nasal colonization model in diabetic mice, which was abolished by the deletion of the SaeRS TCS. Our data indicated that highly virulent S. aureus strains preferentially colonize diabetic patients with poorly controlled FBG through SaeRS TCS. Detection of S. aureus colonization and elimination of colonizing S. aureus are critical in the care of diabetic patients with high FBG.

Regulation of Bacterial Two-Component Systems by Cardiolipin.

The regulation of membrane protein activity for cellular functions is critically dependent on the composition of phospholipid membranes. Cardiolipin, a unique phospholipid found in bacterial membranes and mitochondrial membranes of eukaryotes, plays a crucial role in stabilizing membrane proteins and maintaining their function. In the human pathogen Staphylococcus aureus, the SaeRS two-component system (TCS) controls the expression of key virulence factors essential for the bacterium's virulence. The SaeS sensor kinase activates the SaeR response regulator via phosphoryl transfer to bind its gene target promoters. In this study, we report that cardiolipin is critical for sustaining the full activity of SaeRS and other TCSs in S. aureus. The sensor kinase protein SaeS binds directly to cardiolipin and phosphatidylglycerol, enabling SaeS activity. Elimination of cardiolipin from the membrane reduces SaeS kinase activity, indicating that bacterial cardiolipin is necessary for modulating the kinase activities of SaeS and other sensor kinases during infection. Moreover, the deletion of cardiolipin synthase genes cls1 and cls2 leads to reduced cytotoxicity to human neutrophils and lower virulence in a mouse model of infection. These findings suggest a model where cardiolipin modulates the kinase activity of SaeS and other sensor kinases after infection to adapt to the hostile environment of the host and expand our knowledge of how phospholipids contribute to membrane protein function.

Modulation of MRSA virulence gene expression by the wall teichoic acid enzyme TarO.

Phenol-soluble modulins (PSMs) and Staphylococcal protein A (SpA) are key virulence determinants for community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA), an important human pathogen that causes a wide range of diseases. Here, using chemical and genetic approaches, we show that inhibition of TarO, the first enzyme in the wall teichoic acid (WTA) biosynthetic pathway, decreases the expression of genes encoding PSMs and SpA in the prototypical CA-MRSA strain USA300 LAC. Mechanistically, these effects are linked to the activation of VraRS two-component system that directly represses the expression of accessory gene regulator (agr) locus and spa. The activation of VraRS was due in part to the loss of the functional integrity of penicillin-binding protein 2 (PBP2) in a PBP2a-dependent manner. TarO inhibition can also activate VraRS in a manner independent of PBP2a. We provide multiple lines of evidence that accumulation of lipid-linked peptidoglycan precursors is a trigger for the activation of VraRS. In sum, our results reveal that WTA biosynthesis plays an important role in the regulation of virulence gene expression in CA-MRSA, underlining TarO as an attractive target for anti-virulence therapy. Our data also suggest that acquisition of PBP2a-encoding mecA gene can impart an additional regulatory layer for the modulation of key signaling pathways in S. aureus.

Regulation of Bacterial Two-Component Systems by Cardiolipin.

The composition of phospholipid membranes is critical to regulating the activity of membrane proteins for cellular functions. Cardiolipin is a unique phospholipid present within the bacterial membrane and mitochondria of eukaryotes and plays a role in maintaining the function and stabilization of membrane proteins. Here, we report that, in the human pathogen Staphylococcus aureus, cardiolipin is required for full activity of the SaeRS two-component system (TCS). Deletion of the cardiolipin synthase genes, cls1 , and cls2 , reduces the basal activity of SaeRS and other TCSs. Cardiolipin is an indispensable requisite for Sae activation mediated by human neutrophil peptides (HNPs) in the stationary growth phase but not mandatory for Sae induction in the exponential growth phase. Ectopic expression with cls2 , but not with cls1 , in the cls1 cls2 double mutant fully restores Sae activity. Elimination of cardiolipin from the membranes results in decreased kinase activity of the sensor protein SaeS. Purified SaeS protein directly binds to cardiolipin as well as phosphatidylglycerol. A strain lacking cls2 or cls1cls2 renders S. aureus less cytotoxic to human neutrophils and less virulent in a mouse model of infection. Our findings suggest that cardiolipin enables a pathogen to confer virulence by modulating the kinase activity of SaeS and other sensor kinases upon infection.

A novel copper-sensing two-component system for inducing Dsb gene expression in bacteria.

In nature, bacteria must sense copper and tightly regulate gene expression to evade copper toxicity. Here, we identify a new copper-responsive two-component system named DsbRS in the important human pathogen Pseudomonas aeruginosa; in this system, DsbS is a sensor histidine kinase, and DsbR, its cognate response regulator, directly induces the transcription of genes involved in protein disulfide bond formation (Dsb) (i.e., the dsbDEG operon and dsbB). In the absence of copper, DsbS acts as a phosphatase toward DsbR, thus blocking the transcription of Dsb genes. In the presence of copper, the metal ion directly binds to the sensor domain of DsbS, and the Cys82 residue plays a critical role in this process. The copper-binding behavior appears to inhibit the phosphatase activity of DsbS, leading to the activation of DsbR. The copper resistance of the dsbRS knock-out mutant is restored by the ectopic expression of the dsbDEG operon, which is a DsbRS major target. Strikingly, cognates of the dsbRS-dsbDEG pair are widely distributed across eubacteria. In addition, a DsbR-binding site, which contains the consensus sequence 5'-TTA-N8-TTAA-3', is detected in the promoter region of dsbDEG homologs in these species. These findings suggest that the regulation of Dsb genes by DsbRS represents a novel mechanism by which bacterial cells cope with copper stress.

Influence of Staphylococcus aureus Strain Background on Sa3int Phage Life Cycle Switches.

Staphylococcus aureus asymptomatically colonizes the nasal cavity of mammals, but it is also a leading cause of life-threatening infections. Most human nasal isolates carry Sa3 phages, which integrate into the bacterial hlb gene encoding a sphingomyelinase. The virulence factor-encoding genes carried by the Sa3-phages are highly human-specific, and most animal strains are Sa3 negative. Thus, both insertion and excision of the prophage could potentially confer a fitness advantage to S. aureus. Here, we analyzed the phage life cycle of two Sa3 phages, Φ13 and ΦN315, in different phage-cured S. aureus strains. Based on phage transfer experiments, strains could be classified into low (8325-4, SH1000, and USA300c) and high (MW2c and Newman-c) transfer strains. High-transfer strains promoted the replication of phages, whereas phage adsorption, integration, excision, or recA transcription was not significantly different between strains. RNASeq analyses of replication-deficient lysogens revealed no strain-specific differences in the CI/Mor regulatory switch. However, lytic genes were significantly upregulated in the high transfer strain MW2c Φ13 compared to strain 8325-4 Φ13. By transcriptional start site prediction, new promoter regions within the lytic modules were identified, which are likely targeted by specific host factors. Such host-phage interaction probably accounts for the strain-specific differences in phage replication and transfer frequency. Thus, the genetic makeup of the host strains may determine the rate of phage mobilization, a feature that might impact the speed at which certain strains can achieve host adaptation.

Regulation of the Sae Two-Component System by Branched-Chain Fatty Acids in Staphylococcus aureus.

Staphylococcus aureus is a ubiquitous Gram-positive bacterium and an opportunistic human pathogen. S. aureus pathogenesis relies on a complex network of regulatory factors that adjust gene expression. Two important factors in this network are CodY, a repressor protein responsive to nutrient availability, and the SaeRS two-component system (TCS), which responds to neutrophil-produced factors. Our previous work revealed that CodY regulates the secretion of many toxins indirectly via Sae through an unknown mechanism. We report that disruption of codY results in increased levels of phosphorylated SaeR (SaeR~P) and that codY mutant cell membranes contain a higher percentage of branched-chain fatty acids (BCFAs) than do wild-type membranes, prompting us to hypothesize that changes to membrane composition modulate the activity of the SaeS sensor kinase. Disrupting the lpdA gene encoding dihydrolipoyl dehydrogenase, which is critical for BCFA synthesis, significantly reduced the abundance of SaeR, phosphorylated SaeR, and BCFAs in the membrane, resulting in reduced toxin production and attenuated virulence. Lower SaeR levels could be explained in part by reduced stability. Sae activity in the lpdA mutant could be complemented genetically and chemically with exogenous short- or full-length BCFAs. Intriguingly, lack of lpdA also alters the activity of other TCSs, suggesting a specific BCFA requirement managing the basal activity of multiple TCSs. These results reveal a novel method of posttranscriptional virulence regulation via BCFA synthesis, potentially linking CodY activity to multiple virulence regulators in S. aureus. IMPORTANCE Two-component systems (TCSs) are an essential way that bacteria sense and respond to their environment. These systems are usually composed of a membrane-bound histidine kinase that phosphorylates a cytoplasmic response regulator. Because most of the histidine kinases are embedded in the membrane, lipids can allosterically regulate the activity of these sensors. In this study, we reveal that branched-chain fatty acids (BCFAs) are required for the activation of multiple TCSs in Staphylococcus aureus. Using both genetic and biochemical data, we show that the activity of the virulence activator SaeS and the phosphorylation of its response regulator SaeR are reduced in a branched-chain keto-acid dehydrogenase complex mutant and that defects in BCFA synthesis have far-reaching consequences for exotoxin secretion and virulence. Finally, we show that mutation of the global nutritional regulator CodY alters BCFA content in the membrane, revealing a potential mechanism of posttranscriptional regulation of the Sae system by CodY.

Staphylococcus aureus Does Not Synthesize Arginine from Proline under Physiological Conditions.

The Gram-positive pathogen Staphylococcus aureus is the only bacterium known to synthesize arginine from proline via the arginine-proline interconversion pathway despite having genes for the well-conserved glutamate pathway. Since the proline-arginine interconversion pathway is repressed by CcpA-mediated carbon catabolite repression (CCR), CCR has been attributed to the arginine auxotrophy of S. aureus. Using ribose as a secondary carbon source, here, we demonstrate that S. aureus arginine auxotrophy is not due to CCR but due to the inadequate concentration of proline degradation product. Proline is degraded by proline dehydrogenase (PutA) into pyrroline-5-carboxylate (P5C). Although the PutA expression was fully induced by ribose, the P5C concentration remained insufficient to support arginine synthesis because P5C was constantly consumed by the P5C reductase ProC. When the P5C concentration was artificially increased by either PutA overexpression or proC deletion, S. aureus could synthesize arginine from proline regardless of carbon source. In contrast, when the P5C concentration was reduced by overexpression of proC, it inhibited the growth of the ccpA deletion mutant without arginine. Intriguingly, the ectopic expression of the glutamate pathway enzymes converted S. aureus into arginine prototroph. In an animal experiment, the arginine-proline interconversion pathway was not required for the survival of S. aureus. Based on these results, we concluded that S. aureus does not synthesize arginine from proline under physiological conditions. We also propose that arginine auxotrophy of S. aureus is not due to the CcpA-mediated CCR but due to the inactivity of the conserved glutamate pathway. IMPORTANCE Staphylococcus aureus is a versatile Gram-positive human pathogen infecting various human organs. The bacterium's versatility is partly due to efficient metabolic regulation via the carbon catabolite repression system (CCR). S. aureus is known to interconvert proline and arginine, and CCR represses the synthesis of both amino acids. However, when CCR is released by a nonpreferred carbon source, S. aureus can synthesize proline but not arginine. In this study, we show that, in S. aureus, the intracellular concentration of pyrroline-5-carboxylate (P5C), the degradation product of proline and the substrate of proline synthesis, is too low to synthesize arginine from proline. These results call into question the notion that S. aureus synthesizes arginine from proline.

Unravelling the physiological roles of mazEF toxin-antitoxin system on clinical MRSA strain by CRISPR RNA-guided cytidine deaminase.

BACKGROUND: Curiosity on toxin-antitoxin modules has increased intensely over recent years as it is ubiquitously present in many bacterial genomes, including pathogens like Methicillin-resistant Staphylococcus aureus (MRSA). Several cellular functions of TA systems have been proposed however, their exact role in cellular physiology remains unresolved. METHODS: This study aims to find out the impact of the mazEF toxin-antitoxin module on biofilm formation, pathogenesis, and antibiotic resistance in an isolated clinical ST239 MRSA strain, by constructing mazE and mazF mutants using CRISPR-cas9 base-editing plasmid (pnCasSA-BEC). Transcriptome analysis (RNA-seq) was performed for the mazE antitoxin mutant in order to identify the differentially regulated genes. The biofilm formation was also assessed for the mutant strains. Antibiogram profiling was carried out for both the generated mutants followed by murine experiment to determine the pathogenicity of the constructed strains. RESULTS: For the first time our work showed, that MazF promotes cidA mediated cell death and lysis for biofilm formation without playing any significant role in host virulence as suggested by the murine experiment. Interestingly, the susceptibility to oxacillin, daptomycin and vancomycin was reduced significantly by the activated MazF toxin in the mazE mutant strain. CONCLUSIONS: Our study reveals that activated MazF toxin leads to resistance to antibiotics like oxacillin, daptomycin and vancomycin. Therefore, in the future, any potential antibacterial drug can be designed to target MazF toxin against the problematic multi-drug resistant bug.

Virulence adaption to environment promotes the age-dependent nasal colonization of Staphylococcus aureus.

Staphylococcus aureus is an important human commensal bacteria colonizing the human body, especially the nasal cavity. The nasal carriage can be a source of S. aureus bacteremia. However, the bacterial factors contributing to nasal colonization are not completely understood. By analysing S. aureus strains from the nasal cavity of the children, young adults, and seniors, we found that the low activity of the SaeRS two-component system (TCS) is an important determinant for S. aureus to colonize in seniors. The senior group isolates of S. aureus showed a rather distinct sequence type composition as compared with other age group isolates. The senior group isolates showed not only a lower gene carriage of enterotoxins a, c, and q but also lower hemolytic activity against human red blood cells. Of regulators affecting hemolysin production (i.e. agr, saeRS, rot, rsp, and sarS), only the SaeRS TCS showed an age-dependent decrease of activity. The decreased virulence and better colonization ability of the senior group isolates of S. aureus were confirmed in the mouse model. The senior group isolates showed the lowest survival and the best adhesion and colonizing ability. Also, the senior nasal secretions supported S. aureus survival better than the child and young adult nasal secretions. These results indicated that the senior nasal cavity favours colonization of S. aureus with higher adhesion and lower virulence, to which the reduced SaeRS TCS activity contributes. Taken together, our results illustrate an example of bacterial adaptation to the changing host environment.

Molybdopterin biosynthesis pathway contributes to the regulation of SaeRS two-component system by ClpP in Staphylococcus aureus.

In Staphylococcus aureus, the SaeRS two-component system is essential for the bacterium's hemolytic activity and virulence. The Newman strain of S. aureus contains a variant of SaeS sensor kinase, SaeS L18P. Previously, we showed that, in the strain Newman, SaeS L18P is degraded by the membrane-bound protease FtsH. Intriguingly, the knockout mutation of clpP, encoding the cytoplasmic protease ClpP, greatly reduces the expression of SaeS L18P. Here, we report that, in the strain Newman, the positive regulatory role of ClpP on the SaeS L18P expression is due to its destabilizing effect on FtsH and degradation of MoeA, a molybdopterin biosynthesis protein. Although the transcription of ftsH was not affected by ClpP, the expression level of FtsH was increased in the clpP mutant. The destabilizing effect appears to be indirect because ClpXP did not directly degrade FtsH in an in vitro assay. Through transposon mutagenesis, we found out that the moeA gene, encoding the molybdopterin biosynthesis protein A, suppresses the hemolytic activity of S. aureus along with the transcription and expression of SaeS L18P. In a proteolysis assay, ClpXP directly degraded MoeA, demonstrating that MoeA is a substrate of the protease. In a murine bloodstream infection model, the moeA mutant displayed reduced virulence and lower survival compared with the WT strain. Based on these results, we concluded that ClpP positively controls the expression of SaeS L18P in an FtsH and MoeA-dependent manner, and the physiological role of MoeA outweighs its suppressive effect on the SaeRS TCS during infection.

Comparative Genomic Analysis of a Panton-Valentine Leukocidin-Positive ST22 Community-Acquired Methicillin-Resistant Staphylococcus aureus from Pakistan.

Staphylococcus aureus (S. aureus) ST22 is considered a clinically important clone because an epidemic strain EMRSA-15 belongs to ST22, and several outbreaks of this clone have been documented worldwide. We performed genomic analysis of an S. aureus strain Lr2 ST22 from Pakistan and determined comparative analysis with other ST22 strains. The genomic data show that Lr2 belongs to spa-type t2986 and harbors staphylococcal cassette chromosome mec (SCCmec) type IVa(2B), one complete plasmid, and seven prophages or prophage-like elements. The strain harbors several prophage-associated virulence factors, including Panton-Valentine leukocidin (PVL) and toxic shock syndrome toxin (TSST). The single nucleotide polymorphism (SNPs)-based phylogenetic relationship inferred from whole genome and core genome revealed that strain Lr2 exhibits the nearest identities to a South African community-acquired methicillin-resistant S. aureus (CA-MRSA) ST22 strain and makes a separate clade with an Indian CA-MRSA ST22 strain. Although most ST22 strains carry blaZ, mecA, and mutations in gyrA, the Lr2 strain does not have the blaZ gene but, unlike other ST22 strains, carries the antibiotic resistance genes erm(C) and aac(6')-Ie-aph(2″)-Ia. Among ST22 strains analyzed, only the strain Lr2 possesses both PVL and TSST genes. The functional annotation of genes unique to Lr2 revealed that mobilome is the third-largest Cluster of Orthologous Genes (COGs) category, which encodes genes associated with prophages and transposons. This possibly makes methicillin-resistant S. aureus (MRSA) Lr2 ST22 strain highly virulent, and this study would improve the knowledge of MRSA ST22 strains in Pakistan. However, further studies are needed on a large collection of MRSA to comprehend the genomic epidemiology and evolution of this clone in Pakistan.

In silico designed Staphylococcus aureus B-cell multi-epitope vaccine did not elicit antibodies against target antigens suggesting multi-domain approach.

The vaccine development strategies have evolved from using an entire organism as an immunogen to a single antigen and further towards an epitope. Since an epitope is a relatively tiny and immunologically relevant part of an antigen, it has the potential to stimulate more robust and specific immune responses while causing minimal adverse effects. As a result, the recent focus of vaccine development has been to develop multi-epitope vaccines that can target multiple virulence mechanisms. Accordingly, we designed multi-epitope vaccine candidates B (multi-B-cell epitope immunogen) and CTB-B (an adjuvant - cholera toxin subunit B (CTB) - attached to immunogen B) against S. aureus by employing immunoinformatics approaches. The designed vaccines are composed of B-cell epitope segments (20-mer) of the eight well-characterized S. aureus virulence factors, namely ClfB, FnbpA, Hla, IsdA, IsdB, LukE, SdrD, and SdrE connected in series. The designed vaccines were expressed, purified, and administered to C57BL/6 mice with Freund adjuvant to evaluate the immunogenicity and protective efficacy. The results revealed that the immunized mice showed high IgG titers for the immunogen, and the antibody titers increased significantly following the second immunization. However, the generated antibodies did not protect the mice from infection. The interaction of anti-B antibodies with source virulence factors showed that the generated antibodies have no binding affinity with any of the corresponding virulence factors. Our results demonstrate the limitation of the in silico designed B-cell multi-epitope vaccine and suggest that a protein domain carrying both linear and conformational B-cell epitopes might be a better choice for developing an effective multi-epitope vaccine against S. aureus.

Ftsh Sensitizes Methicillin-Resistant Staphylococcus aureus to ß-Lactam Antibiotics by Degrading YpfP, a Lipoteichoic Acid Synthesis Enzyme.

In the Gram-positive pathogen Staphylococcus aureus, FtsH, a membrane-bound metalloprotease, plays a critical role in bacterial virulence and stress resistance. This protease is also known to sensitize methicillin-resistant Staphylococcus aureus (MRSA) to ß-lactam antibiotics; however, the molecular mechanism is not known. Here, by the analysis of FtsH substrate mutants, we found that FtsH sensitizes MRSA specifically to ß-lactams by degrading YpfP, the enzyme synthesizing the anchor molecule for lipoteichoic acid (LTA). Both the overexpression of FtsH and the disruption of ypfP-sensitized MRSA to ß-lactams were observed. The knockout mutation in ftsH and ypfP increased the thickness of the cell wall. The ß-lactam sensitization coincided with the production of aberrantly large LTA molecules. The combination of three mutations in the rpoC, vraB, and SAUSA300_2133 genes blocked the ß-lactam-sensitizing effect of FtsH. Murine infection with the ypfP mutant could be treated by oxacillin, a ß-lactam antibiotic ineffective against MRSA; however, the effective concentration of oxacillin differed depending on the S. aureus strain. Our study demonstrated that the ß-lactam sensitizing effect of FtsH is due to its digestion of YpfP. It also suggests that the larger LTA molecules are responsible for the ß-lactam sensitization phenotype, and YpfP is a viable target for developing novel anti-MRSA drugs.

Genomic Investigation of Methicillin-Resistant Staphylococcus aureus ST113 Strains Isolated from Tertiary Care Hospitals in Pakistan.

Methicillin-resistant Staphylococcus aureus (MRSA) is a multi-drug resistant and opportunistic pathogen. The emergence of new clones of MRSA in both healthcare settings and the community warrants serious attention and epidemiological surveillance. However, epidemiological data of MRSA isolates from Pakistan are limited. We performed a whole-genome-based comparative analysis of two (P10 and R46) MRSA strains isolated from two provinces of Pakistan to understand the genetic diversity, sequence type (ST), and distribution of virulence and antibiotic-resistance genes. The strains belong to ST113 and harbor the SCCmec type IV encoding mecA gene. Both the strains contain two plasmids, and three and two complete prophage sequences are present in P10 and R46, respectively. The specific antibiotic resistance determinants in P10 include two aminoglycoside-resistance genes, aph(3')-IIIa and aad(6), a streptothrin-resistance gene sat-4, a tetracycline-resistance gene tet(K), a mupirocin-resistance gene mupA, a point mutation in fusA conferring resistance to fusidic acid, and in strain R46 a specific plasmid associated gene ant(4')-Ib. The strains harbor many virulence factors common to MRSA. However, no Panton-Valentine leucocidin (lukF-PV/lukS-PV) or toxic shock syndrome toxin (tsst) genes were detected in any of the genomes. The phylogenetic relationship of P10 and R46 with other prevailing MRSA strains suggests that ST113 strains are closely related to ST8 strains and ST113 strains are a single-locus variant of ST8. These findings provide important information concerning the emerging MRSA clone ST113 in Pakistan and the sequenced strains can be used as reference strains for the comparative genomic analysis of other MRSA strains in Pakistan and ST113 strains globally.

Development of Combination Vaccine Conferring Optimal Protection against Six Pore-Forming Toxins of Staphylococcus aureus.

In the Gram-positive pathogen Staphylococcus aureus, pore-forming toxins (PFTs), such as leukocidins and hemolysins, play prominent roles in staphylococcal pathogenesis by killing host immune cells and red blood cells (RBCs). However, it remains unknown which combination of toxin antigens would induce the broadest protective immune response against those toxins. In this study, by targeting six major staphylococcal PFTs (i.e., gamma-hemolysin AB [HlgAB], gamma-hemolysin CB [HlgCB], leukocidin AB [LukAB], leukocidin ED [LukED], Panton-Valentine leukocidin [LukSF-PV], and alpha-hemolysin [Hla]), we generated 10 recombinant toxins or toxin subunits, 3 toxoids, and their rabbit antibodies. Using the cytolytic assay for RBCs and polymorphonuclear cells (PMNs), we determined the best combination of toxin antibodies conferring the broadest protection against those staphylococcal PFTs. Although anti-HlgA IgG (HlgA-IgG) showed low cross-reactivity to other toxin components, it was essential to protect rabbit and human RBCs and human PMNs. For the protection of rabbit RBCs, HlaH35L toxoid-IgG was also required, whereas for human PMNs, LukS-IgG and LukAE323AB-IgG were essential too. When the toxin/toxoid antigens HlgA, LukS-PV, HlaH35L, and LukAE323AB were used to immunize rabbits, they increased rabbit survival; however, they did not block staphylococcal abscess formation in kidneys. Based on these results, we proposed that the combination of HlgA, LukS, HlaH35L, and LukAE323AB is the optimal vaccine component to protect human RBCs and PMNs from staphylococcal PFTs. We also concluded that a successful S. aureus vaccine requires not only those toxin antigens but also other antigens that can induce immune responses blocking staphylococcal colonization.

The N2N3 domains of ClfA, FnbpA and FnbpB in Staphylococcus aureus bind to human complement factor H, and their antibodies enhance the bactericidal capability of human blood.

In the complement system, the opsonin C3b binds to the bacterial cell surface and mediates the opsonophagocytosis. However, the cell-wall protein SdrE of Staphylococcus aureus inhibits the C3b activity by recruiting the complement regulatory protein factor H (fH). SdrE binds to fH via its N-terminal N2N3 domain, which are also found in six other staphylococcal cell-wall proteins. In this study, we report that not only the N2N3 domain of SdrE but also those of ClfA, FnbpA and FnbpB can bind to fH. When immobilized on a microplate, the N2N3 domains recruited fH and enhanced the factor I (fI)-mediated cleavage of C3b. When mixed with fH and S. aureus cells, the N2N3 domains inhibited the fH binding to S. aureus cells and reduced the fI-mediated C3b cleavage on the bacterial cell surface. The F(ab)'2 fragments of the rabbit N2N3 antibodies also inhibited the fH binding to the S. aureus cell surface. When added to human blood, the N2N3 antibodies or the N2N3 domain proteins significantly increased the bactericidal activity. Based on these results, we conclude that, in S. aureus, not only SdrE but also ClfA, FnbpA and FnbpB can contribute to the inhibition of C3b-mediated opsonophagocytosis.

Histamine activates HinK to promote the virulence of Pseudomonas aeruginosa.

During infections, bacteria stimulate host cells to produce and release histamine, which is a key mediator of vital cellular processes in animals. However, the mechanisms underlying the bacterial cell's ability to sense and respond to histamine are poorly understood. Herein, we show that HinK, a LysR-type transcriptional regulator, is required to evoke responses to histamine in Pseudomonas aeruginosa, an important human pathogen. HinK directly binds to and activates the promoter of genes involved in histamine uptake and metabolism, iron acquisition, and Pseudomonas quinolone signal (PQS) biosynthesis. The transcriptional regulatory activity of HinK is induced when histamine is present, and it occurs when HinK binds with imidazole-4-acetic acid (ImAA), a histamine metabolite whose production in P. aeruginosa depends on the HinK-activated histamine uptake and utilization operon hinDAC-pa0222. Importantly, the inactivation of HinK inhibits diverse pathogenic phenotypes of P. aeruginosa. These results suggest that histamine acts as an interkingdom signal and provide insights into the mechanism used by pathogenic bacteria to exploit host regulatory signals to promote virulence.

Mutation-induced remodeling of the BfmRS two-component system in Pseudomonas aeruginosa clinical isolates.

Genetic mutations are a primary driving force behind the adaptive evolution of bacterial pathogens. Multiple clinical isolates of Pseudomonas aeruginosa, an important human pathogen, have naturally evolved one or more missense mutations in bfmS, which encodes the sensor histidine kinase of the BfmRS two-component system (TCS). A mutant BfmS protein containing both the L181P and E376Q substitutions increased the phosphorylation and thus the transcriptional regulatory activity of its cognate downstream response regulator, BfmR. This reduced acute virulence and enhanced biofilm formation, both of which are phenotypic changes associated with a chronic infection state. The increased phosphorylation of BfmR was due, at least in part, to the cross-phosphorylation of BfmR by GtrS, a noncognate sensor kinase. Other spontaneous missense mutations in bfmS, such as A42E/G347D, T242R, and R393H, also caused a similar remodeling of the BfmRS TCS in P. aeruginosa This study highlights the plasticity of TCSs mediated by spontaneous mutations and suggests that mutation-induced activation of BfmRS may contribute to host adaptation by P. aeruginosa during chronic infections.