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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, andcls2, 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 withcls2, but not withcls1, in thecls1 cls2double 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 lackingcls2orcls1cls2rendersS. aureusless 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.

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.

Phenol-soluble modulins (PSMs) and Staphylococcal protein A (SpA) are key virulence determinants for community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA). 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, at least in part, to the loss of the functional integrity of penicillin-binding protein 2 (PBP2) in a PBP2a-dependent manner. We provide multiple lines of evidence that the accumulation of lipid-linked peptidoglycan precursors is a trigger for the activation of VraRS. In sum, our data reveal an unprecedented and important role for WTA biosynthesis in the regulation of virulence gene expression in CA-MRSA, underlining that acquisition of PBP2a-encoding mecA gene can impart an additional regulatory layer for the modulation of key signaling pathways (e.g., VraRS and agr) in S. aureus.

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.ImportanceStaphylococcus 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 non-preferred 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.

Intracellular protein degradation is essential for the survival of all organisms, but its role in interspecies interaction is unknown. Here, we show that the ClpXP protease of Pseudomonas aeruginosa suppresses its antimicrobial activity against Staphylococcus aureus, a common pathogen co-isolated with P. aeruginosa from polymicrobial human infections. Using proteomic, biochemical, and molecular genetic approaches, we found that this effect is due to the inhibitory effects of ClpXP on the quorum sensing (QS) of P. aeruginosa, mainly by degrading proteins (e.g., PhnA, PhnB, PqsR, and RhlI) which are critical for the production of QS signal molecules PQS and C4-HSL. We provide evidence that co-culturing with S. aureus induces a decrease in the activity of ClpXP in P. aeruginosa, an effect which was also achieved by the treatment of P. aeruginosa with N-acetylglucosamine (GlcNAc), a widespread chemical present on the surface of diverse cell types from bacteria to humans. These findings extend the range of biological events governed by proteolytic machinery to microbial community structure, thus also suggesting that a chemical-induced alteration of protein homeostasis is a mechanism for interspecies interactions.

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, Hla(H35L) toxoid-IgG was also required, whereas for human PMNs, LukS-IgG and LukA(E323A)B-IgG were essential too. When the toxin/toxoid antigens HlgA, LukS-PV, Hla(H35L), and LukA(E323A)B 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, Hla(H35L), and LukA(E323A)B 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.

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.

Background: The first two years of medical student education relies on textbooks and lectures for a foundation of knowledge that must be expounded upon in the latter two years. However, this doesn’t mean that students should be constrained to a narrow usage of their intelligence for exam questions, rather they can begin to think about using their knowledge in a clinical setting from the beginning. We researched eight prevalent pathogens that have a higher probability of being encountered by a physician by using real life examples from case reports from around the world. We condensed the materials into a team-based learning (TBL) activity with thought provoking questions related to the case in particular and to fundamental principles of microbiology and immunology as a whole. Approach: We searched extensively through online databases to locate clinical case reports that weren’t too uncommon or too simple. Next, we built a TBL activity using the case report as the framework with clinical presentation, lab values, and images in a certain order that would resemble the way that the physician encountered it. A mixture of questions from simple identification to complex mechanisms were asked along with review of other similar pathogens and relevant pharmacology. Along with every activity, a small abridged review of the pathogen was written after researching and consulting appropriate literature. The review included the most clinically significant information to supplement the TBL activity such as history, epidemiology, transmission, disease, diagnosis, and treatment of the pathogen. Future implications: These TBL activities can be utilized in future host defense course as a way for students to test their understanding while exploring a more clinically relevant scenario. Hopefully, students will appreciate the real world application of their expansive knowledge, and apply their newly developed understanding in rotations, residency, and even their medical practice.

Genetic mutations are a primary driving force behind the adaptive evolution of bacterial pathogens. Multiple clinical isolates of

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.

In the Gram-positive pathogen Staphylococcus aureus, the membrane-bound ATP-dependent metalloprotease FtsH plays a critical role in resistance to various stressors. However, the molecular mechanism of the FtsH functions is not known. Here, we identified core FtsH target proteins in S. aureus. In the strains Newman and USA300, the abundance of 33 proteins were altered in both strains, of which 11 were identified as core FtsH substrate protein candidates. In the strain Newman and some other S. aureus strains, the sensor histidine kinase SaeS has an L18P (T53C in saeS) substitution, which transformed the protein into an FtsH substrate. Due to the increase of SaeS L18P in the ftsH mutant, Eap, a sae-regulon protein, was also increased in abundance, causing the Newman-specific cell-aggregation phenotype. Regardless of the strain background, however, the ftsH mutants showed lower virulence and survival in a murine infection model. Our study illustrates the elasticity of the bacterial regulatory network, which can be rewired by a single substitution mutation.

Copper is an essential element for biological systems but becomes toxic when present in excess. In Pseudomonas aeruginosa, an important human pathogen, the resistance to copper requires the induction of dsbDEG operon encoding proteins involved in disulfide-bond formation (Dsb). However, it is unknown how the copper stress induces the transcription of the operon. Here, we report that the exogenous copper induces the transcription of the dsbDEG operon through a new copper-sensing two-component system named DsbRS. The dsbRS is divergently transcribed from the dsbDEG operon, and the response regulator DsbR binds to the intergenic region between the operons. In the absence of copper, the sensor kinase DsbS acts as a phosphatase toward DsbR and thus blocks the transcription of the operons. However, in the presence of copper, the metal ion directly binds to the sensor domain of DsbS, for which the Cys82 residue plays a critical role. The copper-binding appears to inhibit the phosphatase activity of DsbS, leading to activation of DsbR. The copper resistance of the dsbRS knock-out mutant was restored by ectopic expression of the dsbDEG operon, confirming the critical role of the operon in the resistance to copper. Strikingly, cognates of dsbRS-dsbDEG pair are widely distributed across eubacteria. Also, a DsbR-binding site, which contains the consensus sequence 5’-TAA-N7-TTAAT-3’, is detected in the promoter region of dsbDEG homologs in those species. Thus, regulation of Dsb genes by DsbRS represents a novel mechanism by which bacterial cells cope with copper stress.ImportanceCopper is an essential redox active cofactor that becomes highly cytotoxic when present in excess. Therefore, in order to evade copper toxicity, bacteria must perceive copper stress and tightly regulate genes expression. In the present study, we identify a new copper-sensing two-component system (designated DsbRS) in Pseudomonas aeruginosa, an important human pathogen. We provide multiple lines of evidence that upon copper binding to the periplasmic domain of DsbS, its phosphatase activity is blocked, and the phosphorylated DsbR directly activates the transcription of a number of copper-induced genes including those involved in protein disulfide-bond formation (Dsb). This study suggests that regulation of Dsb genes by DsbRS may be an underappreciated regulatory mechanism by which bacteria sense and respond to copper.

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.

Staphylococcus aureus, a major human pathogen, has been the cause of serious infectious diseases with a high mortality rate. Although genetics is a key means to study S. aureus physiology, such as drug resistance and pathogenesis, genetic manipulation in S. aureus is always time-consuming and labor-intensive. Here we report a CRISPR/Cas9 system (pCasSA) for rapid and efficient genome editing, including gene deletion, insertion, and single-base substitution mutation in S. aureus. The designed pCasSA system is amenable to the assembly of spacers and repair arms by Golden Gate assembly and Gibson assembly, respectively, enabling rapid construction of the plasmids for editing. We further engineered the pCasSA system to be an efficient transcription inhibition system for gene knockdown and possible genome-wide screening. The development of the CRISPR/Cas9-mediated genome editing and transcription inhibition tools will dramatically accelerate drug-target exploration and drug development.

In Enterococcus faecalis, the site 2 protease Eep generates sex pheromones, including cAM373. Intriguingly, in Staphylococcus aureus, a peptide similar to cAM373, named cAM373_SA, is produced from the camS gene. Here, we report that the staphylococcal Eep homolog is not only responsible for the production of cAM373_SA but also critical for staphylococcal virulence. As with other Eep proteins, the staphylococcal Eep protein has four transmembrane (TM) domains, with the predicted zinc metalloprotease active site (HEXXH) in the first TM domain. eep deletion reduced the cAM373_SA activity in the culture supernatant to the level of the camS deletion mutant. It also markedly decreased the cAM373 peptide peak in a high-performance liquid chromatography (HPLC) analysis. Proteomics analysis showed that Eep affects the production and/or the release of diverse proteins, including the signal peptidase subunit SpsB and the surface proteins SpA, SasG, and FnbA. eep deletion decreased the adherence of S. aureus to host epithelial cells; however, the adherence of the eep mutant was increased by overexpression of the surface proteins SpA, SasG, and FnbA. eep deletion reduced staphylococcal resistance to killing by human neutrophils as well as survival in a murine model of blood infection. The overexpression of the surface protein SpA in the eep mutant increased bacterial survival in the liver. Our study illustrates that in S. aureus, Eep not only generates cAM373_SA but also contributes to the survival of the bacterial pathogen in the host. IMPORTANCE The emergence of multidrug-resistant Staphylococcus aureus makes the treatment of staphylococcal infections much more difficult. S. aureus can acquire a drug resistance gene from other bacteria, such as Enterococcus faecalis. Intriguingly, S. aureus produces a sex pheromone for the E. faecalis plasmid pAM373, raising the possibility that S. aureus actively promotes plasmid conjugation from E. faecalis. In this study, we found that the staphylococcal Eep protein is responsible for sex pheromone processing and contributes to the survival of the bacteria in the host. These results will enhance future research on the drug resistance acquisition of S. aureus and can lead to the development of novel antivirulence drugs.

In bacteria, chromosomal DNA resides in the cytoplasm, and most transcription factors are also found in the cytoplasm. However, some transcription factors, called membrane-bound transcription factors (MTFs), reside in the cytoplasmic membrane. Here, we report the identification of a new MTF in the Gram-positive pathogen Staphylococcus aureus and its regulation by the protease FtsH. The MTF, named MbtS (membrane-bound transcription factor of Staphylococcus aureus), is encoded by SAUSA300_2640 and predicted to have an N-terminal DNA binding domain and three transmembrane helices. The MbtS protein was degraded by membrane vesicles containing FtsH or by the purified FtsH. MbtS bound to an inverted repeat sequence in its promoter region, and the DNA binding was essential for its transcription. Transcriptional comparison between the ftsH deletion mutant and the ftsH mbtS double mutant showed that MbtS could alter the transcription of over 200 genes. Although the MbtS protein was not detected in wild-type (WT) cells grown in a liquid medium, the protein was detected in some isolated colonies on an agar plate. In a murine model of a skin infection, the disruption of mbtS increased the lesion size. Based on these results, we concluded that MbtS is a new S. aureus MTF whose activity is proteolytically regulated by FtsH. IMPORTANCEStaphylococcus aureus is an important pathogenic bacterium causing various diseases in humans. In the bacterium, transcription is typically regulated by the transcription factors located in the cytoplasm. In this study, we report an atypical transcription factor identified in S. aureus. Unlike most other transcription factors, the newly identified transcription factor is located in the cytoplasmic membrane, and its activity is proteolytically controlled by the membrane-bound AAA+ protease FtsH. The newly identified MTF, named MbtS, has the potential to regulate the transcription of over 200 genes. This study provides a molecular mechanism by which a protease affects bacterial transcription and illustrates the diversity of the bacterial transcriptional regulation.

Upon microbial infection, host immune cells recognize bacterial cell envelope components through cognate receptors. Although bacterial cell envelope components function as innate immune molecules, the role of the physical state of the bacterial cell envelope (i.e., particulate versus soluble) in host immune activation has not been clearly defined. Here, using two different forms of the staphylococcal cell envelope of Staphylococcus aureus RN4220 and USA300 LAC strains, we provide biochemical and immunological evidence that the particulate state is required for the effective activation of host innate immune responses. In a murine model of peritoneal infection, the particulate form of the staphylococcal cell envelope (PCE) induced the production of chemokine (C-X-C motif) ligand 1 (CXCL1) and CC chemokine ligand 2 (CCL2), the chemotactic cytokines for neutrophils and monocytes, respectively, resulting in a strong influx of the phagocytes into the peritoneal cavity. In contrast, compared with PCE, the soluble form of cell envelope (SCE), which was derived from PCE by treatment with cell wall-hydrolyzing enzymes, showed minimal activity. PCE also induced the secretion of calprotectin (myeloid-related protein 8/14 [MRP8/14] complex), a phagocyte-derived antimicrobial protein, into the peritoneal cavity at a much higher level than did SCE. The injected PCE particles were phagocytosed by the infiltrated neutrophils and monocytes and then delivered to mediastinal draining lymph nodes. More importantly, intraperitoneally (i.p.) injected PCE efficiently protected mice from S. aureus infection, which was abolished by the depletion of either monocytes/macrophages or neutrophils. This study demonstrated that the physical state of bacterial cells is a critical factor for efficient host immune activation and the protection of hosts from staphylococcal infections.

Background and Hypothesis: Methicillin-resistant Staphylococcus aureus (MRSA) is resistant to almost all beta-lactam antibiotics, including oxacillin. To combat MRSA infections, recently, new classes of drugs are developed, such as oxadiazole DR1 and ESP404. We hypothesize that, if we identify MRSA mutants with altered sensitivity toward the drugs, those mutants will help us understand the antimicrobial mechanisms of the drugs. Experimental Design or Project Methods: The screening was carried out on the Nebraska library consisting of 1,920 non-redundant transposon mutants of USA300 strain in 96 well plates. First, the minimum inhibitory concentration (MIC) of each drug was determined. Then the transposon mutants were grown in tryptic soy broth (TSB) containing either 1/4× MIC or 4× MIC of the drugs for sensitive mutants and resistant mutants respectively. Due to the limited availability of ESP404, the drug was used to screen for sensitive mutants only. Results: The first screening identified 81 mutants with altered drug sensitivity: 23 for oxadiazole DR1 (5 sensitive, 18 resistant); 20 for ESP404 (all sensitive); 38 for oxacillin (18 sensitive, 5 resistant). Conclusion and Potential Impact: Screening of the transposon-mutant library identified multiple mutants that have altered sensitivity toward three drugs against S. aureus. Further verification of the phenotype and characterization of those mutants are expected to shed light on the antimicrobial mechanism of the drugs and open a door for the development of drug-potentiators.

BackgroundStaphylococcus aureus(S. aureus) is a major human pathogen owing to its arsenal of virulence factors, as well as its acquisition of multi-antibiotic resistance. Here we report the identification of a Streptolysin S (SLS) like biosynthetic gene cluster in a highly virulent community-acquired methicillin resistantS. aureus(MRSA) isolate, JKD6159. Examination of the SLS-like gene cluster in JKD6159 shows significant homology and gene organization to the SLS-associated biosynthetic gene (sag) cluster responsible for the production of the major hemolysin SLS in Group AStreptococcus.ResultsWe took a comprehensive approach to elucidating the putative role of the sag gene cluster in JKD6159 by constructing a mutant in which one of the biosynthesis genes (sagB homologue) was deleted in the parent JKD6159 strain. Assays to evaluate bacterial gene regulation, biofilm formation, antimicrobial activity, as well as complete host cell response profile and comparativein vivoinfections in Balb/Cj mice were conducted.ConclusionsAlthough no significant phenotypic changes were observed in our assays, we postulate that the SLS-like toxin produced by this strain ofS. aureusmay be a highly specialized virulence factor utilized in specific environments for selective advantage; studies to better understand the role of this newly discovered virulence factor inS. aureuswarrant further investigation.

Due to the shortage of effective antibiotics against drug-resistant Staphylococcus aureus, new targets are urgently required to develop next-generation antibiotics. We investigated mannitol-1-phosphate dehydrogenase of S. aureus USA300 (SaM1PDH), a key enzyme regulating intracellular mannitol levels, and explored the possibility of using SaM1PDH as a target for developing antibiotic. Since mannitol is necessary for maintaining the cellular redox and osmotic potential, the homeostatic imbalance caused by treatment with a SaM1PDH inhibitor or knockout of the gene encoding SaM1PDH results in bacterial cell death through oxidative and/or mannitol-dependent cytolysis. We elucidated the molecular mechanism of SaM1PDH and the structural basis of substrate and inhibitor recognition by enzymatic and structural analyses of SaM1PDH. Our results strongly support the concept that targeting of SaM1PDH represents an alternative strategy for developing a new class of antibiotics that cause bacterial cell death not by blocking key cellular machinery but by inducing cytolysis and reducing stress tolerance through inhibition of the mannitol pathway., Mannitol-1-phosphate dehydrogenase (M1PDH) is a key enzyme in Staphylococcus aureus mannitol metabolism, but its roles in pathophysiological settings have not been established. We performed comprehensive structure-function analysis of M1PDH from S. aureus USA300, a strain of community-associated methicillin-resistant S. aureus, to evaluate its roles in cell viability and virulence under pathophysiological conditions. On the basis of our results, we propose M1PDH as a potential antibacterial target. In vitro cell viability assessment of ΔmtlD knockout and complemented strains confirmed that M1PDH is essential to endure pH, high-salt, and oxidative stress and thus that M1PDH is required for preventing osmotic burst by regulating pressure potential imposed by mannitol. The mouse infection model also verified that M1PDH is essential for bacterial survival during infection. To further support the use of M1PDH as an antibacterial target, we identified dihydrocelastrol (DHCL) as a competitive inhibitor of S. aureus M1PDH (SaM1PDH) and confirmed that DHCL effectively reduces bacterial cell viability during host infection. To explain physiological functions of SaM1PDH at the atomic level, the crystal structure of SaM1PDH was determined at 1.7-Å resolution. Structure-based mutation analyses and DHCL molecular docking to the SaM1PDH active site followed by functional assay identified key residues in the active site and provided the action mechanism of DHCL. Collectively, we propose SaM1PDH as a target for antibiotic development based on its physiological roles with the goals of expanding the repertory of antibiotic targets to fight antimicrobial resistance and providing essential knowledge for developing potent inhibitors of SaM1PDH based on structure-function studies.

Staphylococcus equorum is a benign bacterium and the predominant species in high-salt fermented food. Some strains of S. equorum contain antibiotic-resistance plasmids, such as pSELNU1 that contains a lincosamide nucleotidyltransferase (lnuA) gene and confers resistance to lincomycin. Previously, we showed that pSELNU1 is transferred to other bacteria under laboratory growth conditions. However, it is not known if the plasmid can be transferred to other bacteria during food fermentation (in situ) or during passage through animal intestines (in vivo). In this study, we examined the in situ and in vivo transfer of pSELNU1 using Staphylococcus saprophyticus as a recipient. During soybean fermentation, pSELNU1 was transferred to S. saprophyticus at a rate of 1.9 × 10−5–5.6 × 10−6 per recipient in the presence of lincomycin. However, during passage through murine intestines, the plasmid was transferred at similar rates (1.3 × 10−5 per recipient) in the absence of lincomycin, indicating that the plasmid transfer is much more efficient under in vivo conditions. Based on these results, we conclude that it is prudent to examine food fermentation starter candidates for the presence of mobile genetic elements containing antibiotic resistance genes and to select candidates lacking these genes.

Cationic antimicrobial peptides (CAMPs) are an essential part of the innate immune system. Some Gram-negative enteric pathogens, such as Salmonella enterica , show intrinsic resistance to CAMPs. However, the molecular basis of intrinsic resistance is poorly understood, largely due to a lack of information about the genes involved. In this study, using a microarray-based genomic technique, we screened the Keio collection of 3,985 Escherichia coli mutants for altered susceptibility to human neutrophil peptide 1 (HNP-1) and identified envC and zapB as novel genetic determinants of intrinsic CAMP resistance. In CAMP killing assays, an E. coli Δ envC Ec or Δ zapB Ec mutant displayed a distinct profile of increased susceptibility to both LL-37 and HNP-1. Both mutants, however, displayed wild-type resistance to polymyxin B and human β-defensin 3 (HBD3), suggesting that the intrinsic resistance mediated by EnvC or ZapB is specific to certain CAMPs. A corresponding Salmonella Δ envC Se mutant showed similarly increased CAMP susceptibility. The envC mutants of both E. coli and S. enterica displayed increased surface negativity and hydrophobicity, which partly explained the increased CAMP susceptibility. However, the Δ envC Ec mutant, but not the Δ envC Se mutant, was defective in outer membrane permeability, excluding this defect as a common factor contributing to the increased CAMP susceptibility. Animal experiments showed that the Salmonella Δ envC Se mutant had attenuated virulence. Taken together, our results indicate that the role of envC in intrinsic CAMP resistance is likely conserved among Gram-negative enteric bacteria, demonstrate the importance of intrinsic CAMP resistance for full virulence of S. enterica , and provide insight into distinct mechanisms of action of CAMPs.

As an alternative strategy to fight antibiotic resistance, two-component systems (TCSs) have emerged as novel targets. Among TCSs, master virulence regulators that control the expression of multiple virulence factors are considered as excellent antivirulence targets. In Staphylococcus aureus, virulence factor expression is tightly regulated by a few master regulators, including the SaeRS TCS. In this study, we used a SaeRS GFP-reporter system to screen natural compound inhibitors of SaeRS, and identified xanthoangelol B 1, a prenylated chalcone from Angelica keiskei as a hit. We have synthesized 1 and its derivative PM-56 and shown that 1 and PM-56 both had excellent inhibitory potency against the SaeRS TCS, as demonstrated by various in vitro and in vivo experiments. As a mode of action, 1 and PM-56 were shown to bind directly to SaeS and inhibit its histidine kinase activity, which suggests a possibility of a broad spectrum inhibitor of histidine kinases.

Cytolytic pore-forming toxins including alpha hemolysin (Hla) and bicomponent leukotoxins play an important role in the pathogenesis of Staphylococcus aureus. These toxins kill the polymorphonuclear phagocytes (PMNs), disrupt epithelial and endothelial barriers, and lyse erythrocytes to provide iron for bacterial growth. The expression of these toxins is regulated by the two-component sensing systems Sae and Agr. Here, we report that a point mutation (L18P) in SaeS, the histidine kinase sensor of the Sae system, renders the S. aureus Newman hemolytic activity fully independent of Hla and drastically increases the PMN lytic activity. Furthermore, this Hla-independent activity, unlike Hla itself, can lyse human erythrocytes. The Hla-independent activity towards human erythrocytes was also evident in USA300, however, under strict agr control. Gene knockout studies revealed that this Hla-independent Sae-regulated activity was entirely dependent on gamma hemolysin A subunit (HlgA). In contrast, hemolytic activity of Newman towards human erythrocytes from HlgAB resistant donors was completely dependent on agr. The culture supernatant from Newman S. aureus could be neutralized by antisera against two vaccine candidates based on LukS and LukF subunits of Panton-Valentine leukocidin but not by an anti-Hla neutralizing antibody. These findings display the complex involvement of Sae and Agr systems in regulating the virulence of S. aureus and have important implications for vaccine and immunotherapeutics development for S. aureus disease in humans.

In Staphylococcus aureus, an important Gram-positive human pathogen, the SaeRS two-component system is essential for the virulence and a good target for the development of anti-virulence drugs. In this study, we screened 12,200 small molecules for Sae inhibitors and identified two anti-cancer drugs, streptozotocin (STZ) and floxuridine (FU), as lead candidates for anti-virulence drug development against staphylococcal infections. As compared with STZ, FU was more efficient in repressing Sae-regulated promoters and protecting human neutrophils from S. aureus-mediated killing. FU inhibited S. aureus growth effectively whereas STZ did not. Intriguingly, RNA-seq analysis suggests that both compounds inhibit other virulence-regulatory systems such as Agr, ArlRS, and SarA more efficiently than they inhibit the Sae system. Both compounds induced prophages from S. aureus, indicating that they cause DNA damages. Surprisingly, a single administration of the drugs was sufficient to protect mice from staphylococcal intraperitoneal infection. Both compounds showed in vivo efficacy in a murine model of blood infection too. Finally, at the experimental dosage, neither compound showed any noticeable side effects on blood glucose level or blood cell counts. Based on these results, we concluded that STZ and FU are promising candidates for anti-virulence drug development against S. aureus infection.

Two Gram-stain-positive, non-motile, non-spore-forming, rod-shaped actinobacterial strains were isolated from the surface-sterilized roots of mugwort (Artemisia princeps) and horse-weed (Conyza canadensis), and subjected to taxonomic characterization. 16S rRNA gene sequence analysis indicated that the isolates, designated MWE 3-5T and HWE 2-02T, should be placed in the genus Nocardioides of the family Nocardioidaceae . The strains were closely related to Nocardioides hankookensis DS-30T, which exhibited 16S rRNA gene sequence similarity values of 97.99 and 99.09 % with strains MWE 3-5T and HWE 2-02T, respectively. The genome relatedness of N. hankookensis DS-30T with strain MWE 3-5T was 35.8 %, and that with strain HWE 2-02T was 36.4 %, whereas that between the two isolates was 43.2 %. Strains MWE 3-5T and HWE 2-02T possessed MK-8(H4) as the major isoprenoid quinone, and ll-diaminopimelic acid in the cell-wall peptidoglycan. The main fatty acids were iso-C16 : 0, iso-C17 : 0 and C18 : 1ω9c for strain MWE 3-5T and iso-C16 : 0, 10-methyl C18 : 0 and C18 : 1ω9c for strain HWE 2-02T. Based on phenotypic, genotypic and phylogenetic studies, the following two novel species are proposed: Nocardioides endophyticus sp. nov. (type strain, MWE 3-5T = KCTC 29122T = JCM 18532T) and Nocardioides conyzicola sp. nov. (type strain, HWE 2-02T = KCTC 29121T = JCM 18531T).