Staphylococcus Aureus Membrane Vesicles Kill Tumor Cells Through a Caspase-1-Dependent Pyroptosis Pathway.

Introduction: Nanosized outer membrane vesicles (OMVs) from Gram-negative bacteria have attracted increasing interest because of their antitumor activity. However, the antitumor effects of MVs isolated from Gram-positive bacteria have rarely been investigated. Methods: MVs of Staphylococcus aureus USA300 were prepared and their antitumor efficacy was evaluated using tumor-bearing mouse models. A gene knock-in assay was performed to generate luciferase Antares2-MVs for bioluminescent detection. Cell counting kit-8 and lactic dehydrogenase release assays were used to detect the toxicity of the MVs against tumor cells in vitro. Active caspase-1 and gasdermin D (GSDMD) levels were determined using Western blot, and the tumor inhibition ability of MVs was determined in B16F10 cells treated with a caspase-1 inhibitor. Results: The vesicular particles of S. aureus USA300 MVs were 55.23 ± 8.17 nm in diameter, and 5 µg of MVs remarkably inhibited the growth of B16F10 melanoma in C57BL/6 mice and CT26 colon adenocarcinoma in BALB/c mice. The bioluminescent signals correlated well with the concentrations of the engineered Antares2-MVs (R2 = 0.999), and the sensitivity for bioluminescence imaging was 4 × 10-3 µg. Antares2-MVs can directly target tumor tissues in vivo, and 20 µg/mL Antares2-MVs considerably reduced the growth of B16F10 and CT26 tumor cells, but not non-carcinomatous bEnd.3 cells. MV treatment substantially increased the level of active caspase-1, which processes GSDMD to trigger pyroptosis in tumor cells. Blocking caspase-1 activation with VX-765 significantly protected tumor cells from MV killing in vitro and in vivo. Conclusion: S. aureus MVs can kill tumor cells by activating the pyroptosis pathway, and the induction of pyroptosis in tumor cells is a promising strategy for cancer treatment.

LysSYL: a broad-spectrum phage endolysin targeting Staphylococcus species and eradicating S. aureus biofilms.

BACKGROUND: Staphylococcus aureus and its single or mixed biofilm infections seriously threaten global public health. Phage therapy, which uses active phage particles or phage-derived endolysins, has emerged as a promising alternative strategy to antibiotic treatment. However, high-efficient phage therapeutic regimens have yet to be established. RESULTS: In this study, we used an enrichment procedure to isolate phages against methicillin-resistant S. aureus (MRSA) XN108. We characterized phage SYL, a new member of the Kayvirus genus, Herelleviridae family. The phage endolysin LysSYL was expressed. LysSYL demonstrated stability under various conditions and exhibited a broader range of efficacy against staphylococcal strains than its parent phage (100% vs. 41.7%). Moreover, dynamic live/dead bacterial observation demonstrated that LysSYL could completely lyse MRSA USA300 within 10 min. Scan and transmission electron microscopy revealed evident bacterial cell perforation and deformation. In addition, LysSYL displayed strong eradication activity against single- and mixed-species biofilms associated with S. aureus. It also had the ability to kill bacterial persisters, and proved highly effective in eliminating persistent S. aureus when combined with vancomycin. Furthermore, LysSYL protected BALB/c mice from lethal S. aureus infections. A single-dose treatment with 50 mg/kg of LysSYL resulted in a dramatic reduction in bacterial loads in the blood, liver, spleen, lungs, and kidneys of a peritonitis mouse model, which resulted in rescuing 100% of mice challenged with 108 colony forming units of S. aureus USA300. CONCLUSIONS: Overall, the data provided in this study highlight the strong therapeutic potential of endolysin LysSYL in combating staphylococcal infections, including mono- and mixed-species biofilms related to S. aureus.

Sub-MIC vancomycin enhances the antibiotic tolerance of vancomycin-intermediate Staphylococcus aureus through downregulation of protein succinylation.

Bacteria develop tolerance after transient exposure to antibiotics, and tolerance is a significant driver of resistance. The purpose of this study is to evaluate the mechanisms underlying tolerance formation in vancomycin-intermediate Staphylococcus aureus (VISA) strains. VISA strains were cultured with sub-minimum inhibitory concentrations (sub-MICs) of vancomycin. Enhanced vancomycin tolerance was observed in VISA strains with distinct genetic lineages. Western blot revealed that the VISA protein succinylation (Ksucc) levels decreased with the increase in vancomycin exposure. Importantly, Ksucc modification, vancomycin tolerance, and cell wall synthesis were simultaneously affected after deletion of SacobB, which encodes a desuccinylase in S. aureus. Several Ksucc sites were identified in MurA, and vancomycin MIC levels of murA mutant and Ksucc-simulated (MurA(K69E) and MurA(K191E)) mutants were reduced. The vancomycin MIC levels of K65-MurA(K191E) in particular decreased to 1 mg/L, converting VISA strain K65 to a vancomycin-susceptible S. aureus strain. We further demonstrated that the enzymatic activity of MurA was dependent on Ksucc modification. Our data suggested the influence of vancomycin exposure on bacterial tolerance, and protein Ksucc modification is a novel mechanism in regulating vancomycin tolerance.

GehB Inactivates Lipoproteins to Delay the Healing of Acute Wounds Infected with Staphylococcus aureus.

Staphylococcus aureus is one of the most prevalent bacteria found in acute wounds. S. aureus produces many virulence factors and extracellular enzymes that contribute to bacterial survival, dissemination, and pathogenicity. Lipase GehB is a glycerol ester hydrolase that hydrolyzes triglycerides to facilitate the evasion of S. aureus from host immune recognition. However, the role and mechanism of lipase GehB in skin acute wound healing after S. aureus infection remain unclear. In this study, we found that the gehB gene deletion mutant (USA300ΔgehB) stimulated significantly higher levels of pro-inflammatory cytokines in RAW264.7 and Toll-like receptor 2 (TLR2)-transfected HEK293 cells than the wild-type USA300 strain did. Recombinant GehB-His treated lipoprotein (Lpp) reduced stimulation of TLR2-dependent TNF-α production by RAW264.7 macrophages. GehB delayed the skin acute wound healing in BALB/c mice infected with S. aureus, while wound healing was similar in C57BL/6 TLR2-/- mice infected with either wild-type USA300 or USA300ΔgehB. In BALB/c mice, we also observed more bacterial survival, less leukocyte recruitment, lower IL-8 production, and adipocyte differentiation in USA300-infected skin acute wound tissues than those in USA300ΔgehB-challenged ones. Our data indicated that GehB inactivates lipoproteins to shield S. aureus from innate immune killing, resulting in delayed the healing of skin acute wounds infected with S. aureus.

Genomic Epidemiology and Phenotypic Characterization of Staphylococcus aureus from a Tertiary Hospital in Tianjin Municipality, Northern China.

Staphylococcus aureus remains a dangerous pathogen and poses a great threat to public health worldwide. The prevalence of the S. aureus clonotype is temporally and geographically variable. The genomic and phenotypic characteristics of S. aureus isolates in Tianjin, which is among the four big municipalities in China, are unclear. In the present study, 201 nonduplicate S. aureus isolates, including 70 methicillin-resistant S. aureus (MRSA) and 131 methicillin-susceptible S. aureus (MSSA), were collected from 2015 to 2021 in a tertiary hospital in Tianjin. Whole-genome sequencing of S. aureus isolates was carried out to investigate bacterial molecular characteristics, genomic phylogeny, antimicrobial resistance (AMR) gene carriage, and virulence factor gene distribution. The antibiotic resistance profiles, hemolytic activities, and biofilm formation abilities of the S. aureus isolates were also determined. In total, 31 distinct sequence types (STs) and 68 spa types were identified. ST59 (15.9%, 32/201) was the predominant clonotype, followed by ST398 (14.9%, 30/201) and several other major STs (ST1, ST5, ST6, ST22, ST25, ST188, and the newly emerging ST5527). ST59 and ST5527 mainly included MRSA isolates, while ST398 and the other major STs mainly included MSSA isolates. The unique characteristics of the S. aureus isolates belonging to the major STs were determined. ST59 isolates exhibited strong hemolytic activity, and ST398 strains had high biofilm formation capacity, while ST5527 isolates presented the greatest AMR. The genomic epidemiology and phenotypic characteristics of S. aureus isolates determined in this study will help in disease control in nosocomial environments. IMPORTANCE Staphylococcus aureus is an important bacterium pathogen in tertiary hospitals, which provide rich medical resources. Tianjin is one of the four municipalities in China with a population of more than 13 million. However, the epidemiology and molecular characteristics of S. aureus isolates in Tianjin are unknown. In this study, the genomic and phenotypic analyses were performed to investigate 201 S. aureus isolates collected from a tertiary hospital in Tianjin over a time span of 6 years. The refined analysis of predominant clones ST59, ST398, the newly emerging clone ST5527, as well as other major clones, will undoubtedly aid in the control and prevention of infections caused by S. aureus in tertiary hospitals.

Optimizing a high-sensitivity NanoLuc-based bioluminescence system for in vivo evaluation of antimicrobial treatment.

Focal and systemic infections are serious threats to human health. Preclinical models enable the development of new drugs and therapeutic regimens. In vivo, animal bioluminescence (BL) imaging has been used with bacterial reporter strains to evaluate antimicrobial treatment effects. However, high-sensitivity bioluminescent systems are required because of the limited tissue penetration and low brightness of the BL signals of existing approaches. Here, we report that NanoLuc (Nluc) showed better performance than LuxCDABE in bacteria. However, the retention rate of plasmid constructs in bacteria was low. To construct stable Staphylococcus aureus reporter strains, a partner protein enolase (Eno) was identified by screening of S. aureus strain USA300 for fusion expression of Nluc-based luciferases, including Nluc, Teluc, and Antares2. Different substrates, such as hydrofurimazine (HFZ), furimazine (FUR), and diphenylterazine (DTZ), were used to optimize a stable reporter strain/substrate pair for BL imaging. S. aureus USA300/Eno-Antares2/HFZ produced the highest number of photons of orange-red light in vitro and enabled sensitive BL tracking of S. aureus in vivo, with sensitivities of approximately 10 CFU from mouse skin and 750 CFU from mouse kidneys. USA300/Eno-Antares2/HFZ was a powerful combination based on the longitudinal evaluation of the therapeutic efficacy of antibiotics. The optimized S. aureus Eno-Antares2/HFZ pair provides a technological advancement for the in vivo evaluation of antimicrobial treatment.

Identification of Lysine Succinylome and Acetylome in the Vancomycin-Intermediate Staphylococcus aureus XN108.

Protein posttranslational modifications (PTMs) play important roles in regulating numerous biological functions of prokaryotic and eukaryotic organisms. Lysine succinylation (Ksucc) and acetylation (Kac) are two important PTMs that have been identified in various bacterial species. However, the biological functions of Ksucc and Kac in vancomycin-intermediate S. aureus (VISA) remain unclear. In this study, we systematically identified 3,260 Ksucc sites in 799 proteins and 7,935 Kac sites across 1,710 proteins in the VISA strain XN108. Functional analyses revealed that both Ksucc and Kac sites were highly enriched in several critical metabolic pathways, including ribosomal metabolism, tricarboxylic acid cycle, and glycolysis. Furthermore, a remarkable cross talk between Ksucc and Kac modifications was observed that almost 75% of the succinylated sites were also frequently acetylated. In addition, we identified SaCobB, a Sirtuin 2-like lysine deacetylase, as a bifunctional enzyme with both deacetylation and desuccinylation activities in S. aureus. We demonstrated the first lysine succinylome and acetylome in a VISA and identified SaCobB, a functional enzyme taking part in the regulation of Ksucc and Kac in S. aureus. Our findings provide valuable information for further study on the regulatory mechanisms of PTMs in S. aureus. IMPORTANCE Lysine succinylation (Ksucc) and acetylation (Kac) are two important protein posttranslational modifications (PTMs) that regulate numerous biological functions in prokaryotes and eukaryotes. However, the functions of Ksucc and Kac in Staphylococcus aureus are seldom described. Understanding of Ksucc and Kac modifications in S. aureus will facilitate the development of new strategies to control infections. Herein, we quantified both Ksucc and Kac in a vancomycin-intermediate S. aureus (VISA) strain XN108, analyzed the interaction between these two PTMs, and identified SaCobB as a bifunctional enzyme with both deacetylation and desuccinylation activities. This study is the first description of dual PTMs, Ksucc and Kac profiles, in the VISA. The findings could provide valuable information for the following researches on the regulatory roles of PTMs in S. aureus.

CcpA Regulates Staphylococcus aureus Biofilm Formation through Direct Repression of Staphylokinase Expression.

Staphylococcus aureus represents a notorious opportunistic pathogen causing various infections in biofilm nature, imposing remarkable therapeutic challenges worldwide. The catabolite control protein A (CcpA), a major regulator of carbon catabolite repression (CCR), has been recognized to modulate S. aureus biofilm formation, while the underlying mechanism remains to be fully elucidated. In this study, the reduced biofilm was firstly determined in the ccpA deletion mutant of S. aureus clinical isolate XN108 using both crystal violet staining and confocal laser scanning microscopy. RNA-seq analysis suggested that sak-encoding staphylokinase (Sak) was significantly upregulated in the mutant ∆ccpA, which was further confirmed by RT-qPCR. Consistently, the induced Sak production correlated the elevated promoter activity of sak and increased secretion in the supernatants, as demonstrated by Psak-lacZ reporter fusion expression and chromogenic detection, respectively. Notably, electrophoretic mobility shift assays showed that purified recombinant protein CcpA binds directly to the promoter region of sak, suggesting the direct negative control of sak expression by CcpA. Double isogenic deletion of ccpA and sak restored biofilm formation for mutant ∆ccpA, which could be diminished by trans-complemented sak. Furthermore, the exogenous addition of recombinant Sak inhibited biofilm formation for XN108 in a dose-dependent manner. Together, this study delineates a novel model of CcpA-controlled S. aureus biofilm through direct inhibition of sak expression, highlighting the multifaceted roles and multiple networks regulated by CcpA.

A vancomycin resistance-associated WalK(S221P) mutation attenuates the virulence of vancomycin-intermediate Staphylococcus aureus.

INTRODUCTION: Vancomycin-intermediate Staphylococcus aureus (VISA) is typically associated with a decline in virulence. We previously reported a WalK(S221P) mutation that plays an important role in mediating vancomycin resistance in VISA XN108. Whether this mutation is implicated in bacterial virulence remains unknown. OBJECTIVES: This study aimed to investigate the effect of WalK(S221P) mutation on the virulence of VISA and the underlying mechanism of this effect. METHODS: The influence of WalK(S221P) mutation on VISA virulence and its underlying mechanism were explored using animal models, RNA-seq analysis, RT-qPCR, hemolytic assay, slide coagulase test, Western blot, ß-galactosidase assay, and electrophoresis mobility shift assay (EMSA). RESULTS: Compared with XN108, WalK(S221P)-reverted strain XN108-R exacerbated cutaneous infections with increased lesion size and extensive inflammatory infiltration in mouse models. The bacterial loads of S. aureus XN108-R in murine kidney increased compared with those of XN108. RNA-seq analysis showed upregulation of a set of virulence genes in XN108-R, which exhibited greater hemolytic and stronger coagulase activities compared with XN108. Introduction of WalK(S221P) to methicillin-resistant S. aureus USA300 and methicillin-susceptible strain Newman increased the vancomycin resistance of the mutants, which exhibited reduced hemolytic activities and decreased expression levels of many virulence factors compared with their progenitors. WalK(S221P) mutation weakened agr promoter-controlled ß-galactosidase activity. EMSA results showed that WalK-phosphorylated WalR could directly bind to the agr promoter region, whereas WalK(S221P)-activated WalR reduced binding to the target promoter. Inactivation of agr in S. aureus did not affect their vancomycin susceptibility but mitigated the virulence alterations caused by WalK(S221P) mutation. CONCLUSION: The results of our study indicate that WalK(S221P) mutation can enhance vancomycin resistance in S. aureus of diverse genetic backgrounds. WalK(S221P)- bearing S. aureus strains exhibit reduced virulence. WalK(S221P) mutation may directly impair the activation of the agr system by WalR, thereby decreasing the expression of virulence factors in VISA.

Accessory Gene Regulator (agr) Allelic Variants in Cognate Staphylococcus aureus Strain Display Similar Phenotypes.

The accessory gene regulator (agr) quorum-sensing system is an important global regulatory system of Staphylococcus aureus and contributes to its pathogenicity. The S. aureus agr system is divided into four agr groups based on the amino acid polymorphisms of AgrB, AgrD, and AgrC. The agr activation is group-specific, resulting in variations in agr activity and pathogenicity among the four agr groups. Strains with divergent agr system always have different phenotypes. In the present report, we, respectively, exchanged the agr system of a certain S. aureus with other three agr alleles and assessed the corresponding phenotypes of these congenic strains. Replacement of the agr system led to significant variations in hemolytic activity, protein expression, and virulence gene expression comparing with that of the parental strain. Interestingly, we found that the biological characteristics of these agr congenic strains in the same strain background were highly similar to each other, and the allele-dependent differences of the agr systems were weakened. These findings indicate that the allele-dependent agr predilections of S. aureus are determined by some factors in addition to the polymorphisms of AgrB, AgrD, and AgrC. Future studies may reveal the novel mechanism to improve our understanding of the agr network.

Pyocyanin biosynthesis protects Pseudomonas aeruginosa from nonthermal plasma inactivation.

Pseudomonas aeruginosa is an important opportunistic human pathogen, which raises a worldwide concern for its increasing resistance. Nonthermal plasma, which is also called cold atmospheric plasma (CAP), is an alternative therapeutic approach for clinical infectious diseases. However, the bacterial factors that affect CAP treatment remain unclear. The sterilization effect of a portable CAP device on different P. aeruginosa strains was investigated in this study. Results revealed that CAP can directly or indirectly kill P. aeruginosa in a time-dependent manner. Scanning electron microscopy and transmission electron microscope showed negligible surface changes between CAP-treated and untreated P. aeruginosa cells. However, cell leakage occurred during the CAP process with increased bacterial lactate dehydrogenase release. More importantly, pigmentation of the P. aeruginosa culture was remarkably reduced after CAP treatment. Further mechanical exploration was performed by utilizing mutants with loss of functional genes involved in pyocyanin biosynthesis, including P. aeruginosa PAO1 strain-derived phzA1::Tn, phzA2::Tn, ΔphzA1/ΔphzA2, phzM::Tn and phzS::Tn, as well as corresponding gene deletion mutants based on clinical PA1 isolate. The results indicated that pyocyanin and its intermediate 5-methyl phenazine-1-carboxylic acid (5-Me-PCA) play important roles in P. aeruginosa resistance to CAP treatment. The unique enzymes, such as PhzM in the pyocyanin biosynthetic pathway, could be novel targets for the therapeutic strategy design to control the growing P. aeruginosa infections.

Protective Effect of the Golden Staphyloxanthin Biosynthesis Pathway on Staphylococcus aureus under Cold Atmospheric Plasma Treatment.

Staphylococcus aureus infection poses a serious threat to public health, and antibiotic resistance has complicated the clinical treatment and limited the solutions available to solve this problem. Cold atmospheric plasma (CAP) is a promising strategy for microorganism inactivation. However, the mechanisms of microbial inactivation or resistance remain unclear. In this study, we treated S. aureus strains with a self-assembled CAP device and found that CAP can kill S. aureus in an exposure time-dependent manner. In addition, the liquid environment can influence the survival rate of S. aureus post-CAP treatment. The S. aureus cells can be completely inactivated in normal saline and phosphate-buffered saline but not in tryptic soy broth culture medium. Scanning and transmission electron microscopy revealed that the CAP-treated S. aureus cells maintained integrated morphological structures, similar to the wild-type strain. Importantly, the CAP-treated S. aureus cells exhibited a reduced pigment phenotype. Deletion of the staphyloxanthin biosynthetic genes crtM and crtN deprived the pigmentation ability of S. aureus Newman. Both the Newman-ΔcrtM and Newman-ΔcrtN mutants presented high sensitivity to CAP treatment, whereas Newman-ΔcrtO exhibited a survival rate comparable to wild-type Newman after CAP treatment. Our data demonstrated that the yellow pigment intermediates of the staphyloxanthin biosynthetic pathway are responsible for the protection of S. aureus from CAP inactivation. The key enzymes, such as CrtM and CrtN, of the golden staphyloxanthin biosynthetic pathway could be important targets for the design of novel sterilization strategies against S. aureus infections.IMPORTANCEStaphylococcus aureus is an important pathogen that can be widely distributed in the community and clinical settings. The emergence of S. aureus with multiple-antibiotic resistance has complicated staphylococcal infection control. The development of alternative strategies with powerful bactericidal effects is urgently needed. Cold atmospheric plasma (CAP) is a promising strategy for microorganism inactivation. Nevertheless, the underlying mechanisms of microbial inactivation or resistance are not completely illustrated. In this study, we validated the bactericidal effects of CAP on S. aureus, including antibiotic-resistant strains. We also found that the golden staphyloxanthin, as well as its yellow pigment intermediates, protected S. aureus against CAP, and blocking the staphyloxanthin synthesis pathway at the early steps could strengthen the sensitivity of S. aureus to CAP treatment. These data provide insights into the germicidal mechanism of CAP from the aspect of bacteria and suggest new targets against S. aureus infections.

ß-Lactam Antibiotics Enhance the Pathogenicity of Methicillin-Resistant Staphylococcus aureus via SarA-Controlled Lipoprotein-Like Cluster Expression.

Methicillin-resistant Staphylococcus aureus (MRSA) resists nearly all ß-lactam antibiotics that have a bactericidal activity. However, whether the empirically used ß-lactams enhance MRSA pathogenicity in vivo remains unclear. In this study, we showed that a cluster of lipoprotein-like genes (lpl, sa2275 to sa2273 [sa2275-sa2273]) was upregulated in MRSA in response to subinhibitory concentrations of ß-lactam induction. The increasing expression of lpl by ß-lactams was directly controlled by the global regulator SarA. The ß-lactam-induced Lpls stimulated the production of interleukin-6 and tumor necrosis factor alpha in RAW 264.7 macrophages. The lpl deletion mutants (N315Δlpl and USA300Δlpl) decreased the proinflammatory cytokine levels in vitro and in vivo Purified lipidated SA2275-his proteins could trigger a Toll-like-receptor-2 (TLR2)-dependent immune response in primary mouse bone marrow-derived macrophages and C57BL/6 mice. The bacterial loads of N315Δlpl in the mouse kidney were lower than those of the wild-type N315. The ß-lactam-treated MRSA exacerbated cutaneous infections in both BALB/c and C57BL/6 mice, presenting increased lesion size; destroyed skin structure; and easily promoted abscess formation compared with those of the untreated MRSA. However, the size of abscesses caused by the ß-lactam-treated N315 was negligibly different from those caused by the untreated N315Δlpl in C57BL/6 TLR2-/- mice. Our findings suggest that ß-lactams must be used carefully because they might aggravate the outcome of MRSA infection compared to inaction in treatment.IMPORTANCE ß-Lactam antibiotics are widely applied to treat infectious diseases. However, certain poor disease outcomes caused by ß-lactams remain poorly understood. In this study, we have identified a cluster of lipoprotein-like genes (lpl, sa2275-sa2273) that is upregulated in the major clinically prevalent MRSA clones in response to subinhibitory concentrations of ß-lactam induction. The major highlight of this work is that ß-lactams stimulate the expression of SarA, which directly binds to the lpl cluster promoter region and upregulates lpl expression in MRSA. Deletion of lpl significantly decreases proinflammatory cytokine levels in vitro and in vivo The ß-lactam-induced Lpls enhance host inflammatory responses by triggering the Toll-like-receptor-2-mediated expressions of interleukin-6 and tumor necrosis factor alpha. The ß-lactam-induced Lpls are important virulence factors that enhance MRSA pathogenicity. These data elucidate that subinhibitory concentrations of ß-lactams can exacerbate the outcomes of MRSA infection through induction of lpl controlled by the global regulator SarA.

Virulence Determinants Are Required for Brain Abscess Formation Through Staphylococcus aureus Infection and Are Potential Targets of Antivirulence Factor Therapy.

Bacterial brain abscesses (BAs) are difficult to treat with conventional antibiotics. Thus, the development of alternative therapeutic strategies for BAs is of high priority. Identifying the virulence determinants that contribute to BA formation induced by Staphylococcus aureus would improve the effectiveness of interventions for this disease. In this study, RT-qPCR was performed to compare the expression levels of 42 putative virulence determinants of S. aureus strains Newman and XQ during murine BA formation, ear colonization, and bacteremia. The alterations in the expression levels of 23 genes were further confirmed through specific TaqMan RT-qPCR. Eleven S. aureus genes that persistently upregulated expression levels during BA infection were identified, and their functions in BA formation were confirmed through isogenic mutant experiments. Bacterial loads and BA volumes in mice infected with isdA, isdC, lgt, hla, or spa deletion mutants and the hla/spa double mutant strain were lower than those in mice infected with the wild-type Newman strain. The therapeutic application of monoclonal antibodies against Hla and SpA decreased bacterial loads and BA volume in mice infected with Newman. This study provides insights into the virulence determinants that contribute to staphylococcal BA formation and a paradigm for antivirulence factor therapy against S. aureus infections.

Reconstruction of the Vancomycin-Susceptible Staphylococcus aureus Phenotype From a Vancomycin-Intermediate S. aureus XN108.

The emergence of vancomycin-intermediate Staphylococcus aureus (VISA) has raised healthcare concerns worldwide. VISA is often associated with multiple genetic changes. However, the relative contributions of these changes to VISA phenotypes are incompletely defined. We have characterized VISA XN108 with vancomycin MIC of 12 µg/ml. Genome comparison revealed that WalK(S221P), GraS(T136I), and RpoB(H481N) mutations possibly contributed to the VISA phenotype of XN108. In this study, the above mutations were stepwise cured, and the phenotypes between XN108 and its derivates were compared. We constructed four isogenic mutant strains, XN108-WalK(P221S) (termed as K65), XN108-GraS(I136T) (termed as S65), XN108-RpoB(N481H) (termed as B65), and XN108-WalK(P221S)/GraS(I136T) (termed as KS65), using the allelic replacement experiments with the native alleles derived from a vancomycin-susceptible S. aureus isolate DP65. Antimicrobial susceptibility test revealed K65 and S65 exhibited decreased vancomycin resistance, whereas B65 revealed negligibly differed when compared with the wild-type XN108. Sequentially introducing WalK(P221S) and GraS(I136T) completely converted XN108 into a VSSA phenotype. Transmission electronic microscopy and autolysis determination demonstrated that cell wall thickening and decreasing autolysis were associated with the change of vancomycin resistance levels. Compared with XN108, K65 exhibited 577 differentially expressed genes (DEGs), whereas KS65 presented 555 DEGs. Of those DEGs, 390 were common in K65 and KS65, including those upregulated genes responsible for citrate cycle and bacterial autolysis, and the downregulated genes involved in peptidoglycan biosynthesis and teichoic acid modification. In conclusion, a VSSA phenotype could be completely reconstituted from a VISA strain XN108. WalK(S221P) and GraS(T136I) mutations may work synergistically in conferring vancomycin resistance in XN108.

A novel SigB(Q225P) mutation in Staphylococcus aureus retains virulence but promotes biofilm formation.

Staphylococcus aureus is an important pathogen that produces abundant virulence factors, which cause various diseases that burden human health worldwide. The stress response regulon called sigma factor B (SigB) is a well-characterized global regulator that is involved in the regulation of S. aureus virulence, pigmentation, and biofilm formation. However, the regulatory network upon SigB in S. aureus is incompletely described. Here, we identified a novel substitution mutation, SigB(Q225P), which contributed the nonpigmented phenotype of S. aureus. The S. aureus mutant carrying SigB(Q225P) substitution lacks staphyloxanthin, a key virulence factor in protecting bacteria from host-oxidant killing, but retains bacterial pathogenicity with pleiotropic alterations in virulence factors, resulting in similar lethality and abscess formation ability in animal models. We also reported the SigB(Q225P) promotion of biofilm formation in S. aureus. Real-time quantitative polymerase chain reaction (RT-qPCR) revealed that the expression of nuc gene, which encodes thermonuclease, was significantly downregulated, resulting in accumulation of eDNA in the biofilm of SigB(Q225P) mutant strain. LacZ reporter assay showed that SigB(Q225P) influenced the activity of nuc promoter. Furthermore, electrophoretic mobility shift assay (EMSA) and Bio-layer interferometry (BLI) assay revealed that both SigB and SigB(Q225P) proteins could directly bind to nuc gene promoter; however, the binding activity decreased for SigB(Q225P). Our data renewed the understanding of the relationship between S. aureus golden pigment and its virulence and suggested that a single substitution mutation in SigB might enhance the biofilm formation of S. aureus by directly downregulating nuc expression.

Safe Staphylococcal Platform for the Development of Multivalent Nanoscale Vesicles against Viral Infections.

Many viruses often have closely related yet antigenically distinct serotypes. An ideal vaccine against viral infections should induce a multivalent and protective immune response against all serotypes. Inspired by bacterial membrane vesicles (MVs) that carry different protein components, we constructed an agr locus deletion mutant of the Staphylococcus aureus strain (RN4220-Δagr) to reduce potential toxicity. Nanoscale vesicles derived from this strain (ΔagrMVs) carry at least four major components that can deliver heterologous antigens. These components were each fused with a triple FLAG tag, and the tagged proteins could be incorporated into the ΔagrMVs. The presentation levels were (3.43 ± 0.73)%, (5.07 ± 0.82)%, (2.64 ± 0.61)%, and (2.89 ± 0.74)% of the total ΔagrMV proteins for Mntc-FLAG, PdhB-FLAG, PdhA-FLAG, and Eno-FLAG, respectively. With two DENV envelope E domain III proteins (EDIIIconA and EDIIIconB) as models, the DENV EDIIIconA and EDIIIconB delivered by two staphylococcal components were stably embedded in the ΔagrMVs. Administration of such engineered ΔagrMVs in mice induced antibodies against all four DENV serotypes. Sera from immunized mice protected Vero cells and suckling mice from a lethal challenge of DENV-2. This study will open up new insights into the preparation of multivalent nanosized viral vaccines against viral infections.

WalK(S221P), a naturally occurring mutation, confers vancomycin resistance in VISA strain XN108.

Objectives: Vancomycin-intermediate Staphylococcus aureus (VISA) strains have spread globally. We previously isolated an ST239 VISA (XN108) with a vancomycin MIC of 12 mg/L. The mechanism for XN108 resistance to vancomycin was investigated in this study. Methods: Genome comparison was performed to characterize mutations that might contribute to the XN108 resistance phenotype. The novel mutation WalK(S221P) was identified and investigated using allelic replacement experiments. Vancomycin susceptibilities, autolytic activities and morphologies of the strains were examined. Autophosphorylation activities of WalK and the WalK(S221P) mutant were determined in vitro with [λ- 32 P]ATP, and binding activity of WalK(S221P)-activated WalR to the promoter region of its target gene lytM was determined by electrophoretic mobility shift assay. Results: Genome comparison revealed three mutations, GraS(T136I), RpoB(H481N) and WalK(S221P), which might be responsible for vancomycin resistance in XN108. The introduction of WalK(S221P) to the vancomycin-susceptible strain N315 increased its vancomycin MIC from 1.5 to 8 mg/L, whereas the allelic replacement of WalK(S221P) with the native N315 WalK allele in XN108 decreased its vancomycin MIC from 12 to 4 mg/L. The VISA strains have thickened cell walls and decreased autolysis, consistent with observed changes in the expression of genes involved in cell wall metabolism and virulence regulation. WalK(S221P) exhibited reduced autophosphorylation, which may lead to reduced phosphorylation of WalR. WalK(S221P)-phosphorylated WalR also exhibited a reduced capacity to bind to the lytM promoter. Conclusions: The naturally occurring WalK(S221P) mutation plays a key role in vancomycin resistance in XN108.

Molecular and Phenotypic Characterization Revealed High Prevalence of Multidrug-Resistant Methicillin-Susceptible Staphylococcus aureus in Chongqing, Southwestern China.

Methicillin-susceptible Staphylococcus aureus (MSSA) accounts for ∼40% of staphylococcal infections in China. However, the molecular characterization of MSSA is not well described. In this study, 124 MSSA strains collected in 2013 from a comprehensive teaching hospital in Chongqing, Southwestern China, were subjected to antibiotics susceptibility testing and molecular typing, including multilocus sequence typing, staphylococcal protein A (spa) gene typing, accessory gene regulator (agr) typing, pulsed-field gel electrophoresis (PFGE) typing, Panton-Valentine leukocidin (pvl) gene detection, and antibiotic-resistant gene detection. MSSA strains exhibited high genetic heterogeneity. A total of 10 PFGE groups, 26 sequence types, and 47 spa types were identified. Type I (62.9%) was the most frequent agr type, followed by type II (15.3%), type IV (11.3%), and type III (10.5%). The prevalence of pvl genes was 27.4% (34/124). Notably, 44.4% (55/124) of MSSA strains were multidrug resistance (MDR), and MDR isolates were mostly resistant to penicillin, erythromycin, and clindamycin. The resistance gene blaZ was present in 84.7% of strains, ermC was present in 85.5% of strains, ermA was present in 28.2% of strains, tetK was present in 16.1% of strains, tetM was present in 6.5% of strains, and aacA-aphD was present in 2.6% of strains. These data demonstrated the high prevalence of MDR MSSA in Chongqing, thereby indicating the need to control MSSA infection.

Superficial vimentin mediates DENV-2 infection of vascular endothelial cells.

Damage to vascular endothelial cells (VECs) is a critical hallmark of hemorrhagic diseases caused by dengue virus (DENV). However, the precise molecular event involved in DENV binding and infection of VECs has yet to be clarified. In this study, vimentin (55 kDa) was identified to be involved in DENV-2 adsorption into VECs. This protein is located on the surface of VECs and interacts with DENV-2 envelope protein domain III (EDIII). The expression level of the superficial vimentin on VECs was not affected by viral infection or siRNA interference, indicating that the protein exists in a particular mode. Furthermore, the rod domain of the vimentin protein mainly functions in DENV-2 adsorption into VECs. Molecular docking results predicted several residues in vimentin rod and DENV EDIII; these residues may be responsible for cell-virus interactions. We propose that the superficial vimentin could be a novel molecule involved in DENV binding and infection of VECs. DENV EDIII directly interacts with the rod domain of vimentin on the VEC surface and thus mediates the infection.