Loaded delta-hemolysin shapes the properties of Staphylococcus aureus membrane vesicles.

Background: Membrane vesicles (MVs) are nanoscale vesicular structures produced by bacteria during their growth in vitro and in vivo. Some bacterial components can be loaded in bacterial MVs, but the roles of the loaded MV molecules are unclear. Methods: MVs of Staphylococcus aureus RN4220 and its derivatives were prepared. Dynamic light scattering analysis was used to evaluate the size distribution, and 4D-label-free liquid chromatography-tandem mass spectrometry analysis was performed to detect protein composition in the MVs. The site-mutation S. aureus RN4220-Δhld and agrA deletion mutant RN4220-ΔagrA were generated via allelic replacement strategies. A hemolysis assay was performed with rabbit red blood cells. CCK-8 and lactate dehydrogenase release assays were used to determine the cytotoxicity of S. aureus MVs against RAW264.7 macrophages. The serum levels of inflammatory factors such as IL-6, IL-1ß, and TNFα in mice treated with S. aureus MVs were detected with an enzyme-linked immunosorbent assay kit. Results: Delta-hemolysin (Hld) was identified as a major loaded factor in S. aureus MVs. Further study showed that Hld could promote the production of staphylococcal MVs with smaller sizes. Loaded Hld affected the diversity of loaded proteins in MVs of S. aureus RN4220. Hld resulted in decreased protein diversity in MVs of S. aureus. Site-mutation (RN4220-Δhld) and agrA deletion (RN4220-ΔagrA) mutants produced MVs (ΔhldMVs and ΔagrAMVs) with a greater number of bacterial proteins than those derived from wild-type RN4220 (wtMVs). Moreover, Hld contributed to the hemolytic activity of wtMVs. Hld-loaded wtMVs were cytotoxic to macrophage RAW264.7 cells and could stimulate the production of inflammatory factor IL-6 in vivo. Conclusion: This study presented that Hld was a major loaded factor in S. aureus MVs, and the loaded Hld played vital roles in the MV-property modification.

Drivers of methicillin-resistant Staphylococcus aureus (MRSA) lineage replacement in China.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) is a major nosocomial pathogen subdivided into lineages termed sequence types (STs). Since the 1950s, successive waves of STs have appeared and replaced previously dominant lineages. One such event has been occurring in China since 2013, with community-associated (CA-MRSA) strains including ST59 largely replacing the previously dominant healthcare-associated (HA-MRSA) ST239. We previously showed that ST59 isolates tend to have a competitive advantage in growth experiments against ST239. However, the underlying genomic and phenotypic drivers of this replacement event are unclear. METHODS: Here, we investigated the replacement of ST239 using whole-genome sequencing data from 204 ST239 and ST59 isolates collected in Chinese hospitals between 1994 and 2016. We reconstructed the evolutionary history of each ST and considered two non-mutually exclusive hypotheses for ST59 replacing ST239: antimicrobial resistance (AMR) profile and/or ability to colonise and persist in the environment through biofilm formation. We also investigated the differences in cytolytic activity, linked to higher virulence, between STs. We performed an association study using the presence and absence of accessory virulence genes. RESULTS: ST59 isolates carried fewer AMR genes than ST239 and showed no evidence of evolving towards higher AMR. Biofilm production was marginally higher in ST59 overall, though this effect was not consistent across sub-lineages so is unlikely to be a sole driver of replacement. Consistent with previous observations of higher virulence in CA-MRSA STs, we observed that ST59 isolates exhibit significantly higher cytolytic activity than ST239 isolates, despite carrying on average fewer putative virulence genes. Our association study identified the chemotaxis inhibitory protein (chp) as a strong candidate for involvement in the increased virulence potential of ST59. We experimentally validated the role of chp in increasing the virulence potential of ST59 by creating Δchp knockout mutants, confirming that ST59 can carry chp without a measurable impact on fitness. CONCLUSIONS: Our results suggest that the ongoing replacement of ST239 by ST59 in China is not associated to higher AMR carriage or biofilm production. However, the increase in ST59 prevalence is concerning since it is linked to a higher potential for virulence, aided by the carriage of the chp gene.

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.

Longitudinal in vivo Diffusion Tensor Imaging Detects Differential Microstructural Alterations in the Hippocampus of Chronic Social Defeat Stress-Susceptible and Resilient Mice.

Background: Microstructural alterations in the hippocampus may underlie stress-related disorders and stress susceptibility. However, whether these alterations are pre-existing stress vulnerability biomarkers or accumulative results of chronic stress remain unclear. Moreover, examining the whole hippocampus as one unit and ignoring the possibility of a lateralized effect of stress may mask some stress effects and contribute to the heterogeneity of previous findings. Methods: After C57BL/6 mice were exposed to a 10-day chronic social defeat stress (CSDS) paradigm, different stress phenotypes, i.e., susceptible (n = 10) and resilient (n = 7) mice, were discriminated by the behavior of the mice in a social interaction test. With in vivo diffusion tensor imaging (DTI) scans that were conducted both before and after the stress paradigm, we evaluated diffusion properties in the left and right, dorsal (dHi) and ventral hippocampus (vHi) of experimental mice. Results: A significantly lower fractional anisotropy (FA) was found in the right vHi of the susceptible mice prior to the CSDS paradigm than that found in the resilient mice, suggesting that pre-existing microstructural abnormalities may result in stress susceptibility. However, no significant group differences were found in the post-stress FA values of any of the hippocampal regions of interest (ROIs). In addition, mean diffusivity (MD) and radial diffusivity (RD) values were found to be significantly greater only in the right dHi of the resilient group compared to those of the susceptible mice. Furthermore, a significant longitudinal decrease was only observed in the right dHi RD value of the susceptible mice. Moreover, the social interaction (SI) ratio was positively related to post-stress left MD, right dHi MD, and right dHi RD values and the longitudinal right dHi MD percent change. Meanwhile, a negative relationship was detected between the SI ratio and bilateral mean of the post-stress left relative to right vHi FA value, highlighting the important role of right hippocampus in stress-resilience phenotype. Conclusion: Our findings demonstrated different longitudinal microstructural alterations in the bilateral dHi and vHi between stress-susceptible and resilient subgroups and indicated a right-sided lateralized stress effect, which may be useful in the diagnosis and prevention of stress-related disorders as well as their intervention.

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.

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.

Positive Feedback Cycle of TNFα Promotes Staphylococcal Enterotoxin B-Induced THP-1 Cell Apoptosis.

Staphylococcal enterotoxin B (SEB) has been demonstrated to be of importance in Staphylococcus aureus related diseases, such as atopic dermatitis (AD). Dysregulated apoptosis in AD is remarkable, and SEB can induce apoptosis of various cell types. However, the mechanisms by which SEB induces apoptosis and influences disease processes remain unclear. In this study, the recombinant SEB-induced THP-1 monocyte apoptosis was demonstrated in the absence of preliminary cell activation in a time- and dose-dependent manner. SEB could up-regulate the expression of tumor necrosis factor alpha (TNFα) in THP-1 cells and induce apoptosis via an extrinsic pathway. TNFα could in turn increase the expression of HLA-DRa, the SEB receptor on the cell surface. As a result, a positive feedback cycle of TNFα was established. TNFα expression and SEB-induced apoptosis were decreased by knocking down the expression of either HLA-DRa or TNFR1. Therefore, the feedback cycle of TNFα is crucial for SEB functions. This work provides insights into the mechanisms of SEB-induced monocyte apoptosis and emphasizes the major role of TNFα in future related studies.

Comparative Fitness and Determinants for the Characteristic Drug Resistance of ST239-MRSA-III-t030 and ST239-MRSA-III-t037 Strains Isolated in China.

Sequence type (ST) 239 with SCCmec type III methicillin-resistant Staphylococcus aureus (ST239-MRSA-III) is the most predominant multidrug-resistant clone in China. The subclone ST239-MRSA-III-t037 has been gradually replaced with ST239-MRSA-III-t030 since 2000. Subclones are characterized by drug resistance profiles. However, the mechanisms of the clonal dynamics and determinants of distinct drug resistance remain poorly understood. In the present study, 12 ST239-MRSA-III-t030 and 12 ST239-MRSA-III-t037 strains were collected from Chongqing, Guangzhou, and Shanghai; these strains were selected and investigated in terms of t030/t037 strain pairs. Independent growth curve assay revealed that the ST239-MRSA-III-t030 strains grew more rapidly, with significantly shorter doubling times, than the ST239-MRSA-III-t037 strains (p < 0.001). The ST239-MRSA-III-t037 strains exhibited slightly to moderately higher (3-13%) fitness cost than the ST239-MRSA-III-t030 strains in a competition assay in vitro. The ST239-MRSA-III-t037 strains yielded lower bacterial loads in the kidneys of the infected mice than the ST239-MRSA-III-t030 rivals in a coinfection assay (p < 0.05). The ST239-MRSA-III-t030 strains were resistant to rifampicin but susceptible to trimethoprim/sulfamethoxazole (SXT). In contrast, the ST239-MRSA-III-t037 strains were susceptible to rifampicin but resistant to SXT. The genetic determinants of the resistance to rifampicin and SXT in the MRSA strains were determined. Our results suggest that the relatively low fitness cost and characteristic drug resistance phenotype can help explain the current predominance of these ST239-MRSA-III-t030 strains in Chinese hospitals.

Panton-Valentine leukocidin (PVL)-positive health care-associated methicillin-resistant Staphylococcus aureus isolates are associated with skin and soft tissue infections and colonized mainly by infective PVL-encoding bacteriophages.

The emergence of Panton-Valentine leukocidin (PVL)-positive methicillin-resistant Staphylococcus aureus (MRSA) is a public health concern worldwide. PVL is associated with community-associated MRSA and is linked to skin and soft tissue infections (SSTIs). However, PVL genes have also been detected in health care-associated (HA) MRSA isolates. The diseases associated with PVL-positive HA-MRSA isolates and the distributions of PVL-encoding bacteriophages in HA-MRSA have not been determined. In this study, a total of 259 HA-MRSA strains isolated between 2009 and 2012 in China from inpatients with SSTIs, pneumonia, and bacteremia were selected for molecular typing, including staphylococcal cassette chromosome mec typing, multilocus sequence typing, and staphylococcal protein A gene typing. The PVL genes and PVL bacteriophages in the MRSA isolates were characterized by PCR. Among the tested MRSA isolates, 28.6% (74/259) were PVL positive. The high prevalence of PVL-carrying HA-MRSA was observed to be associated with SSTIs but not with pneumonia or bacteremia. The PVL-positive HA-MRSA isolates were colonized mainly by infective PVL phages, namely, Φ7247PVL, ΦSLT, and ΦSa2958. The distribution of PVL-carrying bacteriophages differed geographically. Our study highlights the potential risk of the emergence of multidrug-resistant HA-MRSA strains with increased virulence.

First report of a sequence type 239 vancomycin-intermediate Staphylococcus aureus isolate in Mainland China.

Methicillin-resistant Staphylococcus aureus (MRSA) is an important pathogen that causes a wide range of both hospital- and community-acquired infections. The high prevalence of MRSA and the extensive use of vancomycin in Mainland China may lead to the emergence of vancomycin-intermediate S. aureus (VISA) isolates. In this case, we report a VISA isolate from a 34-year-old male patient with steam burn. The isolate was determined to be sequence type 239 staphylococcal cassette chromosome mec type III, the most prevalent MRSA clone in Mainland China.

Molecular and phenotypic evidence for the spread of three major methicillin-resistant Staphylococcus aureus clones associated with two characteristic antimicrobial resistance profiles in China.

OBJECTIVES: The distribution of methicillin-resistant Staphylococcus aureus (MRSA) clones is dynamic and geographically unique. To understand the changing epidemiology of MRSA infections in China, we performed a prospective, multicity surveillance study with molecular typing and phenotypic analysis to determine the association of major prevalent clones with their antimicrobial resistance profiles. METHODS: A total of 517 S. aureus isolates collected between January 2009 and March 2012 from six cities in China were subjected to antibiogram analysis and molecular typing, including staphylococcal cassette chromosome mec typing, multilocus sequence typing, staphylococcal protein A gene typing and PFGE typing. RESULTS: Among the isolates collected, 309 were characterized as MRSA, with a prevalence of 59.8%. Three major clones were found to be prevalent in China: ST239-MRSA-III-t030, ST239-MRSA-III-t037 and ST5-MRSA-II-t002. These three clones were associated with two characteristic resistance profiles, namely, gentamicin/ciprofloxacin/rifampicin/levofloxacin for the first clone and gentamicin/ciprofloxacin/clindamycin/erythromycin/tetracycline/levofloxacin/trimethoprim/sulfamethoxazole for the latter two. Several geographically unique minor clones were also identified. CONCLUSIONS: The predominant MRSA clones in China were associated with characteristic antimicrobial resistance profiles. Antibiotics for treating patients with MRSA infections can be selected based on the strain typing data.

Cell wall thickening is associated with adaptive resistance to amikacin in methicillin-resistant Staphylococcus aureus clinical isolates.

OBJECTIVES: Methicillin-resistant Staphylococcus aureus (MRSA) infection is increasing and causing global concern. The mechanism of MRSA resistance to amikacin is poorly understood. We report on the first matched-pair study to reveal that the phenotypic cell wall thickening of MRSA is associated with adaptive resistance to amikacin. METHODS: Two MRSA strains (CY001 and CY002) were isolated from blood and synovial fluid samples, respectively, from a 12-year-old male patient with osteomyelitis. The strains were subjected to a matched-pair study, including antimicrobial agent susceptibility determination, molecular typing, morphological observation and in vitro resistance induction. RESULTS: Both strains are Panton-Valentine leucocidin-positive, multilocus sequence type 59, staphylococcal cassette chromosome mec type IV and spa type 437 MRSA with identical PFGE profiles. The drug susceptibility spectra of the two isolates are similar. However, CY001 is resistant to amikacin (CY001-AMI(R); MIC = 64 mg/L), contrary to the susceptible CY002 (CY002-AMI(S); MIC = 8 mg/L). CY001-AMI(R) may have developed adaptive resistance, because it lacks aminoglycoside-modifying enzymes and has an altered growth curve. Interestingly, CY001-AMI(R) has a thicker cell wall (36.43 ±â€Š4.25 nm) than CY002-AMI(S) (18.15 ±â€Š3.74 nm) in the presence of amikacin at its MIC. The thickened cell wall can also be observed in an in vitro-induced strain (CY002-AMI(R)) in the presence of amikacin at its MIC (36.78 ±â€Š3.41 nm); this strain was obtained by gradually increasing the amount of amikacin. However, the cell wall-thickened strains cultured in the presence of amikacin are still susceptible to vancomycin. CONCLUSIONS: Cell wall thickening is associated with adaptive resistance in MRSA and alternative antibiotics can be used to treat patients when adaptive resistance to amikacin has developed.