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. This study aimed to investigate the effect of WalK(S221P) mutation on the virulence of VISA and the underlying mechanism of this effect. 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, β-galactosidase assay, and electrophoresis mobility shift assay (EMSA). 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 β-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. 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.

Streptococcus suis serotype 2 (SS2) is an important zoonotic pathogen that infects pigs and sporadically causes serious infections in humans. Two rece

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.

Background: Our previous studies revealed that a new disease form of streptococcal toxic shock syndrome (STSS) is associated with specific Streptococcus suis serotype 2 (SS2) strains. To achieve a better understanding of the pathogenicity and evolution of SS2 at the whole-genome level, comparative genomic analysis of 18 SS2 strains, selected on the basis of virulence and geographic origin, was performed using NimbleGen tiling arrays. Results: Our results demonstrate that SS2 isolates have highly divergent genomes. The 89K pathogenicity island (PAI), which has been previously recognized as unique to the Chinese epidemic strains causing STSS, was partially included in some other virulent and avirulent strains. The ABC-type transport systems, encoded by 89K, were hypothesized to greatly contribute to the catastrophic features of STSS. Moreover, we identified many polymorphisms in genes encoding candidate or known virulence factors, such as PlcR, lipase, sortases, the pilus-associated proteins, and the response regulator RevS and CtsR. On the basis of analysis of regions of differences (RDs) across the entire genome for the 18 selected SS2 strains, a model of microevolution for these strains is proposed, which provides clues into Streptococcus pathogenicity and evolution. Conclusions: Our deep comparative genomic analysis of the 89K PAI present in the genome of SS2 strains revealed details into how some virulent strains acquired genes that may contribute to STSS, which may lead to better environmental monitoring of epidemic SS2 strains.

Streptococcus suis serotype 2 (S. suis 2) has evolved efficient mechanisms to cause streptococcal toxic shock syndrome (STSS), which is a new emerging infectious disease linked to S. suis. We have previously reported that a type IV secretion system (T4SS) harbored by the specific 89K pathogenicity island (PAI) of S. suis 2 contributes to the development of STSS and mediates horizontal transfer of 89K. However, the 89K T4SS machinery assembly in vivo and in vitro is poorly understood, and the component acting directly to digest the bacterial cell wall needs to be identified. The virB1-89K gene product encoded in the 89K PAI is the only one that shows similarity to the Agrobacterium VirB1 component and contains a conserved CHAP domain that may function in peptidoglycan hydrolysis, which makes it a plausible candidate acting as a hydrolase against the peptidoglycan cell wall to allow the assembly of the T4SS apparatus. In the current study, the CHAP domain of VirB1-89K from S. suis 89K PAI was cloned and over-expressed in Escherichia coli, and its peptidoglycan-degrading activity in vitro was determined. The results indicated that the VirB1-89K CHAP domain can degrade the peptidoglycan layer of bacteria. Deletion of virB1-89K reduces significantly, but does not abolish, the virulence of S. suis in a mouse model. The experimental results presented here suggested that VirB1-89K facilitates the assembly of 89K T4SS apparatus by catalyzing the degradation of the peptidoglycan cell wall, thus contributing to the pathogenesis of S. suis 2 infection.