RopB-regulated SpeB cysteine protease degrades extracellular vesicles-associated streptolysin O and bacterial proteins from group A Streptococcus.

Extracellular vesicles (EVs) can be released from gram-positive bacteria and would participate in the delivery of bacterial toxins. Streptococcus pyogenes (group A Streptococcus, GAS) is one of the most common pathogens of monomicrobial necrotizing fasciitis. Spontaneous inactivating mutation in the CovR/CovS two-component regulatory system is related to the increase of EVs production via an unknown mechanism. This study aimed to investigate whether the CovR/CovS-regulated RopB, the transcriptional regulator of GAS exoproteins, would participate in regulating EVs production. Results showed that the size, morphology, and number of EVs released from the wild-type strain and the ropB mutant were similar, suggesting RopB is not involved in controlling EVs production. Nonetheless, RopB-regulated SpeB protease degrades streptolysin O and bacterial proteins in EVs. Although SpeB has crucial roles in modulating protein composition in EVs, the SpeB-positive EVs failed to trigger HaCaT keratinocytes pyroptosis, suggesting that EVs did not deliver SpeB into keratinocytes or the amount of SpeB in EVs was not sufficient to trigger cell pyroptosis. Finally, we identified that EV-associated enolase was resistant to SpeB degradation, and therefore could be utilized as the internal control protein for verifying SLO degradation. This study revealed that RopB would participate in modulating protein composition in EVs via SpeB-dependent protein degradation and suggested that enolase is a potential internal marker for studying GAS EVs.

RopB represses the transcription of speB in the absence of SIP in group A Streptococcus.

RopB is a quorum-sensing regulator that binds to the SpeB-inducing peptide (SIP) under acidic conditions. SIP is known to be degraded by the endopeptidase PepO, whose transcription is repressed by the CovR/CovS two-component regulatory system. Both SIP-bound RopB (RopB-SIP) and SIP-free RopB (apo-RopB) can bind to the speB promoter; however, only RopB-SIP activates speB transcription. In this study, we found that the SpeB expression was higher in the ropB mutant than in the SIP-inactivated (SIP*) mutant. Furthermore, the deletion of ropB in the SIP* mutant derepressed speB expression, suggesting that apo-RopB is a transcriptional repressor of speB Up-regulation of PepO in the covS mutant degraded SIP, resulting in the down-regulation of speB We demonstrate that deleting ropB in the covS mutant derepressed the speB expression, suggesting that the speB repression in this mutant was mediated not only by PepO-dependent SIP degradation but also by apo-RopB. These findings reveal a crosstalk between the CovR/CovS and RopB-SIP systems and redefine the role of RopB in regulating speB expression in group A Streptococcus.

Structural basis underlying the synergism of NADase and SLO during group A Streptococcus infection.

Group A Streptococcus (GAS) is a strict human pathogen possessing a unique pathogenic trait that utilizes the cooperative activity of NAD+-glycohydrolase (NADase) and Streptolysin O (SLO) to enhance its virulence. How NADase interacts with SLO to synergistically promote GAS cytotoxicity and intracellular survival is a long-standing question. Here, the structure and dynamic nature of the NADase/SLO complex are elucidated by X-ray crystallography and small-angle scattering, illustrating atomic details of the complex interface and functionally relevant conformations. Structure-guided studies reveal a salt-bridge interaction between NADase and SLO is important to cytotoxicity and resistance to phagocytic killing during GAS infection. Furthermore, the biological significance of the NADase/SLO complex in GAS virulence is demonstrated in a murine infection model. Overall, this work delivers the structure-functional relationship of the NADase/SLO complex and pinpoints the key interacting residues that are central to the coordinated actions of NADase and SLO in the pathogenesis of GAS infection.

The Bacterial Markers of Identification of Invasive CovR/CovS-Inactivated Group A Streptococcus.

Necrotizing fasciitis is a severe infectious disease that results in significant mortality. Streptococcus pyogenes (group A Streptococcus, GAS) is one of the most common bacterial pathogens of monomicrobial necrotizing fasciitis. The early diagnosis of necrotizing fasciitis is crucial; however, the typical cutaneous manifestations are not always presented in patients with GAS necrotizing fasciitis, which would lead to miss- or delayed diagnosis. GAS with spontaneous inactivating mutations in the CovR/CovS two-component regulatory system is significantly associated with destructive diseases such as necrotizing fasciitis and toxic shock syndrome; however, no specific marker has been used to identify these invasive clinical isolates. This study evaluated the sensitivity and specificity of using CovR/CovS-controlled phenotypes to identify CovR/CovS-inactivated isolates. Results showed that the increase of hyaluronic acid capsule production and streptolysin O expression were not consistently presented in CovS-inactivated clinical isolates. The repression of SpeB is the phenotype with 100% sensitivity of identifying in CovS-inactivated isolates among 61 clinical isolates. Nonetheless, this phenotype failed to distinguish RopB-inactivated isolates from CovS-inactivated isolates and cannot be utilized to identify CovR-inactivated mutant and RocA (Regulator of Cov)-inactivated isolates. In this study, we identified and verified that PepO, the endopeptidase which regulates SpeB expression through degrading SpeB-inducing quorum-sensing peptide, was a bacterial marker to identify isolates with defects in the CovR/CovS pathway. These results also inform the potential strategy of developing rapid detection methods to identify invasive GAS variants during infection. IMPORTANCE Necrotizing fasciitis is rapidly progressive and life-threatening; if the initial diagnosis is delayed, deep soft tissue infection can progress to massive tissue destruction and toxic shock syndrome. Group A Streptococcus (GAS) with inactivated mutations in the CovR/CovS two-component regulatory system are related to necrotizing fasciitis and toxic shock syndrome; however, no bacterial marker is available to identify these invasive clinical isolates. Inactivation of CovR/CovS resulted in the increased expression of endopeptidase PepO. Our study showed that the upregulation of PepO mediates a decrease in SpeB-inducing peptide (SIP) in the covR mutant, indicating that CovR/CovS modulates SIP-dependent quorum-sensing activity through PepO. Importantly, the sensitivity and specificity of utilizing PepO to identify clinical isolates with defects in the CovR/CovS pathway, including its upstream RocA regulator, were 100%. Our results suggest that identification of invasive GAS by PepO may be a strategy for preventing severe manifestation or poor prognosis after GAS infection.

Incidence and Effects of Acquisition of the Phage-Encoded ssa Superantigen Gene in Invasive Group A Streptococcus.

The acquisition of the phage-encoded superantigen ssa by scarlet fever-associated group A Streptococcus (Streptococcus pyogenes, GAS) is found in North Asia. Nonetheless, the impact of acquiring ssa by GAS in invasive infections is unclear. This study initially analyzed the prevalence of ssa+ GAS among isolates from sterile tissues and blood. Among 220 isolates in northern Taiwan, the prevalence of ssa+ isolates increased from 1.5% in 2008-2010 to 40% in 2017-2019. Spontaneous mutations in covR/covS, which result in the functional loss of capacity to phosphorylate CovR, are frequently recovered from GAS invasive infection cases. Consistent with this, Phostag western blot results indicated that among the invasive infection isolates studied, 10% of the ssa+ isolates lacked detectable phosphorylated CovR. Transcription of ssa is upregulated in the covS mutant. Furthermore, in emm1 and emm12 covS mutants, ssa deletion significantly reduced their capacity to grow in human whole blood. Finally, this study showed that the ssa gene could be transferred from emm12-type isolates to the emm1-type wild-type strain and covS mutants through phage infection and lysogenic conversion. As the prevalence of ssa+ isolates increased significantly, the role of streptococcal superantigen in GAS pathogenesis, particularly in invasive covR/covS mutants, should be further analyzed.

Effect of Phosphatase Activity of the Control of Virulence Sensor (CovS) on Clindamycin-Mediated Streptolysin O Production in Group A Streptococcus.

Severe manifestations of group A Streptococcus (GAS) infections are associated with massive tissue destruction and high mortality. Clindamycin (CLI), a bacterial protein synthesis inhibitor, is recommended for treating patients with severe invasive GAS infection. Nonetheless, the subinhibitory concentration of CLI induces the production of GAS virulent exoproteins, such as streptolysin O (SLO) and NADase, which would enhance bacterial virulence and invasiveness. A better understanding of the molecular mechanism of how CLI triggers GAS virulence factor expression will be critical to develop appropriate therapeutic approaches. The present study shows that CLI activates SLO and NADase expressions in the emm1-type CLI-susceptible wild-type strain but not in covS or control of virulence sensor (CovS) phosphatase-inactivated mutants. Supplementation with Mg2+, which is a CovS phosphatase inhibitor, inhibits the CLI-mediated SLO upregulation in a dose-dependent manner in CLI-susceptible and CLI-resistant strains. These results not only reveal that the phosphorylation of response regulator CovR is essential for responding to CLI stimuli, but also suggest that inhibiting the phosphatase activity of CovS could be a potential strategy for the treatment of invasive GAS infection with CLI.

Cytotoxicity and Survival Fitness of Invasive covS Mutant of Group A Streptococcus in Phagocytic Cells.

Group A streptococci (GAS) with spontaneous mutations in the CovR/CovS regulatory system are more invasive and related to severe manifestations. GAS can replicate inside phagocytic cells; therefore, phagocytic cells could serve as the niche to select invasive covS mutants. Nonetheless, the encapsulated covS mutant is resistant to phagocytosis. The fate of intracellular covS mutant in phagocytic cells and whether the intracellular covS mutant contributes to invasive infections are unclear. In this study, capsule-deficient (cap-) strains were utilized to study how intracellular bacteria interacted with phagocytic cells. Results from the competitive infection model showed that the cap- covS mutant had better survival fitness than the cap- wild-type strain in the PMA-activated U937 cells. In addition, the cap- covS mutant caused more cell damages than the cap- wild-type strain and encapsulated covS mutant. Furthermore, treatments with infected cells with clindamycin to inhibit the intracellular bacteria growth was more effective to reduce bacterial toxicity than utilized penicillin to kill the extracellular bacteria. These results not only suggest that the covS mutant could be selected from the intracellular niche of phagocytic cells but also indicating that inactivating or killing intracellular GAS may be critical to prevent invasive infection.

Role of TGF­ß1 expressed in bone marrow­derived mesenchymal stem cells in promoting bone formation in a rabbit femoral defect model.

Bone defects represent a major clinical and socioeconomic problem without suitable treatment options. Previous studies have shown that transforming growth factor ß1 (TGF­ß1) is important in the development of various diseases. The present study aimed to investigate the therapeutic potential of rabbit bone marrow­derived mesenchymal stem cells (BMSCs) expressing TGF­ß1 in the treatment of rabbit femoral defects. First, rabbit BMSCs were identified and cultured. TGF­ß1 was then stably overexpressed in the rabbit BMSCs by lentivirus transfection, which was expressed at a high level in the femoral defects treated with TGF­ß1­overexpressing BMSCs, compared with PBS­treated controls. In addition, the TGF­ß1­overexpressing BMSCs promoted new bone formation in the rabbit femoral defect model, and increased the expression of bone­related markers at week 2 and week 6. Therefore, the study demonstrated that BMSCs overexpressing TGF­ß1 may provide a novel therapeutic option for femoral defects.