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 protein of Rhizobium leguminosarum bv. viciae adopts amyloid state during symbiotic interactions with pea (Pisum sativum L.).

Amyloids represent protein aggregates with highly ordered fibrillar structure associated with the development of various disorders in humans and animals and involved in implementation of different vital functions in all three domains of life. In prokaryotes, amyloids perform a wide repertoire of functions mostly attributed to their interactions with other organisms including interspecies interactions within bacterial communities and host-pathogen interactions. Recently, we demonstrated that free-living cells of Rhizobium leguminosarum, a nitrogen-fixing symbiont of legumes, produce RopA and RopB which form amyloid fibrils at cell surface during the stationary growth phase thus connecting amyloid formation and host-symbiont interactions. Here we focused on a more detailed analysis of the RopB amyloid state in vitro and in vivo, during the symbiotic interaction between R. leguminosarum bv. viciae with its macrosymbiont, garden pea (Pisum sativum L.). We confirmed that RopB is the bona fide amyloid protein since its fibrils exhibit circular x-ray reflections indicating its cross-ß structure specific for amyloids. We found that fibrils containing RopB and exhibiting amyloid properties are formed in vivo at the surface of bacteroids of R. leguminosarum extracted from pea nodules. Moreover, using pea sym31 mutant we demonstrated that formation of extracellular RopB amyloid state occurs at different stages of bacteroid development but is enhanced in juvenile symbiosomes. Proteomic screening of potentially amyloidogenic proteins in the nodules revealed the presence of detergent-resistant aggregates of different plant and bacterial proteins including pea amyloid vicilin. We demonstrated that preformed vicilin amyloids can cross-seed RopB amyloid formation suggesting for probable interaction between bacterial and plant amyloidogenic proteins in the nodules. Taken together, we demonstrate that R. leguminosarum bacteroids produce extracellular RopB amyloids in pea nodules in vivo and these nodules also contain aggregates of pea vicilin amyloid protein, which is able to cross-seed RopB fibrillogenesis in vitro. Thus, we hypothesize that plant nodules contain a complex amyloid network consisting of plant and bacterial amyloids and probably modulating host-symbiont interactions.

Two Novel Amyloid Proteins, RopA and RopB, from the Root Nodule Bacterium Rhizobium leguminosarum.

Amyloids represent protein fibrils with a highly ordered spatial structure, which not only cause dozens of incurable human and animal diseases but also play vital biological roles in Archaea, Bacteria, and Eukarya. Despite the fact that association of bacterial amyloids with microbial pathogenesis and infectious diseases is well known, there is a lack of information concerning the amyloids of symbiotic bacteria. In this study, using the previously developed proteomic method for screening and identification of amyloids (PSIA), we identified amyloidogenic proteins in the proteome of the root nodule bacterium Rhizobium leguminosarum. Among 54 proteins identified, we selected two proteins, RopA and RopB, which are predicted to have ß-barrel structure and are likely to be involved in the control of plant-microbial symbiosis. We demonstrated that the full-length RopA and RopB form bona fide amyloid fibrils in vitro. In particular, these fibrils are ß-sheet-rich, bind Thioflavin T (ThT), exhibit green birefringence upon staining with Congo Red (CR), and resist treatment with ionic detergents and proteases. The heterologously expressed RopA and RopB intracellularly aggregate in yeast and assemble into amyloid fibrils at the surface of Escherichia coli. The capsules of the R. leguminosarum cells bind CR, exhibit green birefringence, and contain fibrils of RopA and RopB in vivo.

Streptococcus pyogenes Causing Skin and Soft Tissue Infections Are Enriched in the Recently Emerged emm89 Clade 3 and Are Not Associated With Abrogation of CovRS.

Although skin and soft tissue infections (SSTI) are the most common focal infections associated with invasive disease caused by Streptococcus pyogenes (Lancefield Group A streptococci - GAS), there is scarce information on the characteristics of isolates recovered from SSTI in temperate-climate regions. In this study, 320 GAS isolated from SSTI in Portugal were characterized by multiple typing methods and tested for antimicrobial susceptibility and SpeB activity. The covRS and ropB genes of isolates with no detectable SpeB activity were sequenced. The antimicrobial susceptibility profile was similar to that of previously characterized isolates from invasive infections (iGAS), presenting a decreasing trend in macrolide resistance. However, the clonal composition of SSTI between 2005 and 2009 was significantly different from that of contemporary iGAS. Overall, iGAS were associated with emm1 and emm3, while SSTI were associated with emm89, the dominant emm type among SSTI (19%). Within emm89, SSTI were only significantly associated with isolates lacking the hasABC locus, suggesting that the recently emerged emm89 clade 3 may have an increased potential to cause SSTI. Reflecting these associations between emm type and disease presentation, there were also differences in the distribution of emm clusters, sequence types, and superantigen gene profiles between SSTI and iGAS. According to the predicted ability of each emm cluster to interact with host proteins, iGAS were associated with the ability to bind fibrinogen and albumin, whereas SSTI isolates were associated with the ability to bind C4BP, IgA, and IgG. SpeB activity was absent in 79 isolates (25%), in line with the proportion previously observed among iGAS. Null covS and ropB alleles (predicted to eliminate protein function) were detected in 10 (3%) and 12 (4%) isolates, corresponding to an underrepresentation of mutations impairing CovRS function in SSTI relative to iGAS. Overall, these results indicate that the isolates responsible for SSTI are genetically distinct from those recovered from normally sterile sites, supporting a role for mutations impairing CovRS activity specifically in invasive infection and suggesting that this role relies on a differential regulation of other virulence factors besides SpeB.

Betulin inhibits virulence and biofilm of Streptococcus pyogenes by suppressing ropB core regulon, sagA and dltA.

The present study demonstrates the antivirulence potential of betulin, an abundantly available triterpenoid against Streptococcus pyogenes, a multivirulent and exclusive human pathogen. Crystal violet assay and microscopic examination revealed that betulin (100 µg mL-1) exhibits surface-independent antibiofilm activity and mitigates extracellular polymeric substance production. Betulin treatment enhanced the rate of auto-aggregation in liquid medium. Results of real-time PCR and biochemical assays demonstrated that betulin suppresses the expression of ropB core regulon, sagA and dltA, which correspondingly affects SpeB production, hemolysis and cell surface hydrophobicity for the observed impairment in virulence and biofilm formation. dltA downregulation also affected the production of M protein, making betulin-treated cells more susceptible to phagocytosis. The non-toxic nature of betulin and its antivirulence potential against S. pyogenes were manifested in vivo in Caenorhabditis elegans This study reveals the prospective role of betulin as therapeutic agent for the prevention and treatment of streptococcal infections.

Prevalence, faecal shedding and genetic characterisation of Yersinia spp. in sheep across four states of Australia.

OBJECTIVES: To develop molecular tools for the investigation of the prevalence, species and faecal shedding of Yersinia in sheep. METHODS: A quantitative PCR (qPCR) targeting the ß subunit of the Yersinia spp. RNA polymerase gene was developed and validated. The prevalence of pathogenic Y. enterocolitica was determined by screening for the virulent yst gene. These qPCR assays were used to determine Yersinia spp. prevalence and faecal shedding concentration from 3412 faecal samples collected from approximately 1189 lambs (100-180 lambs/flock) on eight farms across Australia. This was a longitudinal study, with sheep sampled on three occasions (weaning, post-weaning and pre-slaughter). A subset of up to five positive samples from each sampling on each farm (n = 111) was sequenced. RESULTS: Yersinia spp. (including both pathogenic and non-pathogenic species) were identified in all flocks, with 60.7% of lambs shedding Yersinia spp. on at least one sampling occasion. Point prevalence ranged from 4% to 91% across farms and sampling occasions. Median Yersinia spp. bacterial concentration was 1.1 × 10(6) , 2.8 × 10(6) and 5.6 × 10(5) organisms/g faeces at weaning, post-weaning and pre-slaughter, respectively, across all farms. Pathogenic Y. enterocolitica was identified in all eight flocks sampled, with 14.8% of lambs shedding pathogenic Y. enterocolitica on at least one sampling occasion. CONCLUSION: Yersinia spp. and pathogenic Y. enterocolitica in particular were commonly identified in a sample of Australian sheep flocks using molecular techniques. Further studies into associations between faecal shedding of pathogenic Yersinia spp. and sheep productivity or clinical disease may utilise qPCR in conjunction with other diagnostic tools.