Reduced interleukin-18 secretion by human monocytic cells in response to infections with hyper-virulent Streptococcus pyogenes.

BACKGROUND: Streptococcus pyogenes (group A streptococcus, GAS) causes a variety of diseases ranging from mild superficial infections of the throat and skin to severe invasive infections, such as necrotizing soft tissue infections (NSTIs). Tissue passage of GAS often results in mutations within the genes encoding for control of virulence (Cov)R/S two component system leading to a hyper-virulent phenotype. Dendritic cells (DCs) are innate immune sentinels specialized in antigen uptake and subsequent T cell priming. This study aimed to analyze cytokine release by DCs and other cells of monocytic origin in response to wild-type and natural covR/S mutant infections. METHODS: Human primary monocyte-derived (mo)DCs were used. DC maturation and release of pro-inflammatory cytokines in response to infections with wild-type and covR/S mutants were assessed via flow cytometry. Global proteome changes were assessed via mass spectrometry. As a proof-of-principle, cytokine release by human primary monocytes and macrophages was determined. RESULTS: In vitro infections of moDCs and other monocytic cells with natural GAS covR/S mutants resulted in reduced secretion of IL-8 and IL-18 as compared to wild-type infections. In contrast, moDC maturation remained unaffected. Inhibition of caspase-8 restored secretion of both molecules. Knock-out of streptolysin O in GAS strain with unaffected CovR/S even further elevated the IL-18 secretion by moDCs. Of 67 fully sequenced NSTI GAS isolates, 28 harbored mutations resulting in dysfunctional CovR/S. However, analyses of plasma IL-8 and IL-18 levels did not correlate with presence or absence of such mutations. CONCLUSIONS: Our data demonstrate that strains, which harbor covR/S mutations, interfere with IL-18 and IL-8 responses in monocytic cells by utilizing the caspase-8 axis. Future experiments aim to identify the underlying mechanism and consequences for NSTI patients.

Streptolysin O Deficiency in Streptococcus pyogenes M1T1 covR/S Mutant Strain Attenuates Virulence in In Vitro and In Vivo Infection Models.

Mutation within the Streptococcus pyogenes (Streptococcus group A; Strep A) covR/S regulatory system has been associated with a hypervirulent phenotype resulting from the upregulation of several virulence factors, including the pore-forming toxin, streptolysin O (SLO). In this study, we utilized a range of covR/S mutants, including M1T1 clonal strains (5448 and a covS mutant generated through mouse passage designated 5448AP), to investigate the contribution of SLO to the pathogenesis of covR/S mutant Strep A disease. Up-regulation of slo in 5448AP resulted in increased SLO-mediated hemolysis, decreased dendritic cell (DC) viability post coculture with Strep A, and increased production of tumor necrosis factor (TNF) and monocyte chemoattractant protein 1 (MCP-1) by DCs. Mouse passage of an isogenic 5448 slo-deletion mutant resulted in recovery of several covR/S mutants within the 5448Δslo background. Passage also introduced mutations in non-covR/S genes, but these were considered to have no impact on virulence. Although slo-deficient mutants exhibited the characteristic covR/S-controlled virulence factor upregulation, these mutants caused increased DC viability with reduced inflammatory cytokine production by infected DCs. In vivo, slo expression correlated with decreased DC numbers in infected murine skin and significant bacteremia by 3 days postinfection, with severe pathology at the infection site. Conversely, the absence of slo in the infecting strain (covR/S mutant or wild-type) resulted in detection of DCs in the skin and attenuated virulence in a murine model of pyoderma. slo-sufficient and -deficient covR/S mutants were susceptible to immune clearance mediated by a combination vaccine consisting of a conserved M protein peptide and a peptide from the CXC chemokine protease SpyCEP. IMPORTANCE Streptococcus pyogenes is responsible for significant numbers of invasive and noninvasive infections which cause significant morbidity and mortality globally. Strep A isolates with mutations in the covR/S system display greater propensity to cause severe invasive diseases, which are responsible for more than 163,000 deaths each year. This is due to the upregulation of virulence factors, including the pore-forming toxin streptolysin O. Utilizing covR/S and slo-knockout mutants, we investigated the role of SLO in virulence. We found that SLO alters interactions with host cell populations and increases Strep A viability at sterile sites of the host, such as the blood, and that its absence results in significantly less virulence. This work underscores the importance of SLO in Strep A virulence while highlighting the complex nature of Strep A pathogenesis. This improved insight into host-pathogen interactions will enable a better understanding of host immune evasion mechanisms and inform streptococcal vaccine development programs.

CRISPR-dependent endogenous gene regulation is required for virulence in piscine Streptococcus agalactiae.

The clustered regularly interspaced palindromic repeats (CRISPR)-Cas (CRISPR-associated) system is a prokaryotic defence against invading mobile genetic elements, such as bacteriophages or exogenous plasmids. Beyond this, this system has been shown to play an important role in controlling the virulence of some bacterial pathogens. Streptococcus agalactiae strain GD201008-001, a causative agent of septicemia and meningitis in tilapia, contains a single type II CRISPR-Cas system with Cas9 as a signature protein. In this study, we found that the deletion of CRISPR significantly reduced adhesion, invasion, cytotoxicity and haemolysis, and caused severely attenuated virulence in the piscine S. agalactiae strain. RNA-Seq identified 236 endogenous genes regulated by CRISPR, with 159 genes upregulated and 77 genes downregulated. The resulting change in gene transcription by CRISPR was much more pronounced than that by cas9 in this bacterium, indicating CRISPR-mediated endogenous gene regulation was mostly independently of cas9. Subsequent studies showed that CovR/S two-component system was transcriptionally upregulated due to CRISPR deletion, which repressed the expression of the cylE gene coding for a cytolytic toxin, and thus decreased the activity of ß-haemolysin/cytolysin. However, upregulation of CovR/S was not the contributor to the attenuation phenotype of ΔCRISPR. Further, we demonstrated that CRISPR is capable of repressing the expression of Toll-like receptor 2 (TLR2)-activating lipoprotein Sag0671 and thus dampens the innate immune response. This study revealed that the CRISPR system of S. agalactiae exhibited extraordinary potential capability in the regulation of endogenous transcripts, which contributes to bacterial innate immune evasion and virulence.

Eucalyptol inhibits biofilm formation of Streptococcus pyogenes and its mediated virulence factors.

Introduction. Streptococcus pyogenes is a diverse virulent synthesis pathogen responsible for invasive systemic infections. Establishment of antibiotic resistance in the pathogen has produced a need for new antibiofilm agents to control the biofilm formation and reduce biofilm-associated resistance development.Aim. The present study investigates the in vitro antibiofilm activity of eucalyptol against S. pyogenes.Methodology. The antibiofilm potential of eucalyptol was assessed using a microdilution method and their biofilm inhibition efficacy was visualized by microscopic analysis. The biochemical assays were performed to assess the influence of eucalyptol on virulence productions. Real-time PCR analysis was performed to evaluate the expression profile of the virulence genes.Results. Eucalyptol showed significant antibiofilm potential in a dose-dependent manner without affecting bacterial growth. Eucalyptol at 300 µg ml-1 (biofilm inhibitory concentration) significantly inhibited the initial stage of biofilm formation in S. pyogenes. However, eucalyptol failed to diminish the mature biofilms of S. pyogenes at biofilm inhibitory concentration and it effectively reduced the biofilm formation on stainless steel, titanium, and silicone surfaces. The biochemical assay results revealed that eucalyptol greatly affects the cell-surface hydrophobicity, auto-aggregation, extracellular protease, haemolysis and hyaluronic acid synthesis. Further, the gene-expression analysis results showed significant downregulation of virulence gene expression upon eucalyptol treatment.Conclusion. The present study suggests that eucalyptol applies its antibiofilm assets by intruding the initial biofilm formation of S. pyogenes. Supplementary studies are needed to understand the mode of action involved in biofilm inhibition.

Cellular interactions of covR/S mutant group A Streptococci.

Group A Streptococci (GAS) are responsible for a wide array of non-invasive and invasive diseases and varying immune sequelae with high rates of mortality and morbidity. GAS strains with a mutation in their covR/S regulatory system are hypervirulent with an increased capacity for causing invasive disease. covR/S mutants augment their virulence through the up-regulation of important virulence factors and target host immune surveillance primarily by inhibiting neutrophils. An in-depth understanding of the immunopathogenesis of covR/S mutants will facilitate the development of vaccine strategies and design. Ultimately, by targeting separate virulence mechanisms, multi-component vaccines may provide improved protective efficacy against hypervirulent GAS infections.

Strategic development of the conserved region of the M protein and other candidates as vaccines to prevent infection with group A streptococci.

Group A streptococcal (Streptococcus pyogenes) diseases remain a major public health problem in developing countries as well as in the indigenous populations of developed countries. In view of the large number of Group A streptococcal infections and the potential for sequelae such as rheumatic heart disease, control strategies including the development of an anti-streptococcal vaccine that is able to prevent infection and colonization is important. In this article, we discuss the epidemiology and strain variability of Group A streptococcus and how this is rendering vaccine development more challenging. We discuss vaccine strategies with a focus on the conserved region of the M protein and present a viewpoint for the impediments and the way forward.