Functional Analysis of the Quorum-Sensing Streptococcal Invasion Locus (sil)

Group A streptococcus (GAS) causes a wide variety of human diseases, and at the same time, GAS can also circulate without producing symptoms, similar to its close commensal relative, group G streptococcus (GGS). We previously identified, by transposon-tagged mutagenesis, the streptococcal invasion locus (sil). sil is a quorum-sensing regulated locus which is activated by the autoinducer peptide SilCR through the two-component system SilA-SilB. Here we characterize the DNA promoter region necessary for SilA-mediated activation. This site is composed of two direct repeats of 10 bp, separated by a spacer of 11 bp. Fusion of this site to gfp allowed us to systematically introduce single-base substitutions in the repeats region and to assess the relative contribution of various positions to promoter strength. We then developed an algorithm giving different weights to these positions, and performed a chromosome-wide bioinformatics search which was validated by transcriptome analysis. We identified 13 genes, mostly bacteriocin related, that are directly under the control of SilA. Having developed the ability to quantify SilCR signaling via GFP accumulation prompted us to search for GAS and GGS strains that sense and produce SilCR. While the majority of GAS strains lost sil, all GGS strains examined still possess the locus and ∼63% are able to respond to exogenously added SilCR. By triggering the autoinduction circle using a minute concentration of synthetic SilCR, we identified GAS and GGS strains that are capable of sensing and naturally producing SilCR, and showed that SilCR can be sensed across these streptococci species. These findings suggest that sil may be involved in colonization and establishment of commensal host-bacterial relationships

Type VII secretion system and its effect on group B Streptococcus virulence in isolates obtained from newborns with early onset disease and colonized pregnant women

IntroductionGBS may cause a devastating disease in newborns. In early onset disease of the newborn the bacteria are acquired from the colonized mother during delivery. We characterized type VII secretion system (T7SS), exporting small proteins of the WXG100 superfamily, in group B Streptococci (GBS) isolates from pregnant colonized women and newborns with early onset disease (EOD) to better understand T7SS contribution to virulence in these different clinical scenarios.MethodsGBS genomes [N=33, 17 EOD isolates (serotype III/ST17) and 16 colonizing isolates (12 serotype VI/ST1, one serotype VI/ST19, one serotype VI/ST6, and two serotype 3/ST19)] were analyzed for presence of T7SS genes and genes encoding WXG100 proteins. We also perform bioinformatic analysis. Galleria mellonella larvae were used to compare virulence between colonizing, EOD, and mutant EOD isolates. The EOD isolate number 118659 (III/ST17) was used for knocking out the essC gene encoding a membrane-bound ATPase, considered the driver of T7SS.ResultsMost GBS T7SS loci encoded core component genes: essC, membrane-embedded proteins (essA; essB), modulators of T7SS activity (esaA; esaB; esaC) and effectors: [esxA (SAG1039); esxB (SAG1030)].Bioinformatic analysis indicated that based on sequence type (ST) the clinicalGBS isolates encode at least three distinct subtypes of T7SS machinery. In all ST1isolates we identified two copies of esxA gene (encoding putative WXG100proteins), when only 23.5% of the ST17 isolates harbored the esxA gene. Five ST17isolates encoded two copies of the essC gene. Orphaned WXG100 molecule(SAG0230), distinct from T7SS locus, were found in all tested strains, except inST17 strains where the locus was found in only 23.5% of the isolates. In ST6 andST19 isolates most of the structure T7SS genes were missing. EOD isolates demonstrated enhanced virulence in G. mellonella modelcompared to colonizing isolates. The 118659DessC strain was attenuated in itskilling ability, and the larvae were more effective in eradicating 118659DessC.ConclusionsWe demonstrated that T7SS plays a role during infection. Knocking out the essC gene, considered the driver of T7SS, decreased the virulence of ST17 responsible for EOD, causing them to be less virulent comparable to the virulence observed in colonizing isolates

Invasive Group A Streptococcal Infections, Israel

We conducted a prospective, nationwide, population-based study of invasive group A streptococcal infections in Israel. We identified 409 patients (median age 27 years; range <1-92), for an annual incidence of 3.7/100,000 (11/100,000 in Jerusalem). The mortality rate was 5%. Bacteremia occurred in 125 cases (31%). The most common illnesses were soft-tissue infection (63%) and primary bacteremia (14%). Thirty percent of patients had no identifiable risk factors for infection. Eighty-seven percent of pharyngeal carriers had the same serotype as the index patient. M types included M3 (25%), M28 (10%), and M-nontypable (33%). A marked paucity of M1 serotype (1.2%) was detected. The results highlighted concentrated pockets of invasive disease in the Jewish orthodox community (annual incidence 16/100,000)

Characterization of Biofilm Formation by Clinically Relevant Serotypes of Group A Streptococci

Streptococcus pyogenes (group A streptococcus [GAS]) is a frequent cause of purulent infections in humans. As potentially important aspects of its pathogenicity, GAS was recently shown to aggregate, form intratissue microcolonies, and potentially participate in multispecies biofilms. In this study, we show that GAS in fact forms monospecies biofilms in vitro, and we analyze the basic parameters of S. pyogenes in vitro biofilm formation, using Streptococcus epidermidis as a biofilm-positive control. Of nine clinically important serotype strains, M2, M6, M14, and M18 were found to significantly adhere to coated and uncoated polystyrene surfaces. Fibronectin and collagen types I and IV best supported primary adherence of serotype M2 and M18 strains, respectively, whereas serotype M6 and M14 strains strongly bound to uncoated polystyrene surfaces. Absorption measurements of safranin staining, as well as electron scanning and confocal laser scanning microscopy, documented that primary adherence led to subsequent formation of three-dimensional biofilm structures consisting of up to 46 bacterial layers. Of note, GAS isolates belonging to the same serotype were found to be very heterogeneous in their biofilm-forming behavior. Biofilm formation was equally efficient under static and continuous flow conditions and consisted of the classical three steps, including partial disintegration after long-term incubation. Activity of the SilC signaling peptide as a component of a putative quorum-sensing system was found to influence the biofilm structure and density of serotype M14 and M18 strains. Based on the presented methods and results, standardized analyses of GAS biofilms and their impact on GAS pathogenicity are now feasible

Cross-serotype protection against group A Streptococcal infections induced by immunization with SPy_2191

A high number of serotypes makes vaccine development to group A Streptococcus (GAS) difficult. Here, the authors use a reverse vaccinology approach and identify SPy_2191 as conserved surface protein that inhibits GAS adhesion and invasion and induces cross-protective immunity in mice

Cross-serotype protection against group A Streptococcal infections induced by immunization with SPy_2191

Group A Streptococcus (GAS) infection causes a range of diseases, but vaccine development is hampered by the high number of serotypes. Here, using reverse vaccinology the authors identify SPy_2191 as a cross-protective vaccine candidate. From 18 initially identified surface proteins, only SPy_2191 is conserved, surface-exposed and inhibits both GAS adhesion and invasion. SPy_2191 immunization in mice generates bactericidal antibodies resulting in opsonophagocytic killing of prevalent and invasive GAS serotypes of different geographical regions, including M1 and M49 (India), M3.1 (Israel), M1 (UK) and M1 (USA). Resident splenocytes show higher interferon-γ and tumor necrosis factor-α secretion upon antigen re-stimulation, suggesting activation of cell-mediated immunity. SPy_2191 immunization significantly reduces streptococcal load in the organs and confers ~76-92% protection upon challenge with invasive GAS serotypes. Further, it significantly suppresses GAS pharyngeal colonization in mice mucosal infection model. Our findings suggest that SPy_2191 can act as a universal vaccine candidate against GAS infections

Advanced strategies for development of vaccines against human bacterial pathogens.

Infectious diseases are one of the main grounds of death and disabilities in human beings globally. Lack of effective treatment and immunization for many deadly infectious diseases and emerging drug resistance in pathogens underlines the need to either develop new vaccines or sufficiently improve the effectiveness of currently available drugs and vaccines. In this review, we discuss the application of advanced tools like bioinformatics, genomics, proteomics and associated techniques for a rational vaccine design