The CiaR response regulator in group B Streptococcus promotes intracellular survival and resistance to innate immune defenses.

Group B Streptococcus (GBS) is major cause of invasive disease in newborn infants and the leading cause of neonatal meningitis. To gain access to the central nervous system (CNS), GBS must not only subvert host defenses in the bloodstream but also invade and survive within brain microvascular endothelial cells (BMEC), the principal cell layer composing the blood-brain barrier (BBB). While several GBS determinants that contribute to the invasion of BMEC have been identified, little is known about the GBS factors that are required for intracellular survival and ultimate disease progression. In this study we sought to identify these factors by screening a random GBS mutant library in an in vitro survival assay. One mutant was identified which contained a disruption in a two-component regulatory system homologous to CiaR/CiaH, which is present in other streptococcal pathogens. Deletion of the putative response regulator, ciaR, in GBS resulted in a significant decrease in intracellular survival within neutrophils, murine macrophages, and human BMEC, which was linked to increased susceptibility to killing by antimicrobial peptides, lysozyme, and reactive oxygen species. Furthermore, competition experiments with mice showed that wild-type GBS had a significant survival advantage over the GBS DeltaciaR mutant in the bloodstream and brain. Microarray analysis comparing gene expression between wild-type and DeltaciaR mutant GBS bacteria revealed several CiaR-regulated genes that may contribute to stress tolerance and the subversion of host defenses by GBS. Our results identify the GBS CiaR response regulator as a crucial factor in GBS intracellular survival and invasive disease pathogenesis.

Streptococcus agalactiae CspA is a serine protease that inactivates chemokines.

Streptococcus agalactiae (group B Streptococcus [GBS]) remains a leading cause of invasive infections in neonates and has emerged as a pathogen of the immunocompromised and elderly populations. The virulence mechanisms of GBS are relatively understudied and are still poorly understood. Previous evidence indicated that the GBS cspA gene is necessary for full virulence and the cleavage of fibrinogen. The predicted cspA product displays homology to members of the extracellular cell envelope protease family. CXC chemokines, many of which can recruit neutrophils to sites of infection, are important signaling peptides of the immune system. In this study, we purified CspA and demonstrated that it readily cleaved the CXC chemokines GRO-alpha, GRO-beta, GRO-gamma, neutrophil-activating peptide 2 (NAP-2), and granulocyte chemotactic protein 2 (GCP-2) but did not cleave interleukin-8. CspA did not cleave a panel of other test substrates, suggesting that it possesses a certain degree of specificity. CXC chemokines also underwent cleavage by whole GBS cells in a cspA-dependent manner. CspA abolished the abilities of three representative CXC chemokines, GRO-gamma, NAP-2, and GCP-2, to attract and activate neutrophils. Genetic and biochemical evidence indicated that CspA is a serine protease with S575 at its active site. D180 was also implicated as part of the signature serine protease catalytic triad, and both S575 and D180 were required for both N-terminal and C-terminal autocatalytic processing of CspA.

Global transcriptional profiling reveals Streptococcus agalactiae genes controlled by the MtaR transcription factor.

BACKGROUND: Streptococcus agalactiae (group B Streptococcus; GBS) is a significant bacterial pathogen of neonates and an emerging pathogen of adults. Though transcriptional regulators are abundantly encoded on the GBS genome, their role in GBS pathogenesis is poorly understood. The mtaR gene encodes a putative LysR-type transcriptional regulator that is critical for the full virulence of GBS. Previous studies have shown that an mtaR- mutant transports methionine at reduced rates and grows poorly in normal human plasma not supplemented with methionine. The decreased virulence of the mtaR mutant was correlated with a methionine transport defect; however, no MtaR-regulated genes were identified. RESULTS: Microarray analysis of wild-type GBS and an mtaR mutant revealed differential expression of 12 genes, including 1 upregulated and 11 downregulated genes in the mtaR mutant. Among the downregulated genes, we identified a cluster of cotranscribed genes encoding a putative methionine transporter (metQ1NP) and peptidase (pdsM). The expression of four genes potentially involved in arginine transport (artPQ) and arginine biosynthesis (argGH) was downregulated and these genes localized to two transcriptional units. The virulence factor cspA, which encodes an extracellular protease, was downregulated. Additionally, the SAN_1255 locus, which putatively encodes a protein displaying similarity to plasminogen activators, was downregulated. CONCLUSION: To our knowledge, this is the first study to describe the global influence of MtaR on GBS gene expression. This study implicates the metQ1NP genes as encoding the MtaR-regulated methionine transporter, which may provide a mechanistic explanation for the methionine-dependent growth defect of the mtaR mutant. In addition to modulating the expression of genes involved in metabolism and amino acid transport, inactivation of mtaR affected the expression of other GBS genes implicated in pathogenesis. These findings suggest the possibility that MtaR may play a multifaceted role in GBS pathogenesis by regulating the expression of numerous genes.

Expression of the Streptococcus agalactiae virulence-associated protease CspA in a soluble, active form utilizing the Gram-positive host, Lactococcus lactis.

The heterologous expression of streptococcal genes in common Gram-negative hosts may be complicated by low-level expression, toxicity to the host, formation of inclusion bodies, and mislocalization of the encoded proteins. Biochemical study of the Streptococcus agalactiae virulence-associated cell-envelope protease (CEP) CspA, as well as other CEPs, has been limited by the lack of effective expression systems. In this study, we present a simple strategy to express cspA as a catalytically active exoprotein. A recombinant allele of cspA, cspADeltaCWA, was engineered to eliminate the dispensable cell-wall anchor. The cspADeltaCWA allele was expressed in the Gram-positive organism, Lactococcus lactis, using an established, plasmid-based, nisin-inducible expression system. After induction, nearly all of the exoprotein observable by SDS-PAGE corresponded to CspADeltaCWA. CspADeltaCWA-containing medium exhibited similar fibrinolytic activity as whole cells of GBS, indicating the recombinant protein was active. Characterization of CspADeltaCWA indicated that like some other CEPs, it is N-terminally processed, exists predominantly as a dimer, and has the ability to cleave itself at its C-terminus. Taken together, this work presents an efficient strategy for expression of cspA that could be applied to other streptococcal proteins that are not amenable to expression using common Gram-negative hosts.

MtaR, a regulator of methionine transport, is critical for survival of group B streptococcus in vivo.

The group B streptococcus (GBS) is an important human pathogen that infects newborns as well as adults. GBS also provides a model system for studying adaptation to different host environments due to its ability to survive in a variety of sites within the host. In this study, we have characterized a transcription factor, MtaR, that is essential for the ability of GBS to survive in vivo. An isogenic strain bearing a kanamycin insertion in mtaR was attenuated for survival in a neonatal-rat model of sepsis. The mtaR mutant grew poorly in human plasma, suggesting that its utilization of plasma-derived nutrients was inefficient. When an excess of exogenous methionine (200 microg/ml) was provided to the mtaR mutant, its growth rate in plasma was restored to that of the wild-type strain. The mtaR mutant grew poorly in chemically defined medium (CDM) prepared with methionine at a concentration similar to that of plasma (4 microg/ml) but was able to grow normally in CDM prepared with a high concentration of methionine (400 microg/ml). Both the wild-type strain and the mtaR mutant were incapable of growth in CDM lacking methionine, indicating that GBS cannot synthesize methionine de novo. When the abilities of the strains to incorporate radiolabeled methionine were compared, the mtaR mutant incorporated fivefold less methionine than the wild-type strain during a 10-min period. Collectively, the results from this study suggest that the ability to regulate expression of a methionine transport system is critical for GBS survival in vivo.

A novel streptococcal surface protease promotes virulence, resistance to opsonophagocytosis, and cleavage of human fibrinogen.

Group B streptococcus (GBS) is an important human pathogen. In this study, we sought to identify mechanisms that may protect GBS from host defenses in addition to its capsular polysaccharide. A gene encoding a cell-surface-associated protein (cspA) was characterized from a highly virulent type III GBS isolate, COH1. Its sequence indicated that it is a subtilisin-like extracellular serine protease homologous to streptococcal C5a peptidases and caseinases of lactic acid bacteria. The wild-type strain cleaved the alpha chain of human fibrinogen, whereas a cspA mutant, TOH121, was unable to cleave fibrinogen. We observed aggregated material when COH1 was incubated with fibrinogen but not when the mutant strain was treated similarly. This suggested that the product(s) of fibrinogen cleavage have strong adhesive properties and may be similar to fibrin. The cspA gene was present among representative clinical isolates from all nine capsular serotypes, as revealed by Southern blotting. A cspA(-) mutant was ten times less virulent in a neonatal rat sepsis model of GBS infections, as measured by LD(50) analysis. In addition, the cspA(-) mutant was significantly more sensitive than the wild-type strain to opsonophagocytic killing by human neutrophils in vitro. Taken together, the results suggest that cleavage of fibrinogen by CspA may increase the lethality of GBS infection, potentially by protecting the bacterium from opsonophagocytic killing.