SummaryThe hyaluronic acid capsule of group A Streptococcus (GAS) is an important virulence factor, but little is known about mechanisms that regulate capsule expression. Transposon Tn916 mutagenesis of the poorly encapsulated M-type 3 GAS strain DLS003 produced a transconjugant that exhibited a mucoid colony morphology, reflecting increased hyaluronic acid capsule production. Analysis of chromosomal DNA sequence immediately downstream of the transposon insertion identified two open reading frames, designated csrR and csrS, which exhibited sequence similarity to bacterial two-component regulatory systems. We constructed an in-frame deletion mutation within csrR, which encodes the putative response component. Replacement of the native csrR gene in the DLS003 chromosome with the mutant allele resulted in a sixfold increase in capsule production and a corresponding increase in transcription of the has operon, which contains the essential genes for hyaluronic acid synthesis. Increased capsule production by the csrR mutant strain was associated with enhanced resistance to complement-mediated opsonophagocytic killing in vitro and with a 500-fold increase in virulence in mice. These results establish CsrR as a negative regulator of hyaluronic acid capsule synthesis and suggest that it is part of a twocomponent regulatory system that influences capsule expression and virulence.

Group A streptococci control expression of key virulence determinants via the two-component sensor͞regulator system CsrR͞CsrS. The membrane-bound sensor CsrS is thought to respond to previously unknown environmental signal(s) by controlling phosphorylation of its cognate regulator component CsrR. Phosphorylation of CsrR increases its affinity for binding to the promoter regions of Csr-regulated genes to repress transcription. Here we show that environmental Mg 2؉ concentration is a potent and specific stimulus for CsrR͞CsrS-mediated regulation. We studied the effect of divalent cations on expression of the Csr-regulated hyaluronic acid capsule genes (hasABC) by measuring chloramphenicol acetyltransferase (CAT) activity in a reporter strain of group A Streptococcus carrying a has operon promoter-cat fusion. Addition of Mg 2؉ , but not of Ca 2؉ , Mn 2؉ , or Zn 2؉ , repressed capsule gene expression by up to 80% in a dose-dependent fashion. The decrease in capsule gene transcription was associated with a marked reduction in cell-associated capsular polysaccharide. RNA hybridization analysis demonstrated reduced expression of the Csrregulated hasABC operon, streptokinase (ska), and streptolysin S (sagA) during growth in the presence of 15 mM Mg 2؉ for the wild-type strain 003CAT but not for an isogenic csrS mutant. We propose that Mg 2؉ binds to CsrS to induce phosphorylation of CsrR and subsequent repression of virulence gene expression. The low concentration of Mg 2؉ in extracellular body fluids predicts that the CsrR͞CsrS system is maintained in the inactive state during infection, thereby allowing maximal expression of critical virulence determinants in the human host.

The structure-based design, synthesis, and biological activity of a novel indazole-containing inhibitor series for S-adenosyl homocysteine/methylthioadenosine (SAH/MTA) nucleosidase are described. Use of 5-aminoindazole as the core scaffold provided a structure-guided series of low nanomolar inhibitors with broad-spectrum antimicrobial activity. The implementation of structure-based methodologies provided a 6000-fold increase in potency over a short timeline (several months) and an economy of synthesized compounds.

Adaptive responses of bacteria that involve sensing the presence of other bacteria are often critical for proliferation and the expression of virulence characteristics. The autoinducer II (AI‐2) pathway has recently been shown to be a mechanism for sensing other bacteria that is highly conserved among diverse bacterial species, including Gram‐positive pathogens. However, a role for this pathway in the regulation of virulence factors in Gram‐positive pathogens has yet to be established. In this study, we have inactivated luxS, an essential component of the AI‐2 pathway, in the Gram‐positive pathogen Streptococcus pyogenes. Analyses of the resulting mutants revealed the aberrant expression of several virulence properties that are regulated in response to growth phase, including enhanced haemolytic activity, and a dramatic reduction in the expression of secreted proteolytic activity. This latter defect was associated with a reduced ability to secrete and process the precursor of the cysteine protease (SpeB) as well as a difference in the timing of expression of the protease. Enhanced haemolytic activity of the luxS strain was also shown to be linked with an increased expression of the haemolysin S‐associated gene sagA. Disruptions of luxS in these mutants also produced a media‐dependent growth defect. Finally, an allelic replacement analysis of an S. pyogenes strain with a naturally occurring insertion of IS1239 in luxS suggested a mechanism for modulation of virulence during infection. Results from this study suggest that luxS makes an important contribution to the regulation of S. pyogenes virulence factors.