The accessory gene regulator (agr) of Staphylococcus aureus is a global regulator of the staphylococcal virulon, which includes secreted virulence factors and surface proteins. The agr locus is important for virulence in a variety of animal models of infection, and has been assumed by inference to have a major role in human infection. Although most human clinical S. aureus isolates are agr+, there have been several reports of agr-defective mutants isolated from infected patients. Since it is well known that the agr locus is genetically labile in vitro, we have addressed the question of whether the reported agr-defective mutants were involved in the infection or could have arisen during post-isolation handling. We obtained a series of new staphylococcal isolates from local clinical infections and handled these with special care to avoid post-isolation mutations. Among these isolates, we found a number of strains with non-haemolytic phenotypes owing to mutations in the agr locus, and others with mutations elsewhere. We have also obtained isolates in which the population was continuously heterogeneous with respect to agr functionality, with agr+ and agr− variants having otherwise indistinguishable chromosomal backgrounds. This finding suggested that the agr− variants arose by mutation during the course of the infection. Our results indicate that while most clinical isolates are haemolytic and agr+, non-haemolytic and agr− strains are found in S. aureus infections, and that agr+ and agr− variants may have a cooperative interaction in certain types of infections.

agr is a global regulatory system in the staphylococci, operating by a classical two-component signaling module and controlling the expression of most of the genes encoding extracellular virulence factors. As it is autoinduced by a peptide, encoded within the locus, that is the ligand for the signal receptor, it is a sensor of population density or a quorum sensor and is the only known quorum-sensing system in the genus. agr is conserved throughout the staphylococci but has diverged along lines that appear to parallel speciation and subspeciation within the genus. This divergence has given rise to a novel type of interstrain and interspecies cross-inhibition that represents a fundamental aspect of the organism's biology and may be a predominant feature of the evolutionary forces that have driven it. We present evidence, using a newly developed, luciferasebased agr typing scheme, that the evolutionary divergence of the agr system was an early event in the evolution of the staphylococci and long preceded the development of the nucleotide polymorphisms presently used for genotyping. These polymorphisms developed, for the most part, within different agr groups; mobile genetic elements appear also to have diffused recently and, with a few notable exceptions, have come to reside largely indiscriminately within the several agr groups.The agr operon encodes a global regulatory system in the staphylococci, central to the biology of the organism (reviewed by Novick) (36). It controls a large set of genes, including most of those encoding extracellular virulence factors and many others encoding cytoplasmic proteins with catabolic and other functions. agr is highly conserved throughout the staphylococci but has diverged in a way that closely parallels speciation and subspeciation within the genus. This divergence has given rise to a novel type of interstrain and interspecies cross-inhibition that may represent the selective forces that have driven its evolution.agr operates by a classical two-component signaling module (Fig. 1A). It is autoinduced by a peptide, encoded within the locus, that is the ligand for the histidine kinase component of the signaling module. agr is therefore a sensor of population density or a quorum sensor. The agr variants represent specificity groups that determine the response to cognate or heterologous autoinducing peptides (AIPs). Although an AIP always activates its cognate agr locus, it competitively crossinhibits agr activation in most heterologous combinations. This cross-inhibition results in a novel type of bacterial interference in which the expression of accessory genes, but not growth, is blocked. This interference has potential therapeutic implications that are presently under investigation. In Staphylococcus aureus there are four known agr specificity groups, characterized by major sequence variations in a central region of the locus that encodes the AIP, the enzyme that processes it, and the receptor domain of the histidine kinase (7,19,21). The sequences flanking this central variab...

Mycobacterium tuberculosis uses multiple mechanisms to avoid elimination by the immune system. We have previously shown that M. tuberculosis can inhibit selected macrophage responses to IFN-γ through TLR2-dependent and -independent mechanisms. To specifically address the role of TLR2 signaling in mediating this inhibition, we stimulated macrophages with the specific TLR2/1 ligand Pam3CSK4 and assayed responses to IFN-γ. Pam3CSK4 stimulation prior to IFN-γ inhibited transcription of the unrelated IFN-γ-inducible genes, CIITA and CXCL11. Surface expression of MHC class II and secretion of CXCL11 were greatly reduced as well, indicating that the reduction in transcripts had downstream effects. Inhibition of both genes required new protein synthesis. Using chromatin immunoprecipitation, we found that TLR2 stimulation inhibited IFN-γ-induced RNA polymerase II binding to the CIITA and CXCL11 promoters. Furthermore, TATA binding protein was unable to bind the TATA box of the CXCL11 promoter, suggesting that assembly of transcriptional machinery was disrupted. However, TLR2 stimulation affected chromatin modifications differently at each of the inhibited promoters. Histone H3 and H4 acetylation was reduced at the CIITA promoter but unaffected at the CXCL11 promoter. In addition, NF-κB signaling was required for inhibition of CXCL11 transcription, but not for inhibition of CIITA. Taken together, these results indicate that TLR2-dependent inhibition of IFN-γ-induced gene expression is mediated by distinct, gene-specific mechanisms that disrupt binding of the transcriptional machinery to the promoters.

Mycobacterium tuberculosis (Mtb) uses multiple mechanisms to avoid destruction by the immune system. We have previously shown that Mtb can inhibit selected macrophage responses to IFN‐γ using TLR2‐dependent and –independent mechanisms. To address the role of TLR2 signaling in mediating this inhibition, we used microarray analysis to identify IFN‐γ‐inducible genes whose expression decreased when macrophages were treated with the specific TLR2/1 ligand Pam3CSK4 (Pam). Four of the genes identified—Ciita, Klrk1, Dnase1l3, and Cxcl11—were further characterized. Ciita and Klrk1 induction by IFN‐γ was rapidly inhibited by Pam while Dnase1l3 and Cxcl11 required prolonged Pam pretreatment prior to IFN‐γ. Histone H4 acetylation at the Ciita and Klrk1 promoters was reduced in macrophages treated simultaneously with Pam and IFN‐γ compared to those treated with IFN‐γ alone. This effect was dependent on histone deacetylase activity. Treatment with the NF‐κB inhibitor parthenolide restored IFN‐γ‐induced gene expression and histone H4 acetylation at the promoters of selected Pam‐inhibited genes including Klrk1 and Cxcl11. These results indicate that TLR2‐dependent inhibition of IFN‐γ‐induced gene expression by Mtb depends on the length of exposure to a TLR2 agonist, is mediated by a mechanism affecting chromatin remodeling, and involves multiple signaling pathways including NF‐κB.NIH‐R01‐AI46097