Transcriptional downregulation ofagrexpression inStaphylococcus aureusduring growth in human serum can be overcome by constitutively active mutant forms of the sensor kinase AgrC

Abstract

The temporal and cell density-dependent regulation of expression of virtually all the Staphylococcus aureus virulon is under the control of the agr (accessory gene regulatory) operon. The expression of the agr operon is subject to transcriptional regulation by the AgrA/C two-component response regulator/sensor kinase pair. During bacteraemia, a frequent syndrome caused by methicillin-resistant S. aureus (MRSA), the transcriptional downregulation of agr expression has been attributed to the sequestration of the quorum-signalling molecule auto-inducing peptide (AIP) by the human serum component apolipoprotein B as part of an innate immune response to infection. However, it is not known whether transcriptional downregulation of agr expression during growth in human serum is additionally subjected to regulation by transcription regulatory proteins that either directly or indirectly affect transcription from the agr operon promoters. Here, using chromosomal fluorescence reporters of agr expression in S. aureus, we show that the transcriptional downregulation of agr expression in human serum can be overcome using constitutive active mutant forms of AgrC. Therefore, it seems that the sequestration of the AIP is likely to be the only mechanism by which the host innate immune response limits agr expression at the transcriptional level to maintain the host–pathogen balance towards a noninvasive outcome.

The Agr Quorum-Sensing System Regulates Fibronectin Binding but Not Hemolysis in the Absence of a Functional Electron Transport Chain

“…USA300 was also employed to demonstrate that key phenotypic effects of loss of a functional electron transport chain occur in a clinically relevant strain (43). S. aureus was cultured either in 3 ml tryptic soy broth (TSB) in 30-ml universal tubes at 37°C with orbital shaking (180 rpm) for phenotypic analyses or in 200 l TSB in microtiter plates for growth and Agr activity analyses (see below) (44). In some assays, S. aureus was grown in the presence of 2-n-heptyl-4-hydroxyquinoline N-oxide (HQNO) (10 g ml Ϫ1 ; Santa-Cruz Biotechnology) (45); synthetic type 1 AIP, as described previously (46) (1 to 10 M; Peptide Synthetics); hemin (1 g ml Ϫ1 ; Sigma-Aldrich); or menadione (1 g ml Ϫ1 ; Sigma-Aldrich).…”

“…Reporter constructs, consisting of the agr P2-P3 intergenic region fused with gfp under the control of either the P2 or P3 promoter, in pCL55 were passaged through Escherichia coli DC10B and transformed directly into S. aureus strains by electroporation (44). Transformants were selected for by using chloramphenicol, and insertion of the construct into the geh locus was confirmed by PCR (44).…”

“…Transformants were selected for by using chloramphenicol, and insertion of the construct into the geh locus was confirmed by PCR (44).…”

“…Growth and Agr expression reporter assays were performed as described previously (44). Briefly, stationary-phase cultures were diluted 1:50 in a final volume of 200 l TSB (with or without HQNO, hemin, or sAIP, as described above) and added to the wells of black microtiter plates with clear bottoms (Corning).…”

“…Bacterial growth was measured via optical Real-time PCR analysis of RNAII and RNAIII transcripts. RNA extraction and real-time (RT) PCR were performed as described previously (44). Briefly, S. aureus SH1000 wild type or hemB::Tn was grown to either mid-logarithmic or stationary phase, samples were taken, and bacterial cells were lysed using lysostaphin and SDS.…”