Microarray-based analysis of the transcriptional profiles of the genetically distinctStaphylococcus aureusstrains COL, GP268, and Newman indicate that a total of 251 open reading frames (ORFs) are influenced by σ^Bactivity. While σ^Bwas found to positively control 198 genes by a factor of ≥2 in at least two of the three genetic lineages analyzed, 53 ORFs were repressed in the presence of σ^B. Gene products that were found to be influenced by σ^Bare putatively involved in all manner of cellular processes, including cell envelope biosynthesis and turnover, intermediary metabolism, and signaling pathways. Most of the genes and/or operons identified as upregulated by σ^Bwere preceded by a nucleotide sequence that resembled the σ^Bconsensus promoter sequence ofBacillus subtilis. A conspicuous number of virulence-associated genes were identified as regulated by σ^Bactivity, with many adhesins upregulated and prominently represented in this group, while transcription of various exoproteins and toxins were repressed. The data presented here suggest that the σ^BofS. aureuscontrols a large regulon and is an important modulator of virulence gene expression that is likely to act conversely to RNAIII, the effector molecule of theagrlocus. We propose that this alternative transcription factor may be of importance for the invading pathogen to fine-tune its virulence factor production in response to changing host environments.

The growth phase-dependent activity profile of the alternate transcription factor ς ^B and its effects on the expression of sar and agr were examined in three different Staphylococcus aureus strains by Northern blot analyses and by the use of reporter gene fusion experiments. Significant ς ^B activity was detectable only in the clinical isolates MSSA1112 and Newman, carrying the wild-type rsbU allele, but not in the NCTC8325 derivative BB255, which is defective in rsbU . ς ^B activity peaked in the late exponential phase and diminished towards the stationary phase when bacteria were grown in Luria-Bertani medium. Transcriptional analysis and a sarP1-sarP2-sarP3 ( sarP1-P2-P3 )-driven firefly luciferase ( luc +) reporter gene fusion demonstrated a strong ς ^B activity- and growth phase-dependent increase in sar expression that was totally absent in either rsbU or Δ rsbUVWsigB mutants. In contrast, expression of the agr locus, as measured by RNAIII levels and by an hldp :: luc + fusion, was found to be higher in the absence of ς ^B activity, such as in rsbU or Δ rsbUVWsigB mutants, than in wild-type strains. Overexpression of ς ^B in BB255 derivatives resulted in a clear increase in sarP1-P2-P3 :: luc + expression as well as a strong decrease in hldp :: luc + expression. The data presented here suggest that ς ^B increases sar expression while simultaneously reducing the RNAIII level in a growth phase-dependent manner.

Derivatives of the widely used laboratory strain Staphylococcus aureus NCTC8325, which are natural rsbU mutants, were shown to be unable to produce RsbU, a positive regulator of the alternative sigma factor ς ^B . The lack of RsbU prevented the heat-dependent production of ς ^B -controlled transcripts and resulted in reduced H ₂ O ₂ and UV tolerance, enhanced alpha-hemolysin activity, and the inability to produce the alkaline shock protein Asp23. After 48 h of growth, rsbU mutant strains failed to accumulate staphyloxanthin, the major stationary-phase carotenoid. Transcription of Asp23 was found to be exclusively controlled by ς ^B , making it an excellent target for the study of ς ^B activity in S. aureus . Reporter gene experiments, using the firefly luciferase gene ( luc +) fused to the ς ^B -dependent promoter(s) of asp23 , revealed that ς ^B is almost inactive in 8325 derivatives. cis complementation of the 8325 derivative BB255 with the wild-type rsbU gene from strain COL produced the rsbU ⁺ derivative GP268, a strain possessing a ς ^B activity profile comparable to that of the rsbU ⁺ wild-type strain Newman. In GP268, the heat inducibility of ς ^B -dependent genes, Asp23 production, alpha-hemolysin activity, pigmentation, and susceptibility to H ₂ O ₂ were restored to the levels observed in strain Newman, clearly demonstrating that RsbU is needed for activation of ς ^B in S. aureus .

Staphylococcus aureus is a major human pathogen that causes a range of infections from acute invasive to chronic and difficult-to-treat. Infection strategies associated with persisting S. aureus infections are bacterial host cell invasion and the bacterial ability to dynamically change phenotypes from the aggressive wild-type to small colony variants (SCVs), which are adapted for intracellular long-term persistence. The underlying mechanisms of the bacterial switching and adaptation mechanisms appear to be very dynamic, but are largely unknown. Here, we analyzed the role and the crosstalk of the global S. aureus regulators agr, sarA and SigB by generating single, double and triple mutants, and testing them with proteome analysis and in different in vitro and in vivo infection models. We were able to demonstrate that SigB is the crucial factor for adaptation in chronic infections. During acute infection, the bacteria require the simultaneous action of the agr and sarA loci to defend against invading immune cells by causing inflammation and cytotoxicity and to escape from phagosomes in their host cells that enable them to settle an infection at high bacterial density. To persist intracellularly the bacteria subsequently need to silence agr and sarA. Indeed agr and sarA deletion mutants expressed a much lower number of virulence factors and could persist at high numbers intracellularly. SigB plays a crucial function to promote bacterial intracellular persistence. In fact, ΔsigB-mutants did not generate SCVs and were completely cleared by the host cells within a few days. In this study we identified SigB as an essential factor that enables the bacteria to switch from the highly aggressive phenotype that settles an acute infection to a silent SCV-phenotype that allows for long-term intracellular persistence. Consequently, the SigB-operon represents a possible target to develop preventive and therapeutic strategies against chronic and therapy-refractory infections. Staphylococcus aureus is a frequent pathogen of severe invasive infections that can develop into chronicity and become extremely difficult to eradicate. Chronic infections have been highly associated with altered bacterial phenotypes, i.e., the small colony variants (SCVs) that dynamically appear after bacterial host cell invasion and are highly adapted for intracellular long-term persistence. In this study, we analyzed the underlying mechanisms of the bacterial switching and adaptation process by investigating the functions of the global S. aureus regulators agr, sarA and SigB. We demonstrate that a tight crosstalk between these factors supports the bacteria at any stage of the infection and that SigB is the crucial factor for bacterial adaptation during long-term persistence. In the acute phase, the bacteria require active agr and sarA systems to induce inflammation and cytotoxicity, and to establish an infection at high bacterial numbers. In the chronic stage of infection, SigB downregulates the aggressive bacterial phenotype and mediates the formation of dynamic SCV-phenotypes. Consequently, we describe SigB as a crucial factor for bacterial adaptation and persistence, which represents a possible target for therapeutic interventions against chronic infections.

Carbon catabolite protein A (CcpA) is known to function as a major regulator of gene expression in different gram-positive organisms. Deletion of the ccpA homologue ( saCOL1786 ) in Staphylococcus aureus was found to affect growth, glucose metabolization, and transcription of selected virulence determinants. In liquid culture, deletion of CcpA decreased the growth rate and yield; however, the effect was only transient during the exponential-growth phase as long as glucose was present in the medium. Depletion of glucose and production of lactate was delayed, while the level of excretion of acetate was less affected and was even higher in the mutant culture. On solid medium, in contrast, growth of the Δ ccpA mutant resulted in smaller colonies containing a lower number of CFU per colony. Deletion of CcpA had an effect on the expression of important virulence factors of S. aureus by down-regulating RNAIII , the effector molecule of the agr locus, and altering the transcription patterns of hla , encoding α-hemolysin, and spa , encoding protein A. CcpA inactivation markedly reduced the oxacillin resistance levels in the highly methicillin-resistant S. aureus strain COLn and the teicoplanin resistance level in a glycopeptide-intermediate-resistant S. aureus strain. The presence of CcpA in the capsular polysaccharide serotype 5 (CP5)-producing strain Newman abolished capsule formation and decreased cap operon transcription in the presence of glucose. The staphylococcal CcpA thus not only is involved in the regulation of carbon metabolism but seems to function as a modulator of virulence gene expression as well.

Biofilm formation in Staphylococcus aureus under in vitro growth conditions is generally promoted by high concentrations of sugar and/or salts. The addition of glucose to routinely used complex growth media triggered biofilm formation in S. aureus strain SA113. Deletion of ccpA , coding for the catabolite control protein A (CcpA), which regulates gene expression in response to the carbon source, abolished the capacity of SA113 to form a biofilm under static and flow conditions, while still allowing primary attachment to polystyrene surfaces. This suggested that CcpA mainly affects biofilm accumulation and intercellular aggregation. trans -Complementation of the mutant with the wild-type ccpA allele fully restored the biofilm formation. The biofilm produced by SA113 was susceptible to sodium metaperiodate, DNase I, and proteinase K treatment, indicating the presence of polysaccharide intercellular adhesin (PIA), protein factors, and extracellular DNA (eDNA). The investigation of several factors which were reported to influence biofilm formation in S. aureus ( arlRS , mgrA , rbf , sarA , atl , ica , citZ , citB , and cidABC ) showed that CcpA up-regulated the transcription of cidA , which was recently shown to contribute to eDNA production. Moreover, we showed that CcpA increased icaA expression and PIA production, presumably over the down-regulation of the tricarboxylic acid cycle genes citB and citZ .