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 ⌬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 ␣-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-intermediateresistant 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.Carbon catabolite repression (CCR) in bacteria is a widespread, global regulatory phenomenon that allows modulation of the expression of genes and operons involved in carbon utilization and metabolization in the presence of preferred carbon source(s). In CCR, the presence of a preferred carbon source represses the expression of genes and operons whose products are involved in the metabolism of alternative, lesspreferred carbon sources. In low-GC gram-positive bacteria, CCR is achieved via transcriptional control, inducer exclusion, and induction prevention (reviewed in references 55 and 60). In this group of bacteria, a common mechanism for transcriptional control has evolved that is mediated via the proteins phosphotransferase HPr, the bifunctional HPr kinase-phosphatase (HPrK/P), and the pleiotropic regulator CcpA (catabolite control protein A). CCR in Bacillus subtilis has been studied extensively and is thought to serve as the prototype of CCR-regulated gene expression in gram-positive organisms (reviewed in reference 52). In B. subtilis, regulation of transcription of catabolite-repressive genes is exerted mainly through the binding of CcpA to specific cis-acting DNA sequences called catabolite-responsive elements (CREs). The DNA-binding activity of CcpA itself is triggered by HPr or its regulatory paralog Crh, which, in the presence of glucose, are phosphorylated by HPrK/P on regulatory seryl residues, in which st...

BackgroundThe catabolite control protein A (CcpA) is a member of the LacI/GalR family of transcriptional regulators controlling carbon-metabolism pathways in low-GC Gram-positive bacteria. It functions as a catabolite repressor or activator, allowing the bacteria to utilize the preferred carbon source over secondary carbon sources. This study is the first CcpA-dependent transcriptome and proteome analysis in Staphylococcus aureus, focussing on short-time effects of glucose under stable pH conditions.ResultsThe addition of glucose to exponentially growing S. aureus increased the expression of genes and enzymes of the glycolytic pathway, while genes and proteins of the tricarboxylic acid (TCA) cycle, required for the complete oxidation of glucose, were repressed via CcpA. Phosphotransacetylase and acetate kinase, converting acetyl-CoA to acetate with a concomitant substrate-level phosphorylation, were neither regulated by glucose nor by CcpA. CcpA directly repressed genes involved in utilization of amino acids as secondary carbon sources. Interestingly, the expression of a larger number of genes was found to be affected by ccpA inactivation in the absence of glucose than after glucose addition, suggesting that glucose-independent effects due to CcpA may have a particular impact in S. aureus. In the presence of glucose, CcpA was found to regulate the expression of genes involved in metabolism, but also that of genes coding for virulence determinants.ConclusionThis study describes the CcpA regulon of exponentially growing S. aureus cells. As in other bacteria, CcpA of S. aureus seems to control a large regulon that comprises metabolic genes as well as virulence determinants that are affected in their expression by CcpA in a glucose-dependent as well as -independent manner.

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. transComplementation 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.

The accessory gene regulator (agr) locus has been shown to be important for virulence in several animal models of Staphylococcus aureus infection. However, the role of agr in human infections, and specifically in antibiotic treatment, is controversial. Interestingly, agr dysfunction has been associated with reduced vancomycin responses.

Persistent methicillin-resistant Staphylococcus aureus (MRSA) bacteremia (PB) (positive blood cultures after >7 days of therapy) represents a clinically challenging subset of invasive MRSA infections. In this investigation, we examined the potential correlation of specific virulence signatures with PB versus resolving MRSA bacteremia (RB) (negative blood cultures within 2 to 4 days of therapy) strains. Thirty-six MRSA isolates from patients enrolled in a recent multinational clinical trial were studied for (i) susceptibility to host defense cationic peptides (HDPs) (i.e., thrombin-induced platelet microbicidal proteins [tPMPs] and human neutrophil peptide 1 [hNP-1]); (ii) adherence to host endovascular ligands (fibronectin) and cells (endothelial cells); and (iii) biofilm formation. We found that PB isolates exhibited significantly reduced susceptibilities to tPMPs and hNP-1 (P < 0.001 and P ‫؍‬ 0.023, respectively). There was no significant association between the PB outcome and fibronectin binding, endothelial cell binding, or biofilm formation (P ‫؍‬ 0.25, 0.97, and 0.064 versus RB strains, respectively). However, multiple logistic regression analysis revealed that the PB outcome was significantly associated with the combination of reduced susceptibilities to HDPs and extent of biofilm formation (P < 0.0001). Similar results were obtained in a second analysis using days of bacteremia as a continuous outcome, showing that reduced HDP susceptibilities and increased biofilm formation cocontributed to predict the duration of bacteremia. Our data indicate that PB isolates have specific pathogenic signatures independent of conventional antimicrobial susceptibility. These combinatorial mosaics can be defined and used to prospectively distinguish PB from RB strains in advance and potentially to predict ultimate clinical outcomes.Staphylococcus aureus is a leading cause of bacteremia and infective endocarditis (IE) throughout the industrialized world (18,22,42,54). A growing proportion of these bloodstream infections are due to methicillin-resistant Staphylococcus aureus (MRSA), which is associated with worse clinical outcome, longer hospitalization, and higher net cost than similar infections caused by methicillin-susceptible S. aureus (MSSA) (3,6,14,15,26,49,52,60). Persistent-bacteremia (PB) outcomes comprise 20 to 30% of all episodes of MRSA bacteremia and are especially relevant to endovascular infections (13,22,23,31). Why some MRSA bacteremia strains persist while others resolve (RB) despite similar baseline clinical and microbiologic characteristics and identical medical and surgical therapeutic strategies is poorly understood.In two recent studies, we investigated S. aureus virulence factors that could potentially differentiate PB from RB strains in the context of endovascular infections (23, 56). We initially studied PB or RB isolates from the Duke University Medical Center and delineated several in vitro parameters that appeared to distinguish these two strain cohorts, including enhanced matrix ligand and e...

The alternative transcription factor B of Staphylococcus aureus affects the transcription of the cap gene cluster, required for the synthesis of capsular polysaccharide (CP), although this operon is lacking an apparent B -dependent promoter. Regulation of cap expression and CP production in S. aureus strain Newman was shown here to be influenced by B , the two-component signal transduction regulatory system ArlRS, and the yabJ-spoVG locus to different extents. Inactivation of arlR or deletion of the sigB operon strongly suppressed capA (CP synthesis enzyme A) transcription. Deletion of spoVG had a polar effect on yabJ-spoVG transcription and resulted in a two-to threefold decrease in capA transcription. Interestingly, immunofluorescence showed that CP production was strongly impaired in all three mutants, signaling that the yabJ-spoVG inactivation, despite its only partial effect on capA transcription, abolished capsule formation. trans-Complementation of the ⌬spoVG mutant with yabJ-spoVG under the control of its native promoter restored CP-5 production and capA expression to levels seen in the wild type. Northern analyses revealed a strong impact of B on arlRS and yabJ-spoVG transcription. We hypothesize that ArlR and products of the yabJ-spoVG locus may serve as effectors that modulate B control over B -dependent genes lacking an apparent B promoter.Staphylococcus aureus is a major nosocomial pathogen with the ability to cause a variety of diseases, including life-threatening infections. Like most microorganisms that are able to cause invasive diseases, S. aureus produces extracellular capsular polysaccharides (CPs), which are thought to be of importance in pathogenesis (reviewed in reference 35). Although 11 serologically distinct CPs were identified in S. aureus, the majority of clinical isolates produce CPs of serotype 5 (CP-5) or serotype 8 (CP-8). CPs protect S. aureus against opsonophagocytic killing by polymorphonuclear leukocytes (16,17,25,53,56) and enhance virulence in a number of animal models of staphylococcal infection (34,40,53,54,57). Expression of CPs is known to be influenced by various environmental signals in vitro and in vivo (reviewed in references 35 and 56), and transcription of the cap operon was shown to be modulated by regulatory elements, such as arlRS, agr, ccpA, mgr, sae, and sarA (7,8,23,24,26,27,39,48,52,55). Recent microarray analyses added the alternative factor B to the regulatory network controlling cap operon expression (3, 38) and indicated B to control capA transcription in a growth phasedependent manner (3). However, the lack of an apparent B consensus sequence in the promoter of capA suggested that B regulates cap transcription indirectly. Candidates for such downstream-acting regulators might be ArlRS and SarA, which are positively controlled by B in S. aureus (2, 3), although SarA was previously shown to have only a minor effect on cap expression and CP production in S. aureus (24). RNAIII of the agr locus, known to positively affect capA expression (7, 24, 55), could ...

Our results show that the acidic environment favors formation of nonstable SCVs, which reflect the SCVs found in clinics. They also provide evidence that treatment with alkalinizing agents, together with antibiotics, may provide a novel translational strategy for eradicating persisting intracellular reservoirs of staphylococci. This approach may also be extended to other intracellular bacteria.

These observations suggest that sarA activation is important in persistent MRSA endovascular infection, potentially in the setting of biofilm formation.