Staphylococcus aureus CcpA Affects Virulence Determinant Production and Antibiotic Resistance

Abstract

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...

A Novel Mode of Regulation of the Staphylococcus aureus Catabolite Control Protein A (CcpA) Mediated by Stk1 Protein Phosphorylation

“…5B). In line with our previous findings (7,14), we found a significant increase in citZ transcription in the ccpA mutant KS66 in the early exponential growth phase (i.e. after 3 h of growth in TSB supplemented with 0.1% (w/v) of glucose), whereas transcription of hla, which is expressed mainly during the later stages of growth, was markedly down-regulated in KS66 after 8 h of growth compared with the wild type.…”

“…With the present study, we extend previous work demonstrating the regulatory role of Stk1-mediated phosphorylation in central metabolism and virulence pathways by providing first evidence of a Ser/Thr kinase phosphorylation-dependent regulation of the major transcriptional regulator CcpA. This mechanism is of special relevance as CcpA contributes to the pathogenicity of S. aureus (7,8,12,14,15). We demonstrated that phosphorylation of Thr-18 and Thr-33 residues abrogated CcpA DNA binding activity in vitro.…”

“…To test this hypothesis, we analyzed the binding capacities of the nonphosphorylated CcpA form (CcpA) and that of the hyperphosphorylated CcpA form (CcpA-P) to a couple of gene promoter elements that were recently shown to be affected by this regulatory protein (Fig. 4C) (7,(12)(13)(14). Although the unphosphorylated CcpA interacted with all the double-stranded DNA probes tested in our electrophoretic mobility shift assays (Fig.…”

“…In S. aureus, which is one of the leading causes for nosocomial infections and an important human pathogen responsible for a wide variety of diseases, including soft tissue infections, toxic shock syndrome, and necrotizing pneumonia (11), CcpA was shown to affect antibiotic resistance, biofilm formation, expression of toxins such as hla (encoding ␣-hemolysin) and tst (encoding toxic shock syndrome toxin), and infectivity of this bacterium (7,(12)(13)(14)(15). Recent transcriptome and proteome analyses confirmed the broad effects of CcpA on gene expression in S. aureus and suggested this LacI/GalR type of regulator to be active in the presence and absence of glucose (13).…”

Reduced Vancomycin Susceptibility inStaphylococcus aureus, Including Vancomycin-Intermediate and Heterogeneous Vancomycin-Intermediate Strains: Resistance Mechanisms, Laboratory Detection, and Clinical Implications

“…Perhaps the most consistent transcriptional change observed for VISA strains is the downregulation of the spa gene (74,122,169,199,201,251,303). A possibly incomplete list of genes where the gene product has been shown to affect the transcription of spa includes agr (76,343), sarA (51,76,343), sarS (50), arlSR (85,86), sarT (304), srrAB (262), tcaR (200), mgrA (138), rot (240), ccpA (307), and sarZ (342). Potentially, a mutation in any of these regulatory genes could simultaneously increase vancomycin resistance and decrease protein A production.…”