Opposing roles of σB and σB-controlled SpoVG in the global regulation of esxA in Staphylococcus aureus

BACKGROUND: The production of virulence factors in Staphylococcus aureus is tightly controlled by a complex web of interacting regulators. EsxA is one of the virulence factors that are excreted by the specialized, type VII-like Ess secretion system of S. aureus. The esxA gene is part of the σB-dependent SpoVG subregulon. However, the mode of action of SpoVG and its impact on other global regulators acting on esxA transcription is as yet unknown. RESULTS: We demonstrate that the transcription of esxA is controlled by a regulatory cascade involving downstream σB-dependent regulatory elements, including the staphylococcal accessory regulator SarA, the ArlRS two-component system and SpoVG. The esxA gene, preceding the ess gene cluster, was shown to form a monocistronic transcript that is driven by a σA promoter, whereas a putative σB promoter identified upstream of the σA promoter was shown to be inactive. Transcription of esxA was strongly upregulated upon either sarA or sigB inactivation, but decreased in agr, arlR and spoVG single mutants, suggesting that agr, ArlR and SpoVG are able to increase esxA transcription and relieve the repressing effect of the σB-controlled SarA on esxA. CONCLUSION: SpoVG is a σB-dependent element that fine-tunes the expression of esxA by counteracting the σB-induced repressing activity of the transcriptional regulator SarA and activates esxA transcription

Phylogenetic distribution and membrane topology of the LytR-CpsA-Psr protein family

BACKGROUND: The bacterial cell wall is the target of many antibiotics and cell envelope constituents are critical to host-pathogen interactions. To combat resistance development and virulence, a detailed knowledge of the individual factors involved is essential. Members of the LytR-CpsA-Psr family of cell envelope-associated attenuators are relevant for beta-lactam resistance, biofilm formation, and stress tolerance, and they are suggested to play a role in cell wall maintenance. However, their precise function is still unknown. This study addresses the occurrence as well as sequence-based characteristics of the LytR-CpsA-Psr proteins. RESULTS: A comprehensive list of LytR-CpsA-Psr proteins was established, and their phylogenetic distribution and clustering into subgroups was determined. LytR-CpsA-Psr proteins were present in all Gram-positive organisms, except for the cell wall-deficient Mollicutes and one strain of the Clostridiales. In contrast, the majority of Gram-negatives did not contain LytR-CpsA-Psr family members. Despite high sequence divergence, the LytR-CpsA-Psr domains of different subclusters shared a highly similar, predicted mixed alpha/beta-structure, and conserved charged residues. PhoA fusion experiments, using MsrR of Staphylococcus aureus, confirmed membrane topology predictions and extracellular location of its LytR-CpsA-Psr domain. CONCLUSIONS: The LytR-CpsA-Psr domain is unique to bacteria. The presence of diverse subgroups within the LytR-CpsA-Psr family might indicate functional differences, and could explain variations in phenotypes of respective mutants reported. The identified conserved structural elements and amino acids are likely to be important for the function of the domain and will help to guide future studies of the LytR-CpsA-Psr proteins

Induction kinetics of the Staphylococcus aureus cell wall stress stimulon in response to different cell wall active antibiotics

BACKGROUND: Staphylococcus aureus activates a protective cell wall stress stimulon (CWSS) in response to the inhibition of cell wall synthesis or cell envelope damage caused by several structurally and functionally different antibiotics. CWSS induction is coordinated by the VraSR two-component system, which senses an unknown signal triggered by diverse cell wall active agents. RESULTS: We have constructed a highly sensitive luciferase reporter gene system, using the promoter of sas016 (S. aureus N315), which detects very subtle differences in expression as well as measuring > 4 log-fold changes in CWSS activity, to compare the concentration dependence of CWSS induction kinetics of antibiotics with different cell envelope targets. We compared the effects of subinhibitory up to suprainhibitory concentrations of fosfomycin, D-cycloserine, tunicamycin, bacitracin, flavomycin, vancomycin, teicoplanin, oxacillin, lysostaphin and daptomycin. Induction kinetics were both strongly antibiotic- and concentration-dependent. Most antibiotics triggered an immediate response with induction beginning within 10 min, except for tunicamycin, D-cycloserine and fosfomycin which showed lags of up to one generation before induction commenced. Induction characteristics, such as the rate of CWSS induction once initiated and maximal induction reached, were strongly antibiotic dependent. We observed a clear correlation between the inhibitory effects of specific antibiotic concentrations on growth and corresponding increases in CWSS induction kinetics. Inactivation of VraR increased susceptibility to the antibiotics tested from 2- to 16-fold, with the exceptions of oxacillin and D-cycloserine, where no differences were detected in the methicillin susceptible S. aureus strain background analysed. There was no apparent correlation between the induction capacity of the various antibiotics and the relative importance of the CWSS for the corresponding resistance phenotypes. CONCLUSION: CWSS induction profiles were unique for each antibiotic. Differences observed in optimal induction conditions for specific antibiotics should be determined and taken into account when designing and interpreting CWSS induction studies

Staphylococcus aureus DsbA is a membrane-bound lipoprotein with thiol-disulfide oxidoreductase activity

DsbA proteins, the primary catalysts of protein disulfide bond formation, are known to affect virulence and penicillin resistance in Gram-negative bacteria. We identified a putative DsbA homologue in the Gram-positive pathogen Staphylococcus aureus that was able to restore the motility phenotype of an Escherichia coli dsbA mutant and thus demonstrated a functional thiol oxidoreductase activity. The staphylococcal DsbA (SaDsbA) had a strong oxidative redox potential of −131mV. The persistence of the protein throughout the growth cycle despite its predominant transcription during exponential growth phase suggested a rather long half-life for the SaDsbA. SaDsbA was found to be a membrane localised lipoprotein, supporting a role in disulfide bond formation. But so far, neither in vitro nor in vivo phenotype could be identified in a staphylococcal dsbA mutant, leaving its physiological role unknown. The inability of SaDsbA to interact with the E. coli DsbB and the lack of an apparent staphylococcal DsbB homologue suggest an alternative re-oxidation pathway for the SaDsb

Contribution of SecDF to Staphylococcus aureus resistance and expression of virulence factors

Background SecDF is an accessory factor of the conserved Sec protein translocation machinery and belongs to the resistance-nodulation-cell division (RND) family of multidrug exporters. SecDF has been shown in Escherichia coli and Bacillus subtilis to be involved in the export of proteins. RND proteins can mediate resistance against various substances and might be of relevance in antimicrobial therapy. The role of RND proteins in Staphylococcus aureus has not yet been determined. Results Markerless deletion mutants were constructed to analyze the impact of the so far uncharacterized RND proteins in S. aureus. While the lack of Sa2056 and Sa2339 caused no phenotype regarding growth and resistance, the secDF mutant resulted in a pleiotropic phenotype. The secDF mutant was cold sensitive, but grew normally in rich medium at 37°C. Resistance to beta-lactams, glycopeptides and the RND substrates acriflavine, ethidium bromide and sodium dodecyl sulfate was reduced. The secDF mutant showed an aberrant cell separation and increased spontaneous and Triton X-100 induced autolysis, although the amounts of penicillin-binding proteins in the membrane were unchanged. The impact of secDF deletion on transcription and expression of specific virulence determinants varied: While coagulase transcription and activity were reduced, the opposite was observed for the autolysin Atl. A reduction of the transcription of the cell wall anchored protein A (spa) was also found. The accumulation of SpA in the membrane and lowered amounts in the cell wall pointed to an impaired translocation. Conclusions The combination of different effects of secDF deletion on transcription, regulation and translocation lead to impaired cell division, reduced resistance and altered expression of virulence determinants suggesting SecDF to be of major relevance in S. aureus. Thus SecDF could be a potential target for the control and eradication of S. aureus in the future

Identification of three additional femAB-like open reading frames in Staphylococcus aureus

Three new proteins, FmhA, FmhB and FmhC, with significant identities to FemA and FemB were identified in the Staphylococcus aureus (ATCC 55748) genome database. They were mapped to the SmaI-C, SmaI-H and SmaI-A fragments of the S. aureus 8325 chromosome, respectively. Whereas insertional inactivation of fmhA and fmhC had no effects on growth, antibiotic susceptibility, lysostaphin resistance, or peptidoglycan composition of the strains, fmhB could not be inactivated, strongly suggesting that fmhB may be an essential gene. As deduced from the functions of FemA and FemB which are involved in the synthesis of the peptidoglycan pentaglycine interpeptide, FmhB may be a candidate for the postulated FemX thought to add the first glycine to the nascent interpeptid

Evaluation of Fourier transform infrared spectroscopy for the rapid identification of glycopeptide-intermediate Staphylococcus aureus

Objectives To evaluate Fourier transform infrared (FTIR) spectroscopy as a rapid method for distinguishing glycopeptide-intermediate Staphylococcus aureus (GISA) from glycopeptide-susceptible methicillin-resistant S. aureus (MRSA) and to compare three data analysis methods. Methods First-derivative normalized spectra of dried films of bacterial growth on Que-Bact® Universal Medium No. 2 were examined by singular value decomposition to identify key spectral regions. Region selection was analysed by principal component analysis (PCA), self-organizing maps (SOMs) and the K-nearest neighbour (KNN) algorithm. The initial data set included 35 GISA (including GISA Mu50 and heterogeneous GISA Mu3) and 25 epidemic MRSA. The regions were then tested using enlarged data sets that included 22 sporadic and 85 additional epidemic MRSA. Results Epidemic MRSA and GISA/hGISA were separated into two distinct clusters on the basis of spectral data from regions 1352-1315 and 1480-1460 cm−1, the former providing 100% correct classification by all three analyses and the latter providing 96.67% correct by PCA, 98.34% by SOM and 100% by KNN. The 1480-1460 cm−1 region was more effective for distinguishing GISA/hGISA from a set combining sporadic and epidemic MRSA, with two GISA/hGISA and four sporadic MRSA misclassified by PCA and SOM (92.69% correct), while the KNN method misclassified three of the four sporadic MRSA (93.90% correct). The addition of 85 other epidemic MRSA this set increased the fraction of correctly classified isolates to 96.41% and 97.01% by PCA, SOM and KNN, respectively. Conclusions As only 6 of 167 isolates were misclassified, FTIR spectroscopy may provide means of rapid and accurate identification of GISA and hGISA among isolates of MRS

Effect of a glucose impulse on the CcpA regulon in Staphylococcus aureus

BACKGROUND: The 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. RESULTS: The 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. CONCLUSION: This 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

Vancomycin-induced deletion of the methicillin resistance gene mecA in Staphylococcus aureus

Objective: To elucidate factors that contribute to the development of vancomycin resistance in methicillin-resistant Staphylococcus aureus (MRSA). Methods: Forty-nine MRSA isolates were subjected to passage selection with vancomycin to isolate mutants with reduced susceptibility to vancomycin. One mutant was chosen for detailed molecular and biochemical characterization. Results: Five vancomycin-resistant mutants (vancomycin MICs, 6-12 mg/L) were obtained in vitro from five MRSA parent isolates. Upon acquisition of vancomycin resistance, all mutants showed a concomitant decrease in oxacillin resistance. In one particular MRSA strain, selection for vancomycin resistance repeatedly produced deletions and rearrangements, including loss of the mecA gene. Pleiotropic phenotypical changes, such as yellow pigment formation, loss of haemolysis, thickened cell wall, increased resistance to lysostaphin and reduced cell wall turnover were observed in this mutant. Conclusion: Acquisition of vancomycin resistance in one MRSA strain triggered mecA deletion suggesting that this deletion, coupled to other rearrangements and/or mutations, may be responsible for the increased vancomycin resistance phenotyp

MsrR contributes to cell surface characteristics and virulence in Staphylococcus aureus

MsrR, a factor contributing to methicillin resistance in Staphylococcus aureus, belongs to the LytR-CpsA-Psr family of cell envelope-associated proteins. Deletion of msrR increased cell size and aggregation, and altered envelope properties, leading to a temporary reduction in cell surface hydrophobicity, diminished colony-spreading ability, and an increased susceptibility to Congo red. The reduced phosphorus content of purified cell walls of the msrR mutant suggested a reduction in wall teichoic acids, which may explain some of the observed phenotypes. Microarray analysis of the msrR deletion mutant revealed only minor changes in the global transcriptome, suggesting that MsrR has structural rather than regulatory functions. Importantly, virulence of the msrR mutant was decreased in a nematode-killing assay as well as in rat experimental endocarditis. MsrR is therefore likely to play a role in cell envelope maintenance, cell separation, and pathogenicity of S. aureu