Search

Staphylococcus aureus reacts to changing environmental conditions such as heat, pH, and chemicals through global regulators such as the sae (S. aureus exoprotein expression) two-component signaling system. Subin-hibitory concentrations of some antibiotics were shown to increase virulence factor expression. Here, we investigated the S. aureus stress response to sublethal concentrations of a commonly used biocide (Perform), by real-time quantitative PCR (qRT-PCR), promoter activity assay, sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and a flow cytometric invasion assay. Perform, acting through the production of reactive oxygen species, generally downregulated expression of extracellular proteins in strains 6850, COL, ISP479C but upregulated these proteins in strain Newman. Upregulated proteins were sae dependent. The Perform component SDS, but not paraquat (another oxygen donor), mimicked the biocide effect. Eap (extracellular adherence protein) was most prominently augmented. Upregulation of eap and sae was confirmed by qRT-PCR. Promoter activity of sae P1 was increased by Perform and SDS. Both substances enhanced cellular invasiveness, by 2.5-fold and 3.2-fold, respectively. Increased invasiveness was dependent on Eap and the sae system, whereas agr, sarA, sigB, and fibronectin-binding proteins had no major effect in strain Newman. This unique response pattern was due to a point mutation in SaeS (the sensor histidine kinase), as demonstrated by allele swapping. Newman [saeeQRS.sub.ISP479C] behaved like ISP479C, whereas [saePQRS.sub.Newman] rendered ISP479C equally responsive as Newman. Taken together, the findings indicate that a point mutation in SaeS of strain Newman was responsible for increased expression of Eap upon exposure to sublethal Perform and SDS concentrations, leading to increased Eap-dependent cellular invasiveness. This may be important for understanding the regulation of virulence in S. aureus. doi: 10.1128/JB.00630-09

Copyright of Angewandte Chemie is the property of Wiley-Blackwell and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

The rise of antimicrobial resistance poses a severe threat to public health. The natural product chlorotonil was identified as a new antibiotic targeting multidrug resistant Gram‐positive pathogens and Plasmodium falciparum. Although chlorotonil shows promising activities, the scaffold is highly lipophilic and displays potential biological instabilities. Therefore, we strived towards improving its pharmaceutical properties by semisynthesis. We demonstrated stereoselective epoxidation of chlorotonils and epoxide ring opening in moderate to good yields providing derivatives with significantly enhanced solubility. Furthermore, in vivo stability of the derivatives was improved while retaining their nanomolar activity against critical human pathogens (e.g. methicillin‐resistant Staphylococcus aureus and P. falciparum). Intriguingly, we showed further superb activity for the frontrunner molecule in a mouse model of S. aureus infection. [ABSTRACT FROM AUTHOR]

The alternative sigma factor [[delta].sup.B] of Staphylococcus aureus controls the expression of a variety of genes, including virulence determinants and global regulators. Genetic manipulations and transcriptional start point (TSP) analyses showed that the sigB operon is transcribed from at least two differentially controlled promoters: a putative [[delta].sup.A]-dependent promoter, termed sig[B.sub.p1], giving rise to a 3.6-kb transcript covering sa2059-sa2058-rsbU-rsbV-rsbW-sigB, and a [[delta].sup.B]-dependent promoter, sig[B.sub.p3], initiating a 1.6-kb transcript covering rsbV-rsbW-sigB. TSP and promoter-reporter gene fusion experiments indicated that a third promoter, tentatively termed sig[B.sub.p2] and proposed to lead to a 2.5-kb transcript, including rsbU-rsbV-rsbW-sigB, might govern the expression of the sigB operon. Environmental stresses, such as heat shock and salt stress, induced a rapid response within minutes from promoters sig[B.sub.p1] andsig[B.sub.p3]. In vitro, the sig[B.sub.p1] promoter was active in the early growth stages, while the sig[B.sub.p2] andsig[B.sub.p3] promoters produced transcripts throughout the growth cycle, with sig[B.sub.p3] peaking around the transition state between exponential growth and stationary phase. The amount of sigB transcripts, however, did not reflect the concentration of [[delta].sup.B] measured in cell extracts, which remained constant over the entire growth cycle. In a guinea pig cage model of infection, sigB transcripts were as abundant 2 and 8 days postinoculation as values found in vitro, demonstrating that sigB is indeed transcribed during the course of infection. Physical interactions between staphylococcal RsbU-RsbV, RsbV-RsbW, and RsbW-[[delta].sup.B] were inferred from a yeast (Saccharomyces cerevisiae) two-hybrid approach, indicating the presence of a partner-switching mechanism in the [[delta].sup.B] activation cascade similar to that of Bacillus subtilis. The finding that overexpression of RsbU was sufficient to trigger an immediate and strong activation of [[delta].sup.B], however, signals a relevant difference in the regulation of [[delta].sup.B] activation between B. subtilis and S. aureus in the cascade upstream of RsbU.

Inactivation of hemB in Staphylococcus aureus strain Newman resulted in a small-colony phenotype and was accompanied by an altered expression pattern of global regulators and control of virulence factor production. Transcription profiles followed over 15 h by Northern blot analyses revealed that transcripts of the global regulators arl, rot, sae, sarR, sarS, srr, svrA, and sigB disappeared after the exponential phase and that both agr transcripts were completely absent in the hemB mutant. Apart from a general concentration of transcriptional activity to the exponential phase, premature gene expression was observed for rot, hla, and spa. Nevertheless, reported [[sigma].sup.B]-dependent transcripts, such as sarC and clfA, were produced throughout the 15-h growth period monitored. The absence of these transcripts in a hemB sigB double mutant demonstrated their dependence on [[sigma].sup.B] and indicated an unexpected, permanent [[sigma].sup.B] activity in the hemB mutant. Variations in the extents of the directly [[sigma].sup.B]-controlled asp23, rsbVW-sigB, and sarC transcripts argue for additional factors modulating [[sigma].sup.B] activity. This study provides the first extended synopsis of the transcriptional patterns of different regulators over the entire growth cycle in the widely used Newman strain.

The ability of Staphylococcus aureus to adapt to various conditions of stress is the result of a complex regulatory response. Previously, it has been demonstrated that Clp homologues are important for a variety of stress conditions, and our laboratory has shown that a clpC homologue was highly expressed in the S. aureus strain DSM20231 during biofilm formation relative to expression in planktonic cells. Persistence and long-term survival are a hallmark of biofilm-associated staphylococcal infections, as cure frequently fails even in the presence of bactericidal antimierobials. To determine the role of clpC in this context, we performed metabolic, gene expression, and long-term growth and survival analyses of DSM20231 as well as an isogenic clpC allelic-replacement mutant, a sigB mutant, and a clpC sigB double mutant. As expected, the clpC mutant showed increased sensitivity to oxidative and heat stresses. Unanticipated, however, was the reduced expression of the tricarboxylic acid (TCA) cycle gene citB (encoding aconitase), resulting in the loss of aconitase activity and preventing the eatabolization of acetate during the stationary phase, clpC inactivation abolished post-stationary-phase recovery but also resulted in significantly enhanced stationary-phase survival compared to that of the wild-type strain. These data demonstrate the critical role of the ClpC ATPase in regulating the TCA cycle and implicate ClpC as being important for recovery from the stationary phase and also for entering the death phase. Understanding the stationary- and post-stationary-phase recovery in S. aureus may have important clinical implications, as little is known about the mechanisms of long-term persistence of chronic S. aureus infections associated with formation of biofilms.

Microarray-based analysis of the transcriptional profiles of the genetically distinct Staphylococcus aureus strains COL, GP268, and Newman indicate that a total of 251 open reading frames (ORFs) are influenced by [[sigma].sup.B] activity. While [[sigma].sup.B] was found to positively control 198 genes by a factor of [greater than or equal to] 2 in at least two of the three genetic lineages analyzed, 53 ORFs were repressed in the presence of [[sigma].sup.B]. Gene products that were found to be influenced by [[sigma].sup.B] are 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 [[sigma].sup.B] were preceded by a nucleotide sequence that resembled the [[sigma].sup.B] consensus promoter sequence of Bacillus subtilis. A conspicuous number of virulence-associated genes were identified as regulated by [[sigma].sup.B] activity, 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 [[sigma].sup.B] of S. aureus controls a large regulon and is an important modulator of virulence gene expression that is likely to act conversely to RNAIII, the effector molecule of the agr locus. 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.

TcaR, which shares sequence homology with MarR-like transcriptional regulators, has been identified as a novel Staphylococcus aureus regulator affecting the expression of the global regulatory element SarS (SarH1), as well as that of the cell surface-associated protein SasF (N315-SA2439). Microarray analysis, confirmatory Northern blots, and genetic complementation experiments showed that Tear upregulates sarS and thus spa transcription. In addition, it attenuates whole-length transcription of sasF, thereby producing a truncated transcript lacking the 3' terminus, which codes for the cell wall anchor motif. Hence, in strains containing an intact tcaR gene, TcaR is likely to decrease the amount of the surface-associated protein SasF and to increase that of the surface-associated protein A. The widely used laboratory strains derived from NCTC8325 were found to be natural, truncated mutants of tcaR, harboring an inactive TcaR and therefore expressing very low levels of sarS. The data presented here identified TcaR as a further activator of sarS, and a modulator of sasF expression that has to be taken into account in studies of virulence gene expression in S. aureus.

The Staphylococcus aureus lrg and cid loci are homologous operons that have been shown to regulate murein hydrolase activity and affect sensitivity to penicillin. Although the mode of action of these operons has not been demonstrated, a model based on the similarities of the lrgA and cidA gene products to the bacteriophage holin family of proteins has been proposed. In this study, the transcription organization and regulation of these operons were examined by Northern blot analyses. Unexpectedly, cidB and a gene located immediately downstream, designated cidC, were found to be cotranscribed on a 2.7-kb transcript. Maximal cidBC transcription occurred during early exponential growth, and high-level transcription of cidBC was dependent on the rsbU-mediated activation of the alternative sigma factor B ([[sigma].sup.B]). In contrast, lrgAB transcription in stationary phase was negatively regulated by [[sigma].sup.B]. Although cidABC transcription was not detected by Northern blot analysis, reverse transcriptase PCR revealed that these genes are also cotranscribed as a single RNA message in early exponential growth. Primer extension analysis revealed the presence of two cidBC transcription start sites, but no apparent [[sigma].sup.B]-dependent promoter consensus sequence was identified in these regions. The rsbU gene was also shown to have a positive impact on murein hydrolase activity but a negligible effect on sensitivity to penicillin-induced killing. These results suggest that the lrgAB and cidBC genes may be part of the S. aureus [[sigma].sup.B]-controlled stress regulon.