A point mutation in the sensor histidine kinase SaeS of Staphylococcus aureus strain Newman alters the response to biocide exposure.

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. Subinhibitory 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 saePQRS(ISP479C) behaved like ISP479C, whereas saePQRS(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.

Proteomics fingerprinting of phagosome maturation and evidence for the role of a Galpha during uptake.

Phagocytosis, whether of food particles in protozoa or bacteria and cell remnants in the metazoan immune system, is a conserved process. The particles are taken up into phagosomes, which then undergo complex remodeling of their components, called maturation. By using two-dimensional gel electrophoresis and mass spectrometry combined with genomic data, we identified 179 phagosomal proteins in the amoeba Dictyostelium, including components of signal transduction, membrane traffic, and the cytoskeleton. By carrying out this proteomics analysis over the course of maturation, we obtained time profiles for 1,388 spots and thus generated a dynamic record of phagosomal protein composition. Clustering of the time profiles revealed five clusters and 24 functional groups that were mapped onto a flow chart of maturation. Two heterotrimeric G protein subunits, Galpha4 and Gbeta, appeared at the earliest times. We showed that mutations in the genes encoding these two proteins produce a phagocytic uptake defect in Dictyostelium. This analysis of phagosome protein dynamics provides a reference point for future genetic and functional investigations.

Identification and regulation of cold-inducible factors of Bordetella bronchiseptica.

The expression of bacterial cold-shock proteins (CSPs) is highly induced in response to cold shock, and some CSPs are essential for cells to resume growth at low temperature. Bordetella bronchiseptica encodes five CSPs (named CspA to CspE) with significant amino acid homology to CspA of Escherichia coli. In contrast to E. coli, the insertional knock-out of a single csp gene (cspB) strongly affected growth of B. bronchiseptica independent of temperature. In the case of three of the csp genes (cspA, cspB, cspC) more than one specific transcript could be detected. The net amount of cspA, cspB and cspC transcripts increased strongly after cold shock, while no such effect could be observed for cspD and cspE. The exposure to other stress conditions, including translation inhibitors, heat shock, osmotic stress and nutrient deprivation in the stationary phase, indicated that the csp genes are also responsive to these conditions. The coding regions of all of the cold-shock genes are preceded by a long non-translated upstream region (5'-UTR). In the case of the cspB gene, a deletion of parts of this region led to a significant reduction of translation of the resulting truncated transcript, indicating a role of the 5'-UTR in translational control. The cold-shock stimulon was investigated by 2D-PAGE and mass spectrometric characterization, leading to the identification of additional cold-inducible proteins (CIPs). Interestingly, two cold-shock genes (cspC and cspD) were found to be under the negative control of the BvgAS system, the main transcriptional regulator of Bordetella virulence genes. Moreover, a negative effect of slight overexpression of CspB, but not of the other CSPs, on the transcription of the adenylate cyclase toxin CyaA of Bordetella pertussis was observed, suggesting cross-talk between the CSP-mediated stress response stimulon and the Bordetella virulence regulon.

Experimental proof for a signal peptidase I like activity in Mycoplasma pneumoniae, but absence of a gene encoding a conserved bacterial type I SPase.

Although the annotation of the complete genome sequence of Mycoplasma pneumoniae did not reveal a bacterial type I signal peptidase (SPase I) we showed experimentally that such an activity must exist in this bacterium, by determining the N-terminus of the N-terminal gene product P40 of MPN142, formerly called ORF6 gene. Combining mass spectrometry with a method for sulfonating specifically the free amino terminal group of proteins, the cleavage site for a typical signal peptide was located between amino acids 25 and 26 of the P40 precursor protein. The experimental results were in agreement with the cleavage site predicted by computational methods providing experimental confirmation for these theoretical analyses.

Differential regulation of multiple proteins of Escherichia coli and Salmonella enterica serovar Typhimurium by the transcriptional regulator SlyA.

SlyA is a transcriptional regulator of Escherichia coli, Salmonella enterica, and other bacteria belonging to the ENTEROBACTERIACEAE: The SlyA protein has been shown to be involved in the virulence of S. enterica serovar Typhimurium, but its role in E. coli is unclear. In this study, we employed the proteome technology to analyze the SlyA regulons of enteroinvasive E. coli (EIEC) and Salmonella serovar Typhimurium. In both cases, comparative analysis of the two-dimensional protein maps of a wild-type strain, a SlyA-overproducing derivative, and a corresponding slyA mutant revealed numerous proteins whose expression appeared to be either positively or negatively controlled by SlyA. Twenty of the putative SlyA-induced proteins and 13 of the putative SlyA-repressed proteins of the tested EIEC strain were identified by mass spectrometry. The former proteins included several molecular chaperones (GroEL, GroES, DnaK, GrpE, and CbpA), proteins involved in acid resistance (HdeA, HdeB, and GadA), the "starvation lipoprotein" (Slp), cytolysin ClyA (HlyE or SheA), and several enzymes involved in metabolic pathways, whereas most of the latter proteins proved to be biosynthetic enzymes. Consistently, the resistance of the EIEC slyA mutant to heat and acid stress was impaired compared to that of the wild-type strain. Furthermore, the implication of SlyA in the regulation of several of the identified E. coli proteins was confirmed at the level of transcription with lacZ fusions. Twenty-three of the Salmonella serovar Typhimurium proteins found to be affected by SlyA were also identified by mass spectrometry. With the exception of GroEL these differed from those identified in the EIEC strain and included proteins involved in various processes. The data suggest that gene regulation by SlyA might be crucial for intracellular survival and/or replication of both EIEC and Salmonella serovar Typhimurium in phagocytic host cells.