Staphylococcus aureus is a Gram-positive human pathogen that is readily internalized by professional phagocytes such as macrophages and neutrophils but also by non-professional phagocytes such as epithelial or endothelial cells. Intracellular bacteria have been proposed to play a role in evasion of the innate immune system and may also lead to dissemination within migrating phagocytes. Further, S. aureus efficiently lyses host cells with a battery of cytolytic toxins. Recently, phenol-soluble modulins (PSM) have been identified to comprise a genus-specific family of cytolytic peptides. Of these the PSMα peptides have been implicated in killing polymorphonuclear leucocytes after phagocytosis. We questioned if the peptides were active in destroying endosomal membranes to avoid lysosomal killing of the pathogen and monitored integrity of infected host cell endosomes by measuring the acidity of the intracellular bacterial microenvironment via flow cytometry and by a reporter recruitment technique. Isogenic mutants of the methicillin-resistant S. aureus (MRSA) strains USA300 LAC, USA400 MW2 as well as the strongly cytolytic methicillin-sensitive strain 6850 were compared with their respective wild type strains. In all three genetic backgrounds, PSMα mutants were unable to escape from phagosomes in non-professional (293, HeLa, EAhy.926) and professional phagocytes (THP-1), whereas mutants in PSMβ and δ-toxin as well as β-toxin, phosphatidyl inositol-dependent phospholipase C and Panton Valentine leucotoxin escaped with efficiencies of the parental strains. S. aureus replicated intracellularly only in presence of a functional PSMα operon thereby illustrating that bacteria grow in the host cell cytoplasm upon phagosomal escape.

The Staphylococcus aureus regulatory saePQRS system controls the expression of numerous virulence factors, including extracellular adherence protein (Eap), which amongst others facilitates invasion of host cells. The saePQRS operon codes for 4 proteins: the histidine kinase SaeS, the response regulator SaeR, the lipoprotein SaeP and the transmembrane protein SaeQ. S. aureus strain Newman has a single amino acid substitution in the transmembrane domain of SaeS (L18P) which results in constitutive kinase activity. SDS was shown to be one of the signals interfering with SaeS activity leading to inhibition of the sae target gene eap in strains with SaeSL but causing activation in strains containing SaeSP. Here, we analyzed the possible involvement of the SaeP protein and saePQ region in SDS-mediated sae/eap expression. We found that SaePQ is not needed for SDS-mediated SaeS signaling. Furthermore, we could show that SaeS activity is closely linked to the expression of Eap and the capacity to invade host cells in a number of clinical isolates. This suggests that SaeS activity might be directly modulated by structurally non-complex environmental signals, as SDS, which possibly altering its kinase/phosphatase activity.

PURPOSE: We have demonstrated impaired mitochondrial viability and function during pulmonary ischemia reperfusion injury(IR). Resveratrol(Res) has demonstrated to ameliorate the IR of various organs. This study analyzes the impact of Res-preconditioning on pulmonary IR-induced mitochondrial dysfunction and tissue damage.

ABSTRACTStaphylococcus aureusis a major cause of a variety of both local and systemic infections. It can invade human host cells, a process that may account for disseminated and recurrent infections.S. aureuspostinvasion events in nonprofessional phagocytes are only partially understood. While morphological data suggest a phagosomal escape, there is a lack of corroborating functional data. Using a combination of pH determination and morphological techniques, we have tested the integrity ofStaphylococcus-containing phagosomes in 293 (HEK-293), HeLa, and EA.hy926 cells over time. Rapid acidification ofS. aureus-containing phagosomes occurred and was sustained for up to 24 h. AllS. aureusstrains tested displayed equally sustained intraphagosomal pH levels without exhibiting any correlation with pH level and hemolytic activity. The membrane morphology of the phagosomal compartment was heterogeneous, even under conditions where acidic pH was fully maintained, an observation incompatible with phagolysosomal membrane destruction. As an exception,S. aureusstrain 6850 showed a reduced phagosomal acidification signal 6 h after invasion. Additionally, only strain 6850 failed to localize to LAMP-1-positive vesicles in HeLa cells, although this was observed only rarely. Several other strongly beta-hemolytic strains did not modulate phagolysosomal pH, suggesting thatS. aureusα-toxin and β-toxin are not sufficient for this process. Taken together, our data suggest thatS. aureus-containing phagolysosomes generally remain functionally intact in nonprofessional phagocytes, thereby contrasting with transmission electron micrographic results.

BACKGROUNDThe methicillin-resistant Staphylococcus aureus (MRSA) surface protein Pls (plasmin sensitive) reduces adhesion to host proteins and cellular invasiveness by an unknown mechanism that requires Pls expression. Here, we tested the effect of Pls expression using different pls-negative backgrounds.METHODSThree pls-negative strains (the methicillin-susceptible Staphylococcus aureus strains Cowan I and 6850 and the MRSA strain ST239-635/93, which harbors staphylococcal cassette chromosome [SCC] mec type III) were transformed. Constructs used were full-length pls (pPLS4), pls-DeltaLPDTG (no sortase motif; pPLS5), and pls-DeltaSD (no serine-aspartic acid [SD] repeats; pPLS6). Adherence, invasiveness, gene expression, and surface expression were quantified by photometry, flow cytometry, real-time reverse-transcription polymerase chain reaction, and a modified enzyme-linked immunosorbent assay, respectively.RESULTSIn Pls-expressing strains (those with pPLS4), adherence to immobilized fibronectin (Fn) and binding of soluble Fn was reduced by approximately 20% and approximately 25%, respectively. Invasion of 293 cells and EA.hy 926 cells was reduced by up to 85%. Surprisingly, transcription of fnbA and spa was up-regulated, but transcription of clfA and hla was down-regulated. Pls and Fn-binding protein (FnBP) surface expression was increased. Competition with purified FnBPA, but not with Pls, reduced invasiveness by approximately 90%. The invasiveness of 6850 (pPLS5) and of 6850 (pPLS6) was reduced by only approximately 20% and approximately 15%, respectively.CONCLUSIONExpression of cell wall-anchored Pls reduces adherence and invasiveness independently of the MRSA/SCCmec background. This occurs despite early up-regulation of fnbA transcription and FnBP surface expression. Thus, Pls acts by steric hindrance rather than another mechanism.

ABSTRACT 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 .