Staphylococcus aureus is an important pathogen, causing a wide range of infections including sepsis, wound infections, pneumonia, and catheter-related infections. In several pathogens ClpP proteases were identified by in vivo expression technologies to be important for virulence. Clp proteolytic complexes are responsible for adaptation to multiple stresses by degrading accumulated and misfolded proteins. In this report clpP, encoding the proteolytic subunit of the ATP-dependent Clp protease, was deleted, and gene expression of ⌬clpP was determined by global transcriptional analysis using DNA-microarray technology. The transcriptional profile reveals a strong regulatory impact of ClpP on the expression of genes encoding proteins that are involved in the pathogenicity of S. aureus and adaptation of the pathogen to several stresses. Expression of the agr system and agr-dependent extracellular virulence factors was diminished. Moreover, the loss of clpP leads to a complete transcriptional derepression of genes of the CtsR-and HrcA-controlled heat shock regulon and a partial derepression of genes involved in oxidative stress response, metal homeostasis, and SOS DNA repair controlled by PerR, Fur, MntR, and LexA. The levels of transcription of genes encoding proteins involved in adaptation to anaerobic conditions potentially regulated by an Fnr-like regulator were decreased. Furthermore, the expression of genes whose products are involved in autolysis was deregulated, leading to enhanced autolysis in the mutant. Our results indicate a strong impact of ClpP proteolytic activity on virulence, stress response, and physiology in S. aureus.The Clp proteases were first identified in Escherichia coli and consist of an ATPase specificity factor (ClpA or ClpX in E. coli; ClpX, ClpC, or ClpE in Bacillus subtilis) and a proteolytic domain (ClpP) that contains a consensus serine protease active site (33). In E. coli, ClpP-mediated proteolysis is regulated by heat shock and removes abnormal proteins that accumulate during stress conditions, recycles amino acids from nonessential proteins during starvation, and contributes to the clearance of truncated peptides from stalled ribosomes by the SsrAtagging system (34, 65). Moreover, it has been shown that Clp proteases play a significant role in certain processes regulating cellular functions via proteolysis (33,36,45). For example, in E. coli ClpXP is involved in degradation of regulator proteins including the alternative sigma factor SigS, the UmuD SOS protein, and different phage proteins (23,31,34 (44,69,73,74). In B. subtilis Clp-specific target proteins were recognized which are involved in peptidoglycan synthesis, competence, sporulation, and heat shock regulation (30,48,50).In addition, several studies indicate that ClpP proteolytic activity is critical for virulence of pathogenic bacteria, including S. enterica serovar Typhimurium, Streptococcus pneumoniae, Listeria monocytogenes, and Staphylococcus aureus (26,29,37,58,63,73,75). Interestingly, ClpP seems to be essential for s...

Carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) are used by several human pathogens to anchor themselves to or invade host cells. Interestingly, human granulocytes express a specific isoform, CEACAM3, that participates together with CEACAM1 and CEACAM6 in the recognition of CEACAM-binding microorganisms. Here we show that CEACAM3 can direct efficient, opsonin-independent phagocytosis of CEACAM-binding Neisseria , Moraxella , and Haemophilus species. CEACAM3-but not CEACAM6-mediated uptake is blocked by dominant-negative versions of the small GTPase Rac. Moreover, CEACAM3 engagement triggers membrane recruitment and increased GTP loading of Rac that are not observed upon bacterial binding to CEACAM6. Internalization and Rac stimulation are also inhibited by compromising the integrity of an immunoreceptor tyrosine-based activation motif (ITAM)-like sequence in the cytoplasmic tail of CEACAM3 or by interference with Src family protein tyrosine kinases that phosphorylate CEACAM3. In contrast to interfering with CEACAM6, blockage of CEACAM3-mediated events reduces the ability of primary human granulocytes to internalize and eliminate CEACAM-binding bacteria, indicating an important role of CEACAM3 in the control of human-specific pathogens by the innate immune system.

Genomes of prokaryotes differ significantly in size and DNA composition. Escherichia coli is considered a model organism to analyze the processes involved in bacterial genome evolution, as the species comprises numerous pathogenic and commensal variants. Pathogenic and nonpathogenic E. coli strains differ in the presence and absence of additional DNA elements contributing to specific virulence traits and also in the presence and absence of additional genetic information. To analyze the genetic diversity of pathogenic and commensal E. coli isolates, a whole-genome approach was applied. Using DNA arrays, the presence of all translatable open reading frames (ORFs) of nonpathogenic E. coli K-12 strain MG1655 was investigated in 26 E. coli isolates, including various extraintestinal and intestinal pathogenic E. coli isolates, 3 pathogenicity island deletion mutants, and commensal and laboratory strains. Additionally, the presence of virulenceassociated genes of E. coli was determined using a DNA "pathoarray" developed in our laboratory. The frequency and distributional pattern of genomic variations vary widely in different E. coli strains. Up to 10% of the E. coli K-12-specific ORFs were not detectable in the genomes of the different strains. DNA sequences described for extraintestinal or intestinal pathogenic E. coli are more frequently detectable in isolates of the same origin than in other pathotypes. Several genes coding for virulence or fitness factors are also present in commensal E. coli isolates. Based on these results, the conserved E. coli core genome is estimated to consist of at least 3,100 translatable ORFs. The absence of K-12-specific ORFs was detectable in all chromosomal regions. These data demonstrate the great genome heterogeneity and genetic diversity among E. coli strains and underline the fact that both the acquisition and deletion of DNA elements are important processes involved in the evolution of prokaryotes.

Nosocomial infections by Staphylococcus aureus, a Gram-positive pathogen colonising human skin and mucosal surfaces, are an increasing health care problem. Clinical isolates almost invariably express fibronectin-binding proteins that, by indirectly linking the bacteria with host integrin α5β1, can promote uptake of the microorganisms by eukaryotic cells. Integrin engagement by pathogenic fibronectin-binding S. aureus, but not by non-pathogenic S. carnosus, triggered the recruitment of focal contact-associated proteins vinculin, tensin, zyxin and FAK to the sites of bacterial attachment. Moreover, dominant-negative versions of FAK-blocked integrin-mediated internalisation and FAK-deficient cells were severely impaired in their ability to internalise S. aureus. Pathogen binding induced tyrosine phosphorylation of several host proteins associated with bacterial attachment sites, including FAK and the Src substrate cortactin. In FAK-deficient cells, local recruitment of cortactin still occurred, whereas the integrin- and Src-dependent tyrosine phosphorylation of cortactin was abolished. As siRNA-mediated gene silencing of cortactin or mutation of critical amino acid residues within cortactin interfered with uptake of S. aureus, our results reveal a novel functional connection between integrin engagement, FAK activation and Src-mediated cortactin phosphorylation. Cooperation between FAK, Src and cortactin in integrin-mediated internalisation of bacteria also suggests a molecular scenario of how engagement of integrins could be coupled to membrane endocytosis.

Staphylococcus aureus, a common cause of nosocomial infections, is able to invade eukaryotic cells by indirectly engaging ␤ 1 integrin-containing host receptors, whereas non-pathogenic Staphylococcus carnosus is not invasive. Here, we identify intracellular signals involved in integrin-initiated internalization of S. aureus. In particular, the host cell actin cytoskeleton and Src family protein-tyrosine kinases (PTKs) are essential to mediate S. aureus invasion. Src PTKs are activated in response to pathogenic S. aureus, but not S. carnosus. In addition, pharmacological and genetic interference with Src PTK function reduces bacterial internalization. Importantly, Src PTK-deficient cells are resistant to S. aureus invasion, demonstrating the essentiality of host Src PTKs in integrin-mediated uptake of this pathogen.The Gram-positive microorganism Staphylococcus aureus is a common cause of nosocomial infections (1, 2). Introduction of staphylococci is facilitated by indwelling medical devices that provide a surface for colonization and a platform for further dissemination. Due to widespread and often multiple antibiotic resistance of S. aureus, infections with this microbe are increasingly difficult to treat.A prominent characteristic of different S. aureus isolates associated with disease is the expression of extracellular matrix (ECM) 1 -binding proteins that have been collectively termed MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) (3, 4). In particular, the cell wall-attached fibronectin-binding proteins A and B (FnBPA and FnBPB) confer a tight association of the bacteria with the ECM protein fibronectin (Fn) (5, 6). The interaction of FnBPs with fibronectin is mediated by direct protein-protein interactions between multiple Fn-binding domains of the bacterial proteins connecting as extended antiparallel ␤-strands to the fibronectin type 1 modules in the amino-terminal domain of Fn (7). As Fn is a common constituent of the extracellular matrix in different tissues and is also abundant in serum, it is thought that Fn deposition on introduced materials such as bone-implanted metals promotes the attachment of S. aureus on medical devices (8).Eukaryotic cells also possess specific surface receptors that bind to Fn. Most prominently, the integrin ␣ 5 ␤ 1 serves as a Fn receptor on multiple cell types. Integrin ␣ 5 ␤ 1 recognizes a short peptide motif, the Arg-Gly-Asp (RGD) sequence, found within one of the type III repeats of Fn. As staphylococcal FnBP associates with the N terminus of Fn independently of the RGD sequence (7, 9), simultaneous association of both S. aureus and human cells with Fn should be possible. Indeed, it has been shown recently that Fn acts as a molecular bridge, linking FnBP-expressing S. aureus with integrin ␣ 5 ␤ 1 on the surface of human cells (for review see Ref. 10). This interaction not only tightly anchors S. aureus to its eukaryotic host cell, but also promotes the internalization of the microorganisms by human epithelial and endothelial cells (11-13) ...