Temperate bacteriophages play an important role in the pathogenicity of Staphylococcus aureus, for instance, by mediating the horizontal gene transfer of virulence factors. Here we established a classification scheme for staphylococcal prophages of the major Siphoviridae family based on integrase gene polymorphism. Seventy-one published genome sequences of staphylococcal phages were clustered into distinct integrase groups which were related to the chromosomal integration site and to the encoded virulence gene content. Analysis of three marker modules (lysogeny, tail, and lysis) for phage functional units revealed that these phages exhibit different degrees of genome mosaicism. The prevalence of prophages in a representative S. aureus strain collection consisting of 386 isolates of diverse origin was determined. By linking the phage content to dominant S. aureus clonal complexes we could show that the distribution of bacteriophages varied remarkably between lineages, indicating restriction-based barriers. A comparison of colonizing and invasive S. aureus strain populations revealed that hlb-converting phages were significantly more frequent in colonizing strains.Staphylococcus aureus asymptomatically colonizes the anterior nares of humans but also causes a wide spectrum of acute and chronic diseases. Most of the dissimilarity between S. aureus strains is due to the presence of mobile genetic elements such as plasmids, bacteriophages, pathogenicity islands, transposons, and insertion sequences (2,14,19,23). Many virulence factors are encoded on such mobile elements (3,6,17,26,27,35). In particular, bacteriophages play an important role in the pathogenicity of S. aureus either by carrying accessory virulence factors such as Panton-Valentine leukocidin (PVL) (encoded by the luk-PV operon), staphylokinase (encoded by sak), enterotoxin A (encoded by sea), and exfoliative toxin A (encoded by eta) or by interrupting chromosomal virulence genes such as those for ␤-hemolysin (hlb) and lipase (geh) upon insertion. Additionally, phages are the primary vehicle of lateral gene transfer between S. aureus strains, providing the species with the potential for broad genetic variation. We could show that phages increase the genome plasticity of S. aureus during infection, facilitating the adaptation of the pathogen to various host conditions (11,12).Despite the obvious importance of phages for the biology of S. aureus, epidemiological data on the prevalence of phages in this species are limited (28, 33). More than 80 genome sequences of staphylococcal bacteriophages and prophages are available in the public genome databases. Most published S. aureus phages belong to the Siphoviridae family of temperate, tailed bacterial viruses. Traditionally, S. aureus phages were characterized according to their lytic activity, morphology, and serological properties (1, 28). Today, the temperate phages in clinical S. aureus isolates can by identified with a multiplex PCR strategy, which is based on sequence differences between viral genes codin...

No abstract

Our data suggest that XLA might result in part from genetic inflammasome deficiency and that NLRP3 inflammasome-linked inflammation could potentially be targeted pharmacologically through BTK.

The incidence of vancomycin-resistant Enterococcus faecium isolation was low (

Although Staphylococcus aureus is not a classical intracellular pathogen, it can survive within phagocytes and many other cell types. However, the pathogen is also able to escape from cells by mechanisms that are only partially understood. We analysed a series of isogenic S. aureus mutants of the USA300 derivative JE2 for their capacity to destroy human macrophages from within. Intracellular S. aureus JE2 caused severe cell damage in human macrophages and could efficiently escape from within the cells. To obtain this full escape phenotype including an intermittent residency in the cytoplasm, the combined action of the regulatory systems Sae and Agr is required. Mutants in Sae or mutants deficient in the Sae target genes lukAB and pvl remained in high numbers within the macrophages causing reduced cell damage. Mutants in the regulatory system Agr or in the Agr target gene psmα were largely similar to wild-type bacteria concerning cell damage and escape efficiency. However, these strains were rarely detectable in the cytoplasm, emphasizing the role of phenol-soluble modulins (PSMs) for phagosomal escape. Thus, Sae-regulated toxins largely determine damage and escape from within macrophages, whereas PSMs are mainly responsible for the escape from the phagosome into the cytoplasm. Damage of macrophages induced by intracellular bacteria was linked neither to activation of apoptosis-related caspase 3, 7 or 8 nor to NLRP3-dependent inflammasome activation.

Escherichia coli hemolysin (HlyA) is a membrane-permeabilizing protein belonging to the family of RTX-toxins. Lytic activity depends on binding of Ca 21 to the C-terminus of the molecule. The N-terminus of HlyA harbors hydrophobic sequences that are believed to constitute the membrane-inserting domain. In this study, 13 HlyA cysteine-replacement mutants were constructed and labeled with the polarity-sensitive fluorescent probe 6-bromoacetyl-2-dimethylaminonaphthalene (badan). The fluorescence emission of the label was examined in soluble and membrane-bound toxin. Binding effected a major blue shift in the emission of six residues within the N-terminal hydrophobic domain, indicating insertion of this domain into the lipid bilayer. The emission shifts occurred both in the presence and absence of Ca 21 , suggesting that Ca 21 is not required for the toxin to enter membranes. However, binding of Ca 21 to HlyA in solution effected conformational changes in both the C-terminal and N-terminal domain that paralleled activation. Our data indicate that binding of Ca 21 to the toxin in solution effects a conformational change that is relayed to the N-terminal domain, rendering it capable of adopting the structure of a functional pore upon membrane binding.Keywords: badan; calcium; conformation; Escherichia coli hemolysin, membrane.Escherichia coli hemolysin (HlyA), a 107-kDa protein of 1024 amino acids devoid of cysteine [1], belongs to the family of RTX-toxins [2] that are produced by many Gramnegative organisms. To obtain pore-forming activity, HlyA requires post-translational acylation of two lysine residues, Lys564 and Lys690 [3,4]. The protein harbors a hydrophobic domain between residues 177 and 411, which according to predictions of secondary structure [5] largely assumes helical conformation and might be involved in membrane penetration. The C-terminal half of the molecule contains 12 repeats of the consensus nonapeptide X-Leu-XGly-Gly-X-X-Gly-Asp-Asp-Asp. This repeat sequence spanning residues 739±849 represents a Ca 21 -binding domain and is essential for function [6±9]. In the presence of Ca 21 , HlyA forms pores of < 2 nm diameter in target membranes [10]. Several approaches to study structurefunction analysis of HlyA have been undertaken in the past. CD spectroscopy was used to investigate the effect of Ca 21 on HlyA, but no significant changes in secondary structure could be detected with this method [11]. On the other hand, results obtained from intrinsic fluorescence measurements and trypsin digestion were compatible with a Ca 21 -induced change in conformation [11]. Moayeri and Welch [12] studied the altered accessibility of toxin domains for monoclonal antibodies after the binding of HlyA to membranes. They reported that the far N-terminus and the region between residues 594 and 640 remain accessible in the membrane-bound toxin. No conclusions could be made for the putative transmembrane region and the C-terminal domain, as antibodies for these regions were not available.An important step toward unders...

Geographic variation in the risk of SSTIs in travelers supports a globally heterogeneous distribution of virulent S. aureus. Complicated SSTIs in returnees from nontemperate climates are associated with PVL(+) S. aureus and promote the emergence and spread of virulent and antibiotic-resistant strains. We propose a network for the surveillance of imported S. aureus (www.staphtrav.eu).

Bartonella quintana causes trench fever, endocarditis, and the vasculoproliferative disorders bacillary angiomatosis and peliosis hepatis in humans. Little is known about the interaction of this pathogen with host cells. We attempted to elucidate the interaction of B. quintana with human macrophages (THP-1) and epithelial cells (HeLa 229). Remarkably, only B. quintana strain JK-31 induced secretion of vascular endothelial growth factor (VEGF) from THP-1 and HeLa 229 cells upon infection similar to the secretion induced by B. henselae Marseille, whereas other strains (B. quintana 2-D70, B. quintana Toulouse, and B. quintana Munich) did not induce such secretion. Immunofluorescence testing and electron microscopy revealed that the B. quintana strains unable to induce VEGF secretion did not express the variable outer membrane proteins (Vomps) on their surfaces. Surprisingly, the increase in VEGF secretion mediated by B. quintana JK-31 was not paralleled by elevated host cell adherence rates compared with the rates for Vomp-negative B. quintana strains. Our results suggest that the Vomps play a leading role in the angiogenic reprogramming of host cells by B. quintana but not in the adherence to host cells.Bartonella quintana and Bartonella henselae are reemerging, gram-negative, facultative intracellular bacteria which cause a variety of human diseases. B. henselae is the most common cause of cat scratch disease. In immunocompromised patients (e.g., AIDS patients), both B. henselae and the closely related species B. quintana (1) cause the vasculoproliferative disorders bacillary angiomatosis (BA) and peliosis hepatis (PH) (20). Infections with B. quintana can result in "trench fever," which is characterized by periodic feverish relapses every fifth day due to intraerythrocytic bacteremia (12). B. quintana was most important during World War I, when more than one million soldiers suffered from this disease. The confirmed transmission vector is the human body louse (33). Today, B. quintana is a well-recognized cause of fever, bacteremia, and endocarditis in human immunodeficiency virus-seronegative, inner-city patients living under poor hygienic conditions, such as the homeless or alcoholics (5, 13, 43). Prolonged periods of intracellular erythrocyte parasitism appear to be a crucial aspect of the pathogenicity of Bartonella spp. (21, 38).Very little is known about the pathogenicity of B. quintana. It has been shown that Bartonella spp. replicate within endothelial cells in a Bartonella-containing vacuole (6), adhere to endothelial and epithelial cells (3, 9, 17), and invade erythrocytes (36, 39). There is also evidence that B. quintana might interact with human erythroblast cells (37). Contradictory results about the induction of apoptosis by B. quintana have been obtained; while early during B. quintana infection apoptosis of endothelial cells was detectable, this apoptosis was inhibited at later times (26). Recently, variably expressed outer membrane proteins VompA, VompB, VompC, and VompD mediating collagen bindin...