In most bacteria, nutrient limitations provoke the stringent control through the rapid synthesis of the alarmones pppGpp and ppGpp. Little is known about the stringent control in the human pathogen Staphylococcus aureus, partly due to the essentiality of the major (p)ppGpp synthase/hydrolase enzyme RSH (RelA/SpoT homolog). Here, we show that mutants defective only in the synthase domain of RSH (rsh syn ) are not impaired in growth under nutrient-rich conditions. However, these mutants were more sensitive toward mupirocin and were impaired in survival when essential amino acids were depleted from the medium. RSH is the major enzyme responsible for (p)ppGpp synthesis in response to amino acid deprivation (lack of Leu/Val) or mupirocin treatment. Transcriptional analysis showed that the RSH-dependent stringent control in S. aureus is characterized by repression of genes whose products are predicted to be involved in the translation machinery and by upregulation of genes coding for enzymes involved in amino acid metabolism and transport which are controlled by the repressor CodY. Amino acid starvation also provoked stabilization of the RNAs coding for major virulence regulators, such as SaeRS and SarA, independently of RSH. In an animal model, the rsh syn mutant was shown to be less virulent than the wild type. Virulence could be restored by the introduction of a codY mutation into the rsh syn mutant. These results indicate that stringent conditions are present during infection and that RSH-dependent derepression of CodY-regulated genes is essential for virulence in S. aureus.

The human pathogen Staphylococcus aureus successfully colonizes its primary reservoir, the nasal cavity, most likely by regulatory adaptation to the nose environment. Cotton rats represent an excellent model for the study of bacterial gene expression in the initial phases of colonization. To gain insight into the expression profile necessary for the establishment of colonization, we performed direct transcript analysis by quantitative real-time reverse-transcription polymerase chain reaction on cotton rat noses removed from euthanized animals on days 1, 4, or 10 after instillation of 2 human S. aureus nose isolates. Global virulence regulators (agr, sae) were not active in this early phase, but the essential 2-component regulatory system WalKR seems to play an important role. Accordingly, an elevated expression of walKR target genes (sak, sceD) could be detected. In agreement with previous studies that demonstrated the essential role played by wall teichoic acid (WTA) polymers in nasal colonization, we detected a strongly increased expression of WTA-biosynthetic genes. The expression profile switched to production of the adhesive proteins ClfB and IsdA at later stages of the colonization process. These data underscore the temporal differences in the roles of WTA and surface proteins in nasal colonization, and they provide the first evidence for a regulation of WTA biosynthesis in vivo.

The Yersinia adhesin A (YadA) is a trimeric autotransporter adhesin of enteric yersiniae. It consists of three major domains: a head mediating adherence to host cells, a stalk involved in serum resistance, and an anchor that forms a membrane pore and is responsible for the autotransport function. The anchor contains a glycine residue, nearly invariant throughout trimeric autotransporter adhesins, that faces the pore lumen. To address the role of this glycine, we replaced it with polar amino acids of increasing side chain size and expressed wild-type and mutant YadA in Escherichia coli. The mutations did not impair the YadA-mediated adhesion to collagen and to host cells or the host cell cytokine production, but they decreased the expression levels and stability of YadA trimers with increasing side chain size. Likewise, autoagglutination and resistance to serum were decreased in these mutants. We found that the periplasmic protease DegP is involved in the degradation of YadA and that in an E. coli degP deletion strain, mutant versions of YadA were expressed almost to wild-type levels. We conclude that the conserved glycine residue affects both the export and the stability of YadA and consequently some of its putative functions in pathogenesis.

Yersinia enterocolitica (Ye) evades the immune system of the host by injection of Yersinia outer proteins (Yops) via a type three secretion system into host cells. In this study, a reporter system comprising a YopE-β-lactamase hybrid protein and a fluorescent staining sensitive to β-lactamase cleavage was used to track Yop injection in cell culture and in an experimental Ye mouse infection model. Experiments with GD25, GD25-β1A, and HeLa cells demonstrated that β1-integrins and RhoGTPases play a role for Yop injection. As demonstrated by infection of splenocyte suspensions in vitro, injection of Yops appears to occur randomly into all types of leukocytes. In contrast, upon infection of mice, Yop injection was detected in 13% of F4/80+, 11% of CD11c+, 7% of CD49b+, 5% of Gr1+ cells, 2.3% of CD19+, and 2.6% of CD3+ cells. Taking the different abundance of these cell types in the spleen into account, the highest total number of Yop-injected cells represents B cells, particularly CD19+CD21+CD23+ follicular B cells, followed by neutrophils, dendritic cells, and macrophages, suggesting a distinct cellular tropism of Ye. Yop-injected B cells displayed a significantly increased expression of CD69 compared to non-Yop-injected B cells, indicating activation of these cells by Ye. Infection of IFN-γR (receptor)- and TNFRp55-deficient mice resulted in increased numbers of Yop-injected spleen cells for yet unknown reasons. The YopE-β-lactamase hybrid protein reporter system provides new insights into the modulation of host cell and immune responses by Ye Yops.

Certain strains ofClostridium difficile is the causative pathogen of antibioticassociated diarrhea and pseudomembranous colitis (10, 14). It produces two major protein toxins (toxins A and B), which are the prototypes of the family of large clostridial cytotoxins. Both toxins inhibit the functions of Rho GTPases by monoglucosylation. Beside toxins A and B, some strains of C. difficile produce a binary ADP-ribosylating toxin (CDT) which modifies actin (22). The pathogenic role of CDT in diseases induced by C. difficile is not clear, but about 6 to 12.5% of strains isolated from patients with enteritis contained CDT genes (20,26).The binary CDT is composed of the enzymatic component, CDTa (48 kDa), and the binding and translocation component, CDTb (94 kDa), which mediates cell entry of CDTa (19). CDT belongs to the group of binary actin ADP-ribosylating toxins (4), which can be divided into C2 toxin and iota toxin subfamilies (2, 5, 21). The subfamilies differ with respect to their actin substrate specificities (24). Clostridium botulinum C2 toxin ADP-ribosylates only ␤/␥-nonmuscle actin and ␥-smooth muscle actin, whereas iota-like toxins ADP-ribosylate all actin isoforms, including ␣-actin (16). Furthermore, whereas the binding components of the iota toxin subfamily, including those of iota toxin, Clostridium spiroforme toxin, and CDTa, are interchangeable, no functional complementation between the binding components and the enzymatic components of C2 toxin and the toxins of the iota subfamily was observed (12, 21).Here we studied the enzyme component of CDT and characterized its enzyme activity. We show that the N-terminal part of CDTa is responsible for interaction with the binding component, whereas the C-terminal part harbors transferase activity. For delivery of CDTa into cells we used the Ib binding and translocation component from iota toxin, because CDTb was not expressed as a recombinant protein in Escherichia coli. Recently, we characterized the catalytic center of the ADPribosyltransferase C2I and identified several amino acid residues essential for the transferase activity (8). Here we studied the functional roles of several amino acid residues of CDTa suggested to be conserved among the actin ADP-ribosylating toxins. Moreover, we compared the minimal structural requirements of CDTa with those of the enzyme components of iota toxin (Ia) and C2 toxin (C2I).

Hypersensitivity to nickel (Ni) represents the most common manifestation of contact allergy in humans. The role of metal-specific T cells in this disease is well established, but the molecular interactions involved in their activation are poorly understood. We examined the T cell receptor (TCR) repertoire in T cells activated with either NiSO4 or NiSO4-treated human serum albumin from six allergic patients. For the three most hyperreactive donors, we found a strong over-representation of the TCR BV17 element. TCR sequencing for one of these donors revealed an additional skewing for AV1 as well as a selection for an N region encoded argine at position 95 of the BV17 complementarity determining region (CDR)3. Since Arg is not known to participate in Ni complexing, we suppose that this selection is driven by contacts with peptide rather than nickel. However, the CDR1 of BV17 contains a unique combination of amino acids (HDA) that bears similarities to known motifs in Ni-binding proteins or peptides. We therefore propose that the severe hypersensitivity reactions found in BV17 over-expressors may be the result of Ni2+ ions bridging the germ-line-encoded BV17 CDR1 loop to corresponding sites in the major histocompatibility complex/peptide complex and thereby creating a superantigen-like enhancement of weak TCR-peptide contacts.

Clostridium botulinum C2 toxin belongs to the family of binary actin-ADP-ribosylating toxins. Further members of this toxin family are iota toxin from Clostridium perfringens (1), the Clostridium spiroforme toxin (2), the Clostridium difficile ADPribosyltransferase (3), and the recently crystallized vegetative insecticidal protein (VIP) 1 from Bacillus cereus (4). These toxins are composed of two separated proteins, a binding/translocation component and an enzyme component, which must assemble on the surface of target cells to exhibit cytotoxicity. This mechanism of toxin up-take is also shared by anthrax toxin, whose binding and translocation component protective antigen (PA) is related to the binding components of the actin-ADPribosylating toxins (5). C2 toxin consists of the ADP-ribosylating enzyme component C2I (M r ϳ 50,000) and the binding component C2II (M r ϳ 80,000). For cellular uptake of C2 toxin, trypsin-activated C2IIa forms a heptamer (6) that interacts with the C2I enzyme component and binds to the cellular receptor, a hybrid and/or complex carbohydrate structure (7). Both C2IIa and C2I are internalized via receptor-mediated endocytosis. In an acidic endosomal compartment, the C2IIa oligomers insert in the membrane and form pores (6). Most likely, C2I is translocated through this pore across the endosomal membrane into the cytosol to ADP-ribosylate G-actin at Arg-177 (8). As a consequence, actin polymerization is blocked, and actin filaments disassemble. Site-directed mutagenesis of C2I (9) and of the related ADP-ribosyltransferase iota from C. perfringens (10) indicate that the catalytic site of these enzymes is located in their C-terminal part. This is in line with the recently reported crystal structure and the suggested catalytic mechanism of the ADP-ribosyltransferase VIP2 (4).The N-terminal domain of C2I (amino acid residues 1-225) is enzymatically inactive but interacts with the C2IIa oligomer and mediates cellular uptake of C2I (11). This allows C2I to act as an adaptor for C2IIa-mediated delivery of fusion proteins into cells. In the C2I 1-225 -C3 fusion toxin, C2I 1-225 was used to deliver C3 transferase from Clostridium limosum (M r ϳ 23,000) (11), which inactivates Rho GTPase by ADP-ribosylation at . C3 transferase by its own does not enter cells efficiently because it lacks any specific binding and translocation unit (13). We characterized the C2I/C2IIa contact site in more detail by the use of various truncations of C2I and their fusions with C3 transferase. Here, we report that amino acid residues 1-87 of C2I are sufficient for C2IIa-mediated delivery of C2I fusion toxins into cells. EXPERIMENTAL PROCEDURESMaterials-Cell culture medium was from Biochrom (Berlin, Germany), fetal calf serum was from PAN Systems (Aidenbach, Germany), and cell culture materials were from Falcon (Heidelberg, Germany). The C2II binding component from C. botulinum C2 toxin was purified as recombinant GST fusion protein in Escherichia coli and activated with trypsin as described (6). Antiserum against C2I ...

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