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Viruses and intracellular bacterial pathogens (IBPs) have in common the need of suitable host cells for efficient replication and proliferation during infection. In human infections, the cell types which both groups of pathogens are using as hosts are indeed quite similar and include phagocytic immune cells, especially monocytes/macrophages (MOs/MPs) and dendritic cells (DCs), as well as nonprofessional phagocytes, like epithelial cells, fibroblasts and endothelial cells. These terminally differentiated cells are normally in a metabolically quiescent state when they are encountered by these pathogens during infection. This metabolic state of the host cells does not meet the extensive need for nutrients required for efficient intracellular replication of viruses and especially IBPs which, in contrast to the viral pathogens, have to perform their own specific intracellular metabolism to survive and efficiently replicate in their host cell niches. For this goal, viruses and IBPs have to reprogram the host cell metabolism in a pathogen-specific manner to increase the supply of nutrients, energy, and metabolites which have to be provided to the pathogen to allow its replication. In viral infections, this appears to be often achieved by the interaction of specific viral factors with central metabolic regulators, including oncogenes and tumor suppressors, or by the introduction of virus-specific oncogenes. Less is so far known on the mechanisms leading to metabolic reprogramming of the host cell by IBPs. However, the still scant data suggest that similar mechanisms may also determine the reprogramming of the host cell metabolism in IBP infections. In this review, we summarize and compare the present knowledge on this important, yet still poorly understood aspect of pathogenesis of human viral and especially IBP infections.

Objective: Postoperative ileus (POI) is an inflammation-mediated complication of abdominal surgery, characterized by intestinal dysmotility and leukocyte infiltration into the muscularis externa (ME). Previous studies indicated that interleukin (IL)-10 is crucial for the resolution of a variety of inflammation-driven diseases. Herein, we investigated how IL-10 affects the postoperative ME inflammation and found an unforeseen role of IL-10 in POI. Design: POI was induced by a standardized intestinal manipulation (IM) in C57BL/6 and multiple transgenic mouse strain including C-C motif chemokine receptor 2-/-, IL-10-/-, and LysMcre/IL-10fl/fl mice. Leukocyte infiltration, gene and protein expression of cytokines, chemokines, and macrophage differentiation markers as well as intestinal motility were analyzed. IL-10 serum levels in surgical patients were determined by ELISA. Results: IL-10 serum levels were increased in patient after abdominal surgery. In mice, a complete or leucocyte-restricted IL-10 deficiency ameliorated POI and reduced the postoperative ME neutrophil infiltration. Infiltrating monocytes were identified as main IL-10 producers and undergo IL-10-dependent M2 polarization. Interestingly, M2 polarization is not crucial to POI development as abrogation of monocyte infiltration did not prevent POI due to a compensation of the IL-10 loss by resident macrophages and neutrophils. Organ culture studies demonstrated that IL-10 deficiency impeded neutrophil migration toward the surgically traumatized ME. This mechanism is mediated by reduction of neutrophil attracting chemokines. Conclusion: Monocyte-derived macrophages are the major IL-10 source during POI. An IL-10 deficiency decreases the postoperative expression of neutrophil-recruiting chemokines, consequently reduces the neutrophil extravasation into the postsurgical bowel wall, and finally protects mice from POI.

Enteropathogenic Yersinia enterocolitica is able to grow within or outside the mammalian host. Previous transcriptomic studies have indicated that the regulator OmpR plays a role in the expression of hundreds of genes in enterobacteria. Here, we have examined the impact of OmpR on the production of Y. enterocolitica membrane proteins upon changes in temperature, osmolarity and pH. Proteomic analysis indicated that the loss of OmpR affects the production of 120 proteins, a third of which are involved in uptake/transport, including several that participate in iron or heme acquisition. A set of proteins associated with virulence was also affected. The influence of OmpR on the abundance of adhesin YadA and heme receptor HemR was examined in more detail. OmpR was found to repress YadA production and bind to the yadA promoter, suggesting a direct regulatory effect. In contrast, the repression of hemR expression by OmpR appears to be indirect. These findings provide new insights into the role of OmpR in remodelling the cell surface and the adaptation of Y. enterocolitica to different environmental niches, including the host.

Enterohemorrhagic Escherichia coli (EHEC) is the causative agent of bloody diarrhea and extraintestinal sequelae in humans, most importantly hemolytic-uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP). Besides the bacteriophage-encoded Shiga toxin gene ( stx ), EHEC harbors the locus of enterocyte effacement (LEE), which confers the ability to cause attaching and effacing lesions. Currently, the vast majority of EHEC infections are caused by strains belonging to five O serogroups (the “big five”), which, in addition to O157, the most important, comprise O26, O103, O111, and O145. We hypothesize that these four non-O157 EHEC serotypes differ in their phylogenies. To test this hypothesis, we used multilocus sequence typing (MLST) to analyze a large collection of 250 isolates of these four O serogroups, which were isolated from diseased as well as healthy humans and cattle between 1952 and 2009. The majority of the EHEC isolates of O serogroups O26 and O111 clustered into one sequence type complex, STC29. Isolates of O103 clustered mainly in STC20, and most isolates of O145 were found within STC32. In addition to these EHEC strains, STC29 also included stx -negative E. coli strains, termed atypical enteropathogenic E. coli (aEPEC), yet another intestinal pathogenic E. coli group. The finding that aEPEC and EHEC isolates of non-O157 O serogroups share the same phylogeny suggests an ongoing microevolutionary scenario in which the phage-encoded Shiga toxin gene stx is transferred between aEPEC and EHEC. As a consequence, aEPEC strains of STC29 can be regarded as post- or pre-EHEC isolates. Therefore, STC29 incorporates phylogenetic information useful for unraveling the evolution of EHEC.

The Yersinia outer protein M (YopM) is a type 3 secretion system (T3SS)-dependent effector protein of Yersinia enterocolitica , Yersinia pseudotuberculosis and Yersinia pestis . Although YopM is indispensable for full virulence, its molecular functions still remain largely elusive. Recently, we could identify the recombinant YopM (rYopM) protein derived from the Y. enterocolitica strain 8081 (JB580) as a cell-penetrating protein, which down-regulates the expression of various pro-inflammatory cytokines including TNFα. In this study, we have generated rabbit monoclonal anti-YopM antibodies (RabMabs). RabMabs were characterized by SDS–PAGE and Western blotting using various truncated versions of rYopM to identify epitope-containing domains. RabMabs recognizing either the N- or C-terminus of YopM were characterized further and validated using a collection of 61 pathogenic and non-pathogenic Yersinia strains as well as exemplary strains of major intestinal bacterial pathogens such as Salmonella enterica ssp. enterica , Shigella flexneri and intestinal pathogenic Escherichia coli . RabMab 41.3 directed at the N-terminus of YopM of Y. enterocolitica strain 8081 recognized all YopM-expressing pathogenic Yersinia strains analyzed in this study but failed to recognize non-pathogenic isolates. Thus, RabMab 41.3 might be applicable for the detection of pathogenic Yersinia strains.

Antimicrobial peptides are a promising complement to common antibiotics, development of resistance to which is a growing problem. Here we present a de novo-designed peptide, SP1-1 (RKKRLKLLKRLL-NH2), with antimicrobial activity against multiresistant Staphylococcus aureus (minimal inhibitory concentration: 6.25 μM). Elucidation of the mode of action of this peptide revealed a strong interaction with RsbW kinase (Kd: 6.01±2.73 nM), a serine kinase negatively regulating the activity of the transcription factor σB (SigB). SP1-1 binding and functional modulation of RsbW were shown in vitro by a combination of biochemical, molecular, and biophysical methods, which were further genetically evidenced in vivo by analysis of S. aureus ΔsigB deletion mutants. Intracellular localization of the peptide was demonstrated using nanometer-scaled secondary ion mass spectrometry. Moreover, microarray analysis revealed that transcription of numerous genes, involved in cell wall and amino acid metabolism, transport mechanisms, virulence, and pigmentation, is affected. Interestingly, several WalR binding motif containing genes are induced by SP1-1. In sum, the designed peptide SP1-1 seems to have multiple modes of action, including inhibition of a kinase, and therefore might contribute to the development of new antibacterial compounds, giving bacterial kinase inhibition a closer inspection.-Dangel, A., Ackermann, N., Abdel-Hadi, O., Maier, R., Önder, K., Francois, P., Müller, C. W., Pané-Farré, J., Engelmann, S., Schrenzel, J., Heesemann, J., Lindermayr, C. A de novo-designed antimicrobial peptide with activity against multiresistant Staphylococcus aureus acting on RsbW kinase.

In Enterobacteriaceae, the RNA chaperone Hfq mediates the interaction of small RNAs with target mRNAs, thereby modulating transcript stability and translation. This post-transcriptional control helps bacteria adapt quickly to changing environmental conditions. Our previous mutational analysis showed that Hfq is involved in metabolism and stress survival in the enteropathogen Yersinia enterocolitica. In this study we demonstrate that Hfq is essential for virulence in mice and influences production of surface pathogenicity factors, in particular lipopolysaccharide and adhesins mediating interaction with host tissue. Hfq inhibited the production of Ail, the Ail-like protein OmpX and the MyfA pilin post-transcriptionally. In contrast Hfq promoted production of two major autotransporter adhesins YadA and InvA. While protein secretion in vitro was not affected, hfq mutants exhibited decreased protein translocation by the type III secretion system into host cells, consistent with decreased production of YadA and InvA. The influence of Hfq on YadA resulted from a complex interplay of transcriptional, post-transcriptional and likely post-translational effects. Hfq regulated invA by modulating the expression of the transcriptional regulators rovA, phoP and ompR. Therefore, Hfq is a global coordinator of surface virulence determinants in Y. enterocolitica suggesting that it constitutes an attractive target for developing new antimicrobial strategies.

The type 3 secretion system (T3SS) is a powerful bacterial nanomachine that is able to modify the host cellular immune defense in favor of the pathogen by injection of effector proteins. In this regard, cellular Rho GTPases such as Rac1, RhoA or Cdc42 are targeted by a large group of T3SS effectors by mimicking cellular guanine exchange factors or GTPase-activating proteins. However, functional analysis of one type of T3SS effector that is translocated by bacterial pathogens is challenging because the T3SS effector repertoire can comprise a large number of proteins with redundant or interfering functions. Therefore, we developed the Yersinia toolbox to either analyze singular effector proteins of Yersinia spp. or different bacterial species in the context of bacterial T3SS injection into cells. Here, we focus on the WxxxE guanine exchange factor mimetic proteins IpgB1, IpgB2 and Map, which activate Rac1, RhoA or Cdc42, respectively, as well as the Rho GTPase inactivators YopE (a GTPase-activating mimetic protein) and YopT (cysteine protease), to generate a toolbox module for Rho GTPase manipulation.

Autoagglutination (AA) is a protective phenotypic trait facilitating survival of bacteria in hostile environments and in the host during infection. Autoagglutination factors (AFs) that possess self-associating ability are currently characterized in many Gram-negative bacteria, but Yersinia pestis AFs are still a matter of debate. Previously, we have shown that AF of Hms− strain Y. pestis EV76 is a complex of the 17,485-kDa protein and a low-molecular-weight component with siderophore activity. Here, we identified the protein moiety of AF and examined its role in AA of Hms+ and Hms− Y. pestis strains. Using MALDI-TOF MS of trypsin-hydrolyzed AF, we unambiguously identified the protein as YPO0502, which belongs to a family of Hcp-proteins forming pilus-like structures of the type six secretion system (T6SS). To address the role of YPO0502 in AA, we cloned ypo0502 in E. coli, overexpressed it in Y. pestis and constructed its knock-out mutant in Y. pestis. However, all these approaches failed: YPO0502 was not secreted in E. coli, formed inclusion bodies when overexpressed in Y. pestis, and could probably be compensated by other Hcp-like proteins in Y. pestis. In contrast, downregulation of ypo0502 expression by its antisense RNA supported the contribution of YPO0502 in AA of Hms+ and Hms− Y. pestis strains. The results of the present study indicate that the Hcp-like component of T6SS encoded by ypo502 is involved in Y. pestis AA and suggest that at least one (ypo0499-0516) of the 6 T6SS clusters of Y. pestis is involved in bacterial interaction.

In this study, we report on a novel, highly sensitive IL-10 reporter mouse based on the reporter enzyme β-lactamase and the fluorescence resonance energy transfer substrate coumarin-cephalosporin-fluorescein (4). In contrast to an IL-10 reporter mouse model that we generated by using enhanced GFP as reporter and allowed tracking IL-10 expression only in T cells, the IL-10–β-lactamase reporter (ITIB) mouse enables us to easily analyze and quantify IL-10 production at the single-cell level in all myeloid and lymphoid cell types. Furthermore, the ITIB mouse allows studying of the kinetics of IL-10 expression on a single-cell basis and provides a valuable tool for in vivo screening of cell type-specific IL-10–modulating drugs. Remarkably, the ITIB mouse revealed that, although a significant portion of each myeloid and lymphoid cell type produces IL-10, macrophages represent the major IL-10 producer population in several organs of naive mice. Moreover, using the examples of bacterial infection and transplantable skin melanoma models, we demonstrate the exceptional applicability of the ITIB mouse for the identification of IL-10–producing cells during immune responses in vivo. In this study, we identified tumor-infiltrating F4/80+ macrophages as the major source for IL-10 in B16-F10 melanoma in vivo. During systemic infection with Yersinia enterocolitica, although the proportion of IL-10+ cells increased in each myeloid and lymphoid cell type population, infiltrating CD11b+Ly6G+ neutrophils represent a majority among IL-10–producing cells at the site of infection. We conclude that cells of the innate immune system that are involved in immune homeostasis or immune responses are substantial sources of IL-10.

Meningococci are facultative-pathogenic bacteria endowed with a set of adhesins allowing colonization of the human upper respiratory tract, leading to fulminant meningitis and septicemia. The Neisseria adhesin NadA was identified in about 50% of N. meningitidis isolates and is closely related to the Yersinia adhesin YadA, the prototype of the oligomeric coiled-coil adhesin (Oca) family. NadA is known to be involved in cell adhesion, invasion, and induction of proinflammatory cytokines. Because of the enormous diversity of neisserial cell adhesins the analysis of the specific contribution of NadA in meningococcal host interactions is limited. Therefore, we used a non-invasive Y. enterocolitica mutant as carrier to study the role of NadA in host cell interaction. NadA was shown to be efficiently produced and localized in its oligomeric form on the bacterial surface of Y. enterocolitica. Additionally, NadA mediated a β1 integrin-dependent adherence with subsequent internalization of yersiniae by a β1 integrin-positive cell line. Using recombinant NadA24–210 protein and human and murine β1 integrin-expressing cell lines we could demonstrate the role of the β1 integrin subunit as putative receptor for NadA. Subsequent inhibition assays revealed specific interaction of NadA24–210 with the human β1 integrin subunit. Cumulatively, these results indicate that Y. enterocolitica is a suitable toolbox system for analysis of the adhesive properties of NadA, revealing strong evidence that β1 integrins are important receptors for NadA. Thus, this study demonstrated for the first time a direct interaction between the Oca-family member NadA and human β1 integrins.

The long-term persistance of P. aeruginosa in the cystic fibrosis (CF) lung is characterized by the selection of a variety of genotypes and phenotypes that typically descend from one infecting P. aeruginosa clone, a process known as adaptive radiation. This adaptation process of P. aeruginosa includes complex physiological changes that likely confer a selective advantage to better thrive in the diverse niches and microenvironments of the inflamed and hostile CF airways. The occurrence of P. aeruginosa variants is fixed by mutation and selection. Common loss-of-function mutations in genes such as lasR, mucA and mexT lead to a general adaptation pattern and P. aeruginosa variants with increased antimicrobial resistance, alginate overproduction, reduced acute virulence, and improved metabolic fitness. Strikingly, several virulence-associated traits and immunostimulatory components of P. aeruginosa are turned off. In contrast, other cellular factors are positively selected such as the outer membrane protein OprF, the blue copper protein azurin, the cytochrome c peroxidase c551, and the enzymes of the arginine deiminase pathway ArcA-ArcD. These metabolic components probably are required for the optimal anaerobic or microaerobic growth and viability of P. aeruginosa within CF airways. Besides these common adaptations found by the comparison of P. aeruginosa isolates from different CF patients, the overall diversity of isogenic isolates from one CF patient is extended by variable changes in the expression of regulatory-, transport-, metabolic-, and virulence-associated genes. A better understanding of the microevolution of P. aeruginosa towards niche specialists according the selection pressure in the CF lung is a prerequisite to develop new strategies for the detection of P. aeruginosa variants, the antipseudomonal treatment, the prediction of the infectious disease state, and the development of efficient vaccines.

Neutrophil extracellular traps (NETs) represent a distinct mechanism to control and eliminate microbial infections. Our results show that conidia and germ tubes of the human pathogenic mold Aspergillus fumigatus are able to trigger the formation of NETs. Viable fungal cells are not essentially required for this host-pathogen interaction. Neutrophils engulf conidia and thereby inhibit their germination, a process that is independent of NETosis. In the experimental set-up used in this study neutrophils do not kill germ tubes, but reduce their polar growth and this inhibition depends on NETs as it can be overcome by the addition of DNase-1. The Zn(2+) chelator calprotectin is associated with the Aspergillus-induced NETs and addition of Zn(2+) abrogates the NET-mediated growth inhibition. In summary, our data provide evidence that NETs are not sufficient to kill A. fumigatus, but might be a valuable tool to confine infection.

Summary Farnesol is known for inducing apoptosis in some fungi and mammalian cells. To evaluate its potential role as an antifungal agent, we studied its impact on the human pathogen Aspergillus fumigatus. We found that growth of A. fumigatus wild type is inhibited, but two cell wall mutants, Δmnt1 andΔglfA, are much more susceptible to farnesol. This susceptibility is partially rescued by osmotic stabilization, suggesting that farnesol is a cell wall perturbing agent. However, farnesol does not activate but inhibit the cell wall integrity (CWI) pathway. Remarkably, mutants lacking AfMkk2 or AfMpkA, two kinases essential for CWI signalling, are also highly susceptible to farnesol, suggesting that its mode of action goes beyond inhibition of CWI signalling. Farnesyl derivatives are known for interfering with the function of prenylated proteins. We analysed the subcellular localization of two prenylated Rho family GTPases, AfRho1 and AfRho3, which are implicated in controlling CWI and the cytoskeleton. We found that under normal growth conditions AfRho1 and AfRho3 predominantly localize to the hyphal tip. After farnesol treatment this localization is rapidly lost, which is accompanied by swelling of the hyphal tips. Parallel displacement of tropomyosin from the tips suggests a concomitant disorganization of the apical actin cytoskeleton.

GDP-mannose:inositol-phosphorylceramide (MIPC)-derived glycosphingolipids are important pathogen-associated molecular patterns (PAMP) of Candida albicans and according to recently published data also of Aspergillus fumigatus. MIPC transferases are essential for the synthesis of MIPC, but have so far been studied only in Saccharomyces cerevisiae and C. albicans. Here, we have identified MitA as the only MIPC transferase in A. fumigatus. The DeltamitA mutant lacks MIPC and MIPC-derived glycosphingolipids and accumulates the precursor IPC. The mutant grows normally, shows no defects in cell wall or membrane organization and a normal resistance to different stressors. It is, however, sensitive to high Ca(2+) concentrations, especially during germination. Germination of DeltamitA mutant conidia is also decelerated under normal growth conditions, but neither the virulence of this mutant in a systemic model of infection nor its ability to trigger a cytokine response in macrophages is impaired, arguing against a role of MIPC-derived glycosphingolipids as important A. fumigatus PAMPs.

Yersiniae bearing the Yersinia virulence plasmid pYV impact the transcriptome of J774A.1 macrophage-like cells in two distinct ways: (i) by suppressing, in a Yersinia outer protein P (YopP)-dependent manner, the induction of inflammatory response genes and (ii) by mRNA induction of the silencing transcription factor klf2 . Here we show that klf2 induction by Yersinia enterocolitica occurs in several cell lines of macrophage and squamous and upper gastrointestinal epithelial origin as well as in bone marrow-derived dendritic cells. Several strains of Pseudomonas aeruginosa and Staphylococcus aureus are equally effective as Y. enterocolitica in inducing klf2 expression. Screening of mutant strains or incubation with recombinant toxins identified the rho-inactivating toxins YopT from Yersinia spp., ExoS from Pseudomonas aeruginosa , EDIN-B from Staphylococcus aureus , and C3bot from Clostridium botulinum as bacterial inducers of klf2 mRNA. klf2 mRNA induction by these toxins does not require de novo protein synthesis. Serum response factor or actin depolymerization does not seem to be involved in regulating klf2 expression in response to bacterial infection. Instead, short hairpin RNA-mediated inactivation of RhoA and its effector rhophilin 1 is sufficient to induce long-term klf2 expression. Thus, bacteria exploit the RhoA-rhophilin signaling cascade to mediate sustained expression of the immunosuppressive transcription factor klf2 .

Preexisting antivector immunity can severely compromise the ability ofSalmonella entericaserovar Typhimurium live vaccines to induce protective CD8 T-cell frequencies after type III secretion system-mediated heterologous protein translocation in orally immunized mice. To circumvent this problem, we injected CpG DNA admixed to the immunodominant p60217-225peptide fromListeria monocytogenessubcutaneously into BALB/c mice and coadministered a p60-translocatingSalmonellastrain by the orogastric route. The distribution of tetramer-positive p60217-225-specific effector and memory CD8 T cells was analyzed by costaining of lymphocytes with CD62L and CD127. In contrast to the single oral application of recombinantSalmonellaor single immunization with CpG and p60, in the spleens from mice immunized with a combination of both vaccine types a significantly higher level of p60-specific CD8 T cells with a predominance of the effector memory T-cell subset was detected. In vivo protection studies revealed that this CD8 T-cell population conferred sterile protective immunity against a lethal infection withL. monocytogenes. However, p60-specific central memory CD8 T cells induced by single vaccination with CpG and p60 were not able confer effective protection against rapidly replicating intracellularListeria. In conclusion, we provide compelling evidence that the combination ofSalmonellatype III-mediated antigen delivery and CpG immunization is an attractive novel vaccination strategy to modulate CD8 differentiation patterns toward distinct antigen-specific T-cell subsets with favorable protective capacities.

Summary: Antigens of Candida albicans derived from both growth phases employing five different media and from five different strains using one medium were analysed by gel-electrophoresis and immunoblot-technique. No growth phase or growth-medium dependent differences were noted when commercially available rabbit anti-Candida or human antisera were employed. The SDS-PAGE patterns of the five strains analysed were quite similar. However, one strain isolated from a patient with proven systemic candidiasis showed an additional 60 kd membrane protein which was recognized only by human but not by rabbit antisera. The immunoblot-patterns of patient and blood donor sera were compared. A striking result was that patients sera specifically recognized a 43 kd soluble antigen, whereas blood donor sera did not. Interestingly, early sera of patients with systemic candidiasis showed an isolated IgM-response towards an 80 kd antigen of the soluble fraction. Our results suggest that tests based on these two antigens may facilitate the diagnosis of systemic candidiasis. Zusammenfassung: Wir verglichen durch Gel Elektrophorese und Immunoblot Antigen Prepa-rationen, die von funf verschiedenen Candida albicans Stammen sowie unter Verwendung von funf verschiedenen Anzuchtmedien gewonnen worden waren. Es konnten im Immunoblot keine von der Wachstumsphase des Organismus abhangige Unterschiede festgestellt werden. Die Protein-Bandenmuster der SDS-Gelelektrophorese waren ebenfalls vergleichbar. Bei der Analyse unterschiedlicher Stamme zeigte sich jedoch, das ein Isolat aus einer Blutkultur ein zusatzliches Antigen in der Membranfunktion aufwies, das nur von menschlichen, aber nicht von Kaninchen-Antiseren erkannt wurde. Weiterhin wurden die Immunoblot-Muster von Patienten und Blutspenderseren verglichen. Dabei zeigte sich, das Seren von infizierten Patienten mit einem 43kd Antigen reagierten, wahrend Blutspenderseren diese Reaktivitat nicht zeigten. Ein weiterer interessanter Befund war, das Patienten mit systemischer Candidainfektion in der Fruhphase IgM-Antikorperzeigten, die gegen ein 80 kd Antigen gerichtet waren. Unsere Ergebnisse lassen vermuten, das durch Verwendung dieser Antigene in einem ELISA-Test eine Verbesserung der Diagnostik systemischer Candidainfektionen erreicht werden kann.

The Oca family is a novel class of autotransporter-adhesins with highest structural similarity in their C-terminal transmembrane region, which supposedly builds a beta-barrel pore in the outer membrane (OM). The prototype of the Oca family is YadA, an adhesin of Yersinia enterocolitica and Yersinia pseudotuberculosis . YadA forms a homotrimeric lollipop-like structure on the bacterial surface. The C-terminal regions of three YadA monomers form a barrel in the OM and translocate the trimeric N-terminal passenger domain, consisting of stalk, neck, and head region to the exterior. To elucidate the structural and functional role of the C-terminal translocator domain (TLD) and to assess its promiscuous capability with respect to transport of related passenger domains, we constructed chimeric YadA proteins, which consist of the N-terminal YadA passenger domain and C-terminal TLDs of Oca family members UspA1 ( Moraxella catarrhalis ), EibA ( Escherichia coli ), and Hia ( Haemophilus influenzae ). These constructs were expressed in Y. enterocolitica and compared for OM localization, surface exposure, oligomerization, adhesion properties, serum resistance, and mouse virulence. We demonstrate that all chimeric YadA proteins translocated the YadA passenger domain across the OM. Y. enterocolitica strains producing YadA chimeras or wild-type YadA showed comparable binding to collagen and epithelial cells. However, strains producing YadA chimeras were attenuated in serum resistance and mouse virulence. These results demonstrate for the first time that TLDs of Oca proteins of different origin are efficient translocators of the YadA passenger domain and that the cognate TLD of YadA is essential for bacterial survival in human serum and mouse virulence.

As a first approach to establishing a three-dimensional culture infection model, we studied the growth behavior of the extracellular pathogen Yersinia enterocolitica in three-dimensional collagen gels (3D-CoG). Surprisingly, we observed that plasmidless Y. enterocolitica was motile in the 3D-CoG in contrast to its growth in traditional motility agar at 37°C. Motility at 37°C was abrogated in the presence of the virulence plasmid pYV or the exclusive expression of the pYV-located Yersinia adhesion gene yadA. YadA-producing yersiniae formed densely packed (dp) microcolonies, whereas pYVyadA-carrying yersiniae formed loosely packed microcolonies at 37°C in 3D-CoG. Furthermore, we demonstrated that the packing density of the microcolonies was dependent on the head domain of YadA. Moreover, dp microcolony formation did not depend on the capacity of YadA to bind to collagen fibers, as demonstrated by the use of yersiniae producing collagen nonbinding YadA. By using a yopE-gfp reporter, we demonstrated Ca 2 -dependent expression of this pYVlocalized virulence gene by yersiniae in 3D-CoG. In conclusion, this study revealed unique plasmid-dependent growth behavior of yersiniae in a three-dimensional matrix environment that resembles the behavior of yersiniae (e.g., formation of microcolonies) in infected mouse tissue. Thus, this 3D-CoG model may be a first step to a more complex level of in vitro infection models that mimic living tissue, enabling us to study the dynamics of pathogen-host cell interactions. For decades, cell culture monolayers have successfully been used to study mechanisms of microbial adherence, invasion, and intracellular survival/multiplication. For in vitro infection studies, eukaryotic cells are grown on solid supports as monolayers and then are challenged with the respective pathogen. In spite of the liquid culture medium (third dimension) covering the adherent cell monolayer, this infection model can be considered a two-dimensional system which obviously does not reflect the environment of host tissue and dynamic events, such as cell migration, happening during infection. Host tissue (i) is vascularized, allowing the extravasation and migration of host immune cells toward the invading microbe, and (ii) consists of a network of extracellular matrix (ECM) proteins with diverse resident cells (e.g., fibroblasts, macrophages, etc.). Thus, there are many reasons to leave the two-dimensional system and establish an in vitro three-dimensional infection model by approaching in vivo conditions. In order to simulate a tissue-like environment, immunology and cell biology as well as tumor biology make use of three-dimensional collagen matrices to study cell migration, cell-cell interactions, and cell-matrix interactions (reviewed in reference 13). To develop and assess such a three-dimensional infection model, we started with the well-established, three-dimensional collagen gel (3D-CoG) on microscopic glass supports and with Yersinia enterocolitica as the prototype of an extracellular pathogen. Y. enterocolitica causes food-borne gastrointestinal diseases

In recent years, clusters of Pneumocystis jirovecii (formerly Pneumocystis carinii ) pneumonia (PCP) among immunocompromised individuals have been reported. Mostly, the source of infections was suspected to be within the clinical settings when transplant recipients and PCP patients shared hospital facilities. We report on a cluster of 16 renal transplant recipients positive for P. jirovecii . None of them received anti- Pneumocystis prophylaxis prior to P. jirovecii detection. Epidemiological studies revealed that 15 of them had received kidney transplants at a German university hospital and attended the same inpatient and outpatient clinic from January through September 2006. Multilocus sequence typing (MLST) was performed on the following genes: ITS1 , β- tub, 26S , and mt26S. P. jirovecii DNA was available from 14 patients and showed identical MLST types among these renal transplant recipients. Surprisingly, one patient who was treated at a different nephrological center and reported no personal contact with patients from the renal transplantation cluster harbored an identical P. jirovecii MLST type. Three HIV-positive patients and one bone-marrow-transplanted hematologic malignancy patient—treated at different medical centers—were used as controls, and different MLST types were revealed. Interestingly, in three of the four previously described regions, new alleles were detected, and one new polymorphism was observed in the mt26S region. The epidemiological data and the genotyping results strongly suggest a nosocomial patient-to-patient transmission of P. jirovecii as the predominant transmission route. Therefore, strict segregation and isolation of P. jirovecii -positive/suspected patients in clinical settings seems warranted.

Aspergillus fumigatus is an opportunistic fungal pathogen responsible for severe, life-threatening infections in immunocompromised patients. Airborne conidia are the infectious agent and can reach the lower parts of the respiratory system. In the lung, phagocytes represent the first line of defence. Resident macrophages are able to track down, engulf and kill the invading spores. Phagocytosis of the conidia is therefore a prerequisite for their efficient elimination. Using human and murine macrophages we analysed the phagocytic uptake of A. fumigatus conidia. We found that conidial phagocytosis is an actin-depending process that additionally requires myosin motor, phosphoinositide-3-phosphate kinase and tyrosine kinase activity. Both broad range tyrosine kinase inhibitors and inhibitors that specifically block src kinases had a strong impact on the conidial uptake. Immunofluorescence data demonstrate the recruitment of tyrosine-phosphorylated proteins to the vicinity of engulfed conidia. Uptake of the conidia was accompanied by a strong and local reorganisation of the actin cytoskeleton, whereas no prominent reorganisation was apparent for the microtubules. Both confocal immunofluorescence and electron microscopic data revealed the presence of large ruffle-like structures engaged in the uptake of conidia. This suggests that the internalisation of A. fumigatus spores can be mediated by a process resembling macropinocytosis, which is furthermore supported by the detection of intracellular conidia within spacious vacuoles. Taken together, our data provide new insights into the internalisation of A. fumigatus spores by macrophages, a key process in the early immune defence against an Aspergillus infection.

Background The goal of this pilot study was to design an external quality assessment (EQA) scheme for German cystic fibrosis (CF) clinical microbiology laboratories. Therefore, a multicentre study of 18 German CF laboratories was performed to evaluate their proficiency in analyzing CF respiratory secretions. Methods Simulated clinical specimens containing a set of four frequent CF pathogens, namely two Pseudomonas aeruginosa strains differing in morphotype ( mucoid versus non-mucoid ) and resistotype, one Staphylococcus aureus strain and one Burkholderia multivorans strain, were distributed to each laboratory. Isolation, identification and antimicrobial susceptibility testing (AST) of any bacterial pathogen present and completion of a questionnaire about applied microbiological protocols were requested. Results Three of four strains were isolated and identified correctly by almost all laboratories. B. multivorans was once misidentified as Burkholderia cenocepacia . Fourteen laboratories failed to detect the second multidrug resistant P. aeruginosa isolate. AST errors occurred most often for P. aeruginosa 2 followed by B. multivorans, P. aeruginosa 1 and S. aureus . Evaluation of the questionnaires revealed major differences in cultivation and identification techniques applied by the participating laboratories. Conclusions A periodical EQA programme for German CF laboratories and standardized microbiological procedures seem to be necessary to advance diagnostic microbiology employed on CF respiratory tract specimens and may help to improve anti-infective treatment and infection control practices for CF patients.

Pathogenic Yersinia spp. employ a type III protein secretion system that translocates several Yersinia outer proteins (Yops) into the host cell to modify the host immune response. One strategy of the infected host cell to resist the bacterial attack is degradation and inactivation of injected bacterial virulence proteins through the ubiquitin-proteasome pathway. The cytotoxin YopE is a known target protein of this major proteolytic system in eukaryotic cells. Here, we investigated the sensitivity of YopE belonging to different enteropathogenic Yersinia enterocolitica serogroups to ubiquitination and proteasomal degradation. Analysis of the YopE protein levels in proteasome inhibitor-treated versus untreated cells revealed that YopE from the highly pathogenic Y. enterocolitica serotype O8 was subjected to proteasomal destabilization, whereas the YopE isotypes from serogroups O3 and O9 evaded degradation. Accumulation of YopE from serotypes O3 and O9 was accompanied by an enhanced cytotoxic effect. Using Yersinia strains that specifically produced YopE from either Y. enterocolitica O8 or O9, we found that only the YopE protein from serogroup O8 was modified by polyubiquitination, although both YopE isotypes were highly homologous. We determined two unique N-terminal lysines (K62 and K75) in serogroup O8 YopE, not present in serogroup O9 YopE, that served as polyubiquitin acceptor sites. Insertion of either lysine in serotype O9 YopE enabled its ubiquitination and destabilization. These results define a serotype-dependent difference in the stability and activity of the Yersinia effector protein YopE that could influence Y. enterocolitica pathogenesis.

Five coagulase-negative, novobiocin-susceptible staphylococcal strains were isolated from human blood cultures in different German and Belgian medical facilities. A novel species, ‘Staphylococcus pettenkoferi’ was proposed recently to accommodate two of these strains (B3117T and A6664), although the name was not validly published. All five strains belonged to the genus Staphylococcus because they were non-motile, Gram-positive, catalase-positive cocci with peptidoglycan type (A3α type l-lys–gly2–4–l-Ser–Gly), menaquinone pattern (MK-7, MK-6 and MK-8) and major cellular fatty acids (ai-C15 : 0, ai-C17 : 0 and i-C15 : 0) that corresponded to those of staphylococci. Phenotypically, the isolates most closely resembled Staphylococcus capitis subsp. capitis and Staphylococcus auricularis, but they could be distinguished from these species by physiological tests and chemotaxonomic investigations. The results of DNA–DNA hybridization, chemotaxonomic investigations and 16S rRNA gene and RNA polymerase B gene (rpoB) sequence analysis enabled strains B3117T, K6999, 229 and 230 to be differentiated genotypically and phenotypically from known Staphylococcus species, indicating that these isolates are representatives of a novel species. The name Staphylococcus pettenkoferi sp. nov. is proposed for this novel species, with strain B3117T (=CIP 107711T=CCUG 51270T) as the type strain. Due to differences in the results of physiological and chemotaxonomic investigations and DNA–DNA hybridization data, strain A6664 was not included in the description of the novel species.

Inhalation of resting conidia is usually the first step of a systemic infection caused by the opportunistic fungal pathogen Aspergillus fumigatus. In the lung, the inhaled spores encounter an environment that permits germination. However, the relative importance of certain environmental conditions for conidial activation and subsequent hyphae formation has so far not been analyzed in detail. In this study, we studied the role of oxygen during germination. We found that inhibitors of the respiratory chain were nearly as efficient in blocking germination as cycloheximide, an inhibitor of protein synthesis, which is already known to prevent germination of Aspergillus nidulans. We also found that A. fumigatus is unable to grow or germinate under anaerobic conditions, and using the fluorescent mitotracker dye we detected active mitochondria already at the stage of swollen conidia, which indicates that respiration is an early event during germination. In line with these data, we found that significant oxygen consumption was detectable early during germination, whereas no oxygen consumption was measurable in suspensions of resting conidia. In summary, the present study provides evidence that respiration is absolutely required for the germination of A. fumigatus conidia.

Type III secretion systems (T3SSs) are engaged by a broad number of Gram-negative bacteria to inject proteins into host cells. The simultaneous crossing of three biological membranes by these proteins is a phenomenon which is far from being understood mechanistically. However, recent work from several groups has substantially enriched our conception of how proteins travel from the bacterial into the host cell cytosol. Here, we focus on the role of specific T3SS chaperones, the specific ATPase, and the proton motive force in type III secretion.

Aspergillus fumigatus is a fungal pathogen causing severe infections in immunocompromised patients. For clearance of inhaled conidia, an efficient response of the innate immune system is required. Macrophages represent the first line of defence and ingest and kill conidia. C-type lectins represent a family of receptors, which recognize pathogen-specific carbohydrates. One of them is beta1-3 glucan, a major component of the fungal cell wall. Here we provide evidence that beta1-3 glucan plays an important role for the elimination of A. fumigatus conidia. Laminarin, a soluble beta1-3 glucan and antibodies to dectin-1, a well known beta1-3 glucan receptor, significantly inhibited conidial phagocytosis. On resting conidia low amounts of surface accessible beta1-3 glucan were detected, whereas high amounts were found on small spores that appear early during germination and infection as well as on resting conidia of a pksP mutant strain. Swollen conidia also display larger quantities of beta1-3 glucan, although in an irregular spotted pattern. Resting pksP mutant conidia and swollen wild-type conidia are phagocytosed with high efficiency thereby confirming the relevance of beta1-3 glucans for conidial phagocytosis. Additionally we found that TLR2 and the adaptor protein MyD88 are required for efficient conidial phagocytosis, suggesting a link between the TLR2-mediated recognition of A. fumigatus and the phagocytic response.

Background. Pseudomonas aeruginosa (PA) strains with defective DNA mismatch repair genes generate numerous bacterial variants because of high mutation rates. In cystic fibrosis (CF), such mutator strains may lead to the rapid selection of survivors that are specifically adapted to the hostile environment of the inflamed CF lung. Methods. Genotypes and phenotypes of 111 PA variants descending from 3 distinct mutator strains obtained from 3 patients with CF were systematically characterized. Results. We demonstrated that PA mutS isolates accumulated in the CF lung during the observation period of 3-6 years, with dominance during the final stage of the disease. Mutator strains from the final stage of disease were multiresistant and had lost a set of established virulence-associated traits, including cytotoxicity for bronchial epithelial cells (Calu-3) and macrophages (J774). This pathoadaptation was associated with the loss of survival capacity in a typical environmental habitat, such as tap water. Strikingly, nonmutator strains that maintained their virulence potential persisted as a minority, probably with a preference for the lower airways. Conclusions. Mutator strains may evolve from the initially infecting PA strain and generate numerous variants with a loss of destructive virulence factors, probably because of selection for improved survival in the deteriorated CF lung but at the expense of the ability to live freely.

Enteropathogenic Yersinia enterocolitica is able to grow within or outside the mammalian host. Previous transcriptomic studies have indicated that the regulator OmpR plays a role in the expression of hundreds of genes in enterobacteria. Here, we have examined the impact of OmpR on the production of Y. enterocolitica membrane proteins upon changes in temperature, osmolarity and pH. Proteomic analysis indicated that the loss of OmpR affects the production of 120 proteins, a third of which are involved in uptake/transport, including several that participate in iron or heme acquisition. A set of proteins associated with virulence was also affected. The influence of OmpR on the abundance of adhesin YadA and heme receptor HemR was examined in more detail. OmpR was found to repress YadA production and bind to the yadA promoter, suggesting a direct regulatory effect. In contrast, the repression of hemR expression by OmpR appears to be indirect. These findings provide new insights into the role of OmpR in remodelling the cell surface and the adaptation of Y. enterocolitica to different environmental niches, including the host.

Several bacterial pathogens that cause severe infections in warm-blooded animals, including humans, have the potential to actively invade host cells and to efficiently replicate either in the cytosol or in specialized vacuoles of the mammalian cells. The interaction between these intracellular bacterial pathogens and the host cells always leads to multiple physiological changes in both interacting partners, including complex metabolic adaptation reactions aimed to promote proliferation of the pathogen within different compartments of the host cells. In this chapter, we discuss the necessary nutrients and metabolic pathways used by some selected cytosolic and vacuolar intracellular pathogens and - when available - the links between the intracellular bacterial metabolism and the expression of the virulence genes required for the intracellular bacterial replication cycle. Furthermore, we address the growing evidence that pathogen-specific factors may also trigger metabolic responses of the infected mammalian cells affecting the carbon and nitrogen metabolism as well as defense reactions. We also point out that many studies on the metabolic host cell responses induced by the pathogens have to be scrutinized due to the use of established cell lines as model host cells, as these cells are (in the majority) cancer cells that exhibit a dysregulated primary carbon metabolism. As the exact knowledge of the metabolic host cell responses may also provide new concepts for antibacterial therapies, there is undoubtedly an urgent need for host cell models that more closely reflect thein vivoinfection conditions.

Two genomic islands, namely the high-pathogenicity island (HPI) and Ecoc54N target the same asn tRNA genes to integrate into the bacterial chromosome. The HPI encodes the siderophore yersiniabactin in the highly pathogenic Yersinia group (Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica 1B) whilst the Ecoc54N island possibly encodes a polyketide synthase with an unknown function in the uropathogenic Escherichia coli CFT073 strain. HPI encodes the recombinase that promotes site-specific recombination (both integrative and excisive) with its corresponding attachment targets. A recombinase orthologue is also present in Ecoc54N. In addition, the HPI(Yps) of the Y. pestis/Y. pseudotuberculosis evolutionary lineage encodes the excisionase (recombination directionality factor, Xis(HPI)) that facilitates excision of the island. However, no sequence resembling the excisionase gene could be found in Ecoc54N. The rate of the HPI(Yps) excision estimated by real-time PCR was 10(-6) in Y. pseudotuberculosis. The presence of the excisionase increased the efficiency of the excisive recombination only eight fold. However, the introduction of the xis(HPI) in E. coli CFT073 did not influence the excision of Ecoc54N. The Xis(HPI) is encoded by the variable AT-rich part of the HPI(Yps) and substantially differs from its cognate recombinase in A+T content and codon usage. Also the Xis(HPI)-protected region, defined in the HPI attachment site, has suffered several nucleotide substitutions in Ecoc54N that could influence interaction with the excisionase. We propose that the pathogenicity islands (PAIs) targeting asn tRNA genes (PAIs(asn tRNA)) might have acquired recombinase and excisionase (HPI) genes independently and sequentially.

Yersinia species pathogenic to human benefit from a protein transport machinery, a type three secretion system (T3SS), which enables the bacteria to inject effector proteins into host cells. Several of the transport substrates of the Yersinia T3SS, called Yops (Yersinia outer proteins), are assisted by specific chaperones (Syc for specific Yop chaperone) prior to transport. Yersinia enterocolitica SycD (LcrH in Yersinia pestis and Yersinia pseudotuberculosis) is a chaperone dedicated to the assistance of the translocator proteins YopB and YopD, which are assumed to form a pore in the host cell membrane. In an attempt to make SycD amenable to structural investigations we recombinantly expressed SycD with a hexahistidine tag in Escherichia coli. Combining immobilized nickel affinity chromatography and gel filtration we obtained purified SycD with an exceptional yield of 120mg per liter of culture and homogeneity above 95%. Analytical gel filtration and cross-linking experiments revealed the formation of homodimers in solution. Secondary structure analysis based on circular dichroism suggests that SycD is mainly composed of alpha-helical elements. To prove functionality of purified SycD previously suggested interactions of SycD with Yop secretion protein M2 (YscM2), and low calcium response protein V (LcrV), respectively, were reinvestigated.

In 2002, 119 isolates of the Enterobacter cloacae complex were collected randomly from 11 German laboratories nationwide. Antibiotic susceptibilities were tested by disk-diffusion tests according to CLSI guidelines, and MICs were determined using Etests. PCRs were performed to amplify all TEM and SHV, and most CTX-M and OXA beta-lactamase genes. PCR products were sequenced to identify the precise extended spectrum beta-lactamase (ESBL) types. Isoelectric focusing (IEF) and PM/PML Etests were used to confirm production of the respective ESBLs. According to susceptibility tests and CLSI criteria, 49 (40%) isolates were resistant to extended-spectrum cephalosporins. Seven (5.8%) isolates were positive in at least one of the PCR assays. Sequencing identified production of TEM-1 beta-lactamase genes by three (2.9%) isolates, and ESBL genes of the CTX-M and SHV beta-lactamase families by five (4.2%) isolates. IEF confirmed the production of beta-lactamases in the expected pI ranges of the respective ESBLs, and four of the five ESBL-producers were detected using the PM/PML Etest. All ESBL-producing isolates showed co-resistance to sulphonamides.

In contrast to Salmonella and Shigella, enteropathogenic Yersinia species are extracellular multiplying Gram-negative bacteria. This life style requires a sophisticated anti-host strategy, which is implemented by the Yersinia virulence plasmid. This plasmid encodes the type 3 secretion system (injectisome), at least six microinjected anti-host effector proteins, a trimeric coiled coil outer membrane protein (Yersinia adhesin) with cell adhesin and protective functions against complement and defensins, and the released V antigen, which has Toll-like receptor 2 agonist activity.