Bartonella as emerging pathogens.
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Animals↗
Biomedical subjects
C Dehio
Publications and source records attributed to C Dehio.
Interactions of Bartonella henselae with vascular endothelial cells.
The emerging human pathogen Bartonella henselae has the remarkable capacity to colonise vascular tissues and to stimulate vasoproliferative tumour growth. Although the molecular principle of bacterium-induced neovascularisation (angiogenesis) is still unclear, recent studies have indicated a novel mechanism of endothelial colonisation that involves the formation, engulfment and uptake of a large bacterial aggregate.
Angiogenesis Inducing Agents↗
Molecular analysis of neisserial Opa protein interactions with the CEA family of receptors: identification of determinants contributing to the differential specificities of binding.
The carcinoembryonic antigen (CEA) gene family members, CEACAM1, CEACAM3, CEACAM5 and CEACAM6, are bound by the Opa outer membrane proteins of pathogenic Neisseria spp., whereas CEACAM8 is not. In this study, we demonstrate that the closely related CEACAM4 and CEACAM7, which are also members of the CEA family, are not Opa receptors. We exploited the high conservation between CEACAM6 and CEACAM8 to generate an extensive set of chimeric receptors in order to delineate the sequences necessary for Opa binding. Using a transfection-based infection system, we showed that binding of Opa52 involves residues 27-42, which are predicted to form beta-strand C and short loops adjacent to it, and residues lying between amino acids 60 and 108 in the amino-terminal domain. The replacement of residues 27-29 in CEACAM6 with the CEACAM1 or CEACAM5 sequences generated recombinant CEACAM6 receptors that are bound by CEACAM1/CEACAM5-specific Opa variants. Together, our data demonstrate that Opa proteins bind to residues exposed on the GFCC' face of the N-terminal domain of CEACAM receptors, and identify an amino acid triplet sequence that is responsible for the differential binding of Opa proteins to CEACAM1, CEACAM5 and CEACAM6.
Amino Acid Sequence↗
Bartonella alsatica sp. nov., a new Bartonella species isolated from the blood of wild rabbits.
Bartonella species are considered as emerging human pathogens, with at least six different species pathogenic or possibly pathogenic for humans. However, little is known about Bartonella distribution, species polymorphism and pathogenicity in mammalian species. The objective of this work was to determine the presence, the frequency and the distribution of Bartonella species in wild rabbits (Oryctolagus cuniculus) caught in warrens in Alsace, France. Humans may come into contact with wild rabbits when hunting, especially when they are picked up with bare hands and at time of evisceration. Of 30 blood samples collected and cultured from wild rabbits, nine (30%) were positive for organisms morphologically similar to Bartonella spp. The bacteria appeared as small, fastidious, aerobic, oxidase-negative, Gram-negative rods which could be localized within erythrocytes. Their biochemical properties were similar to those of the genus Bartonella. The sequence of the 16S rRNA gene obtained from the rabbit isolates was highly related to the sequences of the different Bartonella species (97.8-99.3% similarity). The high DNA hybridization rate (81-90% similarity) between the three strains isolated from rabbit blood confirmed that they belong to the same bacterial species. Hybridization values, obtained with the nuclease-TCA method, when testing type strains of recognized Bartonella species (9-14% similarity), support the creation of a new species for the rabbit isolates. The name Bartonella alsatica is proposed for these strains isolated from the blood of wild rabbits. The type strain is IBS 382T (= CIP 105477T).
Animals↗
Ligation of cell surface heparan sulfate proteoglycans by antibody-coated beads stimulates phagocytic uptake into epithelial cells: a model for cellular invasion by Neisseria gonorrhoeae.
Binding of a particular opacity outer membrane protein (Opa) of Neisseria gonorrhoeae to cell surface heparan sulfate proteoglycans (HSPGs) of epithelial cells results in tight bacterial adherence; however, the role of this ligand-receptor interaction in triggering the subsequent bacterial internalization step is uncertain. Here we have used latex beads coated with HSPG-ligating antibodies as an in vitro model to study the role of HSPGs in gonococcal uptake into epithelial cells. Beads and gonococci showed the same cell line-specified adherence patterns and increase in phagocytic uptake mediated by serum or purified vitronectin (Vn). Heparitinase digestion as well as antibody competition experiments indicate that a critical level of HSPG ligation is necessary and sufficient to trigger phagocytic uptake into epithelial cells. Vn was found to specifically enhance HSPG-dependent phagocytic uptake while phagocytosis resulting from the ligation of other cell surface receptors was unaffected in the presence of Vn. Pharmacological studies with PKC inhibitors suggest a role for PKC in phagocytic uptake of HSPG-ligating beads. The use of drugs impairing cytoskeletal functions indicates that HSPG-dependent phagocytosis requires actin polymerization by a process distinct from receptor-mediated endocytosis.
Actins↗
Construction of versatile high-level expression vectors for Bartonella henselae and the use of green fluorescent protein as a new expression marker.
Expression vectors suitable for directing high levels of protein synthesis in Bartonella henselae have been constructed based on the mobilisable broad-host-range (IncQ) plasmidpMMB206. They confer kanamycin resistance and feature the taclac (tac-lacUV5 in tandem) promoters in front of a polylinker followed by the rrnB transcriptional stop point. While expression of genes fused to the taclac promoter is constitutive in one vector, the lacIq gene carried by the other vector allows a controlled, IPTG-inducible gene expression. These vectors were tested by subcloning a mutated gfp gene coding for the green fluorescent protein (GFP) from Aequorea victoria into the multiple cloning site and introducing the resulting plasmids into Escherichia coli and B. henselae. GFP expression was determined by measuring fluorescence via flow cytometry or directly by immunoblotting. Compared to E. coli, expression of GFP in B. henselae was more tightly controlled by lacIq and resulted in much higher levels of both IPTG-induced and constitutive gene expression. In vitro infection of endothelial cells indicated that GFP expression does not adversely affect the interaction of B. henselae with host cells. These data demonstrate that (i) the established expression vectors are useful for directing controlled or constitutive high-level protein synthesis in B. henselae and (ii) that GFP is a valuable expression marker which may has important applications in studying the bacterial genetics and cellular interactions of this emerging human pathogen.
Bartonella henselae↗
Vitronectin-dependent invasion of epithelial cells by Neisseria gonorrhoeae involves alpha(v) integrin receptors.
Binding of vitronectin (VN) to Neisseria gonorrhoeae expressing the heparan sulfate proteoglycan (HSPG) specific Opa50 protein was recently shown to trigger bacterial internalization into distinct epithelial cell lines. We have investigated the role of VN-binding integrin receptors and protein kinase C (PKC) in VN-triggered bacterial uptake. Blocking integrin function by RGDS peptides or by antibodies specific to alpha(v)beta5 or alpha(v)beta3 resulted in an abrogation of VN-triggered bacterial internalization. Moreover, inhibitors of PKC were found to block VN-triggered uptake. The essential role of alpha(v) integrins and the presumable involvement of PKC in VN-triggered gonococcal uptake are discussed.
Antigens, Bacterial↗
The role of neisserial Opa proteins in interactions with host cells.
Pathogenic Neisseria spp. possess a repertoire of phase-variable Opa proteins that mediate various pathogen--host cell interactions, including bacterial engulfment by epithelial cells and opsonin-independent phagocytosis by professional phagocytes. Recent studies have identified cellular targets recognized by defined Opa proteins and have begun to reveal host signalling events involved in mediating these Opa-dependent cellular processes.
Animals↗
Opa binding to cellular CD66 receptors mediates the transcellular traversal of Neisseria gonorrhoeae across polarized T84 epithelial cell monolayers.
We have analysed the capacity of the 11 phase-variable, opacity-associated (Opa) proteins encoded by Neisseria gonorrhoeae MS11 to mediate traversal across polarized monolayers of the human colonic carcinoma T84 cell line. Gonococci expressing either the heparan sulphate proteoglycan (HSPG) binding Opa protein (Opa50) or no Opa protein (Opa-) did not interact with the apical pole of T84 monolayers, whereas the 10 variant Opa proteins previously shown to bind CD66 receptors were found to mediate efficient gonococcal adherence and transepithelial traversal. Consistent with this, T84 cells were shown by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunoblotting to co-express CD66a (BGP), CD66c (NCA) and CD66e (CEA). The recruitment of CD66 receptors by Opa-expressing gonococci indicates their involvement in mediating adherence to the surface of T84 cells, and these bacterial interactions could be inhibited completely using polyclonal antibodies cross-reacting with all of the CD66 proteins co-expressed on T84 cells. Consistent results were obtained when Opa proteins were expressed in Escherichia coli, suggesting that the Opa-CD66 interaction is sufficient to mediate bacterial traversal. Transcytosis of Opa-expressing N. gonorrhoeae or E. coli did not disrupt the barrier function of infected monolayers, as indicated by a sustained transepithelial electrical resistance (TEER) throughout the course of infection, and confocal laser scanning and electron microscopy both suggest a transcellular rather than a paracellular route of traversal across the monolayers. Parallels between the results seen here and previous work done with organ cultures confirm that T84 monolayers provide a valid model for studying neisserial interactions with the mucosal surface, and suggest that CD66 receptors contribute to this process in vivo.
Antibodies↗
Bartonella tribocorum sp. nov., a new Bartonella species isolated from the blood of wild rats.
Two Bartonella strains from blood of two wild rats (Rattus norvegicus) living in a rural environment were isolated. These strains were distinct from all previously known Bartonella species based on phenotypic and genotypic characteristics. This new species is distinguished by its trypsin-like activity, the absence of the ability to hydrolyse proline and tributyrin, its 16S rRNA and citrate synthase gene sequences and by whole-DNA hybridization data. This new species, for which the name Bartonella tribocorum sp. nov. is proposed, seems to be genetically related to Bartonella elizabethae, an agent isolated in a case of human endocarditis. The type strain of Bartonella tribocorum sp. nov. is IBS 506T (CIP 105476T).
Animals↗
CD66 carcinoembryonic antigens mediate interactions between Opa-expressing Neisseria gonorrhoeae and human polymorphonuclear phagocytes.
Colonization of urogenital tissues by the human pathogen Neisseria gonorrhoeae is characteristically associated with purulent exudates of polymorphonuclear phagocytes (PMNs) containing apparently viable bacteria. Distinct variant forms of the phase-variable opacity-associated (Opa) outer membrane proteins mediate the non-opsonized binding and internalization of N. gonorrhoeae by human PMNs. Using overlay assays and an affinity isolation technique, we demonstrate the direct interaction between Opa52-expressing gonococci and members of the human carcinoembryonic antigen (CEA) family which express the CD66 epitope. Gonococci and recombinant Escherichia coli strains synthesizing Opa52 showed specific binding and internalization by transfected HeLa cell lines expressing the CD66 family members BGP (CD66a), NCA (CD66c), CGM1 (CD66d) and CEA (CD66e), but not that expressing CGM6 (CD66b). Bacterial strains expressing either no opacity protein or the epithelial cell invasion-associated Opa50 do not bind these CEA family members. Consistent with their different receptor specificities, Opa52-mediated interactions could be inhibited by polyclonal anti-CEA sera, while Opa50 binding was instead inhibited by heparin. Using confocal laser scanning microscopy, we observed a marked recruitment of CD66 antigen by Opa52-expressing gonococci on both the transfected cell lines and infected PMNs. These data indicate that members of the CEA family constitute the cellular receptors for the interaction with, and internalization of, N. gonorrhoeae.
Antigens, Bacterial↗
Differential Opa specificities for CD66 receptors influence tissue interactions and cellular response to Neisseria gonorrhoeae.
The ability of all 11 variable opacity (Opa) proteins encoded by Neisseria gonorrhoeae MS11 to interact directly with the five CD66 antigens was determined. Transfected HeLa cell lines expressing individual CD66 antigens were infected with recombinant N. gonorrhoeae and Escherichia coli strains expressing defined Opas. Based upon the ability of these bacteria to bind and invade and to isolate specifically CD66 antigens from detergent-soluble extracts of the corresponding cell lines, distinct specificity groups of Opa interaction with CD66 were seen. Defining these specificity groups allowed us to assign a specific function for CD66a in the Opa-mediated interaction of gonococci with two different target cell types, which are both known to co-express multiple CD66 antigens. The competence of individual Opas to interact with CD66a was strictly correlated with their ability to induce an oxidative response by polymorphonuclear neutrophils. The same Opa specificity was observed for the level of gonococcal binding to primary endothelial cells after stimulation with TNFalpha, which was shown to increase the expression of CD66a rather than CD66e. As CD66e alone is expressed on other target tissues of gonococcal pathogenicity, Opa variation probably contributes to the cell tropism displayed by gonococci.
Animals↗
Binding of vitronectin to opa-expressing Neisseria gonorrhoeae mediates invasion of HeLa cells.
Neisseria gonorrhoeae induces local infections in the human genitourinary tract and can disseminate to other organs to cause severe disease. Blood-derived factors present in the genital mucosa have been suggested to facilitate the spread of N. gonorrhoeae in disseminated gonococcal infections. Using gentamicin invasion assays and confocal microscopy, we observed a strong stimulatory effect of fetal calf serum (FCS) on the gonococcal invasion of HeLa cells. FCS-mediated invasion was dependent on the expression of the epithelial cell invasion-associated Opa protein (plasmid-encoded Opa50 or its chromosomal homolog Opa30), while N. gonorrhoeae expressing noninvasive Opa proteins (Opa(51-60)) or no Opa protein (Opa-) was not invasive even in the presence of FCS. Incubation of N. gonorrhoeae MS11 with biotinylated FCS revealed a 78-kDa protein as the prominent protein binding to Opa50- or Opa30-expressing gonococci. This protein was recognized by antibodies against vitronectin (VN) in Western blots. Purified human or bovine VN efficiently bound to Opa50-expressing gonococci, while binding to noninvasive Opa- or Opa52-expressing gonococci was significantly lower. Binding of VN was inhibited by heparin in a concentration-dependent manner, indicating that the heparin binding sites present in VN or Opa50 may play an essential role in this interaction. Based on gentamicin invasion assays and confocal microscopy studies, VN binding was associated with an increased invasion of Opa50- and Opa30-expressing gonococci into HeLa cells. The ability of VN to mediate entry into epithelial cells may constitute an important event in the pathogenesis of local as well as disseminated gonococcal infections.
Animals↗
Maintenance of broad-host-range incompatibility group P and group Q plasmids and transposition of Tn5 in Bartonella henselae following conjugal plasmid transfer from Escherichia coli.
The first demonstration of conjugal plasmid transfer from Escherichia coli to Bartonella henselae is reported. Transconjugants bearing plasmids of incompatibility groups P (IncP) and Q (IncQ), expressing various resistance markers, were generated. Tn5 transposons delivered on suicide plasmids by conjugation showed transpositional insertion into random chromosomal sites.
Bartonella henselae↗
Interaction of Bartonella henselae with endothelial cells results in bacterial aggregation on the cell surface and the subsequent engulfment and internalisation of the bacterial aggregate by a unique structure, the invasome.
Vascular colonisation by Bartonella henselae may cause vaso-proliferative tumour growth with clumps of bacteria found in close association with proliferating endothelial cells. By using B. henselae-infected human umbilical vein endothelial cells as an in vitro model for endothelial colonisation, we report here on a novel mechanism of cellular invasion by bacteria. First, the leading lamella of endothelial cells establishes cellular contact to sedimented bacteria and mediates bacterial aggregation by rearward transport on the cell surface. Subsequently, the formed bacterial aggregate is engulfed and internalised by a unique host cellular structure, the invasome. Completion of this sequence of events requires 24 hours. Cortical F-actin, intercellular adhesion molecule-1 and phosphotyrosine are highly enriched in the membrane protrusions entrapping the bacterial aggregate. Actin stress fibres, which are anchored to the numerous focal adhesion plaques associated with the invasome structure, are typically found to be twisted around its basal part. The formation of invasomes was found to be inhibited by cytochalasin D but virtually unaffected by nocodazole, colchicine or taxol, indicating that invasome-mediated invasion is an actin-dependent and microtubuli-independent process. Bacterial internalisation via the invasome was consistently observed with several clinical isolates of B. henselae, while a spontaneous mutant obtained from one of these isolates was impaired in invasome-mediated invasion. Instead, this mutant showed increased uptake of bacteria into perinuclear localising phagosomes, suggesting that invasome-formation may interfere with this alternative mechanism of bacterial internalisation. Internalisation via the invasome represents a novel paradigm for the invasion of bacteria into host cells which may serve as a cellular colonisation mechanism in vivo, e.g. on proliferating and migrating endothelial cells during Bartonella-induced vaso-proliferative tumour growth.
Actins↗
The secreted Ipa complex of Shigella flexneri promotes entry into mammalian cells.
The bacterial pathogen Shigella flexneri causes bacillary dysentery in humans by invading coloncytes. Upon contact with epithelial cells, S. flexneri elicits localized plasma membrane projections sustained by long actin filaments which engulf the microorganism. The products necessary for Shigella entry include three secretory proteins: IpaB, IpaC, and IpaD. Extracellular IpaB and IpaC associate in a soluble complex, the Ipa complex. We have immunopurified this Ipa complex on latex beads and found that they were efficiently internalized into HeLa cells. Like S. flexneri entry, uptake of the beads bearing the Ipa complex was associated with membrane projections and polymerization of actin at the site of cell-bead interaction and was dependent on small Rho GTPases. These results indicate that a secreted factor can promote S. flexneri entry into epithelial cells.
Actins↗
Bacterial entry into epithelial cells: the paradigm of Shigella.
Shigella flexneri is a model for the entry of bacterial pathogens into nonphagocytic epithelial cells. On contact with the epithelial cell surface, the Ipa proteins are secreted from the bacterium. The Ipa complex then triggers a reorganization of the host-cell cytoskeleton leading to the formation of membrane ruffles, which engulf the bacterium.
Bacterial Adhesion↗
Invasion of epithelial cells by Shigella flexneri induces tyrosine phosphorylation of cortactin by a pp60c-src-mediated signalling pathway.
Shigella flexneri causes bacillary dysentery in humans by invading epithelial cells of the colon. Cell invasion occurs via bacterium-directed phagocytosis, a process requiring polymerization of actin at the site of bacterial entry. We show that invasion of HeLa cells by S.flexneri induces tyrosine phosphorylation of cortactin, a host cell protein previously identified as a cytoskeleton-associated protein tyrosine kinase (PTK) substrate for the proto-oncoprotein pp60c-src. Immunolocalization experiments indicate that cortactin is recruited to submembranous actin filaments formed during bacterial entry. In particular, cortactin is highly enriched in membrane ruffles of the entry structure, which engulf entering bacteria, and also in the periphery of the phagosome early after bacterial internalization. The proto-oncoprotein pp60c-src appears to mediate tyrosine phosphorylation of cortactin, since overexpression of this PTK in HeLa cells specifically increases the level of cortactin tyrosine phosphorylation induced during bacterial entry. Immunolocalization studies in pp60c-src-overexpressing HeLa cells indicate that pp60c-src is recruited to the entry structure and to the periphery of the phagosome, where pp60c-src appears to accumulate in association with the membrane. Our results suggest that epithelial cell invasion by S.flexneri involves recruitment and kinase activation of pp60c-src. Signalling by the proto-oncoprotein pp60c-src may play a role in cytoskeletal changes that facilitate S.flexneri uptake into epithelial cells, since transient overexpression of pp60c-src in HeLa cells can provoke membrane ruffling and appears also to stimulate bacterial uptake of a non-invasive S.flexneri strain.
Actins↗