The Legionella pneumophila F-box protein Lpp2082 (AnkB) modulates ubiquitination of the host protein parvin B and promotes intracellular replication.

The environmental pathogen Legionella pneumophila encodes three proteins containing F-box domains and additional protein-protein interaction domains, reminiscent of eukaryotic SCF ubiquitin-protein ligases. Here we show that the F-box proteins of L. pneumophila strain Paris are Dot/Icm effectors involved in the accumulation of ubiquitinated proteins associated with the Legionella-containing vacuole. Single, double and triple mutants of the F-box protein encoding genes were impaired in infection of Acanthamoeba castellanii, THP-1 macrophages and human lung epithelial cells. Lpp2082/AnkB was essential for infection of the lungs of A/J mice in vivo, and bound Skp1, the interaction partner of the SCF complex in mammalian cells, similar to AnkB from strain AA100/130b. Using a yeast two-hybrid screen and co-immunoprecipitation analysis we identified ParvB a protein present in focal adhesions and in lamellipodia, as a target. Immunofluorescence analysis confirmed that ectopically expressed Lpp2082/AnkB colocalized with ParvB at the periphery of lamellipodia. Unexpectedly, ubiquitination tests revealed that Lpp2082/AnkB diminishes endogenous ubiquitination of ParvB. Based on these results we propose that L. pneumophila modulates ubiquitination of ParvB by competing with eukaryotic E3 ligases for the specific protein-protein interaction site of ParvB, thereby revealing a new mechanism by which L. pneumophila may employ translocated effector proteins to promote bacterial survival.

Prevalence of the lpfO113 gene cluster among Escherichia coli O157 isolates from different sources.

Domestic farm animals represent an important reservoir of infection for Shiga toxin-producing Escherichia coli (STEC). Nevertheless the bacterial factors required to colonise these hosts are poorly defined. In this study, the prevalence of a recently described fimbrial gene cluster, lpfO113, among human and animal isolates of STEC was investigated. lpfO113 has been shown to play a role in the adherence of STEC O113:H21 to epithelial cells. Here the presence of the lpfAO113 gene (predicted to encode a major fimbrial subunit) was examined by PCR in E. coli of serogroups O157 and O26 isolated from pigs (n=38), cattle (n=10), and humans (n=9). In addition, we tested for several other genetic virulence markers including Shiga toxin (stx), intimin (eae), the translocated intimin receptor (tir), EHEC-hemolysin (ehx) and F18 fimbriae (fedA). Overall 45 of the 57 strains (79%) possessed the lpfAO113 gene as determined by the presence of a 573 bp PCR product. Moreover, there was a close correlation between the presence of the lpfAO113 marker and the absence of the eae gene. lpfAO113 was found in all of pig isolates, suggesting a possible role in colonisation of the porcine host. In addition, several E. coli strains isolated from pigs had two fimbrial gene markers, fedA and lpfAO113. lpfAO113 was not present in strains of E. coli O157:H7 as described previously. Overall these results show that lpfAO113 is widely distributed among eae-negative E. coli isolates and thus may represent an important adherence factor in this group of pathogens.

Intimin type influences the site of human intestinal mucosal colonisation by enterohaemorrhagic Escherichia coli O157:H7.

BACKGROUND: Enterohaemorrhagic (EHEC) and enteropathogenic (EPEC) Escherichia coli epithelial cell adhesion is characterised by intimate attachment, and attaching and effacing (A/E) lesion formation. This event is mediated in part by intimin binding to another bacterial protein, Tir (translocated intimin receptor), which is exported by the bacteria and integrated into the host cell plasma membrane. Importantly, EPEC (O127:H6) and EHEC (O157:H7) express antigenically distinct intimin types known as intimin alpha and gamma, respectively. EHEC (O157:H7) colonises human intestinal explants although adhesion is restricted to the follicle associated epithelium of Peyer's patches. This phenotype is also observed with EPEC O127:H6 engineered to express EHEC intimin gamma. AIMS: To investigate the influence of intimin on colonisation of human intestine by E coli O157:H7, and intimin types on tissue tropism in humans. METHODS: Human intestinal in vitro organ culture with wild type and mutant strains of O157:H7 were employed. RESULTS: Introducing a deletion mutation in the eae gene encoding intimin gamma in EHEC (O157:H7) caused the strain (ICC170) to fail to colonise human intestinal explants. However, colonisation of Peyer's patches and A/E lesion formation were restored with intimin gamma expression from a plasmid (ICC170 (pICC55)). In contrast, complementing the mutation with intimin alpha resulted in a strain (ICC170 (pCVD438)) capable of colonising and producing A/E lesions on both Peyer's patch and other small intestinal explants. CONCLUSION: Intimin is necessary for human intestinal mucosal colonisation by E coli O157:H7. Intimin type influences the site of colonisation in a Tir type independent mechanism; intimin gamma appears to restrict colonisation to human follicle associated epithelium.

Coiled-coil domain of enteropathogenic Escherichia coli type III secreted protein EspD is involved in EspA filament-mediated cell attachment and hemolysis.

Many animal and plant pathogens use type III secretion systems to secrete key virulence factors, some directly into the host cell cytosol. However, the basis for such protein translocation has yet to be fully elucidated for any type III secretion system. We have previously shown that in enteropathogenic and enterohemorrhagic Escherichia coli the type III secreted protein EspA is assembled into a filamentous organelle that attaches the bacterium to the plasma membrane of the host cell. Formation of EspA filaments is dependent on expression of another type III secreted protein, EspD. The carboxy terminus of EspD, a protein involved in formation of the translocation pore in the host cell membrane, is predicted to adopt a coiled-coil conformation with 99% probability. Here, we demonstrate EspD-EspD protein interaction using the yeast two-hybrid system and column overlays. Nonconservative triple amino acid substitutions of specific EspD carboxy-terminal residues generated an enteropathogenic E. coli mutant that was attenuated in its ability to induce attaching and effacing lesions on HEp-2 cells. Although the mutation had no effect on EspA filament biosynthesis, it also resulted in reduced binding to and reduced hemolysis of red blood cells. These results segregate, for the first time, functional domains of EspD that control EspA filament length from EspD-mediated cell attachment and pore formation.

Immune response to infection with Mycobacterium ulcerans.

Mycobacterium ulcerans is a slow-growing, acid-fast bacillus that causes chronic necrotizing skin ulcers known as Buruli ulcers. Previously reported information on immunity to this mycobacterium is limited. We examined immune responses to M. ulcerans and M. bovis BCG in patients with M. ulcerans disease and in 20 healthy control subjects (10 tuberculin test positive and 10 tuberculin test negative). Cell-mediated immunity was assessed by stimulating peripheral blood mononuclear cells (PBMC) with whole mycobacteria and then measuring PBMC proliferation and the production of gamma interferon (IFN-gamma). Humoral immunity was assessed by immunoblotting. PBMC from all subjects showed significantly greater proliferation and IFN-gamma production in response to stimulation with living mycobacteria compared with killed cells. However, PBMC from subjects with past or current M. ulcerans disease showed significantly reduced proliferation and production of IFN-gamma in response to stimulation with live M. ulcerans or M. bovis than PBMC from healthy, tuberculin test-positive subjects (P < 0.001) and showed results in these assays comparable to those of tuberculin test-negative subjects (P > 0.2). Serum from 9 of 11 patients with M. ulcerans disease, but no control subject, contained antibodies to M. ulcerans. The results indicate that patients with M. ulcerans infection mount an immune response to M. ulcerans as evidenced by antibody production, but they demonstrate profound systemic T-cell anergy to mycobacterial antigens. These findings may explain some of the distinct clinical and pathological features of M. ulcerans-induced disease.

Expression of intimin gamma from enterohemorrhagic Escherichia coli in Citrobacter rodentium.

The carboxy-terminal 280 amino acids (Int280) of the bacterial adhesion molecule intimin include the receptor-binding domain. At least five different types of Int280, designated alpha, beta, gamma, delta, and epsilon, have been described based on sequence variation in this region. Importantly, the intimin types are associated with different evolutionary branches and contribute to distinct tissue tropism of intimin-positive bacterial pathogens. In this study we engineered a strain of Citrobacter rodentium, which normally displays intimin beta, to express intimin gamma from enterohemorrhagic Escherichia coli. We show that intimin gamma binds to the translocated intimin receptor (Tir) from C. rodentium and has the ability to produce attaching and effacing lesions on HEp-2 cells. However, C. rodentium expressing intimin gamma could not colonize orally infected mice or induce mouse colonic hyperplasia. These results suggest that intimin may contribute to host specificity, possibly through its interaction with a receptor on the host cell surface.

The type III protein translocation system of enteropathogenic Escherichia coli involves EspA-EspB protein interactions.

Enteropathogenic Escherichia coli (EPEC), like many bacterial pathogens, use a type III secretion system to deliver effector proteins across the bacterial cell wall. In EPEC, four proteins, EspA, EspB, EspD and Tir are known to be exported by a type III secretion system and to be essential for 'attaching and effacing' (A/E) lesion formation, the hallmark of EPEC pathogenicity. EspA was recently shown to be a structural protein and a major component of a large, transiently expressed, filamentous surface organelle which forms a direct link between the bacterium and the host cell. In contrast, EspB is translocated into the host cell where it is localized to both membrane and cytosolic cell fractions. EspA and EspB are required for translocation of Tir to the host cell membrane suggesting that they may both be components of the translocation apparatus. In this study, we show that EspB co-immunoprecipitates with the EspA filaments and that, during EPEC infection of HEp-2 cells, EspB localizes closely with EspA. Using a number of binding assays, we also show that EspB can bind and be copurified with EspA. Nevertheless, binding of EspA filaments to the host cell membranes occurred even in the absence of EspB. These results suggest that following initial attachment of the EspA filaments to the target cells, EspB is delivered into the host cell membrane and that the interaction between EspA and EspB may be important for protein translocation.

Antibody response of patients infected with verocytotoxin-producing Escherichia coli to protein antigens encoded on the LEE locus.

Sera from patients infected with verocytotoxin-producing Escherichia coli (VTEC) O157, from patients with antibodies to E. coli O157 lipopolysaccharide (LPS) and from healthy controls were examined for antibodies to proteins involved in expressing the attaching and effacing phenotype. After SDS-PAGE, purified recombinant intimin, EspA-filament structural protein, translocated protein EspB and three separate domains of the translocated intimin receptor (Tir) were tested for reaction with patients' sera by immunoblotting. An ELISA was also used to detect antibodies to intimin in sera from E. coli O157 LPS antibody-positive individuals. Seven of nine culture-positive patients and one control patient had antibodies to EspA. Five of these patients and two controls had serum antibodies to the intimin-binding region of Tir, whereas none of the sera contained antibodies binding to either of the intracellular domains of Tir. By immunoblotting, 10 of 14 culture-positive patients had antibodies to the conserved region of intimin, eight of whom were infected with E. coli O157 phage type 2. Thirty-six of 60 sera from culture-negative but E. coli O157 LPS antibody-positive patients had antibodies to intimin as determined by ELISA. The secreted proteins are expressed in vivo during infection and are considered as pathogenic markers. Antibodies to these proteins may form the basis of a serodiagnostic test for the detection of patients infected with VTEC which carry the locus for the enterocyte effacement pathogenicity island and provide an adjunct test to the established serological tests based on VTEC LPS.

Human colostrum and serum contain antibodies reactive to the intimin-binding region of the enteropathogenic Escherichia coli translocated intimin receptor.

BACKGROUND: In Brazil, enteropathogenic Escherichia coli (EPEC) diarrhoea is endemic in young infants. A characteristic feature of EPEC adhesion to host cells is intimate attachment leading to the formation of distinctive "attaching and effacing" (A/E) lesions on mammalian cells. Two genes directly involved in intimate adhesion, eae and tir, encode the adhesion molecule intimin and its translocated receptor Tir, respectively. The intimin-binding domain of Tir was recently mapped to the middle part of the polypeptide (Tir-M), and the amino (Tir-N) and carboxy (Tir-C) termini were found to be located within infected host cells. Recently, it was shown that colostrum samples from mothers living in Sao Paulo contain IgA-class antibodies reactive with a number of proteins associated with EPEC virulence. It has also been shown that patients infected with verocytotoxin-producing E. coli O157 can produce antibodies to Tir. In the current study antibody responses to the different Tir domains were analyzed in sera and colostrum samples collected in an EPEC-endemic area of Brazil. METHODS: Recombinant Tir, Tir-N, Tir-M, and Tir-C were expressed as His-tagged protein in E. coli BL21a and purified on nickel columns. Western blot analysis was used to investigate colostrum IgA- and serum IgG-class antibodies reactive with the Tir fragments. RESULTS: Anti-Tir IgG antibodies were detected in the serum of children, with (63%) or without (50%) diarrhoea. Anti-Tir IgA-class antibodies were detected in all the colostrum pools tested. With the use of both serum IgG- and colostrum IgA-class antibodies, an immunodominant domain of the Tir-polypeptide, Tir M, was identified. CONCLUSION: The intimin-binding region of Tir (Tir-M) is the immunodominant region of the polypeptide in humans. Both serum IgG-class and colostrum IgA-class antibodies reacted predominantly with the Tir-M domain.

The coiled-coil domain of EspA is essential for the assembly of the type III secretion translocon on the surface of enteropathogenic Escherichia coli.

Enteropathogenic E. coli (EPEC) utilize a type III secretion system to deliver virulence-associated effector proteins to the host cell. Four proteins, EspA, EspB, EspD, and Tir, which are integral to the formation of characteristic "attaching and effacing" (A/E) intestinal lesions, are known to be exported via the EPEC type III secretion system. Recent work demonstrated that EspA is a major component of a filamentous structure, elaborated on the surface of EPEC, which is required for translocation of EspB and Tir. The carboxyl terminus of EspA is predicted to comprise an alpha-helical region, which demonstrates heptad periodicity whereby positions a and d in the heptad repeat unit abcdefg are occupied by hydrophobic residues, indicating a propensity for coiled-coil interactions. Here we demonstrate multimeric EspA isoforms in EPEC culture supernatants and EspA:EspA interaction on solid phase. Non-conservative amino acid substitution of specific EspA heptad residues generated EPEC mutants defective in filament assembly but which retained the ability to induce A/E lesions; additional mutation totally abolished EspA filament assembly and A/E lesion formation. These results demonstrate a similarity to flagellar biosynthesis and indicate that the coiled-coil domain of EspA is required for assembly of the EspA filament-associated type III secretion translocon.

Binding of intimin from enteropathogenic Escherichia coli to Tir and to host cells.

Enteropathogenic Escherichia coli (EPEC) induce characteristic attaching and effacing (A/E) lesions on epithelial cells. This event is mediated, in part, by binding of the bacterial outer membrane protein, intimin, to a second EPEC protein, Tir (translocated intimin receptor), which is exported by the bacteria and integrated into the host cell plasma membrane. In this study, we have localized the intimin-binding domain of Tir to a central 107-amino-acid region, designated Tir-M. We provide evidence that both the amino- and carboxy-termini of Tir are located within the host cell. In addition, using immunogold labelling electron microscopy, we have confirmed that intimin can bind independently to host cells even in the absence of Tir. This Tir-independent interaction and the ability of EPEC to induce A/E lesions requires an intact lectin-like module residing at the carboxy-terminus of the intimin polypeptide. Using the yeast two-hybrid system and gel overlays, we show that intimin can bind both Tir and Tir-M even when the lectin-like domain is disrupted. These data provide strong evidence that intimin interacts not only with Tir but also in a lectin-like manner with a host cell intimin receptor.

Examination of Serpulina pilosicoli for attachment and invasion determinants of Enterobacteria.

The spirochaete, Serpulina pilosicoli, is the agent of intestinal spirochaetosis, a diarrhoeal disease of humans and other species. By mechanisms as yet unknown, large numbers of these spirochaetes intimately attach to the colonic mucosa by one cell end. In some infected individuals, the spirochaetes may invade the lamina propria and adjacent tissues, and they may cause spirochaetaemia. To examine S. pilosicoli for pathogenic determinants homologous with Enterobacteria, DNA was extracted from six strains of S. pilosicoli and hybridised at low stringency with DNA probes derived from the inv, ail and yadA genes of Yersinia enterocolitica, the eae gene from enteropathogenic Escherichia coli and a probe derived from the virulence plasmid of Shigella flexneri. No hybridisation of the enterobacterial probes to S. pilosicoli DNA was detected, indicating that these gene sequences, which are known to be involved in the attachment and invasion processes of the other intestinal pathogens, were not present in the spirochaetes.

In vitro association between the virulence proteins, YopD and YopE, of Yersinia enterocolitica.

The virulence plasmid (pYV) of Y. enterocolitica encodes a set of anti-host proteins, known as Yops, which contribute to survival of the bacteria in the host. Several Yops directly influence the interaction of pathogenic bacteria with host cells which in the case of at least four Yops, YopE, YopH, YopM and YpkA (YopO), involves translocation of the Yop across the eukaryotic cell membrane into the cytoplasm of target cells. Translocation requires the presence of two other pYV-encoded proteins, YopB and YopD, but it is not clear how these proteins mediate translocation of the effector Yops. We have used an affinity blot technique (overlay) to examine potential binding between YopD and other Yops. The results indicated that under the in vitro conditions used in this study, YopD bound preferentially to YopE and YopB. To investigate the interaction of YopD and YopE in more detail we produced different regions of the YopD protein in Escherichia coli by creating fusions of YopD with glutathione S-transferase. These studies showed that YopE bound the central hydrophobic region of YopD. This is the first demonstration of protein interactions between an effector Yop and a putative Yop translocator.

Differential secretion of interleukin-8 by human epithelial cell lines upon entry of virulent or nonvirulent Yersinia enterocolitica.

Epithelial cells of the intestinal mucosa are among the first cells encountered by invasive pathogens. Bacterial invasion of the mucosa gives rise to an inflammatory response, characterized by the influx of polymorphonuclear leukocytes. The chemotactic stimulus responsible for this accumulation is unknown, but several in vitro studies have demonstrated that epithelial cells secrete the chemokine interleukin-8 (IL-8), a potent chemoattractant of polymorphonuclear leukocytes, upon bacterial entry. In this study we analyzed the secretion of IL-8 by human intestinal (T84) and cervical (HeLa) epithelial cell lines in response to infection with the enteric pathogen Yersinia enterocolitica. IL-8 was secreted by T84 and HeLa cells in response to invasion by Y. enterocolitica. Virulent Y. enterocolitica induced a significantly lower level of IL-8 secretion than nonvirulent Y. enterocolitica. Subsequent analysis employing a mutant defective in Yop secretion and various yop mutants showed that the reduced secretion of IL-8 is due to the presence of Yop proteins. Our data suggest that YopB and YopD are required for the suppressive effect.

Contribution of YopB to virulence of Yersinia enterocolitica.

The 70-kb virulence plasmid, pYV, of Yersinia enterocolitica encodes a number of secreted proteins (Yops) which are essential for virulence. YopD, the 33-kDa product of the lcrGVHyopBD operon, appears to be involved in delivering YopE and YopH (the Yersinia protein tyrosine phosphatase) into target cells. These proteins then act in concert to cause cytotoxicity in host cells. Previously, we reported that bacteria carrying transposon insertions in yopD are not cytotoxic for macrophages, show impaired tyrosine phosphatase activity in host cells, and are avirulent for mice (E. L. Hartland, S. P. Green, W. A. Phillips, and R. M. Robins-Browne, Infect. Immun. 62:4445-4453, 1994). trans complementation of yopD mutants of Y. enterocolitica with the yopD gene restores all these properties. In this study, we show that polar mutations in proximal genes of the lcrGVHyopBD operon also abrogated bacterial virulence and the capacity to induce cytotoxicity in mouse bone marrow-derived macrophages and HEp-2 epithelial cells. Moreover, trans complementation of a yopBD mutant with the yopD gene alone was not sufficient to restore the ability of the bacteria to cause cytotoxicity. Further work showed that YopB was required for cytotoxicity, dephosphorylation of host proteins, and virulence for mice. These findings indicate that YopB and YopD may serve a related function in Y. enterocolitica and that they may act together to deliver intracellularly acting Yops to their respective targets in host cells.

Role of YopH in the suppression of tyrosine phosphorylation and respiratory burst activity in murine macrophages infected with Yersinia enterocolitica.

Tyrosine phosphorylation is an important component of the signaling pathways responsible for the activation of the macrophage respiratory burst. Because the virulence plasmid of Yersinia enterocolitica encodes a phosphotyrosine phosphatase, YopH, it is possible that the pathogenic strategy of Y. enterocolitica involves the disruption of tyrosine phosphorylation in the macrophage leading to inhibition of respiratory burst activity. We have investigated the effects of Yersinia infection on tyrosine phosphorylation and respiratory burst activity in murine bone marrow-derived macrophages. Infection of macrophages with virulent [Ye(pYV+)] but not avirulent [Ye(pYV-)] strains of Y. enterocolitica was found to suppress both tyrosine phosphorylation and respiratory burst activity in response to zymosan. Mutational inactivation of YopH reversed the suppressive effect of Ye(pYV+) on zymosan-induced tyrosine phosphorylation, indicating that YopH is responsible for the dephosphorylation of macrophage phosphotyrosine-containing proteins observed in macrophages infected with the virulent strain of Y. enterocolitica. In contrast, mutational loss of YopH failed to reverse the inhibitory effect of Ye(pYV+) on the zymosan-triggered respiratory burst. We conclude that the inhibition of the macrophage respiratory burst by Y. enterocolitica involves a plasmid-encoded virulence protein(s) other than, or in addition to, YopH.

Essential role of YopD in inhibition of the respiratory burst of macrophages by Yersinia enterocolitica.

The respiratory burst is a key element of the bactericidal armamentarium of phagocytes. In this study we have shown that a virulent strain of Yersinia enterocolitica serogroup O:9 completely inhibited the ability of murine bone marrow-derived macrophages to mount a respiratory burst in response to stimulation by zymosan. This property of the bacterium was abrogated by curing the strain of its 71.5-kb virulence plasmid and by transposon mutagenesis of the plasmid-borne yopD gene. Derivatives of the bacterium which were unable to inhibit the respiratory burst were also less able to disrupt cytoskeletal actin and to resist phagocytosis. yopD mutants also showed an impaired ability to dephosphorylate phosphotyrosine residues in macrophage proteins and were completely avirulent for mice. All of these defects were fully or partly restored by trans-complementation of a yopD mutant with a cloned yopD gene. The results of this study and those of previous work with YopD (R. Rosqvist, A. Forsberg, and H. Wolf-Watz, Infect. Immun. 59:4562-4569, 1991) suggest that YopD functions chiefly by facilitating the transport of virulence plasmid-encoded proteins, such as YopE, a cytotoxin, and YopH, a protein tyrosine phosphatase, across the cytoplasmic membrane to their targets within host cells. The combined action of these Yops on cytoplasmic proteins, especially actin, could explain the effects of virulent Y. enterocolitica on macrophage morphology, phagocytic capacity, and respiratory burst activity, all of which rely on cytoskeletal integrity to function normally.