Site-directed mutagenesis of intimin alpha modulates intimin-mediated tissue tropism and host specificity.

The hallmark of enteropathogenic (EPEC) and enterohaemorrhagic (EHEC) Escherchia coli adhesion to host cells is intimate attachment leading to the formation of distinctive 'attaching and effacing' lesions. This event is mediated, in part, by binding of the bacterial adhesion molecule intimin to a second bacterial protein, Tir, delivered by a type III secretion system into the host cell plasma membrane. The receptor-binding activity of intimin is localized to the C-terminal 280 amino acids (Int280) and at least five distinct intimin types (alpha, beta, gamma, delta and epsilon) have been identified thus far.

Salmonella enterica serovar Typhi possesses a unique repertoire of fimbrial gene sequences.

Salmonella enterica serotype Typhi differs from nontyphoidal Salmonella serotypes by its strict host adaptation to humans and higher primates. Since fimbriae have been implicated in host adaptation, we investigated whether the serotype Typhi genome contains fimbrial operons which are unique to this pathogen or restricted to typhoidal Salmonella serotypes. This study established for the first time the total number of fimbrial operons present in an individual Salmonella serotype.

Yersinia pestis pFra shows biovar-specific differences and recent common ancestry with a Salmonella enterica serovar Typhi plasmid.

Population genetic studies suggest that Yersinia pestis, the cause of plague, is a clonal pathogen that has recently emerged from Yersinia pseudotuberculosis. Plasmid acquisition is likely to have been a key element in this evolutionary leap from an enteric to a flea-transmitted systemic pathogen. However, the origin of Y.

Mucosal vaccines: non toxic derivatives of LT and CT as mucosal adjuvants.

Most vaccines are still delivered by injection. Mucosal vaccination would increase compliance and decrease the risk of spread of infectious diseases due to contaminated syringes. However, most vaccines are unable to induce immune responses when administered mucosally, and require the use of strong adjuvant on effective delivery systems.

Immunomodulation using bacterial enterotoxins.

Immunologic unresponsiveness (tolerance) is a key feature of the mucosal immune system, and deliberate vaccination by a mucosal route can effectively induce immune suppression. However, some bacterial-derived proteins, e.g.

Salmonella: immune responses and vaccines.

Salmonella infections are a serious medical and veterinary problem world-wide and cause concern in the food industry. Vaccination is an effective tool for the prevention of Salmonella infections. Host resistance to Salmonella relies initially on the production of inflammatory cytokines leading to the infiltration of activated inflammatory cells in the tissues.

Typhoid fever and genetic polymorphisms at the natural resistance-associated macrophage protein 1.

Control of Salmonella enterica serovar Typhimurium (S. typhimurium) infection in the mouse model of typhoid fever is critically dependent on the natural resistance-associated macrophage protein 1 (Nramp1). In this study, we examined the role of genetic polymorphisms in the human homologue, NRAMP1, in resistance to typhoid fever in southern Vietnam.

Serology of typhoid fever in an area of endemicity and its relevance to diagnosis.

Currently, the laboratory diagnosis of typhoid fever is dependent upon either the isolation of Salmonella enterica subsp. enterica serotype Typhi from a clinical sample or the detection of raised titers of agglutinating serum antibodies against the lipopolysaccharide (LPS) (O) or flagellum (H) antigens of serotype Typhi (the Widal test). In this study, the serum antibody responses to the LPS and flagellum antigens of serotype Typhi were investigated with individuals from a region of Vietnam in which typhoid is endemic, and their usefulness for the diagnosis of typhoid fever was evaluated.

The Escherichia coli gene pool.

Wild Escherichia coli are superbly adapted to survive in the intestines of their mammalian hosts and in the environment. E. coli K12 derivative (MG1655) encodes 4288 potential genes that provide the background genetic framework of this species.

Early responses to Salmonella typhimurium infection in mice occur at focal lesions in infected organs.

Salmonella typhimurium causes an invasive disease in mice that has similarities to human typhoid, with key roles for cytokines and possibly also inducible nitric oxide synthase (iNOS), in mediating host responses to infection. In this paper we demonstrate that iNOS mRNA, protein and enzyme activity is induced within spleens and livers of infected mice as early as 5 h post-infection. Immunohistochemistry and in situ hybridization indicated that iNOS expression occurs predominantly in macrophages in localized, discrete foci in the infected organs.

Mucosal delivery of a respiratory syncytial virus CTL peptide with enterotoxin-based adjuvants elicits protective, immunopathogenic, and immunoregulatory antiviral CD8+ T cell responses.

In an effort to develop a safe and effective vaccine against respiratory syncytial virus (RSV), we used Escherichia coli heat-labile toxin (LT), and LTK63 (an LT mutant devoid of ADP-ribosyltransferase activity) to elicit murine CD8(+) CTL responses to an intranasally codelivered CTL peptide from the second matrix protein (M2) of RSV. M2(82-90)-specific CD8(+) T cells were detected by IFN-gamma enzyme-linked immunospot and (51)Cr release assay in local and systemic lymph nodes, and their induction was dependent on the use of a mucosal adjuvant. CTL elicited by peptide immunization afforded protection against RSV challenge, but also enhanced weight loss.

Interaction of tetanus toxin derived hybrid proteins with neuronal cells.

The non-toxic ganglioside binding domain of tetanus toxin (Hc fragment C or TTC) has been studied as a vector for delivering therapeutic proteins to neurons. There is little information on the cellular processing of proteins delivered by linkage to TTC. We have evaluated the cellular handling of a multi-domain hybrid protein containing TTC and both the human enzyme superoxide dismutase and the maltose binding protein from E.

Genes of the class II and class III major histocompatibility complex are associated with typhoid fever in Vietnam.

The influence of genes of the major histocompatibility complex (MHC) class II and class III loci on typhoid fever susceptibility was investigated. Individuals with blood culture-confirmed typhoid fever and control subjects from 2 distinct geographic locations in southern Vietnam were genotyped for HLA-DRB1 and HLA-DQB1 alleles, the gene that encodes tumor necrosis factor (TNF)-alpha (TNFA [-238] and TNFA [-308]), the gene that encodes lymphotoxin-alpha, and alleles of the TNF-alpha microsatellite. HLA-DRB1*0301/6/8, HLA-DQB1*0201-3, and TNFA*2 (-308) were associated with susceptibility to typhoid fever, whereas HLA-DRB1*04, HLA-DQB1*0401/2, and TNFA*1 (-308) were associated with disease resistance.

Influence of codon usage on the immunogenicity of a DNA vaccine against tetanus.

Two related DNA vaccine vector plasmids, harbouring either wild-type (pcDNA3/ntetC) or synthetic codon optimised (pcDNA3/stetC) DNA encoding fragment C (TetC) of tetanus toxin were constructed. COS-7 cells transformed with pcDNA3/stetC reproducibly expressed higher levels of TetC than similar cells transformed with pcDNA3/ntetC. BALB/c mice immunised intramuscularly with pcDNA3/stetC produced significantly higher levels of anti-TetC antibodies in their serum in the weeks following vaccination compared to mice immunised with pcDNA3/ntetC, even when differences in the CpG content between the two sequences were controlled for using non-expressing DNA.

Inhibition of T-cell response by Escherichia coli heat-labile enterotoxin-treated epithelial cells.

Escherichia coli heat-labile enterotoxin (LT) is an extensively studied adjuvant of mucosal responses. Nevertheless, its mode of action as an adjuvant remains incompletely understood. In this study, we describe a simplified in vitro model with which to look at some aspects of immunoregulation by LT.

Susceptibility of calves to challenge with Salmonella typhimurium 4/74 and derivatives harbouring mutations in htrA or purE.

Salmonella typhimurium 4/74 is highly virulent for cattle after oral challenge, causing severe diarrhoea, which is sometimes associated with systemic spread of the micro-organism. Although susceptible to oral challenge, groups of cattle were found to be relatively resistant to subcutaneous challenge with this strain. The virulence of S.

Salmonella typhimurium as a basis for a live oral Echinococcus granulosus vaccine.

A live attenuated Salmonella typhimurium vaccine candidate, LVR01, was constructed by introducing a null deletion into the aroC gene of the parental canine S. typhimurium isolate, P228067. LVR01 was used to orally deliver to the canine immune system a fatty acid binding protein (FABP) from Echinococcus granulosus (EgDf1), as a fusion protein with fragment C (TetC) of tetanus toxin.

Safety and immune responses to attenuated Salmonella enterica serovar typhi oral live vector vaccines expressing tetanus toxin fragment C.

Attenuated Salmonella enterica serovar Typhi vaccine strain CVD 908-htrA was used as a vector to deliver fragment C of tetanus toxin as a single-dose oral tetanus vaccine candidate to elicit protective levels of serum tetanus antitoxin. Twenty-one healthy adult volunteers received doses of 1.6 x 10(7) to 8.

Protective effect of supplemental superoxide dismutase on survival of neuronal cells during starvation. Requirement for cytosolic distribution.

There is evidence that raising cellular levels of Cu2+/Zn2+ superoxide dismutase (SOD1) can protect neurons from oxidative injury. We compared a novel method of elevating neuronal SOD activity using a recombinant hybrid protein composed of the atoxic neuronal binding domain of tetanus toxin (C fragment or TTC) and human SOD1 (hSOD1) with increasing cellular SOD levels through overexpression. Fetal murine cortical neurons or N18-RE-105 cells were incubated with the TTC-hSOD1 hybrid protein and compared to cells constitutively expressing hSOD1 for level of SOD activity, cellular localization of hSOD1, and capacity to survive glucose and pyruvate starvation.

Interaction of Bordetella pertussis with human respiratory mucosa in vitro.

The human respiratory tract pathogen Bordetella pertussis is the major cause of whooping cough in infants and young children, and also causes chronic cough in adults. B. pertussis infection damages ciliated epithelium in the respiratory tract.

Enterohaemorrhagic Escherichia coli O157:H7 target Peyer's patches in humans and cause attaching/effacing lesions in both human and bovine intestine.

Background: Enterohaemorrhagic Escherichia coli (EHEC) constitute a significant risk to human health worldwide, and infections, particularly with serogroup O157:H7, are associated with consumption of a variety of food and water vehicles, particularly food of bovine origin. EHEC cause acute gastroenteritis, bloody diarrhoea, and haemorrhagic colitis; up to 10% of cases develop severe complications, including the haemolytic uraemic syndrome, with a 5% case fatality. A virulence characteristic of enteropathogenic E coli, the attaching/effacing lesion, is considered to be important in EHEC.

Antimicrobial actions of the NADPH phagocyte oxidase and inducible nitric oxide synthase in experimental salmonellosis. II. Effects on microbial proliferation and host survival in vivo.

The roles of the NADPH phagocyte oxidase (phox) and inducible nitric oxide synthase (iNOS) in host resistance to virulent Salmonella typhimurium were investigated in gp91phox(-/)-, iNOS(-/)-, and congenic wild-type mice. Although both gp91phox(-/)- and iNOS(-/)- mice demonstrated increased susceptibility to infection with S. typhimurium compared with wild-type mice, the kinetics of bacterial replication were dramatically different in the gp91phox(-/)- and iNOS(-/)- mouse strains.

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.

Transmission of IgA and IgG monoclonal antibodies to mucosal fluids following intranasal or parenteral delivery.

Background: The efficacy by which passive antibodies can reach the lungs could be important for the outcome of immunotherapy of respiratory pulmonary infections. We examined how transmission to a number of mucosal sites is affected by the route of inoculation.

Methods: Transmission of newly raised IgA class Mabs against mycobacterial surface antigens to saliva, lung or vaginal lavage, bile and serum of BALB/c mice was compared with existing IgG Mabs.