Identification of in vivo-induced bacterial protein antigens during human infection with Salmonella enterica serovar Typhi

We applied an immunoscreening technique, in vivo-induced antigen technology (IVIAT), to identify immunogenic bacterial proteins expressed during human infection with Salmonella enterica serovar Typhi, the cause of typhoid fever. We were able to assign a functional classification to 25 of 35 proteins identified by IVIAT. Of these 25, the majority represent proteins with known or potential roles in the pathogenesis of S. enterica. These include proteins implicated in fimbrial structure and biogenesis, antimicrobial resistance, heavy metal transport, bacterial adhesion, and extracytoplasmic substrate trafficking as well as secreted hydrolases. The 10 remaining antigens represent proteins with unknown functions. Of the 35 identified antigens, four had no immunoreactivity when probed with control sera from individuals never exposed to serovar Typhi organisms; these four included PagC, TcfB, and two antigens of unknown function encoded by STY0860 and STY3683. PagC is a virulence factor known to be upregulated in vivo in S. enterica serovar Typhimurium infection of mice. TcfB is the major structural subunit of a fimbrial operon found in serovar Typhi with no homolog in serovar Typhimurium organisms. By examining differential immunoreactivities in acute- versus convalescent-phase human serum samples, we found specific anti-PagC and anti-TcfB immunoglobulin G responses in patients with serovar Typhi bacteremia. Serovar Typhi antigens identified by IVIAT warrant further evaluation for their contributions to pathogenesis, and they may have diagnostic, therapeutic, or preventive uses.     

Identification of group A Streptococcus antigenic determinants upregulated in vivo

Group A Streptococcus (GAS) causes a range of diseases in humans, from mild noninvasive infections to severe invasive infections. The molecular basis for the varying severity of disease remains unclear. We identified genes expressed during invasive disease using in vivo-induced antigen technology (IVIAT), applied for the first time in a gram-positive organism. Convalescent-phase sera from patients with invasive disease were pooled, adsorbed against antigens derived from in vitro-grown GAS, and used to screen a GAS genomic expression library. A murine model of invasive GAS disease was included as an additional source of sera for screening. Sequencing DNA inserts from clones reactive with both human and mouse sera indicated 16 open reading frames with homology to genes involved in metabolic activity to genes of unknown function. Of these, seven genes were assessed for their differential expression by quantitative real-time PCR both in vivo, utilizing a murine model of invasive GAS disease, and in vitro at different time points of growth. Three gene products-a putative penicillin-binding protein 1A, a putative lipoprotein, and a conserved hypothetical protein homologous to a putative translation initiation inhibitor in Vibrio vulnificus-were upregulated in vivo, suggesting that these genes play a role during invasive disease.     

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.     

Reactivity of convalescent-phase hemolytic-uremic syndrome patient sera with the megaplasmid-encoded TagA protein of Shiga toxigenic Escherichia coli O157

A cosmid library of Shiga toxigenic Escherichia coli (STEC) O157:H7 strain EDL933 DNA was screened for clones capable of reacting with convalescent-phase serum from a patient with hemolytic-uremic syndrome (HUS), in an attempt to identify candidate virulence genes. One of the immunoreactive clones contained a portion of the large plasmid pO157, and the immunoreactive gene product was identified as TagA. The function of this 898-amino-acid protein is unknown, but it exhibits 42% amino acid sequence identity and 63% similarity to a 312-amino-acid region of a ToxR-regulated lipoprotein of Vibrio cholerae. Antibodies to E. coli O157 TagA were detected in sera from other HUS patients with O157 STEC infection but not in those from patients whose illnesses were caused by other STEC types or in healthy controls. These data demonstrate that TagA is expressed in vivo and provide circumstantial evidence for a role in the pathogenesis of the disease. The tagA gene is present only in STEC strains belonging to serogroup O157, and so antibodies to TagA are a potentially useful serological marker for infections due to such strains.     

Role of intimin-tir interactions and the tir-cytoskeleton coupling protein in the colonization of calves and lambs by Escherichia coli O157:H7

Intimin facilitates intestinal colonization by enterohemorrhagic Escherichia coli O157:H7; however, the importance of intimin binding to its translocated receptor (Tir) as opposed to cellular coreceptors is unknown. The intimin-Tir interaction is needed for optimal actin assembly under adherent bacteria in vitro, a process which requires the Tir-cytoskeleton coupling protein (TccP/EspF(U)) in E. coli O157:H7. Here we report that E. coli O157:H7 tir mutants are at least as attenuated as isogenic eae mutants in calves and lambs, implying that the role of intimin in the colonization of reservoir hosts can be explained largely by its binding to Tir. Mutation of tccP uncoupled actin assembly from the intimin-Tir-mediated adherence of E. coli O157:H7 in vitro but did not impair intestinal colonization in calves and lambs, implying that pedestal formation may not be necessary for persistence. However, an E. coli O157:H7 tccP mutant induced typical attaching and effacing lesions in a bovine ligated ileal loop model of infection, suggesting that TccP-independent mechanisms of actin assembly may operate in vivo.     

Identification and Testing of Porphyromonas gingivalis Virulence Genes with a pPGIVET System

An in vivo expression technology (IVET) system was designed to identify previously unknown virulence genes of Porphyromonas gingivalis. Fourteen ivi (for in vivo induced) genes that are induced during infection in a mouse abscess model were identified in our study. Of these, seven had homology to genes in the NCBI database, and the rest had no homology to reported DNA sequences. In order to determine virulence-related properties of these genes, three mutant strains, deleted of ivi8 (no homology to genes in the database), ivi10 (homologous to a putative TonB-dependent outer membrane receptor protein), and ivi11 (an immunoreactive 33-kDa antigen PG125 in P. gingivalis), were created. The mutants were tested in a mouse abscess model for alterations in virulence relative to the wild type by a competition assay in BALB/c mice. After 5 days we observed the enrichment of the wild-type strain over mutant strains Deltaivi10 and Deltaivi11, which indicated that mutant strains Deltaivi10 and Deltaivi11 are less able to survive in this model than the wild-type strain, while Deltaivi8 survives as well as the wild-type strain. We propose that knockout of these ivi genes reduced the ability of the mutated P. gingivalis to survive and cause infection compared to the wild-type strain at the site of injection. Also, in separate experiments, groups of mice were challenged with subcutaneous injections of each individual mutant strain (Deltaivi8, Deltaivi10, and Deltaivi11) or with the wild-type strain alone and were then examined to assess their general health status. The results showed that knockout of these ivi genes conferred a reduction in virulence. The ability of the mutants to invade KB cells compared to the wild type was also determined. Interestingly, the CFU counts of the mutant strain Deltaivi10 recovered from KB cells were eight times lower than those of the wild type, indicating that this mutant has a lower capacity for invasion. These results demonstrate that IVET is a powerful tool in discovering virulence genes and the significant role that ivi genes play in the pathogenesis of this species.     

Distinct binding properties of eaeA-negative verocytotoxin-producing Escherichia coli of serotype O113:H21.

Infection of humans with verotoxin-producing Escherichia coli (VTEC) O113:H21 is associated with clinical features comparable to those associated with infection with attaching and effacing VTEC strains including those of serotype O157:H7. We have shown previously that the adhesion phenotype of VTEC O157:H7 is influenced by the presence of a homolog of the chromosomal eaeA (for E. coli attaching and effacing) gene. In contrast, by colony blot hybridization, VTEC O113:H21 is negative for the eaeA gene. Therefore, the aim of this study was to define the adhesion phenotype of VTEC O113:H21 strain CL-15 to both cultured epithelial cells (HEp-2) and rabbit intestine in vivo. Under transmission electron microscopy, areas of microvillus effacement were observed in regions directly beneath the organism in CL-15-infected cells both in vitro and in vivo. However, F-actin adhesion pedestals on the host plasma membrane were absent. Failure of CL-15 to induce polymerization of actin was confirmed by using staining of F-actin with fluorescein-labeled phalloidin. Under indirect immunofluorescence microscopy, CL-15-infected HEp-2 cells also failed to demonstrate the recruitment of another cytoskeletal element, alpha-actinin, below foci of bacterial adhesion. In contrast, VTEC O157:H7 infection of HEp-2 cells was associated with increased alpha-actinin immunofluorescence. These findings suggest that bacterial factors distinct from those of EaeA are necessary for the adhesion phenotype of VTEC O113:H21.     

The kinetics of antibody production to antigens of Escherichia coli O157 in a pregnant woman with haemolytic uraemic syndrome

Sequential blood samples taken from a pregnant woman with haemolytic uraemic syndrome caused by verocytotoxin (VT)-producing Escherichia coli O157 were used to examine the kinetics of serum antibody production to E. coli O157 lipopolysaccharide (LPS), intimin and the conserved region of the translocated intimin receptor (Tir-M). Umbilical cord blood and two samples of blood from the newborn baby were also examined for antibodies to these antigens. In the mother, antibodies of the IgM class, specific for E. coli O157 LPS, were produced in the initial stages of the infection, reaching a peak at 9 days after onset of diarrhoea and subsiding 3 days later. High levels of IgG class antibodies, specific for E. coli O157 LPS, were detected 8 days after the onset of diarrhoea and were present at high titres on day 18. Serum antibodies of the IgA class to E. coli O157 LPS were not detected. Antibodies binding to Tir-M were detected 8 days after the onset of diarrhoea and high titres of these antibodies were still present on day 18. Serum antibodies to intimin were not detected in the mother and no antibodies to any of the antigens tested were detected in either the baby's blood or cord blood. This study describes for the first time the kinetics of serum antibody production during pregnancy, to selected antigens expressed by E. coli O157.     

toxB gene on pO157 of enterohemorrhagic Escherichia coli O157:H7 is required for full epithelial cell adherence phenotype

Adherence of enterohemorrhagic Escherichia coli (EHEC) to the intestinal epithelium is critical for initiation of a bacterial infection. An in vitro infection study previously indicated that EHEC bacteria initially adhere diffusely and then proliferate to develop MC, a process that is mediated by various secreted proteins, such as EspA, EspB, EspD, Tir, and intimin, as well as other putative adherence factors. In the present study, we investigated the role of a large 93-kb plasmid (pO157) in the adherence of O157:H7 (O157Sakai) and found the toxB gene to be involved in the full adherence phenotype. A pO157-cured strain of O157Sakai (O157Cu) developed microcolonies on Caco-2 cells; however, the number of microcolonies was lower than that of O157Sakai, as were the production and secretion levels of EspA, EspB, and Tir. Introduction of a mini-pO157 plasmid (pIC37) composed of the toxB and ori regions restored full adherence capacity to O157Cu, including production and secretion of the proteins. In contrast, introduction of a pO157 mutant possessing toxB::Km into O157Cu could not restore the full adherence phenotype. Expression of truncated versions of His-tagged ToxB also promoted EspB production and/or secretion by O157Cu. These results suggest that ToxB contributes to the adherence of EHEC to epithelial cells through promotion of the production and/or secretion of type III secreted proteins.     

Characterization of Escherichia coli O157:H7 and other Shiga toxin-producing E. coli serotypes isolated from sheep.

The isolation and characterization of Escherichia coli O157:H7 and non-O157 Shiga toxin-producing E. coli (STEC) strains from sheep are described. One flock was investigated for E. coli O157:H7 over a 16-month period that spanned two summer and two autumn seasons. Variation in the occurrence of E. coli O157:H7-positive sheep was observed, with animals being culture positive only in the summer months but not in the spring, autumn, or winter. E. coli O157:H7 isolates were distinguished by pulsed-field gel electrophoresis (PFGE) of chromosomal DNA and toxin gene restriction fragment length polymorphism (RFLP) analysis. Ten PFGE patterns and five RFLP patterns, identified among the isolates, showed that multiple E. coli O157:H7 strains were isolated from one flock, that a single animal simultaneously shed multiple E. coli O157:H7 strains, and that the strains shed by individuals changed over time. E. coli O157:H7 was isolated only by selective enrichment culture off 10 g of ovine feces. In contrast, strains of eight STEC serotypes other than O157:H7 were cultured from feces of sheep from a separate flock without enrichment. The predominant non-O157 STEC serotype found was O91:NM (NM indicates nonmotile), and others included O128:NM, O88:NM, O6:H49, and O5:NM. Irrespective of serotype, 98% of the ovine STEC isolates possessed various combinations of the virulence-associated genes for Shiga toxin(s) and the attaching-and-effacing lesion (stx1, stx2, and eae), suggesting their potential for human pathogenicity. The most common toxin-eae genotype was positive for stx1, stx2, and eae. A Vero cell cytotoxicity assay demonstrated that 90% of the representative STEC isolates tested expressed the toxin gene. The report demonstrates that sheep transiently shed a variety of STEC strains, including E. coli O157:H7, that have potential as human pathogens.     

Enterohemorrhagic Escherichia coli O157:H7 gal mutants are sensitive to bacteriophage P1 and defective in intestinal colonization

Enterohemorrhagic Escherichia coli (EHEC), especially E. coli O157:H7, is an emerging cause of food-borne illness. Unfortunately, E. coli O157 cannot be genetically manipulated using the generalized transducing phage P1, presumably because its extensive O antigen obscures the P1 receptor, the lipopolysaccharide (LPS) core subunit. The GalE, GalT, GalK, and GalU proteins are necessary for modifying galactose before it can be assembled into the repeating subunit of the O antigen. Here, we constructed E. coli O157:H7 gal mutants which presumably have little or no O antigen. These strains were able to adsorb P1. P1 lysates grown on the gal mutant strains could be used to move chromosomal markers between EHEC strains, thereby facilitating genetic manipulation of E. coli O157:H7. The gal mutants could easily be reverted to a wild-type Gal(+) strain using P1 transduction. We found that the O157:H7 galETKM::aad-7 deletion strain was 500-fold less able to colonize the infant rabbit intestine than the isogenic Gal(+) parent, although it displayed no growth defect in vitro. Furthermore, in vivo a Gal(+) revertant of this mutant outcompeted the galETKM deletion strain to an extent similar to that of the wild type. This suggests that the O157 O antigen is an important intestinal colonization factor. Compared to the wild type, EHEC gal mutants were 100-fold more sensitive to a peptide derived from bactericidal permeability-increasing protein, a bactericidal protein found on the surface of intestinal epithelial cells. Thus, one way in which the O157 O antigen may contribute to EHEC intestinal colonization is to promote resistance to host-derived antimicrobial polypeptides.     

Escherichia coli O157:H7 colonization in cattle following systemic and mucosal immunization with purified H7 flagellin

Escherichia coli O157:H7 is an important pathogen of humans. Cattle are most frequently identified as the primary source of infection, and therefore, reduction in E. coli O157:H7 prevalence in cattle by vaccination represents an attractive strategy for reducing the incidence of human disease. H7 flagella have been implicated in intestinal-epithelial colonization of E. coli O157:H7 and may represent a useful target for vaccination. In this study, calves were immunized either systemically with H7 flagellin by intramuscular injection or mucosally via the rectum with either H7 or H7 incorporated into poly(DL-lactide-co-glycolide) microparticles (PLG:H7). Systemic immunization resulted in high levels of flagellin-specific immunoglobulin G (IgG) and IgA in both serum and nasal secretions and detectable levels of both antibody isotypes in rectal secretions. Rectal administration of flagellin resulted in levels of rectal IgA similar to those by the intramuscular route but failed to induce any other antibody response, whereas rectal immunization with PLG:H7 failed to induce any H7-specific antibodies. Following subsequent oral challenge with E. coli O157:H7, reduced colonization rates and delayed peak bacterial shedding were observed in the intramuscularly immunized group compared to nonvaccinated calves, but no reduction in total bacterial shedding occurred. Rectal immunization with either H7 or PLG:H7 had no effect on subsequent bacterial colonization or shedding. Furthermore, purified H7-specific IgA and IgG from intramuscularly immunized calves were shown to reduce intestinal-epithelial binding in vitro. These results indicate that H7 flagellin may be a useful component in a systemic vaccine to reduce E. coli O157:H7 colonization in cattle.     

Identification of in vivo induced genes in Actinobacillus pleuropneumoniae.

We have developed an in vivo expression technology (IVET) system to identify Actinobacillus pleuropneumoniae gene promoters that are specifically induced in vivo during infection. This system is based upon an avirulent riboflavin-requiring A. pleuropneumoniae mutant and a promoter-trap vector (pTF86) that contains, in sequence, the T4 terminator, a unique Bam HI site, a promoterless copy of the V. harveyi luxAB genes, and a promoterless copy of the B. subtilis ribBAH genes in the E. coli - A. pleuropneumoniae shuttle vector pGZRS19. Sau 3A fragments of A. pleuropneumoniae genomic DNA were cloned into the Bam HI site in pTF86 and transformed into the A. pleuropneumoniae Rib- mutant. Pigs were infected with pools of 300-600 transformants by endobronchial inoculation and surviving bacteria were isolated from the pigs' lungs at 12-16 h post-infection. Infection strongly selected for transformants containing cloned promoters which drove expression of the vector ribBAH genes and allowed survival of the Rib- mutant in vivo. Strains that survived in vivo, but which minimally expressed luciferase activity in vitro, should contain cloned promoters that are specifically induced in vivo. Ten clones, designated iviA-J, were isolated which contain promoters that are induced in vivo during infection. These ivi clones were shown to be induced in the animal by luminescence of infected tissue and by direct assay of bacteria recovered from bronchoalveolar lavage. Four of these clones were putatively identified by amino acid sequence similarity as ilvI, the ilvDA operon, the secE-nusG operon, and the mrp gene. This is the first report of an IVET system for use in the family Pasteurellaceae, as well as the first report of an IVET system utilizing an infection model of pneumonia in the natural host.     

Characterization of flagella purified from enterohemorrhagic, vero-cytotoxin-producing Escherichia coli serotype O157:H7.

Escherichia coli of the serotype O157:H7 has recently been isolated in human fecal specimens in association with sporadic cases and outbreaks of hemorrhagic colitis and with the hemolytic uremic syndrome. The aim of this study was to characterize the flagellin protein subunit constituents of flagellar filaments from E. coli O157:H7 strain CL-56. Flagellin isolated from a reference Salmonella enteritidis strain was used for comparison. Flagella were dissociated by incubation of bacteria under acidic conditions, centrifugation, and differential ammonium sulfate precipitation. Reconstituted flagellar filaments were demonstrated by three complementary methods: transmission electron microscopy, antigenic reactivity with H7 antiserum by a dot blot immunoassay, and immunogold localization of antiserum raised to the purified antigen to intact flagella on whole E. coli O157:H7. On sodium dodecyl sulfate-polyacrylamide gels flagellin proteins from E. coli O157:H7 demonstrated an apparent Mr of 66,000. The isoelectric point of E. coli O157:H7 flagellin was 5.42. By immunoblotting, H7 flagellin proteins were shown to be immunogenic. They induced a systemic immune response both in rabbits challenged with whole bacteria and in a human previously infected with E. coli O157:H7.