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EspA-Intimin chimeric protein, a candidate vaccine against Escherichia coli O157:H7.

ABSTRACT: BACKGROUND AND OBJECTIVE:Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an important enteric pathogen in human causing bloody or nonbloody diarrhea, which may be complicated by hemolytic uremic syndrome (HUS). Cattle are an important reservoir of EHEC. This research aims at vaccination with a divalent chimer protein composed of EspA120 and Intimin 282 and its preventive effect of EHEC O157 colonization in mice rectal epithelium. MATERIALS AND METHODS:A divalent recombinant EspA-Intimin (EI) protein containing EspA120 and Intimin280 attached with a linker was amplified from a trivalent construct and cloned in pET-28a (+) vector. The immunization was conducted in mice after expression and purification of the recombinant EI (rEI). RESULTS:Mice subcutaneously immunized with rEI, elicited significant rEI specific serum IgG antibodies and showed significantly decreased E.coli O157:H7 shedding compared to the control group. CONCLUSION:The chimeric recombinant protein induced strong humoral response as well as protection against oral challenges with live E.coli O157:H7.

SUBMITTER: Rad HS

PROVIDER: S-EPMC3895562 | biostudies-literature | 2013 Sep

REPOSITORIES: biostudies-literature

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EspA-Intimin chimeric protein, a candidate vaccine against Escherichia coli O157:H7.

Rad Hamid Sedighian HS Mousavi Seyed Latif SL Rasooli Iraj I Amani Jafar J Nadooshan Moohamad Reza Jalali MR

Iranian journal of microbiology 20130901 3

<h4>Background and objective</h4>Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an important enteric pathogen in human causing bloody or nonbloody diarrhea, which may be complicated by hemolytic uremic syndrome (HUS). Cattle are an important reservoir of EHEC. This research aims at vaccination with a divalent chimer protein composed of EspA120 and Intimin 282 and its preventive effect of EHEC O157 colonization in mice rectal epithelium.<h4>Materials and methods</h4>A divalent recombinant E ...[more]

PMID: 24475331

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Project description:Enterohemorrhagic Escherichia coli (EHEC) are important human pathogens, causing hemorrhagic colitis and hemolytic uraemic syndrome in humans. E. coli O157:H7 is the most common serotype associated with EHEC infections worldwide, although other non-O157 serotypes cause life-threatening infections. Cattle are a main reservoir of EHEC and intervention strategies aimed at limiting EHEC excretion from cattle are predicted to lower the risk of human infection. We have previously shown that immunization of calves with recombinant versions of the type III secretion system (T3SS)-associated proteins EspA, intimin and Tir from EHEC O157:H7 significantly reduced shedding of EHEC O157 from experimentally-colonized calves, and that protection could be augmented by the addition of H7 flagellin to the vaccine formulation. The main aim of the present study was to optimize our current EHEC O157 subunit vaccine formulations by identifying the key combinations of these antigens required for protection. A secondary aim was to determine if vaccine-induced antibody responses exhibited cross-reactive potential with antigens from other EHEC serotypes. Immunization with EspA, intimin and Tir resulted in a reduction in mean EHEC O157 shedding following challenge, but not the mean proportion of calves colonized. Removal of Tir resulted in more prolonged shedding compared with all other groups, whereas replacement of Tir with H7 flagellin resulted in the highest levels of protection, both in terms of reducing both mean EHEC O157 shedding and the proportion of colonized calves. Immunization of calves with recombinant EHEC O157 EspA, intimin and Tir resulted in the generation of antibodies capable of cross-reacting with antigens from non-O157 EHEC serotypes, suggesting that immunization with these antigens may provide a degree of cross-protection against other EHEC serotypes. Further studies are now required to test the efficacy of these vaccines in the field, and to formally test the cross-protective potential of the vaccines against other non-O157 EHEC.

Project description:Type III secretion systems of enteric bacteria enable translocation of effector proteins into host cells. Secreted proteins of verotoxigenic Escherichia coli O157 strains include components of a translocation apparatus, EspA, -B, and -D, as well as "effectors" such as the translocated intimin receptor (Tir) and the mitochondrion-associated protein (Map). This research has investigated the regulation of LEE4 translocon proteins, in particular EspA. EspA filaments could not be detected on the bacterial cell surface when E. coli O157:H7 was cultured in M9 minimal medium but were expressed from only a proportion of the bacterial population when cultured in minimal essential medium modified with 25 mM HEPES. The highest proportions of EspA-filamented bacteria were detected in late exponential phase, after which filaments were lost rapidly from the bacterial cell surface. Our previous research had shown that human and bovine E. coli O157:H7 strains exhibit marked differences in EspD secretion levels. Here it is demonstrated that the proportion of the bacterial population expressing EspA filaments was associated with the level of EspD secretion. The ability of individual bacteria to express EspA filaments was not controlled at the level of LEE1-4 operon transcription, as demonstrated by using both beta-galactosidase and green fluorescent protein (GFP) promoter fusions. All bacteria, whether expressing EspA filaments or not, showed equivalent levels of GFP expression when LEE1-4 translational fusions were used. Despite this, the LEE4-espADB mRNA was more abundant from populations with a high proportion of nonsecreting bacteria (low secretors) than from populations with a high proportion of secreting and therefore filamented bacteria (high secretors). This research demonstrates that while specific environmental conditions are required to induce LEE1-4 expression, a further checkpoint exists before EspA filaments are produced on the bacterial surface and secretion of effector proteins occurs. This checkpoint in E. coli O157:H7 translocon expression is controlled by a posttranscriptional mechanism acting on LEE4-espADB mRNA. The heterogeneity in EspA filamentation could arise from phase-variable expression of regulators that control this posttranscriptional mechanism.

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EspA-Intimin chimeric protein, a candidate vaccine against Escherichia coli O157:H7.

Publications

EspA-Intimin chimeric protein, a candidate vaccine against Escherichia coli O157:H7.

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