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Characterization of the C-terminal domains of intimin-like proteins of enteropathogenic and enterohemorrhagic Escherichia coli, Citrobacter freundii, and Hafnia alvei.

ABSTRACT: Surface proteins called intimins (Int), which are homologous to the invasin protein (Inv) of Yersinia spp., play a role in inducing brush border damage, termed attachment and effacement, which follows infection by enteropathogenic and enterohemorrhagic Escherichia coli, Citrobacter freundii biotype 4280, and Hafnia alvei. Maltose-binding protein (MBP) fusions containing the C-terminal 280 amino acids of Int-like proteins of strains of enteropathogenic E. coli, enterohemorrhagic E. coli, H. alvei, and C. freundii biotype 4280 and of Yersinia pseudotuberculosis Inv were constructed and purified. The 3' end of the gene for the H. alvei Int-like protein was sequenced and showed homology to corresponding regions of other Int-encoding genes. Binding of MBP-Int-like and MBP-Inv fusion proteins to HEp-2 cells was demonstrated by immunofluorescence microscopy and by fluorescence-activated cell sorting. MBP-Inv induced attachment and spreading of HEp-2 cells to plastic-coated wells, but MBP-Int-like fusion proteins did not. Preincubation of HEp-2 cells with MBP-Inv, but not with MBP-Int-like fusion proteins, inhibited MBP-Inv-induced cell attachment. Fixed staphylococci and fluorescent polymer microspheres coated with both MBP-Int-like and MBP-Inv fusion proteins showed enhanced adhesion to HEp-2 cells. These fusion proteins will facilitate studies of the role of intimin in the pathogenesis of diarrhea associated with members of the family Enterobacteriaeceae that induce attachment and effacement.

SUBMITTER: Frankel G

PROVIDER: S-EPMC186420 | biostudies-other | 1994 May

REPOSITORIES: biostudies-other

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Characterization of the C-terminal domains of intimin-like proteins of enteropathogenic and enterohemorrhagic Escherichia coli, Citrobacter freundii, and Hafnia alvei.

Frankel G G Candy D C DC Everest P P Dougan G G

Infection and immunity 19940501 5

Surface proteins called intimins (Int), which are homologous to the invasin protein (Inv) of Yersinia spp., play a role in inducing brush border damage, termed attachment and effacement, which follows infection by enteropathogenic and enterohemorrhagic Escherichia coli, Citrobacter freundii biotype 4280, and Hafnia alvei. Maltose-binding protein (MBP) fusions containing the C-terminal 280 amino acids of Int-like proteins of strains of enteropathogenic E. coli, enterohemorrhagic E. coli, H. alvei ...[more]

PMID: 8168946

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Project description:UnlabelledTranslocation of effector proteins via a type III secretion system (T3SS) is a widespread infection strategy among Gram-negative bacterial pathogens. Each pathogen translocates a particular set of effectors that subvert cell signaling in a way that suits its particular infection cycle. However, as effector unbalance might lead to cytotoxicity, the pathogens must employ mechanisms that regulate the intracellular effector concentration. We present evidence that the effector EspZ controls T3SS effector translocation from enteropathogenic (EPEC) and enterohemorrhagic (EHEC) Escherichia coli. Consistently, an EPEC espZ mutant is highly cytotoxic. Following ectopic expression, we found that EspZ inhibited the formation of actin pedestals as it blocked the translocation of Tir, as well as other effectors, including Map and EspF. Moreover, during infection EspZ inhibited effector translocation following superinfection. Importantly, while EspZ of EHEC O157:H7 had a universal "translocation stop" activity, EspZ of EPEC inhibited effector translocation from typical EPEC strains but not from EHEC O157:H7 or its progenitor, atypical EPEC O55:H7. We found that the N and C termini of EspZ, which contains two transmembrane domains, face the cytosolic leaflet of the plasma membrane at the site of bacterial attachment, while the extracellular loop of EspZ is responsible for its strain-specific activity. These results show that EPEC and EHEC acquired a sophisticated mechanism to regulate the effector translocation.ImportanceEnteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) are important diarrheal pathogens responsible for significant morbidity and mortality in developing countries and the developed world, respectively. The virulence strategy of EPEC and EHEC revolves around a conserved type III secretion system (T3SS), which translocates bacterial proteins known as effectors directly into host cells. Previous studies have shown that when cells are infected in two waves with EPEC, the first wave inhibits effector translocation by the second wave in a T3SS-dependent manner, although the factor involved was not known. Importantly, we identified EspZ as the effector responsible for blocking protein translocation following a secondary EPEC infection. Interestingly, we found that while EspZ of EHEC can block protein translocation from both EPEC and EHEC strains, EPEC EspZ cannot block translocation from EHEC. These studies show that EPEC and EHEC employ a novel infection strategy to regulate T3SS translocation.

Dataset Information

Characterization of the C-terminal domains of intimin-like proteins of enteropathogenic and enterohemorrhagic Escherichia coli, Citrobacter freundii, and Hafnia alvei.

Publications

Characterization of the C-terminal domains of intimin-like proteins of enteropathogenic and enterohemorrhagic Escherichia coli, Citrobacter freundii, and Hafnia alvei.

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