CexE Is a Coat Protein and Virulence Factor of Diarrheagenic Pathogens

Abstract

CexE is a 12 kDa protein that was originally reported to be present in just three strains of enterotoxigenic Escherichia coli (ETEC); a frequent cause of diarrheal illnesses worldwide. However, an examination of sequenced genomes has revealed that CexE is actually present in a majority of ETEC strains. In addition, homologs of CexE are present in enteroaggregative E. coli (EAEC), Yersinia enterocolitica, Providencia alcalifaciens, and Citrobacter rodentium. Although it has been hypothesized that CexE and its homologs are virulence factors, this has yet to be tested. Thus the primary aim of this study was to determine if these proteins contribute to pathogenicity. Our secondary aim was determine if they are secreted coat proteins. Here we report that all neonatal mice infected with a wild-type strain of C. rodentium perished. In contrast a cexE mutant was significantly attenuated with 45% neonate survival. In adult mice the wildtype strain reached significantly higher loads in the large intestines and were shed in greater numbers than cexE mutants. Secretion of the CexE homolog in EAEC is dependent upon an atypical Type I secretion system that accepts its client from the periplasm rather than the cytoplasm. Insertion mutants of cexC, the putative ATPase of the CexE secretion system, were attenuated in our murine model. In vitro we found that CexC is required for the secretion of CexE to the outer membranes of both ETEC and C. rodentium. Secretion is not constitutive because CexE accumulates in the periplasm when the two pathogens are cultured under noninducing conditions. Although secretion conditions differ between ETEC and C. rodentium, secreted CexE remains predominantly associated with the outer membranes of both species. In aggregate these findings demonstrate that CexE is a secreted coat protein and virulence factor that promotes colonization of host intestinal tissues by enteric pathogens.

Structure of the master regulator Rns reveals an inhibitor of enterotoxigenic Escherichia coli virulence regulons

“…The λ Red recombinase system 47 was used to generate a rns knockout in ETEC 1392/75-2a similarly to the cfaD::kan H10407 strain previously described 48 . Primers 1242/1150 were used to amplify a tetracycline resistance cassette targeting rns from pAH162.…”

“…PVDF membranes were blocked in TBS-Blotto (25 mM TrisCl pH 7.6, 150 mM NaCl, 5% wt/vol powdered nonfat milk). Antibodies against CexE homologs CexEα (H10407) and CexEε (1392/75-2a) were produced by immunization of rabbits (Proteintech Group, Inc.) with purified antigens and used at a dilution of 1:5000 in TBS-Blotto with 0.05% vol/vol Tween20 45 , 48 . The primary antibody against DnaK (AbCam ab69617) was used at a 1:10,000 dilution.…”

Glycine acylation and trafficking of a new class of bacterial lipoprotein by a composite secretion system

“…The conservation of this system within pathogenic bacterial strains represents a possible new drug target. Previous studies have shown that deletion of aatD, aatC, or aap/cexE reduces bacterial colonisation and disease (11,12). Thus, chemicals targeting the Aat system have the potential to prevent or perhaps reduce the severity of bacterial disease.…”

“…The full Aat system and an Aap or CexE homolog was identified in 826 separate nucleotide accessions (Supplementary Table S1). This revealed that the Aat system is distributed more widely than initially anticipated and is encoded in pathogens with diverse mechanisms of virulence such as ETEC, EAEC, enteropathogenic E. coli, Shiga-toxin producing E. coli, Shigella sp., Salmonella enterica, Citrobacter rodentium, Providencia alcalifaciens and Yersinia entercolitica (10,11,16).…”

“…Previously, we described two outer membrane proteins, CexE and Aap, associated with enterotoxigenic (ETEC) and enteroaggregative Escherichia coli (EAEC), respectively (9,10). CexE has been implicated in prolonged bacterial shedding and increased severity of infection (11) whereas Aap influences biofilm formation and gut colonisation (9,12). Despite Aap and CexE sharing only 18% amino acid identity, both proteins are secreted by the Aat system (11 13).…”