Study of Serine protease autotransporters of Enterobacteriaceae in Citrobacter rodentium pathogenesis

Abstract

Pathogenic E. coli have evolved to encode unique set of virulence genes that define their distinct phenotypes of pathogenesis. However, Serine protease autotransporters of Enterobacteriaceae (SPATEs) are encoded by all members of pathogenic E. coli implicating their role in pathogenesis is evolutionarily significant. Phylogenetically, SPATEs are classified into class 1 that are cytotoxic to epithelial cells by causing actin cytoskeletal damage; and class 2 SPATEs that are cytopathic by cleaving mucin and O-glycoproteins on hematopoietic cells implicating a role in immune evasion. The limitation of host-specificity has challenged in vivo studies to determine the role of SPATEs during intestinal infection. In order to determine the role of SPATEs in the context of natural infection we sought to use the mouse pathogen, C. rodentium that causes transmissible murine colonic hyperplasia. C. rodentium encodes 3 SPATEs, Crc1 which is homologous to EspC from enteropathogenic E. coli; Crc2 homologous to Pic of enteroaggregative E. coli and S. flexneri ; and AdcA which is close to Tsh of avian pathogenic E. coli. We found that upon infecting C57BL/6 mice with C. rodentium, the Δcrc1 infected group exhibited more weight loss and increased mortality in younger animals than C. rodentium wild-type infected mice. This severity in disease and mortality rate was reversed upon restoration of Crc1 in the crc1- repaired infected mice. Histologically modest increase in submucosal edema and significantly more infiltration of PMN, T and B lymphocytes in the distal colon was observed in the Δcrc1 infected mice than the C. rodentium wild-type or crc1-repaired infected groups. Using RT-PCR we studied mRNA expression levels of genes encoding various pro-inflammatory cytokines in the distal colon. There was several fold induction of cytokines IFNγ, TNFα, IL1β, IL6 and iNOS in the Δcrc1 infected in comparison to other infected groups correlating with increased infiltration at this site. These studies have shown that deletion of Crc1 is associated with exacerbated disease and that Crc1 plays an anti-inflammatory role by ameliorating the immune response. Our studies have furthered our understanding of SPATEs beyond bacterial colonization and illuminate their role in host immune modulation, thereby favoring host survival and possibly prolonged transmission time.