Essential Role of the Type III Secretion System Effector NleB in Colonization of Mice by Citrobacter rodentium

Abstract

Attaching and effacing (A/E) pathogens are a significant cause of gastrointestinal illness in humans and animals. All A/E pathogens carry a large pathogenicity island, termed the locus for enterocyte effacement (LEE), which encodes a type III secretion system that translocates several effector proteins into host cells. To identify novel virulence determinants in A/E pathogens, we performed a signature-tagged mutagenesis screen in C57BL/6 mice by using the mouse A/E pathogen Citrobacter rodentium. Five hundred seventy-six derivatives of C. rodentium were tested in pools of 12 mutants. One attenuated mutant carried a transposon insertion in nleB, which encodes a putative effector of the LEE-encoded type III secretion system (T3SS). nleB is present in a genomic pathogenicity island that also encodes another putative effector, NleE, immediately downstream. Using translational fusions with ␤-lactamase (TEM-1), we showed that both NleB and NleE were translocated into host cells by the LEE-encoded T3SS of enteropathogenic Escherichia coli. In addition, deletion of the gene encoding NleB in C. rodentium resulted in reduced colonization of mice in single infections and reduced colonic hyperplasia. In contrast, the deletion of other non-LEE-encoded effector genes in C. rodentium, nleC, nleD, or nleE, had no effect on host colonization or disease. These results suggest that nleB encodes an important virulence determinant of A/E pathogens.

In Vitro and In Vivo Model Systems for Studying Enteropathogenic Escherichia coli Infections

“…Although much of the in vivo work on A/E pathogens has focused on the virulence roles of secreted proteins, others have attempted to determine tissue localization of effectors such as Tir (Deng et al 2003), in an effort to uncover function. Infection of mice with C. rodentium mutants confirms a critical role in colonization and virulence of the LEE-encoded effectors Tir, EspB, and EspZ, as well as the non-LEE-encoded effectors NleA and NleB (Newman et al 1999;Deng et al 2003Deng et al , 2004Gruenheid et al 2004;Mundy et al 2004;Kelly et al 2006), whereas others are thought to contribute collectively (Dean and Kenny 2009). Mice were orally infected with C. rodentium and colonic tissues were harvested and subjected to immunofluorescent staining at 6 days postinfection.…”

Signature tagged mutagenesis in the functional genetic analysis of gastrointestinal pathogens

“…The study examined 576 mutants of which 19 were attenuated for survival at 5-7 d post-infection. 48 Several insertions corresponded to previously identified virulence genes, including the gene cluster cfc and the espI. However one interesting finding was an insertion in the gene encoding a putative translocation effector of A/E pathogens, NleB.…”

“…An STM screen was created in C. rodentium and analyzed in two different strains of mice; C3H/HeJ and C57BL/ 6. [48][49][50] The C3H/HeJ strains of mice are highly susceptible to infection due to a lack of an innate immune response to LPS and are therefore colonized more rapidly and to a higher degree. This results in more extensive colonic hyperplasia and higher mortality rates.…”

“…However one interesting finding was an insertion in the gene encoding a putative translocation effector of A/E pathogens, NleB. 48,54 An nleB deletion mutant was constructed and tested for its ability to colonize the mouse. As with the transposon mutant the nleB deletion mutant was outcompeted by the wild-type in mixed infections.…”

“…In addition in single infections it was also shown to be essential for colonization and virulence. 48 NleB is also present in the EHEC O157:H7 strain indicating that C. rodentium is an invaluable small animal model to represent other A/E pathogens and to test the role of new effector proteins in disease. 48 …”