Citrobacter rodentium possesses a functional type II secretion system necessary for successful host infection

ABSTRACT

Infectious diarrheal diseases are the third leading cause of mortality in young children, many of which are driven by Gram-negative bacterial pathogens. To establish successful host infections these pathogens employ a plethora of virulence factors necessary to compete with the resident microbiota, and evade and subvert the host defenses. The type II secretion system (T2SS) is one such conserved molecular machine that allows for the delivery of effector proteins into the extracellular milieu. To explore the role of the T2SS during natural host infection, we used Citrobacter rodentium, a murine enteric pathogen, as a model of human intestinal disease caused by pathogenic Escherichia coli such as Enteropathogenic and Enterohemorrhagic E. coli (EPEC and EHEC). In this study, we determined that the C. rodentium genome encodes one T2SS and 22 potential T2SS-secreted protein effectors, as predicted via sequence homology. We demonstrated that this system was functional in vitro, identifying a role in intestinal mucin degradation allowing for its utilization as a carbon source, and promoting C. rodentium attachment to a mucus-producing colon cell line. During host infection, loss of the T2SS or associated effectors led to a significant colonization defect and lack of systemic spread. In mice susceptible to lethal infection, T2SS-deficient C. rodentium was strongly attenuated, resulting in reduced morbidity and mortality in infected hosts. Together these data highlight the important role of the T2SS and its effector repertoire during C. rodentium pathogenesis, aiding in successful host mucosal colonization.

KEYWORDS:

• Citrobacter rodentium
• type II secretion system
• T2SS
• mucin
• attaching and effacing pathogen
• mucosal invasion
• mucus degradation
• enteric infection
• bacterial pathogenesis
• host-pathogen interactions

Acknowledgments

The authors would like to thank all of our colleagues in the Finlay laboratory for their support and assistance. We would also like to thank Ingrid Barta for assistance with histological sectioning and analysis. We would like to thank Dr. Mihai Cirstea for help with 16S rRNA gene sequencing library preparation.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Author contributions

Z.K., S.E.W., and B.B.F. conceived the project. Z.K., S.E.W., and B.B.F. wrote the original draft of the manuscript with input and edits from all authors. Z.K. and S.E.W. designed and performed experiments and analyzed data. S.E.W. and A.S.P. performed animal work. S.E.W., A.S.P., and J.P.D. processed samples obtained from in vivo studies. Z.K. constructed the bacterial deletion mutants. A.S.P. constructed the chromosomal complementation strains. Z.K. performed the homology analysis and visualization. K.M.M. assisted with in vitro studies. All authors contributed to a draft of the manuscript. B.B.F. and L.J.F. contributed to supervision and provided funding.

Data availability statement

The authors confirm that the data supporting the findings of this study are available within the article [and/or] its supplementary materials. Raw 16S sequence data from mouse fecal samples have been deposited in the NCBI SRA database with the accession number (PRJNA949387).

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/19490976.2024.2308049

Additional information

Funding

This research was supported by grants from the Canadian Institutes of Health Research (B.B.F.) [FDN-159935]. B.B.F. is a University of British Columbia Peter Wall Distinguished Professor.