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Citrobacter rodentium espB is necessary for signal transduction and for infection of laboratory mice
J V Newman1, B A Zabel, S S Jha
1Division of Bioengineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Abstract:
Citrobacter rodentium is the causative agent of transmissible murine colonic hyperplasia and contains a locus of enterocyte effacement (LEE) similar to that found in enteropathogenic Escherichia coli (EPEC). EPEC espB is necessary for intimate attachment and signal transduction between EPEC and cultured cell monolayers. Mice challenged with wild-type C. rodentium develop a mucosal immunoglobulin A response to EspB. In this study, C. rodentium espB has been cloned and its nucleotide sequence has been determined. C. rodentium espB was found to have 90% identity to EPEC espB. A nonpolar insertion mutation in C. rodentium espB was constructed and used to replace the chromosomal wild-type allele. The C. rodentium espB mutant exhibited reduced cell association and had no detectable fluorescent actin staining activity on cultured cell monolayers. The C. rodentium espB mutant also failed to colonize laboratory mice following experimental inoculation. The espB mutation could be complemented with a plasmid-encoded copy of the gene, which restored both cell association and fluorescent actin staining activity, as well as the ability to colonize laboratory mice. These studies indicate that espB is necessary for signal transduction and for colonization of laboratory mice by C. rodentium.
Insights
Citrobacter rodentium espB is essential for bacterial attachment and colonization in mice. Disabling espB prevents infection, while restoring it allows C. rodentium to infect mice.
Area of Science:
- Microbiology
- Bacterial Pathogenesis
- Molecular Biology
Background:
- Citrobacter rodentium causes murine colonic hyperplasia and shares virulence factors with enteropathogenic Escherichia coli (EPEC).
- The EPEC espB gene is crucial for intimate attachment and host cell signal transduction.
- Mice infected with wild-type C. rodentium mount an immune response against the EspB protein.
Purpose of the Study:
- To clone and characterize the C. rodentium espB gene.
- To investigate the role of C. rodentium espB in host-pathogen interactions and colonization.
- To determine if espB is essential for C. rodentium virulence in a murine model.
Main Methods:
- Cloning and sequencing of the C. rodentium espB gene.
- Construction of a nonpolar espB insertion mutant.
- In vitro assays using cultured cell monolayers to assess cell association and actin staining.
- In vivo colonization experiments in laboratory mice.
- Complementation studies using a plasmid-encoded espB gene.
Main Results:
- C. rodentium espB shares 90% nucleotide identity with EPEC espB.
- The espB mutant showed reduced association with cultured cells and lacked fluorescent actin staining.
- The espB mutant was unable to colonize laboratory mice.
- Complementation with a functional espB gene restored both in vitro and in vivo phenotypes.
Conclusions:
- The espB gene is critical for C. rodentium's ability to associate with host cells.
- espB plays a vital role in signal transduction pathways during infection.
- espB is essential for the colonization and virulence of C. rodentium in laboratory mice.