title: Towards a vaccine for attaching/effacing Escherichia coli: a LEE encoded regulator (ler) mutant of rabbit enteropathogenic Escherichia coli is attenuated, immunogenic, and protects rabbits from lethal challenge with the wild-type virulent strain.
abstract: The ler (LEE encoded regulator) gene product is a central regulator for the genes encoded on the locus of enterocyte effacement (LEE) pathogenicity island of attaching/effacing (A/E) pathogens, including human enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC) as well as animal isolates. Although an in vivo role for Ler in bacterial virulence has not been documented, we hypothesized that a Ler deletion mutant should be attenuated for virulence but might retain immunogenicity. The goals of this study were to genetically characterize ler of a rabbit EPEC (rEPEC) strain (O103:H2), to examine the effect of ler on in vivo virulence, and to determine if intragastric inoculation of an attenuated rEPEC ler mutant was immunogenic and could protect rabbits against subsequent challenge with the wild-type virulent parent strain. The predicted ler gene product of rEPEC strain O103:H2 shares high homology (over 95% amino acid identity) with the Lers of another rEPEC strain RDEC-1 (O15:H-) and human EPEC and EHEC. A defined internal ler deletion mutant of rEPEC O103:H2 showed reduced production of secreted proteins. Although orogastric inoculation of rabbits with the virulent parent O103:H2 strain induced severe diarrhea, significant weight loss and early mortality with adherent mucosal bacteria found at sacrifice, the isogeneic ler mutant strain was well tolerated. Animals gained weight and showed no clinical signs of disease. Examination of histological sections of intestinal segments revealed the absence of mucosal bacterial adherence. This result demonstrates an essential role for Ler in in vivo pathogenicity of A/E E. coli. Single dose orogastric immunization with the rEPEC ler mutant induced serum IgG antibody to whole bacteria (but not to intimin). Immunized animals were protected against enteric infection with the WT virulent parent strain exhibiting normal weight gain, absence of diarrhea and absence of mucosally adherent bacteria at sacrifice. Such attenuated ler mutant strains may have potential for use as oral vaccines, or as vaccine vectors for delivery of foreign antigens. It remains to be determined whether such regulatory mutants can protect against infection with A/E bacteria of differing serotypes affecting different hosts.
authors.affiliation: Center for Vaccine Development, University of Maryland School of Medicine, 685 West Baltimore Street, Baltimore, MD 21201, USA.
authors.forename: Chengru
authors.initials: C
authors.lastname: Zhu
authors.forename: Shuzhang
authors.initials: S
authors.lastname: Feng
authors.forename: Timothy E
authors.initials: TE
authors.lastname: Thate
authors.forename: James B
authors.initials: JB
authors.lastname: Kaper
authors.forename: Edgar C
authors.initials: EC
authors.lastname: Boedeker
mesh_terms: D018829:Adhesins, Bacterial / Q000276:immunology; D000284:Administration, Oral; D000818:Animals; D000907:Antibodies, Bacterial / Q000097:blood; D001422:Bacterial Adhesion; D001426:Bacterial Proteins / Q000378:metabolism; D001835:Body Weight; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D003967:Diarrhea; D004195:Disease Models, Animal; D004926:Escherichia coli / Q000235:genetics / Q000276:immunology* / Q000472:pathogenicity*; D004927:Escherichia coli Infections / Q000473:pathology / Q000517:prevention & control*; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000276:immunology / Q000502:physiology; D022361:Escherichia coli Vaccines / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology*; D017353:Gene Deletion; D007074:Immunoglobulin G / Q000097:blood; D007413:Intestinal Mucosa / Q000382:microbiology; D008969:Molecular Sequence Data; D011817:Rabbits; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D015534:Trans-Activators / Q000235:genetics* / Q000502:physiology; D014613:Vaccines, Attenuated / Q000235:genetics / Q000276:immunology
chemical_list: D018829:Adhesins, Bacterial; D000907:Antibodies, Bacterial; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D022361:Escherichia coli Vaccines; D007074:Immunoglobulin G; C465556:Ler protein, E coli; D015534:Trans-Activators; D014613:Vaccines, Attenuated; C073819:eaeA protein, E coli