Title

Identification and characterization of Salmonella enteriditis outer membrane proteins that mediate attachment to intestinal epithelial cells

Date of Completion

January 1999

Keywords

Agriculture, Food Science and Technology|Biology, Microbiology|Health Sciences, Public Health

Degree

Ph.D.

Abstract

Salmonella enteritidis (SE) is frequently implicated in food-borne disease outbreaks. During the past decade, there has been a dramatic increase in the prevalence of SE infection in humans. Economically SE infection is among the most common bacterial diseases affecting the poultry industry and public health. Annual loss to the U.S. poultry industry due to SE infection ranges from $64–114 million, and $200 million to 1 billion in medical costs and loss of productivity. Attachment of SE to intestinal mucosa is the first step in the process of pathogenesis, is mediated by proteins collectively known as adhesins. Adhesins are conserved proteins which induce antibody responses especially in the mucosal surfaces thereby, preventing bacterial attachment and disease. Inhibition of SE attachment to intestinal mucosa would prevent the disease, therefore, identification of SE adhesins would help to develop effective strategy for control of SE in chickens. The objectives of this study were to: identify and characterize SE outer membrane proteins (OMPs) expressed during attachment to intestinal epithelial cells, evaluate SE OMPs 75.6 and 82.3 KDa to inhibit attachment to chicken intestinal mucosa, and to identify, clone and determine the nucleotide sequence of the gene encoding 82.3 KDa SE attachment proteins. To achieve this objectives, SE was incubated with human intestinal epithelial cells (int-407) and the expressed proteins were labeled by pulse labeling technique. Newly synthesized proteins were characterized by SE specific polyclonal antibody and Tx-114 phase partitioning. Outer membrane proteins, p82.3 and p75.6, were identified and their respective amino acid sequences were determined. Polyclonal antibodies against p82.3 and p75.6 were raised in SPF rabbits and chickens, and used in in vitro attachment inhibition assay. SPF chickens were vaccinated with p82.3 and p75.6 and challenged with a virulent strain of SE. SE colonization in the intestinal mucosa was determined. The genes encoding p82.3 and p75.6 were identified and cloned for nucleotide sequencing. ^ We identified SE OMPs 75.6 and 82.3 KDa involved in the attachment to intestinal epithelial cells. Antibodies against these proteins were able to reduce SE attachment to Int-407 intestinal epithelial cells. Immunization of chickens with the aforementioned OMPs significantly reduced SE attachment to chicken intestinal mucosa. The gene encoding the 82.3 KDa SE attachment protein was identified, cloned and its nucleotide sequence was determined. ^