Date of Award

Spring 1997

Project Type

Dissertation

Program or Major

Microbiology

Degree Name

Doctor of Philosophy

First Advisor

Frank G Rodgers

Abstract

Campylobacter jejuni is a major cause of enteritis in developed and developing countries. A cytotoxic complex was isolated from organism-free filtrates of broth grown organisms by screening for activity in HEp-2 cells following high performance liquid chromatography using a combination of size exclusion and DEAE column chromatography. The toxin was found to coincide with a 45 kDa protein possessing an N-terminus sequence indicative of a bacterial outer membrane porin protein together with a high molecular weight carbohydrate which was determined to be lipopolysaccharide (LPS).

The carbohydrate portion of the LPS had reactivity for the lectins Galanthus nivalis agglutinin, Maakia amurensis agglutinin and Datura stromonium agglutinin. The cytotoxicity of the porin-LPS complex was heat-labile at 70$\sp\circ$C within 30 min. It was also found to be resistant to trypsin and to degradation by the enzymes neuriminidase and glycosidase F as well as to oxidation with sodium meta-periodate. The complex induced DNA fragmentation, cytoplasmic blebbing and nuclear condensation in HEp-2 cells after 24 h of intoxication possibly indicating that the cells were becoming apoptotic.

Oligonucleotides were generated from the N-terminus of the porin protein. Using a combination of vectorette and inverse polymerase chain reaction (PCR) and cloning, a 1.45 kb fragment was sequenced and found to contain a 1275 base pair (bp) open reading frame (ORF). The ORF, designated porA encoded a 424 amino acid protein with a 22 residue leader sequence and had a calculated molecular weight of 45.6 kDa and pI of 4.44. The mature protein was 402 amino acids in length and had a molecular weight of 43.5 kDa and a pI of 4.35. The translated PorA protein had a 50% sequence homology with Haemophilis influenzae major outer membrane protein P2. Thirty-two strains of Campylobacter sp. and closely related organisms were screened for the porA gene and expression of cytotoxin in cell culture. Although all strains examined produced a cytotoxin, 21 of the 32 strains (66%) were genotypically positive for porA and of these 21 of 23 (91%) were C. jejuni strains. It is postulated that this cytotoxic protein is a major virulence factor for C. jejuni and may be responsible for the clinical symptoms usually associated with campylobacteriosis.

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