Identification of proteins from Erwinia chrysanthemi involved in animal pathogenesis



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Texas Tech University


Erwinia chrysanthemi (EC 16) is a Gram-negative bacterial phytopathogen that causes soft rot disease on a variety of fruit and vegetable-producing plants. The pathogenicity of EC 16 is related to its ability to secrete pectolytic enzymes directly onto the cuticle of the host plant via the type HI secretion system. The type III secretion system is among the virulence factors that have made us increasingly aware of the common mechanistic processes utilized by bacterial pathogens to attack and gain nutrients from either plant or animal host organisms. Crucial to EC 16's ability to deliver its degradative enzymes to the target plant is its ability to associate with the plant surface. Several different genera of bacteria utilize attaching and effacing (AE) proteins to achieve an intimate relationship with their host tissue or organism. An oligonucleotide probe related to Escherichia coli (K12) genomic sequence, which bears homology to numerous bacterial attaching and effacing proteins, was used to probe an EC 16 genomic DNA library in order to detect possible genes that may contribute to the virulence of the bacterium. Cosmid pLAFR 2020 was identified as harboring DNA complementary to the probe. A series of oligonucleotide primers was designed to amplify the desired 7.5 kbp fragment from purified pLAFR 2020 DNA via polymerase chain reaction (PCR). The fragment was cloned and sequenced. Nucleotide sequence analysis indicated that the cloned fragment had approximatly 85% homology with a genomic segment from E. coli K12, which bears strong similarity to numerous bacterial AE proteins and invasins. The translation product from this sequence would have a greater than 60% identity to an AE homologue fragment from enteroaggregative enterohemorrhagic (EAEH)E. coli.

Expression of bacterial virulence factors is known to be induced by exposure of the pathogen to host cells or host-cell-nutrient environment. In order to determine if the AE-like DNA is expressed in EC 16, several growth conditions were used as mducers. The proteins from the bacteria grown under the different conditions were separated by SDS-PAGE, and the gels were stained with silver stain. Novel protems were present in each different induced sample (70, 94, and greater than 205 kDa), but most notable of these is the approximately 94- kDa protein present in samples grown in media containing pectin, fetal bovme serum (FBS), or in the presence of human colon adenocarcinoma derived cells. Immuno-blot analysis utilizing anti-Intimm polyclonal antisera revealed that this protein bears immunologic identity to the EPEC Intimin protem.



Enterobacteriaceae -- Genetics, Phytopathogenic bacteria, Membrane proteins