Genetic identification and characterization of Listeria adhesion protein, an alcohol acetaldehyde dehydrogenase homologue in Listeria monocytogenes
Abstract
Listeria adhesion protein (LAP) interacted with a putative host cell receptor or heat shock protein 60 (Hsp60) and was involved in the adhesion of L. monocytogenes to various mammalian cell lines. However, the genetic identification of LAP, a 104 kDa protein, was not accomplished. The goals of this study were: (1) to identify the gene encoding LAP and confirm its function at the molecular level; (2) to determine the effects of environmental and physiological factors on its expression; and (3) to investigate the existence of any heterogeneity in LAP among Listeria species. Protein sequencing of LAP and the database search identified it as an alcohol acetaldehyde dehydrogenase (Aad) homologue in L. monocytogenes. The aad insertion mutant (KB208) did not react with MAb-H7 and exhibited decreased L. monocytogenes adhesion to various intestinal cell lines, which confirmed the identification and function of Aad as an adhesion protein. The mutant also showed impaired pathogenicity in the in vivo mouse assay. Western hybridization, ELISA and transmission electron microscopy analyses revealed that surface expression of Aad was enhanced by the anaerobic condition or high temperature (41°C); however, low temperature (4°C), high salts (5% NaCl), low pH (4.5), ethanol (5%), mucin (1%) and bile salts (0.01%) did not induce Aad expression. The aad gene was present in all Listeria species except L. grayi as shown by PCR and Western blot studies. The existence of heterogeneity in aad sequences from different Listeria species were shown by DNA sequencing and Southern hybridization analyses. Listeria species-specific primer sets were designed based on the aad sequences and successfully used to distinguish the species of the genus Listeria at the species level. In conclusion, LAP in L. monocytogenes was identified to be Aad and its surface expression was affected by an anaerobic environment likely to be encountered by L. monocytogenes in the gastro-intestinal tract. In addition, heterogeneity of aad sequences among different Listeria species implied the possible presence of a unique adhesion property in Aad from L. monocytogenes.
Degree
Ph.D.
Advisors
Bhunia, Purdue University.
Subject Area
Microbiology
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