The biochemical characterization and functional study of the Salmonella effector protein SopA in Salmonella pathogenesis
Abstract
Salmonella translocate bacterial effectors into host cells to confer bacterial entry and survival. It is not known how the host cells cope with the influx of these effectors. During the functional study of SopA, one of SPI-1 effector proteins, we discovered its interaction with a host protein HsRMA1 by yeast two-hybrid screening. HsRma1 (Ring-finger protein with a Membrane Anchor) is a newly identified membrane-bound ubiquitin E3 ligase (Matsuda 2001), belonging to the RING (Really Interesting New Gene) finger protein family. In vitro ubiquitination assays further showed that SopA is ubiquitinated and degraded by the HsRMA1-mediated ubiquitination pathway. Previous studies have indicated that sopA-mutant strain is highly attenuated in inducing the polymorphonuclear leukocytes (PMN) transepithelial migration compared with that of the wild-type strain (Wood et al., 2000). In an effort to study the biological role of SopA-HsRma1 interaction in Salmonella pathogenesis, we found that sopA-mutant escapes less frequently to the cytosol than wild type Salmonella in HeLa cells in an HsRMA1-dependent manner. We hypothesize that efficient bacterial escape into the cytosol of epithelial cells requires HsRMA1-mediated SopA ubiquitination and contributes to Salmonella-induced enteropathogenicity. Interestingly we also discovered that SopA itself possess the ubiquitin E3 ligase activity. A detailed biochemical and structural studies revealed that SopA functionally mimics the mammalian HECT (Homologous to E6AP C-Terminus) E3 ubiquitin ligases. A Salmonella strain expressing a catalytically incompetent SopAC753S mutant had reduced Salmonella -induced polymorphonuclear leukocytes transepithelial migration. We speculate that SopA ubiquitinates bacterial/host proteins involved in Salmonella-induced intestinal inflammation.
Degree
Ph.D.
Advisors
Zhou, Purdue University.
Subject Area
Microbiology
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