A Putative U32 Family Collagenase of Vibrio vulnificus Induced upon Exposure to Oyster

Abstract

Vibrio vulnificus is a foodborne pathogen that frequently contaminates seafood, especially oysters. Ingestion of V. vulnificus-contaminated seafood can cause foodborne diseases such as gastroenteritis and even life-threatening septicemia (Vugia et al., 2013). In the present study, RNA-sequencing was performed to identify differentially expressed genes of V. vulnificus upon exposure to oyster. V. vulnificus CMCP6, a clinical isolate from a patient in South Korea, was grown to mid-log phase and incubated in VFMG (Vibrio fisheri minimal medium containing glycerol, Kim et al., 2013) with or without oyster tissue for 1hr. Then bacterial cells were harvested and total cellular RNA was isolated. cDNA libraries were generated from the enriched mRNA and sequenced using the Illumina Hi-seq platform. As a result, 2,175 genes (more than 47% of the V. vulnificus genome) showing a > 2-fold change in expression with p-values of 0.05 or lower were identified, indicating that a variety of cellular processes of V. vulnificus were influenced by exposure to oyster. In particular, many genes primarily involved in the transport and metabolism of carbohydrates and amino acids, and iron acquisition were induced, implying that V. vulnificus utilizes oyster as a nutrient source. Interestingly, expression of the genes encoding the virulence factors of V. vulnificus including RTX toxin (RtxA1) and its transporter (RtxB), thermolabile hemolysin (Tlh), metalloprotease (VvpE), putative collagenase, and antioxidants were also induced by exposure to oyster. Therefore, the combined results suggest that numerous genes contribute to the V. vulnificus pathogenesis in humans as well as in the bacterials survival in oysters. The survival and growth of pathogenic bacteria in hosts are dependent on the actions of the bacterials proteolytic enzymes (Eckhard et al., 2009). Especially, among the proteolytic enzymes, microbial collagenases are strongly related to bacterial survival and pathogenesis (Matsushita & Okabe, 2001). Because collagen is a component of extracellular matrix and is the most abundant protein in animal tissues, destruction of collagen may serve dual roles by providing rich nutrient source, and give an opportunity of infection for pathogenic bacteria (Harrington, 1996). Transcriptomic analysis of V. vulnificus genes induced upon exposure to oyster indicated the upregulation of putative collagenase gene. This enzyme belongs to the U32 family of collagenases or peptidases and shares some homology with the Porphyromonas gingivalis PrtC one of the representative proteins of the U32 family peptidase (Kato et al., 1992). Considering the potential role of U32 peptidase family in the pathogenicity of Helicobacter pylori (gastric colonization, Kavermann et al., 2003), Streptococcus mutans (dental root decay, han et al., 2006) and P. gingivalis (periodontitis disease, Kato et al., 1992), it is necessary to study how the U32 enzymes contribute to the virulence of pathogenic bacteria. To find out whether V. vulnificus collagenase (VvC) also affects pathogenicity, phenotypic differences between the wild type and the vvc mutant were compared in vitro and in vivo. As a result, VvC may be a player in digesting collagen, especially in the intestinal mucus layer at early infection phase, as well as contributing to nutrient acquirement for bacterial survival. By doing so, V. vulnificus enhances its opportunity to infect the host.