K-099. Expression of ilpA Gene Encoding an Immunogenic Lipoprotein in Pathogenic Bacterium, Vibrio vulnificus: Repression of ilpA Expression by Iron and Fur

Y-S. Han1, S-J. Park2, K-H. Lee1;
1Hankuk Univ. Foreign Studies, Yongin, Kyunggi-Do, REPUBLIC OF KOREA, 2Yonsei Univ Coll Med, Seoul, REPUBLIC OF KOREA.

The ilpA gene was isolated from an immunoscreening assay of surface molecules of Vibrio vulnificus, and its gene product was shown to be involved in cytoadherence to and cytokine production in human cells. The upstream region of the ilpA gene includes two open-reading frames putatively encoding an ATP-binding cassette (ABC)-transporter and an ABC-permease. Reverse transcriptase-PCR and primer extension experiments revealed that ilpA gene was transcribed from the upstream region of an ABC-transporter gene (PL), which suggests that ilpA gene in V. vulnificus is transcribed as a polycistronic mRNA with two upstream genes. In addition, a monocistronic ilpA mRNA was also formed from an additional promoter locating between the 3’-end region of the ABC-permease gene and the 5’-end region of ilpA gene (PS). To characterize the expression pattern of these two ilpA promoters, luxAB-transcriptional fusions containing each promoter region were constructed. The fusion assays showed that PL is a stronger (about 10-folds) promoter than PS. The expression from PS was basal during exponential phase and increased up to 3-folds at the early stationary phase. In contrast, the expression from PL is maximal during the middle of exponential phase. Since bacterial survival in presence of an iron-chelator was severely impaired in the ΔilpA mutant in comparison with wildtype, its expression was also monitored in the Δfur mutant V. vulnificus. Expression from both promoters was derepressed by fur mutation and by iron depletion. Putative Fur-binding sites were discernable in both promoters. Repression of ilpA expression by iron and Fur was confirmed by western blot using anti-IlpA polyclonal antibodies. These results clearly demonstrate that synthesis of IlpA in V. vulnificus is negatively regulated by Fur-iron complex, and thus its expression is induced under the iron-depleted conditions, such as host environment, to provide the bacterial pathogen with increased ability of cytoadherence and cytotoxicity.