Characterization of Transcription Factors That Regulate the Type IV Secretion System and Riboflavin Biosynthesis in Wolbachia of Brugia malayi

The human filarial parasite Brugia malayi harbors an endosymbiotic bacterium Wolbachia ( w Bm) that is required for parasite survival. Consequently, targeting w Bm is a promising approach for anti-filarial drug development. The Type IV secretion system (T4SS) plays an important role in bacteria-host interactions and is under stringent regulation by transcription factors. In w Bm, most T4SS genes are contained in two operons. We show the w Bm is active since the essential assembly factor virB8-1 , is transcribed in adult worms and larval stages, and VirB8-1 is present in parasite lysates. We also identify two transcription factors ( w BmxR1 and w BmxR2) that bind to the promoter region of several genes of the T4SS. Gel shift assays show binding of w BmxR1 to regions upstream of the virB9-2 and wBmxR2 genes, whereas w BmxR2 binds to virB4-2 and wBmxR1 promoter regions. Interestingly, both transcription factors bind to the promoter of the ribA gene that precedes virB8-1 , the first gene in operon 1 of the w Bm T4SS. RT-PCR reveals ribA and virB8-1 genes are co-transcribed as one operon, indicating the ribA gene and T4SS operon 1 are co-regulated by both w BmxR1 and w BmxR2. RibA encodes a bi-functional enzyme that catalyzes two essential steps in riboflavin (Vitamin B2) biosynthesis. Importantly, the riboflavin pathway is absent in B. malayi . We demonstrate the pathway is functional in w Bm, and observe vitamin B2 supplementation partially rescues filarial parasites treated with doxycycline, indicating Wolbachia may supply the essential vitamin to its worm host. This is the first characterization of a transcription factor(s) from w Bm and first report of co-regulation of genes of the T4SS and riboflavin biosynthesis pathway. In addition, our results demonstrate a requirement of vitamin B2 for worm health and fertility, and imply a nutritional role of the symbiont for the filarial parasite host.