Background Armigeres subalbatus is a natural vector of the filarial worm Brugia pahangi, but it kills Brugia malayi microfilariae by melanotic encapsulation. Because B. malayi and B. pahangi are morphologically and biologically similar, comparing Ar. subalbatus-B. pahangi susceptibility and Ar. subalbatus-B. malayi refractoriness could provide significant insight into recognition mechanisms required to mount an effective anti-filarial worm immune response in the mosquito, as well as provide considerable detail into the molecular components involved in vector competence. Previously, we assessed the transcriptional response of Ar. subalbatus to B. malayi, and now we report transcriptome profiling studies of Ar. subalbatus in relation to filarial worm infection to provide information on the molecular components involved in B. pahangi susceptibility.Methodology/Principal FindingsUtilizing microarrays, comparisons were made between mosquitoes exposed to B. pahangi, B. malayi, and uninfected bloodmeals. The time course chosen facilitated an examination of key events in the development of the parasite, beginning with the very start of filarial worm infection and spanning to well after parasites had developed to the infective stage in the mosquito. At 1, 3, 6, 12, 24 h post infection and 2–3, 5–6, 8–9, and 13–14 days post challenge there were 31, 75, 113, 76, 54, 5, 3, 13, and 2 detectable transcripts, respectively, with significant differences in transcript abundance (increase or decrease) as a result of parasite development.Conclusions/SignificanceHerein, we demonstrate that filarial worm susceptibility in a laboratory strain of the natural vector Ar. subalbatus involves many factors of both known and unknown function that most likely are associated with filarial worm penetration through the midgut, invasion into thoracic muscle cells, and maintenance of homeostasis in the hemolymph environment. The data show that there are distinct and separate transcriptional patterns associated with filarial worm susceptibility as compared to refractoriness, and that an infection response in Ar. subalbatus can differ significantly from that observed in Ae. aegypti, a common laboratory model.

To further understand. Experiments were done to characterize the eis promoter in M. smegmatis and M. tuberculosis H37Ra. The putative ؊10 and ؊35 regions matched the Escherichia coli 70 consensus 67 and 83%, respectively, making it a group A/SigA-like mycobacterial promoter. Expression of site-directed variants of the core promoter region, determined by flow cytometry using gfp as a reporter, showed that the putative ؊10 region is essential for eis expression. In addition, site-directed alteration of the eis promoter to the consensus E. coli 70 promoter elements increased gfp transcription to levels similar to that driven by the heat shock promoter, phsp60, of Mycobacterium bovis BCG. Upstream promoter deletion analysis showed that a 200-and 412-bp region of the promoter was necessary for maximum expression of gfp in M. smegmatis and M. tuberculosis H37Ra, respectively. Random mutagenesis of the 412-bp eis promoter, using a catechol 2,3-dioxygenase screen and activity assay, defined nucleotides upstream of the core promoter region that are essential to eis expression in both M. smegmatis and M. tuberculosis H37Ra, including a region homologous to a DinR cis element.Tuberculosis (TB) continues to be the world's most destructive human bacterial infectious disease. Current estimates show that more than two million people die from TB each year, and TB remains a major cause of premature death (18). Mortality due to TB is a major global health crisis due to AIDS and the increasing prevalence of multidrug-resistant strains of Mycobacterium tuberculosis, although effective treatments are available. Despite the elucidation of the genome sequence of several M. tuberculosis strains (5, 14) and available genetic tools to identify genes involved in TB pathogenesis (13,16,23,26), the molecular basis of its ability to survive within host cells and evade host immune responses is unknown. Understanding the molecular mechanisms of pathogenesis is essential for the development of better methods of diagnosis, treatment, and prevention. One way to heighten our understanding of M. tuberculosis pathogenesis is to examine the regulation of potential virulence genes, in particular the promoters and other elements that govern their expression.Approximately 130 mycobacterial promoters have been characterized to date, but only 76 have been categorized into the four groups (A to D) of mycobacterial promoters (15). Identified promoters comprise less than 3% of the potential promoters in the genome of M. tuberculosis. The majority of the categorized promoters are from Mycobacterium smegmatis and Mycobacterium paratuberculosis, with less than 25% derived from M. tuberculosis itself. This indicates that we know little about promoter function in mycobacteria and even less about promoters from M. tuberculosis. Therefore, there is a need for detailed studies on M. tuberculosis promoter characterization and function, especially for genes that may play a role in the survival of M. tuberculosis within macrophages.The eis gene of M. tuberculosis H37Rv...

Reporter systems efficient at monitoring temporal gene expression in slow-growing mycobacteria would significantly aid the characterization of gene expression in specific environments. Bacterial luciferase is a reporter that has not been widely used to study gene expression in mycobacteria. This report describes the determination of the degradation of bacterial luciferase in Mycobacterium tuberculosis H37Ra and its utility as a reporter of temporal gene expression in this slow-growing mycobacterium. The inducible/repressible alanine dehydrogenase promoter of M. tuberculosis H37Rv was used to track the decay kinetics of Vibrio harveyi luciferase in both mid-log phase and stationary phase grown M. tuberculosis H37Ra, which proved to be highly similar during both phases of growth. The luciferase reporter was then used to detect changes in expression from the heat-shock promoter, phsp60, of M. bovis BCG during M. tuberculosis H37Ra growth in culture. Quantitative real-time PCR analysis of groEL2, the hsp60 homologue in M. tuberculosis, displayed a similar pattern of expression to phsp60-driven luciferase. These results strongly suggest that the luciferase reporter can be used to monitor temporal changes in gene expression in M. tuberculosis and may serve as a novel system to examine gene expression under specific conditions.