Triacylglycerol utilization is required for regrowth of in vitro hypoxic nonreplicating Mycobacterium bovis bacillus Calmette-Guerin.

Mycobacteria store triacylglycerols (TGs) under various stress conditions, such as hypoxia, exposure to nitric oxide, and acidic environments. These stress conditions are known to induce nonreplicating persistence in mycobacteria. The importance of TG accumulation and utilization during regrowth is not clearly understood. Here we specifically determined the levels of accumulated TG and TG lipase activity in Mycobacterium bovis bacillus Calmette-Guerin (BCG) in various different physiological states (logarithmic growth, aerated stationary phase, hypoxia-induced dormancy, and regrowth from dormancy). We found extensive accumulation and degradation of TGs in the bacilli during entry into and exit from hypoxia-induced dormancy, respectively. These processes are accompanied by dynamic appearance and disappearance of intracellular TG lipid particles. The reduction in TG levels coincides with an increase in cellular TG lipase activity in the regrowing bacilli. Tetrahydrolipstatin, an inhibitor of TG lipases, reduces total lipase activity, prevents breakdown of TGs, and blocks the growth of mycobacteria upon resuscitation with air. Our results demonstrate that utilization of TGs is essential for the regrowth of mycobacteria during their exit from the hypoxic nonreplicating state.

Prioritizing genomic drug targets in pathogens: application to Mycobacterium tuberculosis.

We have developed a software program that weights and integrates specific properties on the genes in a pathogen so that they may be ranked as drug targets. We applied this software to produce three prioritized drug target lists for Mycobacterium tuberculosis, the causative agent of tuberculosis, a disease for which a new drug is desperately needed. Each list is based on an individual criterion. The first list prioritizes metabolic drug targets by the uniqueness of their roles in the M. tuberculosis metabolome ("metabolic chokepoints") and their similarity to known "druggable" protein classes (i.e., classes whose activity has previously been shown to be modulated by binding a small molecule). The second list prioritizes targets that would specifically impair M. tuberculosis, by weighting heavily those that are closely conserved within the Actinobacteria class but lack close homology to the host and gut flora. M. tuberculosis can survive asymptomatically in its host for many years by adapting to a dormant state referred to as "persistence." The final list aims to prioritize potential targets involved in maintaining persistence in M. tuberculosis. The rankings of current, candidate, and proposed drug targets are highlighted with respect to these lists. Some features were found to be more accurate than others in prioritizing studied targets. It can also be shown that targets can be prioritized by using evolutionary programming to optimize the weights of each desired property. We demonstrate this approach in prioritizing persistence targets.

Use of proteomics to identify novel virulence determinants that are required for Edwardsiella tarda pathogenesis.

Edwardsiella tarda is an important cause of haemorrhagic septicaemia in fish and also of gastro- and extraintestinal infections in humans. Using a combination of comparative proteomics and TnphoA mutagenesis, we have identified five proteins that may contribute to E. tarda PPD130/91 pathogenesis. Lowered protein secretion, impaired autoaggregation and the absence of six proteins were observed only in three highly attenuated mutants when cultured in Dulbecco's modified eagle medium (DMEM). Five out of six proteins could be identified by their mass spectra. Three proteins were identified as putative effector proteins (EseB, EseC and EseD) that are homologous to SseB, SseC and SseD of a type III secretion system (TTSS) in Salmonella species. The other two were EvpA and EvpC, homologous to Eip20 and Eip18 in Edwardsiella ictaluri. The complete sequencing and homology studies of evpA-H indicate that similar gene clusters are widely distributed in other pathogens such as Escherichia, Salmonella, Vibrio and Yersinia species with unknown functions. Insertional inactivation and deletion of evpB or evpC led to lower replication rates in gourami phagocytes, and reduced protein secretion and virulence in blue gourami. Complementation of these deletion mutants showed partial recovery in the above three phenotypes, indicating that these genes are vital for E. tarda pathogenesis. The transport of the EvpC protein may not use the TTSS in E. tarda. The expression of EvpA and EvpC as well as EseB, EseC and EseD was temperature dependent (suppressed at 37 degrees C), and disruption of esrB affected their expression. The present study identifies two possible secretion systems (TTSS and Evp) that are vital for E. tarda pathogenesis.

A type III secretion system is required for Aeromonas hydrophila AH-1 pathogenesis.

Aeromonas hydrophila is a gram-negative opportunistic pathogen in fish and humans. Many bacterial pathogens of animals and plants have been shown to inject anti-host virulence determinants into the hosts via a type III secretion system (TTSS). Degenerate primers based on lcrD family genes that are present in every known TTSS allowed us to locate the TTSS gene cluster in A. hydrophila AH-1. A series of genome walking steps helped in the identification of 25 open reading frames that encode proteins homologous to those in TTSSs in other bacteria. PCR-based analysis showed the presence of lcrD homologs (ascV) in all of the 33 strains of A. hydrophila isolated from various sources. Insertional inactivation of two of the TTSS genes (aopB and aopD) led to decreased cytotoxicity in carp epithelial cells, increased phagocytosis, and reduced virulence in blue gourami. These results show that a TTSS is required for A. hydrophila pathogenesis. This is the first report of sequencing and characterization of TTSS gene clusters from A. hydrophila. The TTSS identified here may help in developing suitable vaccines as well as in further understanding of the pathogenesis of A. hydrophila.