Malonyl coenzyme A (CoA)-acyl carrier protein (ACP) transacylase (MCAT) is an essential enzyme in the biosynthesis of fatty acids in all bacteria, including Mycobacterium tuberculosis. MCAT catalyzes the transacylation of malonate from malonyl-CoA to activated holo-ACP, to generate malonyl-ACP, which is an elongation substrate in fatty acid biosynthesis. To clarify the roles of the mycobacterial acyl carrier protein (AcpM) and MCAT in fatty acid and mycolic acid biosynthesis, we have cloned, expressed, and purified acpM and mtfabD (malonyl-CoA: AcpM transacylase) from M. tuberculosis. According to the culture conditions used, AcpM was produced in Escherichia coli in two or three different forms: apo-AcpM, holo-AcpM, and palmitoylated-AcpM, as revealed by electrospray mass spectrometry. The mtfabD gene encoding a putative MCAT was used to complement a thermosensitive E. coli fabD mutant. Expression and purification of mtFabD resulted in an active enzyme displaying strong MCAT activity in vitro. Enzymatic studies using different ACP substrates established that holo-AcpM constitutes the preferred substrate for mtFabD. In order to provide further insight into the structure-function relationship of mtFabD, different mutant proteins were generated. All mutations (Q9A, R116A, H194A, Q243A, S91T, and S91A) completely abrogated MCAT activity in vitro, thus underlining the importance of these residues in transacylation. The generation and characterization of the AcpM forms and mtFabD opens the way for further studies relating to fatty acid and mycolic acid biosynthesis to be explored in M. tuberculosis. Since a specific type of FabD is found in mycobacterial species, it represents an attractive new drug target waiting to be exploited.
BackgroundMAP is a suspected zoonotic pathogen and the causative agent of Johne’s Disease in cattle and other ruminant animals. With over $1 billion dollars in loss to the dairy industry due to Johne’s Disease, efforts to eliminate or reduce MAP from cattle are of importance. The purpose of this study was to determine if daily intake of probiotics could eliminate or reduce Johne’s Disease associated symptoms and pathogenesis by MAP. Post infection, animals are often asymptomatic carriers with limited shedding of the pathogen, proving early detection to be difficult. Disease and symptoms often appear 3–4 years after infection with antibiotic treatment proving ineffective. Symptoms include chronic gastrointestinal inflammation leading to severe weight-loss from poor feed and water intake cause a wasting disease. These symptoms are similar to those found in individuals with Crohn’s Disease (CD); MAP has been implicated by not proven to be the causative agent of CD. Probiotics administered to livestock animals, including dairy and beef cattle have demonstrated improvements in cattle performance and health. Our objectives included determining the benefits of Lactobacillus animalis (strain name: NP-51) in MAP infected BALB/c mice by evaluating systemic and gastrointestinal response by the host and gut microbiota. Male and female animals were fed 1×106 CFU/g probiotics in sterile, powdered mouse chow daily and infected with 1 × 107 CFU/ml MAP and compared to controls. Animals were evaluated for 180 days to assess acute and chronic stages of disease, with sample collection from animals every 45 days. MAP concentrations from liver and intestinal tissues were examined using real time-PCR methods and the expression of key inflammatory markers were measured during MAP infection (interferon-gamma [IFN-Υ], Interleukin-1α, IL-12, IL-10, IL-6, and Tumor necrosis factor alpha [TNF-α]).ResultsOur results demonstrate administration of probiotics reduces production of IFN-Υ and IL-6 while increasing TNF-α and IL-17 in chronic disease; healthful immune responses that reduce chronic inflammation associated to MAP infection.ConclusionsWe observed that the immune system’s response in the presence of probiotics to MAP contributes towards host health by influencing the activity of the immune system and gut microbial populations.
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