Mycobacterium tuberculosis, the causative agent of tuberculosis, is an intracellular pathogen that shifts to a lipid-based metabolism in the host. Moreover, metabolism of the host lipid cholesterol plays an important role in M. tuberculosis infection. We used transcriptional profiling to identify genes transcriptionally regulated by cholesterol and KstR (Rv3574), a TetR-like repressor. The fadA5 (Rv3546) gene, annotated as a lipidmetabolizing thiolase, the expression of which is upregulated by cholesterol and repressed by KstR, was deleted in M. tuberculosis H37Rv. We demonstrated that fadA5 is required for utilization of cholesterol as a sole carbon source in vitro and for full virulence of M. tuberculosis in the chronic stage of mouse lung infection. Cholesterol is not toxic to the fadA5 mutant strain, and, therefore, toxicity does not account for its attenuation. We show that the wild-type strain, H37Rv, metabolizes cholesterol to androst-4-ene-3,17-dione (AD) and androsta-1,4-diene-3,17-dione (ADD) and exports these metabolites into the medium, whereas the fadA5 mutant strain is defective for this activity. We demonstrate that FadA5 catalyzes the thiolysis of acetoacetyl-coenzyme A (CoA). This catalytic activity is consistent with a ␤-ketoacyl-CoA thiolase function in cholesterol ␤-oxidation that is required for the production of androsterones. We conclude that the attenuated phenotype of the fadA5 mutant is a consequence of disrupted cholesterol metabolism that is essential only in the persistent stage of M. tuberculosis infection and may be caused by the inability to produce AD/ADD from cholesterol.Circumstantial evidence implicates a role for cholesterol in M. tuberculosis infection. How this host lipid affects infection and the role of its metabolism by the bacteria is not clear. Foamy macrophages, laden with lipid droplets, are known to accumulate in lung granulomas in human infection (23) as well as in infected mouse lungs (4). Electron microscopy revealed that M. tuberculosis is in close apposition to lipid bodies in foamy macrophages and that the bacteria eventually merge with these lipid bodies and even accumulate intracellular lipids (22). In fact, since foamy macrophages contain high levels of cholesterol esters (19,32), it is possible that the lipid bodies observed to merge with M. tuberculosis bacteria are composed of cholesterol esters. Macrophages infected with Mycobacterium leprae also accumulate cholesterol esters (17) that are thought to be responsible for the conversion of macrophages into foam cells. Significantly, bacteria from the sputum of human tuberculosis (TB) patients were observed to contain lipid bodies, and transcriptional profiling of bacteria from these samples demonstrated the induction of genes proposed to encode enzymes required for cholesterol utilization (13). These studies implicate host lipids and possibly cholesterol in the development of mycobacterial disease.The first direct evidence suggesting that bacterial cholesterol metabolism is required for M. tuberculosis vir...

New approaches are required to combat M. tuberculosis (Mtb), especially the multi and extremely drug resistant and organisms (MDR-TB and XDR-TB). There are many reports that mycobacteria oxidize 3β-hydroxysterols to 3-ketosteroids, but the enzyme(s) responsible for this activity have not been identified in mycobacterial species. In this work, the Rv1106c gene that is annotated as a 3β-hydroxysteroid dehydrogenase in Mtb has been cloned and heterologously expressed. The purified enzyme was kinetically characterized and found to have a pH optimum between 8.5 and 9.5. The enzyme, which is a member of the short chain dehydrogenase superfamily, uses NAD + as a cofactor and oxidizes cholesterol, pregnenolone and dehydroepiandrosterone to their respective 3-keto-4-ene products. The enzyme forms a ternary complex with NAD + binding before the sterol. The enzyme shows no substrate preference for dehydroepiandrosterone versus pregnenolone with second-order rate constants (k cat /K m ) of 3.2 ± 0.4 μM −1 min −1 and 3.9 ± 0.9 μM −1 min −1 , respectively at pH 8.5, 150 mM NaCl, 30 mM MgCl 2 , and saturating NAD + . Trilostane is a competitive inhibitor of dehydroepiandrosterone with a K i of 197 ± 8 |μM. The expression of the 3β-hydroxysteroid dehydrogenase in Mtb is intracellular. Disruption of the 3β-hydroxysteroid dehydrogenase gene in Mtb abrogates mycobacterial cholesterol oxidation activity. These data are consistent with Rv1106c being the gene responsible for 3β-hydroxysterol oxidation in Mtb.Tuberculosis is an opportunistic infection caused by Mycobacterium tuberculosis (Mtb 1 ) in individuals with HIV-AIDS that is estimated to infect 30% of the world's population (1,2). The World Health Organization estimates that 2 million people die every year from tuberculosis. Drug resistance to front-line Mtb drugs rifampicin and isoniazid has emerged (3,4) and additional resistance to second line drugs is emerging (5,6). It is clear that new approaches are required to combat these multi drug-resistant and extreme (or extensively) drugresistant organisms (7-9).The complete genome sequences of microorganisms are a rich source for mining new drug targets. However, oftentimes, biochemical functions have been assigned to genes purely on the basis of their sequence homology to gene products which are themselves poorly † Financial support from the National Institutes of Health (AI065251 (N.S.S.), AI065997 (I.S.), RR021008, (N.S.S.) and NIAID Contract# HHSN266200400091C, (Colorado State University) and the National Science Foundation (CHE0131146 for NMR spectrometers) is gratefully acknowledged. *corresponding author: Address: Stony Brook University, Stony Brook, New York 11794-3400, Phone: (631) Fax: (631) 632-5731 Nicole.Sampson@StonyBrook.edu. 1 Mtb: Mycobacterium tuberculosis; MDR-TB: multi-drug resistant Mycobacterium tuberculosis; XDR-TB: extremely-drug resistant Mycobacterium tuberculosis; DHEA: dehydroepiandrosterone; rH 6 3BHSD: recombinant 3β-hydroxysteroid dehydrogenase with an Nterminal six histidine tag; rSBHSD...

Mycobacterium tuberculosis can metabolize cholesterol to both acetate and propionate. The mass of isolated phthiocerol dimycoserate, a methyl-branched fatty acylated polyketide, was used as a reporter for intracellular propionate metabolic flux. When Mycobacterium tuberculosis is grown using cholesterol as the only source of carbon, a 42 a.m.u increase in average phthiocerol dimycoserate molecular weight is observed, consistent with the cellular pool of propionate and thus, methylmalonyl CoA increasing upon cholesterol metabolism. In contrast, no shift in phthiocerol dimycoserate molecular weight is observed upon supplementation of medium containing glycerol and glucose with cholesterol. We conclude that cholesterol is only a significant source of propionate in the absence of sugar carbon sources.Mycobacterium tuberculosis (M. tb), the etiologic agent of tuberculosis (TB) in humans, is responsible for the majority of deaths caused by bacterial infections. Although it is estimated that one-third of the world's population is infected with the pathogen, only 10% of immunocompetent individuals carrying the active form of M. tb develop the disease (1) and upon suppression of the immune system, M. tb bacilli may become active. Consequently, TB is responsible for the majority of HIV-related deaths worldwide. The emergence of multidrug resistant tuberculosis (MDR-TB) and extensively drug resistant tuberculosis (XDR-TB) has made the successful treatment of the disease cumbersome, often resulting in death of individuals infected with these strains. Therefore, we have undertaken studies to investigate the metabolic state of M. tb that ensues upon switching to a lipid-based metabolism in the host, in order to better develop new therapies.Unlike most pathogenic bacteria, M. tb lacks common virulence factors like capsules, endotoxins and exotoxins. Its success as a pathogen lies in its ability to elude the host's immune response and persist within the harsh milieu of the macrophage (2,3). Several distinct modes for virulence have been proposed to play important roles in the survival of M. tb within the phagosome including inhibition of phagosome-lysosome fusion (4-6), preventing maturation † This work was supported by the NIH (AI065251 (NSS), AI065987 (IS), DK007521, (NMN)), and the NYSTAR Program (FDP C040076, NSS).

Rv1106c ( hsd ; 3β-hydroxysteroid dehydrogenase) is required by Mycobacterium tuberculosis for growth on cholesterol as a sole carbon source, whereas Rv3409c is not. Mutation of Rv1106c does not reduce Mycobacterium tuberculosis growth in infected macrophages or guinea pigs. We conclude that cholesterol is not required as a nutritional source during infection.

Antibiotic contamination in agroecosystems may cause serious problems, such as the proliferation of various antibiotic resistant bacteria and the spreading of antibiotic resistance genes (ARGs) in the environment or even to human beings. However, it is unclear whether environmental antibiotics, antibiotic resistant bacteria, and ARGs can directly enter into, or occur in, the endophytic systems of plants exposed to pollutants. In this study, a hydroponic experiment exposing pakchoi (Brassica chinensis L.) to tetracycline, cephalexin, and sulfamethoxazole at 50% minimum inhibitory concentration (MIC) levels and MIC levels, respectively, was conducted to explore plant growth, antibiotic uptake, and the development of antibiotic resistance in endophytic systems. The three antibiotics promoted pakchoi growth at 50% MIC values. Target antibiotics at concentrations ranging from 6.9 to 48.1 µg·kg−1 were detected in the treated vegetables. Additionally, the rates of antibiotic-resistant endophytic bacteria to total cultivable endophytic bacteria significantly increased as the antibiotics accumulated in the plants. The detection and quantification of ARGs indicated that four types, tetX, blaCTX-M, and sul1 and sul2, which correspond to tetracycline, cephalexin, and sulfamethoxazole resistance, respectively, were present in the pakchoi endophytic system and increased with the antibiotic concentrations. The results highlight a potential risk of the development and spread of antibiotic resistance in vegetable endophytic systems.