pckA-deficient Mycobacterium bovis BCG shows attenuated virulence in mice and in macrophages

Functional assignment of enzymes encoded by the Mycobacterium tuberculosis genome is largely incomplete despite recent advances in genomics and bioinformatics. Here, we applied an activitybased metabolomic profiling method to assign function to a unique phosphatase, Rv1692. In contrast to its annotation as a nucleotide phosphatase, metabolomic profiling and kinetic characterization indicate that Rv1692 is a D,L-glycerol 3-phosphate phosphatase. Crystal structures of Rv1692 reveal a unique architecture, a fusion of a predicted haloacid dehalogenase fold with a previously unidentified GCN5-related N-acetyltransferase region.Although not directly involved in acetyl transfer, or regulation of enzymatic activity in vitro, this GCN5-related N-acetyltransferase region is critical for the solubility of the phosphatase. Structural and biochemical analysis shows that the active site features are adapted for recognition of small polyol phosphates, and not nucleotide substrates. Functional assignment and metabolomic studies of M. tuberculosis lacking rv1692 demonstrate that Rv1692 is the final enzyme involved in glycerophospholipid recycling/catabolism, a pathway not previously described in M. tuberculosis.haloacid dehalogenase superfamily | enzyme function | pathway discovery E ach year, 1.4 million people succumb to tuberculosis, making Mycobacterium tuberculosis the deadliest bacterium affecting mankind (1). In addition, the dissemination of strains resistant to several antibiotics underscores the need for better understanding of this pathogen and for the development of novel vaccines and therapeutics (2, 3). Our approach to elucidating the unique pervasiveness of M. tuberculosis is through comprehensive discovery and characterization of metabolic pathways, as metabolism underlies survival of the bacteria both inside and outside the host and can contribute to phenotypic and genetic drug resistance.The M. tuberculosis genome encodes for 4,043 genes, of which 3,933 encode proteins (4, 5). Many genes with essential functions, such as DNA replication, protein and RNA synthesis, and cell-division, have close homologs in other bacteria, and their functions are annotated largely on the basis of analysis of their counterparts. However, functions of at least one-third of the genes are unknown or putative (4). In particular, little is known about genes that are not conserved or conditionally important. Characterization of such genes presents a daunting task. M. tuberculosis is thought to be subjected to a myriad of conditions during its life cycle in the host, such as low pH, reactive oxygen and nitrogen species, and so on (6, 7). Understanding how M. tuberculosis adapts to and even thrives in such diverse environments is critical to our understanding of the pathology itself and for the discovery of novel therapeutics to treat tuberculosis.We applied an innovative, activity-based metabolomic profiling (ABMP) approach to assign function to orphan (without a priori known substrates/products) mycobacterial enzymes and to discover unk...

Section: Resultsmentioning

confidence: 99%

Discovery of a glycerol 3-phosphate phosphatase reveals glycerophospholipid polar head recycling in Mycobacterium tuberculosis

Larrouy-Maumus

Biswas

Hunt

et al. 2013

“…Five proteins (19%) of the S-nitroso proteome (phosphoenolpyruvate carboxykinase, Mpa, glutamine synthetase, mycocerosic acid synthase, and acetohydroxyacid synthase) have been individually validated as important for virulence and͞or persistence in animal models of tuberculosis through targeted gene disruption or enzymatic inhibitor studies (24,31,(35)(36)(37)(38). Ten proteins (Ϸ50% of the S-nitroso proteins involved in intermediary or lipid metabolism) are involved in pathways important for the virulence and͞or persistence of Mtb: mycolic acid synthesis (mycocerosic acid synthase, polyketide synthase 13, and fatty acyl-AMP ligase), gluconeogenesis (phosphoenolpyruvate carboxykinase and malate synthase), branched chain amino acid synthesis (acetohydroxyacid synthase), nitrogen assimilation (asparagine synthase, glutamine synthetase, and glutamate synthase), and iron metabolism (mycobacterial ortholog of bacterioferritin) (35)(36)(37)(38)(39)(40)(41).…”

Section: Resultsmentioning

confidence: 99%

“…Ten proteins (Ϸ50% of the S-nitroso proteins involved in intermediary or lipid metabolism) are involved in pathways important for the virulence and͞or persistence of Mtb: mycolic acid synthesis (mycocerosic acid synthase, polyketide synthase 13, and fatty acyl-AMP ligase), gluconeogenesis (phosphoenolpyruvate carboxykinase and malate synthase), branched chain amino acid synthesis (acetohydroxyacid synthase), nitrogen assimilation (asparagine synthase, glutamine synthetase, and glutamate synthase), and iron metabolism (mycobacterial ortholog of bacterioferritin) (35)(36)(37)(38)(39)(40)(41).…”

Section: Resultsmentioning

confidence: 99%

S-nitroso proteome ofMycobacterium tuberculosis: Enzymes of intermediary metabolism and antioxidant defense

Rhee

Erdjument‐Bromage

Tempst

et al. 2004

160

139

The immune response to Mycobacterium tuberculosis (Mtb) includes expression of nitric oxide (NO) synthase (NOS)2, whose products can kill Mtb in vitro with a molar potency greater than that of many conventional antitubercular agents. However, the targets of reactive nitrogen intermediates (RNIs) in Mtb are unknown. One major action of RNIs is protein S-nitrosylation. Here, we describe, to our knowledge, the first proteomic analysis of S-nitrosylation in a whole organism after treating Mtb with bactericidal concentrations of RNIs. The 29 S-nitroso proteins identified are all enzymes, mostly serving intermediary metabolism, lipid metabolism, and͞or antioxidant defense. Many are essential or implicated in virulence, including defense against RNIs. For each of two target enzymes tested, lipoamide dehydrogenase and mycobacterial proteasome ATPase, S-nitrosylation caused enzyme inhibition. Moreover, endogenously biotinylated proteins were driven into mixed disulfide complexes. Targeting of metabolic enzymes and antioxidant defenses by means of protein S-nitrosylation and mixed disulfide bonding may contribute to the antimycobacterial actions of RNIs.nitric oxide ͉ biotin ͉ lipoamide dehydrogenase ͉ mycobacterial proteasome ATPase

“…Second, and conversely, functions that are essential in vivo are not necessarily essential in vitro. For example, the gluconeogenesis pathway is dispensable for growth of M. bovis bacillus Calmette-Guérin on standard medium in vitro, yet this pathway is essential for growth in macrophages and in mice (24). For these reasons, screening for essential functions in vitro is bedeviled by "false-positive" and "false-negative" ascertainment biases.…”

Section: Discussionmentioning

confidence: 99%

Mycobacterium tuberculosis persistence mutants identified by screening in isoniazid-treated mice

Dhar

McKinney

2010

114

106

Tuberculosis (TB) is notoriously difficult to cure, requiring administration of multiple antibiotics for 6 mo or longer. Conventional anti-TB drugs inhibit biosynthetic processes involved in cell growth and division, such as DNA replication, RNA transcription, protein translation, and cell wall biogenesis. Although highly effective against bacteria cultured in vitro under optimal growth conditions, these antibiotics are less effective against bacteria grown in vivo in the tissues of a mammalian host. The factors that contribute to the antibiotic tolerance of bacteria grown in vivo are unknown, although altered metabolism and sluggish growth are hypothesized to play a role. To address this question, we identified mutations in Mycobacterium tuberculosis that impaired or enhanced persistence in mice treated with isoniazid (INH), a front-line anti-TB drug. Disruption of cydC, encoding a putative ATP-binding cassette transporter subunit, accelerated bacterial clearance in INH-treated mice without affecting growth or survival in untreated mice. Conversely, transposon insertions within the rv0096-rv0101 gene cluster attenuated bacterial growth and survival in untreated mice but paradoxically prevented INH-mediated killing of bacteria in treated mice. These contrasting phenotypes were dependent on the interaction of the bacteria with the tissue environment because both mutants responded normally to INH when grown in macrophages ex vivo or in axenic cultures in vitro. Our findings have important implications because persistence-impairing mutations would be missed by conventional genetic screens to identify candidate drug targets. Conversely, persistenceenhancing mutations would be missed by standard diagnostic methods, which are performed on bacteria grown in vitro, to detect drug resistance.chemotherapy | drug tolerance | host environment T uberculosis (TB) has been a treatable disease for more than half a century, yet TB eradication remains a distant and possibly unachievable goal barring the introduction of more effective control strategies (1). A formidable obstacle to successful treatment of TB is the requirement for frequent administration of multiple drugs for 6 mo or longer (2). Unless drug administration is closely supervised, the majority of patients will not adhere to such a protracted therapeutical regimen (2). Consequently, patient nonadherence is responsible for high rates of treatment failure, relapse, and emergent drug resistance (3). To reduce treatment costs and improve patient adherence, there is an urgent need for more effective anti-TB drugs to shorten the treatment time (2, 4).Conventional anti-TB drugs target biosynthetic processes involved in cell growth, including RNA transcription [rifampicin (RIF)], protein translation (streptomycin), and cell wall biogenesis [isoniazid (INH)]. Although these drugs rapidly kill Mycobacterium tuberculosis grown in vitro, they are less active against bacteria grown in vivo in mammalian hosts (4,5). In the mouse model of TB, infection progresses through a brief ac...