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1.  Inactivation of Fructose-1,6-Bisphosphate Aldolase Prevents Optimal Co-catabolism of Glycolytic and Gluconeogenic Carbon Substrates in Mycobacterium tuberculosis 
PLoS Pathogens  2014;10(5):e1004144.
Metabolic pathways used by Mycobacterium tuberculosis (Mtb) to establish and maintain infections are important for our understanding of pathogenesis and the development of new chemotherapies. To investigate the role of fructose-1,6-bisphosphate aldolase (FBA), we engineered an Mtb strain in which FBA levels were regulated by anhydrotetracycline. Depletion of FBA resulted in clearance of Mtb in both the acute and chronic phases of infection in vivo, and loss of viability in vitro when cultured on single carbon sources. Consistent with prior reports of Mtb's ability to co-catabolize multiple carbon sources, this in vitro essentiality could be overcome when cultured on mixtures of glycolytic and gluconeogenic carbon sources, enabling generation of an fba knockout (Δfba). In vitro studies of Δfba however revealed that lack of FBA could only be compensated for by a specific balance of glucose and butyrate in which growth and metabolism of butyrate were determined by Mtb's ability to co-catabolize glucose. These data thus not only evaluate FBA as a potential drug target in both replicating and persistent Mtb, but also expand our understanding of the multiplicity of in vitro conditions that define the essentiality of Mtb's FBA in vivo.
Author Summary
The development of new chemotherapies targeting Mycobacterium tuberculosis (Mtb) will benefit from genetic evaluation of potential drug targets and a better understanding of the pathways required by Mtb to establish and maintain chronic infections. We employed a genetic approach to investigate the essentiality of fructose-1,6-bisphosphate aldolase (FBA) for growth and survival of Mtb in vitro and in mice. A conditional fba mutant revealed that Mtb requires FBA for growth in the acute phase and for survival in the chronic phase of mouse infections. In vitro essentiality of fba was strictly condition-dependent. An FBA deletion mutant (Δfba) required a balanced combination of carbon substrates entering metabolism above and below the FBA-catalyzed reaction for growth and died in media with single carbon sources and in mouse lungs. Death of Δfba in vitro was associated with the perturbation of intracellular metabolites. These studies highlight how a conditional fba mutant helped identify conditions in which FBA is dispensable for growth of Mtb, evaluate FBA as a potential target for eliminating persistent bacilli and offer insight into metabolic regulation of carbon co-catabolism in Mtb.
doi:10.1371/journal.ppat.1004144
PMCID: PMC4031216  PMID: 24851864
2.  Intermediate-Type Vancomycin Resistance (VISA) in Genetically-Distinct Staphylococcus aureus Isolates Is Linked to Specific, Reversible Metabolic Alterations 
PLoS ONE  2014;9(5):e97137.
Intermediate (VISA-type) vancomycin resistance in Staphylococcus aureus has been associated with a range of physiologic and genetic alterations. Previous work described the emergence of VISA-type resistance in two clonally-distinct series of isolates. In both series (the first belonging to MRSA clone ST8-USA300, and the second to ST5-USA100), resistance was conferred by a single mutation in yvqF (a negative regulator of the vraSR two-component system associated with vancomycin resistance). In the USA300 series, resistance was reversed by a secondary mutation in vraSR. In this study, we combined systems-level metabolomic profiling with statistical modeling techniques to discover specific, reversible metabolic alterations associated with the VISA phenotype.
doi:10.1371/journal.pone.0097137
PMCID: PMC4016254  PMID: 24817125
3.  Triosephosphate Isomerase Is Dispensable In Vitro yet Essential for Mycobacterium tuberculosis To Establish Infection 
mBio  2014;5(2):e00085-14.
ABSTRACT
Triosephosphate isomerase (TPI) catalyzes the interconversion of dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G3P). This reaction is required for glycolysis and gluconeogenesis, and tpi has been predicted to be essential for growth of Mycobacterium tuberculosis. However, when studying a conditionally regulated tpi knockdown mutant, we noticed that depletion of TPI reduced growth of M. tuberculosis in media containing a single carbon source but not in media that contained both a glycolytic and a gluconeogenic carbon source. We used such two-carbon-source media to isolate a tpi deletion (Δtpi) mutant. The Δtpi mutant did not survive with single carbon substrates but grew like wild-type (WT) M. tuberculosis in the presence of both a glycolytic and a gluconeogenic carbon source. 13C metabolite tracing revealed the accumulation of TPI substrates in Δtpi and the absence of alternative triosephosphate isomerases and metabolic bypass reactions, which confirmed the requirement of TPI for glycolysis and gluconeogenesis in M. tuberculosis. The Δtpi strain was furthermore severely attenuated in the mouse model of tuberculosis, suggesting that M. tuberculosis cannot simultaneously access sufficient quantities of glycolytic and gluconeogenic carbon substrates to establish infection in mice.
IMPORTANCE
The importance of central carbon metabolism for the pathogenesis of M. tuberculosis has recently been recognized, but the consequences of depleting specific metabolic enzymes remain to be identified for many enzymes. We investigated triosephosphate isomerase (TPI) because it is central to both glycolysis and gluconeogenesis and had been predicted to be essential for growth of M. tuberculosis. This work identified metabolic conditions that make TPI dispensable for M. tuberculosis growth in culture and proved that M. tuberculosis relies on a single TPI enzyme and has no metabolic bypass for the TPI-dependent interconversion of dihydroxyacetone phosphate and glyceraldehyde-3-phosphate in glycolysis and gluconeogenesis. Finally, we demonstrate that TPI is essential for growth of the pathogen in mouse lungs.
doi:10.1128/mBio.00085-14
PMCID: PMC3994511  PMID: 24757211
4.  Para-Aminosalicylic Acid Acts as an Alternative Substrate of Folate Metabolism in Mycobacterium tuberculosis 
Science (New York, N.Y.)  2012;339(6115):88-91.
Folate biosynthesis is an established anti-infective target, and the antifolate para-aminosalicylic acid (PAS) was one of the first anti-infectives introduced into clinical practice based on target-based drug discovery. Fifty years later, PAS continues in use for tuberculosis. PAS is assumed to inhibit dihydropteroate synthase (DHPS) in Mycobacterium tuberculosis (M. tuberculosis) by mimicking the substrate, p-aminobenzoate (PABA). However, we found inhibition of DHPS did not inhibit growth of M. tuberculosis. Instead, PAS, unlike sulfonamides, served as a replacement substrate for DHPS. Products of PAS metabolism at this and subsequent steps in folate metabolism inhibited those enzymes, competing with their substrates. PAS is thus a prodrug that blocks growth of M. tuberculosis when its active forms are generated by enzymes in the pathway that they poison.
doi:10.1126/science.1228980
PMCID: PMC3792487  PMID: 23118010
5.  Metabolomic Analysis of Patient Plasma Yields Evidence of Plant-Like α-Linolenic Acid Metabolism in Plasmodium falciparum 
The Journal of Infectious Diseases  2012;206(2):238-248.
Metabolomics offers a powerful means to investigate human malaria parasite biology and host-parasite interactions at the biochemical level, and to discover novel therapeutic targets and biomarkers of infection. Here, we used an approach based on liquid chromatography and mass spectrometry to perform an untargeted metabolomic analysis of metabolite extracts from Plasmodium falciparum–infected and uninfected patient plasma samples, and from an enriched population of in vitro cultured P. falciparum-infected and uninfected erythrocytes. Statistical modeling robustly segregated infected and uninfected samples based on metabolite species with significantly different abundances. Metabolites of the α-linolenic acid (ALA) pathway, known to exist in plants but not known to exist in P. falciparum until now, were enriched in infected plasma and erythrocyte samples. In vitro labeling with 13C-ALA showed evidence of plant-like ALA pathway intermediates in P. falciparum. Ortholog searches using ALA pathway enzyme sequences from 8 available plant genomes identified several genes in the P. falciparum genome that were predicted to potentially encode the corresponding enzymes in the hitherto unannotated P. falciparum pathway. These data suggest that our approach can be used to discover novel facets of host/malaria parasite biology in a high-throughput manner.
doi:10.1093/infdis/jis339
PMCID: PMC3490690  PMID: 22566569
6.  Central carbon metabolism in Mycobacterium tuberculosis: an unexpected frontier 
Trends in microbiology  2011;19(7):307-314.
Recent advances in liquid chromatography and mass spectrometry have enabled the highly parallel, quantitative measurement of metabolites within a cell and the ability to trace their biochemical fates. In Mycobacterium tuberculosis (Mtb), these advances have highlighted major gaps in our understanding of central carbon metabolism (CCM) that have prompted fresh interpretations of the composition and structure of its metabolic pathways and the phenotypes of Mtb strains in which CCM genes have been deleted. High-throughput screens have demonstrated that small chemical compounds can selectively inhibit some enzymes of Mtb’s CCM while sparing homologs in the host. Mtb’s CCM has thus emerged as a frontier for both fundamental and translational research.
doi:10.1016/j.tim.2011.03.008
PMCID: PMC3601588  PMID: 21561773
7.  Glucose Phosphorylation Is Required for Mycobacterium tuberculosis Persistence in Mice 
PLoS Pathogens  2013;9(1):e1003116.
Mycobacterium tuberculosis (Mtb) is thought to preferentially rely on fatty acid metabolism to both establish and maintain chronic infections. Its metabolic network, however, allows efficient co-catabolism of multiple carbon substrates. To gain insight into the importance of carbohydrate substrates for Mtb pathogenesis we evaluated the role of glucose phosphorylation, the first reaction in glycolysis. We discovered that Mtb expresses two functional glucokinases. Mtb required the polyphosphate glucokinase PPGK for normal growth on glucose, while its second glucokinase GLKA was dispensable. 13C-based metabolomic profiling revealed that both enzymes are capable of incorporating glucose into Mtb's central carbon metabolism, with PPGK serving as dominant glucokinase in wild type (wt) Mtb. When both glucokinase genes, ppgK and glkA, were deleted from its genome, Mtb was unable to use external glucose as substrate for growth or metabolism. Characterization of the glucokinase mutants in mouse infections demonstrated that glucose phosphorylation is dispensable for establishing infection in mice. Surprisingly, however, the glucokinase double mutant failed to persist normally in lungs, which suggests that Mtb has access to glucose in vivo and relies on glucose phosphorylation to survive during chronic mouse infections.
Author Summary
The development of new drugs targeting Mycobacterium tuberculosis (Mtb) will benefit from a better understanding of the mechanisms by which this pathogen establishes and maintains chronic infections. Mtb has to adapt its metabolic needs to the nutritional environment in the host. We investigated the role of glucose phosphorylation and discovered that Mtb expresses two functional glucokinases. Using 13C-tracing experiments we demonstrated that both enzymes are competent to incorporate glucose into central carbon metabolism. In agreement with the view that Mtb metabolizes fatty acids to grow in vivo, both enzymes were dispensable for Mtb replication in mouse lungs and spleens. Surprisingly, however, the glucokinase double mutant was attenuated during the chronic phase of mouse infections. These studies suggest that Mtb metabolizes glucose in vivo and that its survival in chronically infected mice depends on glucose phosphorylation.
doi:10.1371/journal.ppat.1003116
PMCID: PMC3542180  PMID: 23326232
8.  Prevalence, persistence, and microbiology of Staphylococcus aureus nasal carriage among hemodialysis outpatients at a major New York Hospital☆,☆☆ 
The study aimed to determine the natural history of Staphylococcus aureus nasal colonization in hemodialysis outpatients. Surveillance cultures were taken from patients presenting for hemodialysis or routine care to identify S. aureus nasal carriers. A prospective cohort study was performed to identify risks for persistent colonization. Detailed microbiologic and molecular studies of colonizing isolates were performed. Only 23/145 (15.9%) dialysis patients were persistently colonized, and only HIV-positive status was associated with persistence (P = 0.05). Prior hospitalization was the only risk factor for methicillin-resistant S. aureus carriage (OR 2.5, P = 0.03). In isolates from patients with ≤42 days of vancomycin exposure, vancomycin minimum bactericidal concentrations (MBCs) increased with duration of exposure. Among dialysis patients, S. aureus colonization was limited and transient; only HIV status was associated with persistence. Nevertheless, duration of vancomycin exposure was associated with increasing vancomycin MBCs. Vancomycin exposure in S. aureus carriers may be involved in increasing resistance.
doi:10.1016/j.diagmicrobio.2010.12.005
PMCID: PMC3534839  PMID: 21334154
Staphylococcus aureus; Hemodialysis; Colonization; Vancomycin
9.  Nitazoxanide Disrupts Membrane Potential and Intrabacterial pH Homeostasis of Mycobacterium tuberculosis 
ACS Medicinal Chemistry Letters  2011;2(11):849-854.
Nitazoxanide (Alinia), a nitro-thiazolyl antiparasitic drug, kills diverse microorganisms by unknown mechanisms. Here we identified two actions of nitazoxanide against Mycobacterium tuberculosis (Mtb): disruption of Mtb’s membrane potential and pH homeostasis. Both actions were shared by a structurally related antimycobacterial compound, niclosamide. Reactive nitrogen intermediates were reported to synergize with nitazoxanide and its deacetylated derivative tizoxanide in killing Mtb. Herein, however, we could not attribute this to increased uptake of nitazoxanide or tizoxanide as monitored by targeted metabolomics, nor to increased impact of nitazoxanide on Mtb’s membrane potential or intrabacterial pH. Thus, further mechanisms of action of nitazoxanide or tizoxanide may await discovery. The multiple mechanisms of action may contribute to Mtb’s ultralow frequency of resistance against nitazoxanide.
doi:10.1021/ml200157f
PMCID: PMC3215101  PMID: 22096616
Nitazoxanide; tizoxanide; Mycobacterium tuberculosis; niclosamide; pH homeostasis; membrane potential; metabolomics
10.  Correction: Endemic Acinetobacter baumannii in a New York Hospital 
PLoS ONE  2012;7(10):10.1371/annotation/22d2ce95-f6c5-46fa-9e32-0d0fd21e20df.
doi:10.1371/annotation/22d2ce95-f6c5-46fa-9e32-0d0fd21e20df
PMCID: PMC3502304
11.  Expression of the Leptin Receptor Outside of Bone Marrow-derived Cells Regulates Tuberculosis Control and Lung Macrophage MHC Expressioni 
Leptin is a pleiotropic hormone proposed to link nutritional status to the development of strong Th1 immunity. As Mycobacterium tuberculosis (MTb) control is affected by starvation and diabetes, we studied the role of the leptin receptor in regulating distinct immune cells during chronic infection. Infected db/db mice, bearing a natural mutation in the leptin receptor, have a markedly increased bacterial load in their lungs when compared to their wild-type (WT) counterparts. In response to MTb infection, db/db mice exhibited disorganized granulomas, neutrophilia, and reduced B cell migration to the lungs, correlating with dysfunctional lung chemokine responses that include XCL1, CCL2, CXCL1, CXCL2, and CXCL13. In a db/db lung, myeloid cells were delayed in their production of inducible nitric oxide synthase (iNOS) and had reduced expression of MHC I and II. Although the Th1 cell response developed normally in the absence of leptin signaling, production of pulmonary IFNγ was delayed and ineffective. Surprisingly, a proper immune response took place in bone marrow (BM) chimeras lacking leptin receptor exclusively in BM-derived cells, indicating that leptin acts indirectly on immune cells to modulate the anti-tuberculosis response and bacterial control. Together these findings suggest that the pulmonary response to MTb is affected by the host’s nutritional status via the regulation of non-BM derived cells, and not through direct action of leptin on the Th1 immunity.
doi:10.4049/jimmunol.1003226
PMCID: PMC3178734  PMID: 21859958
12.  Extended Spectrum Beta-Lactamase-Producing Enterobacteriaceae in International Travelers and Non-Travelers in New York City 
PLoS ONE  2012;7(9):e45141.
Background
We performed this study 1) to determine the prevalence of community-associated extended spectrum beta-lactamase producing Enterobacteriaceae (ESBLPE) colonization and infection in New York City (NYC); 2) to determine the prevalence of newly-acquired ESBLPE during travel; 3) to look for similarilties in contemporaneous hospital-associated bloodstream ESBLPE and travel-associated ESBLPE.
Methods
Subjects were recruited from a travel medicine practice and consented to submit pre- and post-travel stools, which were assessed for the presence of ESBLPE. Pre-travel stools and stools submitted for culture were used to estimate the prevalence of community-associated ESBLPE. The prevalence of ESBLPE-associated urinary tract infections was calculated from available retrospective data. Hospital-associated ESBLPE were acquired from saved bloodstream isolates. All ESBLPE underwent multilocus sequence typing (MLST) and ESBL characterization.
Results
One of 60 (1.7%) pre- or non-travel associated stool was colonized with ESBLPE. Among community-associated urine specimens, 1.3% of Escherichia coli and 1.4% of Klebsiella pneumoniae were identified as ESBLPE. Seven of 28 travelers (25.0%) acquired a new ESBLPE during travel. No similarities were found between travel-associated ESBLPE and hospital-associated ESBLPE. A range of imported ESBL genes were found, including CTX-M-14 and CTX-15.
Conclusion
ESBL colonization and infection were relatively low during the study period in NYC. A signficant minority of travelers acquired new ESBLPE during travel.
doi:10.1371/journal.pone.0045141
PMCID: PMC3447858  PMID: 23028808
13.  On the Chemical Mechanism of Succinic Semialdehyde Dehydrogenase (GabD1) from Mycobacterium tuberculosis* 
Succinic semialdehyde dehydrogenases (SSADHs) are ubiquitous enzymes that catalyze the NAD(P)+-coupled oxidation of succinic semialdehyde (SSA) to succinate, the last step of the γ-aminobutyrate shunt. Mycobacterium tuberculosis encodes two paralogous SSADHs (gabD1 and gabD2). Here we describe the first mechanistic characterization of GabD1, using steady-state kinetics, pH-rate profiles, 1H-NMR, and kinetic isotope effects. Our results confirmed SSA and NADP+ as substrates and demonstrated that a divalent metal, such as Mg2+, linearizes the time course. pH-rate studies failed to identify any ionizable groups with pKa between 5.5 and 10 involved in substrate binding or rate-limiting chemistry. Primary deuterium, solvent and multiple kinetic isotope effects revealed that nucleophilic addition to SSA is very fast, followed by a modestly rate-limiting hydride transfer and fast thioester hydrolysis. Proton inventory studies revealed that a single proton is associated with the solvent-sensitive rate-limiting step. Together, these results suggest that product dissociation and/or conformational changes linked to it are rate-limiting. Using structural information for the human homolog enzyme and 1H-NMR, we further established that nucleophilic attack takes place at the Si face of SSA, generating a thiohemiacetal with S stereochemistry. Deuteride transfer to the Pro-R position in NADP+ generates the thioester intermediate and [4A-2H, 4B-1H] NADPH. A chemical mechanism based on these data and the structural information available is proposed.
doi:10.1016/j.abb.2011.01.023
PMCID: PMC3094805  PMID: 21303655
succinic semialdehyde dehydrogenase; aldehyde dehydrogenase; chemical mechanism; stereochemistry; hydride transfer; isotope effects
14.  Metabolomics and malaria biology 
Metabolomics has ushered in a novel and multi-disciplinary realm in biological research. It has provided researchers with a platform to combine powerful biochemical, statistical, computational, and bioinformatics techniques to delve into the mysteries of biology and disease. The application of metabolomics to study malaria parasites represents a major advance in our approach towards gaining a more comprehensive perspective on parasite biology and disease etiology. This review attempts to highlight some of the important aspects of the field of metabolomics, and its ongoing and potential future applications to malaria research.
doi:10.1016/j.molbiopara.2010.09.008
PMCID: PMC3004989  PMID: 20970461
Malaria; metabolomics
15.  Virulence of Mycobacterium tuberculosis depends on lipoamide dehydrogenase, a member of three multi-enzyme complexes 
Cell host & microbe  2011;9(1):21-31.
SUMMARY
Mycobacterium tuberculosis (Mtb) adapts to persist in a nutritionally limited macrophage compartment. Lipoamide dehydrogenase (Lpd), the third enzyme (E3) in Mtb’s pyruvate dehydrogenase complex (PDH), also serves as E1 of peroxynitrite reductase/peroxidase (PNR/P), which helps Mtb resist host reactive nitrogen intermediates. In contrast to Mtb lacking dihydrolipoamide acyltransferase (DlaT), the E2 of PDH and PNR/P, Lpd-deficient Mtb is severely attenuated in wild type and immunodeficient mice. This suggests that Lpd has a function that DlaT does not share. When DlaT is absent, Mtb upregulates an Lpd-dependent branched chain keto-acid dehydrogenase (BCKADH) encoded by pdhA, pdhB, pdhC and lpdC. Without Lpd, Mtb cannot metabolize branched chain amino acids and potentially toxic branched chain intermediates accumulate. Mtb deficient in both DlaT and PdhC phenocopies Lpd-deficient Mtb. Thus, Mtb critically requires BCKADH along with PDH and PNR/P for pathogenesis. These findings position Lpd as a potential target for anti-infectives against Mtb.
doi:10.1016/j.chom.2010.12.004
PMCID: PMC3040420  PMID: 21238944
16.  Nitazoxanide Disrupts Membrane Potential and Intrabacterial pH Homeostasis of Mycobacterium tuberculosis 
ACS medicinal chemistry letters  2011;2(11):849-854.
Nitazoxanide (Alinia®), a nitro-thiazolyl antiparasitic drug, kills diverse microorganisms by unknown mechanisms. Here we identified two actions of nitazoxanide against Mycobacterium tuberculosis (Mtb): disruption of Mtb’s membrane potential and pH homeostasis. Both actions were shared by a structurally related anti-mycobacterial compound, niclosamide. Reactive nitrogen intermediates were reported to synergize with nitazoxanide and its deacetylated derivative tizoxanide in killing Mtb. Herein, however, we could not attribute this to increased uptake of nitazoxanide or tizoxanide as monitored by targeted metabolomics, nor to increased impact of nitazoxanide on Mtb’s membrane potential or intrabacterial pH. Thus, further mechanisms of action of nitazoxanide or tizoxanide may await discovery. The multiple mechanisms of action may contribute to Mtb’s ultra-low frequency of resistance against nitazoxanide.
doi:10.1021/ml200157f
PMCID: PMC3215101  PMID: 22096616
Nitazoxanide; tizoxanide; Mycobacterium tuberculosis; niclosamide; pH homeostasis; membrane potential; metabolomics
17.  Endemic Acinetobacter baumannii in a New York Hospital 
PLoS ONE  2011;6(12):e28566.
Background
Acinetobacter baumannii is an increasingly multidrug-resistant (MDR) cause of hospital-acquired infections, often associated with limited therapeutic options. We investigated A. baumannii isolates at a New York hospital to characterize genetic relatedness.
Methods
Thirty A. baumannii isolates from geographically-dispersed nursing units within the hospital were studied. Isolate relatedness was assessed by repetitive sequence polymerase chain reaction (rep-PCR). The presence and characteristics of integrons were assessed by PCR. Metabolomic profiles of a subset of a prevalent strain isolates and sporadic isolates were characterized and compared.
Results
We detected a hospital-wide group of closely related carbapenem resistant MDR A. baumannii isolates. Compared with sporadic isolates, the prevalent strain isolates were more likely to be MDR (p = 0.001). Isolates from the prevalent strain carried a novel Class I integron sequence. Metabolomic profiles of selected prevalent strain isolates and sporadic isolates were similar.
Conclusion
The A. baumannii population at our hospital represents a prevalent strain of related MDR isolates that contain a novel integron cassette. Prevalent strain and sporadic isolates did not segregate by metabolomic profiles. Further study of environmental, host, and bacterial factors associated with the persistence of prevalent endemic A. baumannii strains is needed to develop effective prevention strategies.
doi:10.1371/journal.pone.0028566
PMCID: PMC3236744  PMID: 22180786
18.  Evaluating the Sensitivity of Mycobacterium tuberculosis to Biotin Deprivation Using Regulated Gene Expression 
PLoS Pathogens  2011;7(9):e1002264.
In the search for new drug targets, we evaluated the biotin synthetic pathway of Mycobacterium tuberculosis (Mtb) and constructed an Mtb mutant lacking the biotin biosynthetic enzyme 7,8-diaminopelargonic acid synthase, BioA. In biotin-free synthetic media, ΔbioA did not produce wild-type levels of biotinylated proteins, and therefore did not grow and lost viability. ΔbioA was also unable to establish infection in mice. Conditionally-regulated knockdown strains of Mtb similarly exhibited impaired bacterial growth and viability in vitro and in mice, irrespective of the timing of transcriptional silencing. Biochemical studies further showed that BioA activity has to be reduced by approximately 99% to prevent growth. These studies thus establish that de novo biotin synthesis is essential for Mtb to establish and maintain a chronic infection in a murine model of TB. Moreover, these studies provide an experimental strategy to systematically rank the in vivo value of potential drug targets in Mtb and other pathogens.
Author Summary
We evaluated the biotin synthetic pathway of Mycobacterium tuberculosis (Mtb) as a new drug target by first generating an Mtb deletion mutant, ΔbioA, in which the biotin biosynthetic enzyme 7,8-diaminopelargonic acid synthase (BioA) has been inactivated. This mutant grew in the presence of biotin or des-thiobiotin, but not with an intermediate of the biotin biosynthesis pathway that requires BioA to be converted into biotin. Without exogenous biotin or des-thiobiotin, ΔbioA, was unable to produce biotinylated proteins, which are required for the biosynthesis of fatty acids, and thus died in biotin-free media. Using a regulatable promoter and different ribosome binding sequences we next constructed tightly controlled TetON mutants, in which expression of BioA could be induced with tetracyclines, but was inhibited in their absence. Characterization of these mutants during infections demonstrated that de novo biotin synthesis is not only required to establish infections but also to maintain bacterial persistence. Inhibition of BioA or other enzymes of the biotin biosynthesis pathways could thus be used to kill Mtb during both acute and chronic infections. Biochemical and immunological analyses of different Mtb mutants indicate that drugs targeting BioA would have to inactive approximately 99% of its activity to be effective.
doi:10.1371/journal.ppat.1002264
PMCID: PMC3182931  PMID: 21980288
19.  Dispensability of Surfactant Proteins A and D in Immune Control of Mycobacterium tuberculosis Infection following Aerosol Challenge of Mice▿  
Infection and Immunity  2011;79(3):1077-1085.
Surfactant proteins A and D (SP-A and -D) play a role in many acute bacterial, viral, and fungal infections and in acute allergic responses. In vitro, human SPs bind Mycobacterium tuberculosis and alter human and rat macrophage-mediated functions. Here we report the roles of SP-A and SP-D in M. tuberculosis infection following aerosol challenge of SP-A-, SP-D-, and SP-A/-D-deficient mice. These studies surprisingly identified no gross defects in uptake or immune control of M. tuberculosis in SP-A-, SP-D-, and SP-A/-D-deficient mice. While both SP-A- and SP-D-deficient mice exhibited evidence of immunopathologic defects, the CD11bhigh CD11chigh dendritic cell populations and the gamma interferon (IFN-γ)-dependent CD4+ T cell response to M. tuberculosis were unaltered in all genotypes tested. Together, these data indicate that SP-A and SP-D are dispensable for immune control of M. tuberculosis in a low-dose, aerosol challenge, murine model of tuberculosis (TB).
doi:10.1128/IAI.00286-10
PMCID: PMC3067487  PMID: 21199913
20.  Chronic Disease Surveillance Systems Within the US Associated Pacific Island Jurisdictions 
Preventing Chronic Disease  2011;8(4):A86.
In recent years, illness and death due to chronic disease in the US Associated Pacific Islands (USAPI) jurisdictions have dramatically increased. Effective chronic disease surveillance can help monitor disease trends, evaluate public policy, prioritize resource allocation, and guide program planning, evaluation, and research. Although chronic disease surveillance is being conducted in the USAPI, no recently published capacity assessments for chronic disease surveillance are available. The objective of this study was to assess the quality of existing USAPI chronic disease data sources and identify jurisdictional capacity for chronic disease surveillance. The assessment included a chronic disease data source inventory, literature review, and review of surveillance documentation available from the web or through individual jurisdictions. We used the World Health Organization's Health Metric Network Framework to assess data source quality and to identify jurisdictional capacity. Results showed that USAPI data sources are generally aligned with widely accepted chronic disease surveillance indicators and use standardized data collection methodology to measure chronic disease behavioral risks, preventive practices, illness, and death. However, all jurisdictions need to strengthen chronic disease surveillance through continued assessment and expanded support for valid and reliable data collection, analysis and reporting, dissemination, and integration among population-based and institution-based data sources. For sustained improvement, we recommend investment and technical assistance in support of a chronic disease surveillance system that integrates population-based and institution-based data sources. An integrated strategy that bridges and links USAPI data sources can support evidence-based policy and population health interventions.
PMCID: PMC3136978  PMID: 21672410
21.  Activity-based metabolomic profiling of enzymatic function: Identification of Rv1248c as a mycobacterial 2-hydroxy-3-oxoadipate synthase 
Chemistry & biology  2010;17(4):323-332.
SUMMARY
Activity based metabolomic profiling (ABMP) allows unbiased discovery of enzymatic activities encoded by genes of unknown function. ABMP applies liquid chromatography-mass spectrometry (LC-MS) to analyze the impact of a recombinant enzyme on the homologous cellular extract as a physiologic library of potential substrates and products. The Mycobacterium tuberculosis protein Rv1248c was incompletely characterized as a thiamine diphosphate-dependent α-ketoglutarate decarboxylase. Here, recombinant Rv1248c catalyzed consumption of α–ketoglutarate in a mycobacterial small molecule extract with matched production of 5-hydroxylevulinate (HLA) in a reaction predicted to require glyoxylate. As confirmed using pure substrates by LC-MS, 1H-NMR, chemical trapping, and intracellular metabolite profiling, Rv1248c catalyzes C-C bond formation between the activated aldehyde of α–ketoglutarate and the carbonyl of glyoxylate to yield 2-hydroxy-3-oxoadipate (HOA), which decomposes to HLA. Thus, Rv1248c encodes a HOA synthase (HOAS).
doi:10.1016/j.chembiol.2010.03.009
PMCID: PMC2878197  PMID: 20416504
22.  Innovation Networks for Improving Access and Quality Across the Healthcare Ecosystem 
Telemedicine Journal and e-Health  2010;16(1):107-111.
Abstract
Partnerships between patient communities, healthcare providers, and academic researchers are key to stepping up the pace and public health impact of clinical and translational research supported by the National Institutes of Health. With emphasis shifting toward community engagement and faster translation of research advances into clinical practice, academic researchers have a vital stake in widening the use of health information technology systems and telehealth networks to support collaboration and innovation. However, limited interaction between academic institutions and healthcare providers hinders the ability to form and sustain the integrated networks that are needed to conduct meaningful community-engaged research that improves public health outcomes. Healthcare providers, especially those affiliated with smaller practices, will need sustainable infrastructure and real incentives to utilize such networks, as well as training and additional resources for ongoing technical assistance.
doi:10.1089/tmj.2009.0157
PMCID: PMC3016866  PMID: 20043702
business administration/economics; distance learning; e-health; home health monitoring; policy
23.  Selective Killing of Nonreplicating Mycobacteria 
Cell host & microbe  2008;3(3):137-145.
SUMMARY
Antibiotics are typically more effective against replicating rather than nonreplicating bacteria. However, a major need in global health is to eradicate persistent or nonreplicating subpopulations of bacteria such as Mycobacterium tuberculosis (Mtb). Hence, identifying chemical inhibitors that selectively kill bacteria that are not replicating is of practical importance. To address this, we screened for inhibitors of dihydrolipoamide acyltransferase (DlaT), an enzyme required by Mtb to cause tuberculosis in guinea pigs and used by the bacterium to resist nitric oxide-derived reactive nitrogen intermediates, a stress encountered in the host. Chemical screening for inhibitors of Mtb DlaT identified select rhodanines as compounds that almost exclusively kill nonreplicating mycobacteria in synergy with products of host immunity, such as nitric oxide and hypoxia, and are effective on bacteria within macrophages, a cellular reservoir for latent Mtb. Compounds that kill nonreplicating pathogens in cooperation with host immunity could complement the conventional chemotherapy of infectious disease.
doi:10.1016/j.chom.2008.02.003
PMCID: PMC2423947  PMID: 18329613
24.  A chemical genetic screen in Mycobacterium tuberculosis identifies carbon-source-dependent growth inhibitors devoid of in vivo efficacy 
Nature Communications  2010;1(5):1-8.
Candidate antibacterials are usually identified on the basis of their in vitro activity. However, the apparent inhibitory activity of new leads can be misleading because most culture media do not reproduce an environment relevant to infection in vivo. In this study, while screening for novel anti-tuberculars, we uncovered how carbon metabolism can affect antimicrobial activity. Novel pyrimidine–imidazoles (PIs) were identified in a whole-cell screen against Mycobacterium tuberculosis. Lead optimization generated in vitro potent derivatives with desirable pharmacokinetic properties, yet without in vivo efficacy. Mechanism of action studies linked the PI activity to glycerol metabolism, which is not relevant for M. tuberculosis during infection. PIs induced self-poisoning of M. tuberculosis by promoting the accumulation of glycerol phosphate and rapid ATP depletion. This study underlines the importance of understanding central bacterial metabolism in vivo and of developing predictive in vitro culture conditions as a prerequisite for the rational discovery of new antibiotics.
Candidate anti-tuberculosis drugs are often identified in whole-cell screens. Here, Pethe et al. show that inappropriate carbon-source selection can lead to the identification of compounds devoid of efficacy in vivo, underlining the importance of developing predictive in vitro screens.
doi:10.1038/ncomms1060
PMCID: PMC3220188  PMID: 20975714

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