Robert J. Fenton

Predicting the in Vivo Mechanism of Action for Drug Leads Using NMR Metabolomics

New strategies are needed to circumvent increasing outbreaks of resistant strains of pathogens and to expand the dwindling supply of effective antimicrobials. A common impediment to drug development is the lack of an easy approach to determine the in vivo mechanism of action and efficacy of novel drug leads. Toward this end, we describe an unbiased approach to predict in vivo mechanisms of action from NMR metabolomics data. Mycobacterium smegmatis, a non-pathogenic model organism for Mycobacterium tuberculosis, was treated with 12 known drugs and 3 chemical leads identified from a cell-based assay. NMR analysis of drug-induced changes to the M. smegmatis metabolome resulted in distinct clustering patterns correlating with in vivo drug activity. The clustering of novel chemical leads relative to known drugs provides a mean to identify a protein target or predict in vivo activity.

Metabolomics Analysis Identifies d-Alanine-d-Alanine Ligase as the Primary Lethal Target of d-Cycloserine in Mycobacteria

d-Cycloserine is an effective second line antibiotic used as a last resort to treat multi (MDR)- and extensively (XDR) drug resistant strains of Mycobacterium tuberculosis . d-Cycloserine interferes with the formation of peptidoglycan biosynthesis by competitive inhibition of alanine racemase (Alr) and d-alanine-d-alanine ligase (Ddl). Although the two enzymes are known to be inhibited, the in vivo lethal target is still unknown. Our NMR metabolomics work has revealed that Ddl is the primary target of DCS, as cell growth is inhibited when the production of d-alanyl-d-alanine is halted. It is shown that inhibition of Alr may contribute indirectly by lowering the levels of d-alanine, thus allowing DCS to outcompete d-alanine for Ddl binding. The NMR data also supports the possibility of a transamination reaction to produce d-alanine from pyruvate and glutamate, thereby bypassing Alr inhibition. Furthermore, the inhibition of peptidoglycan synthesis results in a cascading effect on cellular metabolism as there is a shift toward the catabolic routes to compensate for accumulation of peptidoglycan precursors.

Immunogenicity and Reactivity of Novel Mycobacterium avium subsp. paratuberculosis PPE MAP1152 and Conserved MAP1156 Proteins with Sera from Experimentally and Naturally Infected Animals

ABSTRACT Mycobacterium avium subsp. paratuberculosis causes Johnes disease (JD) in ruminants. Development of genetic tools and completion of the M. avium subsp. paratuberculosis genome sequencing project have expanded the opportunities for antigen discovery. In this study, we determined the seroreactivities of two proteins encoded at the 5′ and 3′ regions of the MAP1152-MAP1156 gene cluster. MAP1152 encodes a PPE protein, and MAP1156 encodes a diacylglycerol acyltransferase involved in triglyceride metabolism and classified in the uncharacterized protein family UPF0089. Recombinant MAP proteins were overproduced and purified from Escherichia coli as maltose-binding protein (MBP) fusions. Immunoblotting analysis indicated that both MAP1152 and MAP1156 displayed reactivity against sera of mice and rabbits immunized with live M. avium subsp. paratuberculosis cells and against samples from naturally infected cattle. In immunoblot assays, MAP1156 yielded a stronger positive signal than MAP1152 against sera from cattle with JD. An enzyme-linked immunosorbent assay for the recombinant proteins was developed and used to test preclassified positive and negative serum samples from naturally infected and noninfected cattle. Samples, with one exception, displayed no seroreactivity against the MBP-LacZ fusion protein (P > 0.05), the negative-control antigen. MAP1152 displayed seroreactivity against all positive sera but no seroreactivity to the negative sera (P < 0.01). MAP1156 displayed stronger and more variable reactivity than MAP1152, but significant differences were observed between noninfected and infected cattle (P < 0.05). Otherwise, degrees of reactivity followed the same trend as the positive reference antigen. In conclusion, both proteins are immunogenic in mice and rabbits, and M. avium subsp. paratuberculosis-infected cattle mount a humoral response to both MAP1152 and MAP1156 cross-reactive epitopes. These findings have potential applications to diagnostics, vaccine production, and elucidation of the immunopathogenesis of JD.

Impairment of D-alanine biosynthesis in Mycobacterium smegmatis determines decreased intracellular survival in human macrophages

d-Alanine is a structural component of mycobacterial peptidoglycan. The primary route of d-alanine biosynthesis in eubacteria is the enantiomeric conversion from l-alanine, a reaction catalysed by d-alanine racemase (Alr). Mycobacterium smegmatis alr insertion mutants are not dependent on d-alanine for growth and display a metabolic pattern consistent with an alternative pathway for d-alanine biosynthesis. In this study, we demonstrate that the M. smegmatis alr insertion mutant TAM23 can synthesize d-alanine at lower levels than the parental strain. The insertional inactivation of the alr gene also decreases the intracellular survival of mutant strains within primary human monocyte-derived macrophages. By complementation studies, we confirmed that the impairment of alr gene function is responsible for this reduced survival. Inhibition of superoxide anion and nitric oxide formation in macrophages suppresses the differential survival. In contrast, for bacteria grown in broth, both strains had approximately the same susceptibility to hydrogen peroxide, acidified sodium nitrite, low pH and polymyxin B. In contrast, TAM23 exhibited increased resistance to lysozyme. d-Alanine supplementation considerably increased TAM23 viability in nutritionally deficient media and within macrophages. These results suggest that nutrient deprivation in phagocytic cells combined with killing mediated by reactive intermediates underlies the decreased survival of alr mutants. This knowledge may be valuable in the construction of mycobacterial auxotrophic vaccine candidates.

Generation and screening of a comprehensive Mycobacterium avium subsp. paratuberculosis transposon mutant bank

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of Johnes Disease in ruminants. This enteritis has significant economic impact and worldwide distribution. Vaccination is one of the most cost effective infectious disease control measures. Unfortunately, current vaccines reduce clinical disease and shedding, but are of limited efficacy and do not provide long-term protective immunity. Several strategies have been followed to mine the MAP genome for virulence determinants that could be applied to vaccine and diagnostic assay development. In this study, a comprehensive mutant bank of 13,536 MAP K-10 Tn5367 mutants (P > 95%) was constructed and screened in vitro for phenotypes related to virulence. This strategy was designated to maximize identification of genes important to MAP pathogenesis without relying on studies of other mycobacterial species that may not translate into similar effects in MAP. This bank was screened for mutants with colony morphology alterations, susceptibility to D-cycloserine, impairment in siderophore production or secretion, reduced cell association, and decreased biofilm and clump formation. Mutants with interesting phenotypes were analyzed by PCR, Southern blotting and DNA sequencing to determine transposon insertion sites. These insertion sites mapped upstream from the MAP1152-MAP1156 cluster, internal to either the Mod operon gene MAP1566 or within the coding sequence of lsr2, and several intergenic regions. Growth curves in broth cultures, invasion assays and kinetics of survival and replication in primary bovine macrophages were also determined. The ability of vectors carrying Tn5370 to generate stable MAP mutants was also investigated.

Assessment of Metabolic Changes in Mycobacterium smegmatis Wild-Type and alr Mutant Strains: Evidence of a New Pathway of d-Alanine Biosynthesis

In mycobacteria, d-alanine is an essential precursor for peptidoglycan biosynthesis. The only confirmed enzymatic pathway to form d-alanine is through the racemization of l-alanine by alanine racemase (Alr, EC 5.1.1.1). Nevertheless, the essentiality of Alr in Mycobacterium tuberculosis and Mycobacterium smegmatis for cell survivability in the absence of d-alanine has been a point of controversy with contradictory results reported in the literature. To address this issue, we examined the effects of alr inactivation on the cellular metabolism of M. smegmatis. The M. smegmatis alr insertion mutant TAM23 exhibited essentially identical growth to wild-type mc2155 in the absence of d-alanine. NMR metabolomics revealed drastically distinct phenotypes between mc2155 and TAM23. A metabolic switch was observed for TAM23 as a function of supplemented d-alanine. In the absence of d-alanine, the metabolic response directed carbon through an unidentified transaminase to provide the essential d-alanine required for survival. The process is reversed when d-alanine is available, in which the d-alanine is directed to peptidoglycan biosynthesis. Our results provide further support for the hypothesis that Alr is not an essential function of M. smegmatis and that specific Alr inhibitors will have no bactericidal action.

Development of cyclobutene- and cyclobutane-functionalized fatty acids with inhibitory activity against Mycobacterium tuberculosis

Eleven fatty acid analogues incorporating four‐membered carbocycles (cyclobutenes, cyclobutanes, cyclobutanones, and cyclobutanols) were investigated for the ability to inhibit the growth of Mycobacterium smegmatis (Msm) and Mycobacterium tuberculosis (Mtb). A number of the analogues displayed inhibitory activity against both mycobacterial species in minimal media. Several of the molecules displayed potent levels of inhibition against Mtb, with MIC values equal to or below those observed with the anti‐tuberculosis drugs D‐cycloserine and isoniazid. In contrast, two of the analogues that display the greatest activity against Mtb failed to inhibit E. coli growth under either set of conditions. Thus, the active molecules identified herein may provide the basis for the development of anti‐mycobacterial agents against Mtb.

Sample preparation of Mycobacterium tuberculosis extracts for nuclear magnetic resonance metabolomic studies.

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) drug-resistant strains threatens to derail current disease control efforts. Thus, there is an urgent need to develop drugs and vaccines that are more effective than those currently available. The genome of M. tuberculosis has been known for more than 10 years, yet there are important gaps in our knowledge of gene function and essentiality. Many studies have since used gene expression analysis at both the transcriptomic and proteomic levels to determine the effects of drugs, oxidants, and growth conditions on the global patterns of gene expression. Ultimately, the final response of these changes is reflected in the metabolic composition of the bacterium including a few thousand small molecular weight chemicals. Comparing the metabolic profiles of wild type and mutant strains, either untreated or treated with a particular drug, can effectively allow target identification and may lead to the development of novel inhibitors with anti-tubercular activity. Likewise, the effects of two or more conditions on the metabolome can also be assessed. Nuclear magnetic resonance (NMR) is a powerful technology that is used to identify and quantify metabolic intermediates. In this protocol, procedures for the preparation of M. tuberculosis cell extracts for NMR metabolomic analysis are described. Cell cultures are grown under appropriate conditions and required Biosafety Level 3 containment, harvested, and subjected to mechanical lysis while maintaining cold temperatures to maximize preservation of metabolites. Cell lysates are recovered, filtered sterilized, and stored at ultra-low temperatures. Aliquots from these cell extracts are plated on Middlebrook 7H9 agar for colony-forming units to verify absence of viable cells. Upon two months of incubation at 37 °C, if no viable colonies are observed, samples are removed from the containment facility for downstream processing. Extracts are lyophilized, resuspended in deuterated buffer and injected in the NMR instrument, capturing spectroscopic data that is then subjected to statistical analysis. The procedures described can be applied for both one-dimensional (1D) H NMR and two-dimensional (2D) H-(13)C NMR analyses. This methodology provides more reliable small molecular weight metabolite identification and more reliable and sensitive quantitative analyses of cell extract metabolic compositions than chromatographic methods. Variations of the procedure described following the cell lysis step can also be adapted for parallel proteomic analysis.