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-Dalanine 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

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-Dalanine 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

Assessment of metabolic changes in \u3ci\u3eMycobacterium smegmatis\u3c/i\u3e wild type and \u3ci\u3ealr\u3c/i\u3e mutant strains: evidence for 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

\u3ci\u3eMycobacterium avium\u3c/i\u3e subsp. \u3ci\u3eparatuberculosis\u3c/i\u3e Candidate Vaccine Strains Are Pro-apoptotic in RAW264.7MurineMacrophages

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiological agent of Johne’s disease, a severe gastroenteritis of ruminants. This study developed a model cell culture system to rapidly screen MAP mutants with vaccine potential for apoptosis. Two wild-type strains, a transposon mutant, and two deletion mutant MAP strains (MOI of 10 with 1.2 × 106 CFU) were tested in murine RAW 264.7 macrophages to determine if they induce apoptosis and/or necrosis. Both deletion mutants were previously shown to be attenuated and immunogenic in primary bovine macrophages. All strains had similar growth rates, but cell morphology indicated that both deletion mutants were elongated with cell wall bulging. Cell death kinetics were followed by a real-time cellular assay to measure luminescence (apoptosis) and fluorescence (necrosis). A 6 h infection period was the appropriate time to assess apoptosis that was followed by secondary necrosis. Apoptosis was also quantified via DAPI-stained nuclear morphology and validated via flow cytometry. The combined analysis confirmed the hypothesis that candidate vaccine deletion mutants are pro-apoptotic in RAW 264.7 cells. In conclusion, the increased apoptosis seen in the deletion mutants correlates with the attenuated phenotype and immunogenicity observed in bovine macrophages, a property associated with good vaccine candidates

Novel Amphiphilic Cyclobutene and Cyclobutane \u3ci\u3ecis\u3c/i\u3e-C18 Fatty Acid Derivatives Inhibit \u3ci\u3eMycobacterium avium\u3c/i\u3e subsp. \u3ci\u3eparatuberculosis\u3c/i\u3e Growth

Mycobacterium avium subspecies paratuberculosis (Map) is the etiologic agent of Johne’s disease in ruminants and has been associated with Crohn’s disease in humans. An effective control of Map by either vaccines or chemoprophylaxis is a paramount need for veterinary and possibly human medicine. Given the importance of fatty acids in the biosynthesis of mycolic acids and the mycobacterial cell wall, we tested novel amphiphilic C10 and C18 cyclobutene and cyclobutane fatty acid derivatives for Map inhibition. Microdilution minimal inhibitory concentrations (MIC) with 5 or 7 week endpoints were measured in Middlebrook 7H9 base broth media. We compared the Map MIC results with those obtained previously with Mycobacterium tuberculosis and Mycobacterium smegmatis. Several of the C18 compounds showed moderate effcacy (MICs 392 to 824 μM) against Map, while a higher level of inhibition (MICs 6 to 82 μM) was observed for M. tuberculosis for select analogs from both the C10 and C18 groups. For most of these analogs tested in M. smegmatis, their effcacy decreased in the presence of bovine or human serum albumin. Compound 5 (OA-CB, 1-(octanoic acid-8-yl)-2-octylcyclobutene) was identified as the best chemical lead against Map, which suggests derivatives with better pharmacodynamics may be of interest for evaluation in animal models

Bayesian analyses of multiple epistatic QTL models for body weight and body composition in mice

To comprehensively investigate the genetic architecture of growth and obesity, we performed Bayesian analyses of multiple epistatic quantitative trait locus (QTL) models for body weights at five ages (12 days, 3, 6, 9 and 12 weeks) and body composition traits (weights of two fat pads and five organs) in mice produced from a cross of the F1 between M16i (selected for rapid growth rate) and CAST/Ei (wild-derived strain of small and lean mice) back to M16i. Bayesian model selection revealed a temporally regulated network of multiple QTL for body weight, involving both strong main effects and epistatic effects. No QTL had strong support for both early and late growth, although overlapping combinations of main and epistatic effects were observed at adjacent ages. Most main effects and epistatic interactions had an opposite effect on early and late growth. The contribution of epistasis was more pronounced for body weights at older ages. Body composition traits were also influenced by an interacting network of multiple QTL. Several main and epistatic effects were shared by the body composition and body weight traits, suggesting that pleiotropy plays an important role in growth and obesity

Generation and screening of a comprehensive \u3ci\u3eMycobacterium avium\u3c/i\u3e subsp. \u3ci\u3eparatuberculosis\u3c/i\u3e transposon mutant bank

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of Johne’s Disease in ruminants. This enteritis has significant economic impact and world wide 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 developent. In this study, a comprehensive mutant bank of 13,536 MAP K-10 Tn5367 mutants (P\u3e95% )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 up stream 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

Development of Luminescent \u3ci\u3eMycobacterium avium\u3c/i\u3e subsp. \u3ci\u3eparatuberculosis\u3c/i\u3e for Rapid Screening of Vaccine Candidates in Mice

Mycobacterium avium subsp. paratuberculosis is a slowly growing mycobacterial species, requiring 6 to 8 weeks of culture before colonies can be counted visually. Here, we describe the development of luminescent M. avium subsp. paratuberculosis expressing luxAB genes of Vibrio harveyi and its use for vaccine testing in an experimental mouse model, replacing fastidious CFU counting by rapid luminometry

Identification of D-alanine Transaminase in Mycobacterium smegmatis and Effects of Mutations in Peptidoglycan Enzymes on Canonical and Non-Canonical Tuberculosis Drugs

Tuberculosis is a disease that is one of the leading ten causes of death worldwide and is believed to latently infect one-third of the human population. Tuberculosis is caused by Mycobacterium tuberculosis, which has become increasingly drug resistant. The model organism of M. tuberculosis is Mycobacterium smegmatis. Production of the mycobacterial cell wall is dependent on production of a molecule called peptidoglycan which requires different enantiomers of amino acids. In humans, D-amino acids are practically unavailable. In an effort to more effectively treat drug resistant tuberculosis, we investigated the complementary ability of a proposed mycobacterial enzyme (D-alanine transaminase) coded by Msmeg_5795 on deletion or insertion alr/murI or double mutant strains. We also investigated resistance or susceptibility of strains overexpressing or having null mutations in alr/murI to certain canonical and non-canonical inhibitory agents. We performed growth studies by creating different agar media plates supplemented with different combinations of amino acids or with different inhibitory agents. We then streaked or spotted live cells on these plates and imaged growth over a one or two week period. Growth was subjectively graded. We found evidence that supports the hypothesis that Msmeg_5795 does indeed code for a D-alanine transaminase-like enzyme and that this enzyme is likely multi-functional. Our results also suggest the existence of another D-alanine transaminase as well. We confirmed observations previously made regarding inhibitory agent resistance but found two non-canonical results: First, glutamate racemase (MurI) appears to be inhibited not only by Beta-chloro-D-alanine (BCDA) but also by D-cycloserine (DCS), and L-cycloserine (LCS). Previous research identified MurI as only being inhibited by BCDA. Second, alanine dehydrogenase (Ald) overproduction appears to impart susceptibility to LCS and have no effect on DCS. We confirmed previous research that identified the deletion of Ald as likely having resistance-imparting characteristics. Our non-canonical results indicate the need for further research

Identification of D-alanine Transaminase in Mycobacterium smegmatis and Effects of Mutations in Peptidoglycan Enzymes on Canonical and Non-Canonical Tuberculosis Drugs

Tuberculosis is a disease that is one of the leading ten causes of death worldwide and is believed to latently infect one-third of the human population. Tuberculosis is caused by Mycobacterium tuberculosis, which has become increasingly drug resistant. The model organism of M. tuberculosis is Mycobacterium smegmatis. Production of the mycobacterial cell wall is dependent on production of a molecule called peptidoglycan which requires different enantiomers of amino acids. In humans, D-amino acids are practically unavailable. In an effort to more effectively treat drug resistant tuberculosis, we investigated the complementary ability of a proposed mycobacterial enzyme (D-alanine transaminase) coded by Msmeg_5795 on deletion or insertion alr/murI or double mutant strains. We also investigated resistance or susceptibility of strains overexpressing or having null mutations in alr/murI to certain canonical and non-canonical inhibitory agents. We performed growth studies by creating different agar media plates supplemented with different combinations of amino acids or with different inhibitory agents. We then streaked or spotted live cells on these plates and imaged growth over a one or two week period. Growth was subjectively graded. We found evidence that supports the hypothesis that Msmeg_5795 does indeed code for a D-alanine transaminase-like enzyme and that this enzyme is likely multi-functional. Our results also suggest the existence of another D-alanine transaminase as well. We confirmed observations previously made regarding inhibitory agent resistance but found two non-canonical results: First, glutamate racemase (MurI) appears to be inhibited not only by Beta-chloro-D-alanine (BCDA) but also by D-cycloserine (DCS), and L-cycloserine (LCS). Previous research identified MurI as only being inhibited by BCDA. Second, alanine dehydrogenase (Ald) overproduction appears to impart susceptibility to LCS and have no effect on DCS. We confirmed previous research that identified the deletion of Ald as likely having resistance-imparting characteristics. Our non-canonical results indicate the need for further research