Progress 10/01/11 to 09/30/16
Outputs
Target Audience:To make information available to the research community and public, research results relating to microbiology and microbial genetics were presented at a scientific meeting (ASM Microbe 2016). Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Over the grant period, the PI participated in several face-to-face and teleconference meetings regarding the MDA-CAP. Dr. Barletta and his Ph.D. graduate student, Govardhan Rathnaiah, also participated in one regional and three local microbiology, redox biology and NMR meetings. Denise K. Zinniel (Laboratory Manager) and I trained Govardhan on the molecular genetics of mycobacteria and the generation of Mycobacterium smegmatis cell free extracts. He trained and was approved for work with Mycobacterium tuberculosis in the BSL3 laboratory. Govardhan also trained in Illumina Sequencing technology that required him to travel to the University of Nebraska Medical Center for the use of specialized equipment. We also trained in our laboratory 2 more Ph.D. graduate students from the Department of Chemistry (Darrell Marshall in Dr. Powers Laboratory and Wantanee Sittiwong in Dr. Dussault's Laboratory. Both Darrell and Wantanee trained on the correct procedures to maniuplate and grow bacterial cultures, the determination of minimal inhibitory concentrations (MICs) and the generation of Mycobacterium smegmatis cell free extracts. Darrell also conducted NMR metabolomic lysate collection. In addition, we trained six undergraduate students (Fatimah Barnawi, Daniel Baer, Rachael Higgins, Philion Hoff, Sri Narayanan and Danny Dooling) and 1 research technician (Celso Perez-Bolaños). This involved proper microbiological sterile procedures, media/reagent preparation, how to grow mycobacteria, osmometer instrument utilization, enzyme assay protocols and how to determine MICs. How have the results been disseminated to communities of interest?The primary forms of result dissemination were by posters and presentations at local and regional scientific meetings, as well as by publications in peer-reviewed journals. In addition, we discussed potential patent applications with NU Ventures and contacted various animal health companies to establish collaborations and to explore commercialization of products and materials developed in this project. Specifically, Dr. Barletta gave two presentations to Merck Animal Health regarding the potential collaboration of MAP mutants. However, funding could not be established at this time. Publications and presentations during this entire grant period were the following: Galbadage, T., D. Patel, S. Subbian, D.K. Zinniel, S.L.G. Cirillo, R.G. Barletta and J.D. Cirillo. Virulence Gene Regulator luxR1 (MMAR_1239) in Mycobacterium marinum. Abstract and presentation at the Molecular Basis of Infectious Diseases Retreat. Houston, Texas. March 23, 2012. Halouska, S., R.J. Fenton, R.G. Barletta and R. Powers. 2012. Predicting the in vivo mechanism of action for drug leads using NMR metabolomics. ACS Chem. Biol. 7:166-171. Thacker, N.C., J. Molnár-Tóth, J.L. Miska, R. Barletta and J. Takacs. 2012. Preparation of D-Cycloserine and 13C-Labeled D-Cycloserine. The Japan Institute of Heterocyclic Chemistry Heterocycles 86(2):1575-1582. Zinniel, D.K., R.J. Fenton, S. Halouska, R. Powers and R.G. Barletta. 2012. Sample preparation of Mycobacterium tuberculosis extracts for Nuclear Magnetic Resonance (NMR) metabolomics studies. J Vis Exp. 67:e3673. Sittiwong, W., P.H. Dussault, R. Barletta, D. Zinniel, R. Fenton and R. Powers. Synthesis and antimycobacterial activity of novel fatty acid analogs. Abstract submitted to the 245th American Chemical Society National Meeting. New Orleans, Louisiana. April 7-11, 2013. Sittiwong, W., Zinniel, D. K., Fenton, R. J., Marshall, D. D., Story, C. B., Kim, B., Lee, J.-Y., Powers, R., Barletta, R., Dussault, P. (2014). Development of Cyclobutene- and Cyclobutane- Functionalized Fatty Acids with Inhibitory Activity Against Mycobacterium tuberculosis. ChemMedChem, 9, 1838-1849. Janagamaa, H.K., S. Tounkanga, S.L.G. Cirillo, D.K. Zinniel, R.G. Barletta and J.D. Cirillo*. 2013. Molecular analysis of the Mycobacterium tuberculosis lux-like mel2 operon. Tuberculosis. 93:S83-S87. Halouska, S., R.J. Fenton, D.K. Zinniel, D.D. Marshall, R.G. Barletta* and R. Powers*. 2014. Metabolomics analysis identifies D-Alanine-D-alanine ligase as the primary lethal target of D-cycloserine in mycobacteria. Journal of Proteome Research. 13:1065-1076. Hines II*, M.E., S.E. Turnquist, M.R.S. Ilha, S. Rajeev, A.L. Jones, L. Whittington, J.P. Bannantine, R.G. Barletta, Y.T. Gröhn, R. Katani, A.M. Talaat, L. Li and V. Kapur. 2014. Evaluation of novel oral vaccine candidates and validation of a caprine model of Johne's Disease. Frontiers in Microbiology. 4(Article 26):1-14. Bannantine, J.P., Everman, J., Rose, S.J., Babrak, L., Katani, R., Barletta, R.G., Talaat, A.M., Gröhn, Y.T., Chang, Y.-F., Kapur, V., and Bermudez, L.E. (2014). Evaluation of eight live attenuated Mycobacterium avium subspecies paratuberculosis mutant strains for protection from colonization of tissues in mice. Front Cell Infect Microbiol. 4(Article 88):1-8. Govardhan Rathnaiah, Elise A. Lamont, N. Beth Harris, Robert J. Fenton, Denise K. Zinniel, Xiaofei Liu, Josh Sotos, Zhengyu Feng, Ayala Livneh-Kol, Nahum Y. Shpigel, Charles J. Czuprynski, Srinand Sreevatsan, Raúl G. Barletta. (2014). Generation and screening of a comprehensive Mycobacterium avium subsp. paratuberculosis transposon mutant bank. Front Cell Infect Microbiol. 4(Article 144):1-17. Rathnaiah, G., and R.G. Barletta. Isolation and characterization of Mycobacterium smegmatis murI mutants suggest the existence of a new pathway of D-glutamate biosynthesis. University of Nebraska Redox Biology Center 2015 Retreat Oral Presentation. Ahsland, NE. 3/21/15. Rathnaiah, G., and R.G. Barletta. Isolation and characterization of Mycobacterium smegmatis murI mutants suggest the existence of a new pathway of D-glutamate biosynthesis. Joint Meeting Missouri Valley Branch and Missouri Branch of the American Society for Microbiology Oral Presentation. Lincoln, NE. 3/27/2015. Rathnaiah, G., F. Barnawi, D.K. Zinniel and R.G. Barletta. Isolation and characterization of Mycobacterium smegmatis murI mutants suggest the existence of a new pathway of D-glutamate biosynthesis. University of Nebraska NMR Symposium Poster Presentation. Lincoln, NE. 3/31/2015. Barnawi, F., G. Rathnaiah, D.K. Zinniel and R.G. Barletta. Isolation and characterization of Mycobacterium smegmatis murI mutants suggest the existence of a new pathway of D-glutamate biosynthesis. University of Nebraska Research Fair Undergraduate Student Poster Session. Lincoln, NE. 4/15/2015. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Mycobacterium avium subsp. paratuberculosis (MAP), one of the slowest growing mycobacteria, is the etiologic agent of paratuberculosis (Johne's disease, JD), in ruminants. JD is a chronic enteritis with significant economic impact and worldwide distribution. A JD regression model estimates that losses to the dairy industry range from $40 to $227 per inventoried cow per year. JD has lower prevalence in beef herds but still has a negative impact on trade. MAP has the potential to be a zoonotic and/or food-borne pathogen, as evidenced by its possible linkage to Crohn's disease. Other pathogenic mycobacteria include Mycobacterium tuberculosis (MTB), the agent of human tuberculosis, and Mycobacterium bovis that causes bovine tuberculosis, a zoonotic disease transmissible from and to ruminants/wildlife to and from human hosts. Obective 1: To develop a transposon mutagenesis system, we prepared a high titer phage vector lysate for the himar-1 transposon (MycoMarT7). This phage was propagated on Mycobacterium smegmatis (Msmeg) to a titer of 1.4 x 1011 PFU/ml. We infected Mycobacterium avium subsp. paratuberculosis (MAP) and demonstrated that the phage was able to infect yielding insertions into chromosomal genes. Based on these results, we submitted a grant application to the NIFA foundational program expanding on the objectives proposed in this Animal Health project. The application (Proposal No. 2013-01040) was funded. Thus, this goal was considered completed in 2013. Objective 2: We completed the analysis of the comprehensive mutant bank of 13,536 MAP transposon K-10 Tn5367 mutants (P > 95%). Additional mutants were generated with transposon Tn5370. 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. MAP1152 encodes for a PPE protein. Growth curves in broth cultures, invasion assays and kinetics of survival and replication in primary bovine macrophages were also determined. These mutants were evaluated using a protection challenge model in C57BL/6 mice, demonstrating different levels of protection based on colonization in the liver and spleen. Eighty goat kids were vaccinated orally twice with an experimental vaccine or once subcutaneously with Silirum® (Zoetis), or a sham control oral vaccine. All kids were necropsied at 13 months post-challenge. Results indicated that the goat challenge is a highly efficient and valid model for JD challenge studies. None of the experimental or control vaccines evaluated prevented MAP infection or eliminated fecal shedding. Strain 318 vaccine reduced the presence of JD gross and microscopic lesions. In addition, we performed a bioinformatic analysis of the main PPE targets in Mycobacterium avium, MAP and M. bovis. The key points are: (1) PE and PPE proteins are highly expanded in the microorganisms of the Mycobacterium tuberculosis (MTB) complex, as compared to those of the M. avium complex (only about 1% of the genome coding sequences) and (2) in M. avium and MAP, there are no PE-PGRS (PE proteins with multiple tandem repetitive sequences that are exceptionally rich in glycine and alanine)/PPE-MPTR (PPE proteins with a major polymorphic tandem repeat region). Most PE and PPE gene sequences are linked to the five ESAT-6 (early secretory antigenic target) gene clusters identified in slow-growing mycobacteria. The ESAT-6 gene clusters encode highly immunogenic low molecular weight proteins and the components of their secretion apparatus. In summary, PE/PPE proteins may play a key role in immunopathogenesis and intracellular survival within phagocytic cells. This analysis has furthered our knowledge on targets for vaccine development. Thus Objective 2 was completed in 2015. Objective 3: In collaboration with the UNL Department of Chemistry, we performed NMR metabolomic analysis in Msmeg, MTB and M. bovis wild type and mutant strains that occur in response to antibiotic treatments. We identified the overall physiological process inhibited by new compounds and confirm the mechanism of action of known control antimycobacterial agents. NMR analysis of cell free extracts was performed by capturing spectroscopic data followed by the determination of chemical shifts and statistical analysis. Assignments of NMR spectra to specific metabolites utilized the Madison Metabolomics Consortium Database, the BioMagResBank, and the Human Metabolome Database. It was possible to identify and quantify 40-50 metabolites, including amino acids, nucleotide precursors, and glycolytic and citric acid intermediates. These metabolomic changes are directly correlated with broad mechanisms of action that are associated with each tuberculosis class of antibiotics, disruption of cell walls or membranes, inhibition of transcription, translation, or DNA supercoiling, or the inhibition of mycolic acid biosynthesis. Thus, the impact of these studies is that we have proof of concept for an NMR metabolomics method to elucidate the mechanisms of action of promising antimicrobial agents against mycobacteria. We applied this technology to find the therapeutic targets that inhibit the D-alanine pathway of peptidoglycan biosynthesis. NMR metabolomics revealed that D-alanine-D-alanine ligase (Ddl) is the primary target of D-Cycloserine (DCS), as cell growth is inhibited when the production of D-alanyl-D-alanine is halted. It is shown that inhibition of alanine racemase (Alr) may contribute indirectly by lowering the levels of D-alanine, thus allowing DCS to outcompete D-alanine for Ddl binding. 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. Thus discovery was highlighted in the manuscript that resolved a 50 year old question regarding the in vivo lethal target of DCS and will lay the foundations for developing new tuberculosis therapies. Glutamate racemase (MurI) has been implicated in both the production and maintenance of the D-glutamate pool required for peptidoglycan biosynthesis. To further investigate the role of MurI in mycobacterial physiology, we generated Msmeg murI deletion mutants by allelic exchange. These mutants were able to grow on the standard low osmolality Middlebrook 7H9 media without D-glutamate, though with a significant growth arrest. Ultrastructural analysis by negative staining transmission electron microscopy did not reveal any differences in size or morphology. The viability of our new mutants and independence of D-glutamate for growth indicate that inactivation of murI does not impose an auxotrophic requirement for D-glutamate. These results have a major impact in our knowledge of mycobacterial physiology which will assist in the development of novel drugs and vaccines. In addition, with Dr. J. Takacs, we synthesized 13C DCS in small scale from DL-serine methyl ester. With Dr. Pat Dussault, cyclobutene derivatives were synthesized that showed inhibitory growth activity against Msmeg and MTB inhibitors. These studies developed new compounds to treat tuberculosis and related diseases. With Dr. J. Cirillo, we characterized the MTB mel2 locus. Three genes had similarities to bioluminescence genes that facilitate detoxification of reactive oxygen species. We demonstrated enhanced production of the cell wall virulence lipid, pthiocerol dimycoserosate. These observations provide the first evidence that mel2 plays a critical role in MTB lipid biosynthesis and protection against oxidative stress. In summary, the major goals of this project have been completed in 2016. Avenues opened for further research will be pursued in future internal and/or external projects.
Publications
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2016
Citation:
Rathnaiah, G., F. Barnawi, D.K. Zinniel and R.G. Barletta. Analysis of Mycobacterium smegmatis glutamate racemase mutants suggests a new pathway of D-glutamate biosynthesis. ASM Microbe Annual Meeting. June 2016.
|
Progress 10/01/14 to 09/30/15
Outputs
Target Audience:We targeted the local and regional audiences of microbiologists and redox biologists at various meetings (see Professional Development section). Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The PI participated in various regional and local meetings: University of Nebraska Redox Biology Center 2015 Retreat. Ahsland, NE. 3/21/15. Joint Meeting Missouri Valley Branch and Missouri Branch of the American Society for Microbiology. Lincoln, NE. 3/27/2015. University of Nebraska NMR Symposium. Lincoln, NE. 3/31/2015. University of Nebraska Research Fair. Lincoln, NE. 4/15/2015. Denise K. Zinniel (Laboratory Manager) and I continued training 6 undergraduate students (Fatimah Barnawi, Daniel Baer, Rachael Higgins, Philion Hoff, Sri Narayanan and Danny Dooling) and 1 research technician (Celso Perez-Bolaños). This involved proper microbiological sterile procedures, media/reagent preparation, osmometer instrument utilization and enzyme assay protocols. My Ph.D. graduate student, Govardhan Rathnaiah, continued training on the molecular genetics of mycobacteria and the generation of Mycobacterium smegmatis cell free extracts. How have the results been disseminated to communities of interest?The basic dissemination of the results was by posters and presentations at local and regional scientific meetings (see Products Section). What do you plan to do during the next reporting period to accomplish the goals?As previously stated, Goal 1 is completed and there is no further work planned. The expanded project is currently funded by AFRI-NIFA competitive funding (reported under NEB39-169). For Goal 2, there is no further work planned unless external funding becomes available to support the project. For Goal 3, we plan to continue a major effort on the characterization of Mycobacterium smegmatis mutants related to glutamate racemase and complete the experiments to submit a major manuscript.
Impacts
What was accomplished under these goals?
Mycobacterium avium subsp. paratuberculosis (MAP), one of the slowest growing mycobacteria, is the etiologic agent of paratuberculosis (Johne's disease, JD), in ruminants. JD is a chronic enteritis with significant economic impact and worldwide distribution. A JD regression model estimates that losses to the dairy industry range from $40 to $227 per inventoried cow per year. JD has lower prevalence in beef herds but still has a negative impact on trade. MAP has the potential to be a zoonotic and/or food-borne pathogen, as evidenced by its possible linkage to Crohn's disease. Other pathogenic mycobacteria include Mycobacterium tuberculosis (MTB), the agent of human tuberculosis, and Mycobacterium bovis that causes bovine tuberculosis, a zoonotic disease transmissible from and to ruminants/wildlife to and from human hosts. For Objective 1, there is nothing to report (main tasks are described under NEB39-169 in the context of competitive NIFA funding). For Objective 2, as we were not able to secure external funding to conduct major experiments, we performed a bioinformatic analysis of the main PPE targets in Mycobacterium avium, MAP and M. bovis to plan for future research. Mycobacteria are characterized by the presence of PE (proline-glutamic acid) and PPE (proline-proline-glutamic acid) proteins for their characteristic amino acid residues in defined positions of their conserved N-terminal domains. Approximately 10% of the MTB genome coding capacity is dedicated to encode these protein families. The results from our bioinformatic analysis are described in Table 1. The key points are: (1) PE and PPE proteins are highly expanded in the microorganisms of the MTB complex, as compared to those of the M. avium complex (only about 1% of the genome coding sequences) and (2) in M. avium and MAP, there are no PE-PGRS (PE proteins with multiple tandem repetitive sequences that are exceptionally rich in glycine and alanine)/PPE-MPTR (PPE proteins with a major polymorphic tandem repeat region). Most PE and PPE gene sequences are linked to the five ESAT-6 (early secretory antigenic target) gene clusters identified in slow-growing mycobacteria. The ESAT-6 gene clusters encode highly immunogenic low molecular weight proteins and the components of their secretion apparatus. This finding led to the hypothesis that these PE/PPE/ESAT-6 regions constitute a genomic "immunogenicity island". In summary, PE/PPE proteins may play a key role in immunopathogenesis and intracellular survival within phagocytic cells. However, specific functions of particular members remain mostly unknown. Nonetheless, their potential major role in pathogenesis makes a compelling case for their systematic study in the context of practical applications to disease control. This analysis has furthered our knowledge on targets for vaccine development. Table 1: PE/PPE Proteins in Mycobacteria1 Species/strain #PEs #PPEs Comments M. bovis 91 63 Multiple PE-PGRS/PPE-MPTR (sublineage V) M. bovis BCG strain Pasteur 87 59 Multiple PE-PGRS/ PPE-MPTR (sublineage V) M. bovis BCG strain Tokyo 89 59 Multiple PE-PGRS/ PPE-MPTR (sublineage V) M. tuberculosis 143 84 Multiple PE-PGRS/ PPE-MPTR (sublineage V) M. avium strain 104 8 35 No PE-PGRS/PPE-MPTR; ancestral ESAT-6 Region V M. paratuberculosis 10 39 No PE-PGRS/PPE-MPTR; ancestral ESAT-6 Region V M. smegmatis 2 2 No PE-PGRS/PPE-MPTR; No ESTAT-6 Region V 1Data collected from (http://pfam.sanger.ac.uk/) For Objective 3, we focused on the construction and characterization of glutamate racemase mutants of Mycobacterium smegmatis (Msmeg). Msmeg is a fast-growing nonpathogenic species particularly useful in studying basic cellular processes of relevance to pathogenic mycobacteria, including MTB and MAP. Glutamate racemase (MurI) has been implicated in both the production and maintenance of the D-glutamate pool required for peptidoglycan biosynthesis. MurI has been reported to be essential in both Msmeg mc2155 and MTB H37Rv, but non-essential in MTB Erdman. To further investigate the role of MurI in mycobacterial physiology, we generated Msmeg murI deletion mutants by allelic exchange using a suicide plasmid carrying a sacB gene and a kanamycin-resistant determinant. We selected single crossover recombinants and then counter selected on 2% sucrose media to obtain double crossover recombinants. The deletion of murI was confirmed by PCR and Southern blotting analyses. These mutants were able to grow on the standard low osmolality Middlebrook 7H9 media without D-glutamate, though with a significant lag time. In contrast, previous reports found that Msmeg murI mutants were only able to grow on high osmolality media supporting the growth of cell wall deficient forms. The viability of our new mutants and independence of D-glutamate for growth indicate that inactivation of murI does not impose an auxotrophic requirement for D-glutamate. We hypothesize the existence of another pathway of D-glutamate biosynthesis in Msmeg. These results have a major impact in our knowledge of mycobacterial physiology which will assist in the development of novel drugs and vaccines. In addition for this objective, we also continued the analysis of D-alanine ligase inhibitors. This enzyme is involved in the biosynthesis of the critical dipeptide that is utilized in peptidoglycan crosslinking. We received from collaborators several enzyme batches that unfortunately were inactive due to shipping delays from Australia. We expect to be able to resolve this shipping problem in the future. Inhibitors of this enzyme could be very effect antimycobacterial drugs.
Publications
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2015
Citation:
Rathnaiah, G., and R.G. Barletta. Isolation and characterization of Mycobacterium smegmatis murI mutants suggest the existence of a new pathway of D-glutamate biosynthesis. University of Nebraska Redox Biology Center 2015 Retreat Oral Presentation. Ahsland, NE. 3/21/15.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2015
Citation:
Rathnaiah, G., and R.G. Barletta. Isolation and characterization of Mycobacterium smegmatis murI mutants suggest the existence of a new pathway of D-glutamate biosynthesis. Joint Meeting Missouri Valley Branch and Missouri Branch of the American Society for Microbiology Oral Presentation. Lincoln, NE. 3/27/2015.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2015
Citation:
Rathnaiah, G., F. Barnawi, D.K. Zinniel and R.G. Barletta. Isolation and characterization of Mycobacterium smegmatis murI mutants suggest the existence of a new pathway of D-glutamate biosynthesis. University of Nebraska NMR Symposium Poster Presentation. Lincoln, NE. 3/31/2015.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2015
Citation:
Barnawi, F., G. Rathnaiah, D.K. Zinniel and R.G. Barletta. Isolation and characterization of Mycobacterium smegmatis murI mutants suggest the existence of a new pathway of D-glutamate biosynthesis. University of Nebraska Research Fair Undergraduate Student Poster Session. Lincoln, NE. 4/15/2015.
|
Progress 10/01/13 to 09/30/14
Outputs
Target Audience: During this period for Goal 2, we specifically targeted the Johne’s Disease community of scientists worldwide by submitting three manuscripts to the special peer-reviewed issue on paratuberculosis to the journal Frontiers in Cellular and Infection Microbiology. Two of these manuscripts are published and the other was accepted for publication (in press). For Goal 3, we submitted a major study, now published, on the role of alanine racemase and alanine ligase in the mechanism of resistance to D-cycloserine in Mycobacterium tuberculosis, targeting the audience related to drug development in both academia and industry. Changes/Problems: As indicated previously, we are not further pursuing studies on PPE proteins of M. avium and M. bovis, as we consider this expansion distractive to current efforts. What opportunities for training and professional development has the project provided? The PI participated in several face-to-face and teleconference meetings regarding the MDA-CAP. We continued training in our laboratory of 2 Ph.D. graduate students from our Department (Govardhan Rathnaiah) School of Veterinary Medicine and Biomedical Sciences) and the Department of Chemistry (Darrell Marshall in Dr. Powers Laboratory). Govardhan trained on the molecular genetics of mycobacteria and Darrell on the determination of minimal inhibitory concentrations (MICs) and the generation of Mycobacterium smegmatis cell free extracts. Govardhan also trained and was approved for work with Mycobacterium tuberculosis in the BSL3 laboratory. This training also required Govardhan to travel to the University of Nebraska Medical Center for the use of specialized equipment. In addition, we trained two undergraduates on how to grow mycobacteria and determine MICs. How have the results been disseminated to communities of interest? The basic dissemination of the results were by publications in peer-reviewed journals (see below). What do you plan to do during the next reporting period to accomplish the goals? As previously stated, Goal 1 is completed and there is no further work planned. However, this goal has been expanded into a major grant currently funded by AFRI-NIFA. These studies are curretly reported under NEB39-169. For Goal 2, we plan to continue focusing on new mutants of paratuberculosis (DMAP52 and DMAP56). We will not further pursue the study of PPE proteins in M. avium and M. bovis unless external funding can be obtained. For Goal 3, we plan to continue a major effort on the characterization of M. smegmatis and M. tuberculosis mutants related to alanine racemase, glutmate racemase and alanine dehydrogenase.
Impacts
What was accomplished under these goals?
The main objectives of Goal 1 have been completed and the expansion of this project is reported under NEB39-169. For Goal 2, we focused on the following: (1) A comprehensive mutant bank of 13,536 MAP transposon K-10 Tn5367 mutants (P > 95%) was constructed and screened in vitro for phenotypes related to virulence. Additional mutants were generated with transposon Tn5370. 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. These mutants were evaluated using a C57BL/6 mouse model. The persistence of the vaccine candidates was measured at 6, 12, and 18 weeks post vaccination. These strains demonstrated different levels of protection based on colonization of the challenge strain in liver and spleen tissues at 12 and 18 weeks post vaccination. Based on total MAP burden in both tissues at both time points, strain 315 (MAP1566::Tn5370) was the most protective whereas strain 318 (intergenic Tn5367 insertion between MAP0282c and MAP0283c) had the most colonization. Mice vaccinated with an undiluted commercial vaccine preparation displayed the highest bacterial burden as well as enlarged spleens indicative of a strong infection. Eighty goat kids were vaccinated orally twice at 8 and 10 weeks of age with an experimental vaccine or once subcutaneously at 8 weeks with Silirum® (Zoetis), or a sham control oral vaccine at 8 and 10 weeks. Kids were challenged orally with a total of approximately 1.44 × 109 CFU divided in two consecutive daily doses using MAP ATCC-700535 (K10-like bovine isolate). All kids were necropsied at 13 months post-challenge. Results indicated that the goat challenge model is a highly efficient and valid model for JD challenge studies. None of the experimental or control vaccines evaluated prevented MAP infection or eliminated fecal shedding. The relative performance ranking of vaccine strains from our laboratory was 318, 316 (intergenic Tn5367 insertion between MAP3695 and FadE5), 315 and finally 319 (MAP1566::Tn5367). The subcutaneously injected control vaccine outperformed the orally-delivered mutant vaccine candidates. Strain 318 vaccine reduced the presence of JD gross and microscopic lesions. In summary, we completed the final characterization of MAP transposon mutants in broth cultures, macrophages, mice and goats in collaboration with the members of the Johne's Disease Integrated Program (JDIP)/APHIS testing program. During this period, we determined the growth curves in broth cultures, invasion assays and kinetics of survival and replication in primary bovine macrophages. These mutants were further evaluated for vaccine potential in mice and goats. (2) A proposal on the PPE proteins of M. bovis was sent to the USDA-NIFA Mycobacterial Diseases of Animals-Coordinated Agricultural Project (MDA-CAP), but this project was declined by the MDA-CAP and therefore was not incorporated in the current MDA-CAP submission which is still pending. (3) A proposal dealing with DMAP52 and DMAP56 Mycobacterium paratuberculosis mutants was sent to the USDA-NIFA in collaboration with UK partners, but the proposal was declined. For Goal 3, we focused on the following: (1) Using a high throughput genetic strategy, designated Random Inducible Controlled Expression, we identified the six gene mel2 locus in Mtb and M. marinum. Interestingly, three of the genes present in mel2 have similarities to bioluminescence genes. Similar to other bacterial bioluminescence systems, mel2 facilitates detoxification of reactive oxygen species. Through the use of thin layer chromatography we demonstrate enhanced production of the cell wall virulence lipid, pthiocerol dimycoserosate, in a Mtb mel2 mutant relative to the wild type strain in the presence of both H2O2 and diamide oxidative stresses. Furthermore, propionate toxicity assays revealed increased accumulation of triacylglycerol in the mel2 mutant relative to wild type. These observations provide the first evidence that mel2 plays a critical role in Mtb lipid biosynthesis. (2) NMR metabolomics revealed that D-alanine-D-alanine ligase (Ddl) is the primary target of D-Cycloserine (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 alanine racemase (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. In summary, the studies of M. tuberculosis mel mutants related to oxidative stress and latency were completed and the corresponding manuscript was submitted and accepted for publication. The biochemical studies with Mycobacterium smegmatis and M. tuberculosis wild type strains demonstrated that D-alanine ligase is the most important target of D-cycloserine mechanism of action, a discovery that was highlighted in the manuscript that resolved a 50 year old question regarding the in vivo lethal target of D-cycloserine and will lay the foundations for developing new tuberculosis therapies.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Janagamaa, H.K., S. Tounkanga, S.L.G. Cirillo, D.K. Zinniel, R.G. Barletta and J.D. Cirillo*. 2013. Molecular analysis of the Mycobacterium tuberculosis lux-like mel2 operon. Tuberculosis. 93:S83-S87.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Halouska, S., R.J. Fenton, D.K. Zinniel, D.D. Marshall, R.G. Barletta* and R. Powers*. 2014. Metabolomics analysis identifies D-Alanine-D-alanine ligase as the primary lethal target of D-cycloserine in mycobacteria. Journal of Proteome Research. 13:1065-1076.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Hines II*, M.E., S.E. Turnquist, M.R.S. Ilha, S. Rajeev, A.L. Jones, L. Whittington, J.P. Bannantine, R.G. Barletta, Y.T. Gr�hn, R. Katani, A.M. Talaat, L. Li and V. Kapur. 2014. Evaluation of novel oral vaccine candidates and validation of a caprine model of Johnes Disease. Frontiers in Microbiology. 4(Article 26):1-14.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Bannantine, J.P., Everman, J., Rose, S.J., Babrak, L., Katani, R., Barletta, R.G., Talaat, A.M., Gr�hn, Y.T., Chang, Y.-F., Kapur, V., and Bermudez, L.E. (2014). Evaluation of eight live attenuated Mycobacterium avium subspecies paratuberculosis mutant strains for protection from colonization of tissues in mice. Front Cell Infect Microbiol. 4(Article 88):1-8.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2014
Citation:
Govardhan Rathnaiah, Elise A. Lamont, N. Beth Harris, Robert J. Fenton, Denise K. Zinniel, Xiaofei Liu, Josh Sotos, Zhengyu Feng, Ayala Livneh-Kol, Nahum Y. Shpigel, Charles J. Czuprynski, Srinand Sreevatsan, Ra�l G. Barletta. (2014). Generation and screening of a comprehensive Mycobacterium avium subsp. paratuberculosis transposon mutant bank. Front Cell Infect Microbiol. 4(Article 144):1-17.
|
Progress 10/01/12 to 09/30/13
Outputs
Target Audience: During this period we targeted the greater audience of scientists worldwide by submitting three publications to peer-reviewed journals. As of September 2013, these manuscripts are under review. Changes/Problems: We established a new protocol to determine minimal inhibitory concentrations maintaining the dimethylsulfide concentration constant throughout the experiment. This resulted in a more reliable and consistent minimal inhibitory concentrations while allowing better solubization of the corresponding inhibitors. What opportunities for training and professional development has the project provided? We trained in our laboratory from the University of Nebraska Department of Chemistry (Dr. Dussault and Dr. Powers Laboratory, respectively) Wantanee Sittiwong (Msmeg and MAP minimal inhibitory concentrations) and Darrell Marshall (NMR metabolomic lysate collection) during this reporting period. These graduate students were also trained in the correct procedures to maniuplate and grow Msmeg and MAP cultures. How have the results been disseminated to communities of interest? We discussed potential patent applications with NU Ventures and contacted various animal health companies to establish collaborations and to explore commercializtion of products and materials developed in this project. Specifically, Dr. Barletta gave two presentations to Merck Animal Health regarding the potential collaboration of MAP mutants DMAP52 and DMAP56. However, funding could not be established at this time. As listed under the products heading, we have submitted three manuscripts to peer reviewed journals. What do you plan to do during the next reporting period to accomplish the goals? As the main objectives of Goal 1 have been completed, we will focus on Goals 2 and 3. For Goal 2, we plan to: 1) complete charactization of available MAP transposon mutants in macrophages, mice and goats in collaboration with the members of the JDIP/APHIS testing program 2) perform bioinformatic analysis of PPE proteins in MAP, M. bovis and Mtb 3) continue the charaterization of DMAP52 and DMAP56 (generation of growth curves, kinetics of intracellular macrophages) 4) generate unmarked delection mutants for the genes inactived in DMAP52 and DMAP56 5) generate additional mutants as needed For Goal 3, we plan to: 1) complete biochemical charactization of Msmeg and Mtb mutants impaired in alanine metabolism, including mutants in alanine racemase and alanine dehydrogenase 2) complete analysis of Mtb D-alanine ligase inhibitors 3) determine the in vitro susceptibility of MAP to fatty acid analogs in broth cultures
Impacts
What was accomplished under these goals?
Goal 1: To develop the TraSH mutagenesis system, we prepared a high titer phage vector lysate for the himar-1 transposon (MycoMarT7). This phage was propagated on Mycobacterium smegmatis (Msmeg) to a titer of 1.4 x 1011 PFU/ml. With these materials and the work carried out in the previous funding period, we submitted a grant application to the NIFA foundational program expanding on the objectives proposed in this Animal Health project. The application (Proposal No. 2013-01040) was funded. Thus, this goal is considered completed and any further progress will be reported under Project No. NEB 39-169. Goal 2: Nothing to report. Goal 3: In this goal we focus on the Mycobacterium tuberculosis (Mtb) mel mutants. In collaboration with Texas A&M Health Science Center (Dr. Jeffrey Cirillo), we used a high throughput genetic strategy, designated Random Inducible Controlled Expression (RICE) to identify the six gene mel2 locus in Mtb. Interestingly, three of the genes present in mel2 have similarities to bioluminescence genes. Similar to other bacterial bioluminescence systems, we observed that mel2 facilitates detoxification of reactive oxygen species (ROS). Through the use of thin layer chromatography (TLC) we demonstrate enhanced production of the cell wall virulence lipid, pthiocerol dimycoserosate (PDIM), in a Mtb mel2 mutant relative to the wild type strain in the presence of both H2O2 and diamide oxidative stresses. Furthermore, propionate toxicity assays revealed increased accumulation of triacylglycerol (TAG) in the mel2 mutant relative to wild type. These observations provide the first evidence that mel2 plays a critical role in Mtb lipid biosynthesis. The major focus of the overall project was placed in this goal. In collaboration with Dr. Robert Powers in the Department of Chemistry at the University of Nebraska, we developed a metabolomic approach to study the lethal target of D-cycloserine (DCS) in Msmeg and Mtb. Our NMR metabolomics work revealed that Ddl (D-alanine Ligase) is the primary target of DCS, as cell growth is inhibited when the production of D-alanyl-D-alanine is halted. We showed 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. In addition, we determined the effects of fatty acid analogs on Msmeg and Mtb as potential inhibitors of latent Mtb bacilli in collaboration with Dr. Patrick Dussault in the Department of Chemistry at the University of Nebraska. Mycolic acids are a class of very long chain fatty acids that are present in large quantities in mycobacteria and closely related microorganisms of the order Actinomycetales. As these lipids play essential structural roles in mycobacteria, we hypothesize that compounds inhibiting their synthesis, may act as effective anti-mycobacterial agents. To test this hypothesis, a series of eleven fatty acid analogs incorporating cyclobutenes, cyclobutanes , cyclobutanones, and –cyclobutanols were chemically synthesized maintaining the lengths and cross sections of the parent compounds decenoic acid (C10) and cis-oleic acid (C18). The analogs were tested for their ability to inhibit the growth of Esherichia coli, Msmeg and Mtb in both nutrient-rich and nutrient-deficient media. None of the compounds inhibited E. coli growth under either set of conditions; in contrast a number of the analogs displayed inhibitory activity against both mycobacterial species in minimal media. The inhibitory activity was modest against Msmeg, but quite potent against Mtb with MIC values equal or below those obtained with anti-tuberculosis drugs DCS and Isoniazid. Since growth in minimal media resembles conditions of Mtb in the latent stage, these compounds may serve as candidate adjunctive anti-mycobacterial agents against dormant Mtb bacilli.
Publications
- Type:
Journal Articles
Status:
Submitted
Year Published:
2013
Citation:
Steven Halouska, Robert J. Fenton, Denise K. Zinniel, Darrell D. Marshall, Ofelia Chacon, Raul G. Barletta, and Robert Powers. 2013. Metabolomics Analysis Identifies D-Alanine-D-alanine Ligase as the Primary Lethal Target of D-cycloserine in Mycobacteria. ACS Chemical Biology (submitted).
- Type:
Journal Articles
Status:
Submitted
Year Published:
2013
Citation:
Harish K. Janagama, Sambou Tounkang, Suat L.G. Cirillo, Denise K. Zinniel, Raul G. Barletta and Jeffrey D. Cirillo. 2013. Molecular analysis of the Mycobacterium tuberculosis lux-like mel2 operon. Tuberculosis (submitted).
- Type:
Journal Articles
Status:
Submitted
Year Published:
2013
Citation:
Wantanee Sittiwong, Denise K. Zinnel, Robert J. Fenton, Darrel Marshall, Courtney B. Story, Ji-Young Lee, Robert Powers, Raul G. Barletta, and Patrick H. Dussault. 2013. Development of cyclobutene- and cyclobutane-functionalized fatty acids with inhibitory activity against Mycobacterium tuberculosis. Journal of Medicinal Chemistry (submitted).
|
Progress 10/01/11 to 09/30/12
Outputs
OUTPUTS: During this grant period, we focused on Objective 3. In collaboration with the UNL Department of Chemistry, we developed a method to determine the mechanism of action of drugs based on NMR metabolomic analysis. Using Mycobacterium smegmatis as a model system, we were able to identify the overall physiological process inhibited by these compounds and confirm the mechanism of action of known control antimycobacterial agents (Halouska et al., 2012). The clustering of novel chemical leads relative to known drugs provides a mean to identify a protein target or predict in vivo activity. In addition, we developed the technology to apply metabolomics analysis to Mycobacterium tuberculosis and Mycobacterium bovis (Zinniel et al., 2012). To prepare M. tuberculosis cell extracts for NMR metabolomics, cell cultures were grown under Biosafety Level 3 containment, harvested, and subjected to mechanical lysis, maintaining cold temperatures to preserve metabolites. Cell lysates were recovered, filtered sterilized, and stored at ultra-low temperatures. Upon confirming the absence of viable cells by platings, samples were removed for downstream processing. Extracts were lyophilized, resuspended in deuterated buffer and injected in the NMR instrument, capturing spectroscopic data later subjected to statistical analysis. We obtained a spectrum that identified metabolites directly or indirectly associated with the D-alanine pathway. Assignments of NMR spectra to specific metabolites were performed using chemical shifts reported in the Madison Metabolomics Consortium Database, the BioMagResBank, and the Human Metabolome Database. The spectrum contained ca. 400 peaks, from which it was possible to identify and quantify 40-50 metabolites, including amino acids, nucleotide precursors, and glycolytic and citric acid intermediates. In addition, we continued studying the metabolic pathways involved in mycobacteria alanine biosynthesis and catabolism, isolating the corresponding mutants. Analyses of these mutants are still in progress. To advance these studies, we again collaborated with the Department of Chemistry to synthesize C13 D-cycloserine (Thacker et al., 2012) and determine the minimal inhibitory concentrations of novel cyclobutene and cyclobutane derivatives against mycobacteria (Sittiwong et al., 2013). In preparation for Objective 1, an important consideration is the transduction frequency in Mycobacterium paratuberculosis of the MycoMarT7 phage to be used in TraSH mutagenesis experiments. The frequency previously determined was approximately 8.0 x 10-5 kanamycin resistant colonies/PFU (about 80,000 transductants/ml of transduced culture). We are currently expanding the phage stocks prior to M. paratuberculosis infection assays. In Objective 2, we developed PCR assays to identify the corresponding mutants in infection assays currently being performed as part of the vaccine testing program sponsored by the Johne's Disease Integrated Program. In collaboration with Texas A&M, we constructed mutants in the M. tuberculosis mel locus (Galbadage et al., 2012). We plan to mutate the MAP mel homologue in the PPE mutant MAP1152. PARTICIPANTS: The following people collaborated in this project: Dr. Ofelia Chacon (University of Nebraska) - research interest is to use molecular genetics and biochemical analyses to study the D-alanine pathway in mycobacteria, identify drug targets, and develop novel, effective, safer inhibitors to treat diseases. Dr. Patrick Dussault (University of Nebraska Department of Chemistry) - research interest is to apply organic synthesis and organic oxidation chemistry to develop antimicrobial agents. Dr. Robert Powers (University of Nebraska Department of Chemistry) - research interest is focused on developing technologies that use bioinformatics, NMR spectroscopy, mass spectroscopy, metabolomics, molecular modeling, and structural biology to assign a biological function to novel proteins. Dr. James Takacs (University of Nebraska Department of Chemistry) - research interest involves chemical chiral synthesis. Dr. Jeffrey Cirillo (Texas A&M University) - his research interest is to examine the virulence mechanisms of M. tuberculosis using cellular, molecular and genetic techniques to obtain a better understanding of the roles of the pathogen and host in disease. TARGET AUDIENCES: Research results relating to microbiology, microbial genetics, metabolomics, bioinformatics and NMR spectroscopy were presented at a scientific meeting (Molecular Basis of Infectious Diseases Retreat in Houston, Texas) and will presented at the 245th American Chemical Society National Meeting in New Orleans, Louisiana. In addition, we published a detailed analysis of our experimental results in peer-reviewed scientific journals (ACS Chemical Biology, Heterocycles, and the Journal of Visualized Experiments) to make information available to the research community and public as soon as possible. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The utilization of NMR technology demonstrated that different classes of antibiotics uniquely affect the metabolome of M. smegmatis. These metabolomic changes are directly correlated with broad mechanisms of action that are associated with each tuberculosis class of antibiotics, disruption of cell walls or membranes, inhibition of transcription, translation, or DNA supercoiling, or the inhibition of mycolic acid biosynthesis. We have also developed the methodology to apply this analysis to the agents of bovine and human tuberculosis in the biocontainment laboratory for safe transfer of samples for downstream processing steps in the NMR Facility. Thus, the impact of these studies is that we have proof of concept for an NMR metabolomics method to elucidate the mechanism of action of promising antimicrobial agents against mycobacteria. Finding therapeutic targets that inhibit the D-alanine pathway of peptidoglycan biosynthesis is a highly sought after goal in drug discovery research. D-Cycloserine (DCS) is a second stage drug for the treatment of tuberculosis. In our study, we synthesized DCS in 19.8% overall yield from DL-serine methyl ester. This synthetic route gives both enantiomers of cycloserine via a corrected and improved one pot resolution procedure using D- and L-tartaric acids. The final outcome of this study was the preparation of C13 DCS in a small scale. We plan to expand this procedure to prepare large amounts of isotopomeric labeled DCS for mechanistic studies with live mycobacteria. For M. tuberculosis MIC testing, synthetic compounds were created that incorporated cyclobutene, cyclobutane, cyclobutanol, or cyclobutanone subunits. Preliminary biological activity data indicate that a number of derivatives show good inhibitory activity against Mycobacterium smegmatis and that are more potent inhibitors of growth of Mycobacterium tuberculosis than D-cycloserine, a clinically used drug. The impact of these studies is the development of a new set of compounds to treat tuberculosis and related diseases.
Publications
- Galbadage, T., D. Patel, S. Subbian, D.K. Zinniel, S.L.G. Cirillo, R.G. Barletta and J.D. Cirillo. Virulence Gene Regulator luxR1 (MMAR_1239) in Mycobacterium marinum. Abstract and presentation at the Molecular Basis of Infectious Diseases Retreat. Houston, Texas. March 23, 2012.
- Zinniel, D.K., R.J. Fenton, S. Halouska, R. Powers and R.G. Barletta. 2012. Sample preparation of Mycobacterium tuberculosis extracts for Nuclear Magnetic Resonance (NMR) metabolomics studies. J Vis Exp. 67:e3673.
- Sittiwong, W., P.H. Dussault, R. Barletta, D. Zinniel, R. Fenton and R. Powers. Synthesis and antimycobacterial activity of novel fatty acid analogs. Abstract submitted to the 245th American Chemical Society National Meeting. New Orleans, Louisiana. April 7-11, 2013.
- Halouska, S., R.J. Fenton, R.G. Barletta and R. Powers. 2012. Predicting the in vivo mechanism of action for drug leads using NMR metabolomics. ACS Chem. Biol. 7:166-171.
- Thacker, N.C., J. Molnar-Toth, J.L. Miska, R.G. Barletta and J.M. Takacs. 2012. Preparation of D-cycloserine and 13C-Labeled D-cycloserine. Heterocycles. 86:In press and published online.
|