GENOME WIDE ANALYSIS OF M. PARATUBERCULOSIS PATHOGENESIS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1000784
• Grant No.: 2013-67015-21239
• Project No.: NEB39-169
• Proposal No.: 2013-01040
• Program Code: A1221
• Project Start Date: Sep 1, 2013
• Project End Date: Aug 31, 2019
• Grant Year: 2013

Project Director
Barletta, R.

Recipient Organization
UNIVERSITY OF NEBRASKA
(N/A)
LINCOLN,NE 68583

Performing Department
School Veterinary Med & Biomed

Non Technical Summary
Johne's disease (JD) caused by Mycobacterium avium subsp. paratuberculosis (MAP) is one of the most significant problems in animal health, especially for the dairy industry. Moreover, the potential linkage with Crohn's disease makes MAP a concern as a zoonotic and/or food-borne pathogen. In this study, we will utilize a novel approach to comprehensively identify MAP essential genes, and mine its genome for virulence determinants encoded by non-essential genes involved in intracellular (macrophage) survival or required for infection of calves. Mariner transposon mutagenesis coupled with new generation sequencing technologies for high-resolution phenotypic profiling (Tn-seq) and Transposon Site Hybridization (TraSH) will be applied to analyze fully random mutant banks representing genomic regions that are likely significantly underrepresented from current collections. We hypothesize that these approaches will result in mining of the MAP genome for virulence determinant discovery, independently of their constitutive or regulated expression. To test this hypothesis, we propose to identify: (1) Genes essential for MAP growth in complex and defined media; (2) Genetic determinants required for intracellular survival in primary bovine macrophages; and (3) Genetic determinants required for infection of calves. From these studies, we expect to gain a better understanding of JD, and identify targets for the development of state of the art safe and effective vaccines. We also expect these technologies, developed herein for the first time in a large production animal, would hasten its application to the analysis of host-pathogen interactions in any animal and plant microbial system relevant to United States agriculture.

Animal Health Component

100%

Research Effort Categories

Basic

50%

Applied

20%

Developmental

30%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	4010	1030	20%
311	4010	1040	40%
311	4010	1100	30%
311	4010	2090	10%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
4010 - Bacteria;

Field Of Science
2090 - Statistics, econometrics, and biometrics; 1040 - Molecular biology; 1030 - Cellular biology; 1100 - Bacteriology;

Keywords

johne`s disease

bovine

macrophages

neonatal calf model

pathogenesis

transposon mutagenesis

Goals / Objectives
Johne's Disease (JD) is one of the most significant problems in animal health for the cattle industry, especially dairies. Moreover, the potential for Mycobacterium avium subsp. paratuberculosis (MAP) being a zoonotic and/or food-borne pathogen, as evidenced by its possible linkage to Crohn's disease, creates concerns for both national and international trade. These issues underscore the various initiatives to develop improved management and control strategies including diagnostic tests and vaccines. The significance of JD and MAP has also been highlighted by the National Academies of Sciences and the funding of the Coordinated Agricultural Project JDIP. Significant progress has been made in analyzing disease pathogenesis and developing diagnostic tests and vaccines. However, tests of high sensitivity and specificity have not been developed and most diseased animals escape detection, especially during early infection. Likewise, vaccines are not efficacious and interfere with diagnostic tests against bovine tuberculosis. In this context, completion of the MAP genome sequence ushered in the development of new tools for diagnostics and control. Several attempts have been made to address pathogenesis on a genome-wide scale, including the construction and analysis of mutant banks and transcriptomic studies both in vitro and in vivo. The goal of this application is to implement TraSH mutagenesis and the related technology of Tn-seq (Illumina) for high-resolution phenotypic profiling for MAP. We plan to determine MAP essential genes and those required for pathogenesis. The novelty and uniqueness of this approach for MAP is conceptual and underscored by: i) the creation of fully stable and random insertional mutants; ii) coupling mutant identification to next generation sequencing or microarray technologies; iii) mutant screening in primary bovine macrophages infected at the appropriate conditions (e.g., 39°C) as the most relevant in vitro system; iv) use of the native host (calf) for in vivo screening; and v) use of an unbiased approach to identify genes important in pathogenesis without relying on expression data. In our studies, we will test the hypothesis that TraSH mutagenesis and Tn-seq are the best approaches to mine the MAP genome for virulence determinant discovery independently of their mode of regulation (e.g., constitutive or regulated expression). We also predict that a significant number of genes required for survival in macrophages and calves are non-essential and either constitutively expressed or transiently regulated. To test these hypotheses, we propose to identify: 1. Genes essential for MAP growth in complex and defined media; 2.Genetic determinants required for intracellular survival in primary bovine macrophages; and 3.Genetic determinants required for infection of calves.

Project Methods
Objective 1: Identify Genes Essential for Mycobacterium avium subsp. paratuberculosis (MAP) Growth in Complex and Defined Media High density transposon mutagenesis provides a genome wide approach to identify essential genes in a sequenced microbial genome. Essential genes are required under all growth conditions and their inactivation cannot be overcome by nutrient addition and their function cannot be supplied by another gene located elsewhere in the genome. For this objective, the novelty is that we will use 2 conditions and define essential genes as those for which transposon mutants are significantly negatively selected in: (i) complex media consisting of a modified Middlebrook 7H9 medium supplemented with amino acids and egg yolk; and (ii) a chemically defined media. Genomic DNA will be isolated from mutant libraries selected on either complex or chemically defined medium and processed by Illumina sequencing or microarray analysis. For the Tn-seq Illumina approach, we will identify and quantify sequence reads that contain the Himar-1 sequence and the corresponding TA site. These sequences will be aligned against the MAP genome to map transposon insertion sites. Gene essentiality will be assessed by determining the length of the longest run of TA dinucleotides within a gene that lacks detectable transposon insertions (5 or less reads) within the 5-80% of the ORF assessed from the 5'-end with P < 0.05. For TraSH, instead of comparing the signal of two cDNA TraSH probes as in the general methods, the signals of the in vitro libraries (complex or chemically defined medium) TraSH probes will be compared, as described previously, to the signal of genomic probes (randomly labeled chromosomal DNA that hybridizes to every gene in the array). Essential genes will be defined as those yielding statistically significant normalized log signal ratios of TraSH/genomic probes of< 0.2. We expect that the media with amino acids and egg yolk to be able to sustain growth of almost all mutants in non-essential genes. The chemically defined medium is expected to support the growth of fewer non-essential gene mutants, but it is possible that some particular mutants may be inhibited in the rich medium. We expect TraSH to yield slightly fewer essential genes as some of these genes may allow insertions beyond the 5'end 5-80% as discussed above. This objective will allow us to discriminate between genes essential for physiology from those exclusively involved in virulence. This will result in an increased knowledge in the genetic program initiated and developed by MAP to cause Johne's Disease (JD). In this objective, we also plan to establish that both whole genome sequencingand genetic methodssuperveneupon microarray analysisand gene expression profiling to analyze MAP pathogenesis. Objective 2: Identify Genetic Determinants Required for Intracellular Survival in Primary Bovine Macrophages Macrophage survival is a key feature of mycobacterial pathogenesis. Various virulent determinants and housekeeping functions are required for mycobacteria to survive within phagocytic cells. Thus, their identification is crucial to understand MAP pathogenesis and construct attenuated strains. The surviving mutant pool in non-essential genes will serve to determine which genes are then negatively selected in macrophages. To identify genes required for intracellular survival, the library of transposon mutants will be used to infect blood-monocyte derived macrophages (BMDMs). At various times post-infection, surviving bacteria will be recovered and mutants with a specific growth or survival defect will be identified by Tn-seq or TraSH. The salient issue of this approach is the use of primary cells from the natural host to screen for genes essential for intracellular survival. Key to hypothesis testing is to determine whether genes identified by our transposon mutagenesis studies are constitutively expressed or regulated. Extensive databases of transcriptomic data are available, but our conditions may differ from prior studies. Thus, we will also isolate and store total RNA samples from infected macrophages. As needed, comparison of expression levels in macrophages and broth cultures will be evaluated by Q-RT-PCR for a reduced number of selected genes or the full transcriptome determined as needed. These studies will allow us to further strengthen the outcomes established for Objective 1 and develop new methodologies to dissect gene interactions in macrophages. Objective 3: Identify Genetic Determinants Required for Infection of Calves A successful animal model that mimics the natural disease state is valuable for understanding host immune responses and pathogenesis and for the development of vaccine candidates. Current paratuberculosis vaccine preparations do not prevent infection but reduce fecal shedding and clinical disease thereby slowing the spread of disease. The goal of this objective is to identify in neonatal calves, attenuated mutants that are underrepresented. We expect to cover all aspects of pathogenesis including infection, colonization, mucosal translocation, and spread in addition to intracellular survival in phagocytic cells. An oral neonatal calf infection model will be used to implement Tn-seq or TraSH mutagenesis in vivo. Age-matched calves will be infected with the mutant pool and disease progression will be followed in all infected animals by diagnostic tests already developed in our calf model. We expect similar infectivity for the mutant pool as shown for MTB experiments in mice. Thus, we expect to find ≈ 300 genes on average to be underrepresented in vivo (e.g., 7% of potential mutants). To assure this course, we will use a slightly greater mutant pool inoculum (1.1 x 1010 per inoculation) than in our neonatal calf model study (1.0 x 1010). For Cycle 1 of negative selection, five calves will be infected orally and disease progression followed for 12 months. Bacilli will be collected from mucosal scrapings and other tissues for molecular analysis after expansion in culture. Mucosal scraping bacilli will be inoculated into five new calves by the oral-mucosal route to start Cycle 2 (8 months) of negative selection. A final iteration (Cycle 3) of this process will be repeated in the same manner as for Cycle 2. This design differs from the approach used for the MTB TraSH study in mice with mutant collection at 5 time points post-infection. Our "cycling" procedure seems more appropriate for a slow-growing microorganism such as MAP as implementation of selection cycles may result in an increased number of negatively selected mutants. Analysis of mutants negatively selected in vivo would be expected to identify a larger gene set than that selected in macrophages. This larger pool will be expected to include mutants impaired in pathogenic steps other than intracellular survival in phagocytic cells (e.g, mucosal translocation and adherence, invasion, and translocation). In contrast, most mutants impaired in macrophage survival (Objective 2) would be expected to be attenuated in vivo. Nonetheless, a few mutants with decreased intracellular survival in primary macrophages may be able to survive better in vivo, if compensatory mutations are selected in calves. We expect that most attenuating mutations validated in our collection or other studies will be confirmed by Tn-seq and/or TraSH. This study will allow us to further strengthen the outcomes expected for Objectives 1 and 2, and test this approach for the first time in a large production animal. We expect that the application of these technologies will facilitate the development of new generation vaccines to reduce JD economic losses to the dairy industry. We also expect that our study will hasten the application of these molecular strategies to dissect the pathogenesis of microorganisms of agricultural importance.

Progress 09/01/13 to 08/31/19

Outputs
Target Audience:The primary target audience was faculty, students and research scientists involved in the study of mycobacterial diseases of animals. This included annual meetings at the Conference of Research Workers in Animal Diseases (2014-2018), the Mycobacterial Diseases in Animals Multi-state Meeting (2014 and 2017), the USDA-NIFA AFRI Project Director Workshop (2014-2017) and the American Society of Microbiology Microbe (2018). We also presented research at the University of Nebraska (2016), Universidad de Mexico (2016), Michigan State University (2016), National Animal Disease Center (2017) and University of Georgia (2018). Finally, we were able to reach a greater audience of scientists in microbial pathogenesis by publishing a manuscript in Microbiology (2016), Frontiers in Veterinary Science (2017) and Methods in Molecular Biology (2019). Changes/Problems:Early in the project, we encountered transposon mutant pool biases that were corrected by implementing the analysis with generalized gamma distribution. This required us to modify software programs and obtain new Hidden Markov Model classification results. We decided to expand the calf studies to a "low" and "high" dose infection model and exclude the use of tissue culture systems since the low dose bacterial burdens were below the expected levels to yield an antibody response. This required the creation of a new transduction MAP pool. What opportunities for training and professional development has the project provided?Over the entire period, everyone involved with this project had the opportunity to learn from and interact with the PD, Co-PD's, as well as other researchers in mycobacterial diseases of animals. In addition, all personnel developed their oral and writing skills. Finally, we were able to discuss genomic studies and construction of novel mutants, analysis of essential genes and virulence determinants, Illumina DNA sequencing, global phenotypic profiling, and generation of mutant libraries. The following is a list of events attended: University of Nebraska Bioinformatics Workshop (July 2014) NE1201 Mycobacterial Diseases of Animals Multistate Initiative (October 2014) National Institute of Food and Agriculture Annual Meeting (November 2014) Conference of Research Workers in Animal Diseases (December 2014) University of Nebraska Medical Center Illumina DNA Sequencing (2014) University of Nebraska School of Veterinary Medicine and Biomedical Sciences meeting with co-PDs Drs. John P. Bannantine and Judith R. Stabel (August 2015) USDA-NIFA AFRI Project Director Workshop Annual Meeting (December 2015) Conference of Research Workers in Animal Diseases Meeting (December 2015) Michigan State University Seminar (April 2016) Universidad Nacional Autonoma de Mexico Seminar (June 2016) School of Veterinary Medicine and Biomedical Sciences Seminar (August 2016) USDA-NIFA AFRI Project Director Workshop Annual Meeting (December 2016) Conference of Research Workers in Animal Diseases Meeting (December 2016) National Animal Disease Center Seminar (August 2017) USDA-NIFA AFRI Project Director Workshop Annual Meeting (December 2017) Conference of Research Workers in Animal Diseases Meeting (December 2017) Mycobacterial Diseases in Animals Multistate Meeting (December 2017) University of Georgia Invited Presentation (March 1018) American Society of Microbiology Microbe (June 2018) How have the results been disseminated to communities of interest?In addition to the meetings, seminars, workshops and presentations attended in the training and profession development area, the following is a list of ways that we made the scientific community aware of our research results by poster/powerpoint presentations and manuscripts: Conference of Research Workers in Animal Diseases (December 2014) University of Nebraska Research Fair Graduate Student Poster (April 2015) USDA-NIFA AFRI Project Director Workshop Annual Meeting (December 2015) Conference of Research Workers in Animal Diseases Meeting (December 2015) Michigan State University Seminar (April 2016) Universidad Nacional Autonoma de Mexico Seminar (June 2016) School of Veterinary Medicine and Biomedical Sciences Seminar (August 2016) USDA-NIFA AFRI Project Director Workshop Annual Meeting (December 2016) Microbiology Volume 162 Pages 633-641 Manuscript (2016) National Animal Disease Center Seminar (August 2017) USDA-NIFA AFRI Project Director Workshop Annual Meeting (December 2017) Conference of Research Workers in Animal Diseases Meeting (December 2017) Mycobacterial Diseases in Animals Multistate Meeting (December 2017) Frontiers in Veterinary Science Manuscript (2017) University of Nebraska Doctoral Dissertation (2017) University of Georgia Invited Presentation (March 1018) American Society of Microbiology Microbe (June 2018) Methods in Molecular Biology Manuscript (2019) What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Impact: Annual losses due to JD are estimated in the range of $250 million to $1.5 billion for the United States cattle industry. This project uses a genetic technology to identify molecular targets for the development of state of the art safe and effective vaccines against JD. We expect these technologies would be applied to develop control strategies for other infectious diseases of large production animals and plant microbial systems. Aim 1 The MycoMarT7 Tn, that recognizes TA sites, was delivered with a phagemid vector to obtain ca. one million mutants. Chromosomal DNA was isolated, and Tn junctions were amplified and sequenced with an Illumina HiSeq 2500. The reads for each Tn insertion were mapped to the genome to determine the insertion frequency. To classify genes according to their properties, we applied a 4-state Hidden Markov Model (HMM) analysis normalizing read counts by the Beta Geometric Distribution (BGD) or Generalized Gamma Distribution (GGD). The HMM model assigns each TA site to a "state call" based on the number of reads: ES (essential), GD (growth defect), NE (non-essential) and GA (growth advantage). Individual gene essentiality is assigned based on the most frequent TA site state call within these boundaries. We applied the HMM model with the BGD, resulting in the removal of sets that had considerable skewing. The resulting mutant sets (ca. 700,000) displayed similar quantile patterns regarding TA sites and had a saturation density of 0.58 (30,271 TA sites out of 52,384) corresponding to 87% of the ORFs. The BGD normalized analysis identified 111 ES genes (55 of these genes corresponded to Mycobacterium tuberculosis (MTB) orthologues). However, this resulted in a very low number of ES genes in MAP compared to other microbial species. As we observed significant MAP Tn insertion biases, read counts may not follow the BGD thus we reanalyzed read counts using the R-program and the GGD, and upon normalization, we now included all mutants (ca. 1,000,000) and replaced the BGD for the GGD. This GGD analysis resulted in 221 ES genes with a saturation density of 0.60 (31,388 TA sites out of 52,384) corresponding to 90% of the ORFs. Out of these ES MAP genes with orthologues in MTB, this new analysis yielded 152 genes (69%) that were essential in both species. Many ES genes correspond to well known functions in key macromolecule metabolism, cell processes and conserved hypotheticals (e.g., nadB, inhA, pyrG, dnaE, rpoB, rpoC, MAP_1190 and MAP_1864c). The comparison of the number of ES genes based on GGD with other bacterial genomes indicate that MAP has the same number as B. subtillis (221), is slightly below E. coli (296) and about half of MTB (461). Outcomes - a blueprint (database) of the ES or NE properties of each MAP gene. Aim 2 As stated in the REEport Progress Report for 9-1-15 to 8-31-16, we decided to focus our efforts in identifying the MAP genes required for in vivo infection. This included expanding the calf studies to a "low" and "high" dose infection model. We excluded the use of tissue culture systems, as it would be less informative than expanding on the in vivo approach. Outcomes - none. Aim 3 Calves (n = 5 for each group) were orally inoculated via milk replacer with the MAP mutants on average 5.0 x 105 (low dose) or 1.0 x 109 (high dose) CFU on Days 0, 2 and 4. One control calf was inoculated with live MAP (wild type K-10) with ca. the same inoculum and dosing schedule. Calves were sampled for feces (< 10 g) and blood (30-50 ml) on the day prior to inoculation for baseline values and at various times up to 14 months. We determined fecal shedding, IFN-g responses and serum antibodies (HerdChek ELISA test). Animals were necropsied and tissue sections were obtained and processed for culture, histopathologic, flow cytometry and gene expression analyses. Low Dose - Fecal shedding was transient and passive. Culturing on HEYM media and/or IS900 PCR (MAP specific) resulted in at least one MAP positive calf for all days up to Day 30. For PCR with MycoMarT7 Tn specific primers (785 bp band), fecal samples were positive only during the first week. Serology using the HerdChek ELISA test remained at background levels throughout the entire study (Day 420). Whole blood was treated for 18 hours with either Concanavalin A mitogen (ConA), Pokeweed Mitogen (PWM) or a whole cell MAP sonicate (MPS) antigen preparation. The IFN-g responses were measured by subtracting the stimulated from the non-stimulated (NS) levels. The IFN-g test for the mutant pool was negative up to Day 60 (0.0043), reaching a maximum at Day 180 (3.4). Moreover, all mutant calves were positive on Day 120 and thereafter. For K-10, the test was negative up to Day 120 (0.089), reaching a maximum level at 180 days (3.9). This indicates that the animals are positive for cell-mediated immunity (CMI) consistent with a response to MAP infection. To analyze the CMI response in more detail, we isolated peripheral blood mononuclear cells (PBMCs) to determine the T-cell subpopulations regarding the CD4, CD8, gamma-delta T-cell receptor (gdTCR) and CD28 activation markers. Cells stimulated with the MPS antigen clearly show an increase in the CD4 and CD8 cells that are antigen-specific. We also observed a decrease in the gdTCR population, a phenomenon seen as calves age and the number of immature cells decrease. For CD28, there was an increase in response between Days 0 and 420 demonstrating a recall response to the antigen (most apparent in CD8 and gdTCR). In contrast the non-specific stimulation with ConA and PWM does not show any difference. Animal necropsy was at Day 420 to determine MAP burden by IS900 qPCR for sections of the duodenum, jejunum (JEJ), ileum (IL), cecum, colon (COL), their associated lymph nodes (LN), as well as sections of the ileoceccal valve (ICV), mesenteric lymph nodes, liver and spleen. We extrapolated MAP CFU/g tissue from a standard curve (femtograms of MAP DNA vs. Ct). Ct values < 38 were considered positive (≥ 0.1 CFU/g). Animals infected with mutant and wild type strains were positive for all intestinal sections and spleen. For all tissues, MAP burden results were variable among animals (maximum = 19,569 CFU/g of cecum tissue, calf 82). For the lymph nodes, most areas for the mutant pool and wild type were positive. Likewise, the results varied among animals (maximum = 11,087 CFU/g of transverse COL tissue, calf 79). High Dose - After necropsy at 270 days, the main results are: fecal shedding was observed in calves, as in the low dose, early in the study through day 30 and was likely transient shedding due to the experimental challenge. Some intermittent shedding was observed in calves at later points. High dose calves had a greater number of tissues that were colonized with the mutants, and at a higher CFU, when compared with the lose dose calves. Antigen-specific IFN-g in whole blood and serum antibody (HerdChek) were performed throughout the studies to assess infection. Calves had robust whole blood IFN-g responses by day 90 post-inoculation that extended throughout the study periods. Only one calf in the high dose study had a positive serum antibody response, aligning with the higher level of infection in that group. Interestingly, calves receiving the high dose had higher expression of IFN-g, IL-10, IL-12, IL-13, IL-17 and iNOS compared to calves given the low dose. This aligned with cytokine secretion by PBMCs stimulated for 24 hr with MAP antigen, with notable increases in IFN-g, IL-10, and IL-17 secretion for the high dose calves. Overall, the effects of inoculum dose were notable with increased infection of calves, along with concomitant increases in pro-inflammatory cytokines in the high dose calves. Outcomes - defining MAP pathogenesis in calf infections with a "low" and "high" dose, important for defining novel vaccines.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Rathnaiah, G., J.P. Bannantine, D.K. Zinniel, J.R. Stabel, Y.T. Grohn and R.G. Barletta. Comparative analysis of IS1096- and Himar1- derived transposon insertion sites in Mycobacterium avium subsp. paratuberculosis. Conference of Research Workers in Animal Diseases Meeting Poster Presentation. Chicago, IL. 12/07/2014.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Rathnaiah, G., J.P. Bannantine, D.O. Bayles, D.K. Zinniel, J.R. Stabel, Y.T. Grohn and R.G. Barletta. Analysis of a comprehensive Himar1 transposon library of Mycobacterium avium subsp. paratuberculosis. Proceedings Conference of Research Workers in Animal Diseases; 2015.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Rathnaiah G., D.O. Bayles, J.R. Stabel, D.K. Zinniel, J.P. Bannantine, Y.T. Gr�hn and R.G. Barletta. Molecular Genetic Analysis of a Large Mycobacterium avium subsp. paratuberculosis Mutant Bank. Conference of Research Workers in Animal Diseases Annual Meeting. Chicago, Illinois. December 2017.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Barletta, R.G., J.P. Bannantine, J.R. Stabel, Y.T. Gr�hn. Genome wide analysis of M. paratuberculosis pathogenesis. USDA-NIFA AFRI Project Director Workshop Annual Meeting. Chicago, Illinois. 2015.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Barletta, R.G., J.P. Bannantine, J.R. Stabel, Y.T. Gr�hn. Genome wide analysis of M. paratuberculosis pathogenesis. USDA-NIFA AFRI Project Director Workshop Annual Meeting. Chicago, Illinois. 2016.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Barletta, R.G., J.P. Bannantine, J.R. Stabel, Y.T. Gr�hn. Genome wide analysis of M. paratuberculosis pathogenesis. USDA-NIFA AFRI Project Director Workshop Annual Meeting. Chicago, Illinois. 2017.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Barletta, R.G. Molecular Genomics and Pathogenesis of Mycobacterium avium subspecies paratuberculosis. Mycobacterial Diseases in Animals Multi-state Meeting. Chicago, Illinois. December 2017. Invited Oral Presentation.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Rathnaiah G., D.O. Bayles, J.R. Stabel, D.K. Zinniel, J.P. Bannantine, Y.T. Gr�hn and R.G. Barletta. In vitro and In vivo Analysis of a Comprehensive Mycobacterium avium subsp. paratuberculosis Mutant Bank. Proceedings ASM Microbe Annual Meeting. Atlanta, Ga. June 2018.
• Type: Theses/Dissertations Status: Published Year Published: 2017 Citation: Molecular Genetic Analysis of Mycobacterium avium subsp. paratuberculosis and Mycobacterium smegmatis. Govardhan Rathnaiah. University of Nebraska.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Rathnaiah, G., J.P. Bannantine, D.O. Bayles, D.K. Zinniel, J.R. Stabel, Y.T. Gr�hn and R.G. Barletta 2016. Analysis of Mycobacterium avium subsp. paratuberculosis mutant libraries reveals loci-dependent transposition biases and strategies to novel mutant discovery. Microbiology, 162, 633-641.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Pathogenesis, Molecular Genetics, and Genomics of Mycobacterium avium subsp. paratuberculosis, the etiologic Agent of Johnes Disease. G. Rathnaiah, D.K. Zinniel, J.P. Bannantine, J.R. Stabel, Y.T. Gr�hn, M.T. Collins and R.G. Barletta. Frontiers in Veterinary Science.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Transposon Mutagenesis in Mycobacterium avium subspecies Paratuberculosis. John P. Bannantine, Denise K. Zinniel, and Raul G. Barletta. Methods in Molecular Biology.
• Type: Other Status: Other Year Published: 2015 Citation: Rathnaiah, G., J.P. Bannantine, D.K. Zinniel, J.R. Stabel, Y.T. Grohn and R.G. Barletta. Comparative analysis of IS1096- and Himar1- derived transposon insertion sites in Mycobacterium avium subsp. paratuberculosis. University of Nebraska Research Fair Graduate Student Poster Session. Lincoln, NE. 4/14/2015.
• Type: Other Status: Other Year Published: 2016 Citation: Rathnaiah, G. Molecular genetic analysis of Mycobacterium avium subsp. paratuberculosis. University of Nebraska School of Veterinary Medicine and Biomedical Sciences Seminar. Lincoln, NE. August 29, 2016.
• Type: Other Status: Other Year Published: 2016 Citation: Barletta, R.G. Exploding bugs! Breaking the mycobacterial cell wall towards new vaccines and antibiotics. Department of Microbiology & Molecular Genetics at Michigan State University Seminar. East Lansing, Michigan. April 2016.
• Type: Other Status: Other Year Published: 2016 Citation: Barletta, R.G. Descubrimiento de Nuevos Blancos Moleculares y Ant�genos de Micobacterium paratuberculosis Aplicaciones para el Desarrollo de Vacunas Diferenciales (DIVA). (Discovery of New Mycobacterium paratuberculosis Molecular Targets and Antigens. Applications for the Development of Differential (DIVA) Vaccines. Universidad Nacional Autonoma de Mexico (UNAM) Seminar. Mexico City, Mexico. June 2016.
• Type: Other Status: Other Year Published: 2017 Citation: Barletta, R.G. M. avium paratuberculosis. Invited Seminar. National Animal Disease Center. Ames, Iowa. August 2017.
• Type: Other Status: Other Year Published: 2018 Citation: Barletta, R.G. Development of Attenuated Vaccines against Johnes Disease and Other Mycobacterioses. Invited Presentation. University of Georgia. Athens, Georgia. March 2018.
• Type: Journal Articles Status: Other Year Published: 2020 Citation: Comprehensive Hidden Markov Model Analysis to Classify Mycobacterium avium subsp. paratuberculosis Genes for In-Vitro Growth by Saturation Transposon Mutangenesis. Govardhan Rathnaiah, John P. Bannantine, Darrell O. Bayles, Denise K. Zinniel, Judith R. Stabel, Yrjo T. Gr�hn and Ra�l G. Barletta. In preparation.

Progress 09/01/17 to 08/31/18

Outputs
Target Audience:The PD contributed a required abstract and poster to the USDA-NIFA AFRI Project Director Workshop Annual Meeting held in Chicago, Illinois (December 1, 2017). At the Mycobacterial Diseases in Animals Multi-state Meeting (entitled "Genomics and Pathogenesis of Mycobacterium avium subspecies paratuberculosis") and the Conference of Research Workers in Animal Diseases (entitled "Molecular Genetic Analysis of a Large Mycobacterium avium subsp. paratuberculosis Mutant Bank") Meeting that followed, I was able to discuss the progress on our genome project with Drs. Yrjo T. Grohn and John P. Bannantine. These meetings gave us an opportunity to reach a wider audience working in Animal Health. In November 2017, we reached a greater audience of scientists in microbial pathogenesis by our manuscript that was published in Frontiers in Veterinary Science entitled "Pathogenesis, Molecular Genetics and Genomics of Mycobacterium avium subsp. paratuberculosis, the Etiologic Agent of Johne's Disease". Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?In this reporting period, the PD (Dr. Raul G. Barletta) participated and contributed the required abstract for this project to the USDA-NIFA AFRI Project Director Workshop Annual Meeting (December 2017). Next, further professional and development opportunities for Dr. Barletta were provided by the Conference of Research Workers in Animal Diseases meeting held in Chicago, Illinois (December 2017). He had the opportunity to interact with scientists in the field of animal health and discuss results with Co-PDs, Drs. Yrjo T. Grohn and Dr. John P. Bannantine. Dr. Barletta presented an oral presentation at the Mycobacterial Diseases in Animals Multi-state Meeting (December 2017). In addition, he gave a invited presentationto Georgia scientists entitled "Development of Attenuated Vaccines against Johne's Disease and Other Mycobacterioses" to discuss genomic studies and constructiuon of novel mutants. How have the results been disseminated to communities of interest?- Twoposters werepresented at annual meetings to disseminate the results: Rathnaiah G., D.O. Bayles, J.R. Stabel, D.K. Zinniel, J.P. Bannantine, Y.T. Gröhn and R.G. Barletta. Molecular Genetic Analysis of a Large Mycobacterium avium subsp. paratuberculosis Mutant Bank. Conference of Research Workers in Animal Diseases Annual Meeting. Chicago, Illinois. December 2017. Barletta, R.G., J.P. Bannantine, J.R. Stabel, Y.T. Gröhn. Genome wide analysis of M. paratuberculosis pathogenesis. USDA NIFA AFRI Project Director Workshop Annual Meeting. Chicago, Illinois. December 2017. - A formal manuscript was published ina peer reviewed journal: Rathnaiah, G., D.K. Zinniel, J.P. Bannantine, J.R. Stabel, Y.T. Gröhn, M.T. Collins, and R.G. Barletta. (2017). Pathogenesis, Molecular Genetics and Genomics of Mycobacterium avium subsp. paratuberculosis, the Etiologic Agent of Johne's Disease. Frontiers in Veterinary Science, 4 (Article 187), 1-13. - An invited oralpresentation was givenat an annual meeting: Barletta, R.G. Molecular Genomics and Pathogenesis of Mycobacterium avium subspecies paratuberculosis. Mycobacterial Diseases in Animals Multi-state Meeting. Chicago, Illinois. December 2017. - An invited presentation was givenat the University of Georgia: Barletta, R.G. Development of Attenuated Vaccines against Johne's Disease and Other Mycobacterioses. Invited Presentation. University of Georgia. Athens, Georgia. March 2018. What do you plan to do during the next reporting period to accomplish the goals?The PI's at The University of Nebraska, The National Animal Disease Center and Cornell University will complete the analysis of the in vitro pools for gene essentiality and submit the manuscript that has already been drafted (see Aim 1 above). Future studies for Aim 3 will be to complete the "high dose" calf studies from January and September inoculations and recover the mutants and analyze what is present in the recovered pools (e.g., whole blood IFN-g responses, serum antibodies (Herdchek ELISA test), fecal shedding). In addition, we will sequence DNA from pooled tissue and lymph node calf samples from the "low and high dose" infection experiments.

Impacts
What was accomplished under these goals? Impact: Annual losses due to JD are estimated in the range of $250 million to $1.5 billion for the United States cattle industry. This project uses a genetic technology to identify molecular targets for the development of state of the art safe and effective vaccines against JD. We expect these technologies would be applied to develop control strategies for other infectious diseases of large production animals and plant microbial systems. Aim 1) We constructed a MycoMarT7 transposon mutant library that was given to the National Animal Disease Center that was inoculated into calves at a "high dose". Briefly for construction of the MAP MMT7 transposon mutant library, we grew 20 individual MAP cultures to an OD of approximately 1.0 to 1.5 dividing this up into 5 sets. Next we conducted a transduction with MAP and MMT7 phage (2.1 x 10^10 PFU/ml, based on the cotrol titer on M. smegmatis) plating on ten 500 cm^2 MOADC Kanamycin 50 ug/ml plates for each culture (1 control plate with just MP buffer was also done). The MMT7 phage titer on MAP was 6.0 x 10^9 PFU/ml at the permissive temperature of 30C. All tranductions at the non-permissive temperature of 37C were allowed to grow on agar for 2 months. At that time, we imaged all plates to obtain an estimate of approximately 500,000 MAP MMT7 mutants. These colonies were then scraped and collected. We drafted a manuscript entitlted "Essential Genes for In-Vitro Growth of Mycobacterium avium subsp. paratuberculosis Identified by Saturation Transposon Mutagenesis by Govardhan Rathnaiah, John P. Bannantine, Darrell O. Bayles, Denise K. Zinniel, Judith R. Stabel, Yrjo T. Gröhn and Raúl G. Barletta" that analyzes the transposon insertion sites and their gene classifications according to the Hidden Markov Model Probability Distribution. The manuscript is in internal review by the authors to determine if further analysis or experimentation is needed. Aim 3) For the "low dose" calf infection experiments we have completed the collection of all data except for some of the cytokine determinations. For the "high dose" experiment, calves were infected (with MAP from Aim 1) in January 2018 with approximately 1.25 x 10^9 CFU/ml for the MAP MMT7 mutant pool. For the MAP wild type K-10 strain, the infection dose was 1.5 x 10^9 CFU/ml. These calves were received at the National Animal Disease Center and allowed time to acclimate to their new surroundings before the infections. We have collected blood and fecal samples on regular intervals with the analysis on these samples to occur in late 2018 and early 2019. Furthermore, we are completing the cytokine analyses for the calves infected in January 2018. This includes running cytokine gene expression data on the "low dose" and "high dose" calves at all relevant time points. For calves infected with the "high dose", we collected mucosal tissue scrapings an infected a new set of calves in September 2018. We are currently collecting blood and fecal samples on regular intervals and culturing PBMCs from those calves. These experiments will extend until June 2019 when the calves will be necropsied.

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: Rathnaiah, G., D.K. Zinniel, J.P. Bannantine, J.R. Stabel, Y.T. Gr�hn, M.T. Collins, and R.G. Barletta. (2017). Pathogenesis, Molecular Genetics and Genomics of Mycobacterium avium subsp. paratuberculosis, the Etiologic Agent of Johnes Disease. Frontiers in Veterinary Science, 4 (Article 187), 1-13.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2017 Citation: Rathnaiah G., D.O. Bayles, J.R. Stabel, D.K. Zinniel, J.P. Bannantine, Y.T. Gr�hn and R.G. Barletta. Molecular Genetic Analysis of a Large Mycobacterium avium subsp. paratuberculosis Mutant Bank. Conference of Research Workers in Animal Diseases Annual Meeting. Chicago, Illinois. December 2017.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2017 Citation: Barletta, R.G. Molecular Genomics and Pathogenesis of Mycobacterium avium subspecies paratuberculosis. Mycobacterial Diseases in Animals Multi-state Meeting. Chicago, Illinois. December 2017. Invited Oral Presentation.
• Type: Other Status: Accepted Year Published: 2018 Citation: Barletta, R.G. Development of Attenuated Vaccines against Johnes Disease and Other Mycobacterioses. Invited Presentation. University of Georgia. Athens, Georgia. March 2018.

Progress 09/01/16 to 08/31/17

Outputs
Target Audience:The PD contributed a required abstract and poster to the USDA-NIFA AFRI Project Director Workshop Annual Meeting held in Chicago, Illinois (December 4, 2016). At the Conference of Research Workers in Animal Diseases Meeting that followed, my PhD graduate student Govardhan Rathnaiah, and I were able to discuss the progress on our genome project with Dr. Yrjo T. Grohn. This meeting gave us an opportunity to reach a wider audience working in Animal Health. Mr. Rathnaiah presented his PhD Dissertation seminar to the School of Veterinary Medicine and Biomedical Sciences in August 2017. The members of his dissertation committee, faculty and staff working on infectious and noninfectious diseases attended this seminar. The PI presented a seminar on M. avium paratuberculosis at the National Animal Disease Center in August 2017. This further allowed the PI and co-PD's to discuss the genome project. Finally, we expect to reach a greater audience of scientists in microbial pathogenesis by our submitted manuscript to Frontiers in Veterinary Science once it is published Changes/Problems:Initial results from the calf infection studies revealed that a "low" dose infection model might not be the best strategy for the M. avium paratuberculosis (MAP) mutant library. Therefore, it was decided to re-create a new MAP library that can be inoculated as a "high" dose. The 400 individual transduction with MAP and phage MycoMarT7 have been plated. We will process and collect this library in 2018. What opportunities for training and professional development has the project provided?In this reporting period, the PD (Dr. Raul G. Barletta) participated and contributed the required abstract for this project to the USDA-NIFA AFRI Project Director Workshop Annual Meeting (December 2016). Next, further professional and development opportunities for Dr. Barletta and his PhD Graduate student (Govardhan Rathnaiah) were provided by the Conference of Research Workers in Animal Diseases meeting held in Chicago, Illinois (December 2016). They had opportunities to interact with scientists in the field of animal health and discuss results with Co-PD, Dr. Yrjo T. Grohn. Mr. Rathnaiah completed his dissertation research entitled "Molecular Genetic Analysis of Mycobacterium avium subsp. paratuberculosis and Mycobacterium smegmatis". A portion of this document included the analysis of the MAP genome from the previous report. In addition, a chapter of this dissertation was expanded in a collaborative fashion with the Co-PD's of the grant into a manuscript that was submitted (see below). Dr. Barletta presented a seminar to National Animal Disease Center scientists entitled "Molecular Genetics and Genomics of Mycobacterium avium subspecies paratuberculosis, the Causative Agent of Johne's Disease" to discuss genomic studies regarding the generation of mutant libraries and the analysis of essential genes and virulence determinants. He had the opportunity to interact with the Co-PD's (Drs. Bannantine and Stabel) and other scientists of the Genomics Core. In addition, he visited with Dr. Jesse Hostetter from Iowa State University to discuss future collaborations. How have the results been disseminated to communities of interest?- A poster was presented to disseminate the results: Barletta, R.G., J.P. Bannantine, J.R. Stabel, Y.T. Gröhn. Genome wide analysis of M. paratuberculosis pathogenesis. USDA NIFA AFRI Project Director Workshop Annual Meeting. Chicago, Illinois. December 2016. - A formal manuscript was submitted to a peer reviewed journal (August 2017; Frontiers in Veterinary Science): Molecular Genetics and Genomics of Mycobacterium avium subsp. paratuberculosis, the Etiologic Agent of Johne's Disease by Govardhan Rathnaiah, Denise K. Zinniel, John P. Bannantine, Judith R. Stabel, Yrjö T. Gröhn, and Raúl G. Barletta. - A first draft of a manuscript was prepared (journal submission to be determined at a later date): Essential Genes for In-Vitro Growth of Mycobacterium avium subsp. paratuberculosis Identified by Saturation Transposon Mutagenesis by Govardhan Rathnaiah, John P. Bannantine, Darrell O. Bayles, Denise K. Zinniel, Judith R. Stabel, Yrjo T. Gröhn and Raúl G. Barletta. - A seminar was presented at the National Animal Disease Center: Molecular Genetics and Genomics of Mycobacterium avium subspecies paratuberculosis, the Causative Agent of Johne's Disease by Raúl G. Barletta (August 16, 2017). - A Doctoral Dissertation was submitted and accepted to the University of Nebraska: Molecular Genetic Analysis of Mycobacterium avium subsp. paratuberculosis and Mycobacterium smegmatis by Govardhan Rathnaiah (graduated August 12, 2017). The dissertation seminar took place on July 17, 2017. What do you plan to do during the next reporting period to accomplish the goals?Aim 1) We will reconstruct a MycoMarT7 transposon mutant library so that it can be inoculated into calves at a "high dose". We will continue to analyze the transposon insertion sites to better classify the genes accroding to the Hidden Markov Model. Aim 3) We will infect the calves using the library collected in Aim 1. Calves will be sampled for feces and blood on the day prior to inoculation of the calves for baseline values. Thereafter, calves will be sampled on days 3, 7, 14 and 30, and then will be sampled at regular intervals up to the completion of the experiment at the end of the funding cycle. Whole blood IFN-g responses, serum antibodies (Herdchek ELISA test) and fecal shedding will be determined.

Impacts
What was accomplished under these goals? Impact: Annual losses due to JD are estimated in the range of $250 million to $1.5 billion for the United States cattle industry. This project uses a genetic technology to identify molecular targets for the development of state of the art safe and effective vaccines against JD. We expect these technologies would be applied to develop control strategies for other infectious diseases of large production animals and plant microbial systems. Aim 1) We re-evaluated the Hidden Markov Model data that previously reported 328 essential genes. This was necessary as we found a transposon insertion "hot spot" in the MAP_4116c (mmaA4) gene. This hot spot led to anomalies in the gene classification scheme: 228 genes in the clockwise direction beyond the hot spot were classified as "essential". To solve this problem, the read counts were normalized using geometric means beta distribution. When doing this type of normalization, it became apparent that three out of five of the original transposon mutant sets had the most read counts and best distributions. Therefore, these three sets were combined for the HMM analysis. When the total read counts were summed for these library sets, we obtained a total of 111 essential genes. Most of these genes were also listed as essential for M. tuberculosis (http://tuberculist.epfl.ch/). Aim 3) During this reporting period, we continued the calf M. avium paratuberculosis (MAP) low dose infection model. Calves (n = 5) were fed milk replacer containing on average 5.0 x 105 CFU live MAP from the mutant pool daily on days 0, 2, and 4. One calf (n =1) was inoculated with live MAP (wild type strain K-10) at 1.0 x 104 on the same dosing schedule. Calves were sampled for feces and blood on the day prior to inoculation of the calves for baseline values. Thereafter, calves were sampled on days 3, 7, 14 and 30, and then sampled at regular intervals for 14 months (n = 6). Whole blood IFN-g responses, serum antibodies (Herdchek ELISA test) and fecal shedding were determined. The IFN-g test was negative up to Day 60, reaching a maximum at day 180 for the mutant pool and decreased thereafter. For K-10, the test was negative up to Day 120, reaching a maximum level at 180 days and decreased thereafter. This indicates that the animals are becoming positive for the MAP infection. The serum ELISA remained at background levels throughout the entire study (420 days). To identify which calves were colonized with MAP, fecal calf samples were harvested and subjected to culture and PCR (IS900 and MycoMarT7). Cultures were positive for MAP at Days 0, 3, 7, 14 and 30. For IS900 PCR, fecal samples were positive up to 30 days post-infection. The maximum levels were observed at Days 7 and 14. For MycoMarT7 PCR, fecal samples were positive on Days 3 and 7, indicating shedding of transposon mutants. In general, more shedding was observed by fecal culture than by PCR. This indicates that fecal shedding occurred only after the experimental infection. In addition, animals were necropsied and sections of jejunum, ileum, and their associated lymph nodes, as well as sections of ileoceccal valve, and iliac lymph nodes were obtained and processed for IS900 PCR. Colony forming units were determined by establishing a standard curve to extrapolate back to MAP cfu/g tissue. Animals infected with the mutant pool were positive for all intestinal sections (associated lymph nodes), spleen and liver for at least 2 out of 5 animals. The mid- and distal ileum, as well as the mid-jejunum lymph nodes were positive for all animals. The highest cfu/g results varied from calf to calf consistent with recovery amounts at a "low dose".

Publications

• Type: Journal Articles Status: Submitted Year Published: 2017 Citation: Pathogenesis, Molecular Genetics, and Genomics of Mycobacterium avium subsp. paratuberculosis, the etiologic Agent of Johnes Disease. G. Rathnaiah, D.K. Zinniel, J.P. Bannantine, J.R. Stabel, Y.T. Gr�hn, M.T. Collins and R.G. Barletta. Frontiers in Veterinary Science.
• Type: Theses/Dissertations Status: Accepted Year Published: 2017 Citation: Molecular Genetic Analysis of Mycobacterium avium subsp. paratuberculosis and Mycobacterium smegmatis. Govardhan Rathnaiah. University of Nebraska.

Progress 09/01/15 to 08/31/16

Outputs
Target Audience:The PD contributed a required abstract and poster to the USDA-NIFA AFRI Project Director Workshop Annual Meeting held in Chicago, Illinois (December 4, 2015). We discussed progress on the project with the Co-PD's of this grant. Scientists in the Animal Health, Animal Well-Being and Food Security areas that are funded by AFRI were in attendance. My PhD graduate student, Govardhan Rathnaiah, presented an abstract and poster at the Annual Meeting for the Conference of Research Workers in Animal Diseases held in Chicago, Illinois (December 6, 2015). This meeting gave us an opportunity to reach a wider audience working in Animal Health and discuss project research results with Co-PD, Dr. Yrjo T. Grohn. Mr. Rathnaiah presented a seminar to the University of Nebraska School of Veterinary Medicine and Biomedical Sciences on August 29, 2016 that included faculty and students working on infectious and noninfectious diseases. The PD was the keynote speaker at the IX Congress of the Master and Doctorate Program in Animal Science and Health held at the Universidad Nacional Autonoma de Mexico (June 2016). Faculty and students in animal science and health were in attendance. The PD presented a seminar to the Department of Microbiology and Molecular Genetics at Michigan State University (April 2016). Faculty, students and scientists in the Natural Science College attended. Finally, we reached a greater audience of scientists in microbial pathogenesis by publishing a manuscript in the journal Microbiology (February 2016). Changes/Problems:We decided to focus our efforts in identifying the MAP genes required for in vivo infection. We will curtail the use of tissue culture systems. What opportunities for training and professional development has the project provided?In this reporting period, the PD (Dr. Raul G. Barletta) participated and contributed the required abstract for this project to the USDA-NIFA AFRI Project Director Workshop Annual Meeting (December 4, 2015). Next, further professional and development opportunities for Dr. Barletta and his PhD Graduate student (Govardhan Rathnaiah) were provided by the Conference of Research Workers in Animal Diseases meeting held in Chicago, Illinois (December 2015). They had opportunities to interact with scientists in the field of animal health and discuss results with Co-PD, Dr. Yrjo T. Grohn. Importantly, Mr. Rathnaiah had the opportunity to develop his oral and writing skills by presenting a seminar to the School of Veterinary Medicine and Biomedical Sciences and a manuscript published in Microbiology. How have the results been disseminated to communities of interest?- Two posters were presented to disseminate the results: Rathnaiah, G., J.P. Bannantine, D.O. Bayles, D.K. Zinniel, J.R. Stabel, Y.T. Grohn and R.G. Barletta. Analysis of a comprehensive Himar1 transposon library of Mycobacterium avium subsp. paratuberculosis. Conference of Research Workers in Animal Diseases. Chicago, Illinois. 2015. Barletta, R.G., J.P. Bannantine, J.R. Stabel, Y.T. Gröhn. Genome wide analysis of M. paratuberculosis pathogenesis. USDA-NIFA AFRI Project Director Workshop Annual Meeting. Chicago, Illinois. 2015. - A formal manuscript was published in a peer reviewed journal: Rathnaiah, G., J.P. Bannantine, D.O. Bayles, D.K. Zinniel, J.R. Stabel, Y.T. Gröhn and R.G. Barletta 2016. Analysis of Mycobacterium avium subsp. paratuberculosis mutant libraries reveals loci-dependent transposition biases and strategies to novel mutant discovery. Microbiology, 162, 633-641. - Three oral presentations were given at University of Nebraska, Michigan State and Universidad Nacional Autonoma de Mexico: Rathnaiah, G. Molecular genetic analysis of Mycobacterium avium subsp. paratuberculosis. University of Nebraska School of Veterinary Medicine and Biomedical Sciences Seminar. Lincoln, NE. August 29, 2016. Barletta, R.G. Exploding bugs! Breaking the mycobacterial cell wall towards new vaccines and antibiotics. Department of Microbiology & Molecular Genetics at Michigan State University Seminar. East Lansing, Michigan. April 2016. Barletta, R.G. Descubrimiento de Nuevos Blancos Moleculares y Antígenos de Micobacterium paratuberculosis Aplicaciones para el Desarrollo de Vacunas Diferenciales (DIVA). (Discovery of New Mycobacterium paratuberculosis Molecular Targets and Antigens. Applications for the Development of Differential (DIVA) Vaccines. Universidad Nacional Autonoma de Mexico (UNAM) Seminar. Mexico City, Mexico. June 2016. What do you plan to do during the next reporting period to accomplish the goals?Next Reporting Period 3) Animals will be necropsied at the end of the study and sections of jejunum, ileum, and their associated lymph nodes, as well as sections of ileoceccal valve, and iliac lymph nodes will be obtained and processed for culture, PCR, histopathologic analyses, and gene expression assays. Supernatants were harvested for cytokine secretion and cells were harvested for flow cytometric analyses. Results on these last two assays are still pending until completion of the study.

Impacts
What was accomplished under these goals? Impact: Annual losses due to JD are estimated in the range of $250 million to $1.5 billion for the United States cattle industry. This project uses a genetic technology to identify molecular targets for the development of state of the art safe and effective vaccines against JD. We expect these technologies would be applied to develop control strategies for other infectious diseases of large production animals and plant microbial systems. Results: 1. For this reporting period, we continued the characterization of MAP essential genes in defined media by standard and comparative genomic analysis. To classify genes according to their properties, we applied a 4-state HMM analysis. Each "state call" is considered independent of the calls for adjacent sites and it is revealed by the actual number of reads for the corresponding site. Then, this model assigns each TA site to a "state call" based on the number of sequence reads at that site: ES (essential, no to very few reads), GD (growth defect, few reads), NE (non-essential, significant number of reads) and GA (growth advantage, many reads). This analysis classifies these "state calls" without considering gene boundaries. Individual gene essentiality is then assigned based on the most frequent TA site state call within gene boundaries. We classified the 328 essential genes into different categories. The majority of the essential genes encode for proteins involved in small molecule metabolism (110), followed by those in macromolecule metabolism (92), conserved hypotheticals (64), unknown hypotheticals (34), cell processes (18) and other functions not readily categorized (10). The Database of Essential Genes (DEG) was utilized to identify MAP essential gene homologs in other microbial species: 70% of the essential genes (229/328) had one or more homologs, while 30% (99/328) had no homologs and mostly encode for hypothetical proteins. In addition, we further propagated the MAP mutant pool in the original complete Middlebrook 7H9 media with oleic acid enrichment (control) and the same media containing 5% NaCl or 3% SDS. The goal was to identify genes that encode susceptibility to high osmolarity and cell wall stress, respectively. Cells (ca. 1 x 105 CFU per plate) were spread onto 10 plates of each media, surviving colonies (only recovered from the control and NaCl plates) were harvested at ca. 8 weeks and stocked at -80°C for further analysis. 3. During this reporting period, we started calf MAP infection experiments. Calves (n = 5) were fed milk replacer containing on average 5.0 x 105 CFU live MAP from the mutant pool daily on days 0, 2, and 4. One calf (n =1) was inoculated with live MAP (strain K-10) at 1.0 x 104 on the same dosing schedule. Calves were sampled for feces and blood on the day prior to inoculation of the calves for baseline values. Thereafter, calves were sampled on days 3, 7, 14 and 30, and then will be sampled at regular intervals for 12 months (n = 6). Whole blood IFN-g responses, serum antibodies (Herdchek ELISA test) and fecal shedding were determined. The IFN-g test was positive after 90 days up until day 270 for the mutant pool. For K-10, the test was positive at 180 days. This indicates that the animals are becoming positive for the MAP infection. The serum ELISA remains negative throughout the entire study (270 days) with a slight trend towards positivity, indicating that disease progression is in the early stages. Fecal shedding was only observed for the first 14 days for both K-10 and the mutant pool and remained negative thereafter (up to day 120). This indicates that fecal shedding occurred only after the experimental infection.

Publications

• Type: Journal Articles Status: Published Year Published: 2016 Citation: Rathnaiah, G., J.P. Bannantine, D.O. Bayles, D.K. Zinniel, J.R. Stabel, Y.T. Gr�hn and R.G. Barletta 2016. Analysis of Mycobacterium avium subsp. paratuberculosis mutant libraries reveals loci-dependent transposition biases and strategies to novel mutant discovery. Microbiology, 162, 633-641.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Rathnaiah, G., J.P. Bannantine, D.O. Bayles, D.K. Zinniel, J.R. Stabel, Y.T. Grohn and R.G. Barletta. Analysis of a comprehensive Himar1 transposon library of Mycobacterium avium subsp. paratuberculosis. Proceedings Conference of Research Workers in Animal Diseases; 2015.
• Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Rathnaiah, G. Molecular genetic analysis of Mycobacterium avium subsp. paratuberculosis. University of Nebraska School of Veterinary Medicine and Biomedical Sciences Seminar. Lincoln, NE. August 29, 2016.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Barletta, R.G., J.P. Bannantine, J.R. Stabel, Y.T. Gr�hn. Genome wide analysis of M. paratuberculosis pathogenesis. USDA-NIFA AFRI Project Director Workshop Annual Meeting. Chicago, Illinois. 2015.
• Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Barletta, R.G. Descubrimiento de Nuevos Blancos Moleculares y Ant�genos de Micobacterium paratuberculosis Aplicaciones para el Desarrollo de Vacunas Diferenciales (DIVA). (Discovery of New Mycobacterium paratuberculosis Molecular Targets and Antigens. Applications for the Development of Differential (DIVA) Vaccines. Universidad Nacional Autonoma de Mexico (UNAM) Seminar. Mexico City, Mexico. June 2016.
• Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Barletta, R.G. Exploding bugs! Breaking the mycobacterial cell wall towards new vaccines and antibiotics. Department of Microbiology & Molecular Genetics at Michigan State University Seminar. East Lansing, Michigan. April 2016.

Progress 09/01/14 to 08/31/15

Outputs
Target Audience:The primary target audience was scientists involved in the study of mycobacterial diseases of animals. In this reporting period, we reached these scientists at the Annual Meeting of the NE1201 Mycobacterial Diseases of Animals multistate initiative to be held in Kansas City, Missouri (October 18-19, 2014). We discussed progress on the project with the Co-PD's of this grant. The PD contributed a required abstract to the Annual Meeting of National Institute of Food and Agriculture funded investigators (November 19-21, 2014). An abstract was selected for a poster presentation at the Annual Meeting for the Conference of Research Workers in Animal Diseases to be held in Chicago, Illinois (December 7-9, 2014). This meeting gave us an opportunity to reach a wider audience working in Animal Health and discuss project research results with Co-PD, Dr. Yrjo T. Grohn. Changes/Problems:We have confimed vector sequences in the anaylsis of the mutant pool that was described in the last reporting period. This problem was overcome by collecting a larger pool of independent mutants of approximately 1 million. As the Illumina sequencing as worked exceptionally well, we are no longer planning to do microarray analysis. Instead, we are planning to expand the analysis of the mutant pools in vitro and in vivo using Illumina Sequencing. What opportunities for training and professional development has the project provided?In this reporting period, the PD (Dr. Raul G. Barletta) and his PhD Graduate student (Govardhan Rathnaiah) participated in the 2014 Annual Meeting of the NE1201 Mycobacterial Diseases of Animals multistate initiative held in Kansas City, Missouri (October 18-19, 2014). This meeting provided opportunities to interact with the Co-PD's of this grant, as well as other researchers in mycobacterial diseases of animals. Next, Dr. Barletta participated and contributed the required abstract for this NEB39-169 project to the Annual Meeting of National Institute of Food and Agriculture funded investigators (November 25, 2014). Further professional and development opportunities for both were provided by the Conference of Research Workers in Animal Diseases meeting held in Chicago, Illinois (December 7-9, 2014). They had opportunities to interact with scientists in the field of animal health and discuss results with Co-PD, Dr. Yrjo T. Grohn. Govardhan had opportunities to practice his communication skills winning an award at the graduate student poster competition. Then both the student and the PD visited several times to the University of Nebraska Medical Center Sequencing Facility to discuss the Illumina DNA sequencing protocol based on our goal to identify essential genes in MAP by global phenotypic profiling. Finally, the PD hosted a meeting on August 13th (2015) at the University of Nebraska School of Veterinary Medicine and Biomedical Sciences with co-PDs (Drs. John P. Bannantine and Judith R. Stabel) and laboratory personnel to discuss and learn from each other expertise, discuss results, and exchange materials and data files. How have the results been disseminated to communities of interest?- Two posters were presented to disseminate the results: Rathnaiah, G., J.P. Bannantine, D.K. Zinniel, J.R. Stabel, Y.T. Grohn and R.G. Barletta. Comparative analysis of IS1096- and Himar1- derived transposon insertion sites in Mycobacterium avium subsp. paratuberculosis. Conference of Research Workers in Animal Diseases Meeting Poster Presentation. Chicago, IL. 12/07/2014. The abstract for this research is also published in the Proceedings of this meeting. Rathnaiah, G., J.P. Bannantine, D.K. Zinniel, J.R. Stabel, Y.T. Grohn and R.G. Barletta. Comparative analysis of IS1096- and Himar1- derived transposon insertion sites in Mycobacterium avium subsp. paratuberculosis. University of Nebraska Research Fair Graduate Student Poster Session. Lincoln, NE. 4/14/2015. - In addition, a formal manuscript to report these results is also in preparation. What do you plan to do during the next reporting period to accomplish the goals?We plan to expand the mutant pool in vitro through further cycles of growth to evaluate in more detail genes that may confer either growth defect or advantage. The mutant collection has been already transferred to the NADC where calf infection experiments are scheduled to start in early 2016. The in vivo experiments are expected to define MAP genes required for survival and pathogenesis in the bovine host.

Impacts
What was accomplished under these goals? Impact: Annual losses due to JD are estimated in the range of $250 million to $1.5 billion for the United States cattle industry. This project uses a genetic technology to identify molecular targets for the development of state of the art safe and effective vaccines against JD. We expect these technologies would be applied to develop control strategies for other infectious diseases of large production animals and plant microbial systems. Results: The generation of comprehensive random mutant banks by transposon mutagenesis is a fundamental technology to determine the role of MAP genes in cell physiology and pathogenesis. We constructed a comprehensive Himar1 transposon library consisting of five independent sets comprising a total of ca. 1 million mutants. MAP K-10 was transduced with MycomarT7 phage carrying Himar1 derived MycomarT7 transposon. Kanamycin-resistant transductants were isolated on complete Middlebrook 7H9 medium after a 6-8 week incubation period at 37°C. DNA was isolated from each set and sheared randomly by ultra-sonication to obtain ca. 500 bp fragments. Transposon-chromosomal junctions were amplified by PCR and amplicons subjected to high throughput Illumina sequencing to locate the transposon insertion sites. Bioinformatic analysis of Illumina reads from all sets indicated that the transposon inserted in 31,483 (60.10%) out of 52,384 TA sites, corresponding to 93.1% of the genes containing at least one transposon insertion. Applying a 4-state Hidden Markov Model (DeJesus and Loerger BMC Bioinformatics 2013, 14:303), we identified 328 essential; 1,103 growth-defect; 2,603 nonessential and 258 as growth-advantage genes. These assignments covered 4,292 out of 4,350 total genes in MAP genome. As expected, essential genes identified match essential functions in DNA replication (dnaE), transcription (rpoB) translation (rpmC, rpsL) and essential metabolic pathways as those involved in lipid biosynthesis (fabD2, fadE9). Out of 31 Himar1 and 8 Tn5367 isolated individual mutants previously identified as carrying transposon insertions inactivating coding regions, none (except for MAP1432::Tn5367) were identified as essential in our analysis. These results confirm the robustness of our assignments. This study defines for the first time gene essentiality in MAP on a whole genome basis. In addition, we identified growth-defect and growth-advantage genes whose inactivation limit or promote growth under the conditions tested.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Rathnaiah, G., J.P. Bannantine, D.K. Zinniel, J.R. Stabel, Y.T. Grohn and R.G. Barletta. Comparative analysis of IS1096- and Himar1- derived transposon insertion sites in Mycobacterium avium subsp. paratuberculosis. Conference of Research Workers in Animal Diseases Meeting Poster Presentation. Chicago, IL. 12/07/2014.

Progress 09/01/13 to 08/31/14

Outputs
Target Audience: The primary target audience was scientists involved in the study of mycobacterial diseases of animals. In this reporting period, we will reached these scientists at the 2014 Annual Meeting of the NE1201 Mycobacterial Diseases of Animals multistate initiative to be held in Kansas City, Missouri (October 18-19, 2014). We expect to have the opportunity to discuss progess on the project with the Co-PD's of this grant. The PD will participate and contribute a required abstract to the Annual Meeting of National Institute of Food and Agriculture funded investigators (November 19-21, 2014). An abstract was submitted and selected for a poster presentation at the meeting for the Conference of Research Workers in Animal Diseases to be held in Chicago, Illinois (December 7-9, 2014). This meeting will give us an opportunity to reach a wider audience working in Animal Health. Changes/Problems: We have identified vector sequences from mycobacteriophage TM4 in the analysis of the first mutant pool. We are currently trying to determine the origin of these sequences and whether they will create any bias in the mutant pool representation. To this end, we are analyzing all independent mutant pools for the presence of TM4 sequences using specific primers. In addition, we plan to complete full sequencing and analysis of the pools prior to proceeding with animal experiments. What opportunities for training and professional development has the project provided? In this reporting period, a PhD Graduate student (Govardhan Rathnaiah) participated in the University of Nebraska Bioinformatics Workshop on July 28-30, 2014. This activity will be useful to analyze our incoming DNA sequencing data. He and the PD (Barletta) have also travelled to the University of Nebraska Medical Center (James Eudy) to discuss library preparation for the illumina DNA sequencing protocol based on our goal to identify essential genes in MAP by global phenotypic profiling. Fall 2014 meetings to be attended include the following: a) NE1201 Mycobacterial Diseases of Animals multistate initiative held in Kansas City, Missouri (October 18-19, 2014), b) Conference of Research Workers in Animal Diseases to be held in Chicago, Illinois (December 7-9, 2014) and c) Annual Meeting of National Institute of Food and Agriculture funded investigators (November 19-21, 2014). How have the results been disseminated to communities of interest? We submitted an abstract that is published in the Proceedings of the Conference of Research Workers in Animal Diseases, Chicago, Illinois, December 7-9, 2014. During this meeting, Govardhan Rathnaiah will present a poster entitled “Comparative analysis of IS1096- and Himar1- derived transposon insertion sites in Mycobacterium avium subsp. paratuberculosis” on behalf of the authors: G. Rathnaiah, J.P. Bannantine, D.K. Zinniel, J.R. Stabel, Y.T. Grohn and R.G. Barletta. What do you plan to do during the next reporting period to accomplish the goals? We plan to perform large scale illumina DNA sequencing and the corresponding analysis of transposon insertion sites in all of our MAP mutant pools so far generated. The mutant pool is expected to include multiple independent mutants per gene and each mutant is expected to be stable as the transposed element can no longer excise and move into another gene. Once the representation and stability of the combined mutant pool is confirmed, this collection will be sent to the National Animal Disease Center for animal infection experiments in Holstein dairy calves. To monitor disease progression in animals infected with the mutant pool, fecal and blood samples will be collected on 2 consecutive days prior to initiation of the study, followed by monthly samplings.

Impacts
What was accomplished under these goals? Impact: Annual losses due to JD are estimated in the range of $250 million to $1.5 billion for the United States cattle industry. This project uses a genetic technology to identify molecular targets for the development of state of the art safe and effective vaccines against JD. We expect these technologies would be applied to develop control strategies for other infectious diseases of large production animals and plant microbial systems. Results: The generation of comprehensive random mutant banks by transposon mutagenesis is a fundamental technology to determine the role of MAP genes in cell physiology and pathogenesis. Bioinformatics was applied to perform a comparative analysis of insertion sites for the mycobacterial transposons Tn5367 and Tn5370, both derived from the mycobacterial insertion sequence IS1096 and the mariner transposons carrying the Himar1 transposase. We determined that mariner transposons provide a random representation of insertions in almost any MAP gene. Further analysis of transposon recognition sites indicated that 16.3% (710) and only 0.85% (37) of all 4,350 MAP genes do not possess an IS1096 and Himar1 insertion site, respectively. Thus, a significant number of MAP genes remain underrepresented in Tn5367 or Tn5370 insertion libraries. Experimental confirmation was provided by sequencing 100 Himar1 mutants. Seven Himar1 insertion mutants were identified that lacked IS1096 insertion sites, while 80 mutants had insertions in different MAP genes. In addition, we have generated 5 large transposon mutant pools comprising ca. one million transductants for maximum saturation of the genome with transposon insertions. We are assessing whether these pools constitute a representative mutant library by DNA sequencing. We expect to find ca. 15-20% of genes that are required for survival because actual Himar1 insertions are not found in those genes within the mutant pool. These results will enable a comprehensive list ing of essential genes in MAP which may be targets for therapeutic strategies.

Publications