Genome Wide Analysis of M. Paratuberculosis pathogenesis

<p>NON-TECHNICAL SUMMARY:<br/> 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.
<p>APPROACH:<br/> 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.