Performing Department
Veterinary Population Medicine
Non Technical Summary
Tuberculosis (TB) is a global health emergency. Bovine TB is one of the most damaging diseases in agriculture. M. bovis is a zoonosis and a major concern in the pastoral settings of the developing world where animal-human interface is intimate, and HIV prevalence is high. In the developed world, a wildlife reservoir of Mycobacterium bovis has meant an alarming increase in the incidence of bovine tuberculosis. We propose to study the responses of the bacterium inside the host in granulomatous lesions. We hypothesize that understanding the in-vivo mechanisms of attenuation of MTBC strains in a natural host (cattle) will provide definition of determinants of attenuation not detected via comparative genomics. Our research takes a "one health" approach, providing dual benefits to humans and cattle and our studies will yield a complete host-pathogen interactome that will be the cornerstone for understanding of mechanisms of attenuation of MTBC strains. The functional genomics information generated from our studies will extensively aid in diagnostics and vaccine development, and molecular responses of the pathogen within lesions will aid in discovery of targets for the development of new generation antitubercular drugs.
Animal Health Component
100%
Research Effort Categories
Basic
75%
Applied
20%
Developmental
5%
Goals / Objectives
Tuberculosis (TB) is a global health emergency. Bovine TB is one of the most damaging diseases in agriculture. M. bovis is a zoonosis and a major concern in the pastoral settings of the developing world where animal-human interface is intimate, and HIV prevalence is high. In the developed world, a wildlife reservoir of Mycobacterium bovis has meant an alarming increase in the incidence of bovine tuberculosis.The Mycobacterium tuberculosis complex (MTBC) includes closely related species or biotypes - M. tuberculosis, M. bovis, M. africanum, M. pinnipedi, M. microti, M. caprae, and M. canetti. Despite their close genetic relatedness, MTBC present different phenotypes including host range, ability to transmit between associated hosts, and pathology. A critical limitation to tuberculosis control and eradication is the lack of fundamental knowledge of the genotype-disease phenotype associations, especially as related to virulence/attenuation. Our goal is to address this key missing information by characterizing the host-pathogen "interactomes" in a natural host model to elucidate mechanisms of attenuation among MTBC strains.We have established that the Ravenel strain of M. bovis is attenuated in cattle. Ravenel induces robust cellular immune responses in calves without disease, akin to M. tuberculosis H37Rv infection of cattle. We have also shown that the genome of the M. bovis Ravenel does not carry any large sequence polymorphisms (including RD1, an LSP that is shown to be necessary and sufficient for attenuation in BCG) relative to virulent AF2122/97 suggesting that its attenuation trait to be different than BCG. Thus comparative genomics or mouse models alone do not provide complete information on mechanisms of MTBC attenuation/virulence in natural hosts. We hypothesize that understanding the in-vivo mechanisms of attenuation of MTBC strains in a natural host (cattle) will provide definition of determinants of attenuation not detected via comparative genomics. We propose to test this hypothesis under two specific aims - Aim 1. Characterize the in vitro functional genomes of Ravenel, BCG, and M. bovis 95-1315; Aim 2. Compare ex-vivo interactomes with in-vivo transcriptome of M. bovis 95-1315 in an experimental calf model that has already been performed at NADC.
Project Methods
Bacteria and growth media - MTBC strains will be cultured in Middlebrook 7H9 with and without glycerol and Tyloxapol. Both detergents are generally used in mycobacterial growth media and will serve as in vitro controls for the virulence phenotype variations identified among MTBC strains. Transcriptome and proteome analysis of these treatments will be performed using protocols established in our laboratories (29-32).Macrophage as a model for "omics" of pathogenic mycobacteria - We have been working with pathogenic mycobacteria for numerous years and have developed models to study their intra-macrophage behavior in bovine monocyte derived macrophage model system. We have established models of imaging, invasion, and survival of Mycobacterium avium subspecies paratuberculosis (MAP) in bovine macrophages. We have performed these types of analyses on M. bovis in the BSL-3 labs at NADC, Ames (Joshi et al., 2012; PhD Thesis). We propose to study early molecular events in the cellular life cycle of M. bovis to elucidate mechanisms of attenuation. We have also shown transcriptional activity of pathogenic mycobacteria in primary monocyte-derived macrophages as well as in infected tissues either using selective capture of transcribed sequences (SCOTS) or direct MAP mRNA capture and enrichment from infected tissues and macrophages to perform microarray based transcriptional analyses. In a third study, we established MAP-host interactomes unsing RNA-seq of MAP infecting co-cultures of epithelial cells and macrophages. We will apply SCOTS method to study the transcriptomes of 95-1315 in time-course infection of macrophages.RNA-Seq -Total RNA will be extracted and subjected to RNA-Seq using the Illumina Hi-Seq technology. For M. bovis transcriptomes, Selective Capture of Transcribed Sequences (SCOTs) method established in our laboratory, will be applied and cDNA libraries will be sequenced. For host transcriptomes, strand-specific cDNA libraries for Illumina sequencing will be generated from total RNA. In order to yield cDNAs covering transcripts over their entire length, the RNA is first fragmented by incubation at 95°C for 5 min. RNA fragments are linked to random hexamers and amplified - use of a no poly(A) system is intended to aid in enrichment of pathogen and host mRNAs.The cDNA libraries will be sequenced using 300 paired end cycles on a HiSeq2000 (Illumina) at the University of Minnesota Genomics Center.Sequence mapping, coverage, and statistics - We will use the expertise and supercomputer resources available at the Minnesota Supercomputing Institute for all data analysis. In brief, for mapping of reads, 5'-linker clipped reads will be aligned to the M. bovis genomes (AF2122/97, Ravenel and the two BCG strains) genome using the segemehl program which is based on an error-tolerant suffix array method. A filtering step will be applied to remove all sequences with an A-content of >70% (to eliminate errors generated from poly(A) tails; the poly(A) tail of these sequences will be trimmed separately (macrophage model studies where host RNA sequences are also expected). All cDNA sequences (except sequences <15-bp) will be mapped on the four genomes and transcript profiles will be developed for each strain analyzed. Overall genome fold coverage for all libraries will be calculated separately for the plus strand, the minus strand, and the sum of both strands.Neonatal calf model, Infection and outcomes - Experimental M. bovis challenge. These experiments have already been conducted at NADC and granulomas from forty animals are available for transcriptional profiling for this study. Collaboration letter from Dr. Waters has been obtained for this study. All experimental infections were conducted atthe National Animal Disease Center (Ames, IA). All cattle used in this study were housed according to institutional guidelines and approved animal care and use protocols at the National Animal Disease Center, Ames, Iowa (NADC). For M. bovis challenge studies, cattle were housed in a biosafety level 3 (BL-3) facility. Samples collected from these studies were stored in Trizol for RNA extraction and shipped to Dr. Sreevatsan lab for further analysis.Biomarker identification to index responses that specify attenuation - Serum biomarker discovery studies performed in our laboratories, using iTRAQ analysis have also addressed variations in the circulating peptidome based on the pathogenesis of two biologically similar mycobacterial diseases of cattle (28, 33). Using iTRAQ, we have identified Vitamin D binding protein precursor (DBP), fetuin, and 16 M. bovis peptides in infected animals (28, 33). Thus discovery of these biomarkers has significant impact on the elucidation of pathogenesis of two mycobacterial diseases at the cellular and the molecular level and can be applied in the development of mycobacterium-specific diagnostic tools for the monitoring progression of disease, response to therapy, and/or vaccine based interventions.Mycobacterial responses within lesions - discovery of molecular determinants of attenuation in-vivo: The defining feature of pathogenic mycobacterial infections is a granuloma - yet very little is known about mycobacterial life within this dynamic structure. We propose that a directed approach to uncover common and unique pathways utilized by mycobacteria in lymph nodes or lung lesions using the natural host model of infection. Elucidation of the active transcriptome, proteome, and lipidomes within granulomas in local infection sites is expected to not only augment our knowledge of MTBC pathogenesis. Transcriptomics of the MTBC strain 95-1315 inside host lesions will be performed using standard SCOTS and RNA-Seq approach as described under Aim 1. Challenge procedures were performed at NADC as described above using intratonsilar inoculation (n = 10; lesions from all animals in the inoculated groups will be analyzed). At necropsy, sections from affected portions of the lymph node and/or lungs were harvested, wrapped in aluminum foil and either snap-frozen in liquid nitrogen or fixed in formalin for RNA extraction and histopathological examination, respectively. All samples were stored at -80.0oC until RNA extraction. Lesions are triturated and cultured for of the presence MTBC using standard mycobacterial culture techniques. Sections were taken for microscopy using hematoxylin and eosin staining and acid fast staining. We will use total RNA for host transcriptional responses but the enriched microbial RNA to generate MTBC response patterns. We have extensive experience with lesions and transcriptomic analyses and anticipate no major problems in RNA processing and evaluation. In order to address the lack of progression of nonvirulent strains and their behavior, we have proposed a prospective intratonsillar infection model that is expected to provide at least 1 positive lesion in every infection group. These studies will establish for the first time an interactive network of host-pathogen responses in a natural host.