Performing Department
College of Veterinary Medicine
Non Technical Summary
Johne's disease (JD) is a chronic enteric infection of dairy cattle worldwide and imposes significant economic losses to the industry. To reduce the prevalence of JD we need to better understand the biology of Mycobacterium avium subsp. paratuberculosis (MAP), the causative agent of JD. MAP unlike other mycobacteria appears to have a special iron requirement to grow in laboratory media. This iron requirement makes MAP fastidious, often requiring eight to sixteen weeks to produce colonies in culture - a major hurdle in the diagnosis and therefore in implementation of optimal JD control measures. A major breakthrough in one of our studies was the identification of an in-vivo up-regulated Ferric uptake regulator (Fur)-like element found on the MAP-specific phage-like region as a likely candidate (MAP3773c) suggesting a novel iron regulation mechanismin MAPOur initial in silico analysis on MAP3773c as a putative Fur protein identified 23 pathways directly regulated by Fur protein, pathways that are critical for metabolism and virulence factors. Further analysis using multiple sequence alignments of well studied Fur proteins (E.coli and S. thyphimurium) and MAP's 3773c showed 41% overall amino acid similarity with highly conserved and nearly identical "Fur" binding site. Based on sequence similarity and conserved "Fur" domains, MAP3773c was confirmed as a Fur-like protein that acts as a ferric uptake regulator. We hypothesize that MAP3773c (fur) that was acquired by horizontal gene transfer encodes a functional protein that orchestrates major metabolic pathways required for superior extracellular iron sensing of iron.
Animal Health Component
30%
Research Effort Categories
Basic
100%
Applied
0%
Developmental
0%
Goals / Objectives
It has been over 8 years since Mycobacterium avium subsp. paratuberculosis (MAP) genome was sequenced and annotated - but there still exists a major knowledge gap in our understanding of iron regulation in this agriculturally important pathogen. Iron is indispensible for survival of most living organisms including mycobacteria. MAP unlike other mycobacteria appears to have special iron requirements. MAP requires addition of mycobactin (iron chelating molecule) when grown in vitro while it expresses mycobactin genes inside macrophages. This suggests that, MAP differentially regulates mycobactin genes only in an intracellular milieu and that the current in vitro culture method is perhaps inadequate to induce sufficient mycobactin production. We have recently characterized a transcriptional factor termed Iron Dependent Regulator (IdeR) that functions as a principal regulator maintaining iron homeostasis MAP and M. tuberculosis. Our preliminary studies show that MAP employs a sophisticated repertoire of inter-connected proteins in response to environmental stress. We have identified an iron sparing responses as well as the upregulation of IrtAB and fur elements in-vivo that suggests alternate regulatory pathways are likely employed by MAP, unlike other pathogenic mycobacteria. Thus a deeper analysis is warranted to understand how environmental iron regulation is orchestrated in MAP. We hypothesize that MAP3773c (fur) that was acquired by horizontal gene transfer encodes a functional protein that orchestrates major metabolic pathways required for superior extracellular iron sensing of iron. We will analyze the Fur regulon under 1 aim- 1. Confirm the MAP Fur regulon using chromatin immunoprecipitation (ChIP).
Project Methods
Research Design and MethodsBacterial culture and growth conditions - Bacterial cultures will be grown to logarithmic growth phase in routine laboratory culture medium (MB7H9 medium supplemented with glycerol, mycobactin J and OADC enrichment medium). When cell density reaches >1.0 OD600, the cultures will be pelleted and washed three times in PBS. Washed cells will be re-suspended in freshly prepared minimal essential medium (MEM) containing pre-determined iron concentrations. MEM (2%glycerol, 0.5% w/v Asparagine, 0.05% Tween -80, 0.5% KH2PO4, 50mg/L MgSO4, 5mg/L MnCl2, 5mg/L ZnCl2) is prepared using nanopure water. Identification of the Fur regulon using ChIP sequencing - To obtain independent confirmation of the transcriptional dataset, we will investigate Fur-binding to the chromosome of MAP strains (K-10 and S396) during exponential growth under varying iron conditions by ChIP-Seq, chromatin immunoprecipitation followed by ultra-high throughput DNA sequencing.ChIP-Seq - Chromatin immunoprecipitation will be performed essentially as previously described but with some variations. Briefly, MAP cultures (50 ml) grown to OD600 0.4-0.6 (~106 cfu) will be treated with formaldehyde (final concentration 1%) for 10 min at 37C. Crosslinking will be quenched by addition of glycine to a final concentration of 125 mM. Harvested cells are washed twice with Tris-buffered saline (20 mM Tris-HCl pH 7.5, 150 mM NaCl), re-suspended in 600 ml Immunoprecipitation (IP) buffer (50 mM Hepes-KOH pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, miniprotease inhibitor cocktail (Roche)) and sonicated to shear DNA to an average size of 100-500 bp. Insoluble cellular debris will be removed by centrifugation and the supernatant used as input sample in IP experiments. 300 ?l of input will be incubated with either no antiHis antibody (mock-IP) or 20 ?l of serum containing mouseanti-His polyclonal antibodies. Washing and crosslink reversal of protein-DNA complexes, as well as purification of the resulting DNA will be carried out as previously described [34]. Prior to sequencing, DNA fragment sizes will be checked and enrichment verified by gene-specific quantitative PCR (ChIPqPCR).Fur-ChIP-Seq - Library construction and sequencing DNA fragments (150 to 250 bp) will be selected for library construction and sequencing libraries prepared using the ChIPSeq Sample Preparation Kit (Illumina; San Diego, California, USA; Cat. No. IP-102-1001) according to the protocol supplied with the reagents. Prior and post library construction, chromatin immunoprecipitation products will be quantified using the Qubit fluorometer (Invitrogen; Carlsbad, California, USA). One lane of each library will be sequenced on the Illumina HiSeq 2000. Data will be processed using the Illumina Pipeline Software and using the programs available on the Galaxy suite.ChIP-Seq data analysis - ChIP-Seq experiments, with three independent mid-log phase cultures with and without iron (at concentrations identified above), will be mapped to MAP-K10 and MAP-S396 genomes using Bowtie allowing up to 3 mismatches and up to 10 hits per read. As a control, input DNA will also be sequenced and mapped to the MAP genome to identify sequencing artifacts and calculate enrichment values. In order to estimate the binding location in the enriched regions, we will use a deconvolution algorithm that models the expected tag distribution on both strands. Based on the deconvoluted profile, a score will be calculated for each peak that will be proportional to the read density in the peak. Only peaks with more than 600 reads per position will be selected. Reads mapped to the forward and reverse strands will be shifted (by 80 bp) and merged together to generate single peak profiles, which are visualized on the UCSC Genome Browser database. Annotation of the peaks will be performed using the ChipPeakAnno package from Bioconductor. The read count at each binding region (400 bp width) will be determined as the total number of reads mapping to the region divided by the length of the region normalized to the total number of mapped reads across the whole genome. The enrichment at each locus will be calculated as the ratio of the average read count from the ChIP sample (from three datasets of each strain) to the read count from the input DNA sample. Peaks with an enrichment ratio lower than 1.5 will be filtered out.Motif detection. Sequences that fulfill the peak selection criteria will be extracted and used for motif analysis using MEME (16) with motif occurrence set as ''zero or one per sequence'', minimum width as 5 and other parameters as default.Electrophoretic mobility shift assays (EMSA) and DNase I footprinting - Fur protein will be produced and purified in an M. smegmatis strain (4517) optimized for mycobacterial protein production. This Histag protein will be used in EMSA, DNase I footprinting and quantitative Western Blot experiments, as well as to immunize rats. Fur-DNA gel retardation assays will be performed as recently described by us and others using 5'-biotin-labeled forward primers and unlabeled reverse primers in PCR reactions designed to yield DNA probes of 99 to 120 bp encompassing selected ChIP-Seq peak sequences for analysis. DNase I footprinting assay will be carried out using 6-FAM-labeled probes as described in. The nucleotide sequences of protected regions will be determined by DNA sequencing.Technical and Collaborative approach - The proposed studies will be performed in collaboration with Dr. Raul Barletta (molecular microbiologist at University of Nebraska) and Dr. John Bannantine (Scientist at National Animal Disease Center). Dr. Sreevatsan is an established JD investigator and has an inter-institutional collaborative research program on mycobacterial diseases. The proposed work will further strengthen these collaborations.