ALTERNATE IRON REGULATORY PATHWAYS IN MYCOBACTERIUM AVIUM SUBSPECIES PARATUBERCULOSIS

<b>Project Methods</b><br> Bacterial cultures and growth conditions:We will utilize two strains ofMAP: MAP K10 – representing a common cattleMAPgenotype and MAP?3773c- a Fur transposon mutant generated from MAP K10 by our collaborator Dr. Barletta.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.In this study we will generate an in-frame deletion of MAP3773 in collaboration with Dr. Barletta using his established methods.Identification of iron concentration inducedMAPgene expression:Iron medium: Before addition of metal ions (Mg, Mn, Zn) the medium will be pH adjusted to 6.8 and treated with chelex (Biorad labs) to chelate all metal ions. Chelex will be removed by filtration and metal ions will be added aseptically at desired concentrations. This medium has an iron concentration of 0-1 µM. All the glassware used to prepare medium will be rinsed thoroughly with 50% nitric acid followed by additional rinses in nanopure water. Plastic ware will be used to grow bacterial cultures to avoid leaching of iron from glassware. Finally iron (Ferric Chloride) will be added to MEM at desired concentrations.Sequence mapping, coverage, and statistics-We will use the expertise and supercomputer resources available at the MSU HPCC facilities for all data analysis. In brief, for mapping of reads, 5'-linker clipped reads will be aligned to theMAPgenomes(K-10, S396 [sheep strain], MAP4 [human strain]) using thesegemehlprogram, 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 and in-vivo 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. All hits on the genome will be mapped to specific coordinates of nucleotide sequences to identify percent coverage per region to identify fold coverage of each locus or coding region. Overall genome fold coverage for all libraries will be calculated separately for the plus strand, the minus strand, and the sum of both strands.Intracellularcharacterization of iron dependent networks ofMAP:To verify in-vitro iron concentration induced gene expression is also induced inside macrophages and/or JD positive tissues in-vivoMAPgene profiling will be performed.Macrophages from fresh monocyte derived macrophages (MDMs) of cows (n= 20)free of JD, will be inoculated with logarithmically grownMAPisolates at a multiplicity of infection of 20:1.TotalRNA from the infected macrophages at time pointsviz.3, 6, 12 and 24 hours grown in 25 cm2culture flask will be extracted by TRIzol reagent.The purity and yield of total RNA samples will be examinedby capillary electrophoresis in an Agilent 2100 bioanalyzer (Agilent technologies, Foster City, CA)and will be stored at -80°C until analyzed.All samples will be treated with RNase-free DNase I (Ambion, Inc., Austin, TX) to eliminate genomic DNA contamination and confirmed by standard PCR for at least 3 housekeeping targets.Computational protein network predictions:Iron concentration inducedMAPgene expression revealed by RNA-Seq will be used to draw protein networks. Computational scientists located in BMGC at UMN have expertise in such analyses and their services will be utilized. An approach similar to that employed in predicting protein networks inM. tuberculosiswill be used here. We will use Rosetta Stone, Phylogenetic Profile, Conserved gene Neighbor and Operon computational methods to construct the networks.Identification of iron concentration inducedMAPprotein expression:Bacterial cultures grown under varying iron concentrations will be used for proteome profiling (pellets and culture supernatants) using iTRAQ based approach. We have successfully used iTRAQ to identify serum biomarkers inM. bovisandMAPinfected cattleand also to generate preliminary data for this proposal.Bacterial pellets will be re-suspended in minimal quantity (250 μL) of iTRAQ dissolution buffer (0.5 M TEAB pH 8.5) and 0.1% SDS. The solution will be transferred to a 2 ml screw cap tube containing 0.1mm zirconium beads (Biospec) and disrupted in minibead beater (Biospec) for 4X1 minute pulses with samples kept on ice every minute. The lysate will then be centrifuged at 12,000xg for 10 minutes at 4°C. Supernatant will be transferred to a fresh tube without disturbing the pellet and used in iTRAQ labeling for detection of cell associated proteome. Culture supernatants will be concentrated using 3kDa molecular cut off spin columns (Millipore) and used in iTRAQ labeling for detection of secretome.Several fractions will be collected at frequent intervals and fractions that show mAU280 > 2 will be analyzed by LC-MS/MS.Fractions will be reconstituted in reversed-phase load buffer (10mM phosphate buffer) and analyzed by LC-ESI/MS/MS on a QSTAR Pulsar I-Quadrupole TOF MS using Analyst QS software. Protein pilot Software™ 2.0.1 (Applied Biosystems Inc., Foster city, CA) will be used to identify labeled peptides and determine relative abundance at a > 95% confidence interval (CI). A minimum identification of at least two unique peptides per protein will be used as a cutoff for protein analysis. Relative abundance of identified proteins will be log2transformed for further analysis.Protein networks or interactions will be drawn from the differentially identified proteins (using tools such aswww.string.embl.de) and are compared to predicted protein networks generated under specific aim 1.Intracellular characterization of Fur dependent protein expression ofMAP:Macrophage culturing and cell infections will be performed as detailed under specific aim 1.MAPproteins expressed inside macrophages will be extracted and identified by iTRAQ (to comparein-vitroiron stress versus intra macrophage iron stress). Previous studies reported successful purification of phagosomes and identifiedSalmonellaorMycobacterialproteins expressed within macrophages. Detailed methods to purify bacteria containing phagosomes using a differential centrifugation following macrophage cell lysis are also published. To identifyMAPproteins expressed inside bovine macrophages we will utilize the approach described to identifyM. bovisBCG proteins expressed inside macrophages. Macrophages stimulated withMAPwill be washed vigorously to remove extracellular bacteria. Macrophages containing internalizedMAPwill be lysed with 0.25% SDS at 37°C for 5 minutes. Cell suspension will be passed through 23-gauge needles several times to reduce viscosity. Cell suspensions from 2-3 replicate flasks will be pooled if required to attain sufficient quantities ofMAPproteins for iTRAQ analysis.MAPwithin the cell suspensions will be pelleted at 5000Xg for 20 minutes. Pellets will be washed with PBS to remove any contaminating mycobacterial proteins and finally lysed in a mini bead beater after re-suspending in iTRAQ dissolution buffer carrying 0.1mm zirconium beads (Biospec). Protein concentration will be determined and processed for iTRAQ labeling and identification as detailed above.