Progress 11/01/21 to 04/30/22

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One graduate student and 2 technicians were trained in molecular microbiological methods. The graduate student has completed her PhD and a second graduate student is working toward an MS on this specific study. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The iron regulatory mechanism under the control of Fur has been completed using ChIP-Seq. We have creaed a deletion mutant and developed a complementation strain that is still in culture and growing. The parent strain (K10), sheep strain, and mutant have been exposed to iron restriction and total RNA has been harvested for sequencing. A toal of six replications at 4 different time points post exposure to iron limitation have been extracted. RNA-Seq is underway and data analsysi will be finalized by the middle og June 2022 and prepared for consideration of publication.

Publications

• Type: Theses/Dissertations Status: Accepted Year Published: 2020 Citation: CHARACTERIZATION OF MAP3773C, FERRIC UPTAKE REGULATOR PROTEIN, IN IRON METABOLISM OF MYCOBACTERIUM AVIUM SUBSP. PARATUBERCULOSIS

Progress 11/01/17 to 04/30/22

Outputs
Target Audience:Veterinarians, Veterinary scientists, microbiologists, veterinary diagnostic laboratories Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?1. Fernanda Shoyama completed her PhD on this grant and topic 2. Sajani Thapa is working on completing her MS How have the results been disseminated to communities of interest?Presentations have been made at CRWAD meetings, ASM General meeting, and 2 papers and one thesis have been published What do you plan to do during the next reporting period to accomplish the goals?Finish up the remainder of the transcriptional analysis

Impacts
What was accomplished under these goals? 1. We have created a M3776c in-frame deletion mutant 2. A complementation with an RFP expressing plasmid has also been accomplished 3. A new transcriptional analysis (RNA-Seq) has been completed 4. A final manuscript addressing the regulon of MAP3776cis under preparation. 5. A second graduate student was opartially supported on the funds from this grant to perform lab experiments - she is writing her MS thesis based on RNA-Seq data

Publications

Progress 11/01/20 to 10/31/21

Outputs
Target Audience:Microbiologists at American Society for Microbiology general meeting (Microbe); CRWAD (award winning presentation); Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Fernanda Shoyama obtained her PhD under this grant funding How have the results been disseminated to communities of interest?Results have been published and or or presented at ASM Microbe or CRWAD meetings. What do you plan to do during the next reporting period to accomplish the goals?We plan to replicate transcriptional analyses and submit the last manuscript during the next 2-3 months.

Impacts
What was accomplished under these goals? Functional characterization of iron regulation byMycobacterium aviumsubsp.paratuberculosis(MAP)has revealed thatMAPemploys a complex protein pathway in response to iron starvation in-vitro or ex-vivo. Previous work demonstrated that the iron dependent repressor protein (IdeR) not only controls mycobactin synthesis genes inside macrophages but also regulates transcription of genes involved in iron acquisition (mbtB), iron storage (bfrA) and oxidative stress. While expression of theideRregulon varies with the concentration of intrabacterial iron, there seemed to be an external metal-sensing regulator missing for iron scavenging. It is known thatMAP's genome contains 6 unique genomic islands, absent in other mycobacteria. Transcriptional analysis by our laboratory suggests that two of theselargesequencepolymorphisms, LSP14 and LSP15, form a major genomic island that may play a role in iron homeostasis. However, it is not clear whether the genes encoded in those MAP-specific LSPs contribute to the pathogenicity and phenotype ofMAP.Later it was confirmed that LSP14 and LSP15 are involved in metal homeostasis. Furthermore, using a transposon mutant with a disruption in the LSP15 geneMAP3776c. Corroborating the importance of LSP15 genes during infection, tit was shown that a disruption inMAP3776creduces bacterial fitness by 10-fold in the mouse model. Additionally, by bioinformatic analysis, LSP15 is predicted to encode an ATP-binding cassette (ABC) transporter, a metal uptake regulator and a gene that may be involved in cobalamin synthesis. Previous studies in our lab showed genes LSP15 were differentially regulated in infected bovine tissues, and interestingly,MAP3773c, a predictedferricuptakeregulator (fur) protein, was downregulated in the tissues and upregulated in experimentally infected macrophages. The same result was reported in the ABC transporter operon (LSP14) that contains twofurbinding boxes. Thus, it is already known that both LSP14 and LSP15 are involved in iron homeostasis, however there is no information on whether (or not)Furcontributes to this regulation. Originally described inE. coli, Fur protein is an iron-responsive repressor that controls the expression of genes for siderophore biosynthesis and iron transportation. Additionally, it has been reported that intracellular iron concentrations in many bacteria are under the control ofFur. During infection, in a metal-depleted environment, Fur allows for efficient acquisition of iron leading to enhanced fitness of the pathogen.Supporting this critical role ofFurin virulence, a deletion offurin numerous bacterial pathogensmost often resulted in partial or complete attenuation within animal models of infection. For activation of responsive genes, there is coordination of one Fe2+per monomer that enables the dimericFurprotein to bind a specific 19bp DNA sequence (the "Fur box") that inhibits binding of RNA polymerase to prevent transcription. Beside Fe(II), it has been reported inB. subtilisthat binding of Mn(II) and Zn(II) can also activateFurand are effective at triggering changes needed for DNA binding. Both metals are preferred and widely used in biochemical studies as the activating cofactor, since Fe(II) requires strict anaerobic conditions. It is still unknown howMAP-Furis activated. The overall objectiveof the studies presented in this thesis was to elucidate the mechanism of iron homeostasis inMycobacterium aviumsubsp.paratuberculosis. Characterization of a large sequence polymorphism on MAP genome (LSP15) andMAP3773cphenotype using a transposon mutantMAP3776cwas performed.In-vitro assays showed potential roles ofMAP3776cinMAPcell invasion and further supported the reported involvement of LSP15 in intracellular survival. Characterization of the Fur regulon in MAP using ChIP-seq was performed to identify the regulon under iron-replete and -depleted conditionsin vitro. Sequences were analyzed using CLC Genomics Workbench 12. Confirmation of physical binding ofMAP3773cto the Fur box was carried out by chemiluminescent EMSA, using a labeled Fur box and a recombinantMAPFur-like protein. Acomparative analysis of transcriptomes from MAP K-10 (wild-type), fur deletion mutant and complemented strains under metal-replete and -depleted conditions were evaluated and no major variations were noted.

Publications

• Type: Theses/Dissertations Status: Published Year Published: 2020 Citation: Fernanda Shoyama. CHARACTERIZATION OF MAP3773C, FERRIC UPTAKE REGULATOR PROTEIN, IN IRON METABOLISM OF MYCOBACTERIUM AVIUM SUBSP. PARATUBERCULOSIS. A DISSERTATION Submitted to - Michigan State University in partial fulfillment of the requirements for the degree of Comparative Medicine and Integrative Biology  Doctor of Philosophy

Progress 11/01/19 to 10/31/20

Outputs
Target Audience:American College of Veterinary microbiologists - Presented to this group at the CRWAD meeting in Dec 2019 and 2020. Changes/Problems:Since Covid19 pandemic took over in MArc h of 2020, research labs (including ours) was closed for a period of 6 months and has performed activities on a limited basis after reopening in August. This has set our activity on transcriptional analysis and ex-vivo experiments of the 3773c mutant have been delayed. We will complete all the proposedactivities by the end of October 2021. What opportunities for training and professional development has the project provided?Fernanda Shoyama successfully defended her PhD. She was funded by this grant. How have the results been disseminated to communities of interest?Presentations at CRWAD. What do you plan to do during the next reporting period to accomplish the goals?In the next phase, we will perform global transcriptional analysis of the 3773c mutant, its parent strain as well as a complemented strain within epithelial cells and in-Vitro under limiting iron conditions. This will establish if the regulator is active and confirm the ChiP seqfindings.

Impacts
What was accomplished under these goals? During this current period, we successfully elucidated the regulon of MAP3773c using Chromatin immunoprecipitation (ChIP seq)and corroborated these findings using electrophoretic mobility shift assays. An "in-frame" deletion of MAP3773c was also successfully constructed and characterized. We are currently performing cell invasion and transcriptional analyses with these mutant and parent strains.

Publications

• Type: Journal Articles Status: Published Year Published: 2020 Citation: Shoyama FM, Janetanakit T, Bannantine JP, Barletta RG, Sreevatsan S. Elucidating the Regulon of a Fur-like Protein in Mycobacterium avium subsp. paratuberculosis (MAP). Front Microbiol. 2020 Apr 23;11:598. doi: 10.3389/fmicb.2020.00598. eCollection 2020. PMID: 32390963

Progress 11/01/18 to 10/31/19

Outputs
Target Audience:Researchers in Animal Disease; Mycobacteriologists Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?PhD student working on this project will be able to attend the 100thAnimal Conference of Research Workers in Animal Disease (CRWAD) besides all research funded in this grant is part of her PhD research training. How have the results been disseminated to communities of interest?Current findings were will be presented at the 100thAnimal Conference of Research Workers in Animal Disease (CRWAD) and two manuscripts are under preparation for submission. What do you plan to do during the next reporting period to accomplish the goals?Work is underway on RNA-seq followed by Quantitative RT-PCR to confirm Fur regulon identified by ChiP-seq, using the Fur deletion mutant,MAPK-10 and complemented strain. Additionally, CRISPRi is being applied to develop a Fur box 1 knock down inMAP.Results generated from this project are expected to lead to a better understanding of iron regulation inMAPproviding new insights for an improved culture thus a faster diagnostic of Johne's Disease.?

Impacts
What was accomplished under these goals? Primary goal of this research is to provide a better understanding of iron homeostasis inMycobacterium aviumsubsp.paratuberculosis(MAP). For this, the role of a second global iron regulator,MAP3773c, FUR (Ferric Uptake Regulator) -like protein is being investigated. As reported before, FUR protein was recombinantly expressed to establish that it physically bound to FUR box (a 19-bp consensus region on the promotor sites) carried out by chemiluminescent EMSA. Next, we used - ChIP (Chromatin Immunoprecipitation)-seq experiment to confirm protein-DNA binding andidentify the Fur regulon under iron replete and deplete conditions in-vitro. Sequences were analyzed using CLC Genomic Workbench 12.A total of 5,381 (replete) and 4,960 (deplete) binding sites of Fur on theMAPK10 genome were identified. Applying a false discovery rate at ≤ 10-50we homed in on 43 enriched regions (replete) localized either between (27%) or within ORFs (73%). In contrast, in a deplete condition, 11 enriched binding-sites within ORFs were identified. Four binding sites under both conditions simultaneously, located either between (75%) or within ORF (25%), were also identified. Binding was sensitive to iron availability. Under replete condition, Fur Box 2 (located between 4159132 and 4159456) site presented peak score 33.46 compared to 19.63 in box 1(located between 4158681 and 4158966), while in deplete condition the highest peak score of 38.57 was in Fur box 1, against 12.54 in box 2. EMSA showed that Fur-Fur box 1 binding is regulated by the availability of Mn2+and a competitive binding assay confirmed specificity. Interestingly, only under iron-deplete conditions Fur binds toMAP3638, a hemophore-like protein. The signal-to-noise ratio for peak calling was statistically significant (FDR ≤ 10-50), with a peak score of 17.74. Suggesting that Fur regulation of heme-binding protein is induced by iron deficiency, indicating thatMAPmight be capable of using heme as an additional iron source. Taken together, these data identify the binding locations for direct or indirect targets of Fur under iron replete and deplete conditions, providing a genome-wide view of Fur regulon. To further confirm function of Fur, we constructed a deletion mutant using homologous recombination that will be used in our RNA-seq experiments for characterization of the Fur regulon.?

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Fernanda Miyagaki Shoyama, John Bannantine, Raul Barletta, Srinand Sreevatsan, Characterization of the Fur protein in Mycobacterium avium subsp. paratuberculosis. 99th Annual CRWAD meeting, Chicago, IL
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: Fernanda Miyagaki Shoyama, Taveesak Janetanakit, John Bannantine, Raul Barletta, Srinand Sreevatsan, Elucidating the Regulon of a Fur-like protein in Mycobacterium avium subsp. paratuberculosis (MAP). 100th Annual CRWAD, Chicago, IL
• Type: Book Chapters Status: Awaiting Publication Year Published: 2019 Citation: Govardhan Rathnaiah, Fernanda Miyagakishoyama, Evan P. Brenner, Denise K Zinniel, John P. Bannantine, Srinand Sreevatsan, Ofelia Chacon and Ra�l G. Barletta. Molecular Genetics of Mycobacterium avium subsp. paratuberculosis. In, Paratuberculosis: organism, Disease, Control.

Progress 11/01/17 to 10/31/18

Outputs
Target Audience:Veterinarians, Microbiologists, Animal disease diagnosticians, diagnostic laboratories, Mycobacteriologists Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A PhD student working on this project will be able to attend the 99thAnimal Conference of Research Workers in Animal Disease (CRWAD) besides all research funded in this grant is part of her PhD research training. She also attended the International Colloquium on Paratuberculosis in Cancun to present her work on MAP immune responses. How have the results been disseminated to communities of interest?The current findings will be presented at the 99thAnimal Conference of Research Workers in Animal Disease (CRWAD). What do you plan to do during the next reporting period to accomplish the goals?Next, to validate findings from this report that FUR is involved in iron regulation, we will investigate the second FUR BOX identified in the same genomic island, all conditions tested in the FUR BOX 1 will be repeated for the FUR BOX 2. Furthermore, using the recombinant MAP3736c we will analyzeβ-galactosidase activity in MAPgrown under iron starvation. When cultures reach log phase, we will use RNA-seq to investigate the transcriptomes of MAP wild-type and MAP3773c deletion mutant strains under iron starvation. To determine if the disrupted gene is important for bacterial survival, we will perform an in-vitro growth analysis with mutant and wild-type strains under replete and deplete iron. Together, these data will inform us about the role of FUR iniron utilization in MAP and help us identify critivcal pathways under its control. These will be targeted in future vaccine studies.

Impacts
What was accomplished under these goals? Our goal is to better understand iron regulation in Mycobactyerium acvium subspecies paratuberculosis. We have successfully cloned and expressedMAP3773c, a Ferric Uptake Regulatory protein (FUR)homolog). In order to demonstrate physical binding of FUR to putative regulatory domains on the MAP genome we performed Gel Shift assays using genomic DNA of the cattle and the sheep strain of MAP. We identified two FUR boxes that are potentially involved in the iron regulation. Promoter sequence containing the putative FUR box 1 which lies upstream of an ABC transporter gene on the MAPgenome, was amplified using 5' biotin labeled primers usingPCR. Purified amplification products were used in DNA-FUR interactions. FUR titration was pergfomed at verying concentrations -0nM, 5nM, 10nM, 15nM, 20nM in the presence of 10mM of MnCl2containing 20fmol of DNA. With this assay we were able to confirm that DNA binding is dose dependent, as we increased the concentration of FUR protein there were an increase in the binding activity, with 20mM as an optimal concentration; MnCl2titration: 0mM, 5mM, 10mM, 15mM, 20mM of MnCl2containing 20fmol of DNA and 20nM of FUR protein, revealed that MnCl2 was necessary for binding; Competition assay using excess of unlabeled promoter DNA: 0-4pmol of Unlabeled DNA and 0-20fmol of labeled DNA containing 10mM of MnCl2and 20nM of FUR protein, revealedthat MAP3773c specificity for binding to the FUR box. Next, to confirm biocomputational analysis where we identified 23 pathways controlled by MAP3773c we used protocol generated by Jainiet al. 2018 to prepare ChIP-DNA using the cattle and sheep strain in 3 different conditions: a) MAP grown under sufficient iron conditions b) MAP grown under iron starvation (200mM 2,2′-Bipyridyl) c) MAP with 200ng of FUR protein. All samples were processed the same way; protein-DNA cross-linking was done by adding formaldehyde to a final concentration of 1% and incubating for 30min at room temperature. To stop cross-linking, 250mM of glycine was added and samples were incubated for 15min; cells were washed 2 times with ice-cold 1xPBS and resuspended with freshly made buffer 1+PI (20mM KHEPES ph 7.9, 50mM KCL, 0.5mM DTT, 10% glycerol, complete mini protease inhibitor). Cells were lysed using MagNA Lyser four times at 4,000 rpm and 45sec. Covaris was done for 18min for lysate (amplitude = 20%, intensity = 5, cycles/burst = 200). Salt concentration was adjusted to 10mM Tris HCL, 150mM NaCl, 0.1% NP40. For IP assay, we used a customized FUR antibody and samples were incubated overnight at 4°C on the rotating platform. Next day, samples were incubated with protein-G agarose beads; washed five times with IPP150 buffer (10mM Tris HCl, 150mM NaCl, 0.1% NP40); followed by two washes of 1xTE buffer. Samples were eluted with elution buffer (Tris HCl, 10mM EDTA, 1% SDS) and incubated with proteinase K (1mg/ml) overnight to reverse cross-link. All DNA were purified using the PCR purification kit from Qiagen and submitted to Genewiz for sequencing. Currently we are waiting for sequencing data to identify binding sites and motifs within enriched regions. Functional activity of FUR box was assessed using reporter gene assay. Predicted MAP promoter sequence containing the FUR box was amplified using primers carrying ScaI restriction sites. Amplified products were restriction digested with ScaI and ligated into a predigested promoterless integrative plasmid, pSM128. The recombinant MAP3736c (carrying FUR box and lacZ fusions) was transformed into the cattle (K-10) and sheep strain (S397) and analyzed for β-galactosidase activity. To further confirm function of FUR, we are constructing a FUR mutant using the standarsd allelic exchange method as well as the novelCRISPRi system, where we repress gene expression ofMAP3773cusing the plasmid, optimized for M. tuberculosis, PLJR965 designed by Rock JM et al 2017. We designed sgRNA targeting MAP3773c and as recommended by Rock JM et al we used the "strongest" PAM for maximal gene knockdown (fwd 5'-GGGA GCTGTCAGTTCGTCGCCTGGC-3' rev 5'-AAAC GCCAGGCGACGAACTGACAGC-3'). Oligos were annealed using annealing buffer (50mM Tris pH 7.5, 50mM NaCl, 1mM EDTA) on the following program: 95°C for 2:00, 0.1°C/sec to 25°C, end. Annealed oligos were ligated into a predigested and purified PLJR965 and transformed into chemically competentE.coli(TOP10, Invitrogen). Single colonies were sent for sequencing with primer 1834 (5'-TTCCTGTGAAGAGCCATTGATAATG-3'). We selected one of the clones that carried the insert and transformed into the cattle and sheep strain of MAP. Currently we are waiting for colonies growth.

Publications