Defining the role of Salmonella Typhimurium ArtAB toxin in diarrheal disease to promote bovine vaccine development

DEFINING THE ROLE OF SALMONELLA TYPHIMURIUM ARTAB TOXIN IN DIARRHEAL DISEASE TO PROMOTE BOVINE VACCINE DEVELOPMENT

Sponsoring Institution

National Institute of Food and Agriculture

Project Status

COMPLETE

Funding Source

AFRI COMPETITIVE GRANT

Reporting Frequency

Annual

Accession No.

1015682

Grant No.

2018-67016-28297

Cumulative Award Amt.

$149,997.00

Proposal No.

2017-05752

Multistate No.

(N/A)

Project Start Date

Jul 1, 2018

Project End Date

Jun 30, 2022

Grant Year

2018

Program Code

[A1221]- Animal Health and Production and Animal Products: Animal Health and Disease

Recipient Organization
BOISE STATE UNIVERSITY
1910 UNIVERSITY DRIVE
BOISE,ID 83725

Performing Department
(N/A)

Non Technical Summary
Despite the licensure and use of bovine Salmonella vaccines, the presence of this bacterial pathogen on large beef and dairy operations remains common. Salmonella can be harbored subclinically, but causes significant morbidity and mortality in calves, and reducing or eliminating this pathogen is an economically impactful priority for agriculture. In addition, cows are a significant source of transmission to humans, and concerns over increasingly antibiotic-resistance Salmonella has focused attention on the need for improved methods of prevention. An inexpensive, effective and safe vaccine that can induce mucosal responses and protect cows from systemic disease will improve animal welfare and production, and potentially reduce transmission to humans. Bacterial AB5 toxins represent essential virulence factors in certain Gram negative bacteria and and are important vaccine antigens in licensed vaccines. The proposed studies are based on the identification of a novel AB5 toxin in S. Typhimurium DT014, and the hypothesis that this toxin is expressed during clinical infection, a contibuting virulence factor to disease and a potential vaccine antigen. In the proposed studies we will; 1) determine the expression of ArtAB in clinical disease, 2) define a role for ArtAB in cellular cytotoxicity and barrier dysfunction and 3) quantify the effects of toxin deletion on pathogenicity. These studies are the first that will define contributions of ArtAB to toxicity and virulence, and represent primary steps to the long-term goal of developing a mucosal Salmonella vaccine for use in cows. This project aligns with the priority of disease prevention and control within the USDA-AFRI Animal Health and Disease program area.

Animal Health Component

60%

Research Effort Categories

Basic

20%

Applied

60%

Developmental

20%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	3410	1100	50%
311	3310	1100	50%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3310 - Beef cattle, live animal; 3410 - Dairy cattle, live animal;

Field Of Science
1100 - Bacteriology;

Keywords

Goals / Objectives
Based upon previous studies of toxicity and homology to well characterized AB5-type bacterial toxins, we hypothesize that the S.Typhimurium ArtAB enterotoxin is expressed during infection and promotes Salmonella pathogenicity in bovines. The long-term goal of this research is to develop a mucosal Salmonella vaccine for bovines. The objectives of this proposal will deliver preliminary evidence for the contribution of this toxin to disease, and will provide a foundation for vaccine development. To test this hypothesis we propose the following objectives:Objective 1: Confirm the prevalence and expression of artAB from bovine Salmonella. Clinical and subclinical Salmonella from fecal bovine samples will be genotyped for the presence of artAB. Bovine anti-ArtAB antibody expression will be determined by ELISA to identify in vivo toxin expression, and confirmed by RT-PCR.Objective 2: Assay the contribution of purified ArtAB to Salmonella pathogenicity. Electric cell-substrate impedance and cellular cytotoxicity assays by microscopy will be used with purified ArtAB to quantify effects on cell viability and epithelial barrier function. The mouse ligated loop model will be used to determine contributions to intestinal fluid secretion.Objective 3: Construct a S.Typhimurium artAB targeted deletion mutant. The modified lambda red recombinase will be used to disrupt artAB on the chromosome of bovine S.Typhimurium DT104. The contribution of artAB to Salmonella pathogenicity will be assessed using the mouse ligated loop model.

Project Methods
The methods for this research project are limited to instruction in laboratory research and experimentation. Specific reserch methods for each objective are described below:Objective 1. Confirm the prevalence and expression of artAB from bovine Salmonella. I. Genotyping of clinical Salmonella. 50-100 isolates of bovine Salmonella, with known clinical status, will be obtained from the Washington State Animal Disease Diagnostic Lab (WADDL) and Dr. William Sischo (see attached letter). Bacterial cultures will be stocked and suspended in lysis buffer TZ (2.0% Triton-X 100, 2.5 mg/mL sodium azide, 0.1 M Tris-HCL, pH 8) (28). Lysed samples will be subjected to genomic DNA isolation (DNeasy Blood and Tissue, Qiagen) and stored at -20°C. Multiplex PCR (MP-PCR) will be performed by the method above (Figure 3C), as described (29). This will identify Typhimurium DT104, utilizing primers for the Salmonella specific gene invA, and the DT104-specific sequences intI1 and flostR, with a three-band pattern. The artAB region will also be amplified and results will be correlated to clinical status. II. Expression of ArtAB in vivo. 20-30 clinical S. Typhimurium dairy or beef fecal samples will be obtained from WADDL (see attached letter). Samples will be shipped frozen and suspended in: 1) Bacterial RNA-later (Sigma-Aldrich), and 2) IgA extraction buffer (1X PBS + 0.5% Tween-20 + 0.05% sodium azide). Bovine IgA will be purified from supernatants in extraction buffer after addition of protease inhibitor and centrifugation as described (30). Purified native ArtAB (Figure 4) will be used to coat plates and ELISA will be performed as described utilizing anti-bovine IgA-HRP (24). Controls will include Salmonella negative fecal samples and CT antigen. For samples in RNA-later, total RNA will be isolated (RNeasy, Qiagen), as described (31). RNA will be transcribed to cDNA (cDNA Reverse Transcriptase Kit, Life Technologies), and qPCR on artA performed using SYBR Fast Green (Kappa Biosystems). All qPCR will be reported as relative values to total bacterial 16S rRNA. Experimental results from Objective 1 will include; 1) the prevalence of artAB in clinical bovine Salmonella, and 2) the quantitation of toxin transcript and protein expression in vivo. Outcomes are expected to provide epidemiological evidence in support of ArtAB involvement in Salmonella disease pathology in bovines.Objective 2. Assay the contribution of purified ArtAB to Salmonella pathogenicity. I. Cell viability assay. Native ArtAB will be purified from Clean Coli (Lucigen) as described above using fetuin-agarose and dialyzed into 1 X PBS. Bovine MDBK cells will be purchased (ATCC CRL-2048) and cultured in triplicate at 5 X 105 cells/well in treated 96-well plates in DMEM + 10% FBS at 37°C and 5% CO2. Purified ArtAB will be pre-activated with 50mM DTT at 37°C for 30 minutes. Toxin will be added to cells in 200ml per well at 2-fold dilutions starting at 10mg. At time points of 1 hr, 5 hr and 24 hr cells will be stained with priopidium iodide (PI) and calcein (Sigma-Aldrich). Simultaneous quantification of live/dead cells will be determined by imaging at 490nm in a BioTek Cytation 3 (USDA Tinker 2014-06321 ). II. ECIS assay. Transepithelial Electric Resistance (TEER) and cellular impedance will be measured as an indicator of epithelial cell-cell adhesive barrier properties by Dr. Richard Beard using the Electric Cell-Substrate Impedance Sensor (ECIS; Applied BioPhysics), as described (36, 37, 38). Briefly 2 X 105 MDBK cells will be seeded on ECIS electrode arrays pre-coated with collagen. After 12-16 hrs, ArtAB will be added in triplicate 2-fold dilutions starting at 10mg, and real-time TEER and impedance measurements will be monitored for 24 hrs. The ECIS system contains a 1-V, 4000-Hz alternating current supplied through a 1-MΩ resistor to a constant-current source. In-phase and out of phase voltage will be recorded and real-time tracings expressed as TEER (Ω), and cellular impedance (Z). Controls will include vehicle control (1 X PBS) and identical concentrations of CT. III. Mouse ligated loop model. In conjunction with studies described in Objective 3, we will quantify fluid secretion and inflammation in the mouse ligated loop after injection of purified, pre-activated, ArtAB at 10mg per loop. Experimental results from Objective 2 will determine effects of purified toxin on; 1) cellular toxicity, 2) barrier disruption and 3) in vivo fluid secretion and inflammation. Outcomes are expected to provide mechanistic evidence of ArtAB toxicity.Objective 3 Construct a Salmonella Typhimurium artAB targeted deletion mutant.I. Constuction of an artAB deletion mutation. Bovine artAB + S.Typhimurium DT104 will be transformed with pKD46 containing the recombinase gene and ampicillin marker (Ampr, plasmids kindly donated by S. Clegg, University of Iowa). Strains will be maintained at 30°C. 60bp primers that flank the 1.4kb artAB region on the chromosome will be designed to amplify the chloramphenicol (CMr) marker on plasmid pKD3. The purified CMr amplicon will be electroporated into competent DT104 + pKD46 and selected on CM plates after growth at 37°C. Resistant strains will be assessed for AMP sensitivity and presence of chromosomal deletion by PCR. Cure of the CM marker will occur by transforming with pCP20 and growth at 30°C on AMP. Cultures will then be grown o/n at 42°C and patch plated on LB (growth), AMP (no growth) and CM (no growth). Mutant will be confirmed by PCR and sequencing. II. The mouse ligated loop model. The murine ligated loop model will be performed as described (46, 47, 49). Briefly, one mouse will provide two ileal loops for control and experimental. 7-8 week old C5BL6 mice will be fasted for 8 hours, anesthetized, and kept on a heating pad throughout the experiment. The DT104 deletion and parental strain will be grown o/n and resuspended in 1X PBS. 1cm of abdominal wall will be opened and the small intestine removed. The intestine will be ligated, with care to avoid blood vessels, into two 2cm loops. 50 mL of 108 CFU of bacteria will be injected into each loop with a 27- gauge needle. A group of mice will also be inoculated with 10mg of purified ArtAB or 1X PBS control (Group 1, Table 1). The intestine will be placed back into the animal and closed for a 90 minute incubation. At the end of the experiment, animals will be euthanized, and loops excised, measured and weighed prior to removal of luminal content and fluid quantification. Specimens of intestinal loops containing Peyer's patches will be placed into sterile medium with gentamicin, incubated at 37°C for 30min, homogenized and plated to determine intracellular CFU. Samples will also be flash frozen and stored for histochemistry by hematoxylin/eosin to determine neutrophil migration. Based upon studies measuring fluid in the loop, a sample size of 6 mice per group was determined using G power to achieve a power of 0.95 for a total number of 12 mice as described in Vertebrate Animals (50, 51). Experimental outcomes from Objective 3 will be: 1) a bovine DT104 artAB mutant, and 2) in vivo evidence of ArtAB contribution to diarrheal disease. Outcomes are expected to provide a foundation for future disease studies in bovines.

Progress 07/01/18 to 06/30/22

Outputs
Target Audience:The long term goals of this project support the beef and dairy industries through the prevention of salmonellosis, or diseasecaused bythe bacteria Salmonella,in cattle. Salmonella can infect cattle and result in animal loss, reductions in animal welfare, and decreasesin product qualtity and quantity. In addition, Salmonella can be transmitted to humans, causing infections in farmers and food-borne disease in consumers.Thus, the broadtarget audience includes farmers, veterinarians, the animal vaccine industry, and the food production industry. Translation of this research also has the potential to improve public health through reduced transmission ofSalmonella to the consumer.Efforts during this project period were research based and specifically focused on; student training, experimentation, data collection and publication. Thus thetarget audience that directly benefitted from this funding during the grantperiod included human and animal biomolecularundergraduate and graduate students at Boise State University. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provided funding to support the research of two Ph.D. candidates within the Boise State Biomolecular Graduate Program. One candidate, Rosey Whiting, left the program in 2018, the other candidate, Elise Overgaard, began in the fall of 2018 and hasbeen wellsupported by this project as an RA and with funding for supplies. Elise Overgaard will defend her thesis on September 12, 2022. Two new Ph.D. candidates, Haley Bridgewater and Noah Souza, have not been directly supported on this project, but their work on enterotoxin adjuvants is closely related and synergistic. In addition, there have been six Boise State Biology undergraduates supported as technicians on this project: Omid Mohammad Mousa, Adriana Rodriguez, Kimberly Dueno, Noah Souza, Fallon Cassidy and Gabe Frandsen; and nine student internships/fellowships supported: Morgan Hansen, Elizabeth Heller, Hunter Johnson, Kris Gardner, Leyla Cufurovic, Holly Harper, Noah Souza, Lydia Howell and Sam Butler. A number of these students have presented this work at regional conferences and/or been included as co-authors on a manuscript. Noah Souza is now a Ph.D. student in the Boise State Biomolecular Program and Gabe Frandsen, Adriana Rodriguez and Sam Butler are planning to apply this spring. This project has supported training of graduate and undergraduate students through the Boise State Biomolecular Research Center in tissue culture maintenance, quantitative PCR and confocal microscopy. Dr. Richard Beard (Co-PI) has trained Elise Overgaard in tissue culture and ECIS. Dr. Tinker and Elise Overgaard were trained by the Boise State attending veterinarian (Dr. Stuart Shoemaker) on ligated loop mouse studies in the fall of 2019. Elise Overgaard was able to gain significant Cryo-EM training at PNCC through an accepted Screening Access grant (2022) on this project. Kimberly Dueno, Elizabeth Heller, Morgan Hansen, Kris Gardner, Leyla Cufurovic, Adriana Rodriguez, Fallon Cassidy, Gabe Frandsen, Lydia Howell and Sam Butler have all had the opportunity to present posters on this project at local or regional scientific conferences. How have the results been disseminated to communities of interest?During the project period, this work has been presented locally by a number of undergraduate students at the annual Boise State Undergraduate Research conference, the Idaho Undergraduate Research conference, the Idaho INBRE Annual Research conference and the ASM Intermountain Branch annual meeting. Elise Overgaard (Ph.D. candidate) has been able to present her work on this toxin at the CRWAD Annual Research Conference (December 2020), the ASM Intermountain Research Conference (December 2020 and December 2021), and the Vaccines Summit, Boston 2022 (Enzymatic Activity of the A subunit of the Salmonella Typhimurium DT104 AB5-Type Toxin, ArtAB). Her work was also accepted for a poster at the ASM World Microbe Forum in June 2021. The PD, Dr. Tinker presented this work at CRWAD in 2019 (Prevalence, expression and activity of ArtAB toxin from bovine Salmonella Typhimurium).A manuscript waspublished in the fall 2021 (Overgaard et al., Toxins, 2021). A ,manuscript encompasing the more recent work on ArtA active site mutations and structure function of this toxin will be submitted in late 2022. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Salmonellacauses an estimated 1.35 million infections and over 20,000 hospitalizations per year (CDC.gov). Beef and dairy products are a significant source of human disease, and the incidence of contaminated beef has not decreased. Prevention ofSalmonellain cows would reduce foodborne illness, and would have many positive impacts on animal welfare and the industries that depend on these animals. Vaccines that are currently available in the U.S. to preventSalmonellain cows fall short of complete protection and are highly serovar specific. The outcomes of our objectives have helped to define a keySalmonellaprotein, the ArtAB toxin, as a promising bovine vaccine.Objective 1:Major activities and Data collected. The goal of this objective was to determine the prevalence and expression of the genes encoding the ArtAB toxin (artAB) in bovine strains ofSalmonella. Results indicated thatartAB is present on 16% of these isolates.artAB is commonly associated with Typhimurium phage-type DT104, but not always, as 63% of the DT104 isolates in this sample did not have this toxin.artAB was also found on Typhimurium and non-DT104 isolates (29% ofartAB+). Sequencing analysis of theartAB region indicated it was highly conserved, with greater than 98% nucleic acid homology. A collaboration with the Idaho Bureau of Laboratories has enabled the collection of over 500 WGSof human clinically-relevantSalmonella. Data mining of this repository has revealed thatartAB, or a closely related toxin with a B subunit and pseudoAis present on 13% of these isolates. We have recentlycloned the novel ArtB (73% identity to DT104 ArtB) from pseudo-artAB. In silico data mining has also enabled the discovery of ArtA and ArtB homologs on the bovineSalmonellaserovars Infantis, Dublin and Montevideo. During this project period we have also collected bovine feces from 40 animals infected withSalmonella(Idaho Dept of Agriculture).The samples are stored for DNA and antibody analysis. The toxin was found to be expressed after induction with mitomycin C, and secreted to the bacterial supernatant where it can be purified. A publication (Miura et al. Microbiology, 2020) preempted our studies on the expression ofartAB. This report demonstrated that stress environments trigger the expression of this toxin inS.Typhimurium.Key outcomes and Discussion. Genomic analysis thus far has been very consistent;artAB is present in 10-20% of clinically relevant human and bovineSalmonellaisolates. This prevalence is supportive of a role of this toxin in pathogenicity, and consistent with the genomics of other bacterial toxins. artAB is encoded on an active lytic phage related to Gifsy-1. Under environment stress conditions, such as in the gut, this lytic phage is produced and causes death to its host bacteria, while releasing the ArtAB toxin. This process has been referred to as "bacterial altruism" and hypothesized to occur at a low enough frequency in natural habitats that toxin production stays in the population and remains a positive selective force (Los et al. Front Cell Infect Microbiology 2012).Objective 2:Major activities and Data collected. During the grant period we quantifyedthe effects ofArtAB in vitro and in vivo. We usedE. colipurified ArtAB toxin to perform in vitro tissue culture assays to assess cellular activity on two cell types.Results indicated that ArtAB induces a slow cytotoxicity over 72 hours similar to that induced by CT. ArtAB induces a clustering morphology that is characteristic of PT and elongation of cells, similar to that induced by CT. During the grant period, we purified the ArtB receptor alone, and determined that this protein has distinct effects, including cytotoxicity on CHO cells. These studies have been reported (Overgaard et al. Toxins, 2021). During the project period we also completed two sets of mouse ligated loop assays to assess fluid accumulation induced by purified ArtABin vivo (total n=7). These assays compared purified ArtAB with purified cholera toxin (CT). Combined results indicate that ArtAB triggers fluid accumulation within 1 hour compared to uninoculated loops (average 117% of control) however this accumulation was not as severe as that induced by CT (average 135% of control). Mouse intestinal tissues from ArtAB-incubated loops showed visible histological changes in hematoxylin-eosin stained sections, and a protocol for quantification of villi-crypt ratios was developed. Loop tissue and fluid has been stored for RNA extraction and RT-PCR/ELISA for cytokine production is ongoing. In the final trial within this project period, we injected a fluorescent marker (FITC-ova) along with the purified toxin. Results are consistent with fluid accumulation data andsupportive of disruption of intestinal barrier function.Key outcomes and Discussion.The outcomes during this grant period related to this objective include:1)optimization of toxin affinity purification,2)AB holotoxin and B subunit cellular activity quantification,3)animal ligated loop model development, and 4) quantification of toxin-induced in vivo fluid accumulation. The protocols and expertise developed from this objective will be used to continue efforts to characterize ArtAB and otherSalmonellatoxins.Objective 3:Major activities and Data collected. Objective 3 was to constructa chromosomalSalmonellaTyphimuriumartAB genetic-replacement mutation. During the project period, we obtained the materials and protocols for Lambda Red recombination to construct anartAB gene-replacement mutant, however the large number of antibiotic-resistance genes housed on artAB+ DT104 isolates made construction of a gene-replacement mutant using this method unfeasible. Thus within the scope of this objective, we instead based phenotypic analysis on structure and function ofArtAB. In silico modelling and comparison of ArtAB to pertussis toxin and CT identified amino acid residues that were highly conserved in ADP-ribosylating toxins. We constructed alanine scanning site-directed ArtAB mutants in three of these residues. These mutantshave been confirmed by nucleotide sequencing and mass spectrometry. We have characterized the structure of these mutants using ultracentrifugation and circular dichroism. These mutant toxins have also been compared to native ArtABin vitroin studies that indicate they do not cause cell clustering and have reduced intracellular cAMP. Through a Pacific Northwest CryoEM Center (PNCC) access grant (E. Overgaard), we have been able to make grids of purified ArtAB.Key outcomes and Discussion.The mutational studies performed in this objective provide insight into the cellular target, intracellular activity and evolutionary host adaptation of this toxin that will enable predictions of host disease outcomes.Toxin structure will also determine its potential as a vaccine component by identifying epitope regions and the potential for immunomodulation in comparison to known enterotoxin adjuvants. The ArtAB mutants constructed here may also represent novel vaccines or vaccine adjuvants.The long-term goal of these studies is to develop a mucosal vaccine to prevent salmonellosis in cows. We proposed that the ArtAB enterotoxin is a promising vaccine component and these studies help to define this potential. Combined, our current evidence supports the hypothesis that the ArtAB toxin is a significant virulence factor involved in bovineSalmonellainfection and a candidate fo a novel vaccine. Progress toward vaccine development strongly aligns with the priorities of USDA-AFRI Animal Health and Disease. An effective bovineSalmonellavaccine will have significant impacts on public health as well as economic impacts on the beef and dairy industries.?

Publications

Type: Theses/Dissertations Status: Under Review Year Published: 2022 Citation: Overgaard, E. Structure, function and immunogenic applications of AB5-type ADP-ribosylating bacterial toxins. (Submitted 8-15-22, Ph.D. defense 9-14-22)
Type: Conference Papers and Presentations Status: Accepted Year Published: 2021 Citation: Overgaard, E., and J.K. Tinker. Purification of ArtAB, an AB5-type Salmonella Typhimurium DT104 toxin, with single residue mutations for investigation of the enzymatic activity of the A subunit. American Society for Microbiology (ASM) Intermountain Branch Meeting. December 4, 2021 (Online).
Type: Journal Articles Status: Other Year Published: 2023 Citation: Overgaard, E., Bridgewater, H., Woodbury, L., and J.K. Tinker (2023) Structural analysis of the ADP-ribosylating A subunit of the Salmonella Typhimurium ArtAB toxin. Toxins (in preparation)
Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Howell, L., and J.K. Tinker. Identification of Novel ADPRTs in Salmonella infantis: Data mining shows that pertussis toxin variants found in Salmonella infantis are actually examples of a unique S. infantis toxin. Boise State Annual Undergraduate Research Showcase. April 22, 2022, Boise, I.D.
Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Butler, S. and J.K. Tinker. Eat the raw cookie dough: Salmonella vaccine development by exploring Salmonella toxins. Idaho Conference for Undergraduate Researchers (ICUR) July 20, 2022. Boise, I.D.
Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Butler, S. and J.K. Tinker.Salmonella vaccine development by exploring Salmonella toxins. Idaho INBRE Annual Research Conference. August 1-3, 2022. Moscow, I.D.

Progress 07/01/20 to 06/30/21

Outputs
Target Audience:Efforts during this project period were research based and focused on; student training, experimentation, data collection and publication. The long term goals of this project will support the beef and dairy industries through the prevention of salmonellosis in cattle. Thus, the broadtarget audience includes the farmer, the veterinarian and the animal vaccine industry. Translation of this research also has the potential to improve public health through reduced transmission of Salmonella. The specific target audience during the project period included human and animal biomedical scientists, food scientists and research workers in animal disease. Changes/Problems:Obtaining Salmonella-infected bovine fecal or genomic samples, as described in Objective 1, has been challenging and thus a switch to increased in silco analysis of sequenced genomes is believed to be supportive and also efficient and cost-effective to complete this aim. While we have been unsuccessful in the construction of an artAB deletion mutant in the parental S. Typhimurium DT104, as described in Objective 3, this has lead to the successful construction of site-directed mutants that will enable the study of pathogenesis and also toxin structure/function. Reduced laboratory personnel and research disruptions due to Covid-19 in 2020 and early 2021 have been significant. However, the changes and pivots described have enabled a reduction in direct work with highly resistant Salmonella that requires graduate training and/or significant student oversight, and increased productive work that can be performed away from the laboratory. What opportunities for training and professional development has the project provided?This project has provided funding to support the research of two Ph.D. candidates within the Boise State Biomolecular Graduate Program. One candidate, Rosey Whiting, left the program in 2018, the other candidate, Elise Overgaard, began in the fall of 2018 and was supported for one year on this project. Elise is likely to graduate in the spring or summer of 2022. A new Ph.D. candidate, Haley Bridgewater, is not directly supported on this project, but her work on toxin adjuvants is closely related and stems from this work. In addition, there have been sixBoise State Biology undergraduates supported as technicians on this project: Omid Mohammad Mousa, Adriana Rodriguez, Kimberly Dueno, Noah Souza, Fallon Cassidy and Gabe Frandsen; and seven student internships supported: Morgan Hansen, Elizabeth Heller, Hunter Johnson, Kris Gardner, Leyla Cufurovic, Holly Harper and Noah Souza. A number of these students have presented this work at regional conferences and/or been included as co-authors on a manuscript. Noah Souza is now an M.S. student in the Boise State Biomolecular Program and Gabe Frandsen is planning to apply this spring. This project has supported training of graduate and undergraduate students through the Boise State Biomolecular Research Center in tissue culture maintenance, quantitative PCR and confocal microscopy. Dr. Richard Beard (Co-PI) has trained Elise Overgaard in tissue culture and ECIS. Dr. Tinker and Elise Overgaard were trained by the Boise State attending veterinarian (Dr. Stuart Shoemaker) on ligated loop mouse studies in the fall of 2019. Kimberly Dueno, Elizabeth Heller, Morgan Hansen, Kris Gardner, and Leyla Cufurovic have presented posters on this project at the Boise State Undergraduate Research conference or Idaho Undergraduate Research conference, and Adriana Rodriguez and Fallon Cassidy have had the opportunity to attend and present at the 2018 and 2019 Idaho INBRE Annual Research conference. How have the results been disseminated to communities of interest?During the project period, this work has been presented locally at the Boise State Undergraduate Research conference, the Idaho Undergraduate Research conference and the Idaho INBRE Annual Research conference (Cufurovic et al. BSUURC 4-13-20, Cassidy et al. 2021 INBRE, 8-5-21). Elise Overgaard has presented this work at the CRWAD Annual Research Conference (December 2020) and the ASM Intermountain Research Conference (December 2020). Her work was also accepted for a poster at the ASM World Microbe Forum in June 2021. A manuscript was completed and accepted during the project period (Overgaard et al., Toxins, 2021). What do you plan to do during the next reporting period to accomplish the goals?For Objective 1, we will focus on in silico analysis of sequenced genomes to determine prevalence of artAB and also complete analysis of fecal genomic DNA for the presence of artAB and the novel AB5 toxin. For Objective 2, we will complete analysis of mouse ligated loops and barrier function studies. Possibly confocal microscopy of ArtAB trafficking will be completed. Objective 3 will be completed with our ArtAB site-directed mutations and toxin structure-function analysis. The pandemic slowed expected progress, however experimentation has recentlyrampedup and work is expected to be completed. Studies have formed a basis for many new exciting avenues for future studies and new research proposals regarding this interesting AB5 toxin from Salmonella.

Impacts
What was accomplished under these goals? Objective 1: Confirm the prevalence and expression of artAB from bovine Salmonella. Efforts on this objective during the previous reporting period included the determination of artAB prevalence using multiplex PCR from 90 strains of bovine Salmonella serogoup B, and the initiation of expression studies using RT-PCR and protein purification from these isolates. We have also previously confirmed the expression of artA RNA from DT104 isolates using RT-PCR and initiated quantitative analysis of these results after induction of phage using mitomycin C. A recent publication (Miura et al. Influence of SOS-inducing agents on the expression of ArtAB toxin gene in Salmonella enterica and Salmonella bongori. Microbiology, 2020) completed many of our proposed studies on the expression of artAB, and found that sub-inhibitory levels of quinolone antibiotics, and other SOS-inducing agents, trigger the expression of this toxin in S. Typhimurium. We have since pursued a better understanding of the prevalence of artAB in bovine isolates, and also have assessed the prevalence of a homologous uncharacterized toxin similar to subtylase cytotoxin (E. coli) using our collection of bovine Salmonella. During the project period we have designed primers specific to the novel toxin and have identified two isolates that may express this toxin. Future studies will involve cloning and purification, and comparison to the structure/function of ArtAB. In addition, we have obtained 40 bovine fecal samples suspected to contain Salmonella (Idaho Department of Agriculture) and have isolated total genomic DNA. Currently we are testing these samples using PCR and Salmonella as well as artAB and novel toxin-specific primers. We have also gained access to a large database of sequenced human Salmonella isolates from the Idaho Bureau of Laboratories. Using this database, as well as the rapidly growing number of human and bovine sequences available, we will be able to identify artAB, and the novel toxin in silco to provide an estimate of prevalence of this toxin on pathogenic isolates. Percent completion of Objective 1 = 90%. Percent estimated completed by end of requested no-cost extension =100% Objective 2: Assay the contribution of purified ArtAB to Salmonella pathogenicity. During the last project period we used E. coli purified ArtAB toxin to initiate in vitro tissue culture assays to assess cellular activity. During this period, we have expanded these assays to include: resazurin metabolic assays, crystal violet cytotoxicity assays and microscopic cellular morphology assays. Results indicated that ArtAB induced a slow cytotoxicity over 72 hours similar to that induced by CT. On CHO cells, ArtAB induces a clustering morphology that is characteristic of PT and on Vero cells the toxin promotes the elongation of cells, similar to that induced by CT. During this period, we have also been successful purifying the ArtB receptor subunit alone, and have assayed the effects of this subunit on tissue culture in vitro. These studies have been reported (Overgaard et al. Cellular activity of S. Typhimurium ArtAB toxin and its receptor-binding subunit. Toxins, 2021). Assays using human Caco-2 intestinal epithelial cells to determine toxin effects on transepithelial barrier electric resistance (TEER) have been slowed due to difficulties with Caco-2 growth and consistency. These studies are ongoing and may use a primary intestinal cell line instead. In December of 2019, and again in June of 2021 we have completed sets of mouse ligated loop assays to assess fluid accumulation induced by purified ArtAB in vivo (n=9). These assays compared the fluid-accumulation induced by purified ArtAB with purified cholera toxin (CT). Results have not yet been published, but indicate that ArtAB triggers fluid accumulation in the mouse intestine within 1 hour, and may be more than that induced by CT. In addition, mouse intestinal tissues from ArtAB-incubated loops showed notable histological changes in hematoxylin-eosin stained sections. Analysis of histology and RT-PCR/ELISA for cytokine production from the loop fluid is ongoing. Lastly, our success with the production of purified ArtAB and ArtB will be used to develop polyclonal antibodies to the toxin to promote cellular internalization studies, in conjunction with these mouse ligated loop assays, using confocal microscopy. It is anticipated that the completion of data analysis from mouse studies, barrier resistance and confocal microscopy will lead to a publication within 2022. Percent completion of Objective 2 = 95%. Percent estimated completed by end of requested no-cost extension =100% Objective 3: Construct a S. Typhimurium artAB targeted deletion mutant. During the previous project periods, we obtained the materials and protocols for Lambda Red recombination to construct an artAB gene-replacement mutant, however the high level of antibiotic-resistance associated with DT104 isolates has made construction of a gene-replacement mutant using this method unfeasible. During past project periods we have explored avenues to construct mutant strains. These methods included: 1) the use of non-DT104 artAB isolates, 2) the curing of DT104 prophage to construct an artAB- isolate, and 3) purification of the artAB phage and pro-phage induction into a parental Salmonella isolate. Unfortunately, the curing of DT104 prophage and the purification of the artAB phage have thus-far been unsuccessful. While we will continue to pursue these avenues, as well as the idenfication of DT104 negative artAB + isolates that are not antibiotic-resistant, we will complete this aim instead using constructed site-directed ArtAB mutants. We have constructed, and confirmed by sequencing, three new plasmids for ArtAB expression that contain mutations within the active site of ArtA (T50A, R6A, H32A). These mutant toxins have been purified and compared to native ArtAB in vitro in preliminary studies that indicate they do not cause CHO cell clustering. Mutants will be assessed and compared in additional in vitro assays during the final period of performance. Lastly, while the crystal structure of tagged ArtB has been determined, the structure of ArtAB has not, and thus with our current scale of protein production we will pursue crystallography and/or CryoEM to define the structure of ArtAB and compare to our active-site mutants. It is anticipated that this work will lead to a future publication on the structure of this toxin. Percent completion of Objective 3 = 50%. Percent estimated completed by the end of requested no-cost extension =75% ?

Publications

Type: Journal Articles Status: Accepted Year Published: 2021 Citation: Overgaard, E., Morris, B., Mohammad Mousa, O., Price, E., Rodriguez, A., Cufurovic, L., Beard, R. and J.K. Tinker. 2021. Cellular Activity of Salmonella Typhimurium ArtAB Toxin and Its Receptor-Binding Subunit. Toxins. 13(9) 599. doi.org/10.3390/toxins13090599
Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: Overgaard, E., Mohammad Mousa, O., Beard, R., and J.K. Tinker. Cellular activity of ArtAB toxin from bovine Salmonella Typhimurium. CRWAD/USDA PD Annual Conference 2020. Online. Dec 5-7, 2020.
Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: Overgaard, E., and J.K. Tinker. Cellular activity of ArtAB toxin from Salmonella Typhimurium. ASM Intermountain Annual Research Conference 2020. Online. Dec 5, 2020.
Type: Conference Papers and Presentations Status: Accepted Year Published: 2021 Citation: Cassidy, F., and J.K. Tinker. Cellular biology of ADP-ribosyation. Idaho INBRE Annual Research Conference. 2021. Moscow, ID. Aug 5, 2021.

Progress 07/01/19 to 06/30/20

Outputs
Target Audience:Efforts during this project period were research based and focused on:training, optimizing experimentation, data collection and publication. The long term goals of this project will support the beef and dairy industries through the prevention of salmonellosis in cattle. Thus the main target audience includes the farmer, the veterinarian and the animal vaccine industry. Translation of this research also has the potential to improve public health through reduced transmission of Salmonella and thus the target audience also includes the food industry as well as the beef and dairy consumer. Changes/Problems:Objective 3 has continued to be challenging due to the multiple antibiotic resistance profile of DT104. Work with these highly resistant strains also requires graduate training and/or significant student oversight. However, the identification of two artAB+ DT104- isolates is a promising development to promote chromosomal gene replacement, and the construction of site-directed ArtA mutations is deemed feasible during the last project period. During this project period we overcame a major obstacle and were successful at identifying a consistent cytotoxic phenotype of ArtAB on tissue culture. In addition we now have a rapid morphological assay for toxicity on CHO cells. Overall the pandemic has, of course, been the main problem and has caused a significant reduction in benchwork productivity anddisruption of collaborations for sample collection, however efforts are more recently ramping up and deemed to be on target. What opportunities for training and professional development has the project provided?During this period, this project has supported the research of aPh.D. candidatewithin the Boise State Biomolecular Graduate program, Elise Overgaard. Elise began in the fall of 2018 and is likely to graduate in the spring of 2022. In addition, there have been three Boise State Biology undergraduates supported as technicians on this project: Omid Mohammad Mousa, Adriana Rodriguez and Kimberly Dueno; and seven student internships supported: Morgan Hansen, Elizabeth Heller, Hunter Johnson, Kris Gardner, Leyla Cufurovic, Holly Harper and Noah Souza. This project has continued to supporttraining of graduate and undergraduate students through the Boise State Biomolecular Research Center in tissue culture maintenance, quantitative PCR, histology and confocal microscopy. Dr. Richard Beard (Co-PI) has trained Elise Overgaard in tissue culture and ECIS. Dr. Tinker and Elise Overgaard were trained by the Boise State attending veterinarian (Dr. Stuart Shoemaker) on ligated loop mouse studies in the fall of 2019. Kimberly Dueno, Elizabeth Heller, Morgan Hansen, Kris Gardner and Leyla Cufurovic have presented posters on this project at the Boise State Undergraduate Research conference or Idaho Undergraduate Research conference in 2019 and 2020, and Adriana Rodriguez has had the opportunity to attend and present at the 2018 and 2019 Idaho INBRE Annual Research conference. How have the results been disseminated to communities of interest?Currently this work has been presented locally at the Boise State Undergraduate Research conference, the Idaho Undergraduate Research conference and the Idaho INBRE Annual Research conference (Rodriguez et al. INBRE 8-3-18 and 7-29-19, Dueno et al., BSUURC 4-11-19, Heller et al. BSUURC 4-11-19, Hansen et al, ICUR 7-29-19. Dueno et al, Boise State Biology Conference 11-11-19, Cufurovic et al. BSUURC 4-13-20). Three abstracts on this project were accepted to the ASM Intermountain conference atWeber State University in Ogden, Utah, however the conference was unfortunately cancelled. A manuscript is now complete and will be submitted within the fall of 2020, and a second likely ready for spring submission. Elise Overgaard's abstract on this work has been accepted for a talk at the annual Conference for Research Workers in Animal Disease (CRWAD) conference for the fall of 2020. What do you plan to do during the next reporting period to accomplish the goals?For Objective 1, we will continue to expand our bovine Salmonella strain collection for genomic analysis and collect bovine fecal samples (Dr. Claire Burbick, Washington State WADDL) for antibody analysis. Quantitative RT-PCR of artAB expression will complete this objective. For Objective 2, repeat ECIS barrier assaysand a new set of mouse ligated loop studies using purified holotoxin and ArtB alone will complete this work, and it may be supported by confocal trafficking studies.Objective 3 has proven challenging, however site-directed mutagenesis is deemed realistic for the next project period and alternative methods for chromosomal deletions will continue to be explored. The pandemic has slowed expected progress, including manuscript submission, however, with the approved no-cost extension of this project it is expected that the main goals from each objective will be achieved and disseminated.

Impacts
What was accomplished under these goals? Objective 1: Confirm the prevalence and expression of artAB from bovine Salmonella. Efforts on this objective during the previous reporting period included the determination of artAB prevalence using multiplex PCR from 90 strains of bovine Salmonella serogoup B, and the initiation of expression studies using RT-PCR and protein purification from these isolates. During the current period we have confirmed the expression of artA RNA from DT104 isolates using RT-PCR and initiated quantitative analysis of these results after induction of phage using mitomycin C. RT-PCR is supportive of protein expression studies, indicating that artAB is expressed in the culture supernatant from DT104 in vitro, and at higher levels from phage-induced cultures. Proteins consistent with the expected sizes of ArtAB were purified from the supernatant of mitomycin C induced DT104 (strain 93-1302-4) using fetuin-agarose and, during this project period, these expression studies were expanded to additional strains under alternate conditions. Toxin was found not to be secreted to the Salmonella periplasmic space, and not efficiently purified from Salmonella using D-galactose agarose. We will continue to expand these RNA and protein expression studies to determine expression levels under different conditions, that will include those reported to affect transcription in Salmonella such as: pH, osmolarity, growth phase and magnesium and antibiotic concentrations. We have also constructed a transcriptional reporter plasmid, pEP001, that contains the artA promoter region fused to the lacZ gene. In the next project period this plasmid will be sequenced to determine if it can be used to support expression studies. Continued genomic analysis of bovine Salmonella using primers specific to Salmonella Dublin (vagC) during this project period has also led to the identification of two S. Dublin that are artAB+, as well as a second smaller potential AB5 toxin present on many Salmonella type B isolates. These isolates will be further characterized for resistance and toxicity, and the potential for cloning the novel toxin. For this objective, we remain in need obtaining additional clinical and subclinical bovine Salmonella for genotyping as well as fecal samples for anti-ArtAB humoral analysis. We have initiated a materials transfer agreement with the Washington Animal Disease Diagnostic Laboratory (WADDL, Dr. Claire Burbick) to collect samples over a 6 month period from suspected Salmonella infected cattle. We have initiated collaboration with the local Idaho Department of Agriculture as well to collect additional Salmonella strains and fecal samples. The collection and assay of these strains will enable completion of Objective 1. Percent completion of Objective 1 = 60%. Percent estimated completed in next project period =100% Objective 2: Assay the contribution of purified ArtAB to Salmonella pathogenicity. During the last project period, a new protocol to purify ArtAB from E.coli (Clean Coli®, Lucigen) without requiring a 6XHIS tag (glycan-affinity) was implemented. More recently we have scaled up production and consistently produce 1-2 mg/mL of purified ArtAB per liter of culture. During this project period we have used this purified toxin to complete in vitro tissue culture assays to assess cellular activity. These include: resazurin metabolic assays, crystal violet cytotoxicity assays and microscopic cellular morphology assays. Results indicate that ArtAB induced a slow cytotoxicity over 72 hours similar to that induced by CT. On CHO cells, ArtAB induces a clustering morphology that is characteristic of PT and on Vero cells the toxin promotes the elongation of cells, similar to that induced by CT. These outcomes support previous studies reporting the ADP-ribosylation activity of ArtAB, but indicate that ArtAB binds distinct, and likely broad, host receptors. Assays using human Caco-2 intestinal epithelial cells to determine toxin effects on transepithelial barrier electric resistance (TEER) have indicated that ArtAB reduces resistance and barrier integrity, similar to the effects of CT. These studies will be confirmed in the upcoming project period. Interestingly, we also found that the B subunit of CT (CTB) dramatically increases barrier resistance. This result was unexpected and we have since constructed a plasmid (pLC001) to express the ArtB protein alone and, during this project period, ArtB has been successfully expressed and purified using glycan affinity. Lastly, we have completed a set of mouse ligated loop assays to assess fluid accumulation induced by purified ArtAB the in vivo. These assays indicated that ArtAB triggers fluid accumulation in the mouse intestine within 1 hour, similar to that induced by CT. In addition, mouse intestinal tissues from ArtAB-incubated loops showed notable histological changes in hematoxylin-eosin stained sections. These studies will be repeated in the near future for confirmation prior to publication. Our success with the production of purified ArtAB will be used to develop polyclonal antibodies to the toxin to promote cellular internalization studies, in conjunction with these mouse ligated loop assays, using confocal microscopy. Lastly, while the crystal structure of tagged ArtB has been determined, the structure of ArtAB has not, and thus with our current scale of protein production we also plan to pursue crystallography and/or CryoEM to define the structure of ArtAB. Percent completion of Objective 2 = 85%. Percent estimated completed in next project period =100% Objective 3: Construct a S. Typhimurium artAB targeted deletion mutant. During the previous project period, we obtained the materials and protocols for Lambda Red recombination to construct an artAB gene-replacement mutant, however the high level of antibiotic-resistance associated with DT104 isolates has made construction of a gene-replacement mutant using this method unfeasible. During this project period we have explored avenues to construct mutant strains. These methods include: 1) the use of non-DT104 artAB isolates, 2) the curing of DT104 prophage to construct an artAB- isolate, and 3) purification of the artAB phage and pro-phage induction into a parental Salmonella isolate. Genomic analysis has identified non-DT104 artAB positive Salmonella Type B isolates within our bovine collection of strains (4 of 14 artAB positive bovine Salmonella). An antibiogram of one of these isolates reveals that it has a similar resistance pattern to DT104, however this isolate, while negative for flo conferring florfenicol resistance, was PCR + for the intR regions flanking the resistance cassette. Two additional isolates are flo- int- and artAB+ (94-1118-1,96-0284-9) for which we will extend resistance analysis to determine if cassettes for Lambda Red (kanamycin, chloramphenicol, and ampicillin) can be utilized in these strains. During the current project period we have also begun exploration of the filamentous phage that contains artAB. induction of purification from two DT014 isolates. Phage will be isolated, confirmed for artAB sequences and spotted on a range of potential Salmonella hosts for lysogen-induction. The new artAB+ lysogen and its parental host will be used for comparison in toxicity and bacterial uptake assays in vitro and future in vivo studies. In the process of these studies, we will also work to purify sufficient phage DNA for commercial sequencing analysis. Lastly, during this project period we have prepared plasmids for site-directed mutagenesis of the active site of ArtA in E. coli. Comparison of purified mutant ArtAB to native ArtAB in vitro and in vivo toxicity assays will promote our understanding of the contribution of this toxin to Salmonella pathogenesis. Percent completion of Objective 3 = 25%. Percent estimated completed in next project period =75%

Publications

Type: Journal Articles Status: Other Year Published: 2020 Citation: Overgaard, E., Morris, B., Mohammad Mousa, O., Whiting, R., Rodriguez, A., Hansen, M., and J. K. Tinker. Salmonella Typhimurium ArtAB toxin: glycan affinity purification and cellular activity. Toxins. To be submitted.
Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Cloning and purification of the ArtB binding subunit from a novel toxin in Salmonella enterica Typhimurium. Curfurovic, L and J.K. Tinker. Boise State University Undergraduate Research Conference April 15, 2020.
Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: ArtAB toxin in bovine Salmonella Dublin. Dueno, K. and J.K. Tinker. Boise State Biology Undergraduate Research Conference. Nov 18, 2020.
Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: Binding and activity of ArtAB toxin from bovine Salmonella Typhimurium. Mousa, O.M., Overgaard, E., Rodriguez, A., Morris, B., and J.K. Tinker. ASM Intermountain Branch Annual Conference. Odgen, UT. March 28, 2020.
Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: Cloning and Purification of ArtAB toxin from bovine Salmonella Typhimurium. Rodriguez, A, Mousa, O.M., Overgaard, E., Morris, B., and J.K. Tinker.ASM Intermountain Branch Annual Conference. Odgen, UT. March 28, 2020.
Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: Cloning and purification of the ArtB binding subunit from a novel toxin in Salmonella enterica Typhimurium. Curfurovic, L and J.K. Tinker. ASM Intermountain Branch Annual Conference. Odgen, UT. March 28, 2020.

Progress 07/01/18 to 06/30/19

Outputs
Target Audience:Efforts during this project period were research based and focused on; training, optimizing experimentation, data collection and publication. The target audience for these efforts is Boise State undergraduate and graduate students. The long term goals of this project will support the beef and dairy industries through the prevention of salmonellosis in cattle. Thus the main target audience of outcomes includes the farmer, the veterinarian and the animal vaccine industry. Translation of this research also has the potential to improve public health through reduced transmission of Salmonella in beef and thus the audience also includes the food industry. Changes/Problems:The main obstacle encountered has been the inability to construct an artAB knockout mutation using the protocol described in Objective 3 due to the multiple antibiotic resistance profile of DT104. While other methods for gene replacement will be considered, we will also move in a new direction for this objective to focus on single amino acid replacement mutations within the active site of ArtA. Another significant obstacle has been identifying a consistent cytotoxic phenotype of ArtAB on tissue culture. Optimization of techniques during this initial project period has supported progress however, and it is anticipated that the cytotoxic profile will be established using other cell types during the next project period. What opportunities for training and professional development has the project provided?This project has provided funding to support the research of two Ph.D. candidates within the Boise State Biomolecular Graduate Program. One candidate, Rosey Whiting, left the program in 2018, the other candidate, Elise Overgaard, began in the fall of 2018 and is continuing to be supported by this project. In addition, there have been three Boise State Biology undergraduates supported as technicians on this project: Omid Mohammad Mousa, Adriana Rodriguez and Kimberly Dueno; and three internships supported: Morgan Hansen, Elizabeth Heller and Hunter Johnson. This project has supported training of graduate and undergraduate students through the Boise State Biomolecular Research Center in tissue culture maintenance, quantitative PCR and confocal microscopy. Dr. Richard Beard (Co-PI) has trained Elise Overgaard in tissue culture and ECIS. Dr. Tinker and Elise Overgaard will be trained by the attending veterinarian (Dr. Stuart Shoemaker) prior to proposed ligated loop mouse studies. Elizabeth Heller and Morgan Hansen have presented posters on this project and attended the Boise State Undergraduate Research conference or Idaho Undergraduate Research conference, and Adriana Rodriguez has had the opportunity to attend and present at for the last two years at the Idaho INBRE Annual Research conference. How have the results been disseminated to communities of interest?Within the project period this work has been presented at the Boise State Undergraduate Research conference, the Idaho Undergraduate Research conference and the Idaho INBRE Annual Research conference (Rodriguez et al. INBRE 8-3-18 and 7-29-19, Dueno et al., BSUURC 4-11-19, Heller et al. BSUURC 4-11-19, and Hansen et al, ICUR 7-29-19). An abstract has been accepted to present a poster at CRWAD in November of 2019 in Chicago. A manuscript is currently in preparation. What do you plan to do during the next reporting period to accomplish the goals?For Objective 1, we will expand our bovine Salmonella strain collection to include known subclinical isolates in collaboration with Dr. Bill Sischo (Washington State University). We will also collect bovine fecal samples (Washington State WADDL) for humoral and transcriptional analysis. For Objective 2, we will continue to optimize cytotoxicity assays using different cell types, complete ECIS studies and mouse ligated loop studies on purified toxin. For Objective 3, we will explore new methods of mutant construction as well as complete site-directed mutagenesis. It is also expected that an initial manuscript will be completed in the fall of 2019 and that Elise Overgaard will attend and present work at a national meeting in the spring of 2020.

Impacts
What was accomplished under these goals? Objective 1: Confirm the prevalence and expression of artAB from bovine Salmonella. We have obtained 90 strains of bovine Salmonella serogoup B from the Idaho Caine Veterinary Teaching Center. Serogroup B Salmonella includes the serovar Typhimurium. 27 of these 90 (30%) were determined to be DT104 using multiplex PCR for the int, floSt and inv genes as described (Bolton et al. 1999). 13 of the 27 DT104 were found to be artAB positive for total prevalence of 14.4% artAB in bovine Salmonella Type B. artAB was sequenced in the 13 isolates to determine conservation and sequences were found to be highly homologous, showing 96-100% identity in artA and artB. We have begun to analyze expression of artAB from S. Typhimurium DT014 grown in vitro by toxin purification from the supernatant and quantitative RT-PCR. ArtAB was purified from the supernatant of mytomycin C induced DT104 (strain 93-1302-4) using fetuin-agarose. Primers have been constructed for qPCR of artA and artB separately. RNA was purified from 4 hour and 18 hour cultures of three strains of Salmonella (two artAB positive and one negative), and cDNA constructed. Cultures were grown at 37°C in Luria Broth with and without mitomycin C, which has been shown to induce artAB expression. Current results indicate that artA is expressed in the presence of mitomycin C and can be quantitated (strains SC09039 and 93-1302-4). We will continue this assay to determine expression levels under different conditions, that will include those reported to affect transcription in Salmonella such as: pH, osmolarity, growth phase and magnesium and antibiotic concentrations. For this objective, we need to obtain additional clinical and subclinical bovine Salmonella for genotyping as well as fecal samples for anti-ArtAB humoral analysis. Percent completion of Objective 1 = 40%. Percent estimated completed in next project period =100% Objective 2: Assay the contribution of purified ArtAB to Salmonella pathogenicity. A new protocol to purify ArtAB from E.coli (Clean Coli®, Lucigen) without using an affinity tag on D-galactose agarose was implemented during this project period. Using this protocol we have been able to purify to concentrations as high as 1mg/mL in endotoxin-free buffer. While a percent of the A subunit is consistently nicked and runs at a smaller size, the toxin is otherwise stable for long-term storage after purification. We have used this toxin to complete a number of cytotoxicity assays in vitro using Vero green monkey kidney cells. These assays include: nuclear area factor, alamar blue and crystal violet. To date, assays have confirmed that the E.coli purified ArtAB has a slow cytotoxic effect on these cells at high concentrations (100 mg/mL), resulting in an average of 10% cell death after 24 hours. This cytotoxic effect is similar to that reported for an E.coli pertussis-like toxin and E.coli subtilase cytotoxin (Littler et al. 2017 and Funk et al. 2015). While Vero cells are commonly used to characterize bacterial toxins, we will expand these studies to other cell types, including bovine cells, and extend toxin incubation times up to 72 hours, as reported for SubAB toxin (Funk et al. 2015). We have begun growth of polarized human Caco-2 intestinal epithelial cells to assay transepithelial electric resistance (TEER). These cells are currently the most commonly reported for this system and will be used to perform initial assays, using up to 100 mg/mL purified ArtAB and incubation times up to 72 hours. We have completed IACUC approval and initial training for mouse ligated loop assays using purified ArtAB toxin. These studies are expected to be completed in the fall of 2019. Lastly, we have completed ELISAs to assess the relative affinity of purified ArtAB to fetuin, ganglioside GM1, and D-galactose, in comparison to cholera toxin binding to these substrates. These assays will be used to aid in the confirmation of ArtB receptors to better identify the cellular targets of this toxin. Percent completion of Objective 2 = 30%. Percent estimated completed in next project period =100% Objective 3: Construct a S. Typhimurium artAB targeted deletion mutant. Under this objective we have obtained the plasmids for Lambda Red recombination to create gene-replacement Salmonella chromosomal mutations. We have also performed antibiograms on four DT104 isolates, and unfortunately all are resistant to ampicillin and chloramphenicol; the required sensitivities for use of the Lambda Red system. We will assess other systems for gene-replacement that do not require transformation with ampicillin and chloramphenicol resistant plasmids, such as use of linear fragments (Aranda et al. 2010) and potentially CRISPR-FRT (Swings et al. 2018). In addition, we will construct ArtA site-directed mutants for expression from E.coli. We have identified three amino acids to target as conserved and likely active-site residues in ArtA. Primers have been constructed to modify these residues in our current ArtAB expression plasmid (pBM006) using the Phusion site-directed Mutagenesis Kit (Thermo Scientific). These mutant ArtAB toxins will be assayed using above cytotoxicity and mouse ligated loop assays. These studies will aid in the determination of ArtAB contribution to fluid accumulation in the small intestine. In addition, we have begun in vitro ADP-ribosylation assays to quantify activity of purified toxin. These assays will also be used to characterize site-directed ArtA mutants. Percent completion of Objective 3 = 20%. Percent estimated completed in next project period =50% The current impacts of Objective 1 are: 1) prevalence estimate of artAB toxin positive isolates of bovine Salmonella and, 2) confirmation of toxin protein and RNA expression from bovine clinical isolates. The current impacts of Objective 2 are: 1) the establishment of a cytotoxic profile of purified ArtAB toxin on tissue culture, and 2) confirmation of fetuin receptor-binding preference in vitro. Objective 3 is in progress and thus there are not current impacts for this objective. Current outcomessupport the hypothesis that the ArtAB toxin is a virulence factor involved in bovine Salmonella infection and a potential vaccine antigen

Publications

Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: Heller, E. and J.K. Tinker. Cloning and purification of the A subunit of a novel enterotoxin in Salmonella enterica Typhimurium. Boise State Undergraduate Research Conference. Boise, ID. April 9, 2019.
Type: Conference Papers and Presentations Status: Accepted Year Published: 2018 Citation: Rodriguez, A., Whiting, R., and J.K. Tinker. Characterization of a novel Salmonella toxin via recombinant protein purification. Idaho INBRE Annual Conference, Moscow, ID. August 3, 2018.
Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: Hansen, M., Rodriguez, A., Scarbrough, D. and J.K. Tinker. Characterization of AB-type toxin from Salmonella enterica Typhimurium for the development of a bovine vaccine. Idaho Conference for Undergraduate Research, Boise, ID. July 29, 2019.