E. COLI PROBIOTIC CARRYING A CONJUGATIVE PLASMID ENGINEERED WITH CRISPR-CAS9 TO CONTROL PATHOGENS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1013175
• Project Start Date: Jul 1, 2017
• Project End Date: Jun 30, 2022
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIV OF IDAHO
875 PERIMETER DRIVE
MOSCOW,ID 83844-9803

Performing Department
School of Food Science

Non Technical Summary
This project includes basic research applied to a specific agricultural problem: the ubiquitous presence of the human pathogensEscherichia coli O157:H7 (O157), certain other enterohemorrhagic E. coli (EHEC), and Salmonella in food-producing ruminants.We propose to work on a novel intervention to reduce these human bacterial pathogens from the ruminant reservoir.HarmlessE. coli from the gastrointestinal tract of cattle will be engineered to specifically kill pathogenic E. coli and Salmonella. We willuse naturally occurring bacterial exchange of DNA to create the lethality. The harmless E. coli will be fed to cattle as a probioticand their presence will eliminate the most common disease-causing EHEC and Salmonella. A commensal E. coli will beengineered to carry the CRISPR-Cas9 cassette in an Inc-P broad host range conjugative plasmid to selectively target anddestroy the pathogens and prevent subsequent re-infections. The CRSPR-Cas9 cassette will recognize an EHEC-specific andSalmonella-specific DNA sequence and selectively and efficiently remove these human pathogens from the gastrointestinal tractof cattle without disrupting the cattle microbiome.

Animal Health Component

50%

Research Effort Categories

Basic

50%

Applied

50%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
712	3440	1100	100%

Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
3440 - Meat, dairy cattle;

Field Of Science
1100 - Bacteriology;

Keywords

e. coli o157:h7

salmonella

cattle

crispr-cas9

food safety

Goals / Objectives
Goal 1: Design a probiotic commensal E. coli carrying a specific CRISPR-Cas to reduce or eliminate certain EHEC fromcattle.Goal 2: Expand this intervention approach to control Salmonella species.

Project Methods
Objectives 1.1 and 2.1Intimin is required for EHEC virulence and is localized in the LEE island of pathogenicity for EHEC and the big 6 strains. Using abioinformatics approach, we aligned all intimin family genes from these strains and identified the 30-mer 5-tatctttcgcgtaatcgccgttcagagatc-3 as a unique sequence for the pathogenic E. coli family of intimin genes. This sequence sharesno homology with other enteric bacteria or commensal E. coli. This preliminary data suggests, this sequence is the primecandidate for constructing a CRISPR-cas9 guide sequence directed at EHEC and the big 6. Using a similar approach for nontyphoidalSalmonella, we likewise identified a unique 20-mer in the Salmonella pathogenicity island 1 (Sp1) within the coding region of a type-III secretion system-dependent ATPase.Objective 1.2 and 2.2The CRISPR-cas9 system that allows insertion of guide RNA sequences at a unique Bsa1 site will be obtained from acommercial source. The identified intimin target sequence will be synthesized with Bsa1 restriction sites on the 5' and 3' ends.Complementary target strands will be annealed and cloned into the Bsa1 site on a CRISPR-cas9 containing plasmid.The entire CRISPR-Cas9 cassette with the intimin target guide RNA will be amplified and cloned into a recombinant pKS+containing a 1kb region of pRK24 determined to be nonessential for plasmid maintenance or transfer. This CRISPR-Cas9 regionand pRK24 homologous flanking DNA will then be cloned into pRK24 by homologous recombination. A similar strategy will beused for the Salmonella-specific target.Objectives 1.3 and 2.3Once the pTarget-F is constructed, we will initiate in vitro studies to determine its stability in commensal strains of E. coli and itseffectiveness at killing various strains of O157 and the big 6. For example, pTarget-F constructs will be placed in several bovinecommensal E. coli strains (from our stock collection at the University of Idaho) and will be serially transferred without antibioticselection to determine long-term stability.Conjugation experiments will be conducted to determine the specificity of the plasmid for killing O157 and the big 6 strains.Commensal E. coli with the pTarget-F construct (CmR, nalS) will be mated with O157 and the big 6 nalR CmS wild-type strainsand an O157 Δeae nalR CmS control that lacks the intimin target sequence. Conjugations will be conducted in liquid or on sterilefilters. Equal concentrations of donor and recipients will be combined in broth or onto multiple sterile millipore filters and thenplaced on LB agar plates. We will measure the efficiency of killing over time and genetically analyze survivors. In contrast, O157Δeae control cells should rapidly become CmR, but not display a decrease in cell numbers.Also, we expect survivors to have (i) acquired pTarget-F that have mutations in the CRISPR-cas9 system or (2) mutations in thetarget eae DNA. We will analyze survivors by DNA sequence analysis. If O157 or big 6 survivors have acquired mutations ordeletions in the intimin gene, this may reflect an added benefit to this technique. Because intimin is an essential virulenceattribute, pTarget-F may not only kill O157 and big 6 strains, but may select for attenuated strains.Overnight broth cultures containing the probiotic and commensal enterics including Enterobacter, Serratia, Klebsiella,Pseudomonas, Yersinia, etc. will be used to test the construct stability and conjugative plasmid host range. Standardmicrobiological techniques will be used to plate for antibiotic resistance and sorted on selective medium for identification.Similarly, we will initiate laboratory biofilms experiments to test the stability of plasmid transfer between the probiotic strain andother enteric bacteria. These conditions mimic environmental conditions with low rates of division, induced stress responses,and exopolysaccharide production that may influence transfer and maintenance efficiency. Plasmids that show naturaladaptations that confer persistence under these conditions will be good candidates for animal studies.Experiments with pTarget-Sal will be done to measure, plasmid stability, killing specificity, host-range, and transfer efficiency inthe same conditions tested for pTarget-F.Objectives 1.4 and 2.4:To assure rumen maturity and most closely represent animals that enter our food chain, Holstein steers of at least five monthsof age will be used in all experiments. Animals will be housed individually and challenged with 1010 CFU of a six strain mixture ofO157, the big 6, or Salmonella. Four animals will be used to screen O157, each big 6 serotype, and Salmonella strains. Somechallenges will use multiple pathogens in individual animals. Animals treated or not with the probiotic will be in separate barnfacilities with no contact or cross-contamination of feed, water, or by handling personnel. Statistical justification for the number ofanimals to be tested comes from the calculation that four animals per group will detect a difference of 75% (1 of 4 compared to 4of 4) between the proportion of colonized animals in each group, with a confidence level of 90% and a statistical power of 80%(19). However, we anticipate that the difference between strains that colonize or are cleared will be 100% and that animals nottreated with the probiotic will have 4 of 4 animals colonized. In this situation, four animals per group provide a confidence levelof 95% and a power of 90% (19) to test for an effect from the probiotic.Cattle will receive the probiotic or the non-engineered parent commensal E. coli (control) in daily oral doses of 1010 CFU threetimes during the first and second week (pre- and post-pathogen-challenge) of the trial. The probiotic will not be given on thesame day as oral challenges to prevent inappropriate interaction between the probiotic and challenge doses.All animals will be challenged orally and/or by rectal application of bacteria to create O157-, big 6-, or Salmonella-positive cattle.Bacteria will be suspended in phosphate-buffered saline and administered orally using a large sterile syringe. O157 will beapplied directly to the recto-anal junction (RAJ) mucosa because it is the mucosal colonization site of O157 (21).Animals in O157 trials will be cultured for O157 using the RAMS technique as previously described (20). Similarly, wellestablishedstandard enrichment culture followed by PCR techniques will be used to detect the big 6 serotypes and Salmonella.Briefly, fecal samples will plated directly and after enrichment culture, onto Rainbow agar or Salmonella-Shigella and brilliantgreen agars and positive colonies will be confirmed by serology and PCR for Shiga toxin, eae, sp1 and sp2, respectively.Holstein steers (>5 months old) will be purchased and tested for the pathogens to establish carriage status. Animals will berandomly allocated to one of two experimental groups based on receiving the specific probiotic or the commensal E. coli control strain. Each pathogen will have a total of 8 animals/trial, 4 test and 4 control animals. The experiment will be conducted for fourweeks to accurately assess pathogen carriage and the effects of probiotic intervention.Sampling schedule: Fecal O157 shedding will be detected by RAMS culture on days -1, 1, 4, 7 and twice weekly thereafter for30 days post-inoculation. Samples will also be analyzed for the probiotic CFU density. Outcomes compared will include:prevalence of shedding, total CFU shed (calculated by estimating the best fit curve for the shedding pattern for each animal overthe 14- and 30-day post treatment period, and determining the area under the curve) and the time to cessation of detectablepathogen shedding.

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:Community members reached by presentations. Target audiences are scientists and professionals reached by publications and presentations. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?Yes, the details of our findings have been published in the peer-reveiwed scientific literature. What do you plan to do during the next reporting period to accomplish the goals?We plan to continue research efforts to understand O157, oher EHEC, and enterics. We will give scientific presentaitons at regional/national meetings and prepare manuscripts with our findings for publication.

Impacts
What was accomplished under these goals? We made progress in understanding how Escherichia coli O157:H7 (O157) colonizes and persists in teh bovine gastrointestinal tract. This effort indirectly aids us in accomplishing the goasl of this project. Most strains of O157 are noninvasive and weak biofilm producers; however, a subset of O157 are exceptions. FOr example, O157 ATCC 43895, an isolate from a disease outbreak, forms biofilms and invades epithelial cells. We used Tn5 mutagenesis to identify mutations lacking these traits and interestingly found a mutant that is responsible for both phenotypes. The insertion mapped within the curli csgB fimbriae locus. Screening of O157 strains for biofilm formation and cell invasion identified a bovine and a clinical isolate with those characteristics. This demonstrates that strains with curli fibbriae can colonize cattle and can cause human disease. Also, interestingy, a single base pair A to T transversion, intergenic to the curli divergent operons csgDEFG and csgBAC, was present only in biofilm-producing and invasive strains. We used site-directed mutagenesis to show that if this single base change was introduced into two curli-negative/noninvasive O157 strains, the strains were modified to form biofilms, produce curli, and gain invasive capability. Transmission electron microscopy (EM) and immuno-EM confirmed curli fibers. EM of bovine epithelial cells (MAC-T) co-cultured with curli-expressing O157 showed intracellular bacteria. The role of curli in O157 persistence in cattle was examined by challenging cattle with curli-positive and -negative O157 and comparing carriage. The duration of bovine colonization with the O157 curli-negative mutant was shorter than its curli-positive isogenic parent. These findings definitively demonstrate that a single base pair stably confers biofilm formation, epithelial cell invasion, and persistence in cattle.

Publications

• Type: Journal Articles Status: Published Year Published: 2020 Citation: Sheng, H, Y Xue, W Zhao, CJ Hovde and SA Minnich. 2020. Escherichia coli O157:H7 curli fimbriae promote biofilm formation, epithelial cell invasion, and persistence in cattle. Microorg. 8(4):580. https://doi.org/10.3390/microorganisms8040580.

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:Community members by presentations. Scientists and professionals through publications and presentations Changes/Problems:Although our novel intervention worked well ex vivo, it did not do so in the gastrointestinal tract. We continue our work to understand O157 and how it survives in the bovine gastrointestinal tract and the external environment (on the farm, in the feed, and in our food). What opportunities for training and professional development has the project provided?Both the technical support person and all the students that worked on this projectexpanded their understanding of microbiology,molecular biology, and interventions that could improve food safety. How have the results been disseminated to communities of interest?Scientific and professional groups learn of our findings through scientific publicaitons, scientific poster presentations, and presentations. What do you plan to do during the next reporting period to accomplish the goals?We plan to continue research efforts to understand O157, oher EHEC, and enterics. We will give scientific presentaitons at regional/national meetings and preparemanuscripts with our findings for publication.

Impacts
What was accomplished under these goals? Although we had much success using the commensal microbe with specific CRISPR-Cas mutations toeliminateEscherichiacoli O157 (O157) in vitro, we were unable to expand this to in vivo models. The problem is highly complex and the survival of the modified E. coli in the gastrointestinal tract was not reliable nor was its replication. Both conditions are required for success of this approach. Thus, we turned our efforts to basic understanding of the mechanism by which O157 andother EHEC colonize healthy cattle and even more basic studies of gene regulation in other enterics. Tothis end we have made significant contributions. We analyzed curli. O157is a weak biofilm producer and is considered a non-invasive pathogen. Some O157 strains do not meet this general characterization, however, including ATCC 43895, an outbreak-associated strain that forms biofilms and invades epithelial cells. To identify genes responsible for these traits, random Tn5 mutagenesis was carried out on O157 strain 43895. Mutants that had lost biofilm formation and the ability to invade epithelial cells were identified. The Tn5 mutation responsible for loss of both phenotypes mapped within a locus encoding curli bacterial surface fibers, not normally expressed on O157 at 37 degrees. Screening of laboratory stock O157 strains for biofilm formation and cell invasion resulted in identification of a bovine isolate (FB38) with those characteristics. A single base pair variation (A to T transversion), intergenic to the curli divergent operons csgDEFG and csgBAC, was present in biofilm-producing and invasive strains only. Using site-directed mutagenesis, this single base change was introduced into two curli-negative/noninvasive O157 strains with the result that both modified strains formed biofilms, produced curli and gained invasive capability at 37° C. Transmission electron microscopy (EM) and immuno-EM confirmed the presence of curli amyloid fibers on the surface of these O157 strains. EM of bovine epithelial cells (MAC-T) co-cultured with curli-expressing O157 strains showed intracellular bacteria within membrane-bound vacuoles. O157 naturally colonize and persist at the bovine terminal rectal mucosa so the role of curli in O157 persistence in cattle was examined by inoculating and sampling at that site in comparison groups of cattle. The duration of bovine colonization with the O157 curli-deficient mutant was significantly shorter than its curli-positive isogenic parent. These findings definitively demonstrate that a single base pair stably confers biofilm formation, epithelial cell invasion, and persistence in cattle to O157 bacteria. O157 survives and replicates both in the mammalian gastrointestinal tract and in the outside environment on the farm. How is accomplishes the gene regulation to do this is of interest. Weused Yersinia as a model of gene expression between animal host and the environment. Pathogenic bacteria recognize environmental cues to vary gene expression for host adaptation. Moving from ambient to host temperature, Yersinia enterocolitica responds by immediately repressing flagella synthesis and inducing the virulence plasmid (pYV)-encoded type III secretion system. In contrast, shifting from host to ambient temperature requires 2.5 generations to restore motility suggesting a link to the cell cycle. We hypothesized that differential DNA methylation contributes to temperature-regulated gene expression. We tested this hypothesis by comparing single-molecule real-time (SMRT) sequencing of Y. enterocolitica DNA from cells growing exponentially at 22°C and 37°C. The inter-pulse duration ratio was used to compare DNA from cells grown at each temperature. All 565 YenI restriction sites were fully methylated at both temperatures. Among the 27,118 DNA adenine methylase (Dam) sites, 42 had differential methylation patterns while 17 remained unmethylated regardless of temperature. A subset of the differentially methylated Dam sites localized to promoter regions of predicted regulatory genes including LysR-type and PadR-like transcriptional regulators, and a cyclic-di-GMP phosphodiesterase. The unmethylated Dam sites localized with a bias to the replication terminus, suggesting they were protected from Dam methylase. No cytosine methylation was detected at Dcm sites.Pathogenic bacteria recognize environmental cues to vary gene expression for host adaptation. Moving from ambient to host temperature, Yersinia enterocolitica responds by immediately repressing flagella synthesis and inducing the virulence plasmid (pYV)-encoded type III secretion system. In contrast, shifting from host to ambient temperature requires 2.5 generations to restore motility suggesting a link to the cell cycle. We hypothesized that differential DNA methylation contributes to temperature-regulated gene expression. We tested this hypothesis by comparing single-molecule real-time (SMRT) sequencing of Y. enterocolitica DNA from cells growing exponentially at 22°C and 37°C. The inter-pulse duration ratio was used to compare DNA from cells grown at each temperature. All 565 YenI restriction sites were fully methylated at both temperatures. Among the 27,118 DNA adenine methylase (Dam) sites, 42 had differential methylation patterns while 17 remained unmethylated regardless of temperature. A subset of the differentially methylated Dam sites localized to promoter regions of predicted regulatory genes including LysR-type and PadR-like transcriptional regulators, and a cyclic-di-GMP phosphodiesterase. The unmethylated Dam sites localized with a bias to the replication terminus, suggesting they were protected from Dam methylase. No cytosine methylation was detected at Dcm sites.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2019 Citation: OBrien, AD and CJ Hovde, co-chairs. 2019. National Advisory Committee on Microbiological Criteria For Foods. Virulence Factors and Attributes that Define Foodborne Shiga Toxin-producing Escherichia coli (STEC) as Severe Human Pathogens. J Food Protect 82:724-767.
• Type: Journal Articles Status: Accepted Year Published: 2019 Citation: Van Hofwegen, DJ, CJ Hovde, and SA Minnich. 2019. Comparison of Yersinia enterocolitica DNA methylation at ambient and host temperatures. BioRxiv.10.1101/2019.12.16.878991 Cold Spring Harbor Laboratory https://biorxiv.org/cgi/content/short/2019.12.16.878991v1
• Type: Journal Articles Status: Under Review Year Published: 2020 Citation: Sheng, H, Y Xue, W Zhao, CJ Hovde and SA Minnich. 2019. Escherichia coli O157:H7 curli fimbriae promote biofilm formation, epithelial cell invasion, and persistence in cattle. PLOS Path. Submitted.

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:Our target audience is other scientists in academia, industry, and government and stake holders. These groups are reached through scientific publication in peer-reviewed journals and include novel research results and review articles. In addition, academicians and students are reached through seminar presentations. Changes/Problems:We hypothesize that the conjugation process that works very well in vitro, dos not work efficiently under strict anaerobiosis, conditions that occurr inthe gastrointestinal tract. We are working to isolate mutants to overcome this problem. What opportunities for training and professional development has the project provided?Dr. Haiqing Sheng has had the opportunity to expand his expertise in molecular biology and to collaborate with scinetists with bioniformatics expertise. 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?We will continue or efforts to understand the relationship between healthy cattle and the human pathogens in the entrohemorrhagic E. coli group. We had much success in eliminating O157 in vitro, but were not able to expand the bacterial reduction in vivo. We have turned our efforts to basic understanding of the mechanisms by whichO157 colonizes healthy bovine cattle.

Impacts
What was accomplished under these goals? We were successful in creating a commendal E. coli that had a specifically engineered region to mate with wild-type O157 and eliminate them by cell lysis. This strain reduced O157 in vitro, effectively. We were unable to repeat this reduction in vivo using suckling mice.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Hovde, CJ. 2018.10 Highly Cited Papers Published in Foodborne Pathogens and Disease Authors Perspectives on Their Highly Cited Papers Published in Foodborne Pathogens and Disease No Access Escherichia coli O157:H7: Animal Reservoir and Sources of Human Infection. Foodborne Path Dis. 15(1):3-10.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Haiqing Sheng, H, M Duan, SS Hunter, SA Minnich, ML Settles, D New, JR Chase, and CJ Hovde. 2018. High-Quality Complete Genomes of Three Bovine Shiga Toxin-producing Escherichia coli O177:H- (fliC-H25) Isolates Harboring Virulent stx2 and Multiple Plasmids. Genome Announc. 6(7):e01592-17. doi: 10.1128/genomeA.01592-17. PMID: 29449403 PMCID: PMC5814490.
• Type: Journal Articles Status: Accepted Year Published: 2018 Citation: Duan M, SS Hunter, SA Minnich, MW Fagnan, D New, CJ Hovde, and H Sheng. 2018. Complete Genome Sequence of a Broad Host Range Shiga Toxin-Converting Bacteriophage SH2026Stx1 Isolated from Escherichia coli O157:H7. Genome Announc. 6(25):e00490-18 doi: 10.1128/genomeA.00490-18
• Type: Journal Articles Status: Published Year Published: 2018 Citation: NATIONAL ADVISORY COMMITTEE ON MICROBIOLOGICAL CRITERIA FOR FOODS. 2018. Virulence Factors and Attributes that Define Foodborne Shiga Toxin-producing Escherichia coli (STEC) as Severe Human Pathogens. USDA, FSIS, Office of Public Health Science, PP3, 9-177B, Washington, DC. https://www.fsis.usda.gov/wps/portal/fsis/topics/data-collection-and-reports/nacmcf/nacmcf.

Progress 07/01/17 to 09/30/17

Outputs
Target Audience:The target audience is other scientists, but this reproting period was brief so we did not communicate any results, formally. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported 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?We plan to give scientific presentations at regional/national meetings and to prepare a manuscript to submit for publicaiton.

Impacts
What was accomplished under these goals? The project has begun. For Goal 1. Guide RNAs were designed and constructs kill specific E. coli O157:H7 in vitro.

Publications