Performing Department
VETERINARY MICROBIOLOGY AND PREVENTIVE MEDICINE - CVM
Non Technical Summary
Campylobacter jejuni is a major concern for food safety, animal health, and public health. Two zoonotically important C. jejuni strains will be utilized during this study, C. jejuni clone SA (sheep abortion strain) and reference human pathogenic strain C. jejuni 11168. Small RNAs (sRNAs) have been demonstrated to be expressed by C. jejuni, but little is known about their regulatory mechanisms and physiological functions. Our preliminary research has demonstrated that two C. jejuni sRNAs (CjNC110 and CjNC140) directly affect phenotypes necessary for survival, including phenotypes connected to biofilm formation. The goal of the proposed research is to decipher the regulation of biofilm formation by small RNAs CjNC110 and CjNC140. The central hypothesis is that sRNA CjNC110 and CjNC140 regulate biofilm formation and that their ability to regulate biofilm formation contributes to chicken colonization. To test this hypothesis, I propose the following objectives:Objective # 1:Determine the molecular and phenotypic roles of sRNA CjNC140 and CjNC110 regulation during biofilm formation.Objective # 2:Conduct competitive fitness assays and RNAseq to reveal chicken colonization and environmental survival determinants modulated by CjNC110 and CjNC140.This training grant will support food safety, nutrition, and health, and outcomes will support food and nutrition translation. Critically, training will support the post-doctoral applicant who is interested in microbes afflicting agriculture. Our objectives are innovative because characterizing the molecular mechanisms of C. jejuni biofilm formation in association with sRNAs could provide novel targets for drug development, ultimately reducing Campylobacter persistence pre- and post-harvest of the chicken reservoir.
Animal Health Component
100%
Research Effort Categories
Basic
100%
Applied
0%
Developmental
0%
Goals / Objectives
Campylobacter jejuni is a major concern for food safety, animal health, and public health. Two zoonotically important C. jejuni strains will be utilized during this study, C. jejuni clone SA (sheep abortion strain) and reference human pathogenic strain C. jejuni 11168. Small RNAs (sRNAs) have been demonstrated to be expressed by C. jejuni, but little is known about their regulatory mechanisms and physiological functions. The primary goal of the proposed research is to decipher the regulation of biofilm formation by small RNAs CjNC110 and CjNC140. As a post-doctoral training grant, pursuit of this primary goal will also enable attainment of additional goals of the PD including mentoring, networking, teaching, and scientific training. Critically, training will support the post-doctoral applicant who is interested in microbes afflicting agriculture. The PD will be 100% committed to completing the proposed work, and the training and data generated is expected to be used for pathway to independence grant applications.This training grant will support food safety, nutrition, and health, and outcomes will support food and nutrition translation. Our proposal is relevant to the Food Safety and defense priority area [A1332 (g)] and encompasses basic research aimed at reducing the risk of unintentional contamination of foods. Specifically, our work aims to foster future intervention strategies to control C. jejuni persistence in the environment, host, and within the industry setting pre- and post-harvest. C. jejuni biofilm formation can occur on farms or in storage after harvest encompassing the farm to fork chain, so molecular insights would benefit public health. Additionally, the basic research proposed will lay the foundation for future basic and applied research. The proposed goals are innovative because characterizing the molecular mechanisms of C. jejuni biofilm formation in association with sRNAs could provide novel targets for drug development, ultimately reducing Campylobacter persistance pre- and post-harvest of the chicken resevior.
Project Methods
Objective # 1:Determine the molecular and phenotypic roles of sRNA CjNC140 and CjNC110 regulation during biofilm formation.Study 1.1: Autoagglutination measurement followed by analysis of transcriptional changes during biofilm formation. To begin this investigation, autoagglutination (AGG) assays will be performed under conditions simulating poultry slaughterhouse processing to examine the phenotypic and molecular roles of sRNA CjNC110 and CjNC140 related to biofilm formation. I will use C. jejuni 11168 and C. jejuni IA3902 along with their isogenic sRNA mutants ΔCjNC110, ΔCjNC140, ΔCjNC110ΔCjNC140, and complement strains, which our lab already generated. To mimic industrial condition, temperature, atmosphere, and nutrient acquisition will be examined. The poultry slaughter line can have a wide range of temperatures, but a subset will be tested: 30?C and 42?C (dirty areas), 10?C (putative clean areas), and 4?C (chilling areas where chicken carcasses are stored) (20). Autoagglutination ability will also be tested using microaerophilic (85% N2, 5% O2, 10% CO2) and aerobic (20% O2) conditions under each temperature condition. Lastly, 10% chicken juice will be supplemented within the media, which aids biofilm formation and mimics the nutrients available to C. jejuni during carcass processing. Briefly, 2 mL of 0.5 A600 adjusted C. jejuni culture suspension will be added to: 1) PBS (internal control) and 2) PBS + 10% chicken juice. Next, C. jejuni culture suspension will be incubated under each temperature and atmosphere condition described above. The level of autoagglutination (A600) will be determined at 24 h. This study will include three technical replicates during three separate assays to assess the average A600, which will be statistically analyzed using two-way ANOVA (P<0.05). Additionally, total RNA will be extracted from a subset of treatment conditions (n=72) by using QIAzol lysis reagent and miRNeasy minikit (Qiagen). For RNAseq analysis, PBS + 10% chicken juice under microaerophilic and aerobic conditions at 30?C will be utilized. In each instance, WT controls will be compared to each mutant background. The stranded total RNA library will be prepared and sequenced on an Illumina NovaSeq 6000 machine at the Iowa State University DNA facility. Rockhopper will be used to analyze the statistical differences in gene expression with a fold change cut-off of >1.5.Study 1.2: Biofilm measurement using crystal violet staining and biofilm structure analysis using scanning electron microscopy (SEM). Crystal violet staining as described previously with modification by supplementation with 10% chicken juice to measure biofilm formation. I will use C. jejuni 11168 and C. jejuni IA3902, along with their isogenic sRNA mutant backgrounds, as described in study #1.1. Again, conditions mimicking the industrial environment will be tested, including temperature, atmosphere, and nutrient acquisition, as described in study #1.1. Briefly, 20 μl of 0.5 A600 adjusted C. jejuni culture suspension will be added to 6-well plates containing: 1) 180 ul MH broth (internal control) and 2) 180 μl 10% chicken juice. After incubating under each condition at 48 h, crystal violet staining will be performed (19, 27), and the biofilm production will be assessed by measuring A595. This study will have three biological replicates and three technical replicates per assay, and the averages will be statistically analyzed using two-way ANOVA. Another method to examine biofilm formation includes scanning electron microscopy (SEM). Again, C. jejuni 11168 and C. jejuni IA3902 along with their isogenic sRNA mutant backgrounds will be examined. Briefly, 200 μl of 0.5 A600 adjusted C. jejuni 11168 culture suspension will be added and incubated using microaerophilic atmosphere for 48 h. Next, C. jejuni will be fixed overnight at 30?C. Samples will be prepared and visualized using a JEOL 2100 200kV SEM microscope at the Roy J. Carver High-Resolution Microscopy Facility at Iowa State University.Study 1.3: Determine the mRNA binding partners of sRNAs CjNC110 and CjNC140 associated with biofilm formation using electrophoretic mobility shift assays (EMSA).To evaluate glycan producing systems and molecular interactions with CjNC140 and CjNC110, electrophoretic mobility shift assays (EMSA) will first be used to validate the pairing of sRNA(s) to putative glycan-partner mRNAs (36, 37). Specifically, RNA oligomers of ptmA and CjSA_1234 containing the predicted 5'-UTRs base-pairing regions to CjNC110 and CjNC140 will be generated for this study [Integrated DNA Technologies (IDT)]. Next, glycan gene products that are verified to interact with each sRNA via EMSA will be deleted from the genome of IA3902 WT utilizing Gibson Assembly with synthetic DNA oligos (IDT) and homologous recombination, as previously performed by our lab. The constructs will then be moved into each sRNA mutant background to generate double knockout mutants. The autoagglutination (study #1.1) or biofilm phenotype (study #1.2) of each sRNA mutant alone, and the double knockout mutants will then be compared to IA3902 WT to examine if additive phenotypic effects occur, which would indicate the sRNA regulates ptmA or CjSA_1234.Objective # 2:Conduct competitive fitness assays and RNAseq to reveal chicken colonization and environmental survival determinants modulated by CjNC110 and CjNC140. in vitro shaking and static growth curves will be conducted in MH broth supplemented with chicken juice or chicken cecal extracts using mono-cultures (internal controls) and co-cultures. Co-culture growth is a useful strategy to test strain fitness as each strain must compete for the same limited resources over time. The optical density (A600) will be measured, and CFU/mL determined by the drop-plate method after serial passage at 12h, 24h, 36h, and 48h using positive selection on MH agar plates with appropriate antibiotics. The following co-cultures will be compared: IA3902 WT vs. ΔCjNC110, IA3902 WT vs. ΔCjNC140, ΔCjNC140 vs. ΔCjNC110, IA3902 WT vs. ΔCjNC110c, and IA3902 WT vs. ΔCjNC140c. The growth fitness of each co-culture will then be examined to determine if the presence or absence of CjNC110/CjNC140 enables a colonization fitness advantage or disadvantage. Next, the same mono-cultures and co-cultures described above will be utilized to examine the competitive fitness of the C. jejuni sRNA strains using the chicken host infection model. For each group, 18 3-day-old chicks will be inoculated with 200 µl of bacterial suspension containing 1 x 107 CFU for mono-cultures and a 1:1 ratio equaling 1 x 107 CFU for co-cultures. Groups of chicks will be housed in separate brooders with no contact between the groups. At pre-determined days post-inoculation during weeks 1, 2, and 3, 6 chicks from each group will be randomly selected for humane euthanasia, and cecal contents will be harvested to enumerate CFU/mL to analyze colonization levels, as previously conducted by our lab. Briefly, serial dilutions of ceca contents will be performed, followed by plating on MH agar plates with appropriate antibiotics for positive and negative selection to compare CFU/mL of each strain within a co-culture. For both the growth curve and chicken colonization study, the competitive index (CI) will be calculated by determining the ratio of average colonization (CFU/mL) of each strain over the other for each co-culture comparison.Study 2.2: RNAseq analysis of ceca content colonized by C. jejuni sRNA knockout strains to reveal colonization determinants modulated by CjNC110 and CjNC140.mono-culture cecal extracts from study # 2.1 will be collected and stored in QIAzol for downstream RNA extraction and analysis as described in study# 1.1 with modification to isolate C. jejuni RNA from ceca content followed by RNAseq.