Performing Department
(N/A)
Non Technical Summary
The resident microbiota lining gut and respiratory mucosae provide critical defense against invading pathogens and overgrowth of resident pathobionts, a mechanism termed colonization resistance (CR). This mechanism incorporates both direct pathways of competitive exclusion of pathogens and indirect pathways that enhance host immunity. Although CR is best understood in the context of enteric infection, gut bacteria are also found in healthy lungs where they impact pulmonary cytokine levels and alveolar macrophage (AM) function, thus influencing the risk of pneumonia. Therefore, pulmonary CR may involve both interbacterial antagonism and indirect immunomodulation. There is a critical need to understand how bovine enteropathogens impact CR in the gut and lung. My long-term goal is to understand the relationships among Salmonella spp., the microbiota and host immunity in relation to animal health and food safety. Salmonella enterica threaten both bovine and human health. Salmonella Dublin (SeD) is a cattle-adapted serotype that causes systemic infections and predominantly manifests as pneumonia in calves, in contrast to the generalist serotypes that primarily cause localized enteritis. Salmonella has evolved several mechanisms to overcome CR. Salmonella exploits the inflammatory response to promote gut colonization. In addition, type VI secretion systems (T6SS) inject toxic effector proteins to kill bacteria and eukaryotic cells. Despite years of study, the mechanisms by which SeD overcome CR are poorly described. My preliminary data suggest that gut associated lung bacteria (GALB) may impact pulmonary pathogen colonization in calves. I hypothesize that GALB induce pulmonary immunity to resist SeD infection and SeD uses T6SS to overcome CR in the gut and lung.
Animal Health Component
60%
Research Effort Categories
Basic
40%
Applied
60%
Developmental
(N/A)
Goals / Objectives
The firstmajor goalof this project istoestablish the role of gut-associated lung bacteria (GALB) in pulmonary immune function and resistance against Salmonella Dublin (SeD) infection. This includesevaluatingthe relationship among immune cell populations, cytokine levels and the lung microbiota in bronchoalveolar fluid of healthy calves, asssessing the effects of GALB priming on SeD control in vitro. The hypothesis is that GALB stimulate low-grade inflammation that enhances alveolar macrophageimmune function, promoting pulmonary resistance against SeD.The second major goal is to determine the role of SeD Type VI secretion systems (T6SSs) in subversion of direct colonization resistance (CR). This includes establishing the requirement for T6SS in SeD colonization of the calf gut, determiningthe conditions in which T6SS are expressed and establishingthe role of T6SS in interbacterial competition between SeD and GALB in vitro.The hypothesis is that the2 SeD T6SSs (SPI-6 and SPI-19) allow SeD to overcome CR by disrupting the microbiota.
Project Methods
For Objective 1 (Establish the role of gut-associated lung bacteria in pulmonary immune function and resistance against Salmonella Dublin (SeD) infection), bronchoalveolar lavage fluid (BALF) will first be collected from 19 healthy calves. Calf health will be monitored biweekly; only healthy calves with normal lungs on ultrasound will be used for BALF collection by established sterile technique. I will correlate gut-associated lung bacteria (GALB) taxa with respiratory immune cell presence and cytokine levels. Using a power of 80% with a significance of 5% to detect a Spearman's r=0.6 (moderate correlation) between variables, the minimum required sample size was 19 calves. Samples will be separated for analyses of microbiota, cytokines, and flow cytometry for immune cell characterization. Immune cells of interest include Th17, ????T cells, and NK cells, primary producers of IL- 22 in response to the microbiota, and anti-inflammatory Treg cells, Th17 antagonists with Th plasticity.DNA will be isolated from a portion of BALF for microbiota analysis. I will sequence the V4 region of the 16S rRNA amplicon with the MiSeq Illumina Sequencer and analyze recovered sequences using the Mothur pipeline. The relative abundance of resolved genera and diversity metrics within BALF will be correlated to immune cell populations and cytokine levels using established multivariate analyses. An aliquot of BALF cells in lysis buffer with protease inhibitors will be lysed by sonication. The lysate total protein content will be determined and cytokines measured by ELISA using commercial kits validated for use with bovine samples (MilliporeSigma, MyBiosource). Additionaly, RT-qPCR will be used to quanitfy immune gene expression. I will measure MCP-1, IL-22, IL-23, IL-17A, IL-10, IL-6, IL-8, TNF??, IFN??, IL-1??, IL-1??, and TGF- ??. The cytokines IL-23 and TGF- ?? were selected because they affect Th17 cytokine production and differentiation, respectively; Th17 cells produce IL-22, IL- 17A, and IFN??. IL-22 stimulates IL-10, IL-8, IL-1??, IL-1?? and TNF?? production. Cytokine concentrations will be normalized to total protein content for data analysis. Multi-color flow cytometry will be used to identify alveolar macrophages (AM), Th17 cells, ????T cells, NK cells, and Treg cells from and aliquot of BALF.I will then determine the role of gut-associated lung bacteria (GALB) taxa on AM control of SeD infection in vitro. Cultured AM will be primed with conditioned media from AM infected with GALB species, including Prevotella spp., Blautia spp., and Bacteroides spp.Bacterial strains will be obtained from the ATCC. Primed AM monolayers will be inoculated with SeD at an MOI of 10:1 following established protocols for gentamicin protection assays. Intracellular survival will be determined by AM lysis and enumeration of intracellular CFU. An intracellular survival- deficient mutant (?ssaK; lacking the T3SS-2 needle) will be a negative control. In addition, infection of mock-primed AM will serve as controls. Intracellular survival will be calculated as CFU/viable AM. All in vitro assays will be performed using AM from at least 6 calves.I expect GALB abundance will correlate with BALF cytokine levels and immune cell populations in the healthy calf. Furthermore, I expect that GALB will improve AM control of SeD intracellular replication. I expect that SPI-6 and/or SPI-19 will be needed for SeD to colonize the gut and compete with GALB. One potential pitfall is the 3 GALB taxa may not be abundant in calf BALF microbiota. In this instance, I will identify the dominant genera in BALF (mean relative abundance>1%), using Phyloseq, and stimulate AM with GALB from species of dominant genera. Additionally, if I find the sensitivity of commercial antibodies is too poor to identify bovine cytokines, I will use qPCR to for cytokine gene expression analysis in BALF.For Objective 2 (Determine the role of SeD Type VI Secretion Systems (T6SS) in subversion of direct colonization resistance), SeD T6SS mutants lacking the entire Salmonella Pathogenicity Island (SPI)-6, SPI-19, or both will be made by Lambda Red recombinase following standard procedures. I will execute this objective using the calf ligated ileal loop model already established in the Elfenbein laboratory. Loops will be inoculated with up to 10^9CFU of a 1:1 ratio of WT and mutant (WT:?SPI-6, WT:?SPI-19, WT:?SPI-6?SPI-19). I will also perform competitive infections in the ?SPI-1 genetic background to eliminate inflammation effects on colonization. The competitive index (CI) will be determined as the ratio of WT/mutant after infection as compared to the inoculum. As controls, I will use the WT Salmonella Typhimurium (STm; ST4/74) and a ?SPI-1 mutant, as the STm infection kinetics in the model are documented. I will use 21 loops per calf: 6 competitive infections (duplicate), 6 controls (uninoculated loop, sterile media, STm, STm?SPI-1, SeD, and SeD?SPI-1) and 3 individual infections (?T6SS single and double mutants). A power calculation was performed to establish the mutant fitness using the paired t-test. Using a power of 80% with a significance level of 5% to detect a difference of 5-fold (CI) with a deviation of 3, I must use 5 calves (n=4.826). I expect that SPI-6 and/or SPI-19 will be needed for SeD to colonize the gut and compete with GALB, I anticipate no technical difficulties using the ligated ileal loop model or constructing mutants as the Elfenbein laboratory has >8 years of experience with the ligated ileal loop model and constructing Salmonella mutants.To determine the role of T6SS in interbacterial competition between SeD and GALB in vitro, I will first determine whether SeD uses T6SS to kill GALB using SeD-GALB well-established co-culture methodology. To simulate the lung microenvironment, experiments will be repeated in media supplemented with mucin. The CI of bait recovered will be calculated as SeD/bait after incubation compared with the starting ratio on at least 6 independent occasions. To determine the in vitro conditions in which T6SS (SPI-6 and SPI-19) are expressed, I will clone the promoters of critical structural genes from SPI-6 and SPI-19 into plasmids with reporter genes (GFP, lacZ) using well-established protocols. SeD will be grown in infection-relevant conditions to determine expression levels.