Performing Department
Poultry Science
Non Technical Summary
Understanding the basic physiological properties of Salmonella in terms of its metabolism and how and when the organism expresses its virulent traits, that cause sickness in humans and farm animals, is essential for developing vaccines and other methods to reduce the Salmonella load in the food supply and prevent Salmonellosis in humans. The proposed studies are based on the fact that the gut of the animal host is anaerobic (i.e., has low oxygen concentration) while the food product is aerobic. Clearly the Salmonella cells have to switch their metabolic activities to match the environment they encounter during their life cycle between the food product and the host gut. There are at least three major global regulators that allow the organism to adjust between the aerobic and the anaerobic environments. We believe that these regulators are also involved in the regulation of virulence. We have already exploited one of these global regulators (i.e., FNR) and developed a US-patented live attenuated vaccine strain. In this proposal, we will expand our current studies to discover new and robust Salmonella Vaccines for farm animals to help reduce the incidences of Salmonellosis in humans.
Animal Health Component
30%
Research Effort Categories
Basic
60%
Applied
30%
Developmental
10%
Goals / Objectives
Salmonella enterica serovar Typhimurium (S. Typhimurium) is a facultative, intracellular pathogen responsible for disease across a broad range of hosts and is a useful model for systemic infection. About 4% of the S. Typhimurium genome is involved in virulence in mice and most of the virulence genes are clustered in regions of the chromosome called "Pathogenicity Islands". Salmonella Pathogenicity Island 1 (SPI-1) contains genes important in the invasion of epithelial cells, while SPI-2 contains genes required for survival and replication in macrophages. It is well known that iron, oxygen, and nitric oxide are also important factors in host-microbe interactions and in oxidative stress. Our long-term goal is to elucidate the regulatory networks in S. Typhimurium that are involved in the coordinated regulation of cellular metabolism, oxidative stress defenses, and pathogenesis in order to advance the development of novel strategies and therapeutics for the treatment and prevention of salmonellosis. We hypothesize that coordinated regulation of cellular redox and iron homeostasis in S. Typhimurium plays an important role in its virulence, metabolism, ability to survive sudden environmental changes encountered in the host, and to cause illness. Our long-term goal is to elucidate the regulatory networks in S. Typhimurium that are involved in the coordinated regulation of cellular metabolism, oxidative stress defenses and pathogenesis in order to advance the development of novel strategies and therapeutics (e.g., vaccines) for the treatment and prevention of salmonellosis. We hypothesize that coordinated regulation of cellular redox and iron homeostasis in S. Typhimurium plays an important role in its metabolism, ability to tolerate sudden environmental changes encountered in the host, expression of virulence genes and ability to cause illness. Our hypothesis is supported by our findings (2) that FNR (the master regulator of anaerobic metabolism) also regulates many of the S. Typhimurium virulence genes (i.e., Salmonella pathogenicity island-1, SPI-1, and the virulence operon, srfAB). Furthermore, an FNR mutant was attenuated, both in vivo and ex vivo. Based on these findings and on our previous experience in microbial biology and oxidative stress, we plan to study the combined global effects of the redox and iron regulators on cellular metabolism and pathogenesis in Salmonella.
Project Methods
Specific Aim #1: To test the hypothesis that the global regulators of redox and iron cooperatively coordinate cellular metabolism and virulence in S. Typhimurium.a) Genome-Wide Transcriptional Profiling in response to Combinatorial Mutations in fnr, arcA, and fur using Anaerobic Growth Conditions:• Media and growth conditions: We will use the same conditions as those used before . All strains will be grown at 37°C under anaerobic conditions using MOPS-buffered (100mM, pH 7.4) Luria-Bertani (LB) medium supplemented with 20mM D-xylose to avoid the glucose-based catabolite repression and the indirect effects of pH on gene expression (41). Anaerobic conditions will be obtained using a Coy® anaerobic chamber. All media will be prepared from the same lot of LB supply to avoid possible "lot-to-lot" variations. • Sample collection, RNA extraction, and processing: We will use the same protocols and procedures as was performed in the preliminary studies. Briefly, the cells will be harvested when the cultures reach an optical density (OD600) of 0.35-0.45, unless specified otherwise. For each strain, three independent cultures will be grown. The samples will be treated with RNAlater (Qiagen, Valencia, CA), to fix the cells and preserve the quality of the RNA during extraction and storage. The RNA will be extracted using RNeasy RNA extraction kit (Qiagen). • Data analysis: Spots will be analyzed by adaptive quantification as previously done (2). Confidence intervals and P values (p ≤ 0.05) on the expression change will also be calculated using two systems: Pair wise comparisons, calculated using a two-tailed Student's t test. In this case, microarray data will be analyzed using the MEAN and TTEST procedures of SAS-STAT statistical software (SAS Institute, Cary, NC). b) Assess the Pathogenicity of the Different S. Typhimurium Regulatory Mutants in animal models:Virulence of the combinatorial mutants : The virulence of the S. Typhimurium will be tested in a typhoid mouse model. Thus, the wild-type and the mutants each grown in LB-xylose-MOPS media under aerobic or anaerobic conditions will be administered orally (p.o.) as well as intraperitoneally (i.p.) in C57BL/6 mice as described (2). The surviving mice (after 20-days post-infection) will be sacrificed and the livers and spleens will be harvested, homogenized, and tested for the presence of Salmonella to determine whether the mice were able to clear the infection. Also, the bacteria will be recovered from the dead mice to confirm the stability of the different mutations in vivo. The experiments will be repeated twice.At the end of each experiment animals will be humanely sacrificed using CO2 inhalation. Specific Aim # 2: To Determine the Modes of the Coordinated Interactions Between the Three Global Regulators and their Effects on Virulence. Determinations of Protein-Protein and Protein-DNA Interactions: We plan to use different techniques for the detection of the different protein-protein and protein-DNA complexes. However, the different methods require physiologically active, pure regulatory proteins and their specific antibodies.Production of pure FNR, ArcA, and Fur proteins:We will use the Champion™ pET101 Directional TOPO®. Expression Kit (Invitrogen) to produce pure S. Typhimurium Fur, FNR, and ArcA proteins. This system utilizes expression vectors and a BL21 Star™ E. coli expression strain optimized for producing maximum protein yields. We know that the purification and utilization of ArcA, FNR, and Fur require very special precautions, since these proteins are intrinsically sensitive to changes in the environment (i.e., iron, oxygen, redox, etc.). Therefore, we will grow the cells and isolate the proteins under physiological conditions where they are most active. The purified proteins will be used to raise polyclonal antibodies as well as in protein-protein and in protein-DNA binding studies.Production of antibodies:We will use the purified proteins (ArcA, FNR, and Fur) obtained above to produce antigen-specific antibodies. Each protein (1-2 mg) will be mixed with Hunter's Titer MaxTM adjuvant, sonicated to form an emulsion, and injected into New Zealand white female rabbits. The rabbits will be boosted with ~1 mg protein (in saline or in half the dose of Titer MaxTM) 3-4 times at 2-week intervals. Blood will be collected and the serum will be tested for the presence of the specific antibodies.Gel retardation assays:Gel retardation (Band shift) assays will be performed using the candidate promoter PCR-fragments (~ 200bp) labeled with DIG [using DIG Gel Shift Kit (Roche)]. If needed, we could also use 32P-end labeled PCR-amplified DNA fragments of the selected promoters. We will test the effects of adding different concentrations of the appropriate, pure/activated, transcription factors [Fur, FNR, and ArcA(P)] singularly and in combinations and determine the distance of the shift. We will include sodA and mutM DNA fragments as our positive controls [i.e., GOLD STANDARDS - we know that they are regulated by >1 transcriptional factor in S. Typhimurium (unpublished data)]. The band shifts will be performed in duplicate by a method that we previously used (101).Specific aim #3: To validate the potency of the FNR Vaccine strain (Patents # US 8,101.168 B2 and US 8,435,506 B2 - Titled "Attenuated FNR deficient Enterobacteria), and others (to be developed during the course of the proposed studies), to reduce infection by wild-type S. Typhimurium and related Salmonella serovars in Mice, Poultry and other animals.Assessment of the Immunogenicity (Vaccine Efficacy) of the Combinatorial Mutants:For this part of the study, we will concentrate on the use of the BALB/c strain of mice because of its susceptibility to Salmonella. Mutants that show attenuation in the virulence challenge will be further examined for their efficacy as vaccines for preventing infection by the homologous pathogen, wild-type S. Typhimurium ATCC 14028s. Thus, Salmonella mutants that show a potential for vaccine efficacy in mice will be retested in 6-8 week-old BALB/c mice. We will use oral or nasal routs for administering the potential "vaccine strain", as these are the preferred routs for eliciting mucosal immune responses. About 109 cells will be presented via p.o or i.n. to groups of five mice and a control group with a phosphate-buffered saline. The animals will be given two booster doses (109 organisms each) at two week intervals. After four weeks the experimental animals and the controls will be tested for there ability to survive an oral challenge with 108 to 109 cells of the virulent wild-type strain. The animals will be scored for sign of illness and survival for up to 3 weeks. Parallel groups of mice will be treated with the attenuated mutants (i.e., immunized) and sacrificed for testing for the presence of protecting antibodies in serum (IgG) and intestinal washings (IgA) using ELISA and published protocols. Also, the immunized animals will be checked (at ~ 6-8 weeks after immunization) to determine if the vaccine strain is eliminated from the spleens and livers.Assessment of the Immunogenicity (Vaccine Efficacy) of Candidate Vaccine Strains in Poultry:For this part of the study, we will use chickens and turkeys. The birds (1 day old) will be divided into four (4) groups, 50 birds per group. Group #1 -vaccinated - no challenge; Group #2 -Vaccinated - challenged at 4 weeks with virulent S. Typhimurium (STM); Group #3 - PBS control - no challenge; and Group #4 - PBS control - challenged at 4 wks with virulent STM. The birds in each group will be tested for antibody responses, clinical responses, colonization, and shedding. We will use the same protocol to test for cross-protection against closely related Salmonella serovars (e.g., S. Enteriditis).