Performing Department
(N/A)
Non Technical Summary
Histomonosis is a devastating disease caused by the protozoan,Histomonas meleagridis.Mortality may exceed 80% in affected commercial poultry flocks. Currently, there are no approved interventional or control strategies to mitigateH. meleagridisinfections in poultry. The overall goal of this project is to investigate targetingH. meleagridisvirulence-associated peptidases and relevant inhibitors as novel methods to control histomonosis in poultry. In Objective 1, we will assess the expression pattern and functions of putative virulence-associated cysteine proteases in virulent and highly passaged, or attenuatedH. meleagridisstrains. In Objective 2, the enzymatic parameters of these cysteine proteases will be characterized, and potent inhibitors will be identified through high throughput screening. In Objective 3, we will evaluate the protective efficacy of vaccination with recombinant cysteine proteases generated in Objective 1 and the therapeutic potential of top candidate inhibitors selected in Objective 2 againstH. meleagridischallenge in turkey poults. The results of this project will provide workflow and insights related to the expression of virulence-related proteases in parasite infection as well as the efficacy of recombinant vaccines or inhibitors targeting these proteases in parasite virulence. This proposed project could lead to the development of novel commercially applicable methods to control histomonosis in commercial poultry flocks and provide a better understanding of histomonad biology.
Animal Health Component
100%
Research Effort Categories
Basic
75%
Applied
25%
Developmental
0%
Goals / Objectives
Histomonosis is a disease caused by a Trichomonadida protozoan, H. meleagridis. These protozoa are found in all gallinaceous birds but are especially prevalent in commercial chickens and turkeys. Turkeys are particularly susceptible to disease with morbidity and mortality of histomonosis-affected flocks often exceeding 70%, and frequently causing losses of entire flocks. Currently, there are no approved prophylactics or therapeutics available for histomonosis control, which causes devastating animal welfare and sustainability issues. Although our group showed that vaccination with attenuated H. meleagridis reduced the severity of disease and prevented substantial performance losses associated with virulent H. meleagridis challenge, commercial application is not currently feasible due to the costs related to the large-scale production and application. This highlights an urgent need for an economically viable approach to mitigate histomonosis that can be effectively integrated into a commercial operation. Previous transcriptomics and proteomics studies indicated that cathepsins are a major group of proteases produced by H. meleagridis and are associated with virulence. It is hypothesized that cathepsin proteases secreted by histomonads are involved with parasite attachment, replication, and degradation of cecal tissue promoting translocation to the liver via the hepatic portal vein. Immunization with recombinant H. meleagridis CPL (HmCPL) or inhibition of HmCPL activity may neutralize the secreted HmCPL to block damage to cecal and liver tissue and should be investigated as a method to control H. meleagridis infections in poultry flocks. The main objective of the proposed seed project is to investigate the activity of cathepsin L-like proteases (CPL), a subclass of cysteine proteases, expressed by Histomonas meleagridis to determine if targeting these proteases could be a novel method to control histomonosis in commercial poultry flocks. The long-term goals of the project include: 1) understanding CPL expression in virulent and attenuated H. meleagridis strains and how expression affects parasite infectivity, 2) identifying potent and specific candidates from a library of cysteine protease inhibitors that could be used as potential chemoprophylaxis or therapeutics, and 3) investigating the feasibility of using recombinant vaccines targeting CPL or other cysteine proteases actively expressed by histomonads during infection.
Project Methods
Objective 1. Assess the expression and function of HmCPL proteases in virulent and highly passaged H. meleagridis strains. We will use two classic cysteine protease inhibitors, LHVS and E-64, to suppress H. meleagridis cysteine protease activity in both culture media and tissue culture, specifically HD-11 cells. Our tissue culture model will unravel whether the parasites reside within autophagosomes through endocytosis. We will focus on two HmCPLs, HmCPL-1 and HmCPL-2, that were shown to be upregulated in a virulent H. meleagridis strain. 1A. Evaluate the differential expression of HmCPL-1 and HmCPL-2 in virulent and highly passaged stocks of two H. meleagridis strains. The upregulation of HmCPL-1 and HmCPL-2 in two H. meleagridis strains (Buford and PHL) compared to the corresponding attenuated passages that have been used in our previous challenge and vaccination studies will be evaluated. 1) Overexpress and purify the pro-form of HmCPL-2 in E. coli. As a zymogen, the pro-form of HmCPLs do not show catalytic activity. This is key because the pro-form will be used to produce polyclonal antibodies or as vaccine antigens. HmCPL zymogens will be activated in Obj. 2 for the preparation of active proteases. 2) Generate the polyclonal antibodies against HmCPL-1 and HmCPL-2 individually. To produce polyclonal antibodies, the individual pro-form of the recombinant HmCPLs will be injected into rabbits and mice. 3) Assess the expression, subcellular location, and secretion of HmCPL in virulent and attenuated H. meleagridis strains. Histomonas culture lysates and supernatants will be subjected to a Bicinchoninic Acid assay (BCA) for protein concentration quantification. A subset of the culture will be passed through a 5µm filter to collect the culture bacteria, to be included on immunoblots as a negative control. After protein samples have been collected, 20µg of each protein sample will be separated by SDS-PAGE, followed by immunoblotting. In addition, the partially purified H. meleagridis parasites will be attached to Cell-Tak-treated glass-bottom chamber slides (ibidi). Slides will be stained with individual anti-HmCPL antibodies along with a fixable LysoBrite Red DND-99 dye (AAT Bioquest), a marker used to identify food vacuoles in protozoa, via a standard immunofluorescence assay (IFA). 1B. Determine the roles of cysteine proteases in H. meleagridis infection using HD-11 cells. An in vitro tissue culture model will serve to assess the role of cysteine protease activity in parasite infection. 1) Establish HD-11 chicken macrophage-like cell line. HD-11 cells will be grown in RPMI-1640 medium supplemented with 8% FBS and 2% chicken serum at 37ºC with 5% CO2. 2) Evaluate interaction between H. meleagridis and HD-11 cells using IFA. Partially purified histomonads will be used to inoculate glass-bottom chamber slides (ibidi) coated with HD-11 monolayers. Samples will be collected up to 48h and stained with an antibody targeting H. meleagridis trophozoites and DAPI for parasite and host cell recognition. At each time point, the average number of parasites per host cell will be calculated to compare the infection efficiency for each H. meleagridis strain. In addition, the infected HD-11 cells will be stained with anti-chicken LC3-B antibodies (Novus) to evaluate if parasites localize in an autophagosome. 3) Evaluate the effect of cysteine protease inhibitors on H. meleagridis propagation and infectivity using HD-11 cells. We will individually inoculate HD-11 cells coated on chamber slides, with and without 1µM LHVS or E-64, with virulent or attenuated Buford and PHL trophozoites. Slides will be incubated and sampled as previously described before conducting IFA assays. Objective 2. Characterize the enzymatic parameters of HmCPL and identify potent inhibitors via high throughput screening. Screening cysteine protease libraries will help identify novel potent and specific inhibitors against HmCPL. 2A. Production of active recombinant HmCPL. CPL performs self-activation under an acidic environment. The pro-form of recombinant HmCPL produced in Obj. 1A will be exchanged into activation buffer, followed by the addition of 5mM DTT and incubation at 37ºC for 5h. Samples will be removed every hour and resolved by SDS-PAGE to monitor activation efficacy to choose the optimal activation time. The active form of HmCPL will migrate more quickly than its pro-form on SDS-PAGE due to the removal of inhibitory pro-peptide. Toxoplasma CPL (TgCPL) will be included as a positive control. 2B. Identify potent inhibitors against HmCPL from screening a cysteine protease inhibitor library. We aim to screen a pre-plated library containing 3,200 compounds. 1) Determine the enzymatic parameters for HmCPLs. Parameters used to screen for Toxoplasma CPL will be used to assess HmCPL inhibitors. 2) Quantify the inhibitory constants of LHVS and E-64 against HmCPL. LHVS and E-64 will be used in the screening assay as positive control and quenching reagent, respectively. To determine their inhibitory potencies against HmCPL, the abovementioned enzymatic assays will be conducted at different concentrations. Data will be analyzed by GraphPad Prism 9.0 software for Ki calculation (inhibitory constant). The concentration at 10-fold of Ki will be used in the following screening assay to efficiently block HmCPL activity. 3) Screen a mid-sized cysteine protease inhibitor library. The compounds that inhibit individual HmCPL activity by 99% will be picked for a determination of their inhibitory constants. Their cytotoxicity will be evaluated in HD-11 cell culture using AlamarBlue cell viability assay. The top five candidate inhibitors with the lowest inhibitory constants will be evaluated in vivo in Obj. 3. Objective 3. Evaluate the protective efficacy of vaccination with recombinant HmCPLs or the therapeutic potential of top candidate HmCPL inhibitors against H. meleagridis challenge in turkey poults. Recombinant HmCPLs generated in Obj. 1 and the top five HmCPL inhibitors identified in Obj. 2 will be evaluated in two preliminary in vivo studies. 3A. Assess protective efficacy of recombinant HmCPL proteases against H. meleagridis challenge in turkey poults. HmCPL-1 and HmCPL-2 recombinant proteins generated in Obj. 1 will be evaluated as vaccine candidates. Treatment groups will include: 1) nonchallenged, nonvaccinated control (NC), 2) H. meleagridis challenged, nonvaccinated positive control (PC), 3) challenged + vaccinated with 100 µg HmCPL-1 at hatch and d14 and 4) challenged + vaccinated with 100µg HmCPL-2. Groups 2-4 will be intracloacally challenged at d21 with 1x105 virulent histomonads/mL/turkey poult. Individual turkeys will be weighed pre- and post-challenge. HmCPL-1 or HmCPL-2 recombinant proteins will be individually combined with Seppic Montanide 71RVG and administered subcutaneously in the nape of the neck at d0 (0.25mL/turkey poult) and d14 (0.5mL/turkey poult). Liver and cecal lesions will be recorded from all mortalities following challenge. When mortality approaches 30% in the PC group, all turkeys will be euthanized and liver and cecal lesion scores will be recorded (n=20/group) according to previously established methods. Efficacy will be determined based on body weight gain during the challenge period, lesion score severity, and mortality as compared to the PC group. 3B. Evaluate therapeutic potential of top candidate HmCPL inhibitors against H. meleagridis challenge in turkey poults. Oral administration of up to five top candidates selected in Obj. 2 will be evaluated. Treatment groups will include a 1) NC, 2) PC, 3-7) challenged + candidate inhibitor 1-5 individually, respectively. Inhibitor dosages and frequencies of administration post-challenge will be selected based off previous literature and begin 72h post-challenge. Challenge will be administered, and efficacy will be determined as described in 3A.