Source: OHIO STATE UNIVERSITY submitted to NRP
ANTIMICROBIAL PEPTIDES: NOVEL THERAPEUTICS FOR AVIAN PATHOGENIC E. COLI (APEC) INFECTIONS IN CHICKENS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1027897
Grant No.
2022-67015-36348
Cumulative Award Amt.
$642,000.00
Proposal No.
2021-06968
Multistate No.
(N/A)
Project Start Date
Jan 1, 2022
Project End Date
Jul 19, 2024
Grant Year
2022
Program Code
[A1221]- Animal Health and Production and Animal Products: Animal Health and Disease
Recipient Organization
OHIO STATE UNIVERSITY
1680 MADISON AVENUE
WOOSTER,OH 44691
Performing Department
Center for Food Animal Health
Non Technical Summary
The avian pathogenicE. coli(APEC) is one of the major bacterial pathogens of significant concerns to the US and global poultry industry.APEC causes wide range of localized and systemic infections in poultry, including yolk sac infection, omphalitis, respiratory tract infection, swollen head syndrome, septicemia, polyserositis, coligranuloma, enteritis, cellulitis and salphingitis; collectively referred as colibacillosis.Colibacillosis culminates in multi-million dollars annual losses to all the facets of poultry industry and remains as a serious impediment to the sustainable poultry production worldwide. Current control methods using antibiotic medication and vaccination have limitation as APEC is resistance to multiple antibiotics and the available vaccines fail to confer protection against diverse and heterologous APEC serotypes. Therefore, developing new and potent anti-APEC therapeutics as an alternative to current control measures are urgently needed. We previously identified probiotic-derived antimicrobial peptides (P-1 and P-2) that in small pilot studies reduced the colonization of APEC in cecum and internal organs of chickens. These peptides are heat and proteolysis resistant, effective against diverse APEC serotypes, antibiotic-resistant APEC strains and against biofilm-protected APEC. Further, these peptides function by disrupting outer membrane of APEC, a mechanism to which resistance is less likely to occur. Here, we plan to evaluate the efficacy, safety and applicability of identified anti-APEC peptides (P-1 and P-2) in drinking water of chickens and to elucidate their mechanism(s) of action. Our overarching goal is to develop these peptides as new anti-APEC therapeutics as an alternative to antibiotics. We expect that these peptides will provide a novel therapeutic solution to enhance the control of APEC infections in poultry, which will consequently promote the sustainable poultry production and benefit the public health.
Animal Health Component
40%
Research Effort Categories
Basic
40%
Applied
40%
Developmental
20%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
31132991100100%
Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3299 - Poultry, general/other;

Field Of Science
1100 - Bacteriology;
Goals / Objectives
Here, we plan to determine the therapeutic doses of peptides (P-1 and P-2) for drinking water delivery in chickens followed by testing the efficacy and safety of peptides in large number of chickens under conditions mimicking the field settings and/or natural APEC infections. We will further elucidate and/or validate the mechanism(s) of action (MOA) of peptides by measuring direct drug-target binding using biophysical methods as well as employing affinity-based pulldown and thermal proteome profiling (TPP) approaches.Our overall aim is to evaluate the efficacy, safety and applicability of identified anti-APEC peptides (P-1 and P-2) in drinking water of chickens and to elucidate/validate their mechanism(s) of action.This will be accomplished via conducting the studies described in the following objectives:Objective 1:Determine the therapeutic doses of peptides (P-1 and P-2) in drinking water ofchickens and evaluate efficacy, safety and applicability in large number of chickens under conditions mimicking the field settings and/or natural APEC infections.?Objective 2: Elucidate/validate themechanism(s) of action (MOAs) of peptides by measuring direct drug-target binding, affinity-based pulldown assay, andthermal proteome profiling.Our overarching goal is to develop these peptides as new anti-APEC therapeutics as an alternative to antibiotics. We expect that these peptides will provide a novel therapeutic solution to enhance the control of APEC infections in poultry, which will consequently promote the sustainable poultry production and benefit the public health.We will collaborate with Technology Commercialization Office (TCO), OSU and Ohio State Innovation Foundation to advance these peptides into commercial products and seek FDA approval.
Project Methods
?Objective 1.1: Determine the therapeutic doses of peptides (P-1 and P-2) in drinking water.In this objective, the therapeutic dose of peptides (P-1 and P-2) will be determined in drinking water of chickens. Chickens (N=80) will be divided into 8 groups (n=10/group) Briefly,on day 2, chickens will be challenged orally with rifampicin-resistant (Rifr) APEC O78 (1-2× 109CFU/chicken). Peptides will be administered at three different doses (50 mg/L, 100 mg/L and 200 mg/L) in drinking water for 5 days starting one day after APEC challenge (day 3 to day 7),. The clinical signs and mortality will be monitored until 7 days post-infection (dpi; from day 3to day 9). Chickens that die during this period will be necropsied on the same day, lesions in internal organs (liver, heart, lung, and air-sacs) will be scored, and APEC load will be quantified in cecum and internal organs (liver, heart, lung, and kidney) by plating on MacConkey agar plates supplemented with 50 µg/mL rifampicin. At day 9 (7 dpi), all live birds will be necropsied, lesions will be scored, and the APEC load will be quantified in cecum and internal organs.Objective 1.2: Evaluate efficacy, safety and applicability of peptides.A) Efficacy assessment: The efficacy of peptides (P-1 and P-2) will be evaluated under field simulated conditions using the most effective dose identified fromobj 1.1.A total of 360 one-day-old broiler chickenswill be raised on built-up floor litter in six different groups (n=60/group). The chickens will be infected, and peptides will be administered in drinking water. To compare the efficacy with currently used antibiotic, sulfadimethoxine will be administered for 5 days following manufacturer's recommendation (0.05%). The efficacy of the combination of peptides (1:1) will be also assessed. Clinical signs and mortality will be monitored until day 42,chickens will be necropsied at day 9 (7 dpi; n=30 chickens/group; short-term efficacy), 12 (5 days post-treatment of peptides for residue analysis; n=5 chickens/group), and 42 (40 dpi; slaughter age; n=25 chickens/group; long-term efficacy) and internal organs will be collected for quantification of APEC load. The APEC lesions will be scored.B) Safety assessment:I) Impact on performance parameters:The body weight gain (BWG) and feed conversion ratio (FCR) are the key indices of safety of drugs in chickens. The body weight will be measured weekly [day 1, 7, 14, 21, 28, 35, and 42] and feed intake will be recorded daily in order to determine weekly and total BWG and FCR.II) Evaluation of resistance to peptides:APEC colonies isolated from different organs of peptides treated chickens at day 9 or day 42 (n=20 isolates/group, 5 isolates from each organ) will be used for testing whether APEC acquire resistance to peptides.III) Impact on gut microbiome: The effect of peptides on the gut microbiome will be analyzed using 16S rRNA V4-V5 sequencing using illumina platform. Briefly, DNA will be extracted from the collected cecal samples (10 samples/group) and sequenced at the MCIC, OSU.IV) Quantitation of peptides residues in chicken's tissues:We will quantify the peptidesresidues in muscle, liver, and kidney at day 9 (2 days post-treatment) and day 12 (5 days post-treatment) to determine the drug withdrawal period and day 42 to confirm the absence of peptide's residues. Liver (n=5/group), muscle (n=5/group), and kidney (n=5/group) will be collected from peptides treated groups. All the tissues will be snap-frozen, and shipped to MS&P facility at OSU to quantify the peptide's residues in the tissues using HPLC-MS triple quadrupole (QQQ) Quantiva. Code of Federal Regulations (CFR) Title 21 #21CFR556) will provide the basis for the peptide residue limits.C) Pharmacokinetics (PK) and stability assessment of peptides:For PK study, blood (2 mL, n=5 chickens each time point) will be collected at 0, 1, 2, 4, 8, 12, 16 and 24 h post administration of peptides. Peptides' concentrations in plasma will be quantitated using HPLC-MS triple QQQ Quantiva.For the stability study, samples (2 mL, n=3) of drinking water will be collected at 0, 4, 8, 12, 16, 20 and 24 h post administration of peptides. The concentrations of the peptides in drinking water will be quantitated using HPLC-MS triple QQQ Quantiva.Objective 2: Elucidate themechanism(s) of action (MOAs) of peptides.A) Drug-target binding validation using biophysical methods: ThemlaA andompC genes will be cloned into pET-21b vector and expression of MlaAHisand OmpCHiswill be achieved by addition of IPTG in PlysS. The OM proteins will be purified using Ni-affinity chromatography. and further purified by gel filtration using Superdex 200, and confirmed by immunoblot using anti-MlaA and -OmpC antibodies. The binding affinities of peptides with MlaAHisand OmpCHiswill be determined by surface plasmon resonance (SPR) using NiHC1000 sensor chip with a BIAcore T100 instrument.B) Additional Target identification:I)Affinity-based pulldown assay:Biotin probes will be synthesized linking non-essential amino acid residues of these peptides. Biotinylated peptides will be loaded onto streptavidin column and incubated to allow binding with streptavidin. The APEC O78 whole-cell lysate, will be added to the columns and incubated to allow binding with biotinylated peptides. The binding lysate will be eluted using elution buffer. The eluted protein(s) will be then examined using SDS-PAGE and quantified using HPLC-MS/MS. The proteins will be identified using Mascot in Proteome Discoverer 2.2 andE.coliUniprot database will be used with false discovery rate of below 1%.The protein(s) highly abundant in the eluted buffer compared to the control will be considered as potential targets.II)Thermal proteome profiling (TPP):Logarithmic phase APEC O78 will be treated with two different concentrations (high: 4X, low: 1X of MBC) of peptides for 20 minutes followed by thermal treatment of bacterial cultures at 11 different temperatures (42°C to 72°C with 3°C successive increments). APEC unexposed to peptide and non-heat treated (room temperature, 25°C) will be used as controls. The soluble/non-denatured proteins will be extracted and the quantitative proteomics will be performed using Bruker maXis electrospray ionization (ESI) quadrupole LC-MS/MS coupled with 3000 RSLCnano system. The proteins will be identified using Mascot in Proteome Discoverer 2.2 andE.coliUniprot database will be used with false discovery rate of below 1%.Data analysis:The statistical significance of reduction in mortality, APEC lesions and APEC load in the peptide treated groups will be determined by one-way ANOVA followed by post-hoc Tukey's test (P<0.05). The comparison of mortality, APEC lesions, APEC load, BWG and FCR between peptide and antibiotic treated groups will be made by Kruskal-Wallis test (P<0.05).For the microbiota analysis, QIIME 2 bioinformatics platform will be used. The taxonomic analysis will be performed usingNaive Bayes classifiers trained on Silva 132 99% OTUs (silva-132-99-nb-classifier.qza) database. The phylogenetic diversity will be analyzed using align-to-tree-mafft-fasttreepipelineand alpha (Shannon's diversity index) and beta diversity (Bray-Curtis distance) will be analyzed using core-metrics-phylogenetic pipeline. The statistical comparison (P<0.05) of the taxonomic composition between untreated andpeptide and antibiotic treatedgroups will be made usingKruskal-Wallis test. The alpha and beta diversity will be analyzed using Kruskal-Wallis and PERMANOVA tests (P<0.05), respectively.For proteomic analysis from TPP studies, TPP package using R pipeline in Bioconductor software will be used. The proteins significantly (P<0.05) altered in their abundance across the thermal treatments will be identified through built-in statistical analysis in the TPP package.

Progress 01/01/22 to 07/09/24

Outputs
Target Audience:Veterinarians, Veterinary Daignosticians, Poultry Practitioners, Poultry Producers, Researchers, and Veterinary, Bacteriologists, Pharmaceutical companies, and Immunologists Changes/Problems:PI, Gireesh Rajashekara has accepted a positoin as the Associate Dean for Research and Advanced Studies and will be moving to University of Illinois -Urbana Champaign in August 2024; therefore, transfer of the project to new institue is requested. What opportunities for training and professional development has the project provided?This project support training of graduate students, post-docs, research assistants. In addition to hand-on experiences, students and post-docs presented the results at the local and national meetings and published the results in scientific journals. How have the results been disseminated to communities of interest?The results from this study are disseminated through conferences, publications and meetings What do you plan to do during the next reporting period to accomplish the goals?1. Conduct further evaluation of the efficacy, safety, and applicability of peptides under conditions that mimic field settings or natural APEC infections. 2. Utilize biophysical methods to determine the binding affinity of peptides with potential targets, including MlaA, OmpC, and OmpF. 3. Employ the pulldown assay to gain insight into the interaction of peptides with cellular proteins. 4. Perform thermal proteome profiling to elucidate additional proteins interacting with the peptides.

Impacts
What was accomplished under these goals? Objective 1: Determine the therapeutic doses of peptides (P-1 and P-2) in the drinking water of chickens and evaluate the efficacy, safety, and applicability in a large number of chickens under conditions mimicking the field settings and/or natural APEC infections. The peptide dose optimization study: We performed a peptide dose optimization (50, 100, and 200 mg/liter) study for peptides (P-1 and P-2) by administering them through drinking water to one-day-old SPF layers (n= 10/group) for five days. A significant reduction of APEC load in the cecum was observed in both P1 and P2 at 50 mg/liter by 1.089 (p<0.05) and 1.561 (p<0.001) Log CFU/g, respectively. Microbiota analysis revealed significant differences in observed richness and Chao1 indices among groups, with P1, P2, and NC displaying higher diversity than PC. Interestingly, while Shannon and Simpson's indices remained consistent across groups, suggesting similar microbial community evenness and dominance, the gut's structural integrity assessed through villi height, crypt depth, and the VH: CD ratio, remained unaffected. Efficacy of the in-house synthesized P2 peptide: P2 was synthesized in-house and conducted a comparative study with commercially prepared P2. Results showed a 1.5 log reduction in cecal load with commercially prepared P2 and a 0.9 log reduction with in-house synthesized P2, both demonstrating significant reductions (p-value <= 0.0001 and p <= 0.05, respectively) compared to positive control birds, which were infected but not treated. Sgnificant variations were found in the Shannon and Simpson diversity indices. PC exhibited higher Shannon and Simpson diversity compared to P2, suggesting potential differences in microbial community structure and evenness. Effects of LGG and Peptide Treatments on Avian Bacterial Infections in Chickens We conducted a study to assess their effectiveness in combination of peptideswith the probiotic LGG.Treatment groups received LGG treatment from Day 0 to 13, while peptide treatment commenced on Day 9 and persisted until the study's conclusion. On Day 8, all groups, except the negative control, were orally infected with avian pathogenic Escherichia coli (APEC). On Day 15, birds were euthanized, and their body weights were measured. Samples from the cecum and other organs were collected to estimate the reduction in APEC load. Analysis of APEC load in the cecum after treatment with LGG and peptide combinations revealed significant reductions in the P1, LGG_P1, and LGG_P2 groups compared to the positive control (PC). Conversely, no significant reduction was observed in the birds treated with LGG and P2 alone. The LGG_ P1 and LGG _P2 showed increased effect by reducing 1.67 and 1.48 log CFU/gm, respectively, compared to birds treated with LGG alone (1.233). Microbiota analysis indicated significant differences in species richness, as evidenced by the Chao1 index (p = 0.0386), with higher diversity observed in the treated groups compared to the negative control. However, diversity levels, indicated by the Shannon and Simpson indices, remained consistent across the groups (p > 0.05), suggesting that overall diversity levels were unaffected by the treatments and infection. Physiological parameters, including villi height, crypt depth, and the VH: CD ratio, showed no significant alterations, indicating that the treatments and infection did not impact intestinal morphology in the chickens. Ac-NPSRQERR (P1) and Ac-PDENK (P2) Large Scale Synthesis We have successfully utilized standard solid-phase peptide synthesis to generate P2 with an Fmoc protecting strategy. P2 has been characterized using 1H NMR, 13C NMR, COSY, HPLC, and LC/MS to confirm the desired structure. Initial methodology included a pre-loaded Lys-Wang resin (0.61 mmol/g) to establish the coupling, deprotection, acetylation, and cleavage procedures. These methods were scaled to generate multiple grams of crude peptide for both P1 and P2, with the exception of incorporating an additional resin loading procedure. To increase the cost-effectiveness of this route, Wang resin was used without the pre-loaded amino acid; therefore, coupling to the resin began with the lysine or arginine amino acid for P2 and P1, respectively. Assessment of Modified P2 Against APEC O78: We investigated the activity of modified tetra and tri peptides against APEC O78 by assessing their impact on bacterial growth patterns. These peptides were tested at a concentration of 12 mM alongside controls including DMSO, commercially synthesized peptide (PDENK), and acetylated peptide (ac-PDENK). Our findings indicate that the modified tri and tetra peptides exhibited either no effect or were less effective compared to the full-length peptide at the tested concentration of 12 mM. Objective 2: Elucidate/validate the mechanism(s) of action (MOAs) of peptides by measuring direct drug-target binding, affinity-based pulldown assay, and thermal proteome profiling. We identified the potential targets of peptides through bacterial cytological profiling, gene expression, immunoblot and in silico approaches. Peptides exhibit anti-APEC activity by disrupting the APEC membrane. Particularly, peptides downregulated the expression of ompC, ompF and mlaA genes responsible for maintenance of outer membrane lipid asymmetry in APEC. Further, immunoblot analysis showed that peptides decreased the level of OmpC and MlaA proteins. In silico binding prediction using PEP-SiteFinder revealed that peptides bind with higher affinity to OmpC compared to OmpF. Overall, our results suggest that peptides target the MlaA-OmpC/F system in APEC responsible for regulating phospholipid trafficking to maintain lipid asymmetry at the OM. To validate this further, we cloned the ompC and mlaA genes from APEC O78 with gene-specific primers in E. coli and expressed the proteins in BL21PlysS. MlaA was induced with IPTG, separated the membrane protein from cytosolic protein, and purified the proteins from both (cytosolic faction and membrane fraction) using a Ni-NTA affinity purification column separately. Cytosolic MlaA was purified using size exclusion chromatography and the membrane fraction MlaA via ion exchange chromatography. Presently, we are standardizing the protocol for interaction using isothermal calorimetry. Also, we have obtained a partially purified OmpC from affinity chromatography. Furthermore, we plan to perform gel filtration or ion exchange chromatography to eliminate background proteins and perform protein-ligand interaction studies using isothermal titration calorimetry. Other Antimicrobial peptides derived from LGG and BB12 We identified Lacticaseibacillus rhamnosus GG and Bifidobacterium lactis Bb12 as producing strong zones of inhibition against APEC. In co-culture assays, both LGG and Bb12 completely inhibited APEC growth within 24 hours. Further analysis revealed that antibacterial products in the culture supernatants of these probiotics were responsible for this activity. Using LC-MS/MS analysis of the culture supernatants, we identified several novel bioactive peptides, including VQAAQAGDTKPIEV, AFDNTDTSLDSTFKSA, VTDTSGKAGTTKISNV, and AESSDTNLVNAKAA. We evaluated the effectiveness of these antimicrobial peptides (AMPs), particularly VQAAQAGDTKPIEV and VTDTSGKAGTTKISNV, against APEC serotypes, extraintestinal pathogenic E. coli (ExPEC), and other E. coli pathotypes. Our research demonstrated that two antimicrobial peptides (AMPs) derived from LGG and Bb12 are potent and effective against various E. coli pathotypes, including APEC, ExPEC, and other intestinal and Shiga-toxin-producing strains.

Publications

  • Type: Journal Articles Status: Published Year Published: 2022 Citation: 1. Kathayat, D., Closs, G., Helmy, Y. A., Deblais, L., Srivastava, V., & Rajashekara, G. (2022). In vitro and in vivo evaluation of Lacticaseibacillus rhamnosus GG and Bifidobacterium lactis Bb12 against avian pathogenic Escherichia coli and identification of novel probiotic-derived bioactive peptides. Probiotics and Antimicrobial Proteins, 1-17.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: 1. Gireesh Rajashekara, Yosra A. Helmy, Dipak Kathayat, Dhanashree Lokesh, Oluwatosin R. Ayinde, Katie Galgozy, Antonia D. Duran, Mark Foster, James Fuchs. Novel Therapeutic Leads; Demonstration of efficacy, safety, and applicability of anti-APEC molecules in chickens. CRWAD, Jan 21 -24, Chicago, 2023.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: 2. Katherine A. Galgozy, Dhanashree Lokesh, Menuka Bhandari, Dipak Kathayat, Gireesh Rajashekara, James R. Fuchs. Synthesis and biological evaluation of novel antibacterial small molecules and peptides against APEC. ACS Spring 2023 Crossroads of Chemistry meeting. Indianapolis, IN and Hybrid, March 26-30.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: 3. Galgozy, K.A., Lokesh, D., Bhandari, M., Kathayat, D., Rajashekara G., Fuchs, J.R. Membrane-Targeting Novel Antibacterial Small Molecules and Peptides Against APEC: Synthesis and Biological Evaluation. Mid-Atlantic Graduate Student Symposium (MAGSS) 2023. Columbus, OH.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: 5. Dhanashree Lokesh, Menuka Bhandari, Dipak Kathayat, Yosra A Helmy, James R Fuchs, Antonia Duran, Katie Galgozy, Mark Foster, Gireesh Rajashekara Novel peptides derived from probiotics as an antibiotic alternative to control avian pathogenic Escherichia coli (APEC) infection in poultry. Conference of Research Workers in Animal Diseases, January 20 -23, 2024, Chicago, Illinois
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: 4. Dhanashree Lokesh, Menuka Bhandari, Gireesh Rajashekara, LGG-derived Antimicrobial Peptides As Effective Antibiotic Alternatives To Treat ExPEC, American Society Of Microbiology 2023-EA-2120 Microbe June 15-19, 2023 Houston,Tx.


Progress 01/01/23 to 12/31/23

Outputs
Target Audience: Nothing Reported 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?Presentations: Gireesh Rajashekara, Yosra A. Helmy, Dipak Kathyat, Dhanashree Lokesh, Oluwatosin R. Ayinde, Katie Galgozy, Antonia D. Duran, Mark Foster, James Fuchs. Novel Therapeutic Leads; Demonstration of efficacy, safety, and applicability of anti-APEC molecules in chickens. CRWAD, Jan 21 -24, Chicago, 2023. Katherine A. Galgozy1, Dhanashree Lokesh2, Menuka Bhandari2, Dipak Kathayat2, Gireesh Rajashekara2, James R. Fuchs1Synthesis and biological evaluation of novel antibacterial small molecules and peptides against APEC. ACS Spring 2023 Crossroads of Chemistry meeting. Indianapolis, IN and Hybrid, March 26-30. Galgozy, K.A.,Lokesh, D., Bhandari, M., Kathayat, D., Rajashekara G., Fuchs, J.R. Membrane-Targeting Novel Antibacterial Small Molecules and Peptides Against APEC: Synthesis and Biological Evaluation. Mid-Atlantic Graduate Student Symposium (MAGSS) 2023. Columbus, OH. What do you plan to do during the next reporting period to accomplish the goals?Future Plans: Evaluate efficacy, safety and applicability of peptides in large number of chickens under conditions mimicking the field settings and/or natural APEC infections. Determine the impact of peptides on cecal microbiota of chickens. Determine the impact of peptides on immune responses of chickens. Determine Pharmacokinetics (PK) and stability of peptides: Determine the binding affinity of peptides with potential targets (MlaA, OmpC & OmpF) using isothermal titration calorimetry. We plan to perform gel filtration or ion exchange chromatography to eliminate background proteins and perform protein-ligand interaction studies using isothermal titration calorimetry. Determine additional targets using affinity-based pulldown assay to understand the interaction of these peptides with proteins. we plan to perform the pulldown assays with whole-cell protein lysate and the purified proteins.

Impacts
What was accomplished under these goals? Accomplishments Objective 1: Determine the therapeutic doses of peptides (P-1 and P-2) in drinking water of chickens and evaluate efficacy, safety and applicability in large number of chickens under conditions mimicking the field settings and/or natural APEC infections. The dose optimization study identified 50 mg/liter of drinking wateras the optimum therapeutic dose for P-1 and P-2. Peptide were administered at (50, 100, and 200 mg/liter through drinking water to one-day-old SPF layers for five days. A significant reduction of APEC load in the cecum was observed in both P1 and P2 at 50 mg/liter by 1.089 (p<0.05) and 1.561 (p<0.001) Log CFU/g, respectively. Additionally, no significant difference was observed in body weight gain compared to untreated birds. Ac-NPSRQERR (P1) and Ac-PDENK (P2) Large Scale Synthesis As previously reported, we have successfully utilized standard solid phase peptide synthesis to generate P2 with an Fmoc protecting strategy. P2 has been characterized using 1H NMR, 13C NMR, COSY, HPLC, and LC/MS to confirm the desired structure. Initial methodology included a pre-loaded Lys-Wang resin (0.61 mmol/g) to establish the coupling, deprotection, acetylation, and cleavage procedures. These methods were scaled to generate multiple grams of crude peptide for both P1 and P2, with the exception of incorporating an additional resin loading procedure. To increase the cost-effectiveness of this route, Wang resin was used without the pre-loaded amino acid; therefore, coupling to the resin began with the lysine or arginine amino acid for P2 and P1, respectively. This updated procedure was scaled to 2.5 g batches of resin and was repeated to generate several grams of crude P1 and P2. Upon purity assessment using HPLC methodologies, P2 was approximately 91% desired material. To increase the purity to the 95% standard, multiple purification methods have been evaluated. Standard solid phase peptide synthesis methods often utilize HPLC for purification; however, due to the amount of crude material needing purified and a semi-preparative column and instrument being unavailable, alternate chromatographic methods were considered. First, using normal phase silica was tested using the polar solvent system (EtOAc:ACN:MeOH:H2O), but was not utilized based on inefficient separation and elution off the column. Therefore, reverse phase chromatography was investigated using H2O and ACN as the mobile phase and a C18 stationary phase. This method resulted in better separation and elution and was scalable to purify multigram batches of crude material by use of a CombiFlash. Approximately one gram of P2 was recovered with the desired purity (>95%) using this methodology. This yield could potentially be improved by incorporating an additive to the solvent system, such as TFA or formic acid. Due to the several acidic and basic amino acid side chains present in this short peptide, charged species may elute separately from a neutral peptide, decreasing the efficiency of the separation and the appearance of a lower yield. Upon HPLC purity analysis of the P1 crude material, the major product represented 41% of the total mixture. Similar reverse phase purification methods were utilized to isolate this product; however, upon characterization (NMR and LC/MS) it was determined to not consist of the desired product. Work has begun to troubleshoot the synthesis of P1 at a smaller scale, which includes the loading of the first amino acid onto the Wang resin. Inefficient loading can result in shortened sequences, further decreasing purity of the cleaved peptide. Using the initial loading procedure, loading of lysine (P2) was determined to be 0.041 mmol/g, well below the expected loading (0.6-1.2 mmol/g). This is potentially due to racemization and/or dipeptide side-product formation caused by premature Fmoc cleavage in the presence of DIC/DMAP. Therefore, optimizing the loading procedure is currently being conducted for both P1 and P2, which would likely increase both purity and yield. Small scale synthesis of P1 will also ensure complete coupling of each amino acid, and fully characterize the material generated to confirm product formation prior to additional scale up. Objective 2: Elucidate/validate the mechanism(s) of action (MOAs) of peptides by measuring direct drug-target binding, affinity-based pulldown assay, and thermal proteome profiling. To validate this further, we cloned the OmpC and MlaA genes from APEC O78 with gene-specific primers in E. coli and expressed the proteins in BL21PlysS. MlaA was induced with IPTG. The expression of recombinant proteins were confirmed by SDS PAGE and western blot using antibodies specific to OmpC and MlaA. Proteins were purified from both (cytosolic faction and membrane fraction) using a Ni-NTA affinity purification column. Cytosolic MlaA was purified using size exclusion chromatography and the membrane fraction MlaA via ion exchange chromatography. Presently, we are standardizing the protocol for interaction using isothermal calorimetry. Also, we have obtained a partially purified OmpC from affinity chromatography. Furthermore, we plan to perform gel filtration or ion exchange chromatography to eliminate background proteins and perform protein-ligand interaction studies using isothermal titration calorimetry. Alternatively, to understand the interaction of these peptides with proteins, we plan to perform the pulldown assays with whole-cell protein lysate and the purified proteins.

Publications

  • Type: Journal Articles Status: Accepted Year Published: 2023 Citation: Helmy YA, Kathayat D, Closs G Jr, Galgozy K, Fuchs JR, Rajashekara G. Efficacy of quorum sensing and growth inhibitors alone and in combination against avian pathogenic Escherichia coli infection in chickens. Poult Sci. 2023 Apr;102(4):102543. doi: 10.1016/j.psj.2023.102543. Epub 2023 Feb 1. PMID: 36863122; PMCID: PMC10011511.


Progress 01/01/22 to 12/31/22

Outputs
Target Audience:Veterinarians, Veterinary Daignosticians, Poultry Practitioners, Poultry Producers, Researchers, and Veterinary, Bacteriologists, Pharmaceutical companies, and Immunologists Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Graduate student, technician and post-doc are currently working on this project How have the results been disseminated to communities of interest?The results from this study are being disseminated through conferences, publications and meetings What do you plan to do during the next reporting period to accomplish the goals?1.Evaluate efficacy, safety and applicability of peptides in large number of chickens under conditions mimicking the field settings and/or natural APEC infections. 2.Determine Pharmacokinetics (PK) and stability of peptides: 3.Determine the binding affinity of peptides with potential targets (MlaA, OmpC & OmpF) using biophysical methods. 4.Determine additional targets using affinity-based pulldown assay and Thermal proteome profiling.

Impacts
What was accomplished under these goals? Objective 1: Determine the therapeutic doses of peptides (P-1 and P-2) in drinking water of chickens and evaluate efficacy, safety and applicability in large number of chickens under conditions mimicking the field settings and/or natural APEC infections. We assessed the efficacy of peptides (P-1, P-2, and P-3) by administering orally at 50 mg/kg and 100 mg/kg doses in commercial broiler chickens (n= 10/group). All peptides reduced the colonization of APEC in the cecum of chickens at both doses. At 50 mg/kg dose, P-1, P-2 and P-3 reduced the colonization by 0.5, 0.9 (P<0.05) and 1.1 (P<0.01) logs, respectively. Consistently, at 100 mg/kg dose, P-1 (1.3 logs, P<0.001) and P-2 (1.3 logs, P<0.001) showed a better effect in reducing APEC colonization. In comparison, the efficacy of P-3 (0.6 logs) did not improve with the increased dose. At 50 mg/kg dose, peptides also reduced the recovery of APEC from internal organs (lung, kidney, liver and heart) of chickens, particularly lung (P<0.05). Whereas, at 100 mg/kg dose all peptides except P-1, also reduced the number of chickens positive for APEC in internal organs. Overall, P-2 showed the most effective effect against APEC in chickens. Based on these results, we performed a peptide dose optimization (50, 100, and 200 mg/liter) study for peptides (P-1 and P-2) by administering them through drinking water to one-day-old SPF layers (n= 10/group) for five days. Chickens were orally challenged with rifampicin-resistant APEC (2.3 x 109 CFU/mL on day 3 of age. On day 7, the birds were euthanized, body weight was recorded, and kidney, liver, lung, heart, and cecum were aseptically collected to determine if peptides administered in drinking water could retain anti-APEC activity. A significant reduction of APEC load in the cecum was observed in both P1 and P2 at 50 mg/liter by 1.089 (p<0.05) and 1.561 (p<0.001) Log CFU/g, respectively, and we did not recover the APEC from organs from the treated birds. Additionally, no significant difference was observed in body weight gain compared to untreated birds. Ac-NPSRQERR (P1) and Ac-PDENK (P2) Large Scale Synthesis The first large scale synthesis of Ac-PDENK (P2) was completed using standard solid phase peptide synthesis with the Fmoc protecting strategy. The peptide was built from a pre-loaded Lys-Wang resin (0.61 mmol/g). Conditions for amide bond coupling utilized 3 equiv. of the amino acid being introduced, 2.9 equiv. HATU, and 3.5 equiv. DIPEA in a 0.2 M solution of DMF, relative to the amino acid. Coupling each amino acid was run in duplicate to ensure completion. Upon successful coupling, Fmoc deprotection was completed using a solution of 10% piperidine in DMF, and the resin was washed with DMF and DCM. This cycle was repeated until the desired sequence was completed. A final Fmoc deprotection of the Pro residue allowed for acetylation of the N-terminus to provide added stability to the peptide. Acetylation was preformed using a solution of acetic anhydride and pyridine in DMF. To cleave the finished sequence from the resin and remove any side chain protecting groups, the resin was subjected to the cleavage cocktail: TFA, Et3SiH, DCM (1:0.1:0.9) and stirred for 4-6 h. The resin beads were filtered from the peptide solution and washed with DCM, MeOH, and TFA. The solution was concentrated, and the peptide was precipitated using cold ether. The precipitate was filtered, washed with a cold ether:hexanes (50:50) solution, and dried in vacuo. This procedure was scaled to 0.5 grams of resin per batch and was repeated 16 times to generate approximately 1g of material for in vivo studies. Characterization data obtained for the completed sequence includes 1H NMR, 13C NMR, COSY, HPLC, and LC/MS. Since in vivo studies require more quantities of peptides. To increase the cost- and time-effectiveness of the synthesis, a solution phase approach was attempted. Rather than building the peptide from a resin bead at the C-terminus, an additional protecting was employed at the C-terminal carboxy group. However, when moving forward to the next amino acid coupling, reaction completion was not achieved, increasing the difficulty to cleanly separate the desired product. Therefore, to produce the desired quantity of peptide for upcoming in vivo experiments, we reverted to a solid phase approach. To lower the cost of this approach, Wang resin without the pre-loaded amino acid was utilized. To load the first amino acid, 4 equiv. of Lys or Arg, 4 equiv. HATU, 4 equiv. DIC, and 0.1 equiv. DMAP was combined in a minimal amount of DMF and subjected to swelled resin to agitate overnight. The resin was then treated with a solution of acetic anhydride and pyridine in a minimal amount of DCM to cap any remaining free resin. Upon completion, the resin was washed with DMF and DCM before starting the remaining deprotection and coupling cycles. N-terminal acetylation, cleavage, and precipitation were also repeated as previously described. This procedure was scaled to 2.5 grams batches of resin and was repeated 19 times to generate over 7 grams of crude material of P2 and was repeated 5 times to generate over 7 grams of crude material of P1. Upon HPLC analysis to determine purity, P2 was approximately 91% desired material and P1 was about 41% desired material. The synthesis for P1 is currently being repeated to generate enough crude material to provide the desired amount of product following purification. Various chromatographic and purification approaches are being explored to provide products with >95% purity. Following purification, characterization data will be obtained for the completed sequences including 1H NMR, 13C NMR, COSY, HPLC, and LC/MS. Objective 2: Elucidate/validate the mechanism(s) of action (MOAs) of peptides by measuring direct drug-target binding, affinity-based pulldown assay, and thermal proteome profiling. We identified the potential targets of peptides through bacterial cytological profiling, gene expression, immunoblot and in silico approaches. Peptides exhibit anti-APEC activity by disrupting the APEC membrane. Particularly, peptides downregulated the expression of ompC, ompF and mlaA genes responsible for maintenance of outer membrane lipid asymmetry in APEC. Further, immunoblot analysis showed that peptides decreased the level of OmpC and MlaA proteins. In silico binding prediction using PEP-SiteFinder revealed that peptides bind with higher affinity to OmpC compared to OmpF. Overall, our results suggest that peptides target MlaA-OmpC/F system in APEC responsible for regulating phospholipid trafficking to maintain lipid asymmetry at the OM. To validate this further, we cloned the OmpC and MlaA genes from APEC O78 with gene-specific primers in E. coli and expressed the proteins in BL21PlysS. MlaA was induced with IPTG, separated the membrane protein from cytosolic protein, and purified the proteins from both (cytosolic faction and membrane fraction) using a Ni-NTA affinity purification column separately. Cytosolic MlaA was purified using size exclusion chromatography and the membrane fraction MlaA via ion exchange chromatography. Presently, we are standardizing the protocol for interaction using isothermal calorimetry. Also, we have obtained a partially purified OmpC from affinity chromatography. Furthermore, we plan to perform gel filtration or ion exchange chromatography to eliminate background proteins and perform protein-ligand interaction studies using isothermal titration calorimetry. Alternatively, to understand the interaction of these peptides with proteins, we plan to perform the pulldown assays with whole-cell protein lysate and the purified proteins.

Publications

  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Helmy YA, Kathayat D, Deblais L, Srivastava V, Closs G Jr, Tokarski RJ 2nd, Ayinde O, Fuchs JR, Rajashekara G. Evaluation of Novel Quorum Sensing Inhibitors Targeting Auto-Inducer 2 (AI-2) for the Control of Avian Pathogenic Escherichia coli Infections in Chickens. Microbiol Spectr. 2022 Jun 29;10(3):e0028622. doi: 10.1128/spectrum.00286-22.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: 1. Gireesh Rajashekara, Yosra A. Helmy, Dipak Kathyat, Dhanashree Lokesh, Oluwatosin R. Ayinde, Katie Galgozy, Antonia D. Duran, Mark Foster, James Fuchs. Novel Therapeutic Leads; Demonstration of efficacy, safety, and applicability of anti-APEC molecules in chickens.
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Helmy YA, Kathayat D, Closs G Jr, Galgozy K, Fuchs JR, Rajashekara G. Efficacy of quorum sensing and growth inhibitors alone and in combination against avian pathogenic Escherichia coli infection in chickens. Poult Sci. 2023 Feb 1;102(4):102543. doi: 10.1016/j.psj.2023.102543.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: 2. Katherine A. Galgozy1, Dhanashree Lokesh2, Menuka Bhandari2, Dipak Kathayat2, Gireesh Rajashekara2, James R. Fuchs1Synthesis and biological evaluation of novel antibacterial small molecules and peptides against APEC. ACS Spring 2023 Crossroads of Chemistry meeting. Indianapolis, IN and Hybrid, March 26-30.