Bovine TLR2 Research Articles

Immunoinformatics design of novel multi-epitope vaccine against Trueperella Pyogenes using collagen adhesion protein, fimbriae, and pyolysin.

Trueperella pyogenes (T. pyogenes) is an opportunistic pathogen that causes infertility, mastitis, and metritis in animals. T. pyogenes is also a zoonotic disease and is considered an economic loss agent in the livestock industry. Therefore, vaccine development is necessary. Using an immunoinformatics approach, this study aimed to construct a multi-epitope vaccine against T. pyogenes. The collagen adhesion protein, fimbriae, and pyolysin (PLO) sequences were initially retrieved. The HTL, CTL, and B cell epitopes were predicted. The vaccine was designed by binding these epitopes with linkers. To increase vaccine immunogenicity, profilin was added to the N-terminal of the vaccine construct. The antigenic features and safety of the vaccine model were investigated. Docking, molecular dynamics simulation of the vaccine with immune receptors, and immunological simulation were used to evaluate the vaccine's efficacy. The vaccine's sequence was then optimized for cloning. The vaccine construct was designed based on 18 epitopes of T. pyogenes. The computational tools validated the vaccine as non-allergenic, non-toxic, hydrophilic, and stable at different temperatures with acceptable antigenic features. The vaccine model had good affinity and stability to bovine TLR2, 4, and 5 as well as stimulation of IgM, IgG, IL-2, IFN-γ, and Th1 responses. This vaccine also increased long-lived memory cells, dendritic cells, and macrophage population. In addition, codon optimization was done and cloned in the E. coli K12 expression vector (pET-28a). For the first time, this study introduced a novel multi-epitope vaccine candidate based on collagen adhesion protein, fimbriae, and PLO of T. pyogenes. It is expected this vaccine stimulates an effective immune response to prevent T. pyogenes infection.

Synthesis of 1,2,3-triazole-piperazin-benzo[b][1,4]thiazine 1,1-dioxides: antibacterial, hemolytic and in silico TLR4 protein inhibitory activities.

In this study, we designed and synthesized a number of novel 1,2,3-triazole-piperazin-benzo[b][1,4]thiazine 1,1-dioxide derivatives and investigated their in vitro antibacterial and hemolytic activity. When compared to the lead chemical, dicloxacillin, the majority of the compounds demonstrated acceptable activity. Among them, the most promising compounds 6e, 6g, 6i, 8d, and 8e exhibited excellent antibacterial activity against the methicillin-susceptible S. aureus (MSSA), methicillin-resistant S. aureus (MRSA), and vancomycin-resistant S. aureus (VRSA) with MIC values of 1.56 ± 0.22 to 12.5 ± 1.75 μg mL-1, respectively, The percentage of hemolysis ranged from 21.3 μg mL-1 to 33.8 μg mL-1. Out of the six compounds (6i, 6e, 6f, 6g, 8e, 8d) tested compound 8e and 8d displayed minimal or negligible hemolytic activity across all the tested concentrations 29.6% and 30.2% recorded at 100 μg mL-1 concentration respectively. In silico docking studies were performed to evaluate the molecular interactions of 6e, 6f, 6g, 6i, 8d, and 8e compounds with Human, Mouse and Bovine TLR4 proteins (PDB: 3FXI, 3VQ1, 3RG1) and observed that three of the compounds (6i, 8d, and 8i) had appreciable binding energies ranging from -8.5 to -9.0 Kcal mol-1. Finally, the in silico pharmacokinetic profile was predicted for potent compounds 8d, 8e and 6i using SWISS/ADME, All compounds investigated in this study adhered to Lipinski's rule of five with slight deviation in molecular weight (8d and 8e).

Association of Variants in Innate Immune Genes TLR4 and TLR5 with Reproductive and Milk Production Traits in Czech Simmental Cattle.

Bovine genes TLR4 and TLR5, which encode antibacterial toll-like receptors, were screened for polymorphisms in Czech Red Pied (Czech Simmental) cattle to identify variants associated with reproduction, udder health, and milk production traits. Variants were discovered by hybrid resequencing of 164 bulls using HiSeq X-Ten and PacBio technologies and then individually genotyped. Nominal p-values < 0.05 for associations were detected in 18 combinations between 14 polymorphisms and 15 traits using one-way analysis of variance (ANOVA). The TLR4 variants g.610C>T (rs43578094) and g.10310T>G (rs8193072) in reference AC000135.1 were strictly associated with the index of early reproductive disorders and maternal calving ease, respectively, at false discovery rate (FDR) < 0.05. A highly permissive false discovery rate cutoff of 0.6 separated seventeen combinations in both genes comprising eight positives. In the case of the TLR4 variant g.9422T>C (rs8193060), indications were obtained for the association with as many as four reproductive traits: incidence of cystic ovaries, early reproductive disorders, calving ease, and production longevity. The permissive FDR interpretation for the TLR5 data indicated associations with cyst incidence and early reproduction disorders with maternal calving ease. Moreover, three TLR5 polymorphisms correlated with milk production traits. The discrepancy of the observed associations with the predicted impacts of the SNPs on protein function points to the role of haplotypes. Nevertheless, this question should be resolved on a larger scale. The observed associations are endorsed by independent evidence from the published functional roles in other species and by the published QTL mapping data.

Proteomic profiling of membrane vesicles from Mycobacterium avium subsp. paratuberculosis: Navigating towards an insilico design of a multi-epitope vaccine targeting membrane vesicle proteins

Bacteria typically produce membrane vesicles (MVs) at varying levels depending on the surrounding environments. Gram-negative bacterial outer membrane vesicles (OMVs) have been extensively studied for over 30 years, but MVs from Gram-positive bacteria only recently have been a focus of research. In the present study, we isolated MVs from Mycobacterium avium subsp. paratuberculosis (MAP) and analyzed their protein composition using LC-MS/MS. A total of 316 overlapping proteins from two independent preparations were identified in our study, and topology prediction showed these cargo proteins have different subcellular localization patterns. When MVs were administered to bovine-derived macrophages, significant up-regulation of pro-inflammatory cytokines was observed via qRT-PCR. Proteome functional annotation revealed that many of these proteins are involved in the cellular protein metabolic process, tRNA aminoacylation, and ATP synthesis. Secretory proteins with high antigenicity and adhesion capability were mapped for B-cell and T-cell epitopes. Antigenic, Immunogenic and IFN-γ inducing B-cell, MHC-I, and MHC-II epitopes were stitched together through linkers to form multi-epitope vaccine (MEV) construct against MAP. Strong binding energy was observed during the docking of the 3D structure of the MEV with the bovine TLR2, suggesting that the putative MEV may be a promising vaccine candidate against MAP. However, in vitro and in vivo analysis is required to prove the immunogenic concept of the MEV which we will follow in our future studies. SignificanceJohne's disease is a chronic infection caused by Mycobacterium avium subsp. paratuberculosis that has a potential link to Crohn's disease in humans. The disease is characterized by persistent diarrhea and enteritis, resulting in significant economic losses due to reduced milk yield and premature culling of infected animals. The dairy industry in the United States alone experiences losses of approximately USD 250 million due to Johne's disease. The current vaccine against Johne's disease is limited by several factors, including variable efficacy, limited duration of protection, interference with diagnostic tests, inability to prevent infection, and logistical and cost-related challenges. Nevertheless, a multiepitope vaccine design approach targeting M. avium subsp. paratuberculosis has the potential to overcome these challenges and offer improved protection against Johne's disease.

Novel multi-epitope vaccine against bovine brucellosis: approach from immunoinformatics to expression

Brucellosis is a zoonotic caused by the Brucella which is a well-known infectious disease agent in domestic animals and if transmitted, it can cause infection in humans. Because brucellosis is contagious, its control depends on the eradication of the animal disease in farms. There are two vaccines based on the killed and/or weakened bacteria against B. melitensis and B. abortus, but no recombinant vaccine is available for preventing the disease. The present study was designed to develop a multi-epitope vaccine against of B. melitensis and B. abortus using virB10, Omp31 and Omp16 antigens by the prediction of T lymphocytes, T cell cytotoxicity and IFN-γ epitopes. 50S L7/L12 Ribosomal protein from Mycobacterium tuberculosis was used as a bovine TLR4 and TLR9 agonist. GPGPG, AAY and KK linkers were used as a linker. Brucella construct was well-integrated in the pET-32a Shuttle vector with BamHI and HindIII restriction enzymes. The final construct contained 769 amino acids, that it was soluble protein of about ∼82 kDa after expression in the Escherichia coli SHuffle host. Modeled protein analysis based on the tertiary structure validation, molecular docking studies, molecular dynamics simulations results like RMSD, Gyration and RMSF as well as MM/PBSA analysis showed that this protein has a stable construct and is capable being in interaction with bovine TLR4 and TLR9. Analysis of the data obtained suggests that the proposed vaccine can induce the immune response by stimulating T- and B-cells, and may be used for prevention and remedial purposes, against B. melitensis and B. abortus. Communicated by Ramaswamy H. Sarma

Molecular characterization of CRBR2 fragment of TLR4 gene in association with mastitis in Vrindavani cattle

The bovine TLR4 gene is an interesting candidate marker for mastitis resistance, since it is involved in neutrophil migration to and from the mammary gland during mastitis. TLR4 detects pathogen ligands, such as the Escherichia coli lipopolysaccharide (LPS) endotoxin and facilitates innate and adaptive immune responses. In the current study, a total of 130 crossbred cows (74 mastitis tolerant and 56 with clinical mastitis) kept at the Cattle and Buffalo Farm, IVRI, Izatnagar, were selected to explore the polymorphism in the co-receptor binding region 2 (CRBR2) fragment of the TLR4 gene. PCR-SSCP and sequence analysis showed two genotypes of the TLR4 gene's CRBR2 fragment, AA and AB, which were polymorphic in both the afflicted and tolerant groups. Sequencing revealed eight single nucleotide polymorphisms (SNPs) in allele A and ten SNPs in allele B. This genotype had no significant effect on the incidence of clinical mastitis according to the logistic regression model. Our study found insufficient evidence linking SNP variants in the CRBR2 region of the TLR4 gene to mastitis susceptibility in crossbred cattle.

Host defense against Neospora caninum infection via IL-12p40 production through TLR2/TLR3-AKT-ERK signaling pathway in C57BL/6 mice

Neospora caninum is an intracellular parasite which can cause neosporosis and significant economic losses in both dairy and beef industries worldwide. A better understanding of the immune response by host cells against N. caninum could help to design better strategies for the prevention and treatment of neosporosis. Although previous studies have shown TLR2/TLR3 were involved in controlling N. caninum infection in mice, the precise mechanisms of the AKT and MAPK pathways controlled by TLR2/TLR3 to regulate N. caninum-induced IL-12p40 production and the role of TLR2/TLR3 in anti-N. caninum infection in bovine macrophages remain unclear. In the present study, TLR2−/− mice displayed more parasite burden and lower level of IL-12p40 production compared to TLR3−/− mice. N. caninum could activate AKT and ERK signaling pathways in WT mouse macrophages, which were inhibited in TLR2−/− and TLR3−/− mouse macrophages. In N. caninum-infected WT mouse macrophages, AKT inhibitor or AKT siRNA could decrease the phosphorylation of ERK. AKT or ERK inhibitors reduced the production of IL-12p40 and increased the number of parasites. The productions of ROS, NO, and GBP2 were significantly reduced in TLR2−/− and TLR3−/− mouse macrophages. Supplementation of rIL-12p40 inhibited N. caninum proliferation and rescued the productions of IFN-γ, NO, and GBP2 in WT, TLR2−/−, and TLR3−/− mouse macrophages. In bovine macrophages, the expressions of TLR2, TLR3, and IL-12p40 mRNA were significantly enhanced by N. caninum, and N. caninum proliferation was inhibited by TLR2/TLR3 agonists. Taken together, the proliferation of N. caninum in mouse macrophages was controlled by the TLR2/TLR3-AKT-ERK signal pathway via increased IL-12p40 production, which in turn lead to the productions of NO, GBP2, and IFN-γ during N. caninum infection. And in bovine macrophages, TLR2 and TLR3 contributed to inhibiting N. caninum proliferation via increased IL-12p40 production.

Sequence-Based Structural and Evolution of Polymorphisms in Bovine Toll-Like Receptor2 Gene in Dhanni and Jersey Cattle Breeds. Genetic polymorphisms in bovine TLR2

Host defense against infectious disease is a tormenting task considering not only the enormous pathogens diversity, but also their rapid genome replication and mutation rates. Front line and anti-microbial defense is accomplished by the innate immune system with the help of pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs) in prompt pathogens detection. In this study, distribution of single nucleotide polymorphisms (SNPs) in TLR2 gene was studied in two cattle breeds Dhanni and locally adapted exotic Jersey. The complete sequences of TLR2 gene composed of 5'UTR 136 bp, coding sequence 2355 bp and 3'UTR 1316 bp. Phylogenetic analysis revealed clustering of Dhanni with Bos indicus and Jersey with Bos taurus as the nearest neighbor. Sequencing of TLR2 in Dhanni cattle showed the occurrence of 35 variable sites within coding region including 37% non-synonymous and 63% synonymous, while in Jersey cattle, 21 SNPs were identified within coding region including 48% non-synonymous and 52% synonymous. Mostly SNPs fell within the leucine-rich repeats (LRR) of extracellular domain, regions responsible for ligand recognition. The ratio of dS/dN substitutions was <1 at polymorphic-sites indicating purifying selection. The SNPs at position 681 (p.Leu227Phe in Jersey) and 521(p.Thr174Ile in Dhanni) were presumed to have possible damaging or functional altering effect. The predicted bovine TLR2 is a solenoid-like (coil-like) built from 20 LRRs bend into a horseshoe shaped structure. The polymorphisms in TLR2 can be useful in future research exploring its role in immunity and may be used as markers for disease resistance under selective breeding.

Correlations between single nucleotide polymorphisms in bovine CD209, SLC11A1, SP110 and TLR2 genes and estimated breeding values for several traits in Spanish Holstein cattle

Little is known about the correlations between the genetic susceptibility/resistance to Mycobacterium avium subsp. paratuberculosis (Map) infection and the estimated breeding values for type, production and functional traits. Previously, we identified 70 combinations of five single nucleotide polymorphisms (SNPs) in four bovine innate immune genes (SLC11A1, SP110, TLR2, CD209) that are associated with the genetic risk of paratuberculosis (PTB) or Johne's disease progression, which can be graded as low (LOWIN), latent (LATIN), or patent (PATIN) risk. Other possible combinations of these 5 SNPs were grouped in the average group (AVERIN). In the current study, differences in estimated breeding values (EBVs) for several traits were analyzed using linear models in a large cohort of Holstein cows (N = 15656) genotyped across Spain in 2016 or 2017. After the assignment of each genotyped cow to a risk group, cows within the PATIN risk group (N = 1448) had a superior combined genetic index (2797.57), type genetic index (524.62), milk yield (653.92 kg), protein yield (21.77 kg), fat yield (24.82 kg) and economic merit index (125 Euros) compared with the other three risk groups. Statistically significant differences in the longevity scores between the cows that were included in the PATIN risk group (108.85) and the LOWIN (107.82) and AVERIN (107.92) groups were also observed. The associations between the genetic risk groups and PTB diagnostic results were validated in a population of 99 cows from a Spanish farm with a high prevalence of PTB. Significant differences in ELISA readings between the PATIN (65.49 %) and the AVERIN (15.97 %), LATIN (2.11 %), and LOWIN (3.27 %) groups were observed. In addition, significant differences in Map DNA copies/gram of feces were observed between the PATIN and the other three risk groups. These results together with the substantial economic impact of PTB in dairy cattle support the selection of the animals with less susceptibility to PTB in the Spanish breeding program.

Single-nucleotide polymorphisms in CLEC7A, CD209 and TLR4 gene and their association with susceptibility to paratuberculosis in Indian cattle

The aim of this study was to identify the single-nucleotide polymorphisms (SNPs) in bovine candidate genes CLEC7A, CD209 and TLR4, and explore the association between these SNPs with the occurrence of bovine paratuberculosis (PTB) disease. For this purpose, 549 animals were screened by a panel of four diagnostic tests, namely Johnin PPD test, ELISA test, faecal microscopy and IS900 blood PCR against Mycobacterium avium ssp. paratuberculosis (MAP) to develop case-control populations. SNPs were genotyped by polymerase chain reaction-restriction fragment length polymorphism method. Genotypic-phenotypic associations were assessed by the PROCLOGISTIC procedure of SAS 9.3. Of the seven SNPs; rs110353594 in CLEC7A gene and rs8193046 in TLR4 gene were found to be associated with PTB. For SNP rs110353594, odds of CC and CT genotypes vs TT genotype was 1.543 (0.420-5.667; 95% CI) and 0.284 (0.104-0.774; 95% CI), respectively which means that CT genotype was more resistant than TT and CC genotypes against bovine PTB. For SNP rs8193046, odds of AA and AG genotypes versus GG genotype was 0.947 (0.296-3.034; 95% CI) and 3.947 (1.555-10.022; 95% CI), respectively, i.e. probability for getting an infection in animals with AG genotype was 3.94 times more as compared to GG genotype. Hence, a selection programme favouring CT genotype for rs110353594 and against AG genotype for rs8193046 may be beneficial for conferring resistance against bovine PTB.

Association of TLR gene variants in a Czech Red Pied cattle population with reproductive traits

The bovine genes TLR1, TLR2 and TLR6, which encode Toll-like receptors, key components of the innate immune system, were screened for polymorphisms in Czech Red Pied (Czech Simmental) cattle, and the different variants present in the population were tested for association with reproductive and fitness traits. Diversity was investigated in a group of 164 bulls using hybrid resequencing of pooled amplicons with PacBio technology and of pooled genomic DNA using HiSeq X-Ten technology. The validated single nucleotide polymorphisms (SNPs) were genotyped in individual animals using the primer extension technique. The association of genotypic classes of 16 polymorphisms with six phenotypic traits were estimated with one-way analysis of variance (ANOVA) and with restricted maximum likelihood (REML) algorithm. The evaluated traits included the incidence of cystic ovaries, index of early reproductive disorders, paternal and maternal indicators of calving ease, production longevity and calf vitality index. The estimated breeding values were used for combined trait quantification. Early traits, namely, cystic ovaries and early reproductive disorders, were not associated with any of the tested polymorphisms according to the general ANOVA test. By contrast, five variants of all three genes were associated with calving ease, both paternal and maternal. The production longevity correlated with two variants of TLR1 and the calf vitality index correlated with the 1044 T > C (rs68268249) polymorphism in TLR2. The false discovery rate (FDR) according to Benjamini-Hochberg was favourable for the calving ease trait (0.221) and maternal calving ease (0.214), which allows to consider the observed associations real, regardless of the error arising from the multiple comparisons. These results were supported by REML only partially, probably in view of the additivity assumption. Two mechanisms of action on calving are conceivable, either via infection resistance or via the involvement of TLR2 in signalling in the myometrium. The known formation of heterodimers by the TLR1, -2 and -6 products might be responsible for the shared pattern of action in these genes. The association of the calf vitality index with TLR2 variation might reflect the increased role of infections in calves compared to adult animals.

Distinct evolution of toll-like receptor signaling pathway genes in cetaceans.

The relatively rapid spread and diversity of marine pathogens posed an initial and ongoing challenge for cetaceans (whales, dolphins, and porpoises), descendants of terrestrial mammals that transitioned from land to sea approximately 56 million years ago. Toll-like receptors (TLRs) play important roles in regulating immunity against pathogen infections by detecting specific molecular patterns and activating a wide range of downstream signaling pathways. The ever-increasing catalogue of mammalian genomes offers unprecedented opportunities to reveal genetic changes associated with evolutionary and ecological processes. This study aimed to explore the molecular evolution of TLR signaling pathway genes in cetaceans. Genes involved in the TLR signaling pathway were retrieved by BLAST searches using human coding sequences as queries. We tested each gene for positive selection along the cetacean branches using PAML and Hyphy. Physicochemical property changes of amino acids at all positively selected residues were assessed by TreeSAAP and visualized with WebLogo. Bovine and dolphin TLR4 was assessed using human embryonic kidney cell line HEK293, which lacks TLR4 and its co-receptor MD-2. We demonstrate that eight TLR signaling pathway genes are under positive selection in cetaceans. These include key genes in the response to Gram-negative bacteria: TLR4, CD14, and LY96 (MD-2). Moreover, 41 out of 65 positively selected sites were inferred to harbor substitution that dramatically changes the physicochemical properties of amino acids, with most of them situated in or adjacent to functional regions. We also found strong evidence that positive selection occurred in the lineage of the Yangtze finless porpoise, likely reflecting relatively recent adaptions to a freshwater milieu. Species-specific differences in TLR4 response were observed between cetacean and terrestrial species. Cetacean TLR4 was significantly less responsive to lipopolysaccharides from a terrestrial E. coli strain, possibly a reflection of the arms race of host-pathogen co-evolution faced by cetaceans in an aquatic environment. This study provides further impetus for studies on the evolution and function of the cetacean immune system.

Genetic polymorphism in TLR2 genes of HF cross bred cattle through PCR SSCP

TLRs are cell-surface receptors which recognize a broad class of pathogen-associated molecular patterns (PAMPs) that activates innate and adaptive immune responses. Thorough perusal of literature suggests a possible role of TLR2 gene in resistance to infection due to gram positive bacteria. Bovine TLR2 assigns to Bos taurus (BTA) chromosome 17 it spans over 13.2 kb of genomic DNA which includes 2 exons. The first exon is spread over 179bp while the second one is 3333bp. Molecular characterization of TLR2 gene, carried out by PCR- SSCP analysis, revealed presence of six SNPs viz., A827G (exon 2.2), C1088G (exon 2.3), C2155A (exon 2.5), G2281C (exon 2.5), G2410A (exon 2.6) and C2600T (exon 2.6). Gene and genotypic frequencies were determined by values for allele frequencies and hetrozygosity. The degree of heterozygosities, c2 (Chi-square) test for Hardy Weinberg (HW) equilibrium and F statistics were performed for the HF crossbred population. Population genetic analysis demonstrated high degree of genetic differentiation in the population. The studied population displayed a deviation from the HW equilibrium.

Genetic association of polymorphisms in bovine TLR2 and TLR4 genes with Mycobacterium avium subspecies paratuberculosis infection in Indian cattle population.

Toll like receptors (TLRs) are pattern recognition molecules involved in cellular recognition of Mycobacterium avium subspecies paratuberculosis (MAP), the infectious agent causing Paratuberculosis (PTB), a notified disease of domestic and wild ruminants. The present study was undertaken to investigate the presence of single nucleotide polymorphisms (SNPs) in TLR2 and TLR4 gene and to evaluate association of these SNPs with occurrence of PTB in Indian cattle. A total of 213 cattle, were subjected to multiple diagnostic tests viz. Johnin PPD, ELISA test (Indigenous and Parachek kit method), fecal microscopy and fecal culture for detection of MAP infection. Based on screening results 51 animals each were assigned to case and control population. Two SNPs viz. rs55617172, rs41830058 in TLR2 gene and two SNPs viz. rs8193046, rs8193060 in TLR4 gene and were genotyped by PCR-RFLP method. All SNPs were found to be polymorphic except rs41830058 in the case-control population. Both SNPs in TLR4 gene but none in TLR2 genes were significantly associated with the occurrence of PTB in our population. The genotypes in SNP rs8193046 and SNP rs8193060 were significantly (P < 0.01) different in case-control population. These findings suggest that SNPs rs8193046 and rs8193060 are likely a potential marker against MAP infection and a selection programme eliminating AG genotype for rs8193046 and CT genotype for rs8193060 might be beneficial in conferring resistance to MAP infection in Indian cattle population.

Molecular basis determining species specificity for TLR2 inhibition by staphylococcal superantigen-like protein 3 (SSL3)

Staphylococcus aureus is a versatile opportunistic pathogen, causing disease in human and animal species. Its pathogenicity is linked to the ability of S. aureus to secrete immunomodulatory molecules. These evasion proteins bind to host receptors or their ligands, resulting in inhibitory effects through high affinity protein–protein interactions. Staphylococcal evasion molecules are often species-specific due to differences in host target proteins between species. We recently solved the crystal structure of murine TLR2 in complex with immunomodulatory molecule staphylococcal superantigen-like protein 3 (SSL3), which revealed the essential residues within SSL3 for TLR2 inhibition. In this study we aimed to investigate the molecular basis of the interaction on the TLR2 side. The SSL3 binding region on murine TLR2 was compared to that of other species through sequence alignment and homology modeling, which identified interspecies differences. To examine whether this resulted in altered SSL3 activity on the corresponding TLR2s, bovine, equine, human, and murine TLR2 were stably expressed in HEK293T cells and the ability of SSL3 to inhibit TLR2 was assessed. We found that SSL3 was unable to inhibit bovine TLR2. Subsequent loss and gain of function mutagenesis showed that the lack of inhibition is explained by the absence of two tyrosine residues in bovine TLR2 that play a prominent role in the SSL3–TLR2 interface. We found no evidence for the existence of allelic SSL3 variants that have adapted to the bovine host. Thus, within this paper we reveal the molecular determinants of the TLR2–SSL3 interaction which adds to our understanding of staphylococcal host specificity.

Immuno-informatics Analysis to Identify Novel Vaccine Candidates and Design of a Multi-Epitope Based Vaccine Candidate Against Theileria parasites.

Theileriosis poses a serious threat to ruminants in tropical and subtropical countries. It is a tick-borne disease, caused by an apicomplexan parasite, Theileria. The high disease burden in animals causes huge economic losses to marginal farmers. Further, with increasing cases of resistance to commonly used drugs, it is highly desirable to develop better and cost-effective vaccines against theileriosis. The only available vaccine, live attenuated parasite vaccine, has many drawbacks and hence is unsuitable for controlling this disease. Immuno-informatics has emerged as a useful tool in down selection of potential molecules for vaccine development. In this study, we have used an immuno-informatics driven genome-wide screening strategy to identify potential vaccine targets containing important and effective dominant immunogens against Theileria. The proteome of Theileria annulata was screened for proteins with probability of plasma membrane localization or GPI anchor. The proteins non-homologous to the host (bovine) were selected and their antigenicity was analyzed. The B-cell epitopes were identified in the selected proteins and mapped in the modeled structure of the proteins. A total of 19 linear epitopes in 12 proteins, exposed in the extracellular space and having the potential to induce protective antibodies were obtained. Additionally, CTL epitopes which are peptides with 9-mer core sequence, were also identified, modeled and docked with bovine MHC-I structures. The CTL epitopes showing high binding energy with the bovine MHC-I were further engineered in silico to design a putative multi-epitope vaccine candidate against Theileria parasites. The docking studies and molecular dynamics studies with the predicted multi-epitope vaccine candidate and modeled bovine TLR4 exhibited strong binding energy, suggesting that the complex is stable and the putative multi-epitope vaccine candidate can be a potentially good candidate for vaccine development.

Single Nucleotide Polymorphisms in 5’ Upstream Region of Bovine TLR4 Gene Affecting Expression Profile and Transcription Factor Binding Sites

ABSTRACTThe present study in the 5’ upstream region of TLR4 gene revealed four Single Nucleotide Polymorphisms (SNPs) in Vrindavani and Tharparkar cattle. The polymorphic information content (PIC), heterozygosity and allelic diversity values were low to moderate for these SNPs. In Vrindavani cattle, one SNP was found to be in Hardy-Weinberg Equilibrium (HWE) and the remaining three were found to be in linkage disequilibrium (LD) as indicated statistically (P > 0.05). In Tharparkar cattle, two SNPs were found to be in HWE and were not in LD as indicated statistically (P > 0.05). These SNPs were used for construction of haplotypes. In-silico analysis of these SNPs predicted abolition of eight transcription factor binding sites and creation of eight new sites. The quantitative real time PCR analysis did not show any significant variation of gene expression among haplotypes. However, gene expression between breed was found to be significant (P < 0.05) which suggested that upstream region of bovine TLR4 gene has a crucial role in its expression. These findings in TLR4 gene offer essential evidence that can be useful in future research exploring its role in immunity. TLR4 can be used as a marker for selection for disease resistance in bovines.

Comparison of cellular assays for TLR activation and development of a species-specific reporter cell line for cattle.

PRRs are sentinels of the innate immune system, with TLRs being the most important. Assays for TLR ligand interactions have been used to gain insights into their function and signaling pathways. As significant differences exist between species with regard to ligand recognition, it is necessary to adapt these tools for TLRs of other species. In the present work, we describe a species-specific cell-based assay adapted for the analysis of single PRRs. Human embryonic kidney 293T cells were stably transfected with the NF-κB-inducible reporter gene secreted embryonic alkaline phosphatase (SEAP) together with bovine TLR2. We compared the SEAP response with an existing luciferase NF-κB reporter assay for correlation with IL-8 production. A dose-dependent response was detected upon stimulation using both methods with good correlation to IL-8 secretion. Lower stimulant concentrations were detected by SEAP assay than IL-8 secretion. The luciferase assay produced high non-specific background for all ligand concentrations. Of all assays tested, we found the bovine-specific SEAP reporter assay to be the most convenient and delivered results in the shortest time. The developed reporter cell line would lend well to rapid, high-throughput TLR ligand screening for cattle.

Predicting the effect of non synonymous SNPs in bovine TLR4 gene

The toll like receptor 4 is part of the innate immune system involved in the control of Gram negative pathogens mostly. Any polymorphism in this gene has been associated with susceptibility to diseases. Here we used 4 computational tools based on different approaches to predict the damage-associated non-synonymous single nucleotide polymorphisms (nsSNPs) in the coding region of bovine TLR4 gene. Among the 110 nsSNPs, arranged according to the predictions, 17.2% were classified as damaging in all the tools. The classification was also correlated with the scores of PredictSNP consensus programs. The computational analysis proved capable of identifying the potentially damaging nsSNPs in TLR4, which are candidates for further laboratory studies of the functional significance of the alterations in the receptor and the phenotypic outcomes.

Evidence for gene-gene epistatic interactions between susceptibility genes for Mycobacterium avium subsp. paratuberculosis infection in cattle

Johne's disease is a chronic granulomatous inflammatory disease caused by Mycobacterium avium subsp. paratuberculosis (MAP), with a particularly negative impact on the economy of the dairy industry. In recent years, several whole genome and candidate gene association studies have been published reporting MAP susceptibility genes, but the putative interaction between them remains unknown. Here, twenty-four single nucleotide polymorphisms in the bovine SLC11A1, NOD2, SP110, TLR2, TLR4, and CD209 genes, previously related with paratuberculosis disease, have been analyzed. Several significant (P<0.05) pair-wise genetic interactions were detected: CD209-TLR4, CD209-TLR2, TLR4-TLR2, SP110-SLC11A1, SP110-TLR2, and SP110-NOD2. The statistical interaction described here between bovine CD209 and TLR4 genes may be indicative of the biological interaction between their protein products upon infection by mycobacteria, as has been reported to occur in humans. Overall, this is the first evidence of epistasis among bovine innate immunity genes affecting susceptibility to MAP infection, corroborating, in turn, their implication in the disease.