Brenda Allan

Chicken TLR21 acts as a functional homologue to mammalian TLR9 in the recognition of CpG oligodeoxynucleotides

Similar to mammalian species, chickens show marked immunological responses to CpG oligodeoxynucleotides (ODNs) both in vivo and in vitro. In mammals, the receptor for ODNs has been demonstrated to be TLR9; however, an orthologue to mammalian TLR9 is absent in the chicken genome. In this study, chicken TLRs 7, 15 and 21 were expressed in mammalian HEK-293T cells; expression of TLR21 but not TLR7 or 15 resulted in marked NF-kappaB activation upon stimulation with exogenous ODN. This activation was not observed when cells were stimulated by TLR agonists other than ODNs. In addition, responsiveness of the ectopically expressed TLR21 demonstrated similar kinetics of activation as reported for mammalian TLR9 and was dependent on the nucleotide sequence of the ODN. The same ODN specificity was observed for chicken HD11 macrophage when ODN mediated activation was monitored by up-regulation of IL1, IL6 and iNOS transcripts. Furthermore, when TLR21, but not TLR15, was partially silenced in HD11 cells by RNA interference, ODN mediated responses were reduced. TLR21-mediated NF-kappaB activation in HEK-293T cells was inhibited by bafilomycin A suggesting that endosomal maturation is required for TLR21 activation and observations by confocal microscopy and digestion with endoglycosidase H suggest TLR21 localizes to the endoplasmic reticulum (ER) of resting cells. Expression of TLR21 transcripts was found in all chicken tissues examined but was significantly less in the lung and small intestine of newly hatched birds. Two of the leucine rich repeat regions (LRRs) of TLR21 showed homology with a LRR conserved within mammalian TLR9 and implicated in ligand binding. We hypothesize that avian TLR21 plays a similar role to that of mammalian TLR9 and enables recognition of microbial DNA as a danger signal resulting in downstream innate and adaptive immune responses.

Induction of a Novel Chicken Toll-Like Receptor following Salmonella enterica Serovar Typhimurium Infection

ABSTRACT Toll-like receptors (TLRs) are a group of highly conserved molecules that initiate the innate immune response to pathogens by recognizing structural motifs expressed by microbes. We have identified a novel TLR, TLR15, by bioinformatic analysis of the chicken genome, which is distinct from any known vertebrate TLR and thus appears to be avian specific. The gene for TLR15 was sequenced and is found on chromosome 3, and it has archetypal TIR and transmembrane domains and a distinctive arrangement of extracellular leucine-rich regions. mRNA for TLR15 was detected in the spleen, bursa, and bone marrow of healthy chickens, suggesting a role for this novel receptor in constitutive host defense. Following in vivo Salmonella enterica serovar Typhimurium infection, quantitative real-time PCR demonstrated significant upregulation of TLR15 in the cecum of infected chickens. Interestingly, similar induction of TLR2 expression following infection was also observed. In vitro studies revealed TLR15 upregulation in chicken embryonic fibroblasts stimulated with heat-killed S. enterica serovar Typhimurium. Collectively, these results suggest a role for the TLR in avian defense against bacterial infection. We hypothesize that TLR15 may represent an avian-specific TLR that has been either retained in chicken and lost in other taxa or gained in the chicken.

Protection of chickens against Escherichia coli infections by DNA containing CpG motifs.

ABSTRACT Synthetic oligodeoxynucleotides (ODN) containing CpG motifs (CpG-ODN) have been shown to be effective immunoprotective agents in murine models for a variety of viral, intracellular bacterial, and protozoan infections. Until now, the use of CpG-ODN to protect against extracellular bacterial infections has not been reported. The objective of this study was to investigate the effect of CpG-ODN against cellulitis and colibacillosis in broiler chickens, using a well-established model. At 22 days of age, birds received CpG-ODN by either the subcutaneous or intramuscular route. Three days later, a virulent isolate of Escherichia coli was applied to a scratch site on the caudal abdominal skin. Birds were examined for 10 days after the E. coli challenge, and pathological and bacteriological assessments were conducted on all birds. The control group of birds receiving no CpG-ODN(2007) had a survival rate of 15%. In contrast, groups that received CpG-ODN(2007), by either subcutaneous or intramuscular injection, had significantly higher survival rates (P < 0.0001). Furthermore, the size of the cellulitis lesion was significantly smaller in groups that received CpG-ODN(2007) by the subcutaneous route (P < 0.01). A dose of as little as 3.16 μg of CpG-ODN(2007), delivered 3 days prior to challenge by either the subcutaneous or intramuscular route, significantly protected birds against E. coli infection (P < 0.01). This study demonstrates that CpG-ODN(2007) has both local and systemic protective effects in broiler chickens. This is the first time that CpG-ODN(2007) has been demonstrated to have an immunoprotective effect against an extracellular bacterial infection in any food animal species.

Avian toll-like receptors

Analysis of the genomes of two distantly related bird species, chicken and zebra finch (divergence of about 100 million years), indicate that there are ten avian toll-like receptors and that five of these, TLR2a, 2b, 3, 4, 5 and 7, are clear orthologs to TLRs found in mammals. Duplication of genes has led to TLR1La and 1Lb, TLR2a and 2b, and two TLR7s in the zebra finch. Avian TLR21 may be orthologous to TLR21 found in fish and amphibians, and avian TLR15, which is phylogenetically related to the TLR2 family, appears to be unique to avian species. While TLR2 is conserved between mammalian and avian species, the other TLR2 family members evolved independently. Dimerization between either of the two avian TLR2 species and TLR1La or 1Lb permits recognition of the same broad range of molecules as recognized by mammalian TLR2 dimerized with either TLR1, 6 and 10. Similarly, while TLR9 has been lost from the avian genome, DNA high in unmethylated CpG motifs is immunostimulatory through avian TLR21 which is absent in mammals. Thus, while some TLR members were commonly retained in both mammals and birds, others were separately lost or gained, or diverged independently; but broadly speaking the TLRs of the two classes of vertebrates evolved to recognize very similar spectra of microbial products. Components of downstream TLR signaling are also mostly conserved but with some losses in avian species; notably, TRAM is absent in avian genomes and, hence, the TRIF/TRAM-dependent signaling pathway utilized by mammals in LPS activation appears to be absent in birds.

Biosynthesis of the N-Linked Glycan in Campylobacter jejuni and Addition onto Protein through Block Transfer

In eukaryotes, N-linked protein glycosylation is a universal modification involving addition of preformed oligosaccharides to select Asn-Xaa-Ser/Thr motifs and influencing multiple biological events. We recently demonstrated that Campylobacter jejuni is the first member of the Bacteria to possess an N-linked glycan pathway. In this study, high-resolution magic angle spinning nuclear magnetic resonance (HR-MAS NMR) was applied to probe and quantitate C. jejuni N-glycan biosynthesis in vivo. To confirm HR-MAS NMR findings, glycosylation mutants were screened for chicken colonization potential, and glycoproteins were examined by mass spectrometry and lectin blotting. Consistent with the mechanism in eukaryotes, the combined data indicate that bacterial glycans are assembled en bloc, emphasizing the evolutionary conservation of protein N glycosylation. We also show that under the conditions examined, PglG plays no role in glycan biosynthesis, PglI is the glucosyltransferase and the putative ABC transporter, and WlaB (renamed PglK) is required for glycan assembly. These studies underpin the mechanism of N-linked protein glycosylation in Bacteria and provide a simple model system for investigating protein glycosylation and for exploitation in glycoengineering.

The CprS sensor kinase of the zoonotic pathogen Campylobacter jejuni influences biofilm formation and is required for optimal chick colonization

Campylobacter jejuni, a prevalent cause of bacterial gastroenteritis, must adapt to different environments to be a successful pathogen. We previously identified a C. jejuni two‐component regulatory system (Cj1226/7c) as upregulated during cell infections. Analyses described herein led us to designate the system CprRS (Campylobacterplanktonic growth regulation). While the response regulator was essential, a cprS sensor kinase mutant was viable. The ΔcprS mutant displayed an apparent growth defect and formed dramatically enhanced and accelerated biofilms independent of upregulation of previously characterized surface polysaccharides. ΔcprS also displayed a striking dose‐dependent defect for colonization of chicks and was modestly enhanced for intracellular survival in INT407 cells. Proteomics analyses identified changes consistent with modulation of essential metabolic genes, upregulation of stress tolerance proteins, and increased expression of MOMP and FlaA. Consistent with expression profiling, we observed enhanced motility and secretion in ΔcprS, and decreased osmotolerance and oxidative stress tolerance. We also found that C. jejuni biofilms contain a DNase I‐sensitive component and that biofilm formation is influenced by deoxycholate and the metabolic substrate fumarate. These results suggest that CprRS influences expression of factors important for biofilm formation, colonization and stress tolerance, and also add to our understanding of C. jejuni biofilm physiology.

Polyphosphate Kinase 1 Is a Pathogenesis Determinant in Campylobacter jejuni

Campylobacter jejuni is the leading cause of bacterial gastroenteritis in the developed world. Despite its prevalence, relatively little is known about C. jejunis precise pathogenesis mechanisms, particularly in comparison to other well-studied enteric organisms such as Escherichia coli and Salmonella spp. Altered expression of phosphate genes in a C. jejuni stringent response mutant, together with known correlations between the stringent response, polyphosphate (poly-P), and virulence in other bacteria, led us to investigate the role of poly-P in C. jejuni stress survival and pathogenesis. All sequenced C. jejuni strains harbor a conserved putative polyphosphate kinase 1 predicted to be principally responsible for poly-P synthesis. We generated a targeted ppk1 deletion mutant (Deltappk1) in C. jejuni strain 81-176 and found that Deltappk1, as well as the DeltaspoT stringent response mutant, exhibited low levels of poly-P at all growth stages. In contrast, wild-type C. jejuni poly-P levels increased significantly as the bacteria transitioned from log to stationary phase. Phenotypic analyses revealed that the Deltappk1 mutant was defective for survival during osmotic shock and low-nutrient stress. However, certain phenotypes associated with ppk1 deletion in other bacteria (i.e., motility and oxidative stress) were unaffected in the C. jejuni Deltappk1 mutant, which also displayed an unexpected increase in biofilm formation. The C. jejuni Deltappk1 mutant was also defective for the virulence-associated phenotype of intraepithelial cell survival in a tissue culture infection model and exhibited a striking, dose-dependent chick colonization defect. These results indicate that poly-P utilization and accumulation contribute significantly to C. jejuni pathogenesis and affect its ability to adapt to specific stresses and stringencies. Furthermore, our study demonstrates that poly-P likely plays both similar and unique roles in C. jejuni compared to its roles in other bacteria and that poly-P metabolism is linked to stringent response mechanisms in C. jejuni.

Innate immune gene expression differentiates the early avian intestinal response between Salmonella and Campylobacter

Salmonella enterica serovar Typhimurium and Campylobacter jejuni are major human pathogens, yet colonise chickens without causing pathology. The aim of this study was to compare intestinal innate immune responses to both bacterial species, in a 4-week-old broiler chicken model. Challenged and control birds were sacrificed and tissue samples taken for histopathology and RNA extraction. No significant clinical or pathological changes were observed in response to infection with either bacterial species. Expression of selected genes involved in pathogen detection and the innate immune response were profiled in caecal tissues by quantitative real-time PCR. TLR4 and TLR21 gene expression was transiently increased in response to both bacterial species (P<0.05). Significant increases in TLR5 and TLR15 gene expression were detected in response to S. Typhimurium but not to C. jejuni. Transient increases of proinflammatory cytokine (IL6 and IFNG) and chemokine (IL8 and K60) genes increased as early as 6h in response to S. Typhimurium. Minimal cytokine gene expression was detected in response to C. jejuni after 20h. IL8 gene expression however, was significantly increased by 24-fold (P<0.01). The differential expression profiles of innate immune genes in both infection models shed light on the tailored responses of the host immune system to specific microbes. It is further evidence that innate regulation of these responses is an important prerequisite to preventing development of disease.

Protection of Neonatal Chicks Against a Lethal Challenge of Escherichia coli Using DNA Containing Cytosine-Phosphodiester-Guanine Motifs

Oligodeoxynucleotides (ODN) containing cytosine-phosphodiester-guanine (CpG) motifs have been shown to be effective immunoprotective agents in murine models for a variety of viral, intracellular bacterial, and protozoan infections. We recently have shown that CpG ODN protects against extracellular bacterial infections in mature chickens. The objective of this study was to investigate the effect of CpG ODN on Escherichia coli septicemia in neonatal broiler chicks. Two-day-old chicks, or embryonated eggs that had been incubated for 18 or 19 days, received 50 microg CpG ODN. Three days after exposure to CpG ODN, a virulent isolate of E. coli was inoculated subcutaneously in the neck of each bird. Birds were examined for 7 days post-E. coli challenge and dinical, pathologic, and bacteriologic assessments were conducted. The control group of birds that received no CpG ODN had a survival rate of 0% to 20%. In contrast, groups that received CpG ODN, either by intramuscular or in ovo routes, had significantly higher survival rates (P < 0.0001). Bacterial counts in air sacs were significantly lower when birds or embryos were treated with CpG ODN as compared with controls. A dose as low as 10 microg of CpG ODN, administered intramuscularly, was able to protect birds significantly against E. coli challenge. Formulation of CpG ODN with 30% Emulsigen did not enhance the protection. This study demonstrates that CpG ODN has systemic protective effects in broiler chicks against E. coli infections. This is the first time that CpG ODN has been demonstrated to have an immunoprotective effect against a bacterial infection in chicks following in ovo delivery.

Protection of Neonatal Broiler Chicks Against Salmonella Typhimurium Septicemia by DNA Containing CpG Motifs

Abstract Oligodeoxynucleotides (ODN) containing cytosine-phosphodiester-guanine (CpG-ODN) motifs have been shown to stimulate the innate immune system against a variety of bacterial and protozoan infections in a variety of vertebrate species. The objective of this study was to investigate the immunostimulatory effect of CpG-ODN in neonatal broilers against Salmonella Typhimurium septicemia. Day-old broiler chicks, or embryonated eggs that had been incubated for 18 days, received 50 µg of CpG-ODN, 50 µg of non-CpG-ODN, or saline. Four days after exposure to CpG-ODN or day 2 posthatch, 1 × 106 or 1 × 107 colony-forming units (cfu) of a virulent isolate of Salmonella Typhimurium was inoculated by the subcutaneous route in the neck. Clinical signs, pathology, bacterial isolations from the air sacs, and mortality were observed for 10 days following challenge with Salmonella Typhimurium. The survival rate of birds in groups receiving either non-CpG-ODN or saline following Salmonella Typhimurium infection was 40%–45%. In contrast, birds receiving CpG-ODN had significantly higher survival rate of 80%–85% (P < 0.0001). Bacterial loads and pathology were low in groups treated with CpG-ODN compared to the groups receiving saline or non-CpG-ODN. Colony-forming units of Salmonella Typhimurium in the peripheral blood were significantly lower in birds treated with CpG-ODN compared to the group that received saline. This is the first time that CpG-ODN has been demonstrated to have an immunoprotective effect against an intracellular bacterial infection in neonatal broiler chickens following in ovo delivery. Protección de pollos de engorde recién nacidos contra septicemia causada por Salmonella Typhimurium mediante ADN con motivos CpG. Se ha demostrado que los oligodeoxinucleótidos que contienen motivos CpG (por sus siglas en Inglés CpG-ODN), estimulan el sistema inmune innato contra una variedad de infecciones bacterianas y de protozoarios en varias especies de vertebrados. El objetivo de este estudio fue investigar el efecto inmunoestimulatorio de CpG-ODN contra Salmonella Typhimurium en pollos de engorde recién nacidos. Pollitos de engorde de un día de edad o huevos fértiles en el día 18 de la etapa embrionaria, recibieron 50 µg de CpG-ODN, 50 µg de oligodeoxinucleótidos sin motivos CpG (No-CpG-ODN) o solución salina. Cuatro días después de la exposición a los CpG-ODN o dos días después del nacimiento, las aves se inocularon subcutáneamente en el cuello con 1 × 106 o 1 × 107 unidades formadoras de colonias de un aislamiento virulento de Salmonella Typhimurium. Posterior al desafío, las aves se observaron durante 10 días para la aparición de signos clínicos, alteraciones patológicas, aislamiento bacteriano de los sacos aéreos y mortalidad. La tasa de supervivencia posterior a la infección con Salmonella Typhimurium en los grupos que recibieron No-CpG-ODN o solución salina fue de un 40 a 45%. En contraste, las aves que recibieron CpG-ODN mostraron una tasa de supervivencia significativamente mas alta de 80 a 85% (P < 0.0001). En comparación con los grupos que recibieron No-CpG-ODN o solución salina, las cargas bacterianas y la patología de las aves que recibieron CpG-ODN fueron bajas. Las unidades formadoras de colonia de Salmonella Typhimurium en sangre periférica fueron significativamente menores en las aves tratadas con CpG-ODN que en las aves tratadas con solución salina. Esta es la primera vez que se demuestra que los CpG-ODN administrados in ovo tienen un efecto contra una infección bacteriana intracelular en pollos de engorde recién nacidos. Abbreviations: CCS = cumulative clinical score; cfu = colony-forming units; CpG-ODN = cytosine-phosphodiester-guanine oligodeoxynucleotides; IFN-γ = interferon gamma; IL = interleukin; NK = natural killer cells; PAMPs = pathogen-associated molecular patterns; TLR = Toll-like receptor; TSIA = triple-sugar iron agar