Purpose-built multicomponent supramolecular silver(I)-hydrogels as membrane-targeting broad-spectrum antibacterial agents against multidrug-resistant pathogens.

Membrane-targeting compounds are of immense interest to counter complicated multi-drug resistant infections. However, the broad-spectrum effect of such compounds is often unmet due to the surges of antibiotic resistance among majority of Gram-negative bacteria compared to Gram-positive species. Though amphiphiles, synthetic mimics of antimicrobial peptides etc, have been extensively explored for their potential to perturb bacterial membranes, small molecule-based supramolecular hydrogels have remained unexplored. The design of supramolecular hydrogels can be tuned on-demand, catering to desired applications, including facile bacterial membrane perturbation. Considering the strong biocidal properties of Ag-based systems and the bacterial membrane-targeting potential of appended primary amine groups, we designed self-assembled multicomponent supramolecular Ag(I)-hydrogels with urea and DATr (3,5-diamino-1,2,4-triazole) as ligands, which are predisposed for hydrogen bonding and interacting with negatively charged bacterial membranes at physiological pH. The synthesized supramolecular Ag(I)-hydrogels exhibited almost similar antibacterial activity against both Gram-negative (Campylobacter jejuni; C. jejuni) and Gram-positive (Staphylococcus aureus; S. aureus) bacteria, with minimal inhibitory concentration (MIC) of ∼60 µg mL-1. Ag(I)-hydrogels facilitated the disruption of the negatively charged bacterial membrane due to electrostatic interaction and complementary hydrogen bonding facilitated by DATr and urea. Sustained intracellular ROS generation in the presence of Ag(I)-hydrogel further expedited cell lysis. We envisage that the multicomponent supramolecular Ag(I)-hydrogels studied herein can be employed in designing effective antibacterial coatings on a range of medical devices, including surgical instruments. Moreover, the present form of the hydrogels has the potential to improve the antibacterial functionality of conventional antimicrobials, thus revitalizing the effective targeting of hard-to-treat multi-drug-resistant (MDR) bacterial infections in a clinical set up.

Targeted Bioimaging of Microencapsulated Recombinant LAB Vector Expressing Fluorescent Reporter Protein: A Non-invasive Approach for Microbial Tracking.

Lactococcus lactis (L. lactis), the first genetically modified Generally Recognized As Safe (GRAS) category Lactic Acid producing Bacteria (LAB), is best known for its generalized health-promoting benefits and ability to express heterologous proteins. However, achieving the optimal probiotic effects requires a selective approach that would allow us to study in vivo microbial biodistribution, fate, and immunological consequences. Although the chemical conjugation of fluorophores and chromophores represent the standard procedure to tag microbial cells for various downstream applications, it requires a high-throughput synthesis scheme, which is often time-consuming and expensive. On the contrary, the genetic manipulation of LAB vector, either chromosomally or extra-chromosomally, to express bioluminescent or fluorescent reporter proteins has greatly enhanced our ability to monitor bacterial transit through a complex gut environment. However, with faster passage and quick washing out from the gut due to rhythmic contractions of the digestive tract, real-time tracking of LAB vectors, particularly non-commensal ones, remains problematic. To get a deeper insight into the biodistribution of non-commensal probiotic bacteria in vivo, we bioengineered L. lactis to express fluorescence reporter proteins, mCherry (bright red monomeric fluorescent protein) and mEGFP (monomeric enhanced green fluorescent protein), followed by microencapsulation with a mucoadhesive and biodegradable polymer, chitosan. We show that coating of recombinant Lactococcus lactis (rL. lactis) with chitosan polymer, cross-linked with tripolyphosphate (TPP), retains their ability to express the reporter proteins stably without altering the specificity and sensitivity of fluorescence detection in vitro and in vivo. Further, we provide evidence of enhanced intragastric stability by chitosan-TPP (CS) coating of rL. lactis cells, allowing us to study the spatiotemporal distribution for an extended time in the gut of two unrelated hosts, avian and murine. The present scheme involving genetic modification and chitosan encapsulation of non-commensal LAB vector demonstrates great promise as a non-invasive and intensive tool for active live tracking of gut microbes.

Activation of Antiviral Host Responses against Avian Influenza Virus and Remodeling of Gut Microbiota by rLAB Vector Expressing rIL-17A in Chickens.

Low-pathogenic avian influenza virus (LPAIV) remains the most common subtype of type-A influenza virus that causes moderate to severe infection in poultry with significant zoonotic and pandemic potential. Due to high mutability, increasing drug resistance, and limited vaccine availability, the conventional means to prevent intra- or interspecies transmission of AIV is highly challenging. As an alternative to control AIV infections, cytokine-based approaches to augment antiviral host defense have gained significant attention. However, the selective application of cytokines is critical since unregulated expression of cytokines, particularly proinflammatory ones, can cause substantial tissue damage during acute phases of immune responses. Moreover, depending on the type of cytokine and its impact on intestinal microbiota, outcomes of cytokine-gut microflora interaction can have a critical effect on overall host defense against AIV infections. Our recent study demonstrated some prominent roles of chicken IL-17A (ChIL-17A) in regulating antiviral host responses against AIV infection, however, in an in vitro model. For more detailed insights into ChIL-17A function, in the present study, we investigated whether ChIL-17A-meditated elevated antiviral host responses can translate into effective immune protection against AIV infection in an in vivo system. Moreover, considering the role of gut health in fostering innate or local host responses, we further studied the contributory relationships between gut microbiota and host immunity against AIV infection in chickens. For this, we employed a recombinant lactic acid-producing bacterial (LAB) vector, Lactococcus lactis, expressing ChIL-17A and analyzed the in vivo functionality in chickens against an LPAIV (A/H9N2) infection. Our study delineates that mucosal delivery of rL. lactis expressing ChIL-17A triggers proinflammatory signaling cascades and can drive a positive shift in phylum Firmicutes, along with a marked decline in phylum Actinobacteriota and Proteobacteria, favoring effective antiviral host responses against AIV infection in chickens. We propose that ChIL-17A-mediated selective expansion of beneficial gut microbiota might form a healthy microbial community that augments the effective immune protection against AIV infections in chickens.

Selective depletion of Campylobacter jejuni via T6SS dependent functionality: an approach for improving chickens gut health.

The targeted depletion of potential gut pathogens is often challenging because of their intrinsic ability to thrive in harsh gut environments. Earlier, we showed that Campylobacter jejuni (C. jejuni) exclusively uses the Type-VI Secretion System (T6SS) to target its prey such as Escherichia coli (E. coli), and phenotypic differences between T6SS-negative and T6SS-positive C. jejuni isolates toward bile salt sensitivity. However, it remains unclear how the target-driven T6SS functionality prevails in a polymicrobial gut environment. Here, we investigated the fate of microbial competition in an altered gut environment via bacterial T6SS using a T6SS-negative and -positive C. jejuni or its isogenic mutant of the hemolysin-coregulated protein (hcp). We showed that in the presence of bile salt and prey bacteria (E. coli), T6SS-positive C. jejuni experiences enhanced intracellular stress leading to cell death. Intracellular tracking of fluorophore-conjugated bile salts confirmed that T6SS-mediated bile salt influx into C. jejuni can enhance intracellular oxidative stress, affecting C. jejuni viability. We further investigated whether the T6SS activity in the presence of prey (E. coli) perturbs the in vivo colonization of C. jejuni. Using chickens as primary hosts of C. jejuni and non-pathogenic E. coli as prey, we showed a marked reduction of C. jejuni load in chickens cecum when bile salt solution was administered orally. Analysis of local antibody responses and pro-inflammatory gene expression showed a reduced risk of tissue damage, indicating that T6SS activity in the complex gut environment can be exploited as a possible measure to clear the persistent colonization of C. jejuni in chickens.

Biologically Active Micropatterns of Biomolecules and Living Matter Using Microbubble Lithography.

In situ patterning of biomolecules and living organisms while retaining their biological activity is extremely challenging, primarily because such patterning typically involves thermal stresses that could be substantially higher than the physiological thermal or stress tolerance level. Top-down patterning approaches are especially prone to these issues, while bottom-up approaches suffer from a lack of control in developing defined structures and the time required for patterning. A microbubble generated and manipulated by optical tweezers (microbubble lithography) is used to self-assemble and pattern living organisms in continuous microscopic structures in real-time, where the material thus patterned remains biologically active due to their ability to withstand elevated temperatures for short exposures. Successful patterns of microorganisms (Escherichia coli, Lactococcus. lactis and the Type A influenza virus) are demonstrated, as well as reporter proteins such as green fluorescent protein (GFP) on functionalized substrates with high signal-to-noise ratio and selectivity. Together, the data presented herein may open up fascinating possibilities in rapid in situ parallelized diagnostics of multiple pathogens and bioelectronics.

Application of Mono and Trinuclear Cyclometalated Iridium (III) Complexes in Differential Bacterial Imaging and Antimicrobial Photodynamic Therapy.

The application of transition metal complexes for antimicrobial photodynamic therapy (PDT) has emerged as an attractive alternative in mitigating a broad range of bacterial pathogens, including multidrug-resistant pathogens. In view of their photostability, long excited-state lifetimes, and tunable emission properties, transition metal complexes also contribute as bioimaging agents. In the present work, we designed mono and trinuclear cyclometalated iridium (III) complexes to explore their imaging application and antibacterial potential. For this, we used Methicillin-resistant S. aureus (MRSA), the most prevalent of community-associated (CA) multidrug-resistant (MDR) bacteria (CA MDR) and Lactococcus lactis (L. lactis) as Gram-positive while Campylobacter jejuni (C. jejuni) and E. coli as Gram-negative bacteria. In addition to differential bioimaging of these bacteria, we assessed the antibacterial effects of both mono and trinuclear Ir(III) complexes under exposure to 427 nm LED light. The data presented herein strongly suggest better efficacy of trinuclear Ir(III) complex over the mononuclear complex in imparting photoinduced cell death of MRSA. Based on the safety profile of these complexes, we propose that trinuclear cyclometalated iridium(III) complex holds great promise for selective recognition and targeting MDR bacteria with minimal off-target effect.

Antibacterial activity of hydrophobicity modulated cationic polymers with enzyme and pH-responsiveness.

The membrane lipid compositions of prokaryotic and eukaryotic cells are inherently different in many aspects, although some similarities exist in their structure and composition. Therefore, selective targeting of membrane lipids with a compound of therapeutic value, such as an antibacterial copolymer, is often challenging. Hence, developing an ideal copolymer with antibacterial properties demands hydrophobicity/hydrophilicity balance with a high biosafety profile. To integrate hydrophobic/hydrophilic balance and cationic charge in an alternating antibacterial copolymer with enzyme and pH-responsiveness, a lysine appended styrenic monomer was copolymerized with a fatty acid (octanoic acid (OA) or myristic acid (MA)) tethered maleimide monomer via reversible addition-fragmentation chain transfer (RAFT) polymerization. A range of microscopic analyses, including dynamic light scattering (DLS), confirmed the formation of nanoaggregates (size ∼30-40 nm) by these polymers in aqueous solution with positive zeta potential (cationic surface charge). Hydrophobic Nile red (NR) dye was successfully encapsulated in the nanoaggregates, and the in vitro release kinetics of the NR dye were monitored at different pHs and in the presence or absence of esterase/lipase. The in vitro release kinetics of NR revealed ∼85% dye release in the presence of pH 5.5 and lipase, suggesting their suitability for pH/enzyme-triggered therapeutic payload delivery. The standard broth microdilution assay showed significant bactericidal activity against both Gram-positive (Bacillus subtilis) and Gram-negative (Escherichia coli) bacteria with an MIC50 value <30 µg mL-1. The effect of polymeric nanoaggregates on bacterial morphology and in vitro survival was further confirmed by field emission scanning electron microscopy (FESEM), agar gel disk diffusion assay, and bacterial live/dead cell count. The significantly low hemolytic activity against red blood cells (RBCs) (HC50 >103 µg mL-1) and nontoxic effect on human intestinal epithelial cells (INT 407) (EC50 >500 µg mL-1) ensure that the polymer nanoaggregates are safe for in vivo use and can serve as a potent antibacterial polymer.

Identification and functional characterization of putative ligand binding domain(s) of JlpA protein of Campylobacter jejuni.

Among the major Surface Exposed Colonization Proteins (SECPs) of Campylobacter jejuni (C. jejuni), Jejuni lipoprotein A (JlpA) plays a crucial role in host cell adhesion specifically by binding to the N-terminal domain of the human heat shock protein 90α (Hsp90α-NTD). Although the JlpA binding to Hsp90α activates NF-κB and p38 MAP kinase pathways, the underlying mechanism of JlpA association with the cellular receptor remains unclear. To this end, we predicted two potential receptor binding sites within the C-terminal domain of JlpA: one spanning from amino acid residues Q332-A354 and the other from S258-T295; however, the latter exhibited weaker binding. To assess the functional attributes of these predicted sequences, we generated two JlpA mutants (JlpAΔ1: S258-T295; JlpAΔ2: Q332-A354) and assessed the Hsp90α-binding affinity-kinetics by in vitro and ex vivo experiments. Our findings confirmed that the residues Q332-A354 are of greater importance in host cell adhesion with a measurable impact on cellular responses. Further, thermal denaturation by circular dichroism (CD) confirmed that the reduced binding affinity of the JlpAΔ2 to Hsp90α is not associated with protein folding or stability. Together, this study provides a possible framework for determining the molecular function of designing rational inhibitors efficiently targeting JlpA.

Tuning the Organelle-Specific Imaging and Photodynamic Therapeutic Efficacy of Theranostic Mono- and Trinuclear Organometallic Iridium(III) Complexes.

The organelle-specific localization of mononuclear and trinuclear iridium(III) complexes and their photodynamic behavior within the cells are described herein, emphasizing their structure-activity relationship. Both the IrA2 and IrB2 complexes possess a pair of phenyl-benzothiazole derived from the -CHO moieties of mononuclear organometallic iridium(III) complexes IrA1 and IrB1, which chelates IrCp*Cl (Cp* = 1,2,3,4,5-pentamethylcyclopentadiene) to afford trinuclear complexes IrA3 and IrB3. Insights into the photophysical and electrochemical parameters of the complexes were obtained by a time-dependent density functional theory study. The synthesized complexes IrA2, IrA3, IrB2, and IrB3 were found to be nontoxic to human MCF7 breast carcinoma cells. However, the photoexcitation of complexes using LED light could effectively trigger intracellular reactive oxygen species (ROS) generation, leading to cell death. Furthermore, to check the organelle-specific localization of IrA2 and IrB2, we observed that both complexes could selectively localize in the endoplasmic reticulum. In contrast, trinuclear IrA3 and IrB3 accumulate in the nuclei. The photoexcitation of complexes using LED light could effectively trigger intracellular reactive oxygen species (ROS) generation, leading to cell death.

The immune-adjunctive potential of recombinant LAB vector expressing murine IFNλ3 (MuIFNλ3) against Type A Influenza Virus (IAV) infection.

BACKGROUND: The conventional means of controlling the recurring pandemics of Type A Influenza Virus (IAV) infections remain challenging primarily because of its high mutability and increasing drug resistance. As an alternative to control IAV infections, the prophylactic use of cytokines to drive immune activation of multiple antiviral host factors has been progressively recognized. Among them, Type III Interferons (IFNs) exhibit a pivotal role in inducing potent antiviral host responses by upregulating the expression of several antiviral genes, including the Interferon-Stimulated Genes (ISGs) that specifically target the virus replication machinery. To harness the immuno-adjunctive potential, we examined whether pre-treatment of IFNλ3, a Type III IFN, can activate antiviral host responses against IAV infections. METHODS: In the present study, we bioengineered a food-grade lactic acid-producing bacteria (LAB), Lactococcus lactis (L. lactis), to express and secrete functional murine IFNλ3 (MuIFNλ3) protein in the extracellular milieu. To test the immune-protective potential of MuIFNλ3 secreted by recombinant L. lactis (rL. lactis), we used murine B16F10 cells as an in vitro model while mice (BALB/c) were used for in vivo studies. RESULTS: Our study demonstrated that priming with MuIFNλ3 secreted by rL. lactis could upregulate the expression of several antiviral genes, including Interferon Regulatory Factors (IRFs) and ISGs, without exacerbated pulmonary or intestinal inflammatory responses. Moreover, we also showed that pre-treatment of B16F10 cells with MuIFNλ3 can confer marked immune protection against mice-adapted influenza virus, A/PR/8/1934 (H1N1) infection. CONCLUSION: Since the primary target for IAV infections is the upper respiratory and gastrointestinal tract, immune activation without affecting the tissue homeostasis suggests the immune-adjunctive potential of IFNλ3 against IAV infections.

Multimodal Biofilm Inactivation Using a Photocatalytic Bismuth Perovskite-TiO2-Ru(II)polypyridyl-Based Multisite Heterojunction.

Infectious bacterial biofilms are recalcitrant to most antibiotics compared to their planktonic version, and the lack of appropriate therapeutic strategies for mitigating them poses a serious threat to clinical treatment. A ternary heterojunction material derived from a Bi-based perovskite-TiO2 hybrid and a [Ru(2,2'-bpy)2(4,4'-dicarboxy-2,2'-bpy)]2+ (2,2'-bpy, 2,2'-bipyridyl) as a photosensitizer (RuPS) is developed. This hybrid material is found to be capable of generating reactive oxygen species (ROS)/reactive nitrogen species (RNS) upon solar light irradiation. The aligned band edges and effective exciton dynamics between multisite heterojunctions are established by steady-state/time-resolved optical and other spectroscopic studies. Proposed mechanistic pathways for the photocatalytic generation of ROS/RNS are rationalized based on a cascade-redox processes arising from three catalytic centers. These ROS/RNS are utilized to demonstrate a proof-of-concept in treating two elusive bacterial biofilms while maintaining a high level of biocompatibility (IC50 > 1 mg/mL). The in situ generation of radical species (ROS/RNS) upon photoirradiation is established with EPR spectroscopic measurements and colorimetric assays. Experimental results showed improved efficacy toward biofilm inactivation of the ternary heterojunction material as compared to their individual/binary counterparts under solar light irradiation. The multisite heterojunction formation helped with better exciton delocalization for an efficient catalytic biofilm inactivation. This was rationalized based on the favorable exciton dissociation followed by the onset of multiple oxidation and reduction sites in the ternary heterojunction. This together with exceptional photoelectric features of lead-free halide perovskites outlines a proof-of-principle demonstration in biomedical optoelectronics addressing multimodal antibiofilm/antimicrobial modality.

A combined protocol for isolation of T6SS-positive Campylobacter jejuni and assessment of interspecies interaction.

The bacterial Type VI Secretion System (T6SS) functions as a nanomachine used by many gut pathogens. In the present protocol, we outlined how such molecular activities during interspecies interaction can be demonstrated at a population level. To this end, we first present a comprehensive protocol for isolation, identification, and functional characterization of T6SS-positive Campylobacter jejuni. Further, we developed straightforward techniques for unraveling how the T6SS targets prey populations and host cells when growing with or without environmental stressors. For complete details on the use and execution of this protocol, please refer to Gupta et al. (2021).

The cost of bacterial predation via type VI secretion system leads to predator extinction under environmental stress.

As a common gut pathogen, Campylobacter jejuni (C. jejuni) harbors the Type VI Secretion System (T6SS) that injects toxic effectors into neighboring cells, modulating microbial competitions in the harsh gut environment. Using bile salt as a natural stressor and T6SS-positive C. jejuni as a predator, we show that T6SS activity could entail a cost during bacterial predation under environmental stress. Our data suggest bile salt influx and subsequent DNA damage due to the prey-driven activation of the T6SS. We further combined experiments and mathematical modeling to explore how the stress-induced "predation cost" determines ecological outcomes. Consistent with a population-dynamics model, we found predator extinction above a critical bile salt concentration and prey-predator coexistence below this level. Moreover, we utilized the predation cost as an effective strategy facilitating host defense against C. jejuni infection. Together, we elucidate how predator dominance versus extinction emerges from the interplay between environmental stress and the T6SS machinery.

Pre-treatment with chicken IL-17A secreted by bioengineered LAB vector protects chicken embryo fibroblasts against Influenza Type A Virus (IAV) infection.

The recent advances in our understanding of the host factors in orchestrating qualitatively different immune responses against influenza Type A virus (IAV) have changed the perception of conventional approaches for controlling avian influenza virus (AIV) infection in chickens. Given that infection-induced pathogenicity and replication of influenza virus largely rely on regulating host immune responses, immunoregulatory cytokine profiles often determine the disease outcomes. However, in contrast to the function of other inflammatory cytokines, interleukin-17A (IL-17A) has been described as a 'double-edged sword', indicating that in addition to antiviral host responses, IL-17A has a distinct role in promoting viral infection. Therefore, in the present study, we investigated the chicken IL-17A mediated antiviral immune effects on IAVs infection in primary chicken embryo fibroblasts cells (CEFs). To this end, we first bioengineered a food-grade Lactic Acid Producing Bacteria (LAB), Lactococcus lactis (L. lactis), secreting bioactive recombinant chicken IL-17A (sChIL-17A). Next, the functionality of sChIL-17A was confirmed by transcriptional upregulation of several genes associated with antiviral host responses, including granulocyte-monocyte colony-stimulating factor (GM-CSF) (CSF3 in the chickens), interleukin-6 (IL-6), interferon-α (IFN-α), -ß and -γ genes in primary CEFs cells. Consistent with our hypothesis that such a pro-inflammatory state may translate to immunoprotection against IAVs infection, we observed that sChIL-17A pre-treatment could significantly limit the viral replication and protect the primary CEFs cells against two heterotypic IAVs such as A/turkey/Wisconsin/1/1966(H9N2) and A/PR/8/1934(H1N1). Together, the data presented in this work suggest that exogenous application of sChIL-17A secreted by modified LAB vector may represent an alternative strategy for improving antiviral immunity against avian influenza virus infection in chickens.

Bioengineering of LAB vector expressing Haemolysin co-regulated protein (Hcp): a strategic approach to control gut colonization of Campylobacter jejuni in a murine model.

BACKGROUND: Campylobacter jejuni (C. jejuni) is accountable for more than 400 million cases of gastroenteritis each year and is listed as a high-priority gut pathogen by the World Health Organization (WHO). Although the acute infection of C. jejuni (campylobacteriosis) is commonly treated with macrolides and fluoroquinolones, the emergence of antibiotic resistance among C. jejuni warrants the need for an alternative approach to control campylobacteriosis in humans. To this end, vaccines remain a safe, effective, and widely accepted strategy for controlling emerging and re-emerging infectious diseases. In search of a suitable vaccine against campylobacteriosis, recently, we demonstrated the potential of recombinant Haemolysin co-regulated protein (Hcp) of C. jejuni Type VI secretion system (T6SS) in imparting significant immune-protection against cecal colonization of C. jejuni; however, in the avian model. Since clinical features of human campylobacteriosis are more complicated than the avians, we explored the potential of Hcp as a T6SS targeted vaccine in a murine model as a more reliable and reproducible experimental host to study vaccine-induced immune-protection against C. jejuni. Because C. jejuni primarily utilizes the mucosal route for host pathogenesis, we analyzed the immunogenicity of a mucosally deliverable bioengineered Lactic acid bacteria (LAB), Lactococcus lactis (L. lactis), expressing Hcp. Considering the role of Hcp in both structural (membrane-bound) and functional (effector protein) exhibition of C. jejuni T6SS, a head-to-head comparison of two different forms of recombinant LAB vectors (cell wall anchored and secreted form of Hcp) were tested and assessed for the immune phenotypes of each modality in BALB/c mice. RESULTS: We show that regardless of the Hcp protein localization, mucosal delivery of bioengineered LAB vector expressing Hcp induced high-level production of antigen-specific neutralizing antibody (sIgA) in the gut with the potential to reduce the cecal load of C. jejuni in mice. CONCLUSION: Together with the non-commensal nature of L. lactis, short gut transit time in humans, and the ability to express the heterologous protein in the gut, the present study highlights the benefits of bioengineered LAB vectors based mucosal vaccine modality against C. jejuni without the risk of immunotolerance.

Molecular characterization of Bu-1 and TLR2 gene in Haringhata Black chicken.

Haringhata Black is the only registered indigenous poultry genetic resource of West Bengal till date. Molecular characterization of HB revealed that Bu-1 to be highly glycoylated transmembrane protein unlike mammalian Bu-1, whereas TLR2 of HB chicken was observed to be rich in Leucine rich repeat. HB chicken was observed to be genetically close to chicken of Japan, while distant to chicken breed of UK and Chicago. Avian species wise evolution study indicates genetic closeness of HB chicken with turkey. Differential mRNA expression profile for the immune response genes (TLR2, TLR4 and Bu1 gene) were studied for HB chicken with respect to other chicken breed and poultry birds, which reveals that HB chicken were better in terms of B cell mediated immunity and hence better response to vaccination. Hence HB chicken is one of the best poultry genetic resources to be reared under backyard system where biosecurity measures are almost lacking.

Intragastric delivery of recombinant Lactococcus lactis displaying ectodomain of influenza matrix protein 2 (M2e) and neuraminidase (NA) induced focused mucosal and systemic immune responses in chickens.

Compounding with the problem of frequent antigenic shift and occasional drift of the segmented genome of Avian Influenza Virus (AIV), vaccines based on major surface glycoproteins such as haemagglutinin (HA) to counter heterosubtypic AIV infection in chickens remain unsuccessful. In contrast, neuraminidase (NA), the second most abundant surface glycoprotein present in viral capsid is less mutable and, in some instances, successful in eliciting inter-species cross-reactive antibody responses. However, without selective activation of B-cells and T-cells, the ability of NA to induce strong cell mediated immune responses is limited, thus NA based vaccines cannot singularly address the risk of virus escape from host defence. To this end, the highly conserved ectodomain of influenza matrix protein-2 (M2e) has emerged as an attractive cross-protective vaccine target. The present study describes the potential of recombinant Lactococcus lactis (rL. lactis) in expressing functional influenza NA or M2e proteins and conferring effective mucosal and systemic immune responses in the intestine as well as in the upper respiratory airways (trachea) of chickens. In addition, lavages collected from trachea and intestine of birds administered with rL. lactis expressing influenza NA or M2e protein were found to protect MDCK cells against avian influenza type A/PR/8/34 (H1N1) virus challenge. Although minor, the differences in the expression of pro-inflammatory cytokines gene transcripts targeted in this study among the birds administered with either empty or rL. lactis could be attributed to the activation of innate response by L. lactis.

Immunogenicity and protective efficacy of mucosal delivery of recombinant hcp of Campylobacter jejuni Type VI secretion system (T6SS) in chickens.

The type VI secretion system (T6SS) has recently emerged as a new pattern of protein secretions in Campylobacter jejuni (C. jejuni). Within the T6SS cluster, hemolysin co-regulated protein (hcp) is considered as a hallmark of functional T6SS and holds key role in bacterial virulence. As poultry is the primary reservoir of C. jejuni and the major sources for human infection, we evaluated the capacity of recombinant hcp (rhcp) immunization in blocking C. jejuni colonization in chickens with an aim to control bacterial transmission to humans via poultry food chain. Considering the mucosal route is the primary portal for C. jejuni entry and gut mucosa offers the apposite site for C. jejuni adherence, we investigated the immune-protective potential of intra-gastric administration of rhcp using chitosan-based nanoparticles. To achieve this goal, full length coding sequence of hcp gene from C. jejuni was cloned and expressed in E. coli. Purified rhcp was entrapped in chitosan-Sodium tripolyphosphate nanoparticles (CS-TPP NPs) and orally gavaged in chickens. Our results suggest that intra-gastric immunization of CS-TPP-rhcp induces consistent and steady increase in intestinal (sIgA) and systemic antibody (IgY) response against rhcp with significant reduction in cecal load of C. jejuni. The protection afforded by rhcp associated cellular responses with Th1 and Th17 profile in terms of increased expression of NFkB, IL-1ß, IL-8, IL-6, IFN-γ and IL-17 A genes. Though systemic immunization of rhcp with IFA resulting in a robust systemic (IgY) and local (sIgA) antibody response, mucosal administration of rhcp loaded CS-TPP NPs was found to be superior in terms of bacterial clearance. Altogether, present study suggests that chitosan based intra-gastric delivery of rhcp have several advantages over the injectable composition and could be a promising vaccine approach to effectively control C. jejuni colonization in chickens.

Role of putative virulence traits of Campylobacter jejuni in regulating differential host immune responses.

Among the major enteric pathogens, Campylobacter jejuni is considered an important source of diarrheal illness in humans. In contrast to the acute gastroenteritis in humans, C. jejuni exhibits prolonged cecal colonization at a high level with little or no pathology in chickens. Although several known virulence determinants of C. jejuni have been found to be associated with a higher degree of pathogenesis in humans, to date, little is known about their functions in the persistent colonization of chickens. The present study was undertaken to assess the role of C. jejuni in imparting differential host immune responses in human and chicken cells. Based on the abundance of major genes encoding virulence factors (GEVFs), we used a particular isolate that harbors the cadF, flaA, peb1, racR, ciaB, cdtB, and hcp genes. This study showed that hypervirulent C. jejuni isolate that encodes a functional type VI secretion system (T6SS) has a greater ability to invade and create characteristic "attaching and effacing" lesions in human INT407 compared to primary chicken embryo intestinal cells (CEICs). Furthermore, we demonstrated that the higher bacterial invasion in human INT407 triggered higher levels of expression of major proinflammatory cytokines, such as IL- 1ß and IL-6, and significant downregulation of IL-17A gene expression (P ≤ 0.05). The findings of the present study suggest that the enhanced ability of C. jejuni to invade human cells is tightly regulated by proinflammatory cytokines in the gut and possibly holds the keys to the observed differences in pathogenesis between human and chicken cells.

The effects of administration of ligands for Toll-like receptor 4 and 21 against Marek's disease in chickens.

Ligands for Toll-like receptors (TLRs) are known to stimulate immune responses, leading to protection against bacterial and viral pathogens. Here, we aimed to examine the effects of various TLR ligands on the development of Marek's disease in chickens. Specific-pathogen free chickens were treated with a series of TLR ligands that interact with TLR3, TLR9 and TLR21. In a pilot study, it was determined that TLR4 and TLR21 ligands are efficacious, in that they could reduce the incidence of Marek's disease tumors in infected birds. Hence, in a subsequent study, chickens were treated with lipopolysaccharide (LPS) as a TLR4 and CpG oligodeoxynucleotides (ODN) as TLR21 agonists before being challenged with the RB1B strain of Marek's disease virus (MDV) via the respiratory route. The results demonstrated that the administration of LPS or CpG ODN, but not PBS or non-CpG ODN, delayed disease onset and reduced MDV genome copy number in the spleens of infected chickens. Taken together, our data demonstrate that TLR4 and 21 agonists modulate anti-virus innate immunity including cytokine responses in MD-infected chicken and this response can only delay, but not inhibit, disease progression.