INVOLVEMENT OF PEPTIDOGLYCAN RECOGNITION PROTEIN L6 IN ACTIVATION OF IMMUNE DEFICIENCY PATHWAY IN THE IMMUNE RESPONSIVE SILKWORM CELLS

The immune deficiency (Imd) signaling pathway is activated by Gram‐negative bacteria for producing antimicrobial peptides (AMPs). In Drosophila melanogaster, the activation of this pathway is initiated by the recognition of Gram‐negative bacteria by peptidoglycan (PGN) recognition proteins (PGRPs), PGRP‐LC and PGRP‐LE. In this study, we found that the Imd pathway is involved in enhancing the promoter activity of AMP gene in response to Gram‐negative bacteria or diaminopimelic (DAP) type PGNs derived from Gram‐negative bacteria in an immune responsive silkworm cell line, Bm‐NIAS‐aff3. Using gene knockdown experiments, we further demonstrated that silkworm PGRP L6 (BmPGRP‐L6) is involved in the activation of E. coli or E. coli‐PGN mediated AMP promoter activation. Domain analysis revealed that BmPGRP‐L6 contained a conserved PGRP domain, transmembrane domain, and RIP homotypic interaction motif like motif but lacked signal peptide sequences. BmPGRP‐L6 overexpression enhances AMP promoter activity through the Imd pathway. BmPGRP‐L6 binds to DAP‐type PGNs, although it also binds to lysine‐type PGNs that activate another immune signal pathway, the Toll pathway in Drosophila. These results indicate that BmPGRP‐L6 is a key PGRP for activating the Imd pathway in immune responsive silkworm cells.     

Fitness and microbial networks of the common wasp,Vespula vulgaris (Hymenoptera: Vespidae), in its native and introduced ranges

1. Variation in microbial communities between populations is increasingly hypothesised to affect animal fitness and performance, including for invasive species. Pathogenic species may be lost during the introduction process, enhancing invader fitness and abundance. 2. This study assessed fitness, immune gene expression, and microbial network complexity of invasive common wasps, Vespula vulgaris. Microbial networks were assayed using 16S and 18S sequencing and gene expression arrays in the native (Belgium) and introduced range (New Zealand). The immune gene expression of the wasp Down syndrome cell adhesion molecule (Dscam) gene homologue was examined. Dscam expression can be induced by viruses, Gram‐positive and Gram‐negative bacteria, and parasites. 3. Individual nest fitness was higher in the native range of Belgium than in the introduced New Zealand range. Microbial communities of wasps in the introduced range were more diverse with more complex networks, although some microorganisms were range‐specific. Microbial networks in the introduced range showed higher clustering coefficients, number of connected paths, network centralisation, number of neighbours and network density. 4. Larvae, workers, virgin and foundress queens had higher expression of Dscam in the New Zealand samples. These immune gene expression patterns were associated with higher pathogen pressure and lower relative fitness. 5. Epidemiological theory predicts that a high density of pathogen and microbial hosts should result in a high rate of disease infection, prevalence, and highly connected microbial networks. The results of this study support these predictions. Wasps displayed lower relative fitness and more highly connected microbial networks in New Zealand than in Belgium.     

Rho 1 participates in parasitoid wasp eggs maturation and host cellular immunity inhibition

Endoparasitoid wasps introduce venom into their host insects during the egg-laying stage. Venom proteins play various roles in the host physiology, development, immunity, and behavior manipulation and regulation. In this study, we identified a venom protein, MmRho1, a small guanine nucleotide-binding protein derived from ovary in the endoparasitoid wasp Microplitis mediator and found that knockdown of its expression by RNA interference caused down-regulation of vitellogenin and juvenile hormone, egg production, and cocoons formation in the female wasps. We demonstrated that MmRho1 entered the cotton bollworm's (host) hemocytes and suppressed cellular immune responses after parasitism using immunofluorescence staining. Furthermore, wasp MmRho1 interacted with the cotton bollworm's actin cytoskeleton rearrangement regulator diaphanous by yeast 2-hybrid and glutathione s-transferase pull-down. In conclusion, this study indicates that MmRho1 plays dual roles in wasp development and the suppression of the host insect cellular immune responses.     

Natural variation in the contribution of microbial density to inducible immune dynamics

Immune responses evolve to balance the benefits of microbial killing against the costs of autoimmunity and energetic resource use. Models that explore the evolution of optimal immune responses generally include a term for constitutive immunity, or the level of immunological investment prior to microbial exposure, and for inducible immunity, or investment in immune function after microbial challenge. However, studies rarely consider the functional form of inducible immune responses with respect to microbial density, despite the theoretical dependence of immune system evolution on microbe‐ versus immune‐mediated damage to the host. In this study, we analyse antimicrobial peptide (AMP) gene expression from seven wild‐caught flour beetle populations (Tribolium spp.) during acute infection with the virulent bacteria Bacillus thuringiensis (Bt) and Photorhabdus luminescens (P.lum) to demonstrate that inducible immune responses mediated by the humoral IMD pathway exhibit natural variation in both microbe density‐dependent and independent temporal dynamics. Beetle populations that exhibited greater AMP expression sensitivity to Bt density were also more likely to die from infection, while populations that exhibited higher microbe density‐independent AMP expression were more likely to survive P. luminescens infection. Reduction in pathway signalling efficiency through RNAi‐mediated knockdown of the imd gene reduced the magnitude of both microbe‐independent and dependent responses and reduced host resistance to Bt growth, but had no net effect on host survival. This study provides a framework for understanding natural variation in the flexibility of investment in inducible immune responses and should inform theory on the contribution of nonequilibrium host‐microbe dynamics to immune system evolution.     

Bactericidal peptidoglycan recognition protein induces oxidative stress in Escherichia coli through a block in respiratory chain and increase in central carbon catabolism

Mammalian Peptidoglycan Recognition Proteins (PGRPs) kill both Gram‐positive and Gram‐negative bacteria through simultaneous induction of oxidative, thiol and metal stress responses in bacteria. However, metabolic pathways through which PGRPs induce these bactericidal stress responses are unknown. We screened Keio collection of Escherichia coli deletion mutants and revealed that deleting genes for respiratory chain flavoproteins or for tricarboxylic acid (TCA) cycle resulted in increased resistance of E. coli to PGRP killing. PGRP‐induced killing depended on the production of hydrogen peroxide, which required increased supply of NADH for respiratory chain oxidoreductases from central carbon catabolism (glycolysis and TCA cycle), and was controlled by cAMP‐Crp. Bactericidal PGRP induced a rapid decrease in respiration, which suggested that the main source of increased production of hydrogen peroxide was a block in respiratory chain and diversion of electrons from NADH oxidoreductases to oxygen. CpxRA two‐component system was a negative regulator of PGRP‐induced oxidative stress. By contrast, PGRP‐induced thiol stress (depletion of thiols) and metal stress (increase in intracellular free Zn²⁺ through influx of extracellular Zn²⁺) were mostly independent of oxidative stress. Thus, manipulating pathways that induce oxidative, thiol and metal stress in bacteria could be a useful strategy to design new approaches to antibacterial therapy.     

Combined use of herbivore‐induced plant volatiles and sex pheromones for mate location in braconid parasitoids

Herbivore‐induced plant volatiles (HIPVs) are important cues for female parasitic wasps to find hosts. Here, we investigated the possibility that HIPVs may also serve parasitoids as cues to locate mates. To test this, the odour preferences of four braconid wasps – the gregarious parasitoid Cotesia glomerata (L.) and the solitary parasitoids Cotesia marginiventris (Cresson), Microplitis rufiventris Kokujev and Microplitis mediator (Haliday) – were studied in olfactometers. Each species showed attraction to pheromones but in somewhat different ways. Males of the two Cotesia species were attracted to virgin females, whereas females of M. rufiventris were attracted to virgin males. Male and female M. mediator exhibited attraction to both sexes. Importantly, female and male wasps of all four species were strongly attracted by HIPVs, independent of mating status. In most cases, male wasps were also attracted to intact plants. The wasps preferred the combination of HIPVs and pheromones over plant odours alone, except M. mediator, which appears to mainly use HIPVs for mate location. We discuss the ecological contexts in which the combined use of pheromones and HIPVs by parasitoids can be expected. To our knowledge, this is the first study to show that braconid parasitoids use HIPVs and pheromones in combination to locate mates.     

Pefabloc – A sulfonyl fluoride serine protease inhibitor blocks induction of Diptericin in Drosophila l(2)mbn cells

Abstract Insects protect themselves against microbial infection by an efficient innate immune system that is activated by recognition of invariant microbial surface molecules. In the fruit fly Drosophila melanogaster the presence of bacteria is associated with expression of antimicrobial peptides in host immune‐competent tissues. Host receptors detect infection and relay the signal to mount the appropriate immune response. In Drosophila hemocyte‐like l(2)mbn cells pre‐infection treatment with Pefabloc, a commonly used serine protease inhibitor, induced two major effects: it blocked expression of the antibacterial peptide Diptericin in response to live Gram‐negative bacteria and bacterial surface molecules (crude lipopolysaccharide contaminated by peptidoglycans) and it induced morphological changes.     

Bacterial membrane vesicles deliver peptidoglycan to NOD1 in epithelial cells

Gram‐negative bacterial peptidoglycan is specifically recognized by the host intracellular sensor NOD1, resulting in the generation of innate immune responses. Although epithelial cells are normally refractory to external stimulation with peptidoglycan, these cells have been shown to respond in a NOD1‐dependent manner to Gram‐negative pathogens that can either invade or secrete factors into host cells. In the present work, we report that Gram‐negative bacteria can deliver peptidoglycan to cytosolic NOD1 in host cells via a novel mechanism involving outer membrane vesicles (OMVs). We purified OMVs from the Gram‐negative mucosal pathogens: Helicobacter pylori, Pseudomonas aeruginosa and Neisseria gonorrhoea and demonstrated that these peptidoglycan containing OMVs upregulated NF‐κB and NOD1‐dependent responses in vitro. These OMVs entered epithelial cells through lipid rafts thereby inducing NOD1‐dependent responses in vitro. Moreover, OMVs delivered intragastrically to mice‐induced innate and adaptive immune responses via a NOD1‐dependent but TLR‐independent mechanism. Collectively, our findings identify OMVs as a generalized mechanism whereby Gram‐negative bacteria deliver peptidoglycan to cytosolic NOD1. We propose that OMVs released by bacteria in vivo may promote inflammation and pathology in infected hosts.     

Toll/IMD signal pathways mediate cellular immune responses via induction of intracellular PLA 2 expression

Phospholipase A₂ (PLA₂) hydrolyzes fatty acids from phospholipids at the sn‐2 position. Two intracellular PLA₂s, iPLA₂A and iPLA₂B, have been found in Spodoptera exigua. Both are calcium‐independent cellular PLA₂. Their orthologs have been found in other insects. These two iPLA₂s are different in ankyrin motif of N terminal region. The objective of this study was to determine whether Toll/immune deficiency (IMD) signal pathways could mediate cellular immune responses via induction of iPLA₂ expression. Both iPLA ₂s were expressed in all developmental stages of S. exigua, showing the highest expression in the adult stage. During larval stage, hemocyte is the main tissue showing expression of these iPLA₂s. Both iPLA₂s exhibited similar expression patterns after immune challenge with different microbial pathogens such as virus, bacteria, and fungi. Promoter component analysis of orthologs encoded in S. frugiperda indicated nuclear factor‐κB‐ and Relish‐responsible elements on their promoters, suggesting their expression in S. exigua under Toll/IMD immune signaling pathways. RNA interference (RNAi) of MyD88 or Pelle under Toll pathway suppressed inducible expression levels of both iPLA₂s in response to Gram‐positive bacteria containing Lys‐type peptidoglycan or fungal infection. In contrast, RNAi against Relish under IMD pathway suppressed both iPLA₂s in response to infection with Gram‐negative bacteria. Under RNAi conditions, hemocytes significantly lost cellular immune response measured by nodule formation. However, addition of arachidonic acid (a catalytic product of PLA₂) rescued such immunosuppression. These results suggest that Toll/IMD signal pathways can mediate cellular immune responses via eicosanoid signaling by inducing iPLA₂ expression.     

Immunosuppression induced by expression of a viral RNase enhances susceptibility of Plutella xylostella to microbial pesticides

Abstract Polydnaviruses are a group of insect DNA viruses and are characterized in their segmented genome that is located in the chromosome(s) of host wasps. A polydnavirus, Cotesia plutellae bracovirus (CpBV), encodes a viral ribonuclease (RNase) T2 in a specific segment #3 (CpBV‐S3). This study tested its effect on gene expression associated with host immune responses in the diamondback moth, Plutella xylostella. Micro‐injection of CpBV‐S3 into nonparasitized larvae induced expression of its two encoded genes, CpBV‐ORF301 (=CpBV‐RNase T2) and CpBV‐ORF302. In response to a bacterial challenge, four antimicrobial peptide genes (hemolin, gloverin, cecropin and lysozyme) and six phenoloxidase (PO)–associated genes (proPO‐activating proteinase, PO, serine proteinase homolog and serpins 1–3) were up‐regulated in their expressions. However, the transient expression of CpBV‐S3 suppressed the expressions of cecropin, PO and serpin 1. Double‐stranded RNA specific to the viral RNase T2 could specifically knockdown the viral gene expression and restored the three gene expressions suppressed in the larvae injected with CpBV‐S3. The inhibitory activity of the viral RNase T2 on the target genes was further proven by the suppression of PO activation in response to bacterial challenge in the larvae injected with CpBV‐S3. This immunosuppression by the expression of the viral RNase T2 resulted in significant increase of pathogen susceptibility of P. xylostella against Bacillus thuringiensis or baculovirus infection.     

Identification of two clip domain serine proteases involved in the pea aphid's defense against bacterial and fungal infection

Phenoloxidases (POs) are required for the pea aphid's defense against bacterial and fungal infection. Prophenoloxidases (PPOs) are proteolytically converted to its active form PO through a clip domain serine protease cascade. In this study, we identified five clip domain serine proteases in the pea aphids. The messenger RNA levels of two of them, Ap_SPLP and Ap_VP, were upregulated by Gram‐positive bacterium Staphylococcus aureus and fungus Beauveria bassiana infections. Double‐stranded RNA‐based expression knockdown of these two genes resulted in reduced PO activity of the aphid hemolymph, higher loads of S. aureus and B. bassiana in the aphids, and lower survival rates of the aphids after infections. Our data suggest that Ap_SPLP and Ap_VP are involved in PPO activation pathway in the pea aphid.     

IDENTIFICATION AND FUNCTIONAL CHARACTERIZATION OF A PEPTIDOGLYCAN RECOGNITION PROTEIN FROM THE COTTON BOLLWORM,Helicoverpa armigera

Peptidoglycan recognition proteins (PGRPs) specifically bind to peptidoglycans, and play crucial roles as pattern recognition receptors (PRRs) in mediating innate immune responses. In this study, we identified and characterized a PGRP (HaPGRP‐D) from the cotton bollworm, Helicoverpa armigera. Sequence analysis indicated that HaPGRP‐D is an amidase‐type PGRP. Expression of HaPGRP‐D was upregulated in the hemocytes of H. armigera larvae after injecting Gram‐negative Escherichia coli, Gram‐positive Staphylococcus aureus, or chromatography beads. To test the biological activity of HaPGRP‐D, purified recombinant protein was prepared. Subsequent analysis showed that rHaPGRP‐D (i) could bind and agglutinate Gram‐negative E. coli and Gram‐positive S. aureus in a zinc‐dependent manner, (ii) functioned as an amidase to degrade peptidoglycans in the presence of Zn²⁺, (iii) strongly inhibited the growth of E. coli and S. aureus in the presence of Zn²⁺, (iv) could bind to the surface of hemocytes, (v) increased the phagocytosis of E. coli cells by hemocytes in vitro, and (vi) promoted hemocyte encapsulation on chromatography beads in vitro. These results suggest that HaPGRP‐D plays important roles as PRR, amidase, and opsonin in H. armigera humoral and cellular immune responses.     

Why is Listeria monocytogenes such a potent inducer of CD8+ T‐cells?

Listeria monocytogenes is a rapidly growing, Gram‐positive, facultative intracellular pathogen that has been used for over 5 decades as a model to study basic aspects of infection and immunity. In a murine intravenous infection model, immunisation with a sublethal infection of L. monocytogenes initially leads to rapid intracellular multiplication followed by clearance of the bacteria and ultimately culminates in the development of long‐lived cell‐mediated immunity (CMI) mediated by antigen‐specific CD8+ cytotoxic T‐cells. Importantly, effective immunisation requires live, replicating bacteria. In this review, we summarise the cell and immunobiology of L. monocytogenes infection and discuss aspects of its pathogenesis that we suspect lead to robust CMI. We suggest five specific features of L. monocytogenes infection that positively impact the development of CMI: (a) the bacteria have a predilection for professional antigen‐presenting cells; (b) the bacteria escape from phagosomes, grow, and secrete antigens into the host cell cytosol; (c) bacterial‐secreted proteins enter the major histocompatibility complex (MHC) class I pathway of antigen processing and presentation; (d) the bacteria do not induce rapid host cell death; and (e) cytosolic bacteria induce a cytokine response that favours CMI. Collectively, these features make L. monocytogenes an attractive vaccine vector for both infectious disease applications and cancer immunotherapy.     

Streptococcus mutans requires mature rhamnose‐glucose polysaccharides for proper pathophysiology,morphogenesis and cellular division

The cell wall of Gram‐positive bacteria has been shown to mediate environmental stress tolerance, antibiotic susceptibility, host immune evasion and overall virulence. The majority of these traits have been demonstrated for the well‐studied system of wall teichoic acid (WTA) synthesis, a common cell wall polysaccharide among Gram‐positive organisms. Streptococcus mutans, a Gram‐positive odontopathogen that contributes to the enamel‐destructive disease dental caries, lacks the capabilities to generate WTA. Instead, the cell wall of S. mutans is highly decorated with rhamnose‐glucose polysaccharides (RGP), for which functional roles are poorly defined. Here, we demonstrate that the RGP has a distinct role in protecting S. mutans from a variety of stress conditions pertinent to pathogenic capability. Mutant strains with disrupted RGP synthesis failed to properly localize cell division complexes, suffered from aberrant septum formation and exhibited enhanced cellular autolysis. Surprisingly, mutant strains of S. mutans with impairment in RGP side chain modification grew into elongated chains and also failed to properly localize the presumed cell wall hydrolase, GbpB. Our results indicate that fully mature RGP has distinct protective and morphogenic roles for S. mutans, and these structures are functionally homologous to the WTA of other Gram‐positive bacteria.     

Dose‐dependent fate of GFP‐expressing Escherichia coli in the alimentary canal of adult house flies

The adult house fly Musca domestica (L.) (Diptera: Muscidae) can disseminate bacteria from microbe‐rich substrates to areas in which humans and domesticated animals reside. Because bacterial abundance fluctuates widely across substrates, flies encounter and ingest varying amounts of bacteria. This study investigated the dose‐dependent survival of bacteria in house flies. Flies were fed four different ‘doses’ of green fluorescent protein (GFP)‐expressing Escherichia coli (GFP E. coli) (very low, low, medium, high) and survival was determined at 1, 4, 10 and 22 h post‐ingestion by culture and epifluorescent microscopy. Over 22 h, the decline in GFP E. coli was significant in all treatments (P < 0.04) except the very low dose treatment (P = 0.235). Change in survival (ΔS) did not differ between flies fed low and very low doses of bacteria across all time‐points, although ΔS in both treatments differed from that in flies fed high and medium doses of bacteria at several time‐points. At 4, 10 and 22 h, GFP E. coli ΔS significantly differed between medium and high dose‐fed flies. A threshold dose, above which bacteria are detected and destroyed by house flies, may exist and is likely to be immune‐mediated. Understanding dose‐dependent bacterial survival in flies can help in predicting bacteria transmission potential.     

Modulation of immune responses of Rhynchophorus ferrugineus (Insecta: Coleoptera) induced by the entomopathogenic nematode Steinernema carpocapsae (Nematoda: Rhabditida)

Aim of this study was to investigate relationships between the red palm weevil (RPW) Rhynchophorus ferrugineus (Olivier) and the entomopathogenic nematode Steinernema carpocapsae (EPN); particularly, the work was focused on the immune response of the insect host in naive larvae and after infection with the EPN. Two main immunological processes have been addressed: the activity and modulation of host prophenoloxidase‐phenoloxidase (proPO) system, involved in melanization of not‐self and hemocytes recognition processes responsible for not‐self encapsulation. Moreover, immune depressive and immune evasive strategies of the parasite have been investigated. Our results suggest that RPW possess an efficient immune system, however in the early phase of infection, S. carpocapsae induces a strong inhibition of the host proPO system. In addition, host cell‐mediated mechanisms of encapsulation, are completely avoided by the parasite, the elusive strategies of S. carpocapsae seem to be related to the structure of its body‐surface, since induced alterations of the parasite cuticle resulted in the loss of its mimetic properties. S. carpocapsae before the release of its symbiotic bacteria, depress and elude RPW immune defenses, with the aim to arrange a favorable environment for its bacteria responsible of the septicemic death of the insect target.     

Outer membrane protein OmpV mediates Salmonella enterica serovar typhimurium adhesion to intestinal epithelial cells via fibronectin and α1β1 integrin

Salmonella typhimurium is an invasive Gram‐negative enteric bacterium, which causes salmonellosis, a type of gastroenteritis in humans and typhoid‐like symptoms in mice. Upon entering through the contaminated food and water, S. typhimurium adheres, colonises, and invades intestinal epithelial cells (IECs) of the small intestine. In this study, we have shown that upon deletion of the outer membrane protein OmpV, there is a significant decrease in adherence of S. typhimurium to the IECs, indicating that OmpV is an important adhesin of S. typhimurium. Further, our study showed that OmpV binds to the extracellular matrix component fibronectin and signals through α1β1 integrin receptor on the IECs and OmpV‐mediated activation of α1β1, resulting in the activation of focal adhesion kinase and F‐actin modulation. Actin modulation is crucial for bacterial invasion. To the best of our knowledge, this is the first report of an adhesin mediated its effect through integrin in S. typhimurium. Further, we have observed a decrease in pathogenicity in terms of increased LD₅₀ dose, lesser bacterial numbers in stool, and less colonisation of bacteria in different organs of mice infected with Δompv mutant compared with the wild‐type bacteria, thus confirming the crucial role of OmpV in the pathogenesis of S. typhimurium.     

Involvement of the MyD88‐independent pathway in controlling the intracellular fate of Burkholderia pseudomallei infection in the mouse macrophage cell line RAW 264.7

Burkholderia pseudomallei is a facultative intracellular Gram‐negative bacterium which is capable of surviving and multiplying inside macrophages. B. pseudomallei strain SRM117, a LPS mutant which lacks the O‐antigenic polysaccharide moiety, is more susceptible to macrophage killing during the early phase of infection than is its parental wild type strain (1026b). In this study, it was shown that the wild type is able to induce expression of genes downstream of the MyD88‐dependent (iκbζ, il‐6 and tnf‐α), but not of the MyD88‐independent (inos, ifn‐β and irg‐1), pathways in the mouse macrophage cell line RAW 264.7. In contrast, LPS mutant‐infected macrophages were able to express genes downstream of both pathways. To elucidate the significance of activation of the MyD88‐independent pathway in B. pseudomallei‐infected macrophages, the expression of TBK1, an essential protein in the MyD88‐independent pathway, was silenced prior to the infection. The results showed that silencing the tbk1 expression interferes with the gene expression profile in LPS mutant‐infected macrophages and allows the bacteria to replicate intracellularly, thus suggesting that the MyD88‐independent pathway plays an essential role in controlling intracellular survival of the LPS mutant. Moreover, exogenous IFN‐γ upregulated gene expression downstream of the MyD88‐independent pathway, and interfered with intracellular survival in both wild type and tbk1‐knockdown macrophages infected with either the wild type or the LPS mutant. These results suggest that gene expression downstream of the MyD88‐independent pathway is essential in regulating the intracellular fate of B. pseudomallei, and that IFN‐γ regulates gene expression through the TBK1‐independent pathway.