A Short-Type Peptidoglycan Recognition Protein 1 (PGRP1) Is Involved in the Immune Response in Asian Corn Borer, Ostrinia furnacalis (Guenée).

The insect immune response is initiated by the recognition of invading microorganisms. Peptidoglycan recognition proteins (PGRPs) function primarily as pattern recognition receptors by specifically binding to peptidoglycans expressed on microbial surfaces. We cloned a full-length cDNA for a PGRP from the Asian corn borer Ostrinia furnacalis (Guenée) and designated it as PGRP1. PGRP1 mRNA was mainly detected in the fat bodies and hemocytes. Its transcript levels increased significantly upon bacterial and fungal challenges. Purified recombinant PGRP1 exhibited binding activity to the gram-positive Micrococcus luteus, gram-negative Escherichia coli, entomopathogenic fungi Beauveria bassiana, and yeast Pichia pastoris. The binding further induced their agglutination. Additionally, PGRP1 preferred to bind to Lys-type peptidoglycans rather than DAP-type peptidoglycans. The addition of recombinant PGRP1 to O. furnacalis plasma resulted in a significant increase in phenoloxidase activity. The injection of recombinant PGRP1 into larvae led to a significantly increased expression of several antimicrobial peptide genes. Taken together, our results suggest that O. furnacalis PGRP1 potentially recognizes the invading microbes and is involved in the immune response in O. furnacalis.

Physiological costs of chemical defence: repeated reflex bleeding weakens the immune system and postpones reproduction in a ladybird beetle.

In insects, external chemical defences, such as reflex bleeding, have been proved to be an efficient strategy against various predators. At the same time, significant costs of reflex bleeding can be expected because bled haemolymph is lost and all valuable components included have to be renewed. Interestingly, this issue has rarely been investigated for adult insects. In this study, we examined the immune and fitness costs of repeated reflex bleeding in adults of the invasive ladybird Harmonia axyridis, investigating several haemolymph parameters. Reflex bleeding induced twice a week for three weeks resulted in a significant reduction in haemocyte concentration, total protein content, and antimicrobial activity against Micrococcus luteus, and a marginally non-significant decrease in antimicrobial activity against Escherichia coli. Repeated reflex bleeding did not result in significant body mass reduction. Interestingly, resource limitation in the form of complete food absence did not significantly interact with reflex bleeding, even though starvation itself had a strong negative effect on all haemolymph parameters investigated and individual body mass. Daily reflex bleeding did not result in decreased fecundity of young ladybirds during the first 30 days of their adult life, but the start of ladybird reproduction was delayed by about two days. Moreover, ladybirds bleeding larger amounts of haemolymph started their reproduction significantly later. Overall, our results indicate that repeated reflex bleeding weakens a ladybird's immune system and can increase their susceptibility to pathogens, but a ladybird's reproductive potential remains almost unaffected, even by very intensive reflex bleeding.

A Universal Stress Protein That Controls Bacterial Stress Survival in Micrococcus luteus.

Bacteria have remarkable mechanisms to survive severe external stresses, and one of the most enigmatic is the nonreplicative persistent (NRP) state. Practically, NRP bacteria are difficult to treat, and so inhibiting the proteins underlying this survival state may render such bacteria more susceptible to external stresses, including antibiotics. Unfortunately, we know little about the proteins and mechanisms conferring survival through the NRP state. Here, we report that a universal stress protein (Usp) is a primary regulator of bacterial survival through the NRP state in Micrococcus luteus NCTC 2665, a biosafety level 1 (BSL1) mycobacterial relative. Usps are widely conserved, and bacteria, including Mycobacterium tuberculosis, Mycobacterium smegmatis, and Escherichia coli, have multiple paralogs with overlapping functions that have obscured their functional roles. A kanamycin resistance cassette inserted into the M. luteus universal stress protein A 616 gene (ΔuspA616::kanM. luteus) ablates the UspA616 protein and drastically impairs M. luteus survival under even short-term starvation (survival, 83% wild type versus 32% ΔuspA616::kanM. luteus) and hypoxia (survival, 96% wild type versus 48% ΔuspA616::kanM. luteus). We observed no detrimental UspA616 knockout phenotype in logarithmic growth. Proteomics demonstrated statistically significant log-phase upregulation of glyoxylate pathway enzymes isocitrate lyase and malate synthase in ΔuspA616::kanM. luteus We note that these enzymes and the M. tuberculosis UspA616 homolog (Rv2623) are important in M. tuberculosis virulence and chronic infection, suggesting that Usps are important stress proteins across diverse bacterial species. We propose that UspA616 is a metabolic switch that controls survival by regulating the glyoxylate shunt.IMPORTANCE Bacteria tolerate severe external stresses, including antibiotics, through a nonreplicative persistent (NRP) survival state, yet the proteins regulating this survival state are largely unknown. We show a specific universal stress protein (UspA616) controls the NRP state in Micrococcus luteus Usps are widely conserved across bacteria, but their biological function(s) has remained elusive. UspA616 inactivation renders M. luteus susceptible to stress: bacteria die instead of adapting through the NRP state. UspA616 regulates malate synthase and isocitrate lyase, glyoxylate pathway enzymes important for chronic Mycobacterium tuberculosis infection. These data show that UspA616 regulates NRP stress survival in M. luteus and suggest a function for homologous proteins in other bacteria. Importantly, inhibitors of UspA616 and homologs may render NRP bacteria more susceptible to stresses, including current antibiotics.

Social amoebae establish a protective interface with their bacterial associates by lectin agglutination.

Both animals and amoebae use phagocytosis and DNA-based extracellular traps as anti-bacterial defense mechanisms. Whether, like animals, amoebae also use tissue-level barriers to reduce direct contact with bacteria has remained unclear. We have explored this question in the social amoeba Dictyostelium discoideum, which forms plaques on lawns of bacteria that expand as amoebae divide and bacteria are consumed. We show that CadA, a cell adhesion protein that functions in D. discoideum development, is also a bacterial agglutinin that forms a protective interface at the plaque edge that limits exposure of vegetative amoebae to bacteria. This interface is important for amoebal survival when bacteria-to-amoebae ratios are high, optimizing amoebal feeding behavior, and protecting amoebae from oxidative stress. Lectins also control bacterial access to the gut epithelium of mammals to limit inflammatory processes; thus, this strategy of antibacterial defense is shared across a broad spectrum of eukaryotic taxa.

Relationship between Intrauterine Bacterial Infection and Early Embryonic Developmental Arrest.

BACKGROUND: Early embryonic developmental arrest is the most commonly understudied adverse outcome of pregnancy. The relevance of intrauterine infection to spontaneous embryonic death is rarely studied and remains unclear. This study aimed to investigate the relationship between intrauterine bacterial infection and early embryonic developmental arrest. METHODS: Embryonic chorion tissue and uterine swabs for bacterial detection were obtained from 33 patients who underwent artificial abortion (control group) and from 45 patients who displayed early embryonic developmental arrest (trial group). RESULTS: Intrauterine bacterial infection was discovered in both groups. The infection rate was 24.44% (11/45) in the early embryonic developmental arrest group and 9.09% (3/33) in the artificial abortion group. Classification analysis revealed that the highest detection rate for Micrococcus luteus in the early embryonic developmental arrest group was 13.33% (6/45), and none was detected in the artificial abortion group. M. luteus infection was significantly different between the groups (P < 0.05 as shown by Fisher's exact test). In addition, no correlation was found between intrauterine bacterial infection and history of early embryonic developmental arrest. CONCLUSIONS: M. luteus infection is related to early embryonic developmental arrest and might be one of its causative factors.

Peptidoglycan recognition protein genes and their roles in the innate immune pathways of the red flour beetle, Tribolium castaneum.

We have previously demonstrated that the functional Toll and IMD innate immune pathways indeed exist in the model beetle, Tribolium castaneum while the beetle's pathways have broader specificity in terms of microbial activation than that of Drosophila. To elucidate the molecular basis of this broad microbial activation, we here focused on potential upstream sensors of the T. castaneum innate immune pathways, peptidoglycan recognition proteins (PGRPs). Our phenotype analyses utilizing RNA interference-based comprehensive gene knockdown followed by bacterial challenge suggested: PGRP-LA functions as a pivotal sensor of the IMD pathway for both Gram-negative and Gram-positive bacteria; PGRP-LC acts as an IMD pathway-associated sensor mainly for Gram-negative bacteria; PGRP-LE also has some roles in Gram-negative bacterial recognition of the IMD pathway. On the other hand, we did not obtain clear phenotype changes by gene knockdown of short-type PGRP genes, probably because of highly inducible nature of these genes. Our results may collectively account for the promiscuous bacterial activation of the T. castaneum innate immune pathways at least in part.

Lactoferrin, myeloperoxidase, and ceruloplasmin: complementary gearwheels cranking physiological and pathological processes.

Copper-containing plasma protein ceruloplasmin (Cp) forms a complex with lactoferrin (Lf), an iron-binding protein, and with the heme-containing myeloperoxidase (Mpo). In case of inflammation, Lf and Mpo are secreted from neutrophil granules. Among the plasma proteins, Cp seems to be the preferential partner of Lf and Mpo. After an intraperitoneal injection of Lf to rodents, the "Cp-Lf" complex has been shown to appear in their bloodstream. Cp prevents the interaction of Lf with protoplasts of Micrococcus luteus. Upon immunoprecipitation of Cp, the blood plasma becomes depleted of Lf and in a dose-dependent manner loses the capacity to inhibit the peroxidase activity of Mpo, but not the Mpo-catalyzed oxidation of thiocyanate in the (pseudo)halogenating cycle. Antimicrobial effect against E. coli displayed by a synergistic system that includes Lf and Mpo-H2O2-chloride, but not thiocyanate, as the substrate for Mpo is abrogated when Cp is added. Hence, Cp can be regarded as an anti-inflammatory factor that restrains the halogenating cycle and redirects the synergistic system Mpo-H2O2-chloride/thiocyanate to production of hypothiocyanate, which is relatively harmless for the human organism. Structure and functions of the "2Cp-2Lf-Mpo" complex and binary complexes Cp-Lf and 2Cp-Mpo in inflammation are discussed.

Acanthopanax senticosus extracts have a protective effect on Drosophila gut immunity.

ETHNOPHARMACOLOGICAL RELEVANCE: Aanthopanax senticosus (A. senticosus) Harms is a classical adaptogenic agent used in China. It has been applied as an analeptic aid to improve weakened physical status. However, little is known about the effects of A. senticosus on inflammatory disease processes. MATERIALS AND METHODS: Flies fed with standard cornmeal-yeast medium were used as controls, and the treatment groups contained 10% of A. senticosus aqueous extracts (root or fruit) in standard medium. Survival rate was performed by feeding a vial containing five layers of filter paper hydrated with 5% sucrose solution contaminated with pathogenic or toxic compounds. Imaging of the guts was viewed under the microscope. Death cells were detected by 7-AAD staining. RESULTS: The A. senticosus extract improved the survival rate, attenuated the death of intestinal epithelial cells, promoted the expression of antimicrobial peptide genes, and decreased the formation of melanotic masses. Moreover, our results indicated that the protective effect of fruit is much higher than that of root extracts. CONCLUSIONS: A. senticosus extracts have a protective effect on Drosophila gut immunity and stress response, and may contribute to the prevention of inflammatory diseases induced by pathogenic and toxic compounds.

Culturable bacterial microbiota of Plagiodera versicolora (L.) (Coleoptera: Chrysomelidae) and virulence of the isolated strains.

Plagiodera versicolora (Laicharting, 1781) (Coleoptera: Chrysomelidae) is an important forest pest which damages many trees such as willow, poplar, and hazelnut. In order to find new microbes that can be utilized as a possible microbial control agent against this pest, we investigated the culturable bacterial flora of it and tested the isolated bacteria against P. versicolora larvae and adults. We were able to isolate nine bacteria from larvae and adults. The isolates were characterized using a combination of morphological, biochemical, and physiological methods. Additionally, we sequenced the partial sequence of the 16S rRNA gene to verify conventional identification results. Based on characterization studies, the isolates were identified as Staphylococcus sp. Pv1, Rahnella sp. Pv2, Rahnella sp. Pv3, Rahnella sp. Pv4, Rahnella sp. Pv5, Pantoea agglomerans Pv6, Staphylococcus sp. Pv7, Micrococcus luteus Pv8, and Rahnella sp. Pv9. The highest insecticidal activity against larvae and adults was obtained from M. luteus Pv8 with 50 and 40 % mortalities within 10 days after treatment, respectively. Extracellular enzyme activity of the bacterial isolates such as amylase, proteinase, lipase, cellulose, and chitinase was also determined. Consequently, our results show that M. luteus Pv8 might be a good candidate as a possible microbial control agent against P. versicolora and were discussed with respect to biocontrol potential of the bacterial isolates.

A different repertoire of Galleria mellonella antimicrobial peptides in larvae challenged with bacteria and fungi.

To date, functioning of insect humoral immune response is especially well described in Diptera. The mechanisms of pathogen recognition, activation of signaling pathways and regulation of antimicrobial defense peptide expression are relatively well known. The present paper demonstrates evidence that the immune system of the Lepidoptera moth, Galleria mellonella, is also able to distinguish between different classes of microorganisms and responds to the invading pathogen accordingly. G. mellonella larvae were challenged with Gram-negative and Gram-positive bacteria as well as with yeast and filamentous fungus cells. Subsequently, 24, 48 and 72 h after immunization, the concentrations of lysozyme and six defense peptides were determined in the hemolymph by the HPLC technique. The compounds studied demonstrated variability both in the kinetics of the increase as well as in the concentrations reached. The Gram-negative bacterium and filamentous fungus were particularly effective immunogens, especially affecting the levels of lysozyme, Galleria defensin, proline-rich peptide 2 and cecropin D-like peptide.

AiC1qDC-1, a novel gC1q-domain-containing protein from bay scallop Argopecten irradians with fungi agglutinating activity.

The globular C1q-domain-containing (C1qDC) proteins are a family of versatile pattern recognition receptors via their globular C1q (gC1q) domain to bind various ligands including several PAMPs on pathogens. In this study, a new gC1q-domain-containing protein (AiC1qDC-1) gene was cloned from Argopecten irradians by rapid amplification of cDNA ends (RACE) approaches and expressed sequence tag (EST) analysis. The full-length cDNA of AiC1qDC-1 was composed of 733bp, encoding a signal peptide of 19 residues and a typical gC1q domain of 137 residues containing all eight invariant amino acids in human C1qDC proteins and seven aromatic residues essential for effective packing of the hydrophobic core of AiC1qDC-1. The gC1q domain of AiC1qDC-1, which possessed the typical 10-stranded beta-sandwich fold with a jelly-roll topology common to all C1q family members, showed high homology not only to those of C1qDC proteins in mollusk but also to those of C1qDC proteins in human. The AiC1qDC-1 transcripts were mainly detected in the tissue of hepatopancreas and also marginally detectable in adductor, heart, mantle, gill and hemocytes by fluorescent quantitative real-time PCR. In the microbial challenge experiment, there was a significant up-regulation in the relative expression level of AiC1qDC-1 in hepatopancreas and hemocytes of the scallops challenged by fungi Pichia pastoris GS115, Gram-positive bacteria Micrococcus luteus and Gram-negative bacteria Listonella anguillarum. The recombinant AiC1qDC-1 (rAiC1qDC-1) protein displayed no obvious agglutination against M. luteus and L. anguillarum, but it aggregated P. pastoris remarkably. This agglutination could be inhibited by d-mannose and PGN but not by LPS, glucan or d-galactose. These results indicated that AiC1qDC-1 functioned as a pattern recognition receptor in the immune defense of scallops against pathogens and provided clues for illuminating the evolution of the complement classical pathway.

Analysis of bacteria-challenged wild silkmoth, Antheraea mylitta (lepidoptera) transcriptome reveals potential immune genes.

BACKGROUND: In the recent years a strong resemblance has been observed between the insect immune system and the mammalian innate immune mechanisms suggesting their common origin. Among the insects, only the dipterans (Drosophila and various mosquito species) have been widely investigated for their immune responses towards diverse pathogens. In the present study we constructed and analysed the immune transcriptome of the lepidopteran Antheraea mylitta, an economically important Indian tasar silkmoth with a view to unravel the potential immune-related genes and pathways. RESULTS: An expressed sequence tag (EST) library was constructed from mRNA obtained from fat bodies of A. mylitta larvae that had been challenged by infection with Escherichia coli cells. We identified 719 unique ESTs from a total of 1412 sequences so generated. A third of the transcriptome showed similarity with previously characterized immune-related genes that included both the known and putative immune genes. Of the four putative novel defence proteins (DFPs) annotated by PSI-BLAST three showed similarity to extracellular matrix proteins from vertebrates implicated in innate immunity, while the fourth was similar to, yet distinct from, the anti-microbial protein cecropin. Finally, we analysed the expression profiles of 15 potential immune-related genes, and the majority of them were induced more prominently with E. coli compared to Micrococcus luteus. We also identified several unknown proteins, some of which could have probable immune-related functions based on the results of the ProDom analysis. CONCLUSION: The present study has identified many potential immune-related genes in A. mylitta some of which are vertebrate homologues and others are hitherto unreported putative defence proteins. Several genes were present as members of gene families, as has also been observed in other insect species.

New insights into Drosophila larval haemocyte functions through genome-wide analysis.

Drosophila blood cells or haemocytes comprise three cell lineages, plasmatocytes, crystal cells and lamellocytes, involved in immune functions such as phagocytosis, melanisation and encapsulation. Transcriptional profiling of activities of distinct haemocyte populations and from naive or infected larvae, was performed to find genes contributing to haemocyte functions. Of the 13 000 genes represented on the microarray, over 2500 exhibited significantly enriched transcription in haemocytes. Among these were genes encoding integrins, peptidoglycan recognition proteins (PGRPs), scavenger receptors, lectins, cell adhesion molecules and serine proteases. One relevant outcome of this analysis was the gain of new insights into the lamellocyte encapsulation process. We showed that lamellocytes require betaPS integrin for encapsulation and that they transcribe one prophenoloxidase gene enabling them to produce the enzyme necessary for melanisation of the capsule. A second compelling observation was that following infection, the gene encoding the cytokine Spatzle was uniquely upregulated in haemocytes and not the fat body. This shows that Drosophila haemocytes produce a signal molecule ready to be activated through cleavage after pathogen recognition, informing distant tissues of infection.

Increased inflammation in lysozyme M-deficient mice in response to Micrococcus luteus and its peptidoglycan.

More than 70 years ago, Alexander Fleming discovered lysozyme and proposed that nonpathogenic bacteria fail to cause disease because they are very susceptible to destruction by lysozyme, an enzyme that is one of the principal proteins of phagocytes. Although much has been learned about the effects of lysozyme in vitro, its biological role in vivo has not been determined. We examined transgenic mice deficient in lysozyme M after challenge by the normally nonpathogenic and highly lysozyme-sensitive bacterium Micrococcus luteus. Despite partial compensation by newly expressed lysozyme P in macrophages, lysozyme M-deficient mice developed much more severe lesions than wild-type mice. The tissue injury was due to the failure of lysozyme M-deficient mice to inactivate peptidoglycan, resulting in an intense and prolonged inflammatory response. Our data indicate that tissue injury is normally limited by prompt degradation of bacterial macromolecules that trigger innate immunity and inflammation.

Micrococcus luteus: a putative cause of hepatic abscess?

Micrococcus luteus was repeatedly isolated in blood cultures during a prolonged feverish syndrome in a patient who presented with multiple hepatic abscesses as well. In contrast to the literature, this case is not related to prosthetic devices; an untreated limb wound may have been the site of microbial entry.

Analysis of the Drosophila host defense in domino mutant larvae, which are devoid of hemocytes.

We have analyzed the Drosophila immune response in domino mutant larvae, which are devoid of blood cells. The domino mutants have a good larval viability, but they die as prepupae. We show that, on immune challenge, induction of the genes encoding antimicrobial peptides in the fat body is not affected significantly in the mutant larvae, indicating that hemocytes are not essential in this process. The hemocoele of domino larvae contains numerous live microorganisms, the presence of which induces a weak antimicrobial response in the fat body. A full response is observed only after septic injury. We propose that the fat body cells are activated both by the presence of microorganisms and by injury and that injury potentiates the effect of microorganisms. Survival experiments after an immune challenge showed that domino mutants devoid of blood cells maintain a wild-type resistance to septic injury. This resistance was also observed in mutant larvae in which the synthesis of antibacterial peptides is impaired (immune deficiency larvae) and in mutants that are deficient for humoral melanization (Black cells larvae). However, if domino was combined with either the immune deficiency or the Black cell mutation, the resistance to septic injury was reduced severely. These results establish the relevance of the three immune reactions: phagocytosis, synthesis of antibacterial peptides, and melanization. By working in synergy, they provide Drosophila a highly effective defense against injury and/or infection.