Mosquito phenoloxidase and defensin colocalize in melanization innate immune responses.

Mosquitoes mount strong humoral and cellular immune responses against foreign organisms. Two components of the mosquito immune response that have received much attention are the phenoloxidase cascade that leads to melanization and antimicrobial peptides. The purpose of the current study was to use immunocytochemistry and transmission electron microscopy to identify the location of the melanization rate-limiting enzyme phenoloxidase and the antimicrobial peptide defensin in innate immune reactions against Escherichia coli and Micrococcus luteus by the mosquito Aedes aegypti. Our results show that both phenoloxidase and defensin are present at the sites of melanin biosynthesis in immune reactions against bacteria. Furthermore, both proteins are often present inside the same melanotic capsules. When hemocytes were analyzed, phenoloxidase was present in the cytosol of oenocytoids, but no significant amounts of defensin were detected inside any hemocytes. In summary, these data show that phenoloxidase and defensin colocalize in melanization reactions against bacteria and argue for further studies into the potential role of defensin in phenoloxidase-based melanization innate immune responses in mosquitoes.     

Migration of Myeloid Cells during Inflammation Is Differentially Regulated by the Cell Surface Receptors Slamf1 and Slamf8

Previous studies have demonstrated that the cell surface receptor Slamf1 (CD150) is requisite for optimal NADPH-oxidase (Nox2) dependent reactive oxygen species (ROS) production by phagocytes in response to Gram- bacteria. By contrast, Slamf8 (CD353) is a negative regulator of ROS in response to Gram+ and Gram- bacteria. Employing in vivo migration after skin sensitization, induction of peritonitis, and repopulation of the small intestine demonstrates that in vivo migration of Slamf1^-/- dendritic cells and macrophages is reduced, as compared to wt mice. By contrast, in vivo migration of Slamf8^-/- dendritic cells, macrophages and neutrophils is accelerated. These opposing effects of Slamf1 and Slamf8 are cell-intrinsic as judged by in vitro migration in transwell chambers in response to CCL19, CCL21 or CSF-1. Importantly, inhibiting ROS production of Slamf8^-/- macrophages by diphenyleneiodonium chloride blocks this in vitro migration. We conclude that Slamf1 and Slamf8 govern ROS–dependent innate immune responses of myeloid cells, thus modulating migration of these cells during inflammation in an opposing manner.     

Rapid phagocytosis and melanization of bacteria and Plasmodium sporozoites by hemocytes of the mosquito Aedes aegypti

Mosquitoes are vectors of many deadly and debilitating pathogens. In the current study, we used light and electron microscopies to study the immune response of Aedes aegypti hemocytes to bacterial inoculations, Plasmodium gallinaceum natural infections, and latex bead injections. After challenge, mosquitoes mounted strong phagocytic and melanization responses. Granulocytes phagocytosed bacteria singly or pooled them inside large membrane-delimited vesicles. Phagocytosis of bacteria, Plasmodium sporozoites, and latex beads was extensive; we estimated that individual granulocytes have the capacity to phagocytose hundreds of bacteria and thousands of latex particles. Oenocytoids were also seen to internalize bacteria and latex particles, although infrequently and with low capacity. Besides phagocytosis, mosquitoes cleared bacteria and sporozoites by melanization. Interestingly, the immune response toward 2 species of bacteria was different; most Escherichia coli were phagocytosed, but most Micrococcus luteus were melanized. Similar to E. coli, most Plasmodium sporozoites were phagocytosed. The immune response was rapid; phagocytosis and melanization of bacteria began as early as 5 min after inoculation. The magnitude and speed of the cellular response suggest that hemocytes, acting in concert with the humoral immune response, are the main force driving the battle against foreign invaders.     

Structure of the dnaA region of Micrococcus luteus: conservation and variations among eubacteria

A phylogenetic tree constructed by 5S rRNA analysis is composed of three major branches in eubacteria: high G + C Gram+, low G + C Gram+ and Gram- [Hori and Osawa, Mol. Biol. Evol. 4 (1987) 445-472]. We have shown that the characteristic dnaA region is common among Escherichia coli (Gram-), Pseudomonas putida (Gram-), and Bacillus subtilis (low G + C Gram+). We have now determined the structure of the dnaA region of Micrococcus luteus, as a representative of the last branch, high G + C Gram+. The dnaA gene and at least three other genes, rnpA, rpmH and dnaN were found to be conserved in M. luteus. Large nontranslatable regions were found flanking the dnaA gene. The upstream region is conserved in the four bacteria so far examined. On the other hand, the downstream region is conserved only in Gram+ bacteria, M. luteus and B. subtilis. The consensus sequence of the DnaA box in M. luteus seems to be TTGTCCACA, in contrast to TTATCCACA of other bacteria. These results confirm our hypothesis that the dnaA region is the replication origin of the ancestral bacteria and that the essential feature of the DnaA protein and DnaA-box combination is conserved in eubacteria.     

Structure of the dnaA and DnaA-box region in the Mycoplasma capricolum chromosome: conservation and variations in the course of evolution.

We have previously shown that the dnaA gene and the DnaA-box region were conserved in bacteria representative of all three major branches of the eubacterial phylogenic tree: high G + C Gram+, low-G + C Gram+ and Gram-. In the present work, we determined the structure of the dnaA region of Mycoplasma capricolum and found that the dnaA gene and at least two other genes, rpmH and dnaN, were conserved in this bacterium. An unusually high level of amino acid (aa) substitutions was observed in M. capricolum DnaA. It was the case even in those aa which were well conserved in other bacterial species. The nontranslatable region upstream from the dnaA gene was also conserved in this bacterium, as it was universally found in both Gram+ and Gram- bacteria. An additional nontranslatable region downstream from the dnaA gene, which is common to Gram+ bacteria, was also found in M. capricolum, consistent with the proposal that M. capricolum is Gram+ in origin. These regions were rich in A + T and contained ten DnaA-box-like sequences (9-mers that differ from TTATCCACA by one or two bases).     

The Wound Healing and Antibacterial Activity of Five Ethnomedical Calophyllum inophyllum Oils: An Alternative Therapeutic Strategy to Treat Infected Wounds

Background

Calophyllum inophyllum L. (Calophyllaceae) is an evergreen tree ethno-medically used along the seashores and islands of the Indian and Pacific Oceans, especially in Polynesia. Oil extracted from the seeds is traditionally used topically to treat a wide range of skin injuries from burn, scar and infected wounds to skin diseases such as dermatosis, urticaria and eczema. However, very few scientific studies reported and quantified the therapeutic properties of Calophyllum inophyllum oil (CIO). In this work, five CIO from Indonesia (CIO1), Tahiti (CIO2, 3), Fiji islands (CIO4) and New Caledonia (CIO5) were studied and their cytotoxic, wound healing, and antibacterial properties were presented in order to provide a scientific support to their traditional use and verify their safety.

Methods

The safety of the five CIO was ascertained using the Alamar blue assay on human keratinocyte cells. CIO wound healing properties were determined using the scratch test assay on human keratinocyte cells. CIO-stimulated antibacterial innate immune response was evaluated using ELISA by measuring β defensin-2 release in human derivative macrophage cells. CIO antibacterial activity was tested using oilogramme against twenty aerobic Gram- bacteria species, twenty aerobic Gram+ bacteria species, including a multi-drug resistant Staphylococcus aureus strain and two anaerobic Gram+ bacteria species e.g. Propionibacterium acnes and Propionibacterium granulosum. To detect polarity profile of the components responsible of the antibacterial activity, we performed bioautography against a Staphylococcus aureus strain.

Results

Based on Alamar Blue assay, we showed that CIO can be safely used on keratinocyte cells between 2.7% and 11.2% depending on CIO origin. Concerning the healing activity, all the CIO tested accelerated in vitro wound closure, the healing factor being 1.3 to 2.1 higher compared to control when keratinocytes were incubated after scratch with CIO at 0.1%. Furthermore, our results showed that CIO exhibit two distinct antibacterial effects: one against Gram+ bacteria by direct inhibition of mitotic growth and another potent effect against Gram- bacteria due to increased release of β-defensin 2 peptide by macrophages. Interestingly, the needed concentrations of CIO to inhibit bacteria growth and to promote wound healing are lower than concentrations exhibiting cytotoxic effects on keratinocyte cells. Finally, we performed bioautography assay against Staphylococcus aureus to determine polarity profile of the components responsible for CIO antibacterial activity. Our results showed for the five tested CIO that components responsible of the bacterial growth inhibition are the more polar one on the TLC chromatographic profile and are contained in the resinous fraction of the oil.

Conclusions

This study was conducted to evaluate cytotoxicity, wound healing and antibacterial properties of five CIO traditionally used to treat infected wounds. Using cell and bacteria cultures, we confirmed the pharmacological effects of CIO as wound healing and antimicrobial agent. Moreover, we showed that concentration of CIO needed to exhibit therapeutic effects are lower than concentrations exhibiting cytotoxic effects in vitro. For the first time, this study provides support for traditional uses of CIO. These wound healing and antibiotic properties make CIO a valuable candidate to treat infected wounds especially in tropical areas.     

Characterization of the intracellular melanization response in Anopheles quadrimaculatus against subperiodic Brugia malayi larvae.

Intracellular melanization, a defense or an immune response in the thoracic muscle cells, was investigated in a refractory strain of Anopheles quadrimaculatus infected with larvae of Brugia malayi. In mosquitoes fed on B. malayi-infected jirds, intracellular melanization against first-stage larvae (L1) was better expressed when fewer than 40 microfilariae reached the thoracic muscle cells than when more than 40 microfilariae reached the thoracic muscle cells. This result suggests that when large numbers of microfilariae invade the thoracic muscle cells, the immune response of the mosquito may become overloaded. Intracellular melanization response against L1 in the thoracic muscle cells also showed a significant decrease in older females (14-16-day-old) as compared to the younger ones (4-9-day-old). A comparison is made between intracellular and extracellular responses of mosquitoes to filarial larvae. It is significant that in both cases high rate of infection can reduce both the number and percentage of larvae melanized.     

Hemocyte-mediated phagocytosis and melanization in the mosquito<Emphasis Type="Italic"> Armigeres subalbatus</Emphasis> following immune challenge by bacteria

Mosquitoes are important vectors of disease. These insects respond to invading organisms with strong cellular and humoral immune responses that share many similarities with vertebrate immune systems. The strength and specificity of these responses are directly correlated to a mosquito's ability to transmit disease. In the current study, we characterized the hemocytes (blood cells) of Armigeres subalbatus by morphology (ultrastructure), lectin binding, enzyme activity, immunocytochemistry, and function. We found four hemocyte types: granulocytes, oenocytoids, adipohemocytes, and thrombocytoids. Granulocytes contained acid phosphatase activity and bound the exogenous lectins Helix pomatia agglutinin, Galanthus nivalis lectin, and wheat germ agglutinin. Following bacteria inoculation, granulocytes mounted a strong phagocytic response as early as 5 min postexposure. Bacteria also elicited a hemocyte-mediated melanization response. Phenoloxidase, the rate-limiting enzyme in the melanization pathway, was present exclusively in oenocytoids and in many of the melanotic capsules enveloping bacteria. The immune responses mounted against different bacteria were not identical; gram(–) Escherichia coli were predominantly phagocytosed and gram(+) Micrococcus luteus were melanized. These studies implicate hemocytes as the primary line of defense against bacteria.This work was supported by NIH grant AI19769 to B.M.C. and NIH grant F31 AI50252 to J.F.H.     

无脊椎动物先天免疫模式识别受体研究进展

免疫系统的基本功能是“自己”与“非己”识别.对入侵物的识别是免疫防御的起始,最终引发效应物反应系统,包括吞噬作用、包被作用、激活蛋白酶级联反应和黑化作用以及诱导抗菌肽的合成等,从而清除或消灭入侵物.研究证明,这种“非己”识别是因为存在某些特异性的、可溶的或与细胞膜结合的模式识别受体,可以识别或结合微生物表面保守的、而在宿主中又不存在的病原相关分子模式.模式识别受体通过对病原相关分子的识别启动先天免疫防御.近年来这方面的研究进展很快,已经在无脊椎动物中确定了多种模式识别受体,包括肽聚糖识别蛋白、含硫酯键蛋白、革兰氏阴性菌结合蛋白、清除受体、C型凝集素、硫依赖型凝集素、Toll样受体和血素等,并对其性质和功能进行了研究.     

Hemocyte cell surface changes in Aedes aegypti in response to microfilariae of Dirofilaria immitis

This study involved the assessment of surface changes on hemocytes of Aedes aegypti black-eyed Liverpool strain in association with the melanization response against intrathoracically inoculated Dirofilaria immitis microfilariae (mff). Surface changes on hemocytes were identified using fluorescein-labeled wheat germ agglutinin (WGA). In mosquitoes eliciting a melanization response against inoculated mff, there was a 5-fold increase in the percentages of hemocytes exhibiting WGA binding compared with saline inoculated controls. Relationships of this hemocyte activation in relation to cell-mediated melanization responses of adult mosquitoes against mff are discussed.     

Selective anti-tubercular purines: synthesis and chemotherapeutic properties of 6-aryl- and 6-heteroaryl-9-benzylpurines

6-Aryl- and 6-heteroaryl-9-benzylpurines have been synthesized employing palladium-catalyzed coupling reactions in the step forming the C-C or C-N bond between the aryl- or heteroaryl and the purine. The compounds were screened for activity against Mycobacterium tuberculosis as well as representative Gram+ and Gram- bacteria, and for cytotoxic effects on mammalian cells. Several potent antimycobacterials were identified. These compounds probably act by a novel and selective mechanism; they exhibit low toxicity toward other bacteria as well as mammalian cells.     

CLIP proteases and Plasmodium melanization in Anopheles gambiae

Melanization is a potent immune response mediated by phenoloxidase (PO). Multiple Clip-domain serine proteases (CLIP) regulate PO activation as part of a complex cascade of proteases that are cleaved sequentially. The role of several CLIP as key activators or suppressors of the melanization responses of Anopheles gambiae to Plasmodium berghei (murine malaria) has been established recently using a genome-wide reverse genetics approach. Important differences in regulation of PO activation between An. gambiae strains were also identified. This review summarizes these findings and discusses our current understanding of the An. gambiae melanization responses to Plasmodium.     

A Signaling Protease Required for Melanization in Drosophila Affects Resistance and Tolerance of Infections

Organisms evolve two routes to surviving infections—they can resist pathogen growth (resistance) and they can endure the pathogenesis of infection (tolerance). The sum of these two properties together defines the defensive capabilities of the host. Typically, studies of animal defenses focus on either understanding resistance or, to a lesser extent, tolerance mechanisms, thus providing little understanding of the relationship between these two mechanisms. We suggest there are nine possible pairwise permutations of these traits, assuming they can increase, decrease, or remain unchanged in an independent manner. Here we show that by making a single mutation in the gene encoding a protease, CG3066, active in the melanization cascade in Drosophila melanogaster, we observe the full spectrum of changes; these mutant flies show increases and decreases in their resistance and tolerance properties when challenged with a variety of pathogens. This result implicates melanization in fighting microbial infections and shows that an immune response can affect both resistance and tolerance to infections in microbe-dependent ways. The fly is often described as having an unsophisticated and stereotypical immune response where single mutations cause simple binary changes in immunity. We report a level of complexity in the fly's immune response that has strong ecological implications. We suggest that immune responses are highly tuned by evolution, since selection for defenses that alter resistance against one pathogen may change both resistance and tolerance to other pathogens.     

Shuttle vectors containing a multiple cloning site and a lacZ alpha gene for conjugal transfer of DNA from Escherichia coli to gram-positive bacteria

The mobilizable shuttle cloning vectors, pAT18 and pAT19, are composed of: (i) the replication origins of pUC and of the broad-host-range enterococcal plasmid pAM beta 1; (ii) an erythromycin-resistance-encoding gene expressed in Gram- and Gram+ bacteria; (iii) the transfer origin of the IncP plasmid RK2; and (iv) the multiple cloning site and the lacZ alpha reporter gene of pUC18 (pAT18) and pUC19 (pAT19). These 6.6-kb plasmids contain ten unique cloning sites that allow screening of derivatives containing DNA inserts by alpha-complementation in Escherichia coli carrying the lacZ delta M15 deletion, and can be efficiently mobilized by self-transferable IncP plasmids co-resident in the E. coli donors. Plasmids pAT18, pAT19 and recombinant derivatives have been successfully transferred by conjugation from E. coli to Bacillus subtilis, Bacillus thuringiensis, Listeria monocytogenes, Enterococcus faecalis, Lactococcus lactis, and Staphylococcus aureus at frequencies ranging from 10(-6) to 10(-9). The presence of a restriction system in the recipient dramatically affects (by three orders of magnitude) the efficiency of conjugal transfer of these vectors from E. coli to Gram+ bacteria.     

Regulators and signalling in insect haemocyte immunity

The innate immune system of insects relies on both humoral and cellular immune responses that are mediated via activation of several signalling pathways. Haemocytes are the primary mediators of cell-mediated immunity in insects, including phagocytosis, nodulation, encapsulation and melanization. The last years, research has focused on the mechanisms of microbial recognition and activation of haemocyte intracellular signalling molecules in response to invaders. The powerful tool, RNA interference gene silencing, helped several regulators involved in immune responses, to be identified. In this review, we summarize recent advances in understanding the role(s) of receptors and intracellular signalling molecules involved in immune responses.     

Cross reactivity of S. aureus to murine cytokine assays: A source of discrepancy

Staphylococcus aureus is one of the versatile Gram positive bacteria causing a range of diseases. Upon challenge, host immune cells recognize S. aureus and mount diverse immune responses including production of pro-inflammatory cytokines such as IL-1β and TNF-α. These cytokines are important mediators of inflammation which can be detected via various immunological methods such as enzyme linked immunosorbent assay (ELISA) and immunoblotting. In the current study, we found that a number of clinical isolates as well as laboratory strains of S. aureus exhibited cross reactivity with ELISA antibodies for murine IL-1β and TNF-α assays. This cross reactivity generates exaggerated false positive signals which can be a source of discrepancy for the understanding of real immune responses against S. aureus infection by host immune cells.     

Parasitism-linked block of host plasma melanization

When parasitized by the Ichneumonid parasitoid Campoletis sonorensis, larvae of the Noctuid moth, Heliothis virescens, are unable to mount an effective immune response against parasitoid eggs. Defensive melanization of plasma and cellular encapsulation of parasite eggs are dramatically inhibited by infection with the symbiotic immunosuppressive C. sonorensis ichnovirus (CsIV). This study demonstrates that the CsIV-mediated inhibition of melanization is associated with reduction in the enzymatic activity and protein titer of key enzymes in the melanization pathway, phenoloxidase, dopachrome isomerase, and DOPA decarboxylase. Inhibition of the synthesis of key melanization enzymes leads to reductions in the melanization substrates l-dihydroxyphenylalanine, N-acetyldopamine, and N-beta-alanyl dopamine from millimolar to nanomolar levels in parasitized larvae. By contrast, concentration of a precursor catecholamine, dopamine, rises fourfold in these larvae. Thus in CsIV-infected larvae, enzymatic deficiencies in the melanization pathway lead to reduced concentrations of specific enzyme substrates, causing failure of melanization in parasitized insects.