Effect of prophenoloxidase expression knockout on the melanization of microfilariae in the mosquito Armigeres subalbatus

Melanization is an effective defence reaction used by mosquito hosts to kill malarial and filarial worm parasites. Although phenoloxidase (PO) has long been considered to be the key enzyme in the biosynthesis of melanotic material in insects, there is no direct evidence verifying its role in parasite melanization. To elucidate the role of PO in the melanization of microfilariae (mf) by mosquitoes, a double subgenomic Sindbis (dsSIN) recombinant virus was used to transduce Armigeres subalbatus mosquitoes with a 600 base antisense RNA targeted to the highly conserved copper-binding region of an Ar. subalbatus PO gene. Compared with controls, haemolymph PO activity in mosquitoes transduced with antisense RNA was significantly reduced. When these mosquitoes were challenged with Dirofilaria immitis mf, the melanization of mf was almost completely inhibited. These data verify that PO is an essential component of the biochemical pathway required for the melanization of parasites, and that the dsSIN expression system represents a useful tool in the functional analysis of endogenous gene expression in mosquitoes.     

Ectopic expression of an endoparasitic wasp venom protein in Drosophila melanogaster affects immune function,larval development and oviposition

Endoparasitic hymenoptera inject maternal factors into the host, along with their eggs, to subvert the host immune system. The venom protein, Vn50, previously characterized from the wasp Cotesia rubecula inhibits prophenoloxidase activation in its host Pieris rapae and in another lepidopteran, Manduca sexta. We generated a stable line in the model insect, Drosophila melanogaster, which ectopically expresses Vn50. Results indicated that Vn50 expression accelerates larval development, increases oviposition and reduces melanization in the haemolymph of the transgenic flies. Since melanization is known to be an important facet of the insect immune response, we examined the impact of Vn50 expression on susceptibility to pathogens. Transgenic Vn50 flies challenged with the fungus Beauveria bassiana had increased mortality compared with control flies, but there was no significant change in survival in flies challenged with the pathogenic bacteria, Serratia marcescens. Interestingly, mortality induced by the natural pathogen Drosophila C virus was significantly delayed in Vn50 expressing flies. This indicates a wider range of potential hosts that may be affected by Vn50 and its potential for manipulation of immune system in insects.     

Eater and draper are involved in the periostial haemocyte immune response in the mosquito Anopheles gambiae

Haemocytes respond to infection by phagocytosing pathogens, producing the enzymes that drive the phenoloxidase‐based melanization cascade, secreting lytic factors, and producing other humoral proteins. A subset of haemocytes, called periostial haemocytes, aggregate on the surface of the heart of mosquitoes and kill pathogens in areas of high haemolymph flow. Periostial haemocytes are always present, but an infection induces the recruitment of additional haemocytes to these regions. Here, we tested whether members of the Nimrod gene family are involved in the periostial immune response of the African malaria mosquito, Anopheles gambiae. Using organismal manipulations, RNA interference (RNAi) and microscopy, we show that, following an infection with Escherichia coli, nimrod – the orthologue of Drosophila NimB2 – is not involved in periostial responses. At 4 h postinfection, however, RNAi‐based knockdown of draper results in a marginal increase in the number of periostial haemocytes and a doubling of E. coli accumulation at the periostial regions. Finally, at 24 h postinfection, knockdown of eater decreases the number of periostial haemocytes and decreases the phagocytosis of E. coli on the surface of the heart. Phagocytosis of bacteria is more prevalent in the periostial regions of the mid abdominal segments, and knockdown of draper, nimrod or eater does not alter this distribution. Finally, knockdown of Nimrod family genes did not have a meaningful effect on the accumulation of melanin at the periostial regions. This study identifies roles for eater and draper in the functional integration of the mosquito immune and circulatory systems.     

Two-dimensional gel analysis of haemolymph proteins from Plasmodium-melanizing and -non-melanizing strains of Anopheles gambiae

Haemolymph polypeptides from Plasmodium‐refractory and ‐susceptible mosquitoes were compared by one‐ and two‐dimensional gel electrophoresis. The refractory strain of Anopheles gambiae kills malaria parasites by a humoral melanization mechanism whereas the parasites develop normally in susceptible mosquitoes. The two strains respond in a similar manner to carboxy‐methyl‐Sephadex beads that have been injected into the thoracic haemocoel, i.e. beads are strongly melanized in refractory but not susceptible mosquitoes. Protein profiles were compared between strains following cold shock (naïve control), saline injection and Sephadex bead injection. Using the susceptible naïve control as the standard, eight constitutively expressed polypeptides were specific to naïve susceptible mosquitoes while twelve other spots were reduced, enhanced or specific to refractory mosquitoes. Several of the strain‐specific spots probably comprise related pairs (one in each strain) which vary only in isoelectric focusing point. Nine spots were induced by sham injection or by an injection of beads or saline, but none was reproducibly different between the strains. Amino acid sequence analysis of one of the refractory strain‐specific spots identified it as AgSp14D1, an A. gambiae infection‐responsive serine protease that is most similar to the Drosophila gene easter and Manduca prophenoloxidase activating enzyme. This gene maps to polytene chromosome division 14, which has been implicated in the melanization phenotype by quantitative trait loci mapping.     

Phenoloxidases are required for the pea aphid's defence against bacterial and fungal infection

The pea aphid, Acyrthosiphon pisum, has an incomplete immune system compared to those of other insect species; some conserved components and pathways in other species are missing in its genome. As a core component of the insect immune system, prophenoloxidase (PPO) genes are retained in the pea aphid. Early studies have also shown the presence of phenoloxidase activity in specific tissues or cells in the pea aphid and suggested its involvement in response to immune challenges. In this study, we knocked down the expression of PPO genes in the pea aphid using double‐stranded RNA‐based interference, and quantitative PCR analysis and an enzyme activity assay confirmed our success in the PPO gene knockdown. In bacterial and fungal infection experiments, we observed that the knockdown of PPO resulted in more live bacterial cells and fungal spores in the body of the aphids and higher mortality of the aphids after infection. Our study provides evidence supporting a critical role of PPO in the defence of the pea aphid.     

Potato tuber proteins efficiently inhibit human faecal proteolytic activity: implications for treatment of peri-anal dermatitis

Background Frequent diarrhoea after intestinal resections and faecal incontinence in healthy infants may lead to perianal injury. A causative agent may be a high concentration of pancreatic proteases in faeces. The aim of the present study was to assess whether protease inhibitors are applicable for treating and preventing peri‐anal dermatitis by inhibiting the initial cause of the inflammation, the faecal proteases. Design Proteolytic activity was estimated in faeces of subjects frequently suffering from peri‐anal dermatitis: patients with intestinal resections and healthy infants. The development of perianal dermatitis was studied after the construction of a reservoir with ileoanal anastomosis. The inhibitory effect of crude and partly purified potato juice on proteolytic activity of faecal output from patients with intestinal resections and healthy infants was investigated in vitro and in vivo (skin tests). Results Faecal protease activity in faeces from patients with intestinal resections and healthy infants was found to be significantly higher than in healthy adults. After the construction of an ileum reservoir, 46 of 48 patients developed a protease‐related peri‐anal dermatitis. The partly purified protein fraction from potatoes inhibited the larger part of faecal proteases in vitro and completely prevented skin irritation by pancreatic proteases dissolved in sterilized faecal fluid, in a 24‐h skin test, on the back of healthy human volunteers. Conclusions Potato proteins contain protease inhibitors, which suppress almost the complete proteolytic activity in faeces. Topical application of potato protease inhibitors might be a novel approach in preventing protease‐induced peri‐anal dermatitis, and therapeutic studies are needed to confirm our results.     

The use of a double subgenomic Sindbis virus expression system to study mosquito gene function: effects of antisense nucleotide number and duration of viral infection on gene silencing efficiency

Recently we established a simple, effective antisense strategy using a double subgenomic Sindbis (dsSIN) virus expression system to study gene function in mosquitoes. In this study, we further elucidate the effects of antisense nucleotide number and duration of viral infection on mosquito gene silencing efficiency by the dsSIN virus expression system. Over 15 days post virus infection, the degree of parasite melanization was progressively reduced by more than 95%, 75% and 55% in the mosquito Armigeres subalbatus transduced with 600, 147 or 36 bases antisense RNA, targeted to the highly conserved copper binding region of the Ar. subalbatus prophenoloxidase I gene (As-pro-POI), respectively. As the duration of viral infection increased from day 3-15, the degree of parasite melanization progressively decreased in all mosquitoes transduced with antisense RNA, irrespective of the lengths of antisense RNA. Progressive loss of parasite melanization function was found to correlate with down regulation of As-pro-PO expression at both the mRNA and protein activity levels, and reductions in virus titres in mosquitoes transduced with antisense RNA. A small pro-PO RNA (c. twenty-five nucleotides) was identified in mosquitoes transduced with antisense RNA. These data suggest that As-pro-POI gene expression is knocked down by degrading the As-pro-POI mRNA through the RNAi pathway. In conclusion, our study demonstrates that even a short antisense RNA (thirty-six bases) can cause silencing of the As-pro-POI gene, and the effects of endogenous gene silencing by dsSIN expression system on mosquito gene functions can be accumulative.     

Promising immunotherapy against fungal diseases

Introduction: Despite the relatively high efficacy of antifungal drugs, invasive fungal infections (IFIs) are still associated with tremendous morbidity and mortality, since late diagnosis makes an antifungal drug therapy inefficient. Therefore, antifungal immunotherapies to specifically strengthen the host´s own immune mechanisms constitute an additional promising strategy in taking action against fungal pathogens.

Areas covered: The authors summarize efforts in research and clinical trials to provide safe and efficient immunotherapeutic options against invasive fungal diseases. Treatment of IFIs is challenging as the number of available antifungals is limited and further complications include: toxicity, drug interactions and the emergence of drug resistance. Susceptibility is determined by the impaired immune status of the host. Hence, augmenting immunity by immunotherapeutic interventions may offer future directions to treat IFI.

Expert opinion: A much better understanding of fungus and host cell interactions is essential for the development of safe and successful immunotherapeutic strategies. Indeed, there is encouraging preliminary data available that such approaches are possible; however, current data is too limited to allow solid conclusions on the risks and benefits in the clinical setting. Clinical trials focusing on the role of adjuvant immunotherapeutics with or without a combination of antifungals are highly needed for further evaluation.     

Combination antifungals: an update

Invasive fungal infections are an important cause of morbidity and mortality in specific patient populations. There has been an impressive increase in the antifungal armamentarium, yet optimal therapies for many invasive fungal infections remain unknown. Genomic sequencing of a number of pathogenic fungi will pave the way to discovering additional newer targets for antifungal drug design. These new discoveries, plus the existing repertoire of antifungal agents, create the need to effectively model single and combination antifungal agents. Future therapies may also include the use of cell-stress pathway inhibitors in combination with existing antifungal agents. This review focuses on combination antifungal therapy against Cryptococcus neoformans, Candida and Aspergillus species. Combination therapy is only supported by randomized clinical trials for cryptococcal meningitis. We review data from in vitro and animal model studies as well as insights from clinical trials to discuss current thoughts and highlight the gaps in our knowledge surrounding combination antifungal therapy.     

Is there a role for statins in fungal infections?

It has been hypothesized that statins, HMG-CoA reductase inhibitors, may be used to treat fungal infections. Here we review data on antifungal properties of statins, effects on the host inflammatory response as well as available clinical evidence. We conclude that: statins exhibit antifungal properties in vitro although at supraphysiological concentrations; statins appear to have anti-inflammatory effects on host cells in vitro; statins have effects on fungal physiology beyond direct growth inhibition; clinical studies are scarce (n = 5), and their design is retrospective and observational, which is associated with a high risk of bias. Given the limited evidence for a beneficial effect of statins in fungal infection, randomized and controlled trials are highly warranted in this field.     

Oxidized von Willebrand factor is efficiently cleaved by serine proteases from primary granules of leukocytes: divergence from ADAMTS‐13

To cite this article: Lancellotti S, De Filippis V, Pozzi N, Oggianu L, Rutella S, Scaglione GL, Maset F, Peyvandi F, Mannucci PM, De Cristofaro R. Oxidized von Willebrand factor is efficiently cleaved by serine proteases from primary granules of leukocytes: divergence from ADAMTS‐13. J Thromb Haemost 2011; 9 : 1620–7. Summary. Background: The leukocyte serine proteases (LSPs) elastase, proteinase 3 and cathepsin G cleave von Willebrand factor (VWF) near or at the same cleavage site (Tyr1605–Met1606) as ADAMTS‐13, the metalloprotease that specifically controls the proteolytic processing of VWF. Recent studies have shown that oxidation of VWF at Met1606 with formation of methionine sulfoxide (MetSO) severely impairs its proteolysis by ADAMTS‐13. Methods: This study was aimed at assessing whether or not oxidation of VWF by reactive oxygen species (ROS) can also affect its cleavage by elastase, proteinase 3, and cathepsin G. In this study, the catalytic specificity of hydrolysis by LSPs of the VWF peptide substrate VWF74 and full‐length VWF, both unaltered and in the oxidized form, was measured by RP‐HPLC, electrophoretic and mass spectrometry methods. Results: LSPs cleaved both VWF multimers and VWF74 near or at the same peptide bond as is cleaved by ADAMTS‐13, with k_cat/K_m values similar to those of the metalloprotease. However, unlike ADAMTS‐13, cathepsin G cleaved VWF74 containing a MetSO residue at position 1606 with a k_cat/K_m value higher than that for VWF74, whereas the catalytic efficiencies of both elastase and proteinase 3 were unaffected by the replacement of Met1606 with MetSO. Likewise, oxidation of VWF multimers by hypochlorous acid and ROS, produced by activated leukocytes, improved their hydrolysis by LSPs. Conclusions: Oxidation by leukocyte ROS has a net positive effect on the cleavage of VWF multimers by LSPs, under conditions where high concentrations of oxidant species would severely reduce the proteolytic efficiency of ADAMTS‐13.     

Insulin-mediated secretion of ecdysteroids from mosquito ovaries and molecular cloning of the insulin receptor homologue from ovaries of bloodfed Aedes aegypti

The reproductive cycle of female mosquitoes is activated by ingestion of blood from vertebrate hosts. Shortly after feeding, neurohormones are released from the brain neurosecretory system and stimulate the ovaries to secrete ecdysteroids, which are necessary for vitellogenesis by the fat body. Because bombyxins, which are insulin-like peptides, stimulate ecdysteroidogenesis in silkworm larvae, we tested porcine insulin and found that it activates ecdysteroidogenesis and protein synthesis in ovaries isolated from unfed mosquitoes. To further characterize the regulation of ecdysteroidogenesis in female mosquitoes, we cloned the mosquito insulin receptor (MIR) homologue from ovarian mRNA. The sequence of the extracellular domain shows moderate homologies with vertebrate and Drosophlla Insulin receptor homologues, as well as with the insulin receptor-related receptor, the latter being an ‘orphan’ receptor with an unknown function. In the intracellular domain, high homologies are observed, particularly in those subdomains that are responsible for ATP binding and kinase activity. Northern blot analysis of MIR demonstrated a highly specific expression in ovaries, and cloning experiments indicated its presence in the brain. Recombinant MIR extracts from a baculovirus expression system contained high constitutive kinase activity in the presence of manganese or magnesium. Activation was independent of a ligand. SDS-gel analysis suggested that the recombinant receptor was not post-translationally processed into an α and β-subunit as was expected from a putative cleavage sinal. Enzymatic properties of the propreceptor are presented: the K_m for ATP was between 15 and 50 μM in the presence of a synthetic substrate: maximal kinase activity to 100-fold over basic activity was reached in the presence of 1 mn managanese. Stimulation of key oogenic processes by porcine insulin and identification of a MIR indicate that insulin-like neurophormonones may have an important regulatory role in mosquito oogenesis.     

Cracking the Toll-like receptor code in fungal infections

Innate control of fungal infection requires the specific recognition of invariant fungal molecular structures by a variety of innate immune receptors, including Toll-like receptors. In addition to the role in inducing protective immune responses, Toll-like receptor engagement may paradoxically favor fungal infections, by inducing inflammatory pathology and impairing antifungal immunity. Although the dissection of complex genetic traits modulating susceptibility to fungal infections is complex, the contribution of host genetics may hold the key to elucidating new risk factors for these severe, often fatal diseases. Understanding host–pathogen interactions at the innate immune interface will eventually lead to the development of new therapeutics and genetic markers in fungal infections.     

Serine protease activity of Per a 10 augments allergen-induced airway inflammation in a mouse model

Background We recently reported an immunodominant serine protease allergen (Per a 10) from Periplaneta americana. This study investigates the role of its proteolytic activity in driving the immune responses towards self and other allergens. Materials and methods Groups of Balb/c mice were sensitized intraperitoneally and subcutaneously with proteolytically active Per a 10 or inactivated Per a 10 (using aminoethyl benzenesulfonyl fluoride hydrochloride) or whole body P. americana extract and subsequently challenged intranasally with the respective antigens. Mice were also sensitized and challenged with ovalbumin (OVA) alone or co‐administered with active or inactive Per a 10. The immune‐inflammatory responses were measured by airway hyperresponsiveness (AHR) and cellular infiltration of lungs i.e. eosinophil counts, eosinophil peroxidase (EPO) activity, myeloperoxidase activity (MPO), lung histopathology, serum levels of specific‐antibodies and levels of Th1/Th2 interleukins in bronchoalveolar lavage fluid (BALF) and in spleen cells culture supernatant. Results Mice challenged with active Per a 10/P. americana extract showed a significant airway inflammation demonstrated by enhanced AHR and increased cellular infiltration of lungs as evidenced by high eosinophil counts, EPO activity, IL‐4 and IL‐5 in BALF. Active Per a 10 also induced a significant proliferation of spleen cells, increased secretion of IL‐4 and IL‐5 in the spleen cells culture supernatant and systemic production of specific‐IgE and IgG1. However, exposure with inactive Per a 10 elicited a low cellular infiltration and systemic antibody production. Exposure to OVA with active Per a 10 demonstrated a significantly high cellular infiltration and production of OVA‐specific IgE and IgG1, than exposure to OVA alone or with inactive Per a 10. Conclusions Proteolytic activity of Per a 10 plays an important role in driving the allergic immune response by providing an adjuvant effect, towards self and other potential allergens present in the same microenvironment.