Biochemical Characterization of Anopheles gambiae SRPN6, a Malaria Parasite Invasion Marker in Mosquitoes

An, Chunju; Hiromasa, Yasuaki; Zhang, Xin; Lovell, Scott; Żółkiewski, Michał; Tomich, John M.; Michel, Kristin

doi:10.1371/journal.pone.0048689

Cited by 19 publications

(17 citation statements)

References 64 publications

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“…These data indicate that the transient temperature switch increases the sensitivity of screens involving double stranded RNA (dsRNA)-mediated gene silencing, so this protocol was used in all subsequent experiments. The kinetics of ookinete invasion under this experimental set-up was also evaluated by measuring the changes in expression of Serpin 6 (SRPN6) mRNA, a gene highly induced in response to Plasmodium infection and a very sensitive marker of midgut invasion (39), at 24 and 28 h PF (Fig. S4D).…”

Section: Resultsmentioning

confidence: 99%

Plasmodium falciparum evades mosquito immunity by disrupting JNK-mediated apoptosis of invaded midgut cells

Ramphul

Garver

Molina-Cruz

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

110

113

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The malaria parasite, Plasmodium, must survive and develop in the mosquito vector to be successfully transmitted to a new host. The Plasmodium falciparum Pfs47 gene is critical for malaria transmission. Parasites that express Pfs47 (NF54 WT) evade mosquito immunity and survive, whereas Pfs47 knockouts (KO) are efficiently eliminated by the complement-like system. Two alternative approaches were used to investigate the mechanism of action of Pfs47 on immune evasion. First, we examined whether Pfs47 affected signal transduction pathways mediating mosquito immune responses, and show that the Jun-N-terminal kinase (JNK) pathway is a key mediator of Anopheles gambiae antiplasmodial responses to P. falciparum infection and that Pfs47 disrupts JNK signaling. Second, we used microarrays to compare the global transcriptional responses of A. gambiae midguts to infection with WT and KO parasites. The presence of Pfs47 results in broad and profound changes in gene expression in response to infection that are already evident 12 h postfeeding, but become most prominent at 26 h postfeeding, the time when ookinetes invade the mosquito midgut. Silencing of 15 differentially expressed candidate genes identified caspase-S2 as a key effector of Plasmodium elimination in parasites lacking Pfs47. We provide experimental evidence that JNK pathway regulates activation of caspases in Plasmodium-invaded midgut cells, and that caspase activation is required to trigger midgut epithelial nitration. Pfs47 alters the cell death pathway of invaded midgut cells by disrupting JNK signaling and prevents the activation of several caspases, resulting in an ineffective nitration response that makes the parasite undetectable by the mosquito complement-like system.

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Section: Resultsmentioning

confidence: 99%

Plasmodium falciparum evades mosquito immunity by disrupting JNK-mediated apoptosis of invaded midgut cells

Ramphul

Garver

Molina-Cruz

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

110

113

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“…1.5 mg aliquots of the purified rflCLIPB8 and rflCLIPB9 were further resolved by preparative SDS-PAGE and used for polyclonal rabbit antiserum production (Cocalico Biologicals, Reamstown, PA, USA). Antibodies were purified as described previously (An et al, 2012). …”

Section: Methodsmentioning

confidence: 99%

CLIPB8 is part of the prophenoloxidase activation system in Anopheles gambiae mosquitoes

Zhang

Sprigg

et al. 2016

Insect Biochemistry and Molecular Biology

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In insects and other arthropods the formation of eumelanin (melanization) is a broad spectrum and potent immune response that is used to encapsulate and kill invading pathogens. This immune response is regulated by the activation of prophenoxidase (proPO), which is controlled by proteinase cascades and its serpin inhibitors, together forming the proPO activation system. While the molecular composition of these protease cascades are well understood in insect model systems, major knowledge gaps remain in mosquitoes. Recently, a regulatory unit of melanization in Anopheles gambiae was documented, comprised of the inhibitory serpin-clip-serine proteinase, CLIPB9 and its inhibitor serpin-2 (SRPN2). Partial reversion of SRPN2 phenotypes in melanotic tumor formation and adult survival by SRPN2/CLIPB9 double knockdown suggested other target proteinases of SRPN2 in regulating melanization. Here we report that CLIPB8 supplements the SRPN2/CLIPB9 regulatory unit in controlling melanization in An. gambiae. As with CLIPB9, knockdown of CLIPB8 partially reversed the pleiotropic phenotype induced by SRPN2 silencing with regards to adult survival and melanotic tumor formation. Recombinant SRPN2 protein formed an SDS-stable protein complex with activated recombinant CLIPB8, however did not efficiently inhibit CLIPB8 activity in vitro. CLIPB8 did not directly activate proPO in vitro nor was it able to cleave and activate proCLIPB9. Nevertheless, epistasis analysis using RNAi placed CLIPB8 and CLIPB9 in the same pathway leading to melanization, suggesting that CLIPB8 either acts further upstream of CLIPB9 or is required for activation of a yet to be identified serine proteinase homolog. Taken together, this study identifies CLIPB8 as an additional player in proPO activation cascade and highlights the complexity of the proteinase network that regulates melanization in An. gambiae.

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“…The protein encodes a signal peptide, and is secreted from cell lines that naturally express SRPN6, suggesting its target is either present in the secretory pathway or is a hemolymph proteinase [66]. Based on biochemical characterization of recombinant protein, SRPN6 is functional and inhibits kallikrein in vitro [71]. Like most insect serpins however, SRPN6 remains an orphan with no known physiological target.…”

Section: Biological Functions Of Arthropod Serpins In Insect Immunitymentioning

confidence: 99%

Serpins in arthropod biology

Meekins

Kanost

Michel

2017

Seminars in Cell & Developmental Biology

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150

136

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Serpins are the largest known family of serine proteinase inhibitors and perform a variety of physiological functions in arthropods. Herein, we review the field of serpins in arthropod biology, providing an overview of current knowledge and topics of interest. Serpins regulate insect innate immunity via inhibition of serine proteinase cascades that initiate immune responses such as melanization and antimicrobial peptide production. In addition, several serpins with anti-pathogen activity are expressed as acute-phase serpins in insects upon infection. Parasitoid wasps can downregulate host serpin expression to modulate the host immune system. In addition, examples of serpin activity in development and reproduction in Drosophila have also been discovered. Serpins also function in host-pathogen interactions beyond immunity as constituents of venom in parasitoid wasps and saliva of blood-feeding ticks and mosquitoes. These serpins have distinct effects on immunosuppression and anticoagulation and are of interest for vaccine development. Lastly, the known structures of arthropod serpins are discussed, which represent the serpin inhibitory mechanism and provide a detailed overview of the process.

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Biochemical Characterization of Anopheles gambiae SRPN6, a Malaria Parasite Invasion Marker in Mosquitoes

Cited by 19 publications

References 64 publications

Plasmodium falciparum evades mosquito immunity by disrupting JNK-mediated apoptosis of invaded midgut cells

Plasmodium falciparum evades mosquito immunity by disrupting JNK-mediated apoptosis of invaded midgut cells

CLIPB8 is part of the prophenoloxidase activation system in Anopheles gambiae mosquitoes

Serpins in arthropod biology

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