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S M Paskewitz

Publications and source records attributed to S M Paskewitz.

At least 19 recordsLinked to original sources
PubMed2006

Cloning and characterization of a putative inhibitor of melanization from Anopheles gambiae.

Phenoloxidases, including tyrosinases and laccases, are enzymes involved in the synthesis of melanin, a process that can be elicited during insect immune responses, cuticle maturation, wound healing and egg chorion development. We cloned a putative inhibitor of melanization (POI) from Anopheles gambiae on the basis of homology with a functionally characterized peptide from Musca domestica (Daquinag et al., Proc Natl Acad Sci USA 1995; 92: 2964-2968). The 335 amino acid protein predicted from the A. gambiae cDNA consists of five tandemly arranged inhibitor motifs. The A. gambiae POI gene was expressed in all mosquito stages from egg to adult. POI transcript levels were high in the fat body and were measurable but comparatively reduced in the midgut. The POI transcript level increased after wounding or Sephadex bead injection. Gene knockdown did not result in faster or more extensive bead melanization but did result in more extensive melanization of wound sites following a thoracic bead injection.

Amino Acid Sequence↗
PubMed2004

Identification and molecular characterization of two immune-responsive chitinase-like proteins from Anopheles gambiae.

Two haemolymph proteins that are processed rapidly and specifically in response to exposure to bacteria have been identified from Anopheles gambiae. Both proteins, Anopheles gambiae bacteria-responsive 1 (AgBR1) and AgBR2, are similar to chitinases but belong to a family of proteins that have lost chitinolytic activity. AgBR1 and AgBR2 are converted to smaller forms in vivo or in vitro on exposure to bacteria, and AgBR2 also can be processed on exposure to peptidoglycan alone. AgBR1 and AgBR2 do not bind to bacteria or chitin beads. The AgBR1 and AgBR2 genes are expressed in all developmental stages. In adults, AgBR1 expression is restricted to the fat body, whereas AgBR2 is expressed in many tissues.

Amino Acid Sequence↗
PubMed2001

Serine proteases as mediators of mosquito immune responses.

Serine proteases regulate several invertebrate defense responses, including hemolymph coagulation, antimicrobial peptide synthesis, and melanization of pathogen surfaces. These processes require the presence of serine proteases in the hemolymph where they can rapidly activate immune pathways in response to pathogen detection. Hemolymph coagulation in the horseshoe crab is controlled by several serine proteases, including two that are pathogen recognition molecules and two in the clip domain family of serine proteases. The antimicrobial peptide synthesis and melanization pathways include clip domain proteases as well as other, uncharacterized serine proteases. We have identified five serine proteases from the hemolymph of the mosquito, Anopheles gambiae. One, Sp22D, is a large protease with potential pathogen binding domains. Sp22D is expressed in three tissues that have immune functions (midgut epithelium, fat body, and hemocytes), and its transcript abundance increases after immune challenge. Sp14A, Sp14D1, and Sp14D2 are clip domain serine proteases that are similar to enzymes with presumed roles in melanization or antimicrobial peptide synthesis. They undergo changes in transcript abundance in response to infection with bacteria or malaria parasites, and they reside in a chromosomal region that has been associated with melanization of parasites. Sp18D, also a clip domain protease, is similar to a Manduca protease with a likely role in immunity, but immune challenge does not affect its mRNA abundance.

Amino Acid Sequence↗
PubMed2001

Ixodes scapularis (Acari: Ixodidae): abundance and rate of infection with Borrelia burgdorferi in four state parks in Wisconsin.

Four state parks located in Lyme disease endemic regions of Wisconsin were surveyed for the presence of Ixodes scapularis Say during May and June of 1998 by drag sampling along hiking trails. Nymphal abundance varied between parks, with the average number of nymphs encountered in 1 h ranging from 6.2 +/- 3.8-47.1 +/- 36.3 (mean +/- SD). Questing nymphs were tested for the presence of Borrelia burgdorferi by culture in BSK medium and 7-12% was found to be infected. The average risk of encountering an infected nymph (entomologic risk index) ranged from 0.5 to 5.2 infected nymphs per hour. The highest entomological risk index was recorded from a small island park in northwestern Wisconsin during the last week in May (8.0 infected nymphs per hour). These results indicate a lower risk for human Lyme disease exposure in Wisconsin state parks in comparison with highly endemic areas of the northeastern United States.

Animals↗
PubMed2000

Sp22D: a multidomain serine protease with a putative role in insect immunity.

Serine proteases play critical roles in a variety of insect immune responses; however, few of the genes that code for these enzymes have been cloned. Here, we describe the molecular characterization of a serine protease gene from the mosquito Anopheles gambiae. Sp22D codes for a 1322 amino acid polypeptide with a complex domain organization. In addition to the carboxy terminal serine protease catalytic domain, Sp22D contains two putative chitin binding domains, a mucin-like domain, two low density lipoprotein receptor class A domains, and two scavenger receptor cysteine rich domains. A typical signal peptide sequence and a lack of potential transmembrane helices suggest that Sp22D is secreted. Sp22D is expressed constitutively in three immune-related cell types: adult hemocytes, fat body cells, and midgut epithelial cells. Wounding induces no changes in transcript abundance, but within 1h after injection of bacteria, Sp22D mRNA increases 1.5-fold. Based on domain organization, tissue distribution, and transcriptional up-regulation in response to immune challenge, we suggest that Sp22D has an immune function. In addition, we predict that Sp22D is secreted into the hemolymph where it may interact with pathogen surfaces and initiate an immune response.

Amino Acid Sequence↗
PubMed2000

Molecular characterization of five serine protease genes cloned from Anopheles gambiae hemolymph.

We identified five new serine protease cDNAs from the hemolymph of the malaria vector, Anopheles gambiae. All five show sequence similarity to genes thought to be involved in vertebrate or invertebrate defense responses. Sp14A, Sp14D2 and Sp22D demonstrate changes in transcript abundance in response to bacteria injections. Sp14A and Sp14D2, as well as the previously characterized Sp14D1, are induced by infection with the malaria parasite, Plasmodium berghei. These three proteases, along with Sp18D, are related to a group of secreted proteases that have amino-terminal clip domains and trypsin-like substrate specificity. BLAST results and phylogenetic analyses group Sp14A, Sp14D1 and Sp14D2 with the Drosophila protease EASTER, and three prophenoloxidase activating enzymes from other insects. EASTER's substrate is SPAETZLE, a ligand involved in embryogenesis but also in activating anti-microbial peptide synthesis. Their similarity to EASTER and immune inducibility suggest that one of these proteases may activate a SPAETZLE-like ligand during anti-parasite responses in mosquitoes. Alternatively, as potential prophenoloxidase activators, Sp14A, Sp14D1 or Sp14D2 may play a role in melanotic encapsulation of Plasmodium.

Amino Acid Sequence↗
PubMed2000

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.

Animals↗
PubMed1999

An easter-like serine protease from Anopheles gambiae exhibits changes in transcript abundance following immune challenge.

The nucleotide and deduced amino acid sequence of a serine protease (AgSp14D1) from the human malaria vector, Anopheles gambiae, is presented. The gene product is a 360 amino acid protein that contains two domains and has the highest sequence similarity to the Drosophila melanogaster serine protease easter and to prophenol oxidase activating enzyme (pPAE) from Manduca sexta. The catalytic domain is at the carboxy terminus and has the conserved serine, histidine and aspartic acid residues found in serine proteases as well as six cysteines common to invertebrate enzymes. The amino terminus contains critical cysteines that define a clip (=disulphide knot) domain which places this gene product in a subfamily of regulatory serine proteases that includes not only easter and pPAE but also the Drosophila proteins masquerade, stubble and snake as well as proclotting enzyme and factor B from the horseshoe crab. In situ hybridization to the polytene chromosomes detects a single band at 14D and Southern analysis with a probe from the 5' end of the gene confirms the single copy status of this gene. Northern analysis reveals changes in transcript abundance during development and following blood feeding. Interestingly, this analysis also shows an increase in transcript levels following wounding or injection of bacteria.

Amino Acid Sequence↗
PubMed1998

A factor preventing melanization of sephadex CM C-25 beads in Plasmodium-susceptible and refractory anopheles gambiae.

One major quantitative trait locus controls melanization of both malaria ookinetes and Sephadex CM beads in a refractory strain of the mosquito, Anopheles gambiae. Hemolymph transferred from a nonmelanizing, Plasmodium-susceptible strain (4arr) to a melanizing, Plasmodium-refractory strain (L35) caused a reduction in the melanization of CM beads. In addition, when beads were first incubated in vivo in susceptible mosquitoes and then recovered, washed, and transferred to refractory mosquitoes, a strong reduction in melanization was observed. No changes in melanization were observed when beads or hemolymph were transferred in the opposite direction or within a strain. Incubation of beads in vitro in refractory or susceptible hemolymph resulted in a reduction of melanization when these beads were subsequently transferred to refractory mosquitoes. This reduction was significantly stronger when susceptible hemolymph was used as the incubating medium. Protection from melanization was observed after 3-, 6-, and 24-h incubations of beads in susceptible mosquitoes with longer incubations resulting in greater protection. Treatment of protected beads with 1 M NaOH resulted in the loss of the protection but treatment with 1% sodium dodecyl sulfate (SDS), 1% SDS/DTT/boiling, or 1 M NaOAc (pH 8.9) did not. These results show that a melanization-preventing factor covalently binds to the surface of CM beads in susceptible mosquitoes and can subsequently prevent melanization in refractory mosquitoes.

Animals↗
PubMed1998

Effects of larval nutrition, adult body size, and adult temperature on the ability of Anopheles gambiae (Diptera: Culicidae) to melanize sephadex beads.

A Plasmodium-refractory strain (L35) of the mosquito Anopheles gambiae Giles melanizes late ookinetes on the basal surface of the midgut, resulting in the death of the parasites. This strain also melanizes CM C-25 Sephadex beads, which serve as a model system for investigating the melanization response. The effects of larval nutrition, adult body size, and temperature of the adult environment on the ability of refractory females to melanize CM C-25 beads were studied. Nutritional deprivation during the larval stages significantly decreased the ability of adults to melanize beads. In addition, bead melanization decreased progressively as the environmental temperature of the adults increased from 24 to 30 degrees C. We conclude that environmental stress may affect the immune responses of An. gambiae.

Animal Nutritional Physiological Phenomena↗
PubMed1998

The lethal effects of Cyperus iria on Aedes aegypti.

The sedge Cyperus iria, a common weed in rice, contains large amounts of the insect hormone (10R) juvenile hormone III (JH III). Given its widespread distribution in Asia and Africa, we examined the possibility that C. iria could be used as a safe, inexpensive, and readily available mosquito larvicide. Plants of varying ages were harvested and leaves tested for lethal effects on larvae of the yellow fever mosquito, Aedes aegypti. The median lethal doses (LD50s) for frozen leaves from 1- and 2-month-old plants were 267 and 427 mg/100 ml of water, respectively. Leaves from 1-month-old C. iria contained 193 micrograms JH III/g fresh weight, whereas leaves from 2-month-old plants contained 143 micrograms JH III/g fresh weight. Larval sensitivity to the plant differed with age; 4-day-old larvae displayed the greatest mortality followed in decreasing sensitivity by larvae 5, 6, 3, and 2 days old. Six Cyperus species (C. albostriatus, C. alternifolius, C. esculentus, C. iria, C. miliifolius, and C. papyrus) of similar developmental stage were assayed for JH III content. Only C. iria was found to contain significant levels of JH III.

Aedes↗
PubMed1997

Cloning and characterization of a serine protease from the human malaria vector, Anopheles gambiae.

The nucleotide and deduced amino acid sequence of a serine protease (AgSp24D) from the human malaria vector, Anopheles gambiae, is presented. The gene product is a 271 amino acid protein that contains the conserved serine, histidine and aspartic acid residues found in serine proteases, and has the highest identity to a serine protease of unknown function from Drosophila melanogaster. In situ hybridization to the polytene chromosomes detects a single band at 24D. Northern analysis reveals only low levels of transcripts in larvae and pupae, but more abundant transcription products occur in adults. Interestingly, this analysis also shows that adult males express much higher levels of AgSp24D mRNA than females. In addition, Plasmodium-refractory mosquitoes express higher levels of AgSp24D mRNA than susceptible mosquitoes although the biological significance of this remains to be examined. The thorax is the primary site for expression in the adults. The lack of a dramatic increase in AgSp24D mRNA levels following blood feeding suggests that this protease is not involved in digestive processes. Transcriptional induction does not follow cold shock, septic wounding, bacterial injection, laminarin injection or CM-Sephadex bead injection.

Amino Acid Sequence↗
PubMed1997

Progress in the map-based cloning of the Anopheles gambiae genes responsible for the encapsulation of malarial parasites.

A genetically selected strain of the mosquito Anopheles gambiae, the major vector of malaria in sub-Saharan Africa, is able to encapsulate and kill Plasmodium ookinetes after they have penetrated the midgut cells and come to rest between the midgut epithelial cells and the surrounding basal lamina. The genetic basis of this phenotype has now been examined by high-resolution mapping using microsatellite loci. Results of this mapping indicate that three genes contribute to this phenotype, with one gene on the left arm of chromosome 2 accounting for the most of the effect. These genes, called Pen1, Pen2, and Pen3 (for Plasmodium encapsulation genes 1, 2 and 3) have also been physically localized to relatively small and well defined regions of the polytene chromosome complement. Strategies for cloning these genes by genetic and physical mapping methods are discussed.

Africa South of the Sahara↗
PubMed1997

Mapping a quantitative trait locus involved in melanotic encapsulation of foreign bodies in the malaria vector, Anopheles gambiae.

A Plasmodium-refractory strain of Anopheles gambiae melanotically encapsulates many species of Plasmodium, whereas wild-type mosquitoes are usually susceptible. This encapsulation trait can also be observed by studying the response of refractory and susceptible strains to intrathoracically injected CM-Sephadex beads. We report the results of broad-scale quantitative trait locus (QTL) mapping of the encapsulation trait using the bead model system. Interval mapping using the method of maximum likelihood identified one major QTL, Pen1. The 13.7-cM interval containing Pen1 was defined by marker AGH157 at 8E and AGH46 at 7A on 2R. Pen1 was associated with a maximum LOD score of 9.0 and accounted for 44% of the phenotypic variance in the distribution of phenotypes in the backcross. To test if this QTL is important for encapsulation of Plasmodium berghei, F2 progeny were infected with P. berghei and evaluated for degree of parasite encapsulation. For each of the two markers that define the interval containing Pen1, a significant difference of encapsulation was seen in progeny with at least one refractory allele in contrast with homozygous susceptible progeny. These results suggest that Pen1 is important for melanotic encapsulation of Plasmodium as well as beads.

Animals↗
PubMed1997

A genetic study of a melanization response to Sephadex beads in Plasmodium-refractory and -susceptible strains of Anopheles gambiae.

A previously selected Plasmodium-refractory strain of Anopheles gambiae melanotically encapsulates many species of Plasmodium. Genetic studies of this strain have shown that this refractory phenotype is controlled by a limited number of genes, and the existence of two such genes, Pif-B and Pif-C, has been demonstrated. Further work to determine the molecular basis for this mode of refractoriness led to the discovery that the host-parasite interaction is mimicked by the mosquito's response to carboxymethyl (CM)-Sephadex beads injected into the thorax. These small beads are melanized within 24 hr in refractory mosquitoes but are rarely melanized in susceptible ones. Because of the considerable potential in using bead melanization as a model for Plasmodium refractoriness, we performed a genetic analysis of the differential response to beads. Reciprocal crosses of susceptible (4arr) and refractory (L35) mosquitoes and an analysis of F1 phenotypes were done. The F1 progeny had a phenotype similar to that of the parental refractory mosquitoes; therefore, dominant refractory allele(s) must be present in the refractory strain. Males from the reciprocal crosses had identical phenotypes, indicating that X-linked loci did not have a visible effect on the melanizing phenotype. To further study the mode of inheritance of the melanizing trait, a backcross of F1 females to 4arr males was done. The phenotypic distribution of the backcross progeny was bimodal, and the melanization phenotypes were similar to those of the susceptible and refractory parents. These data suggest that a small number of loci are responsible for the differential response to CM-Sephadex beads, and that one chromosomal region contributes strongly to the melanizing trait. Because the 4arr strain carries mutations in the pink eye (X) and red eye (III) genes, possible linkage of the melanizing phenotype to these two genes was tested. No linkage with either marker was detected. The pattern of inheritance of the melanizing phenotype is similar to that of the refractory phenotype of Pif-B; therefore, the genetic basis of the two responses may be the same.

Animals↗
PubMed1996

A shared genetic mechanism for melanotic encapsulation of CM-Sephadex beads and a malaria parasite, Plasmodium cynomolgi B, in the mosquito, Anopheles gambiae.

A Plasmodium-refractory strain of Anopheles gambiae that melanizes ookinetes and intrathoracically inoculated CM-Sephadex beads was mated to a Plasmodium-susceptible strain that does not melanize the parasite or the beads. The F1 progeny were then backcrossed to the susceptible strain. Backcross progeny were given a blood meal containing infective Plasmodium cynomolgi B, and the parasites were allowed to develop for 6-7 days, at which time the infected mosquitoes were injected with CM-Sephadex beads. The next day the mosquitoes were dissected and the beads were scored for degree of melanization while the parasites were scored for degree of encapsulation. A Spearman rank order correlation test of the degree of correlation between the bead melanization phenotype and the parasite encapsulation phenotype gave a correlation coefficient of 0.74 (P < 0.01). This strong correlation between the two melanization responses suggests that the mechanisms for differential bead and parasite melanization of these two mosquito strains share at least one major gene.

Animals↗