Susan M. Paskewitz

Evolutionary dynamics of immune-related genes and pathways in disease-vector mosquitoes

Mosquitoes are vectors of parasitic and viral diseases of immense importance for public health. The acquisition of the genome sequence of the yellow fever and Dengue vector, Aedes aegypti (Aa), has enabled a comparative phylogenomic analysis of the insect immune repertoire: in Aa, the malaria vector Anopheles gambiae (Ag), and the fruit fly Drosophila melanogaster (Dm). Analysis of immune signaling pathways and response modules reveals both conservative and rapidly evolving features associated with different functional gene categories and particular aspects of immune reactions. These dynamics reflect in part continuous readjustment between accommodation and rejection of pathogens and suggest how innate immunity may have evolved.

Predicting the Risk of Lyme Disease: Habitat Suitability for Ixodes scapularis in the North Central United States

The distribution and abundance of Ixodes scapularis were studied in Wisconsin, northern Illinois, and portions of the Upper Peninsula of Michigan by inspecting small mammals for ticks and by collecting questing ticks at 138 locations in state parks and natural areas. Environmental data were gathered at a local level (i.e., micro and meso levels), and a geographic information system (GIS) was used with several digitized coverages of environmental data to create a habitat profile for each site and a grid map for Wisconsin and Illinois. Results showed that the presence and abundance of I. scapularis varied, even when the host population was adequate. Tick presence was positively associated with deciduous, dry to mesic forests and alfisol-type soils of sandy or loam-sand textures overlying sedimentary rock. Tick absence was associated with grasslands, conifer forests, wet to wet/mesic forests, acidic soils of low fertility and a clay soil texture, and Precambrian bedrock. We performed a discriminant analysis to determine environmental differences between positive and negative tick sites and a regression equation to examine the probability of I. scapularis presence per grid. Both analyses indicated that soil order and land cover were the dominant contributors to tick presence. We then constructed a risk map indicating suitable habitats within areas where I. scapularis is already established. The risk map also shows areas of high probability the tick will become established if introduced. Thus, this risk analysis has both explanatory power and predictive capability.

Emergence of a New Pathogenic Ehrlichia Species, Wisconsin and Minnesota, 2009

BACKGROUND Ehrlichiosis is a clinically important, emerging zoonosis. Only Ehrlichia chaffeensis and E. ewingii have been thought to cause ehrlichiosis in humans in the United States. Patients with suspected ehrlichiosis routinely undergo testing to ensure proper diagnosis and to ascertain the cause. METHODS We used molecular methods, culturing, and serologic testing to diagnose and ascertain the cause of cases of ehrlichiosis. RESULTS On testing, four cases of ehrlichiosis in Minnesota or Wisconsin were found not to be from E. chaffeensis or E. ewingii and instead to be caused by a newly discovered ehrlichia species. All patients had fever, malaise, headache, and lymphopenia; three had thrombocytopenia; and two had elevated liver-enzyme levels. All recovered after receiving doxycycline treatment. At least 17 of 697 Ixodes scapularis ticks collected in Minnesota or Wisconsin were positive for the same ehrlichia species on polymerase-chain-reaction testing. Genetic analyses revealed that this new ehrlichia species is closely related to E. muris. CONCLUSIONS We report a new ehrlichia species in Minnesota and Wisconsin and provide supportive clinical, epidemiologic, culture, DNA-sequence, and vector data. Physicians need to be aware of this newly discovered close relative of E. muris to ensure appropriate testing, treatment, and regional surveillance. (Funded by the National Institutes of Health and the Centers for Disease Control and Prevention.).

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.

Identification of a novel pathogenic Borrelia species causing Lyme borreliosis with unusually high spirochaetaemia: a descriptive study

BACKGROUND Lyme borreliosis is the most common tick-borne disease in the northern hemisphere. It is a multisystem disease caused by Borrelia burgdorferi sensu lato genospecies and characterised by tissue localisation and low spirochaetaemia. In this study we aimed to describe a novel Borrelia species causing Lyme borreliosis in the USA. METHODS At the Mayo clinic, from 2003 to 2014, we tested routine clinical diagnostic specimens from patients in the USA with PCR targeting the oppA1 gene of B burgdorferi sensu lato. We identified positive specimens with an atypical PCR result (melting temperature outside of the expected range) by sequencing, microscopy, or culture. We collected Ixodes scapularis ticks from regions of suspected patient tick exposure and tested them by oppA1 PCR. FINDINGS 100 545 specimens were submitted by physicians for routine PCR from Jan 1, 2003 to Sept 30, 2014. From these samples, six clinical specimens (five blood, one synovial fluid) yielded an atypical oppA1 PCR product, but no atypical results were detected before 2012. Five of the six patients with atypical PCR results had presented with fever, four had diffuse or focal rash, three had symptoms suggestive of neurological inclusion, and two were admitted to hospital. The sixth patient presented with knee pain and swelling. Motile spirochaetes were seen in blood samples from one patient and cultured from blood samples from two patients. Among the five blood specimens, the median oppA1 copy number was 180 times higher than that in 13 specimens that tested positive for B burgdorferi sensu stricto during the same time period. Multigene sequencing identified the spirochaete as a novel B burgdorferi sensu lato genospecies. This same genospecies was detected in ticks collected at a probable patient exposure site. INTERPRETATION We describe a new pathogenic Borrelia burgdorferi sensu lato genospecies (candidatus Borrelia mayonii) in the upper midwestern USA, which causes Lyme borreliosis with unusually high spirochaetaemia. Clinicians should be aware of this new B burgdorferi sensu lato genospecies, its distinct clinical features, and the usefulness of oppA1 PCR for diagnosis. FUNDING US Centers for Disease Control and Prevention Epidemiology and Laboratory Capacity for Infectious Diseases (ELC) Cooperative Agreement and Mayo Clinic Small Grant programme.

Response of Plasmodium refractory and susceptible strains of Anopheles gambiae to inoculated Sephadex beads.

A refractory strain of the mosquito, Anopheles gambiae, melanotically encapsulates and destroys malaria parasites in the midgut. Normal development of parasites is observed in a closely related susceptible strain. To examine the basis for the difference in response, the two strains were compared for responses to inoculated Sephadex beads of varying charge. Negatively charged C-25 beads elicited a much stronger reaction in the refractory strain where 49% of the beads were strongly melanized by 24 h, compared with only 5% in the susceptible strain. Male mosquitoes of each strain responded similarly, with 100% of the beads strongly melanized by 24 h in the refractory strain compared with only 5% in the susceptible strain males. A time course revealed that the melanization in refractory but not susceptible mosquitoes increases substantially over time; 91% of C-25 beads were melanized in refractory females at 72 h compared with 9% in the susceptible sample. Neutral G-25 beads and positively charged A-25 beads were melanized in both strains, demonstrating that the capacity to melanize foreign particles is present in susceptible mosquitoes.

Ultrastructure of the encapsulation of Plasmodium cynomolgi (B strain) on the midgut of a refractory strain of Anopheles gambiae.

Using transmission electron microscopy, we investigated the encapsulation of the simian malaria parasite, Plasmodium cynomolgi, in a refractory strain of the mosquito, Anopheles gambiae. After the ookinete penetrates the mosquito midgut epithelium and lodges between the basal membrane and the basal lamina, an electron-dense, melanin-like substance begins to coalesce around the parasite. Completely encapsulated parasites were found as early as 16 hr after the blood meal. Granules of the melanin-like substance often appeared to condense onto the parasite from the fluid in the extracellular spaces of the basal membrane labyrinth. Melanin granules also appeared to condense from the hemolymph onto the basal lamina underlying the parasite. In addition, groups of tubules, vesicles, and membranous whorls often were found in midgut cells that were located next to or were enclosing parasites. These structures were unusually electron-dense, and may have been associated with melanization. Hemocytes rarely were observed near completed capsules and neither hemocytes nor their remnants were components of the capsules. During later stages of encapsulation, parasites appeared abnormal and often were infiltrated with melanin. Although late-stage capsules were usually located basally, completed capsules enclosed by membranes were occasionally observed near the apical border of the midgut. Other capsules associated with cellular debris, were found in the lumen of the midgut from 1 to 6 days after the blood meal.

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. EASTERs 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.

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.

Ultrastructural localization of phenoloxidase in the midgut of refractory Anopheles gambiae and association of the enzyme with encapsulated Plasmodium cynomolgi.

A melanogenic enzyme, phenoloxidase, was localized ultrastructurally in the midgut epithelia of 2 strains of Anopheles gambiae, a refractory strain that melanotically encapsulates Plasmodium cynomolgi ookinetes on the midgut, and a susceptible strain that does not. Midguts were incubated with either dopa or dopamine, and the resultant electron-dense product of phenoloxidase activity was localized on the basal lamina (BL) and cellular basal membrane labyrinth (BML) in uninfected mosquitoes of both strains. In infected refractory mosquitoes, the reaction products still were observed on the BL and BML but were especially dense in the BML of midgut cells near encapsulated ookinetes and in the capsule itself. In infected susceptible mosquitoes, phenoloxidase localization was reduced or absent in the BL and BML and was not observed near parasites. Phenylthiourea (PTU) inhibited the phenoloxidase reaction, indicating that the reaction product deposited in the absence of PTU resulted from enzyme activity and not autooxidation of the substrates. It is concluded that higher levels of phenoloxidase in the refractory strain following a blood meal may contribute to the ability to encapsulate ookinetes.