Molecular and functional characterization of vacuolar-ATPase from the American dog tick Dermacentor variabilis.

Vacuolar (V)-ATPase is a proton-translocating enzyme that acidifies cellular compartments for various functions such as receptor-mediated endocytosis, intracellular trafficking and protein degradation. Previous studies in Dermacentor variabilis chronically infected with Rickettsia montanensis have identified V-ATPase as one of the tick-derived molecules transcribed in response to rickettsial infection. To examine the role of the tick V-ATPase in tick-Rickettsia interactions, a full-length 2887-bp cDNA (2532-bp open reading frame) clone corresponding to the transcript of the V0 domain subunit a of D. variabilis V-ATPase (DvVATPaseV0a) gene encoding an 843 amino acid protein with an estimated molecular weight of ~96 kDa was isolated from D. variabilis. Amino acid sequence analysis of DvVATPaseV0a showed the highest similarity to VATPaseV0a from Ixodes scapularis. A potential N-glycosylation site and eight putative transmembrane segments were identified in the sequence. Western blot analysis of tick tissues probed with polyclonal antibody raised against recombinant DvVATPaseV0a revealed the expression of V-ATPase in the tick ovary. Transcriptional profiles of DvVATPaseV0a demonstrated a greater mRNA expression in the tick ovary, compared with the midgut and salivary glands; however, the mRNA level in each of these tick tissues remained unchanged after infection with R. montanensis for 1 h. V-ATPase inhibition bioassays resulted in a significant decrease in the ability of R. montanensis to invade tick cells in vitro, suggesting a role of V-ATPase in rickettsial infection of tick cells. Characterization of tick-derived molecules involved in rickettsial infection is essential for a thorough understanding of rickettsial transmission within tick populations and the ecology of tick-borne rickettsial diseases.

Analysis of Rickettsia typhi-infected and uninfected cat flea (Ctenocephalides felis) midgut cDNA libraries: deciphering molecular pathways involved in host response to R. typhi infection.

Murine typhus is a flea-borne febrile illness that is caused by the obligate intracellular bacterium, Rickettsia typhi. The cat flea, Ctenocephalides felis, acquires R. typhi by imbibing a bloodmeal from a rickettsemic vertebrate host. To explore which transcripts are expressed in the midgut in response to challenge with R. typhi, cDNA libraries of R. typhi-infected and uninfected midguts of C. felis were constructed. In this study, we examined midgut transcript levels for select C. felis serine proteases, GTPases and defence response genes, all thought to be involved in the fleas response to feeding or infection. An increase in gene expression was observed for the serine protease inhibitors and vesicular trafficking proteins in response to feeding. In addition, R. typhi infection resulted in an increase in gene expression for the chymotrypsin and rab5 that we studied. Interestingly, R. typhi infection had little effect on expression of any of the defence response genes that we studied. We are unsure as to the physiological significance of these gene expression profiles and are currently investigating their potential roles as it pertains to R. typhi infection. To our knowledge, this is the first report of differential expression of flea transcripts in response to infection with R. typhi.

PATRIC: the VBI PathoSystems Resource Integration Center.

The PathoSystems Resource Integration Center (PATRIC) is one of eight Bioinformatics Resource Centers (BRCs) funded by the National Institute of Allergy and Infection Diseases (NIAID) to create a data and analysis resource for selected NIAID priority pathogens, specifically proteobacteria of the genera Brucella, Rickettsia and Coxiella, and corona-, calici- and lyssaviruses and viruses associated with hepatitis A and E. The goal of the project is to provide a comprehensive bioinformatics resource for these pathogens, including consistently annotated genome, proteome and metabolic pathway data to facilitate research into counter-measures, including drugs, vaccines and diagnostics. The project's curation strategy has three prongs: 'breadth first' beginning with whole-genome and proteome curation using standardized protocols, a 'targeted' approach addressing the specific needs of researchers and an integrative strategy to leverage high-throughput experimental data (e.g. microarrays, proteomics) and literature. The PATRIC infrastructure consists of a relational database, analytical pipelines and a website which supports browsing, querying, data visualization and the ability to download raw and curated data in standard formats. At present, the site warehouses complete sequences for 17 bacterial and 332 viral genomes. The PATRIC website (https://patric.vbi.vt.edu) will continually grow with the addition of data, analysis and functionality over the course of the project.

Differential expression of two glutathione S-transferases identified from the American dog tick, Dermacentor variabilis.

Reciprocal signalling and gene expression play a cardinal role during pathogen-host molecular interactions and are prerequisite to the maintenance of balanced homeostasis. Gene expression repertoire changes during rickettsial infection and glutathione-S-transferases (GSTs) were among the genes found up-regulated in Rickettsia-infected Dermacentor variabilis. GSTs are well known to play an important part in cellular stress responses in the host. We have cloned two full-length GSTs from D. variabilis (DvGST1 and DvGST2). Comparison of these two DvGST molecules with those of other species indicate that DvGST1 is related to the mammalian class theta and insect class delta GSTs, while DvGST2 does not seem to fall in the same family. Northern blotting analyses revealed differential expression patterns, where DvGST1 and DvGST2 transcripts are found in the tick gut, with DvGST2 transcripts also present in the ovaries. Both DvGST transcripts are up-regulated upon tick feeding. Challenge of fed adult ticks with Escherichia coli injection showed decreased transcript amounts compared with ticks injected with phosphate-buffered saline (sham) and naïve ticks.

Stress and transcriptional regulation of tick ferritin HC.

We previously identified a partial Dermacentor variabilis cDNA encoding ferritin HC (HC) subunit homolog (DVFER) that was differentially upregulated in Rickettsia montanensis infected ticks (Mulenga et al., 2003a). We have used rapid amplification of cDNA ends to clone full-length DVFER cDNA and its apparent ortholog from the wood tick, D. andersoni (DAFER), both of which show high sequence similarity to vertebrate than insect ferritin. Both DVFER and DAFER contain the stem-loop structure of a putative iron responsive element in the 5' untranslated region (nucleotide positions, 16-42) and the feroxidase centre loop typical for vertebrate ferritin HC subunits. Quantitative Western and Northern blotting analyses of protein and RNA from unfed and partially fed whole tick as well as dissected tick tissues demonstrated that DVFER is constitutively and ubiquitously expressed. Based on densitometric analysis of detected protein and mRNA bands, DVFER is predominantly expressed in the midgut, and to a lesser extent in the salivary glands, ovary and fatbody. Sham treatment (mechanical injury) and Escherichia coli challenge of D. variabilis ticks stimulated statistically significant (approximately 1.5- and approximately 3.0-fold, respectively) increases in DVFER mRNA abundance over time point matched naive control ticks. These data suggest that DVFER mRNA is nonspecifically up regulated in response to mechanical injury or bacterial infection induced stress.

An immune responsive factor D-like serine proteinase homologue identified from the American dog tick, Dermacentor variabilis.

A Dermacentor variabilis cDNA encoding a clip-domain serine proteinase homologue with glycine replacing the catalytic serine was identified from tick haemocytes. The D. variabilis product was most similar to Tachypleus tridentatus haemocyte antimicrobial factor D and shared significant homologies with a number of immune-responsive gene products of arthropods, including insect prophenoloxidase-activating cofactors. Northern blotting analyses confirmed that the tick serine proteinase homologue expression levels were highest in haemocytes, and to lesser degrees in ovaries and then salivary glands whereas steady-state levels of expression in whole ticks were found to be slightly higher in fed versus unfed adults or eggs. Challenge of fed adults by Escherichia coli injection demonstrated that transcript abundance was significantly increased above those of naive controls in a temporal fashion. Additionally, an apparent orthologue of the D. variabilis clip-domain molecule was cloned, and expression detected, from a Dermacentor andersoni cell line indicating cross species conservation.

Detection of R. felis and R. typhi in fleas using monoclonal antibodies.

Rickettsia typhi and R. felis are flea-transmitted human pathogenic rickettsial species. To investigate the distributional dynamics of these rickettsiae we designed a micro-immunofluorescence assay (MIF) using species-specific monoclonal antibodies (MAbs) applied to flea cryosections. Our assay was performed in less than 3 h and its applicability was demonstrated by the detection of R. typhi in 50 artificially infected human body lice but in none of 50 uninfected lice. With MIF, we identified 31 positive among 32 fleas proven with PCR to be naturally infected with R. felis; and 7 positive among 32 fleas proven with PCR to be naturally infected with R. typhi. No cross-detection was observed with both MAbs. Fresh R. felis-infected fleas were significantly more MIF-positive than long conserved R. typhi-infected fleas (31/32 vs. 7/32, P < 0.01). This discrepancy may be linked to degradation of antigens by long-term freezing. For R. typhi-infected fleas, our assay was significantly more efficient when applied to fleas in early stages of infection (less than 15 days) by comparison with fleas frozen more than 20 days after infection (7/15 vs. 0/17, P = 0.01). This difference may be related to an antigenic modification caused by selection pressure in the vector and host process. The sensitivity of the described method did not exceed 47% (7/15) for R. typhi but, in contrast, was 97% for R. felis. Thus, our method appears to be useful for surveillance in R. felis infections, but requires further studies for the detection of R. typhi.

Dynamics of Rickettsia-tick interactions: identification and characterization of differentially expressed mRNAs in uninfected and infected Dermacentor variabilis.

To begin to explore the molecular dynamics of rickettsial tick interaction, subtractive hybridization was used to screen mRNAs in Rickettsia montanensis-infected and uninfected Dermacentor variabilis. We isolated 30 cDNA fragments, 22 of which were up-regulated and eight were down-regulated in response to rickettsial infection. Based on a putative identity of 11 cDNA fragments with similarity to known protein families, the tick genetic factors have been assigned into three groups including, the tick immune response factors (alpha-2 macroglobulin and IgE-dependent histamine release factor), the receptor/adhesion molecules (the signal transducer and activator of transcription-1/3 protein inhibitor, the clathrin adaptor protein and tetraspanin) and the stress response proteins (aldose reductase, glutathione-S transferase, ferritin, nucleosome assembly protein and cyclin A protein). Density analyses of semiquantitative RT-PCR amplified products demonstrated differential expression for 18 of the 23 tested genetic factors, apparently representing a 78% agreement with results obtained by subtractive hybridization. Additionally, mRNA transcripts of 17 of the 23 tested genetic factors were amplified from tick haemocytes/circulatory cells demonstrating that their expression is not restricted to the ovaries and suggesting a potential involvement in the immune response.

Anti-mosquito midgut antibodies block development of Plasmodium falciparum and Plasmodium vivax in multiple species of Anopheles mosquitoes and reduce vector fecundity and survivorship.

The mosquito midgut plays a central role in the sporogonic development of malaria parasites. We have found that polyclonal sera, produced against mosquito midguts, blocked the passage of Plasmodium falciparum ookinetes across the midgut, leading to a significant reduction of infections in mosquitoes. Anti-midgut mAbs were produced that display broad-spectrum activity, blocking parasite development of both P. falciparum and Plasmodium vivax parasites in five different species of mosquitoes. In addition to their parasite transmission-blocking activity, these mAbs also reduced mosquito survivorship and fecundity. These results reveal that mosquito midgut-based antibodies have the potential to reduce malaria transmission in a synergistic manner by lowering both vector competence, through transmission-blocking effects on parasite development, and vector abundance, by decreasing mosquito survivorship and egg laying capacity. Because the intervention can block transmission of different malaria parasite species in various species of mosquitoes, vaccines against such midgut receptors may block malaria transmission worldwide.

Rickettsia felis: molecular characterization of a new member of the spotted fever group.

In this report, placement of Rickettsia felis in the spotted fever group (SFG) rather than the typhus group (TG) of Rickettsia is proposed. The organism, which was first observed in cat fleas (Ctenocephalides felis) by electron microscopy, has not yet been reported to have been cultivated reproducibly, thereby limiting the standard rickettsial typing by serological means. To overcome this challenge, several genes were selected as targets to be utilized for the classification of R. felis. DNA from cat fleas naturally infected with R. felis was amplified by PCR utilizing primer sets specific for the 190 kDa surface antigen (rOmpA) and 17 kDa antigen genes. The entire 5,513 bp rompA gene was sequenced, characterized and found to have several unique features when compared to the rompA genes of other Rickettsia. Phylogenetic analysis of the partial sequence of the 17 kDa antigen gene indicated that R. felis is less divergent from the SFG rickettsiae than from the TG rickettsiae. The data corroborate results from previous reports that analysed the citrate synthase, 16S rRNA, rompB (135 kDa surface antigen), metK, ftsY, polA and dnaE genes that placed R. felis as a member of the SFG. The organism is passed trans-stadially and transovarially, and infection in the cat flea has been observed in the midgut, tracheal matrix, muscle, hypodermis, ovaries and testes.

Detection of Plasmodium yoelii stage mRNA in BALB/c mice.

Relatively little information is available concerning the expression of parasite genes during the liver stage of Plasmodium infection, mostly because of low-level infection of host hepatocytes and the lack of purification techniques for the liver stage parasites. We have determined the optimal dosage of Plasmodium yoelii sporozoite inoculum and routes of inoculation, which are intravenous tail vein and the intrahepatic portal circulation. To determine which route was optimal, BALB/c mice were inoculated via 1 of these routes, and parasitemia was detected by reverse transcription polymerase chain reaction (RT-PCR) detecting both murine beta-actin and P. yoelii-specific 28S ribosomal RNA in the liver samples. Murine beta-actin was detected after 15 cycles of PCR, and its expression levels did not differ between treatment groups. However, P. yoelii-specific 28S ribosomal RNA gene product was detected after 15 cycles of PCR in animals inoculated via the tail vein but was not detected until 25 cycles in animals inoculated via the intrahepatic portal circulation. Experiments were then performed to identify the smallest inoculum required to initiate a liver stage infection that would yield sufficient parasite RNA for analysis. Inoculation with different doses of sporozoite inocula was followed by RT-PCR on the livers of the inoculated animals. The P. yoelii-specific 28S ribosomal RNA gene product was first detected in both treatment groups after 15 cycles, suggesting that both doses of sporozoite inocula provided relatively the same level of liver infection rate. We also have analyzed infected mouse liver for parasite-specific mRNA, which was detectable as early as 24 hr postinfection.

Host responses to Plasmodium yoelii hepatic stages: a paradigm in host-parasite interaction.

The liver stage of malaria, caused by the genus Plasmodium, is clinically silent, but immunologically significant. Ample evidence exists for an effective CD8(+) T cell response to this stage as well as the involvement of gammadeltaT cells and NK1.1(int) cells in immunized animal models. In contrast, there is little information concerning responses in a naive host. Here we report that several host gene expressions in the liver, spleen, and kidney of BALB/c mice are altered during the liver stage of Plasmodium yoelii infection. Really interesting new gene 3 (Ring3), semaphorin subclass 4 member G, glutamylcysteine synthetase, and p45 NF erythroid 2 were all up-regulated 24 h after infection with P. yoelii. Semaphorin subclass 4 member G expression was elevated in the kidney, whereas Ring3 was elevated in both spleen and kidney. The expression of TNF-alpha (TNF-alpha and IFN-gamma) were down-regulated in all three tissues tested except in infected spleen where IFN-gamma was elevated. P. yoelii-related host gene changes were compared with those in Toxoplasma gondii-infected livers. Ring3 expression increased 5-fold over control values, whereas expression of the other transcripts remained unchanged. TNF-alpha and IFN-gamma expressions were increased in the Toxoplasma-infected livers. The uniform increase of Ring3 expression in both Plasmodium- and Toxoplasma-infected livers suggests an innate immune response against parasitic infections, whereas the other gene expression changes are consistent with Plasmodium parasite-specific responses. Taken together, these changes suggest the immune responses to P. yoelii infection are both parasite and organ specific.

Infection and transovarial transmission of rickettsiae in Dermacentor variabilis ticks acquired by artificial feeding.

In this study we examined the efficiency of an in vitro feeding technique using glass microcapillaries as a method of establishing rickettsiae-infected lines of ticks. To quantify the volume ingested by ticks during microcapillary feeding, the incorporation of radiolabeled amino acids in tick gut and hemolymph was calculated. Fifteen of 18 ticks consumed between 0.06 and 6.77 microl. However, ingestion of fluid was not correlated to weight gain during capillary feeding. Uninfected and partially fed laboratory-reared female Dermacentor variabilis ticks were exposed to either Rickettsia montana- or Rickettsia rhipicephali-infected Vero cells via microcapillary tubes, returned to rabbit hosts, and allowed to feed to repletion. All tissues collected from ticks allowed to feed overnight on rickettsiae-infected fluids were found to be infected when examined by IFA. When rickettsiae-infected and uninfected capillary-fed ticks were allowed to feed to repletion and lay eggs, no significant differences in mean engorgement weight or fecundity was observed. When we assessed the efficiency of transovarial transmission of rickettsiae by ticks that imbibed rickettsiae-infected cells by polymerase chain reaction (PCR) and IFA, infection was detected by PCR in the eggs from 85% of the ticks exposed to R. montana and 69% of the ticks exposed to R. rhipicephali. Rickettsial genes were not amplified in samples of the uninfected controls. Examination by IFA of egg samples from females exposed to rickettsiae-infected cells identified rickettsiae in 100% of the samples tested, while the uninfected controls were negative.

Mercury exposure and murine response to Plasmodium yoelii infection and immunization.

Malaria has re-emerged in Amazonia over the past two decades. Many factors have been proposed for this, among them changes in population distribution, failures of vector control and pharmacologic management, and local as well as global environmental changes. Among the latter factors, we have studied the potential role of increasing exposures to the immunotoxic metal mercury, which is widely used in Amazonia for artisanal extraction of alluvial gold deposits. We report here that Hg impairs host resistance to malaria infection at exo-erythrocytic stages. Hg exposed mice have higher parasitemia following infection with sporozoites, but not after transfusion of infected red cells. In mice inoculated with irradiated sporozoites, Hg blocks acquisition of immunity. In addition Hg affects immunologic parameters that are known to be involved in host response to malaria infection. These results have potential implications for the incidence and prevalence of malaria among populations exposed to mercury from artisanal goldmining and consumption of contaminated fish regions with high rates of malaria and other infectious diseases.

Retrieving parasite specific liver stage gene products in Plasmodium yoelii infected livers using differential display.

Differential display (DD) has been routinely used to identify genes whose expression pattern is altered by changes in the cellular environment and/or at different stages of development. Most reports utilizing DD contain conventional DD primers that have high guanine and cytosine content and would not be expected to be optimal for Plasmodium which has approximately 30-40% G+C. In an attempt to accommodate the high adenine and thymidine rich genome of Plasmodium yoelii, we utilized PCR primers containing 40, 50 and 60% G+C and modified the existing DD technique. Thus 40% G+C appeared to be the most suitable to amplify Plasmodium genome. Gene specific primers were generated from the sequences of selected DD bands amplified using the 40% G+C primers and were used to verify that the DD clones were of parasite origin by PCR and sequence alignment. Additional data on five of the selected DD clones, designated P2T1L5, P2T1L6, P2T6L11, P2T7L12 and P2T7L13, suggested that all are expressed during the P. yoelii liver stage infection. Interestingly, P2T1L5 is also expressed during the sporozoite stage of the life cycle and both P2T1L6 and P2T6L11 are present as blood stage antigens. The results of this study suggest that DD incorporating primers with low G+C content allows the identification of P. yoelii messages from infected mouse livers.

gammadelta T cells are a component of early immunity against preerythrocytic malaria parasites.

We tested the hypothesis that gammadelta T cells are a component of an early immune response directed against preerythrocytic malaria parasites that are required for the induction of an effector alphabeta T-cell immune response generated by irradiated-sporozoite (irr-spz) immunization. gammadelta T-cell-deficient (TCRdelta(-/-)) mice on a C57BL/6 background were challenged with Plasmodium yoelii (17XNL strain) sporozoites, and then liver parasite burden was measured at 42 h postchallenge. Liver parasite burden was measured by quantification of parasite-specific 18S rRNA in total liver RNA by quantitative-competitive reverse transcription-PCR and by an automated 5' exonuclease PCR. Sporozoite-challenged TCRdelta(-/-) mice showed a significant (P < 0.01) increase in liver parasite burden compared to similarly challenged immunocompetent mice. In support of this result, TCRdelta(-/-) mice were also found to be more susceptible than immunocompetent mice to a sporozoite challenge when blood-stage parasitemia was used as a readout. A greater percentage of TCRdelta(-/-) mice than of immunocompetent mice progressed to a blood-stage infection when challenged with five or fewer sporozoites (odds ratio = 2.35, P = 0.06). TCRdelta(-/-) mice receiving a single irr-spz immunization showed percent inhibition of liver parasites comparable to that of immunized immunocompetent mice following a sporozoite challenge. These data support the hypothesis that gammadelta T cells are a component of early immunity directed against malaria preerythrocytic parasites and suggest that gammadelta T cells are not required for the induction of an effector alphabeta T-cell immune response generated by irr-spz immunization.

Identification and molecular analysis of the gene encoding Rickettsia typhi hemolysin.

Rickettsia typhi, the causative agent of murine typhus, grows directly within the host cell cytoplasm, accumulating a large number of progeny, and eventually lyses the cells. Typhus group rickettsiae (R. typhi and R. prowazekii) adhere to and lyse human and sheep erythrocytes. However, the molecular mechanism underlying erythrocyte lysis by R. typhi has not been defined. Here we describe the cloning and nucleotide sequence analysis of the gene (tlyC) encoding a hemolysin from R. typhi. DNA sequence analysis of R. typhi tlyC revealed an open reading frame of 912 bp, which encodes a protein of 304 amino acids with a predicted molecular mass of 38 kDa. To associate the R. typhi tlyC gene product with hemolytic activity, we performed complementation studies with hemolysin-negative Proteus mirabilis WPM111 (a HpmA(-) mutant of BA6163) transformed with R. typhi tlyC or R. typhi GFPuv-tlyC constructs. We demonstrated that the cloned tlyC gene conferred a hemolytic phenotype on an otherwise nonhemolytic mutant of P. mirabilis. The availability of the cloned R. typhi tlyC will permit further characterization and definition of its role in rickettsial virulence.

Infectivity of Plasmodium berghei sporozoites delivered by intravenous inoculation versus mosquito bite: implications for sporozoite vaccine trials.

Plasmodium berghei sporozoites delivered by mosquito bite were more infectious to outbred CD-1 mice than were sporozoites delivered by intravenous inoculation. The route of challenge also affected vaccine efficacy. In view of these findings and the fact that mosquito bites are the natural mode of sporozoite delivery, infectious mosquito bites should be considered the challenge protocol of choice for sporozoite vaccine efficacy trials.

Green fluorescent protein as a marker in Rickettsia typhi transformation.

Transformation of rickettsiae is a recent accomplishment, but utility of this technique is limited due to the paucity of selectable markers suitable for use in this intracellular pathogen. We chose a green fluorescent protein variant optimized for fluorescence under UV lights (GFPUV) as a fluorometric marker and transformed Rickettsia typhi with an rpoB-GFPUV fusion construct. The rickettsiae were subsequently grown in Vero cells, and cultures were screened by PCR and restriction fragment length polymorphism (RFLP) to confirm incorporation of the rpoB-GFPUV construct. Cultures were then analyzed by flow cytometry for detection of GFPUV expression, and transformed R. typhi were isolated in a fluorescence-activated cell sorter. This is the first report of transformation of rickettsiae with a nonrickettsial (GFPUV) gene.