Salivary Gland Invasion Research Articles

Anopheles Gambiae PRS1 Modulates Plasmodium Development at Both Midgut and Salivary Gland Steps

BackgroundInvasion of the mosquito salivary glands by Plasmodium is a critical step for malaria transmission. From a SAGE analysis, we previously identified several genes whose expression in salivary glands was regulated coincident with sporozoite invasion of salivary glands. To get insights into the consequences of these salivary gland responses, here we have studied one of the genes, PRS1 (Plasmodium responsive salivary 1), whose expression was upregulated in infected glands, using immunolocalization and functional inactivation approaches.Methodology/Principal Findings PRS1 belongs to a novel insect superfamily of genes encoding proteins with DM9 repeat motifs of uncharacterized function. We show that PRS1 is induced in response to Plasmodium, not only in the salivary glands but also in the midgut, the other epithelial barrier that Plasmodium has to cross to develop in the mosquito. Furthermore, this induction is observed using either the rodent parasite Plasmodium berghei or the human pathogen Plasmodium falciparum. In the midgut, PRS1 overexpression is associated with a relocalization of the protein at the periphery of invaded cells. We also find that sporozoite invasion of salivary gland cells occurs sequentially and induces intra-cellular modifications that include an increase in PRS1 expression and a relocalization of the corresponding protein into vesicle-like structures. Importantly, PRS1 knockdown during the onset of midgut and salivary gland invasion demonstrates that PRS1 acts as an agonist for the development of both parasite species in the two epithelia, highlighting shared vector/parasite interactions in both tissues.Conclusions/SignificanceWhile providing insights into potential functions of DM9 proteins, our results reveal that PRS1 likely contributes to fundamental interactions between Plasmodium and mosquito epithelia, which do not depend on the specific Anopheles/P. falciparum coevolutionary history.

Interaction of the monoxenic trypanosomatid Blastocrithidia culicis with the Aedes aegypti salivary gland

Blastocrithidia culicis is a monoxenous trypanosomatid that inhabits mosquitoes. Although its life cycle in these insects has not been described, we recently demonstrated that B. culicis colonizes the Aedes aegypti digestive tract and is also able to reach the mosquito's haemocoel. In this work, we describe the interaction of B. culicis with the A. aegypti salivary gland in vitro and in vivo. Ultrastructural analysis reveals different steps of the invasion process, beginning with the insertion of the B. culicis anterior flagellum into the basal lamina of the gland and ending with the protozoan inside the salivary gland acini compartment. Carbohydrates are involved in the initial adhesion of B. culicis cells to the salivary glands, as demonstrated by protozoan binding inhibition assays and fluorescent lectin labeling of the trypanosomatid–salivary gland interaction. B. culicis is able to survive after incubation in vitro in the mosquito haemolymph, and trypanosomatid binding to salivary glands was confirmed by the injection of radioactively labeled protozoa into the mosquito haemocoel. These results suggest that salivary gland invasion could be part of the B. culicis life cycle in A. aegypti, raising the possibility that B. culicis can be transmitted by these mosquitoes.

Malaria Parasite Invasion of the Mosquito Salivary Gland Requires Interaction between the Plasmodium TRAP and the Anopheles Saglin Proteins

SM1 is a twelve-amino-acid peptide that binds tightly to the Anopheles salivary gland and inhibits its invasion by Plasmodium sporozoites. By use of UV-crosslinking experiments between the peptide and its salivary gland target protein, we have identified the Anopheles salivary protein, saglin, as the receptor for SM1. Furthermore, by use of an anti-SM1 antibody, we have determined that the peptide is a mimotope of the Plasmodium sporozoite Thrombospondin Related Anonymous Protein (TRAP). TRAP binds to saglin with high specificity. Point mutations in TRAP's binding domain A abrogate binding, and binding is competed for by the SM1 peptide. Importantly, in vivo down-regulation of saglin expression results in strong inhibition of salivary gland invasion. Together, the results suggest that saglin/TRAP interaction is crucial for salivary gland invasion by Plasmodium sporozoites.

Distinct Roles of Plasmodium Rhomboid 1 in Parasite Development and Malaria Pathogenesis

Invasion of host cells by the malaria parasite involves recognition and interaction with cell-surface receptors. A wide variety of parasite surface proteins participate in this process, most of which are specific to the parasite's particular invasive form. Upon entry, the parasite has to dissociate itself from the host-cell receptors. One mechanism by which it does so is by shedding its surface ligands using specific enzymes. Rhomboid belongs to a family of serine proteases that cleave cell-surface proteins within their transmembrane domains. Here we identify and partially characterize a Plasmodium berghei rhomboid protease (PbROM1) that plays distinct roles during parasite development. PbROM1 localizes to the surface of sporozoites after salivary gland invasion. In blood stage merozoites, PbROM1 localizes to the apical end where proteins involved in invasion are also present. Our genetic analysis suggests that PbROM1 functions in the invasive stages of parasite development. Whereas wild-type P. berghei is lethal to mice, animals infected with PbROM1 null mutants clear the parasites efficiently and develop long-lasting protective immunity. The results indicate that P. berghei Rhomboid 1 plays a nonessential but important role during parasite development and identify rhomboid proteases as potential targets for disease control.

Efficiency of salivary gland invasion by malaria sporozoites is controlled by rapid sporozoite destruction in the mosquito haemocoel

For successful transmission to the vertebrate host, malaria sporozoites must migrate from the mosquito midgut to the salivary glands. Here, using purified sporozoites inoculated into the mosquito haemocoel, we show that salivary gland invasion is inefficient and that sporozoites have a narrow window of opportunity for salivary gland invasion. Only 19% of sporozoites invade the salivary glands, all invasion occurs within 8h at a rate of approximately 200 sporozoites per hour, and sporozoites that fail to invade within this time rapidly die and are degraded. Then, using natural release of sporozoites from oocysts, we show that haemolymph flow through the dorsal vessel facilitates proper invasion. Most mosquitoes had low steady-state numbers of circulating sporozoites, which is remarkable given the thousands of sporozoites released per oocyst, and suggests that sporozoite degradation is a rapid immune process most efficient in regions of high haemolymph flow. Only 2% of Anopheles gambiae haemocytes phagocytized Plasmodium berghei sporozoites, a rate insufficient to explain the extent of sporozoite clearance. Greater than 95% of haemocytes phagocytized Escherichia coli or latex particles, indicating that their failure to sequester large numbers of sporozoites is not due to an inability to engage in phagocytosis. These results reveal the operation of an efficient sporozoite-killing and degradation machinery within the mosquito haemocoel, which drastically limits the numbers of infective sporozoites in the mosquito salivary glands.

SAGE analysis of mosquito salivary gland transcriptomes during Plasmodium invasion

Invasion of the vector salivary glands by Plasmodium is a critical step for malaria transmission. To describe salivary gland cellular responses to sporozoite invasion, we have undertaken the analysis of Anopheles gambiae salivary gland transcriptome using Serial Analysis of Gene Expression (SAGE). Statistical analysis of the more than 160000 sequenced tags generated from four libraries, two from glands infected by Plasmodium berghei, two from glands of controls, revealed that at least 57 Anopheles genes are differentially expressed in infected salivary glands. Among the 37 immune-related genes identified by SAGE tags, four (Defensin1, GNBP, Serpin6 and Cecropin2) were found to be upregulated during salivary gland invasion, while five genes encoding small secreted proteins display induction patterns strongly reminiscent of that of Cecropin2. Invasion by Plasmodium has also an impact on the expression of genes involved in transport, lipid and energy metabolism, suggesting that the sporozoite may exploit the metabolism of its host. In contrast, protein composition of saliva is predicted to be only slightly modified after infection. This study, which is the first transcriptome analysis of the salivary gland response to Plasmodium infection, provides a basis for a better understanding of Plasmodium/Anopheles salivary gland interactions.

Robust salivary gland‐specific transgene expression in Anopheles stephensi mosquito

Malaria sporozoites invade the mosquito salivary glands and wait in the salivary duct until the next blood feeding. The mechanisms of the process and molecules involved in the salivary gland invasion remain largely unknown. To establish a robust salivary gland-specific transgene expression in Anopheles stephensi, we obtained a salivary gland-specific promoter for a gene encoding anopheline antiplatelet protein (AAPP). The aapp promoter is a female salivary gland-specific and blood meal-inducible strong promoter. Using this promoter, we generated a transgenic An. stephensi expressing abundant Discosoma sp. red fluorescent protein (DsRed) in the distal-lateral lobes of the glands, where the sporozoites invade preferentially. These results open up the possibilities of elucidating salivary gland-parasite interactions and generating transgenic mosquitoes refractory to parasites.

Analysis of the Plasmodium and Anopheles Transcriptomes during Oocyst Differentiation

Understanding the life cycle of the malaria parasite in its mosquito vector is essential for developing new strategies to combat this disease. Subtractive hybridization cDNA libraries were constructed that are enriched for Plasmodium berghei and Anopheles stephensi genes expressed during oocyst differentiation on the midgut. Sequencing of 1485 random clones led to the identification of 1137 unique expressed sequence tags. Of the 608 expressed sequence tags with data base hits, 320 (53%) had significant matches to the non-redundant protein data base, whereas 288 (47%) with matches only to genomic data bases represent novel Plasmodium and Anopheles genes. Transcription of six novel parasite genes and two previously identified asexual stage genes was up-regulated during oocyst differentiation. In addition, the mRNA for an Anopheles fibrinogen domain gene was induced on day 2 after an infectious blood meal, at the time of ookinete to oocyst differentiation. The subcellular distribution of MAEBL, a sporozoite surface protein, is developmentally regulated from presumed storage organelles in day 15 oocysts to uniform distribution on the surface in day 22 oocysts. This redistribution may reflect a sporozoite maturation program in preparation for salivary gland invasion. Furthermore, apical membrane antigen 1, another parasite surface molecule, is translationally regulated late in sporozoite development, suggesting a role during infection of the vertebrate host. The present results and those of an accompanying report (Abraham, E. G., Islam, S., Srinivasan, P., Ghosh, A. K., Valenzuela, J., Ribeiro, J. M., Kafatos, F. C., Dimopoulos, G., & Jacobs-Lorena, M. (2003) J. Biol. Chem. 279, 5573-5580) provide the foundation for studies seeking to understand at the molecular level Plasmodium development and its interactions with the mosquito.

The Plasmodium circumsporozoite protein is involved in mosquito salivary gland invasion by sporozoites.

Plasmodium sporozoites develop in oocysts on the midgut wall of the mosquito and are released into the hemocoel. Approximately 15-20% of oocyst sporozoites will successfully attach to and invade salivary glands, their target organ. We have previously shown that the major surface protein of sporozoites, the circumsporozoite (CS) protein, binds specifically to salivary glands and not to other mosquito organs exposed to circulating hemolymph. In addition, a peptide from the N-terminal portion of CS protein inhibits binding of the protein to the glands. In this study, we have extended these findings and show that both the protein and the peptide can inhibit sporozoite invasion of salivary glands.

Complete development of mosquito phases of the malaria parasite in vitro.

Methods for reproducible in vitro development of the mosquito stages of malaria parasites to produce infective sporozoites have been elusive for over 40 years. We have cultured gametocytes of Plasmodium berghei through to infectious sporozoites with efficiencies similar to those recorded in vivo and without the need for salivary gland invasion. Oocysts developed extracellularly in a system whose essential elements include co-cultured Drosophila S2 cells, basement membrane matrix, and insect tissue culture medium. Sporozoite production required the presence of para-aminobenzoic acid. The entire life cycle of P. berghei, a useful model malaria parasite, can now be achieved in vitro.

Species diversity and flagellate infections in the sand fly fauna near Porto Grande, State of Amapá, Brazil (Diptera: Psychodidae. Kinetoplastida: Trypanosomatidae).

Forty-six species of Lutzomyia and one species of Brumptomyia were identified among 20,008 sand flies collected in central Amapá. L. squamiventris maripaensis, L. infraspinosa, L. umbratilis, and L. ubiquitalis accounted for 66% of the specimens caught in light traps, and L. umbratilis was the commonest of the 16 species found on tree bases. Seven species of Lutzomyia including L. umbratilis were collected in a plantation of Caribbean pine. Sixty out of 511 female sand flies dissected were positive for flagellates. Among the sand flies from which Leishmania was isolated, promastigotes were observed in the salivary glands and foregut of 13 out of 21 females scored as having very heavy infections in the remainder of the gut, reinforcing the idea that salivary gland invasion may be part of the normal life cycle of Leishmania in nature. Salivary gland infections were detected in specimens of L. umbratilis, L. whitmani and L. spathotrichia. Parasites isolated from L. umbratilis, L. whitmani and also from one specimen of L. dendrophyla containing the remains of a bloodmeal, were compatible with Le. guyanensis by morphology and behaviour in hamsters.

Plasmodium falciparum: binding studies of peptide derived from the sporozoite surface protein 2 to Hep G2 cells.

Plasmodium falciparum sporozoite surface protein 2 (Pf SSP2), also called thrombospondin related anonymous protein (TRAP), is involved in the process of sporozoite invasion of hepatocytes. Pf SSP2/TRAP possesses two different adhesion domains sharing sequences and structural homology with von Willebrand factor A-domains and human repeat I thrombospondin (TSP). Pf SSP2/TRAP has also been implicated in sporozoite mobility and in mosquito salivary gland invasion processes. We tested 15-mer long synthetic peptides having five overlapping residues covering the complete protein Pf SSP2 sequence in binding assays to Hep G2 cells. In these 57 peptides, 21 high-activity binding peptides (HABPs) were identified; five were in the adhesion domains already described and 16 were in two regions toward the protein's carboxy and middle terminal part. Six HABPs showed conserved amino acid sequences: 3243 (21FLVNGRDVQNNIVDE35), 3279 (201FLVGCHPSDGKCNLY215), 3287 (241TASCGVWDEWSPCSV255), 3289 (251SPCSVTCGKGTRSRK265), 3327 (441ERKQSDPQSQDNNGNY455) and 3329 (451DNNGNRHVPNSEDREY465). The HABPs show saturable binding and dissociation constants between 140 and 900 nm with 40 000-855 000 binding sites per cell. The 3279 (201FLVGCHPSDGKCNLY215), 3323 (421NDKSDRYIPYSPLSP435) and 3331 (461SEDRETRPHGRNNENY475) HABPs have B epitopes in their sequences; these have previously been recognized by antibodies partially inhibiting hepatocyte invasion and development of the hepatic state. The 3287 (241TASCGVWDEWSPCSV255) and 3289 (251SPCSVTCGKGTRSRK265) HABPs share common sequences with the Pf SSP2/TRAP region II plus, which is present in a great number of adhesion proteins. Based on this information, six new peptides covering the high binding regions identified previously were synthesized and, using a competition assay, the amino acid involved in the binding were determined.

Adenoid cystic carcinoma of the salivary glands: a review of 10 years.

A retrospective analysis was performed of 50 patients with adenoid cystic carcinoma who were seen in the Department of Radiation Oncology, University of Witwatersrand, Johannesburg, South Africa, in the past 10 years. There were 25 men and 25 women with a mean age of 52 years (age range, 21 to 88 years). Five patients had metastatic disease, and 17 had neural invasion. Thirty-four patients had surgery (11, complete; 23, microscopic residual). Sixteen patients had radiotherapy as initial management. The disease-free survival was 26%, overall survival was 29%, and local control was 30% at 10 years. Most recurrences occurred in the first 3 years. Nine patients had metastasis following treatment. The mean survival after metastasis was 15 months. Seven prognostic variables were analyzed using the log-rank test. There was no impact of age, site, type of salivary gland (major vs. minor), tumor stage, node positivity, or neural invasion on disease-free survival, overall survival, or local control. Extent of surgical resection (complete vs. microscopic residual) had a significant impact on disease-free survival and local control (P < 0.05) but no impact on overall survival (P > 0.05) because of the slow-growing nature of these tumors. Similarly, patients who had microscopic residual after surgery and were treated with radiotherapy did better than those who had biopsy and radiotherapy, although this was not significant statistically (P > 0.05). Thus, whenever possible, every attempt must be made to remove all microscopic tumor by surgery. Addition of postoperative radiotherapy with high-energy photons did not improve the locoregional control or survival in our series. There is a place for neutrons in the treatment of adenoid cystic carcinomas in advanced cases of inoperable or recurrent tumors, as a review of literature shows.

Borrelia burgdorferi OspA is an arthropod-specific transmission-blocking Lyme disease vaccine.

Borrelia burgdorferi, the spirochetal agent of Lyme disease, is transmitted by Ixodes ticks. A vaccine based on B. burgdorferi outer surface protein (Osp) A protects mice from spirochete infection. Here we report on the expression of OspA on spirochetes inside engorging ticks and relate OspA expression to antispirochetal immunity. Spirochetes in the gut of unfed nymphal ticks were stained by an OspA antibody, whereas in feeding ticks, the majority of spirochetes in the gut and salivary glands did not stain with the antibody. Thus, OspA was not expressed on most spirochetes during transmission from the vector to the vertebrate host. To examine the mechanism of protection afforded by OspA antibody, mice were passively immunized with OspA antibody at different times relative to tick attachment. When OspA antibody was administered to mice before or at the time of tick attachment, spirochetal development events in the vector, such as growth and salivary gland invasion, were blocked and the mice were protected from B. burgdorferi infection. When OspA antibody was administered to mice 48 h after tick attachment, spirochetes persisted in the nymphs and the mice were not protected despite the presence of circulating antibodies in the host as well as in the tick blood meal. Thus, OspA immunity appears to be effective only during a narrow window time at the beginning of the blood meal when antibodies bind to OspA-expressing spirochetes in the tick gut and block transmission from the vector to the host.

Developmentally regulated infectivity of malaria sporozoites for mosquito salivary glands and the vertebrate host.

Sporozoites are an invasive stage of the malaria parasite in both the mosquito vector and the vertebrate host. We developed an in vivo assay for mosquito salivary gland invasion by preparing Plasmodium gallinaceum sporozoites from infected Aedes aegypti mosquitoes under physiological conditions and inoculating them into uninfected female Ae. aegypti. Sporozoites from mature oocysts were isolated from mosquito abdomens 10 or 11 d after an infective blood meal. Salivary gland sporozoites were isolated 13 or 14 d after an infective blood meal. Purified oocyst sporozoites that were inoculated into uninfected female mosquitoes invaded their salivary glands. Using the same assay system, sporozoites derived from salivary glands did not reinvade the salivary glands after inoculation. Conversely, as few as 10 to 50 salivary gland sporozoites induced infection in chickens, while only 2 of 10 chickens inoculated with 5,000 oocyst sporozoites were infected. Both sporozoite populations were found to express a circumsporozoite protein on the sporozoite surface as determined by immunofluorescence assay and circumsporozoite precipitation test using a circumsporozoite protein-specific monoclonal antibody. We conclude that molecules other than this circumsporozoite protein may be responsible for the differential invasion of mosquito salivary glands or infection of the vertebrate host.

Observations on human, monkey and bat trypanosomes and their vectors in Colombia

In all huts in 36 localities in central Colombia Rhodnius prolixus harboured Trypanosoma cruzi, and about 55% of 1000 bugs examined carried flagellates. The incidence of infection in these localities was more or less equal for T. cruzi and T. rangeli and more than one-fifth of the bugs had mixed infections of both flagellates. An area in the eastern part of central Colombia was heavily infested with Rhodnius prolixus carrying Trypanosoma cruzi and T. rangeli. T. cruzi-like organisms were found in a high percentage of several monkey and bat species and in several cases proved to be T. cruzi. A summary is given of the records for T. cruzi-like, T. rangeli-like and T. minasense flagellates in Colombian mammals. T. minasense was recovered from 5 monkey species. 3 of these are new host records for the country and one ( Ateles geoffroyi grisescens) is a completely new host record. T. rangeli was recovered from 2 Cebus capucinus monkeys, and haemolymph or salivary gland invasion of Rhodnius prolixus was observed after xenodiagnosis. Trypanosomes resembling Trypanosoma rangeli were isolated from 3 bats (2 Artibeus lituratus and 1 Glossophaga s. sorcina). After xenodiagnosis flagellates were found in the rectal ampulla of Rhodnius prolixus and Cavernicola pilosa, but did not invade the haemolymph of the insects. Isolation of Trypanosoma cruzi was made in at least 6 cases from the faeces of Cavernicola pilosa. T. rangeli culture forms, fed to Cavernicola pilosa via an artificial xenodiagnosis method, resulted in haemolymph infection in the bugs, but no salivary gland infection. Chagas's disease is discussed as a national public health problem.

Biological Factors Influencing Transmission of Trypanosoma rangeli by Rhodnius prolixus

Certain biological factors which might influence transmission of a Venezuelan strain of Trypanosoma rangeli by Rhodnius prolixus are presented with statistical evaluation of their significance. T. rangeli must invade salivary glands of R. prolixus before transmission is possible. When 1st instar nymphs were exposed to an infected meal, survival during nymphal stages decreased. This decrease was statistically significant and indicates that T. rangeli is pathogenic for R. prolixus nymphs. There was no indication that blood from different vertebrate hosts influenced survival. In adult survivors exposed as nymphs as well as in those exposed as adults cycle progressed to salivary glands in a significantly greater percentage of males than females. There was no significant difference in percentage of mature infections between those exposed as 1st instar nymphs and those exposed as adults. There appeared to be no advantage in multiple infective feedings. Following ingestion of Trypanosoma rangeli by Rhodnius prolixus, cyclical process begins with trypanosomes changing form and multiplying in alimentary canal. This first stage of development is a constant feature; second, passage into hemolymph, is not. However, once hemolymph is invaded, development progresses to salivary glands. Transmission of Trypanosoma rangeli by invertebrate host is not possible until salivary gland invasion has occurred. Groot (1954) in a comparative study of biological behavior of four strains of T. rangeli in Rhodnius prolixus reported that in one strain (ariarii) invasion of salivary glands always occurred when flagellates were found in hemolymph and his figures indicated at least 33% with salivary gland involvement. This high percentage of invasion was not observed in all strains. Tobie (1961), working with a cyclically maintained strain, obtained only a 12.5% invasion of salivary glands. The percentage, however, increased after a number of cyclical passages (Tobie, 1964). Grewal (1957) studying pathogenicity of T. rangeli to R. prolixus reported that most of bugs which died or could not molt had hemolymph infections. Only 41 out of 120 survived to become adults and only one of these had a salivary gland infection. This great loss he attributes to heavy parasitosis of insect, the entire system being blocked by