Dataset Information

In-depth characterization of trypsin-like serine peptidases in the midgut of the sugar fed Culex quinquefasciatus.

ABSTRACT: Culex quinquefasciatus is a hematophagous insect from the Culicidae family that feeds on the blood of humans, dogs, birds and livestock. This species transmits a wide variety of pathogens between humans and animals. The midgut environment is the first location of pathogen-vector interactions for blood-feeding mosquitoes and the expression of specific peptidases in the early stages of feeding could influence the outcome of the infection. Trypsin-like serine peptidases belong to a multi-gene family that can be expressed in different isoforms under distinct physiological conditions. However, the confident assignment of the trypsin genes that are expressed under each condition is still a challenge due to the large number of trypsin-coding genes in the Culicidae family and most likely because they are low abundance proteins.We used zymography for the biochemical characterization of the peptidase profile of the midgut from C. quinquefasciatus females fed on sugar. Protein samples were also submitted to SDS-PAGE followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis for peptidase identification. The peptidases sequences were analyzed with bioinformatics tools to assess their distinct features.Zymography revealed that trypsin-like serine peptidases were responsible for the proteolytic activity in the midgut of females fed on sugar diet. After denaturation in SDS-PAGE, eight trypsin-like serine peptidases were identified by LC-MS/MS. These peptidases have structural features typical of invertebrate digestive trypsin peptidases but exhibited singularities at the protein sequence level such as: the presence of different amino acids at the autocatalytic motif and substrate binding regions as well as different number of disulfide bounds. Data mining revealed a group of trypsin-like serine peptidases that are specific to C. quinquefasciatus when compared to the culicids genomes sequenced so far.We demonstrated that proteomics approaches combined with bioinformatics tools and zymographic analysis can lead to the functional annotation of trypsin-like serine peptidases coding genes and aid in the understanding of the complexity of peptidase expression in mosquitoes.

SUBMITTER: Borges-Veloso A

PROVIDER: S-EPMC4502911 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

In-depth characterization of trypsin-like serine peptidases in the midgut of the sugar fed Culex quinquefasciatus.

Borges-Veloso André A Saboia-Vahia Leonardo L Dias-Lopes Geovane G Domont Gilberto B GB Britto Constança C Cuervo Patricia P De Jesus Jose B JB

Parasites & vectors 20150716

<h4>Background</h4>Culex quinquefasciatus is a hematophagous insect from the Culicidae family that feeds on the blood of humans, dogs, birds and livestock. This species transmits a wide variety of pathogens between humans and animals. The midgut environment is the first location of pathogen-vector interactions for blood-feeding mosquitoes and the expression of specific peptidases in the early stages of feeding could influence the outcome of the infection. Trypsin-like serine peptidases belong to ...[more]

PMID: 26174750

Similar Datasets

Project description:The mosquito Culex quinquefasciatus is a ubiquitous species that serves as a major vector for west nile virus and lymphatic filariasis. Ingestion of bloodmeal by females triggers a series of physiological processes in the midgut and also exposes them to infection by these pathogens. The bacteria normally harbored in the midgut are known to influence physiology and can also alter the response to various pathogens. The midgut bacteria in female Cx. quinquefasciatus mosquitoes collected over a large geographical area from India was studied. Examination of 16S ribosomal DNA amplicons from culturable microflora revealed the presence of 83 bacterial species belonging to 31 bacterial genera. All of these species belong to three phyla i.e. Proteobacteria, Firmicutes and Actinobacteria. Phylum Proteobacteria was the most dominant phylum (37 species), followed by Firmicutes (33 species) and Actinobacteria (13 species). Phylum Proteobacteria, was dominated by members of γ-proteobacteria class. The genus Staphylococcus was the largest genus represented by 11 species whereas Enterobacter was the most prevalent genus and recovered from all the field stations except Leh. Highest bacterial prevalence was observed from Bhuj (22 species) followed by Nagrota (18 species), Masimpur (18 species) and Hathigarh (16 species). Whereas, least species were observed from Leh (8 species). It has been observed that individual mosquito harbor extremely diverse gut bacteria and have very small overlap bacterial taxa in their gut. This variation in midgut microbiota may be one of the factors responsible for variation in disease transmission rates or vector competence within mosquito population. The present data strongly encourage further investigations to verify the potential role of the detected bacteria in mosquito for the transmission of lymphatic filariasis and west nile virus. To the best of our knowledge this is the first study on midgut microbiota of wild Cx. quinquefasciatus from over a large geographical area.

Project description:Culex quinquefasciatus is a cosmopolitan species widely distributed in the tropical and subtropical areas of the world. Due to its long history of close association with humans, the transmission of arboviruses and parasites have an important role in veterinary and public health. Adult females feed mainly on birds although they can also feed on humans and other mammals. On the other hand, larvae are able to feed on a great diversity of microorganisms, including microalgae, present in natural or artificial breeding sites with a high organic load. These two particularities, mentioned above, are some of the reasons why this mosquito is so successful in the environment. In this work, we report the identification of a microalga found during field sampling in artificial breeding sites, in a group of discarded tires with accumulated rainwater. Surprisingly, only one of them had a bright green culture without mosquito larvae while the other surrounding tires contained a large number of mosquito larvae. We isolated and identified this microorganism as Neochloris aquatica, and it was evaluated as a potential biological control agent against Cx. quinquefasciatus. The oviposition site preference in the presence of the alga by gravid females, and the effects on larval development were analyzed. Additionally, microalga effect on Cx. quinquefasciatus wild type, naturally infected with the endosymbiotic bacterium Wolbachia (w+) and Wolbachia free (w-) laboratory lines was explored. According to our results, even though it is chosen by gravid females to lay their eggs, the microalga had a negative effect on the development of larvae from both populations. Additionally, when the larvae were fed with a culture of alga supplemented with balanced fish food used as control diet, they were not able to reverse its effect, and were unable to complete development until adulthood. Here, N. aquatica is described as a biological agent, and as a potential source of bioactive compounds for the control of mosquito populations important in veterinary and human health.

Project description:BackgroundIn vector-borne diseases such as leishmaniasis, the sand fly midgut is considered to be an important site for vector-parasite interaction. Digestive enzymes including serine peptidases such as trypsin and chymotrypsin, which are secreted in the midgut are one of the obstacles for Leishmania in establishing a successful infection. The presence of some natural inhibitors of serine peptidases (ISPs) has recently been reported in Leishmania. In the present study, we deciphered the role of these ISPs in the survival of Leishmania donovani in the hostile sand fly midgut environment.MethodsIn silico and co-immunoprecipitation studies were performed to observe the interaction of L. donovani ISPs with trypsin and chymotrypsin. Zymography and in vitro enzyme assays were carried out to observe the inhibitory effect of purified recombinant ISPs of L. donovani (rLdISPs) on trypsin, chymotrypsin and the sand fly midgut peptidases. The expression of ISPs in the amastigote to promastigote transition stages were studied by semi-quantitative RT-PCR and Western blot. The role of LdISP on the survival of ISP overexpressed (OE) and ISP knocked down (KD) Leishmania parasites inside the sand fly gut was investigated by in vitro and in vivo cell viability assays.ResultsWe identified two ecotin-like genes in L. donovani, LdISP1 and LdISP2. In silico and co-immunoprecipitation results clearly suggest a strong interaction of LdISP molecules with trypsin and chymotrypsin. Zymography and in vitro enzyme assay confirmed the inhibitory effect of rLdISP on trypsin, chymotrypsin and the sand fly midgut peptidases. The expression of LdISP2 was found to be strongly associated with the amastigote to promastigote phase transition. The activities of the digestive enzymes were found to be significantly reduced in the infected sand flies when compared to uninfected. To our knowledge, our study is the first report showing the possible reduction of chymotrypsin activity in L. donovani infected sand flies compared to uninfected. Interestingly, during the early transition stage, substantial killing was observed in ISP2 knocked down (ISP2KD) parasites compared to wild type (WT), whereas ISP1 knocked down (ISP1KD) parasites remained viable. Therefore, our study clearly indicates that LdISP2 is a more effective inhibitor of serine peptidases than LdISP1.ConclusionOur results suggest that the lack of ISP2 is detrimental to the parasites during the early transition from amastigotes to promastigotes. Moreover, the results of the present study demonstrated for the first time that LdISP2 has an important role in the inhibition of peptidases and promoting L. donovani survival inside the Phlebotomus argentipes midgut.

Project description:Simple Summary Mosquito midgut microbiota has become an interesting field in mosquito vector biology, as it has been shown to form an integral part of the mosquito life history. But less is known about seasonal variation of midgut bacterial diversity of Culex quinquefasciatus. Our results illustrate that the Bacteroidetes (Bacterial Phyla) communities have been well observed in autumn and winter seasons, suggesting that this might participate in the nutritional supply of adult mosquitoes when temperatures drop. This discovery provides a new perspective for the control of Cx. quinquefasciatus to reduce the transmission of diseases. There is much sufficiently practical significance to reduce the density of Cx. quinquefasciatus in autumn and winter when their activities are weakened, which is of absolute benefit to human beings and the natural environment. Abstract Culex quinquefasciatus, one of the most significant mosquito vectors in the world, is widespread in most parts of southern China. A variety of diseases including Bancroft’s filariasis, West Nile disease, and St. Louis encephalitis could be transmitted by the vector. Mosquitoes have been shown to host diverse bacterial communities that vary depending on environmental factors such as temperature and rainfall. In this work, 16S rDNA sequencing was used to analyze the seasonal variation of midgut bacterial diversity of Cx. Quinquefasciatus in Haikou City, Hainan Province, China. Proteobacteria was the dominant phylum, accounting for 79.7% (autumn), 73% (winter), 80.4% (spring), and 84.5% (summer). The abundance of Bacteroidetes in autumn and winter was higher than in others. Interestingly, Epsilonbacteraeota, which only exists in autumn and winter, was discovered accidentally in the midgut. We speculated that this might participate in the nutritional supply of adult mosquitoes when temperatures drop. Wolbachia is the most abundant in autumn, accounting for 31.6% of bacteria. The content of Pantoea was highest in the summer group, which might be related to the enhancement of the ability of mosquitoes as temperatures increased. Pseudomonas is carried out as the highest level in winter. On the contrary, in spring and summer, the genus in highest abundance is Enterobacter. Acinetobacter enriches in the spring when it turns from cold to hot. By studying the diversity of midgut bacteria of Cx. quinquefasciatus, we can further understand the co-evolution of mosquitoes and their symbiotic microbes. This is necessary to discuss the seasonal variation of microorganisms and ultimately provide a new perspective for the control of Cx. quinquefasciatus to reduce the spread of the diseases which have notably vital practical significance for the effective prevention of Cx. quinquefasciatus.

Dataset Information

In-depth characterization of trypsin-like serine peptidases in the midgut of the sugar fed Culex quinquefasciatus.

Publications

In-depth characterization of trypsin-like serine peptidases in the midgut of the sugar fed Culex quinquefasciatus.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets