RESUMO
BACKGROUND: Rhodnius prolixus is a major vector of Trypanosoma cruzi, the causative agent of Chagas disease in Latin America. In natural habitats, these insects are in contact with a variety of bacteria, fungi, virus and parasites that they acquire from both their environments and the blood of their hosts. Microorganism ingestion may trigger the synthesis of humoral immune factors, including antimicrobial peptides (AMPs). The objective of this study was to compare the expression levels of AMPs (defensins and prolixicin) in the different midgut compartments and the fat body of R. prolixus infected with different T. cruzi strains. The T. cruzi Dm 28c clone (TcI) successfully develops whereas Y strain (TcII) does not complete its life- cycle in R. prolixus. The relative AMP gene expressions were evaluated in the insect midgut and fat body infected on different days with the T. cruzi Dm 28c clone and the Y strain. The influence of the antibacterial activity on the intestinal microbiota was taken into account. METHODS: The presence of T. cruzi in the midgut of R. prolixus was analysed by optical microscope. The relative expression of the antimicrobial peptides encoding genes defensin (defA, defB, defC) and prolixicin (prol) was quantified by RT-qPCR. The antimicrobial activity of the AMPs against Staphylococcus aureus, Escherichia coli and Serratia marcescens were evaluated in vitro using turbidimetric tests with haemolymph, anterior and posterior midgut samples. Midgut bacteria were quantified using colony forming unit (CFU) assays and real time quantitative polymerase chain reaction (RT-qPCR). RESULTS: Our results showed that the infection of R. prolixus by the two different T. cruzi strains exhibited different temporal AMP induction profiles in the anterior and posterior midgut. Insects infected with T. cruzi Dm 28c exhibited an increase in defC and prol transcripts and a simultaneous reduction in the midgut cultivable bacteria population, Serratia marcescens and Rhodococcus rhodnii. In contrast, the T. cruzi Y strain neither induced AMP gene expression in the gut nor reduced the number of colony formation units in the anterior midgut. Beside the induction of a local immune response in the midgut after feeding R. prolixus with T. cruzi, a simultaneous systemic response was also detected in the fat body. CONCLUSIONS: R. prolixus AMP gene expressions and the cultivable midgut bacterial microbiota were modulated in distinct patterns, which depend on the T. cruzi genotype used for infection.
Assuntos
Peptídeos Catiônicos Antimicrobianos/biossíntese , Corpo Adiposo/imunologia , Expressão Gênica , Insetos Vetores , Rhodnius/imunologia , Trypanosoma cruzi/imunologia , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/farmacologia , Contagem de Colônia Microbiana , Escherichia coli/efeitos dos fármacos , Corpo Adiposo/parasitologia , Trato Gastrointestinal/imunologia , Trato Gastrointestinal/microbiologia , Trato Gastrointestinal/parasitologia , Perfilação da Expressão Gênica , Microscopia , Reação em Cadeia da Polimerase em Tempo Real , Rhodnius/genética , Rhodnius/parasitologia , Serratia marcescens/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Trypanosoma cruzi/efeitos dos fármacosRESUMO
The Rhipicephalus microplus tick is responsible for losses in the livestock production estimated in 2 billions USD. Despite its economical importance the knowledge in tick's physiology is sparse. In order to contribute to this scenario we describe the characterization of a cysteine proteinase inhibitor named Rmcystatin-3. Purified recombinant Rmcystatin-3 was able to inhibit cathepsin L (Ki = 2.5 nM), BmCl1 (Ki = 1.8 nM) and cathepsin B (Ki = 136 nM). Western blot and quantitative PCR analysis revealed the presence of Rmcystatin-3 in fat body, salivary gland but mainly in hemocytes. The mRNA levels of Rmcystatin-3 during bacterial challenge are drastically down-regulated. In order to define the Rmcystatin-3 possible role in tick immunity, the cystatin gene was knockdown by RNA interference with and without Escherichia coli infection. Our results showed that the Rmcystatin-3 silenced group was more immune competent to control bacterial infection than the group injected with non-related dsRNA. Taking together, our data strongly suggested an important role of Rmcystatin-3 in tick immunity.
Assuntos
Inibidores de Cisteína Proteinase/imunologia , Resistência à Doença/imunologia , Hemócitos/imunologia , Rhipicephalus/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Catepsina B/antagonistas & inibidores , Catepsina B/metabolismo , Catepsina L/antagonistas & inibidores , Catepsina L/metabolismo , Inibidores de Cisteína Proteinase/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Resistência à Doença/genética , Escherichia coli/imunologia , Escherichia coli/fisiologia , Corpo Adiposo/imunologia , Corpo Adiposo/metabolismo , Expressão Gênica/imunologia , Hemócitos/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Dados de Sequência Molecular , Interferência de RNA/imunologia , Proteínas Recombinantes/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rhipicephalus/genética , Rhipicephalus/microbiologia , Glândulas Salivares/imunologia , Glândulas Salivares/metabolismo , Homologia de Sequência de AminoácidosRESUMO
The innate immune response of insects is one of the factors that may dictate their susceptibility to viral infection. Two immune signaling pathways, Toll and JAK-STAT, and the RNA interference (RNAi) pathway are involved in Aedes aegypti responses against dengue virus (DENV), however natural differences in these antiviral defenses among mosquito populations have not been studied. Here, two field Ae. aegypti populations from distinct ecological environments, one from Recife and the other from Petrolina (Brazil), and a laboratory strain were studied for their ability to replicate a primary isolate of dengue virus serotype 2 (DENV-2). Virus infectivity and replication were determined in insect tissues collected after viral exposure through reverse-transcription real time PCR (RT-PCR). The expression of a transcript representing these defense mechanisms (Toll, JAK-STAT and RNAi) in the midgut and fat body was studied with RT-PCR to evaluate variations in innate immune mechanisms possibly employed against DENV. Analyses of infection rates indicated that the field populations were more susceptible to DENV-2 infection than the lab strain. There were distinct expression patterns among mosquito populations, in both control and infected insects. Moreover, lower expression of immune molecules in DENV-2-infected insects compared to controls was observed in the two field populations. These results suggest that natural variations in vector competence against DENV may be partly due to differences in mosquito defense mechanisms, and that the down-regulation of immune transcripts after viral infection depends on the insect strain.