RESUMO
Since lectins have been proposed to function as non-self recognizing molecules, it is important to access the specificity and diversity of lectin molecules that play an important role as pattern recognition receptors (PRRs). Like vertebrates, several lectins containing fibrinogen-like (FBG) domains have been reported as PRRs from different invertebrate species, however, in case of mosquito, very little information is available. In this study, we report identification and characterization of a novel family of fibrinogen-related proteins which exhibits (43-68%) similarities to that of A. gambiae FREPs, isolated from the immune challenged cell line of mosquito Aedes albopictus. Analysis of the longest (790bp) cDNA (AF-1) appears to encode a 263 deduced amino acid sequence that contains a C-terminal fibrinogen-like domain. Amino acid sequence similarity and phylogenetic analysis showed a high degree of conservation with other known insect fibrinogen-related proteins, suggesting that mosquito A. albopictus FREPs may play a similar role in an innate immunity as pattern recognition receptor molecules; however, further characterization is needed to understand their transcriptional regulation and behavior during microbial infection.
Assuntos
Aedes/genética , Proteínas de Insetos/genética , Lectinas/genética , Filogenia , Aedes/citologia , Aedes/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , DNA Complementar/química , DNA Complementar/genética , Fibrinogênio/genética , Proteínas de Insetos/classificação , Proteínas de Insetos/imunologia , Lectinas/imunologia , Dados de Sequência Molecular , Pseudomonas aeruginosa/imunologia , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
Manipulating the endogenous immune responses of the mosquito such as temporal and spatial expression of antimicrobial peptides may help in the development of a refractory mosquito, unable to transmit malaria. In mosquito several small antimicrobial peptides are activated locally in the midgut and salivary glands upon Plasmodium infection. Anopheles stephensi, the major urban malaria vector in India, has been considered as an important insect model to study vector-parasite interactions; however, so far no reports are available on the antimicrobial peptides from this mosquito species. In the present study, we report identification and molecular characterization of a novel cDNA encoding defensin like peptide, isolated from the salivary gland subtractive hybridization cDNA library of mosquito A. stephensi. Defensin cDNA is 396 base pair long, bearing an open reading frame of 96 amino acids. Deduced amino acid sequence of A. stephensi defensin (Astp_def) contains a signal peptide sequence of 24 amino acids followed by 32-amino acids long putative propeptide domain and a 40-amino acid mature peptide domain carrying 23-amino acid long consensuses sequence signature of insect defensin. Mature peptide of Astp_def carries six conserved cysteine residues, with a predicted molecular weight of 4.20kDa, and isoelectric point of 8.30, characteristic features of cationic defensins. Amino acid sequence similarity and phylogenetic analysis indicated a higher variation in the pre-propeptide region, as compared to the mature defensin peptide, assuring the presence of finely tuned immune responses to counter pathogens.
Assuntos
Anopheles/genética , DNA Complementar/isolamento & purificação , Defensinas/genética , Glândulas Salivares/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA Complementar/genética , Defensinas/química , Defensinas/imunologia , Índia , Ponto Isoelétrico , Dados de Sequência Molecular , Peso Molecular , Fases de Leitura Aberta , Filogenia , Sinais Direcionadores de Proteínas , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
The coding sequence for hexokinase enzyme was cloned from Leishmania major. The sequence was found to encode an enzyme with a molecular mass of 51.74 kDa. Amino acid sequence showed maximum homology with known trypanosome and plant hexokinases. It has a calculated isoelectric point of 8.46 and contains an N-terminal peroxisome-targeting signal, the characteristics frequently associated with glycosomal proteins. The sequence indicated the presence of conserved amino acid residues and motifs that are present in plant and mammalian hexokinases; these are apparently involved in the binding of different substrates. The L. major genome was found to have 2 copies of hexokinase coding sequences in tandem with an intergenic spacer of 2.58 kb. Both the genes in the hexokinase locus were transcribed as individual transcripts in a monocistronic form, having the same size as seen by Northern blot analysis. The hexokinase gene was transcribed in large amounts in the promastigote stage, whereas there is only weak expression in the amastigote stage as determined by RT-PCR analysis. Sequencing of hexokinase loci from different Leishmania species (e.g., L. donovani, L. infantum, L. tropica, and L. mexicana) revealed that the hexokinase locus is highly conserved at the DNA and protein levels among species of Leishmania compared with trypanosomes.
Assuntos
Hexoquinase/genética , Leishmania major/genética , Sequência de Aminoácidos , Animais , Northern Blotting , Southern Blotting , Clonagem Molecular , Sequência Conservada , DNA Intergênico , Eletroforese em Gel de Campo Pulsado , Hexoquinase/química , Leishmania major/classificação , Leishmania major/enzimologia , Dados de Sequência Molecular , Filogenia , RNA de Protozoário/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de AminoácidosRESUMO
We report identification of putative innate immune related genes from a cell line of the mosquito Aedes albopictus challenged with heat-killed bacteria. Using a subtractive hybridization and sequencing approach, we analyzed a total 309 expressed sequence tags (ESTs) which clustered in 40 contigs. Thirty-five percent of genes yielded homology to known immune genes corresponding to antimicrobial peptides (AMPs), pathogen-associated molecular patterns, protease and immune signaling cascades. Interestingly, most of the genes have not been previously described from this mosquito and thus represent a class of novel immune genes. Further, 25% sequences did not match to any known species in the non-redundant databases, appear to be specific to the mosquito A. albopictus and merit further study.
Assuntos
Aedes/imunologia , Perfilação da Expressão Gênica , Imunidade Inata/genética , Pseudomonas aeruginosa/imunologia , Aedes/citologia , Aedes/genética , Sequência de Aminoácidos , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Proteínas de Transporte/genética , Linhagem Celular , Análise por Conglomerados , Biologia Computacional , DNA Complementar/genética , Defensinas/genética , Fibrinogênio/genética , Imunidade Inata/imunologia , Proteínas de Insetos/genética , Dados de Sequência Molecular , Filogenia , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação , Receptores de Reconhecimento de Padrão/genética , Alinhamento de Sequência , Serina Proteases/genética , Serpinas/genética , Regulação para Cima/genética , alfa-Macroglobulinas/genéticaRESUMO
BACKGROUND: Understanding the tissue-specific molecular cross-talk mechanism during the mosquito-parasite interaction is of prime importance in the design of new strategies for malaria control. Because mosquito salivary glands are the final destination for the parasite maturation and transmission of vector-borne diseases, identification and characterization of salivary genes and their products are equally important in order to access their effect on the infectivity of the parasite. During the last five years there have been several studies on the sialomes of Anopheles mosquitoes, however very limited information is available on the changes in the salivary gland transcriptome in the presence of Plasmodium, and this information is limited to the mosquito Anopheles gambiae. METHODS: In this study we aimed to explore and identify parasite-induced transcripts from the salivary glands of Anopheles stephensi, using a subtractive hybridization protocol. RESULTS: Ninety-four percent of expressed sequence tags (ESTs) showed close homology to previously known families of mosquito salivary gland secretary proteins, representing the induced expression of alternative splicing and/or additional new members of the protein family. The remaining 6% of ESTs did not yield significant homology to any known proteins in the non-redundant database and thus may represent a class of unknown/novel salivary proteins. Primary analysis of the ESTs also revealed identification of several novel immune-related transcripts, including defensin and cecropins, probably involved in counter-activation of the antagonistic defense system. A comprehensive description of each family of proteins has been discussed in relation to the tissue-specific mosquito-parasite interaction. CONCLUSION: This is the first report on the identification of new putative salivary genes, presumably activated during parasite infection.