Rhodnius prolixus interaction with Trypanosoma rangeli: modulation of the immune system and microbiota population.

BACKGROUND: Trypanosoma rangeli is a protozoan that infects a variety of mammalian hosts, including humans. Its main insect vector is Rhodnius prolixus and is found in several Latin American countries. The R. prolixus vector competence depends on the T. rangeli strain and the molecular interactions, as well as the insect's immune responses in the gut and haemocoel. This work focuses on the modulation of the humoral immune responses of the midgut of R. prolixus infected with T. rangeli Macias strain, considering the influence of the parasite on the intestinal microbiota. METHODS: The population density of T. rangeli Macias strain was analysed in different R. prolixus midgut compartments in long and short-term experiments. Cultivable and non-cultivable midgut bacteria were investigated by colony forming unit (CFU) assays and by 454 pyrosequencing of the 16S rRNA gene, respectively. The modulation of R. prolixus immune responses was studied by analysis of the antimicrobial activity in vitro against different bacteria using turbidimetric tests, the abundance of mRNAs encoding antimicrobial peptides (AMPs) defensin (DefA, DefB, DefC), prolixicin (Prol) and lysozymes (LysA, LysB) by RT-PCR and analysis of the phenoloxidase (PO) activity. RESULTS: Our results showed that T. rangeli successfully colonized R. prolixus midgut altering the microbiota population and the immune responses as follows: 1 - reduced cultivable midgut bacteria; 2 - decreased the number of sequences of the Enterococcaceae but increased those of the Burkholderiaceae family; the families Nocardiaceae, Enterobacteriaceae and Mycobacteriaceae encountered in control and infected insects remained the same; 3 - enhanced midgut antibacterial activities against Serratia marcescens and Staphylococcus aureus; 4 - down-regulated LysB and Prol mRNA levels; altered DefB, DefC and LysA depending on the infection (short and long-term); 5 - decreased PO activity. CONCLUSION: Our findings suggest that T. rangeli Macias strain modulates R. prolixus immune system and modifies the natural microbiota composition.

Humoral responses in Rhodnius prolixus: bacterial feeding induces differential patterns of antibacterial activity and enhances mRNA levels of antimicrobial peptides in the midgut.

BACKGROUND: The triatomine, Rhodnius prolixus, is a major vector of Trypanosoma cruzi, the causative agent of Chagas disease in Latin America. It has a strictly blood-sucking habit in all life stages, ingesting large amounts of blood from vertebrate hosts from which it can acquire pathogenic microorganisms. In this context, the production of antimicrobial peptides (AMPs) in the midgut of the insect is vital to control possible infection, and to maintain the microbiota already present in the digestive tract. METHODS: In the present work, we studied the antimicrobial activity of the Rhodnius prolixus midgut in vitro against the Gram-negative and Gram-positive bacteria Escherichia coli and Staphylococcus aureus, respectively. We also analysed the abundance of mRNAs encoding for defensins, prolixicin and lysozymes in the midgut of insects orally infected by these bacteria at 1 and 7 days after feeding. RESULTS: Our results showed that the anterior midgut contents contain a higher inducible antibacterial activity than those of the posterior midgut. We observed that the main AMP encoding mRNAs in the anterior midgut, 7 days after a blood meal, were for lysozyme A, B, defensin C and prolixicin while in the posterior midgut lysozyme B and prolixicin transcripts predominated. CONCLUSION: Our findings suggest that R. prolixus modulates AMP gene expression upon ingestion of bacteria with patterns that are distinct and dependent upon the species of bacteria responsible for infection.

TcI/TcII co-infection can enhance Trypanosoma cruzi growth in Rhodnius prolixus.

BACKGROUND: Rhodnius prolixus is an obligate haematophagous insect and one of the most important vectors of Trypanosoma cruzi, the causative agent of Chagas disease in the Americas. T. cruzi is a highly variable parasite which is not transmitted in the same efficiency by the different triatomine vectors. Because different T. cruzi genotypes are aetiopathologically divergent, further elucidation of the transmission abilities of different Chagas disease vectors is extremely important. FINDINGS: In the present study, the growth behaviour of two T. cruzi isolates, MDID/BR/1993/C45 (TcI) and TBRA/BR/1999/JCA3 (TcII), sharing the same microhabitat (intestinal tract) in single and mixed infections, was examined. The distribution patterns and parasite population densities were evaluated at 7, 14 and 21 days after feeding (daf) by quantification of parasites using Neubauer haemocytometric measurements and mini-exon PCR to identify TcI and TcII subpopulations. Parasitic colonization in the small intestine was more successful in the mixed infection model than the single infection models at 21 daf. In the rectal lumen and wall, the growth behaviour of the mixed infection was similar to that of the TcI group, although the total parasite number was lower. In the TcII group, no metacyclic trypomastigote forms were found. PCR analysis of the contents of each dissected region showed different genotype fractions in the mixed infection model, in which TcI seemed to be the predominant isolate. CONCLUSION: The different growth behaviour of the TcI and TcII isolates in single and mixed infection models demonstrated that possibly an intraspecific factor modulates parasitic development in the intestine of R. prolixus.

Serine carboxypeptidases of Triatoma brasiliensis (Hemiptera, Reduviidae): Sequence characterization, expression pattern and activity localization.

Using specific oligonucleotides, 5'- and 3'-RACE and sequencing, two cDNAs encoding serine carboxypeptidases (tbscp-1 and tbscp-2) from the midgut of the blood sucking heteropteran Triatoma brasiliensis were identified. Both cDNAs with an open reading frame of 1389bp, encode serine carboxypeptidase precursors of 463 amino acid residues, which possess a signal peptide cleavage site after Ala19. Analysis of tbscp-1 and tbscp-2 genomic DNA showed an absence of introns in both sequences and the presence of a further intron-free SCP encoding gene (tbscp-2b). By reverse transcription polymerase chain reaction (RT-PCR), tbscp-1 and tbscp-2 transcript abundance was found similarly in fifth instar nymphs at different days after feeding (daf), high in the posterior midgut (small intestine), lower in the anterior midgut (stomach) and fat body and almost undetectable in the salivary glands. In the anterior, middle and posterior regions of the small intestine at 5daf the transcript abundance of both genes was almost identical. Also in adult female and male insects at 5daf both genes showed the strongest signal in the posterior midgut. Molecular modeling suggested that TBSCP-1 has carboxypeptidase D activity; activities against Hippuryl-Phenylalanine and Hippuryl-Arginine were also located at the posterior midgut, both were induced after blood feeding. Treatment of the posterior midgut extracts with the serine protease inhibitor PMSF strongly reduced carboxypeptidase activity. These findings suggest that triatomines might use serine carboxypeptidases, which are usually found in lysosomes, as digestive enzymes in the posterior midgut lumen, from which TBSCP-1 and TBSCP-2 are possible candidates to fulfill this function.

Isolation and molecular characterization of a major hemolymph serpin from the triatomine, Panstrongylus megistus.

BACKGROUND: Chagas disease kills 2.5 thousand people per year of 15 million persons infected in Latin America. The disease is caused by the protozoan, Trypanosome cruzi, and vectored by triatomine insects, including Panstrongylus megistus, an important vector in Brazil. Medicines treating Chagas disease have unpleasant side effects and may be ineffective, therefore, alternative control techniques are required. Knowledge of the T. cruzi interactions with the triatomine host needs extending and new targets/strategies for control identified. Serine and cysteine peptidases play vital roles in protozoan life cycles including invasion and entry of T. cruzi into host cells. Peptidase inhibitors are, therefore, promising targets for disease control. METHODS: SDS PAGE and chromatograpy detected and isolated a P. megistus serpin which was peptide sequenced by mass spectrometry. A full amino acid sequence was obtained from the cDNA and compared with other insect serpins. Reverse transcription PCR analysis measured serpin transcripts of P. megistus tissues with and without T. cruzi infection. Serpin homology modeling used the Swiss Model and Swiss-PDB viewer programmes. RESULTS: The P. megistus serpin (PMSRP1) has a ca. 40 kDa molecular mass with 404 amino acid residues. A reactive site loop contains a highly conserved hinge region but, based on sequence alignment, the normal cleavage site for serine proteases at P1-P1' was translocated to the putative position P4'-P5'. A small peptide obtained corresponded to the C-terminal 40 amino acid region. The secondary structure of PMSRP1 indicated nine α-helices and three ß-sheets, similar to other serpins. PMSRP1 transcripts occurred in all tested tissues but were highest in the fat body and hemocytes. Levels of mRNA encoding PMSRP1 were significantly modulated in the hemocytes and stomach by T. cruzi infection indicating a role for PMSRP1 in the parasite interactions with P. megistus. CONCLUSIONS: For the first time, a constitutively expressed serpin has been characterized from the hemolymph of a triatomine. This opens up new research avenues into the roles of serine peptidases in the T. cruzi/P. megistus association. Initial experiments indicate a role for PMSRP1 in T. cruzi interactions with P. megistus and will lead to further functional studies of this molecule.

Cathepsin L of Triatoma brasiliensis (Reduviidae, Triatominae): sequence characterization, expression pattern and zymography.

Triatoma brasiliensis is considered one of the main vectors of Chagas disease commonly found in semi-arid areas of northeastern Brazil. These insects use proteases, such as carboxypeptidase B, aminopeptidases and different cathepsins for blood digestion. In the present study, two genes encoding cathepsin L from the midgut of T. brasiliensis were identified and characterized. Mature T. brasiliensis cathepsin L-like proteinases (TBCATL-1, TBCATL-2) showed a high level of identity to the cathepsin L-like proteinases of other insects, with highest similarity to Rhodnius prolixus. Both cathepsin L transcripts were highly abundant in the posterior midgut region, the main region of the blood digestion. Determination of the pH in the whole intestine of unfed T. brasiliensis revealed alkaline conditions in the anterior midgut region (stomach) and acidic conditions in the posterior midgut region (small intestine). Gelatine in-gel zymography showed the activity of at least four distinct proteinases in the small intestine and the cysteine proteinase inhibitors transepoxysuccinyl-l-leucylamido-(4-guanidino)butane (E-64) and cathepsin B inhibitor and N-(l-3-trans-propylcarbamoyl-oxirane-2-carbonyl)-l-isoleucyl-l-proline (CA-074) were employed to characterize enzymatic activity. E-64 fully inhibited cysteine proteinase activity, whereas in the samples treated with CA-074 residual proteinase activity was detectable. Thus, proteolytic activity could at least partially be ascribed to cathepsin L. Western blot analysis using specific anti cathepsin L antibodies confirmed the presence of cathepsin L in the lumen of the small intestine of the insects.

Trypanosoma cruzi infection modulates the expression of Triatoma brasiliensis def1 in the midgut.

Antimicrobial peptides are an essential component of the insect immune system. One of the most ubiquitous is defensin, which has been identified in all examined insect orders. Triatoma brasiliensis (Heteroptera, Triatominae), the main Trypanosoma cruzi vector in semi-arid regions of north-eastern Brazil, expresses def1, a defensin encoding gene, predominantly in the anterior region (cardia and stomach) of the midgut. In the present study, we compared the transcript abundance of T. brasiliensis def1 in the anterior (stomach) and posterior midgut (small intestine) regions of naïve bugs with those infected with a familiar T. cruzi isolate. In the stomach, only slight differences between the two insect groups were visible, whereas in the small intestine wide differences (up to 9.6-fold) between infected and noninfected bugs become apparent. The highly increased def1 transcript abundance at 20 days after the infective blood meal might be a response to the T. cruzi infection, suggesting a potential function of intestinal defensin in the parasite population control.

An overview of Chagas disease and the role of triatomines on its distribution in Brazil.

Chagas disease is caused by the parasitic protozoan Trypanosoma cruzi and affects about 15 million people in the Americas. In the Brazilian Amazon Basin, the disease is enzootic, with bugs from the genus Rhodnius serving as the main vectors, while in the northeastern region, mainly the genus Triatoma is naturally infected with T. cruzi. Oral infections appear sporadically in some regions of Brazil. Even though the illness is typically present in Latin America, autochthonous cases have been reported in the United States, mainly in the south of the country. The Triatominae subfamily comprises a large number of insect species that are potential vectors of Chagas disease. Triatomines are hematophagous insects, ingesting blood in all life stages. Since the insects play an important role in parasite selection, they also influence the geographical distribution of T. cruzi. The globalization process is one of the most important elements influencing the outcome of Chagas diseases in Brazil. This article outlines the status of reemerging Chagas disease in different regions of Brazil, the distribution of its vectors, and the consequences of global climate changes with respect to interactions of the pathogen with triatomines.

Two novel defensin-encoding genes of the Chagas disease vector Triatoma brasiliensis (Reduviidae, Triatominae): gene expression and peptide-structure modeling.

Defensins are cysteine-rich peptides involved in the innate immunity of insects and many other organisms. In the present study, two novel defensin-encoding cDNAs and the respective genomic DNAs (def3 and def4) of Triatoma brasiliensis were identified and their tissue-specific and temporal expression was characterized. Both of the deduced mature peptides consisted of 43 amino acid residues and were highly similar to previously identified triatomine defensins (81.4-100%). Semi-quantitative RT-PCR data showed that def3 was constitutively expressed in the fat body and was induced in salivary glands and the small intestine at 5 and 3 days after feeding (daf), respectively. The def4 mRNA level was highly up-regulated in the stomach and fat-body tissues at 5 and 3 daf, respectively. The three-dimensional structures of these defensins were predicted using a homology modeling approach with Def-AAA, the defensin from Anopheles gambiae, as template (62-74% identity). A map of the electrostatic potential of these models revealed that, despite their similar folding patterns, mature Def2 and Def4 have a more cationic structure than is the case for Def1 and Def3. Such differences may orient the antimicrobial profile of these defensins against distinct targets in different organs of the insect.

Development of a Trypanosoma cruzi (TcI) isolate in the digestive tract of an unfamiliar vector, Triatoma brasiliensis (Hemiptera, Reduviidae).

Triatoma brasiliensis is an important vector of Trypanosoma cruzi, commonly found in semi-arid areas of north-eastern Brazil. T. cruzi (TcI) is a widely distributed genotype in all biomes of Brazil. To evaluate selective pressures exerted by a vector species on the development of TcI derived from a different biome (Atlantic Rainforest), T. brasiliensis larvae were infected with the MDID/BR/1994/C48 isolate. Parasite densities of T. cruzi were determined in three regions of the gut at 3, 5 and 10 days after feeding. Percentages of the different stages of the flagellate were identified in Giemsa stained smears. The TcI isolate possessed always significantly higher densities in the rectum than in the small intestine. Epimastigotes reached their highest percentage at 3 days after feeding in the small intestine and trypomastigotes at 10 days after feeding in the rectal wall. Additionally, high metacyclogenesis rates in the T. brasiliensis gut showed competence of this TcI strain to complete its life cycle in this unfamiliar vector species.

Serine proteinases of the human body louse (Pediculus humanus): sequence characterization and expression patterns.

After the previous characterization of one trypsin gene (Try1) of the human body louse Pediculus humanus, genes encoding a second trypsin (Try2) and a chymotrypsin (Chy1) have been cloned using degenerate serine proteinase primers and 5'- and 3'-RACE, and sequenced. The deduced 259 and 267 amino acid sequences of Try2 and Chy1 show an identity of 33%-40% to trypsinogens and chymotrypsinogens of other insects. Considering previously published partial sequences, P. humanus possesses at least one Try1 gene, five variants/isoforms of Try2 and six variants/isoforms of Chy1. The genomic DNA of Try2 contains three introns and Chy1 contains five introns. Using whole mount in situ hybridization, gene expression of Try1, Try2 and Chy1 has been localized not only in the distensible anterior region of the midgut of lice but sometimes also in the area following the distensible region. The Try2 gene was always expressed at much lower levels than Try1 or Chy1. This lower expression, the constitutive expression of Try1 and Chy1 at 1, 2, 6, 12 and 24 h after feeding of adults and the regional differences have been verified in quantitative real-time PCR.

Isolation and characterization of a cDNA encoding for a lysozyme from the gut of the reduviid bug Triatoma infestans.

We have isolated and characterised a Triatoma infestans cDNA encoding a lysozyme. A 174-bp fragment was amplified by PCR using degenerate oligodeoxyribonucleotide primers derived from the known amino acid sequences of lysozyme from other insects. This PCR fragment was used to screen a cDNA gut library of T. infestans. A clone containing the 3'-end of the lysozyme cDNA (219 bp) was isolated and sequenced. RACE was used to amplify the 5'-end of the lysozyme cDNA. After sequencing the complete lysozyme cDNA, the deduced 417 amino acid sequence showed high identity (40-50%) with other chicken-type lysozymes. The amino acid residues responsible for the catalytic activity and the binding of the substrate were essentially conserved. The expression pattern of the lysozyme gene in bugs at different molting and feeding states showed that this gene was upregulated in the digestive tract directly after the molt and after feeding. Additionally, this lysozyme gene was expressed differently in the different regions of the digestive tract, strongly in the cardia and stomach, the anterior regions of the midgut, and only traces of lysozyme mRNA could be detected in the small intestine, the posterior region of the midgut.