Effects of dietary Bacillus subtilis and Shewanella algae in expression profile of immune-related genes from hemolymph of Litopenaeus vannamei challenged with Vibrio parahaemolyticus.

B. subtilis and S. algae effects in growth, survival and innate immunity were assessed on L. vannamei juveniles. During 60 days, shrimp were reared in three treatments: Bs, fed with 106 CFU of B. subtilis per gram of commercial feed, Sa, fed with 106 CFU of S. algae per gram of commercial feed and Control (without bacterial addition). Then, the animals were subjected to a V. parahaemolyticus challenge. For this purpose, four treatments were established: Control (shrimp not submitted to probiotic treatments), Vibrio (Vibrio challenged shrimp), Vibrio + Bs (Bs challenged shrimp) and Vibrio + Sa (Sa challenged shrimp). Shrimp hemolymph was sampled 45-days after rearing and 24 h post-challenge for quantification of prophenoloxidase (proPO), lipopolysaccharide and ß-1,3-glucan-binding protein (LGBP) and hemocyanin (HEM) transcripts by qPCR. Moreover, shrimp final weight and survival were also verified. B. subtilis administration enhanced shrimp growth and improved proPO, LGBP and HEM expression levels before and after challenge. After 60-days of feeding, Sa final weight was higher than the Control, whereas Vibrio + Sa cumulative mortality after 48 h of Vibrio challenge was lower than Vibrio group. These results could be correlated with the proPO and LGBP up regulation in Vibrio + Sa compared to Vibrio group, protecting L. vannamei from the bacterial infection. Together, these results suggest the probiotic potential of B. subtilis e S. algae in the modulation of immune-related genes as a tool to control V. parahaemolyticus infection inside shrimp.

A new antimicrobial protein from the anterior midgut of Triatoma infestans mediates Trypanosoma cruzi establishment by controlling the microbiota.

The Reduviid Triatoma infestans is a vector for the protozoan Trypanosoma cruzi, the etiological agent of Chagas disease. The parasite must address the defense molecules and microbiota that colonize the anterior midgut of T. infestans. To obtain insight into T. cruzi - microbiota interactions in triatomine insects, we characterized a new antimicrobial product from the anterior midgut of T. infestans (TiAP) that may be involved in these relationships. The TiAP DNA fragment was cloned and expressed in a bacterial system, and the effect of the protein on bacteria and T. cruzi was evaluated by RNAi, qPCR and antimicrobial experiments. The number of T. cruzi in T. infestans anterior midguts was significantly lower in TiAP knockdown insects than in unsilenced groups. We also verified that the amount of bacteria in silenced T. infestans is approximately 600-fold higher than in unsilenced insects by qPCR. The 327-bp cDNA fragment that encodes mature TiAP was cloned into the pET-14b vector and expressed fused to a His-tag in Escherichia coli C43. The recombinant protein (rTiAP) was purified using an Ni-NTA column, followed by a HiTrap SP column. According to a trypanocidal assay, rTiAP did not interfere with the viability of T. cruzi trypomastigotes. Moreover, in antimicrobial experiments using E. coli and Micrococcus luteus, the protein was only bacteriostatic for Gram-negative bacteria. The data indicate that infection by T. cruzi increases the expression of TiAP to modulate the microbiota. The inhibition of microbiota growth by TiAP is important for parasite establishment in the T. infestans anterior midgut.

A Kazal-type inhibitor is modulated by Trypanosoma cruzi to control microbiota inside the anterior midgut of Rhodnius prolixus.

The triatomine insect, Rhodnius prolixus, is a vector of Trypanosoma cruzi, a protozoan parasite that causes Chagas disease. The parasite must overcome immune response and microbiota to develop inside the midgut of triatomines. In this study, we expressed, purified and characterized a Kazal-type inhibitor from the midgut of R. prolixus, named RpTI, which may be involved in microbiota - T. cruzi interactions. The qPCR showed that the RpTI transcript was primarily expressed in tissues from the intestinal tract and that it was upregulated in the anterior midgut after T. cruzi infection. A 315-bp cDNA fragment encoding the mature protein was cloned into the pPIC9 vector and expressed in Pichia pastoris system. Recombinant RpTI (rRpTI) was purified on a trypsin-Sepharose column and had a molecular mass of 11.5 kDa as determined by SDS-PAGE analysis. This protein inhibited trypsin (Ki = 0.42 nM), whereas serine proteases from the coagulation cascade were not inhibited. Moreover, trypanocidal assays revealed that rRpTI did not interfere in the viability of T. cruzi trypomastigotes. The RpTI transcript was also knocked down by RNA interference prior to infection of R. prolixus with T. cruzi. The amount of T. cruzi in the anterior midgut was significantly lower in RpTI knockdown insects compared to the non-silenced groups. We also verified that the bacterial load is higher in the anterior midgut of silenced and infected R. prolixus compared to non-silenced and infected insects. Our results suggest that T. cruzi infection increases the expression of RpTI to mediate microbiota modulation and is important for parasite immediately after infection with R. prolixus.

RmKK, a tissue kallikrein inhibitor from Rhipicephalus microplus eggs.

Rhipicephalus microplus is an important ectoparasite that is responsible for transmission of anaplasmosis and babesiosis to cattle. Tissue kallikrein inhibitors might play an important role in R. microplus eggs. In the present work, we purified and characterized, a tissue kallikrein inhibitor presents in R. microplus eggs (RmKK), a protein which contains two Kunitz domain in tandem. Purified inhibitor was confirmed by amino terminal determination and its dissociation constant (Ki) for bovine trypsin and porcine pancreatic kallikrein were 0.6 nM and 91.5 nM, respectively. Using a cDNA library from R. microplus midgut, we cloned the cDNA fragment encoding mature RmKK and expressed the protein in Pichia pastoris system. Recombinant RmKK was purified by ion exchange chromatography and presented molecular mass of 16.3 kDa by MALDI-TOF analysis. Moreover, RmKK showed a tight binding inhibition for serine proteases as bovine trypsin (Ki=0.2 nM) and porcine pancreatic kallikrein (PPK) (Ki=300 nM). We performed, for the first time, the characterization of a tissue kallikrein inhibitor presents in R. microplus eggs, which the transcript is produced in the adult female gut. BmKK seems to be the strongest PPK inhibitor among all BmTIs present in the eggs and larvae (Andreotti et al., 2001; Sasaki et al., 2004). This data suggests that BmKK may participate in the development of tick egg and larvae phase.

Differential expression profiles in the midgut of Triatoma infestans infected with Trypanosoma cruzi.

Chagas disease, or American trypanosomiasis, is a parasitic disease caused by the protozoan Trypanosoma cruzi and is transmitted by insects from the Triatominae subfamily. To identify components involved in the protozoan-vector relationship, we constructed and analyzed cDNA libraries from RNA isolated from the midguts of uninfected and T. cruzi-infected Triatoma infestans, which are major vectors of Chagas disease. We generated approximately 440 high-quality Expressed Sequence Tags (ESTs) from each T. infestans midgut cDNA library. The sequences were grouped in 380 clusters, representing an average length of 664.78 base pairs (bp). Many clusters were not classified functionally, representing unknown transcripts. Several transcripts involved in different processes (e.g., detoxification) showed differential expression in response to T. cruzi infection. Lysozyme, cathepsin D, a nitrophorin-like protein and a putative 14 kDa protein were significantly upregulated upon infection, whereas thioredoxin reductase was downregulated. In addition, we identified several transcripts related to metabolic processes or immunity with unchanged expressions, including infestin, lipocalins and defensins. We also detected ESTs encoding juvenile hormone binding protein (JHBP), which seems to be involved in insect development and could be a target in control strategies for the vector. This work demonstrates differential gene expression upon T. cruzi infection in the midgut of T. infestans. These data expand the current knowledge regarding vector-parasite interactions for Chagas disease.

Tigutcystatin, a cysteine protease inhibitor from Triatoma infestans midgut expressed in response to Trypanosoma cruzi.

The insect Triatoma infestans is a vector of Trypanosoma cruzi, the etiological agent of Chagas disease. A cDNA library was constructed from T. infestans anterior midgut, and 244 clones were sequenced. Among the EST sequences, an open reading frame (ORF) with homology to a cystatin type 2 precursor was identified. Then, a 288-bp cDNA fragment encoding mature cystatin (lacking signal peptide) named Tigutcystatin was cloned fused to a N-terminal His tag in pET-14b vector, and the protein expressed in Escherichia coli strain Rosetta gami. Tigutcystatin purified and cleaved by thrombin to remove His tag presented molecular mass of 11 kDa and 10,137 Da by SDS-PAGE and MALDI-TOF mass spectrometry, respectively. Purified Tigutcystatin was shown to be a tight inhibitor towards cruzain, a T. cruzi cathepsin L-like enzyme (K(i)=3.29 nM) and human cathepsin L (K(i)=3.78 nM). Tissue specific expression analysis showed that Tigutcystatin was mostly expressed in anterior midgut, although amplification in small intestine was also detected by semi quantitative RT-PCR. qReal time PCR confirmed that Tigutcystatin mRNA is significantly up-regulated in anterior midgut when T. infestans is infected with T. cruzi. Together, these results indicate that Tigutcystatin may be involved in modulation of T. cruzi in intestinal tract by inhibiting parasite cysteine proteases, which represent the virulence factors of this protozoan.

The first pacifastin elastase inhibitor characterized from a blood sucking animal.

Pacifastin-like protease inhibitors belong to a recent classified protease inhibitor family and they are the smallest protease inhibitors described in animals. In this work, we purified and characterized, for the first time, two neutrophil elastase inhibitors belonging to the pacifastin family from the blood sucking insect Triatoma infestans eggs. The inhibitors showed the same N-terminal sequences, molecular masses of 4257 and 4024Da by MALDI-TOF mass spectrometry and dissociation constants (Ki) for neutrophil elastase of 0.52 and 0.29nM, respectively. Using a fat body cDNA library, we cloned a pacifastin precursor containing two protease inhibitor domains similar to locust pacifastins. The first pacifastin domain translated to T. infestans purified protein, named TIPI1. Recombinant TIPI1 expressed in Pichia pastoris system showed similar inhibitory activities compared to the native inhibitor. Its precursor, called TiPP1, is mainly expressed in fat body, and it is up-regulated after blood feeding. The immune challenges of 1(a) instar T. infestans nymph with bacteria or dsRNA strongly stimulated TiPP1 expression in fat body, suggesting a possible role of TiPP1 in T. infestans immunity. This work is the first to characterize a blood feeding insect pacifastin inhibitor.