BackgroundChagas disease is caused by Trypanosoma cruzi, which is transmitted by triatomine vectors. The northeastern region of Brazil is endemic for Chagas disease and has the largest diversity of triatomine species. T. cruzi development in its triatomine vector depends on diverse factors, including the composition of bacterial gut microbiota.MethodsWe characterized the triatomines captured in the municipality of Russas (Ceará) by sequencing the cytochrome c oxidase subunit I (COI) gene. The composition of the bacterial community in the gut of peridomestic Triatoma brasiliensis and Triatoma pseudomaculata was investigated using culture independent methods based on the amplification of the 16S rRNA gene by polymerase chain reaction (PCR), denaturing gradient gel electrophoresis (DGGE), DNA fragment cloning, Sanger sequencing and 454 pyrosequencing. Additionally, we identified TcI and TcII types of T. cruzi by sequencing amplicons from the gut metagenomic DNA with primers for the mini-exon gene.ResultsTriatomines collected in the peridomestic ecotopes were diagnosed as T. pseudomaculata and T. brasiliensis by comparing their COI sequence with GenBank. The rate of infection by T. cruzi in adult triatomines reached 80% for T. pseudomaculata and 90% for T. brasiliensis. According to the DNA sequences from the DGGE bands, the triatomine gut microbiota was primarily composed of Proteobacteria and Actinobacteria. However, Firmicutes and Bacteroidetes were also detected, although in much lower proportions. Serratia was the main genus, as it was encountered in all samples analyzed by DGGE and 454 pyrosequencing. Members of Corynebacterinae, a suborder of the Actinomycetales, formed the next most important group. The cloning and sequencing of full-length 16S rRNA genes confirmed the presence of Serratia marcescens, Dietzia sp., Gordonia terrae, Corynebacterium stationis and Corynebacterium glutamicum.ConclusionsThe study of the bacterial microbiota in the triatomine gut has gained increased attention because of the possible role it may play in the epidemiology of Chagas disease by competing with T. cruzi. Culture independent methods have shown that the bacterial composition of the microbiota in the guts of peridomestic triatomines is made up by only few bacterial species.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-015-0836-z) contains supplementary material, which is available to authorized users.

BackgroundChagas disease is a trypanosomiasis whose agent is the protozoan parasite Trypanosoma cruzi, which is transmitted to humans by hematophagous bugs known as triatomines. Even though insecticide treatments allow effective control of these bugs in most Latin American countries where Chagas disease is endemic, the disease still affects a large proportion of the population of South America. The features of the disease in humans have been extensively studied, and the genome of the parasite has been sequenced, but no effective drug is yet available to treat Chagas disease. The digestive tract of the insect vectors in which T. cruzi develops has been much less well investigated than blood from its human hosts and constitutes a dynamic environment with very different conditions. Thus, we investigated the composition of the predominant bacterial species of the microbiota in insect vectors from Rhodnius, Triatoma, Panstrongylus and Dipetalogaster genera.Methodology/Principal FindingsMicrobiota of triatomine guts were investigated using cultivation-independent methods, i.e., phylogenetic analysis of 16s rDNA using denaturing gradient gel electrophoresis (DGGE) and cloned-based sequencing. The Chao index showed that the diversity of bacterial species in triatomine guts is low, comprising fewer than 20 predominant species, and that these species vary between insect species. The analyses showed that Serratia predominates in Rhodnius, Arsenophonus predominates in Triatoma and Panstrongylus, while Candidatus Rohrkolberia predominates in Dipetalogaster.Conclusions/SignificanceThe microbiota of triatomine guts represents one of the factors that may interfere with T. cruzi transmission and virulence in humans. The knowledge of its composition according to insect species is important for designing measures of biological control for T. cruzi. We found that the predominant species of the bacterial microbiota in triatomines form a group of low complexity whose structure differs according to the vector genus.

SUMMARYThe activation of HPV-specific T cells within the cervical microenvironment is likely to play an important part in the natural history of cervical intraepithelial neoplasia (CIN). The extent and the type of T cell activation will depend critically on the expression of MHC, costimulatory cell surface molecules and cytokines by keratinocytes and Langerhans cells within the cervical lesion. Expression of MHC class II (HLA-DR and -DQ), costimulatory/adhesion molecules (CD11a/18, CD50, CD54, CD58 and CD86) and cytokines (tumour necrosis factor-alpha (TNF-a) and IL-10) was therefore investigated by immunohistochemistry in normal squamous epithelium (n ¼ 12), low-grade (n ¼ 23) and high-grade (n ¼ 18) squamous intraepithelial lesions of the cervix. CIN progression was associated with de novo expression of HLA-DR and CD54, and increased expression of CD58 by keratinocytes. However, significantly, there was no expression of any adhesion/costimulation molecule by epithelial Langerhans cells in any cervical biopsy studied. Furthermore, TNF-a, a potent activator of Langerhans cells, was expressed constitutively by basal keratinocytes in normal cervix (12þ/12), but expression of this cytokine was absent in a number of CIN samples (20þ/23 for low-grade, 12þ/18 for high-grade CIN). Conversely, the suppressive cytokine IL-10 was absent in normal epithelium (0þ/12), but was upregulated in a number of CIN lesions (12þ/23 for low-grade, 8þ/18 for high-grade CIN). The restricted expression of costimulation/adhesion molecules and the nature of the cytokine microenvironment within the epithelium may act to limit effective immune responses in some CIN lesions.

BackgroundTrypanosoma 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.MethodsThe 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.ResultsOur 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.ConclusionOur findings suggest that T. rangeli Macias strain modulates R. prolixus immune system and modifies the natural microbiota composition.

In this paper, we aimed to explore the effects of the calpain inhibitor III (MDL28170) and to detect calpain-like molecules (CALPs) in epimastigote forms of Trypanosoma cruzi isolate Dm28c. MDL28170 at 70 microM promoted a powerful reduction in the growth rate after 48 h. The IC50 value was calculated to be 31.7 microM. This inhibitor promoted an increase in the cellular volume, but not cell lysis, resulting in a trypanostatic effect. T. cruzi CALPs presented a strong cross-reactivity with anti-Drosophila melanogaster calpain and anti-cytoskeleton-associated protein from Trypanosoma brucei antibodies, and labelling was found mainly intracellularly. Furthermore, an 80 kDa reactive protein was detected by Western blotting assays. No significant cross-reactivity was found with anti-human brain calpain antibody. The expression of CALPs was decreased in cells kept for long periods in axenic cultures in comparison to a strain recently isolated from mice, as well as in MDL28170-treated cells, the latter being paralleled by an increased expression of cruzipain. Different levels of CALPs expression were also detected in distinct phylogenetic lineages, like Y strain (lineage TcII), Dm28c (lineage TcI) [corrected] and INPA6147 strain (Z3 zymodeme). These results may contribute for the investigation of the functions of CALPs in trypanosomatids.

Aim: To avoid the limitations of 16S rRNA-based phylogenetic analysis for Paenibacillus species, the usefulness of the RNA polymerase beta-subunit encoding gene (rpoB) was investigated as an alternative to the 16S rRNA gene for taxonomic studies. Methods and Results: Partial rpoB sequences were generated for the type strains of eight nitrogen-fixing Paenibacillus species. The presence of only one copy of rpoB in the genome of P. graminis strain RSA19T was demonstrated by denaturing gradient gel electrophoresis and hybridization assays. A comparative analysis of the sequences of the 16S rRNA and rpoB genes was performed and the eight species showed between 91AE6-99AE1% (16S rRNA) and 77AE9-97AE3% (rpoB) similarity, allowing a more accurate discrimination between the different species using the rpoB gene. Finally, 24 isolates from the rhizosphere of different cultivars of maize previously identified as Paenibacillus spp. were assigned correctly to one of the nitrogen-fixing species. Conclusions, Significance and Impact of the Study: The data obtained in this study indicate that rpoB is a powerful identification tool, which can be used for the correct discrimination of the nitrogen-fixing species of agricultural and industrial importance within the genus Paenibacillus.

The digestive tract of triatomines (DTT) is an ecological niche favored by microbiota whose enzymatic profile is adapted to the specific substrate availability in this medium. This report describes the molecular enzymatic properties that promote bacterial prominence in the DTT. The microbiota composition was assessed previously based on 16S ribosomal DNA, and whole sequenced genomes of bacteria from the same genera were used to calculate the GC level of rare and prominent bacterial species in the DTT. The enzymatic reactions encoded by coding sequences of both rare and common bacterial species were then compared and revealed key functions explaining why some genera outcompete others in the DTT. Representativeness of DTT microbiota was investigated by shotgun sequencing of DNA extracted from bacteria grown in liquid Luria-Bertani broth (LB) medium. Results showed that GC-rich bacteria outcompete GC-poor bacteria and are the dominant components of the DTT microbiota. In addition, oxidoreductases are the main enzymatic components of these bacteria. In particular, nitrate reductases (anaerobic respiration), oxygenases (catabolism of complex substrates), acetate-CoA ligase (tricarboxylic acid cycle and energy metabolism), and kinase (signaling pathway) were the major enzymatic determinants present together with a large group of minor enzymes including hydrogenases involved in energy and amino acid metabolism. In conclusion, despite their slower growth in liquid LB medium, bacteria from GC-rich genera outcompete the GC-poor bacteria because their specific enzymatic abilities impart a selective advantage in the DTT.

Cathepsin E is an aspartic proteinase which has been implicated in antigen processing in the class II major histocompatibility complex pathway. In this study we show that cathepsin E, measured at both the protein and message level, is up-regulated late in human B cell activation. The implications of this observation in terms of cathepsin E function are discussed.