An Investigation into the Protein Composition of the Teneral Glossina morsitans morsitans Peritrophic Matrix.

BACKGROUND Tsetse flies serve as biological vectors for several species of African trypanosomes. In order to survive, proliferate and establish a midgut infection, trypanosomes must cross the tsetse fly peritrophic matrix (PM), which is an acellular gut lining surrounding the blood meal. Crossing of this multi-layered structure occurs at least twice during parasite migration and development, but the mechanism of how trypanosomes do so is not understood. In order to better comprehend the molecular events surrounding trypanosome penetration of the tsetse PM, a mass spectrometry-based approach was applied to investigate the PM protein composition using Glossina morsitans morsitans as a model organism. METHODS PMs from male teneral (young, unfed) flies were dissected, solubilised in urea/SDS buffer and the proteins precipitated with cold acetone/TCA. The PM proteins were either subjected to an in-solution tryptic digestion or fractionated on 1D SDS-PAGE, and the resulting bands digested using trypsin. The tryptic fragments from both preparations were purified and analysed by LC-MS/MS. RESULTS Overall, nearly 300 proteins were identified from both analyses, several of those containing signature Chitin Binding Domains (CBD), including novel peritrophins and peritrophin-like glycoproteins, which are essential in maintaining PM architecture and may act as trypanosome adhesins. Furthermore, 27 proteins from the tsetse secondary endosymbiont, Sodalis glossinidius, were also identified, suggesting this bacterium is probably in close association with the tsetse PM. CONCLUSION To our knowledge this is the first report on the protein composition of teneral G. m. morsitans, an important vector of African trypanosomes. Further functional analyses of these proteins will lead to a better understanding of the tsetse physiology and may help identify potential molecular targets to block trypanosome development within the tsetse

A new perspective on cutaneous leishmaniasis-Implications for global prevalence and burden of disease estimates

This article considers the current public health perspective on cutaneous leishmaniasis (CL) and its implications for incidence, prevalence, and global burden of disease calculations. CL is the most common form of leishmaniasis and one of a small number of infectious diseases increasing in incidence worldwide [1] due to conflict and environmental factors in the Middle East (“Old World”) and the Americas (“New World”)—regions where it is most prevalent. Recently, the disease has reached hyperendemic levels in the conflict zones of the Syrian Arab Republic, Iraq, and Afghanistan while simultaneously affecting refugees from those regions [2]. Nevertheless, CL is not seen as a priority for policymakers because it is not life limiting. This is evidenced by a lack of commitment in recent years to preventive campaigns and patient provision (limited diagnostic capacity, knowledge of treatment, drug availability) in a number of endemic countries

Characterization of an African trypanosome mutant refractory to lectin-induced death

Incubation of African trypanosomes with the lectin concanavalin A (conA) leads to alteration in cellular DNA content, DNA degradation, and surface membrane blebbing. Here, we report the generation and characterization of a conA-refractory Trypanosoma brucei line. These insect stage parasites were resistant to conA killing, with a mediun lethal dose at least 50-fold greater than the parental line. Fluorescence-based experiments revealed that the resistant cells bound less lectin when compared to the parental line. Western blotting and mass spectrometry confirmed that the resistant line lacked an N-glycan required for conA binding on the cellular receptors, EP procyclin proteins. The failure to N-glycosylate the EP procyclins was not the consequence of altered N-glycan precursor biosynthesis, as another glycosylated protein (Fla1p) was normally modified. These findings support the likelihood that resistance to conA was a consequence of failure to bind the lectin trigger

Trypanosomiasis: Skin deep

Trypanosome parasites are hiding in human skin, a discovery that may undermine efforts to eliminate sleeping sickness by 2020

RFT1 Protein Affects Glycosylphosphatidylinositol (GPI) Anchor Glycosylation

The membrane protein RFT1 is essential for normal protein N‑glycosylation, but its precise function is not known. RFT1 was originally proposed to translocate the glycolipid Man5GlcNAc2-PP-dolichol (needed to synthesize N-glycan precursors) across the endoplasmic reticulum membrane, but subsequent studies showed that it does not play a direct role in transport. In contrast to the situation in yeast, RFT1 is not essential for growth of the parasitic protozoan Trypanosoma brucei, enabling the study of its function in a null background. We now report that lack of T. brucei RFT1 (TbRFT1) not only affects protein N‑glycosylation but also glycosylphosphatidylinositol (GPI) anchor side chain modification. Analysis by immunoblotting, metabolic labeling and mass spectrometry demonstrated that the major GPI-anchored proteins of T. brucei procyclic forms have truncated GPI anchor side chains in TbRFT1 null parasites compared to wild-type cells, a defect that is corrected by expressing a tagged copy of TbRFT1 in the null background. In vivo and in vitro labeling experiments using radiolabeled GPI precursors showed that GPI underglycosylation was not the result of decreased formation of the GPI precursor lipid or defective galactosylation of GPI intermediates in the endoplasmic reticulum, but rather due to modifications that are expected to occur in the Golgi apparatus. Unexpectedly, immunofluorescence microscopy localized TbRFT1 to both the endoplasmic reticulum and the Golgi, consistent with the proposal that TbRFT1 plays a direct or indirect role in GPI anchor glycosylation in the Golgi apparatus. Our results implicate RFT1 in a wider range of glycosylation processes than previously appreciated

Variant surface glycoproteins from Venezuelan trypanosome isolates are recognized by sera from animals infected with either Trypanosoma evansi or Trypanosoma vivax

Salivarian trypanosomes sequentially express only one variant surface glycoprotein (VSG) on their cell surface from a large repertoire of VSG genes. Seven cryopreserved animal trypanosome isolates known as TeAp-ElFrio01, TEVA1 (or TeAp-N/D1), TeGu-N/D1, TeAp-Mantecal01, TeGu-TerecayTrino, TeGu-Terecay03 and TeGu-Terecay323, which had been isolated from different hosts identified in several geographical areas of Venezuela were expanded using adult albino rats. Soluble forms of predominant VSGs expressed during the early infection stages were purified and corresponded to concanavalin A-binding proteins with molecular masses of 48–67 kDa by sodium dodecyl sulfate-polyacrylamide gel electropohoresis, and pI values between 6.1 and 7.5. The biochemical characterization of all purified soluble VSGs revealed that they were dimers in their native form and represented different gene products. Sequencing of some of these proteins yielded peptides homologous to VSGs from Trypanosoma (Trypanozoon) brucei and Trypanosoma (Trypanozoon) evansi and established that they most likely are mosaics generated by homologous recombination. Western blot analysis showed that all purified VSGs were cross-reacting antigens that were recognized by sera from animals infected with either T. evansi or Trypanosoma (Dutonella) vivax. The VSG glycosyl-phosphatidylinositol cross-reacting determinant epitope was only partially responsible for the cross-reactivity of the purified proteins, and antibodies appeared to recognize cross-reacting conformational epitopes from the various soluble VSGs. ELISA experiments were performed using infected bovine sera collected from cattle in a Venezuelan trypanosome-endemic area. In particular, soluble VSGs from two trypanosome isolates, TeGu-N/D1 and TeGu-TeracayTrino, were recognized by 93.38% and 73.55% of naturally T. vivax-infected bovine sera, respectively. However, approximately 70% of the sera samples did not recognize all seven purified proteins. Hence, the use of a combination of various VSGs for the diagnosis of animal trypanosomosis is recommended

Understanding the transmission dynamics of Leishmania donovani to provide robust evidence for interventions to eliminate visceral leishmaniasis in Bihar, India

Visceral Leishmaniasis (VL) is a neglected vector-borne disease. In India, it is transmitted to humans by Leishmania donovani-infected Phlebotomus argentipes sand flies. In 2005, VL was targeted for elimination by the governments of India, Nepal and Bangladesh by 2015. The elimination strategy consists of rapid case detection, treatment of VL cases and vector control using indoor residual spraying (IRS). However, to achieve sustained elimination of VL, an appropriate post elimination surveillance programme should be designed, and crucial knowledge gaps in vector bionomics, human infection and transmission need to be addressed. This review examines the outstanding knowledge gaps, specifically in the context of Bihar State, India. The knowledge gaps in vector bionomics that will be of immediate benefit to current control operations include better estimates of human biting rates and natural infection rates of P. argentipes, with L. donovani, and how these vary spatially, temporally and in response to IRS. The relative importance of indoor and outdoor transmission, and how P. argentipes disperse, are also unknown. With respect to human transmission it is important to use a range of diagnostic tools to distinguish individuals in endemic communities into those who: 1) are to going to progress to clinical VL, 2) are immune/ refractory to infection and 3) have had past exposure to sand flies. It is crucial to keep in mind that close to elimination, and post-elimination, VL cases will become infrequent, so it is vital to define what the surveillance programme should target and how it should be designed to prevent resurgence. Therefore, a better understanding of the transmission dynamics of VL, in particular of how rates of infection in humans and sand flies vary as functions of each other, is required to guide VL elimination efforts and ensure sustained elimination in the Indian subcontinent. By collecting contemporary entomological and human data in the same geographical locations, more precise epidemiological models can be produced. The suite of data collected can also be used to inform the national programme if supplementary vector control tools, in addition to IRS, are required to address the issues of people sleeping outsid

On Real-Time AER 2-D Convolutions Hardware for Neuromorphic Spike-Based Cortical Processing

In this paper, a chip that performs real-time image convolutions with programmable kernels of arbitrary shape is presented. The chip is a first experimental prototype of reduced size to validate the implemented circuits and system level techniques. The convolution processing is based on the address–event-representation (AER) technique, which is a spike-based biologically inspired image and video representation technique that favors communication bandwidth for pixels with more information. As a first test prototype, a pixel array of 16x16 has been implemented with programmable kernel size of up to 16x16. The chip has been fabricated in a standard 0.35- m complimentary metal–oxide–semiconductor (CMOS) process. The technique also allows to process larger size images by assembling 2-D arrays of such chips. Pixel operation exploits low-power mixed analog–digital circuit techniques. Because of the low currents involved (down to nanoamperes or even picoamperes), an important amount of pixel area is devoted to mismatch calibration. The rest of the chip uses digital circuit techniques, both synchronous and asynchronous. The fabricated chip has been thoroughly tested, both at the pixel level and at the system level. Specific computer interfaces have been developed for generating AER streams from conventional computers and feeding them as inputs to the convolution chip, and for grabbing AER streams coming out of the convolution chip and storing and analyzing them on computers. Extensive experimental results are provided. At the end of this paper, we provide discussions and results on scaling up the approach for larger pixel arrays and multilayer cortical AER systems.Commission of the European Communities IST-2001-34124 (CAVIAR)Commission of the European Communities 216777 (NABAB)Ministerio de Educación y Ciencia TIC-2000-0406-P4Ministerio de Educación y Ciencia TIC-2003-08164-C03-01Ministerio de Educación y Ciencia TEC2006-11730-C03-01Junta de Andalucía TIC-141