Oligopeptide signaling through TbGPR89 drives Trypanosome Quorum sensing

K.R.M. is funded by a Wellcome Trust Investigator Award (103740/Z14/Z) and a Royal Society Wolfson Research merit award (WM140045). The Medical Research Council (MR/M020118/1) supported T.K.S., and the Wellcome Trust supported J.T. (202094/Z/16/Z). M.A. received financial support from the Scottish Universities Life Sciences Alliance (SULSA; https://www.sulsa.ac.uk) and a Medical Research Council strategic grant (J54359).Trypanosome parasites control their virulence and spread by using quorum sensing (QS) to generate transmissible “stumpy forms” in their host bloodstream. However, the QS signal “stumpy induction factor” (SIF) and its reception mechanism are unknown. Although trypanosomes lack G protein-coupled receptor signaling, we have identified a surface GPR89-family protein that regulates stumpy formation. TbGPR89 is expressed on bloodstream “slender form” trypanosomes, which receive the SIF signal, and when ectopically expressed, TbGPR89 drives stumpy formation in a SIF-pathway-dependent process. Structural modeling of TbGPR89 predicts unexpected similarity to oligopeptide transporters (POT), and when expressed in bacteria, TbGPR89 transports oligopeptides. Conversely, expression of an E. coli POT in trypanosomes drives parasite differentiation, and oligopeptides promote stumpy formation in vitro. Furthermore, the expression of secreted trypanosome oligopeptidases generates a paracrine signal that accelerates stumpy formation in vivo. Peptidase-generated oligopeptide QS signals being received through TbGPR89 provides a mechanism for both trypanosome SIF production and reception.Publisher PDFPeer reviewe

Institutional capacity for health systems research in East and Central Africa schools of public health: enhancing capacity to design and implement teaching programs

BACKGROUND: The role of health systems research (HSR) in informing and guiding national programs and policies has been increasingly recognized. Yet, many universities in sub-Saharan African countries have relatively limited capacity to teach HSR. Seven schools of public health (SPHs) in East and Central Africa undertook an HSR institutional capacity assessment, which included a review of current HSR teaching programs. This study determines the extent to which SPHs are engaged in teaching HSR-relevant courses and assessing their capacities to effectively design and implement HSR curricula whose graduates are equipped to address HSR needs while helping to strengthen public health policy. METHODS: This study used a cross-sectional study design employing both quantitative and qualitative approaches. An organizational profile tool was administered to senior staff across the seven SPHs to assess existing teaching programs. A self-assessment tool included nine questions relevant to teaching capacity for HSR curricula. The analysis triangulates the data, with reflections on the responses from within and across the seven SPHs. Proportions and average of values from the Likert scale are compared to determine strengths and weaknesses, while themes relevant to the objectives are identified and clustered to elicit in-depth interpretation. RESULTS: None of the SPHs offer an HSR-specific degree program; however, all seven offer courses in the Master of Public Health (MPH) degree that are relevant to HSR. The general MPH curricula partially embrace principles of competency-based education. Different strengths in curricula design and staff interest in HSR at each SPH were exhibited but a number of common constraints were identified, including out-of-date curricula, face-to-face delivery approaches, inadequate staff competencies, and limited access to materials. Opportunities to align health system priorities to teaching programs include existing networks. CONCLUSIONS: Each SPH has key strengths that can be leveraged to design and implement HSR teaching curricula. We propose networking for standardizing HSR curricula competencies, institutionalizing sharing of teaching resources, creating an HSR eLearning platform to expand access, regularly reviewing HSR teaching content to infuse competency-based approaches, and strengthening staff capacity to deliver such curricula.DFI

Analysis of released peptidases and their role in the transmission biology of African trypanosomes

The protozoan parasite, Trypanosoma brucei causes devastating diseases in both humans and animals in sub-Saharan Africa. They live extracellularly and undergo a complex life cycle involving the mammalian host and the tsetse fly vector. Two developmental forms of the parasite exist in the bloodstream of the mammalian host; the long proliferative slender forms and the cell-cycle arrested stumpy forms. The slender forms differentiate into stumpy forms upon reaching a density threshold through a quorum-sensing like mechanism. During a trypanosome infection, the parasites release peptidases into the bloodstream of their mammalian host which accumulates as parasitaemia increases. These released peptidases hydrolyse their respective host protein substrates resulting in the generation of oligopeptides. At peak parasitaemia, these oligopeptides accumulate and trigger the slender forms to differentiate into non-proliferative stumpy forms. This helps regulate the parasitaemia in the host as well as prepare the parasites for development in the tsetse fly vector. Two peptidases whose activities can provoke the quorum-sensing response have been identified previously in our lab. In this study, proteins secreted/released by the parasites in the bloodstream and early during differentiation to the tsetse fly midgut procyclic forms were analysed by detailed mass spectrometry. This identified twelve peptidases, belonging to different classes of peptidases, enriched at these developmental stages of the parasite. Each peptidase was then validated for its release from parasites using individually epitope-tagged cell lines. Systematic ectopic overexpression and gene knockout using CRISPR/Cas9 of each peptidase gene and their analysis in vivo in mice revealed that two of the peptidases, oligopeptidase B and metallocarboxypeptidase 1, significantly contribute to the generation of the trypanosome’s quorum-sensing signal. Further analysis of peptidases enriched in the secretome of parasites differentiating from stumpy forms to procyclic forms in tsetse flies, however, showed that these peptidases may not be involved in establishing infection in the midgut of the flies but may assist proventricular infection. This work analysed for the first time the contribution of a set of released peptidases by T. brucei involved in the important differentiation from proliferative slender forms into the cell-cycle arrested stumpy forms

Extracellular release of two peptidases dominates generation of the trypanosome quorum-sensing signal

Trypanosomes causing African sleeping sickness use quorum-sensing (QS) to generate transmission-competent stumpy forms in mammalian hosts. This density-dependent process is signalled by oligopeptides that stimulate the signal transduction pathway leading to stumpy formation. Here, using mass spectrometry analysis, we identify peptidases released by trypanosomes and, for 12 peptidases, confirm their extracellular delivery. Thereafter, we determine the contribution of each peptidase to QS signal production using systematic inducible overexpression in vivo, and confirm this activity operates through the physiological QS signalling pathway. Gene knockout of the QS-active peptidases identifies two enzymes, oligopeptidase B and metallocarboxypeptidase 1, that significantly reduce QS when ablated individually. Further, combinatorial gene knockout of both peptidases confirms their dominance in the generation of the QS signal, with peptidase release of oligopeptidase B mediated via an unconventional protein secretion pathway. This work identifies how the QS signal driving trypanosome virulence and transmission is generated in mammalian hosts

Pathogenicity and virulence of African trypanosomes: From laboratory models to clinically relevant hosts

ABSTRACT African trypanosomes are vector-borne protozoa, which cause significant human and animal disease across sub-Saharan Africa, and animal disease across Asia and South America. In humans, infection is caused by variants of Trypanosoma brucei, and is characterized by varying rate of progression to neurological disease, caused by parasites exiting the vasculature and entering the brain. Animal disease is caused by multiple species of trypanosome, primarily T. congolense, T. vivax, and T. brucei. These trypanosomes also infect multiple species of mammalian host, and this complexity of trypanosome and host diversity is reflected in the spectrum of severity of disease in animal trypanosomiasis, ranging from hyperacute infections associated with mortality to long-term chronic infections, and is also a main reason why designing interventions for animal trypanosomiasis is so challenging. In this review, we will provide an overview of the current understanding of trypanosome determinants of infection progression and severity, covering laboratory models of disease, as well as human and livestock disease. We will also highlight gaps in knowledge and capabilities, which represent opportunities to both further our fundamental understanding of how trypanosomes cause disease, as well as facilitating the development of the novel interventions that are so badly needed to reduce the burden of disease caused by these important pathogens

Environmental Factors and their Influence on Seasonal Variations of Schistosomiasis Intermediate Snail Hosts Abundance in Weija Lake, Ghana

Schistosomiasis, which remains a key Neglected Tropical Disease, is facilitated by the population dynamics of the intermediate snail host that is reported to be influenced by environmental factors. In Ghana fewer studies on environmental factors have been carried out with the advent of climate change and it predicted influence on the ecology of vectors of diseases that tend to be focal. This study therefore sought to investigate the influence of environmental factors on seasonal variations on intermediate snail hosts abundance. Snails were sampled monthly at a demarcated zone on the Weija Lake near Tomefa, a schistosomiasis endemic community using the scoop net and hand picking techniques. A total of 2,612 snails including 739 dead/empty shells were collected throughout the sampling period. Of this number, 1, 367 (inclusive of 600 dead) Biomphalaria pfeifferi and 1, 245 (inclusive of 139 dead) Bulinus truncatus were collected. Total dissolved solids, temperature and turbidity significantly influenced snail abundance (p<0.05). Five aquatic plant species were found to support both snail species, with Ceratophyllum spp being the most common. Snail abundance varied seasonally with TDS, turbidity and temperature identified as important limiting environmental factors to intermediate snail hosts abundance. Aquatic plant species influenced snail abundance by providing shelter, food and sites for oviposition

In Vitro Assessment of Anthelmintic Activities of Rauwolfia vomitoria (Apocynaceae) Stem Bark and Roots against Parasitic Stages of Schistosoma mansoni and Cytotoxic Study

Schistosomiasis is a Neglected Tropical Diseases which can be prevented with mass deworming chemotherapy. The reliance on a single drug, praziquantel, is a motivation for the search of novel antischistosomal compounds. This study investigated the anthelmintic activity of the stem bark and roots of Rauwolfia vomitoria against two life stages of Schistosoma mansoni. Both plant parts were found to be active against cercariae and adult worms. Within 2 h of exposure all cercariae were killed at a concentration range of 62.5–1000 µg/mL and 250–1000 µg/mL of R. vomitoria stem bark and roots, respectively. The LC50 values determined for the stem bark after 1 and 2 h of exposure were 207.4 and 61.18 µg/mL, respectively. All adult worms exposed to the concentrations range of 250–1000 µg/mL for both plant parts died within 120 h of incubation. The cytotoxic effects against HepG2 and Chang liver cell assessed using MTT assay method indicated that both plant extracts which were inhibitory to the proliferation of cell lines with IC50 > 20 μg/mL appear to be safe. This report provides the first evidence of in vitro schistosomicidal potency of R. vomitoria with the stem bark being moderately, but relatively, more active and selective against schistosome parasites. This suggests the presence of promising medicinal constituent(s)