High-speed, three-dimensional imaging reveals chemotactic behaviour specific to human-infective Leishmania parasites.

Cellular motility is an ancient eukaryotic trait, ubiquitous across phyla with roles in predator avoidance, resource access, and competition. Flagellar motility is seen in various parasitic protozoans, and morphological changes in flagella during the parasite life cycle have been observed. We studied the impact of these changes on motility across life cycle stages, and how such changes might serve to facilitate human infection. We used holographic microscopy to image swimming cells of different Leishmania mexicana life cycle stages in three dimensions. We find that the human-infective (metacyclic promastigote) forms display 'run and tumble' behaviour in the absence of stimulus, reminiscent of bacterial motion, and that they specifically modify swimming direction and speed to target host immune cells in response to a macrophage-derived stimulus. Non-infective (procyclic promastigote) cells swim more slowly, along meandering helical paths. These findings demonstrate adaptation of swimming phenotype and chemotaxis towards human cells.

Predicting and Experimentally Validating Hot-Spot Residues at Protein-Protein Interfaces.

Protein-protein interactions (PPIs) are vital to all biological processes. These interactions are often dynamic, sometimes transient, typically occur over large topographically shallow protein surfaces, and can exhibit a broad range of affinities. Considerable progress has been made in determining PPI structures. However, given the above properties, understanding the key determinants of their thermodynamic stability remains a challenge in chemical biology. An improved ability to identify and engineer PPIs would advance understanding of biological mechanisms and mutant phenotypes and also provide a firmer foundation for inhibitor design. In silico prediction of PPI hot-spot amino acids using computational alanine scanning (CAS) offers a rapid approach for predicting key residues that drive protein-protein association. This can be applied to all known PPI structures; however there is a trade-off between throughput and accuracy. Here we describe a comparative analysis of multiple CAS methods, which highlights effective approaches to improve the accuracy of predicting hot-spot residues. Alongside this, we introduce a new method, BUDE Alanine Scanning, which can be applied to single structures from crystallography and to structural ensembles from NMR or molecular dynamics data. The comparative analyses facilitate accurate prediction of hot-spots that we validate experimentally with three diverse targets: NOXA-B/MCL-1 (an α-helix-mediated PPI), SIMS/SUMO, and GKAP/SHANK-PDZ (both ß-strand-mediated interactions). Finally, the approach is applied to the accurate prediction of hot-spot residues at a topographically novel Affimer/BCL-xL protein-protein interface.

Novel Wolbachia strains in Anopheles malaria vectors from Sub-Saharan Africa.

Background:  Wolbachia, a common insect endosymbiotic bacterium that can influence pathogen transmission and manipulate host reproduction, has historically been considered absent from the  Anopheles (An.) genera, but has recently been found in  An. gambiae s.l. populations in West Africa.  As there are numerous  Anopheles species that have the capacity to transmit malaria, we analysed a range of species across five malaria endemic countries to determine  Wolbachia prevalence rates, characterise novel  Wolbachia strains and determine any correlation between the presence of  Plasmodium,  Wolbachia and the competing bacterium  Asaia. Methods:  Anopheles adult mosquitoes were collected from five malaria-endemic countries: Guinea, Democratic Republic of the Congo (DRC), Ghana, Uganda and Madagascar, between 2013 and 2017.  Molecular analysis was undertaken using quantitative PCR, Sanger sequencing,  Wolbachia multilocus sequence typing (MLST) and high-throughput amplicon sequencing of the bacterial  16S rRNA gene.  Results: Novel  Wolbachia strains were discovered in five species:  An. coluzzii,  An. gambiae s.s.,  An. arabiensis,  An. moucheti and  An. species A, increasing the number of  Anopheles species known to be naturally infected. Variable prevalence rates in different locations were observed and novel strains were phylogenetically diverse, clustering with  Wolbachia supergroup B strains.  We also provide evidence for resident strain variants within  An. species A.  Wolbachia is the dominant member of the microbiome in  An. moucheti and  An. species A but present at lower densities in  An. coluzzii.  Interestingly, no evidence of  Wolbachia/Asaia co-infections was seen and  Asaia infection densities were shown to be variable and location dependent.  Conclusions: The important discovery of novel  Wolbachia strains in  Anopheles provides greater insight into the prevalence of resident  Wolbachia strains in diverse malaria vectors.  Novel  Wolbachia strains (particularly high-density strains) are ideal candidate strains for transinfection to create stable infections in other  Anopheles mosquito species, which could be used for population replacement or suppression control strategies.

Development of an urban molecular xenomonitoring system for lymphatic filariasis in the Recife Metropolitan Region, Brazil.

INTRODUCTION: Molecular xenomonitoring (MX)-pathogen detection in the mosquito rather than human-is a promising tool for lymphatic filariasis (LF) surveillance. In the Recife Metropolitan Region (RMR), the last LF focus in Brazil, Culex quinquefasciatus mosquitoes have been implicated in transmitting Wuchereria bancrofti parasites. This paper presents findings on the ideal mosquito collection method, mosquito dispersion, W. bancrofti infection in mosquitoes and W. bancrofti antigen in humans to aid MX development. METHODS: Experiments occurred within two densely populated urban areas of Olinda, RMR, in July and August 2015. U.S. Centers for Disease Control and Prevention (CDC) light traps were compared to battery-powered aspirators as collection methods, and mosquito dispersion was measured by mosquito mark release recapture (MMRR). Female Cx. quinquefasciatus were tested by PCR for W. bancrofti infection, and study area residents were screened by rapid tests for W. bancrofti antigen. RESULTS: Aspirators caught 2.6 times more total Cx. quinquefasciatus, including 38 times more blood-fed and 5 times more gravid stages, than CDC light traps. They also collected 123 times more Aedes aegypti. Of the 9,644 marked mosquitoes released, only ten (0.01%) were recaptured, nine of which were < 50m (34.8m median, 85.4m maximum) from the release point. Of 9,169 unmarked mosquitoes captured in the MMR, 38.3% were unfed, 48.8% blood-fed, 5.5% semi-gravid, and 7.3% gravid. PCR on 182 pools (1,556 mosquitoes) found no evidence of W. bancrofti infection in Cx. quinquefasciatus. Rapid tests on 110 of 111 eligible residents were all negative for W. bancrofti antigen. CONCLUSIONS: Aspirators were more effective than CDC light traps at capturing Ae. aegypti and all but unfed stages of Cx. quinquefasciatus. Female Cx. quinquefasciatus traveled short (< 86m) distances in this urban area. Lack of evidence for W. bancrofti infection in mosquitoes and antigen in humans in these fine-scale studies does not indicate that LF transmission has ceased in the RMR. A MX surveillance system should consider vector-specific collection methods, mosquito dispersion, and spatial scale but also local context, environmental factors such as sanitation, and host factors such as infection prevalence and treatment history.