Biology subjects

Coutinho-Abreu, I. V.

Publications and source records attributed to Coutinho-Abreu, I. V..

7 recordsLinked to original sources

bioRxiv2023

Engineered Skin Microbiome Reduces Mosquito Attraction to Mice

The skin microbiome plays a pivotal role in the production of attractive cues detected by mosquitoes. Here we leveraged recent advances in genetic engineering to significantly reduce the production of L-(+)-lactic acid as a strategy to reduce mosquito attraction to the highly prominent skin commensals Staphylococcus epidermidis and Corynebacterium amycolatum. Engraftment of these engineered bacteria onto the skin of mice reduced mosquito attraction and feeding for up to 11 uninterrupted days, which is considerably longer than the several hours of protection conferred by the leading chemical repellent DEET. Taken together, our findings demonstrate engineering the skin microbiome to reduce attractive volatiles represents an innovative untapped strategy to reduce vector attraction, preventing bites, and pathogen transmission setting the stage for new classes of long-lasting microbiome-based repellent products. One-Sentence SummaryModified microbes make skin less attractive to mosquitoes

microbiology↗

bioRxiv2023

Identification of human skin microbiome odorants that manipulate mosquito landing behavior - towards engineering a repellent skin flora

The resident human skin microbiome is responsible for the production of most of the human scents that are attractive to mosquitoes. Hence, engineering the human skin microbiome to synthesize less of mosquito attractants or produce repellents could potentially reduce bites and prevent the transmission of deadly mosquito-borne pathogens. In order to further characterize the human skin volatilome, we quantified the major volatiles of 39 strains of skin commensals (Staphylococci and Corynebacterium). Importantly, to validate the behavioral activity of these volatiles, we first assessed landing behavior triggered by human skin bacteria volatiles. We demonstrated that this behavioral step is gated by the presence of carbon dioxide and L-(+)-lactic acid, similar to the combinatorial coding triggering short range attraction. Repellency behavior to selected skin volatiles and the geraniol terpene was tested in the presence of carbon dioxide and L-(+)-lactic acid. In a 2-choice landing behavior context, the skin volatiles 2- and 3-methyl butyric acids reduced mosquito landing by 62.0-81.6% and 87.1-99.6%, respectively. Similarly, geraniol was capable of reducing mosquito landing behavior by 74.9%. We also tested the potential repellency effects of geraniol on mosquitoes at short-range using a 4-port olfactometer. In these assays, geraniol reduced mosquito attraction (69-78%) to a mixture of key human kairomones carbon dioxide, L-(+)-lactic acid, and ammonia. These findings demonstrate that carbon dioxide and L-(+)-lactic acid changes the valence of other skin volatiles towards mosquito landing behavior. Moreover, this study offers candidate odorants to be targeted in a novel strategy to reduce attractants or produce repellents by the human skin microbiota that may curtail mosquito bites, and subsequent mosquito-borne disease.

zoology↗

bioRxiv2022

Genetic exchange in Leishmania is facilitated by IgM natural antibodies

Host factors mediating Leishmania genetic exchange are not well defined. Here, we demonstrate that IgM antibodies, but not IgG or IgA, facilitate parasite genetic hybridization in vitro and in vivo. IgM induces the gradual and transient formation of a structured parasite clump in a process essential for L. major and L. tropica hybridization in vitro. Parasite hybrids and 3-nucleated parasites were observed inside this structure, named the Leishmania mating clump. IgM was also required for or significantly increased Leishmania hybrid formation in vivo. At minimum, we observed a 12-fold increase in the proportion of hybrids recovered from sand flies provided a second blood meal containing IgM compared to controls. Notably, genetic backcross events in sand flies were only observed in the presence of IgM, and were reproducibly recovered, reinforcing the relevance of IgM for Leishmania genetic exchange in vivo. The in vitro and in vivo Leishmania crosses from these studies resulted in full genome hybrids. Leishmania co-option of a host antibody to facilitate mating in the insect vector establishes a new paradigm of parasite-host-vector coevolution that promotes parasite diversity and fitness through genetic exchange.

microbiology↗

bioRxiv2020

Binding of Leishmania infantum LPG to the midgut is not sufficient to define vector competence in Lutzomyia longipalpis sand flies

The major surface lipophosphoglycan (LPG) of Leishmania parasites is critical to vector competence in restrictive sand fly vectors by mediating Leishmania attachment to the midgut epithelium, considered essential to parasite survival and development. However, the relevance of LPG for sand flies that harbor multiple species of Leishmania remains elusive. We tested binding of Leishmania infantum wild type (WT), LPG-defective ({Delta}lpg1 mutants) and add-back lines ({Delta}lpg1 + LPG1) to sand fly midguts in vitro and their survival in Lutzomyia longipalpis sand flies in vivo. Le. infantum WT parasites attached to the Lu. longipalpis midgut in vitro with late-stage parasites binding to midguts in significantly higher numbers compared to early-stage stage promastigotes. {Delta}lpg1 mutants did not bind to Lu. longipalpis midguts, and this was rescued in the {Delta}lpg1 + LPG1 lines, indicating that midgut binding is mediated by LPG. When Lu. longipalpis sand flies were infected with either Le. infantum WT, {Delta}lpg1, or {Delta}lpg1 + LPG1 of the BH46 or BA262 strains, the BH46 {Delta}lpg1 mutant, but not the BA262 {Delta}lpg1 mutant, survived and grew to similar numbers as the WT and {Delta}lpg1 + LPG1 lines. Exposure of BH46 and BA262 {Delta}lpg1 mutants to blood engorged midgut extracts led to the mortality of the BA262 {Delta}lpg1 but not the BH46 {Delta}lpg1 parasites. These findings suggest that Le. infantum LPG protects parasites on a strain-specific basis early in infection, likely against toxic components of blood digestion, however, it is not necessary to prevent Le. infantum evacuation along with the feces in the permissive vector Lu. longipalpis. IMPORTANCEIt is well established that LPG is sufficient to define the vector competence of restrictive sand fly vectors to Leishmania parasites. However, the permissiveness of other sand flies to multiple Leishmania species suggests that other factors might define vector competence for these vectors. In this study, we investigated the underpinnings of Leishmania infantum survival and development in its natural vector Lutzomyia longipalpis. We found out that LPG-mediated midgut binding persists in late-stage parasites. This observation is paradigm-changing and suggests that only a subset of infective metacyclics lose their ability to attach to the midgut with implications for parasite transmission dynamics. However, our data also demonstrate that LPG is not a determining factor in Leishmania infantum retention in the midgut of Lutzomyia longipalpis, a permissive vector. Rather, LPG appears to be more important in protecting some parasite strains from the toxic environment generated during blood meal digestion in the insect gut. Thus, the relevance of LPG in parasite development in permissive vectors appears to be complex and should be investigated on a strain-specific basis.

microbiology↗

bioRxiv2020

A Second Generation Leishmanization Vaccine with a Markerless Attenuated Leishmania major Strain using CRISPR gene editing

Leishmaniasis is a debilitating and often fatal neglected tropical disease caused by Leishmania protozoa transmitted by infected sand flies. Vaccination through leishmanization with live Leishmania major has been used successfully but is no longer practiced because it resulted in unacceptable skin lesions. A second generation leishmanization is described here using a CRISPR genome edited L. major strain (LmCen-/-). Notably, LmCen-/- is the first genetically engineered gene deleted Leishmania strain that is antibiotic resistant marker free and does not have any off-target mutations. Mice immunized with LmCen-/- had virtually no visible lesions following challenge with L. major-infected sand flies while non-immunized animals developed large and progressive lesions with a 2-log fold higher parasite burden. LmCen-/- immunization showed protection and an immune response comparable to leishmanization. LmCen-/- is safe since it was unable to cause disease even in immunocompromised mice, induces robust host protection against vector sand fly challenge and because it is marker free, can be advanced to human vaccine trials.

microbiology↗

bioRxiv2019

Leishmania infection induces a limited differential gene expression in the sand fly midgut

BackgroundPhlebotomine sand flies are the vectors of Leishmania worldwide. To develop in the sand fly midgut, Leishmania multiplies and undergoes multiple stage differentiations leading to the infective form, the metacyclic promastigotes. To gain a better understanding of the influence of Leishmania infection in midgut gene expression, we performed RNA-Seq comparing uninfected Lutzomyia longipalpis midguts and Leishmania infantum-infected Lutzomyia longipalpis midguts at seven time points which cover the various developmental Leishmania stages including early time points when blood digestion is taking place and late time points when the parasites are undergoing metacyclogenesis. ResultsOut of over 13,841 transcripts assembled de novo, only 113 sand fly transcripts, about 1%, were differentially expressed. Further, we observed a low overlap of differentially expressed sand fly transcripts across different time points suggesting a specific influence of each Leishmania stage on midgut gene expression. Two main patterns of sand fly gene expression modulation were noticed. At early time points (days 1-4), more transcripts were down-regulated by Leishmania infection at large fold changes (> -32 fold). Among the down-regulated genes, the transcription factor Forkhead/HNF-3 and hormone degradation enzymes were differentially regulated on day 4 and appear to be the upstream regulators of nutrient transport, digestive enzymes, and peritrophic matrix proteins. Conversely, at later time points (days 6 onwards), most of the differentially expressed transcripts were up-regulated by small fold changes (< 32 fold), and the molecular function of such genes are associated with the metabolism of lipids and detoxification of xenobiotics (P450). ConclusionOverall, it appears that Leishmania modulates sand fly gene expression early on in order to overcome the barriers imposed by the midgut, yet it behaves like a commensal at later time points, when modest midgut gene expression changes correlate with a massive amount of parasites in the anterior midgut.

microbiology↗

bioRxiv2019

Distinct gene expression patterns in vector-residing Leishmania infantum identify parasite stage-enriched markers

Promastigotes of Leishmania infantum undergo a series of extracellular developmental stages inside the natural sand fly vector Lutzomyia longipalpis to reach the infectious stage, the metacyclic promastigote. There is limited information regarding the expression profile of L. infantum developmental stages inside the sand fly vector, and molecular markers that can distinguish the different parasite stages are lacking. We performed RNAseq on unaltered midguts of the sand fly Lutzomyia longipalpis after infection with L. infantum parasites. RNAseq was carried out at various time points throughout parasite development. Principal component analysis mapped the sequences corresponding to the procyclic, nectomonad, leptomonad or metacyclic promastigote stage into distinct positions, with the procyclic stage being the most divergent population. Transcriptional levels across genes varied on average between 10- to 100-fold. Comparison between procyclic and nectomonad promastigotes resulted in 836 differentially expressed (DE) genes; between nectomonad and leptomonad promastigotes in 113 DE genes; and between leptomonad and metacyclic promastigotes in 302 DE genes. Most of the DE genes do not overlap across stages, highlighting the uniqueness of each stage. Furthermore, the different stages of Leishmania parasites exhibited specific transcriptional enrichment across chromosomes. Using the transcriptional signatures exhibited by distinct Leishmania stages during their development in the sand fly midgut, we determined the genes predominantly enriched in each stage, identifying multiple stage-specific markers for L. Infantum. Leading stage-specific marker candidates include genes encoding a zinc transporter in procyclics, a beta-fructofuranidase in nectomonads, a surface antigen-like protein in leptomonads, and an amastin-like surface protein in metacyclics. Overall, these findings demonstrate the transcriptional plasticity of the Leishmania parasite inside the sand fly vector and provide a repertoire of stage-specific markers for further development as molecular tools for epidemiological studies.

microbiology↗