sand-fly-phlebotomus-papatasi-embryo-microinjection-for-crisprcas9

Sand Fly (Phlebotomus papatasi) Embryo Microinjection for CRISPR/Cas9 Mutagenesis

Sand flies are the natural vectors for Leishmania species, protozoan parasites producing a broad spectrum of symptoms ranging from cutaneous lesions to ...

National Institutes of Health

For many arthropod vectors, the diverse bacteria and fungi that inhabit the gut can negatively impact pathogen colonization. Our attempts to exploit antibiotic treatment of colonized Phlebotomus duboscqi sand flies in order to improve their vector competency for Leishmania major resulted instead in flies that were refractory to the development of transmissible infections due to the inability of the parasite to survive and to colonize the anterior midgut with infective, metacyclic stage promastigotes. The parasite survival and development defect could be overcome by feeding the flies on different symbiont bacteria but not by feeding them on bacterial supernatants or replete medium. The inhibitory effect of the dysbiosis was moderated by lowering the concentration of sucrose (<30% w/v) used in the sugar feeds to maintain the colony. Exposure of promastigotes to 30% sucrose was lethal to the parasite in vitro. Confocal imaging revealed that the killing in vivo was confined to promastigotes that had migrated to the anterior plug region, corresponding to the highest concentrations of sucrose. The data suggest that sucrose utilization by the microbiota is essential to promote the appropriate osmotic conditions required for the survival of infective stage promastigotes in vivo.

The midgut microbiota plays an essential role in sand fly vector competence for Leishmania major.

Sand flies are the natural vectors for the  species that produce a spectrum of diseases in their mammalian hosts, including humans. Studies of sand fly/ interactions have been limited by the absence of genome editing techniques applicable to these insects. In this report, we adapted CRISPR (clustered regularly interspaced palindromic repeat)/Cas9 (CRISPR-associated protein 9) technology to the  sand fly, a natural vector for , targeting the sand fly immune deficiency (IMD) pathway in order to decipher its contribution to vector competence. We established a protocol for transformation in  and were able to generate transmissible  mutant alleles for Relish (Rel), the only transcription factor of the IMD pathway. While the maintenance of a homozygous mutant stock was severely compromised, we were able to establish in an early generation their greater susceptibility to infection with  Flies carrying different heterozygous mutant alleles variably displayed a more permissive phenotype, presenting higher loads of parasites or greater numbers of infective-stage promastigotes. Together, our data show (i) the successful adaptation of the CRISPR/Cas9 technology to sand flies and (ii) the impact of the sand fly immune response on vector competence for  parasites. Sand flies are the natural vectors of  parasites. Studies of sand fly/ interactions have been limited by the lack of successful genomic manipulation of these insects. This paper shows the first example of successful targeted mutagenesis in sand flies via adaptation of the CRISPR/Cas9 editing technique. We generated transmissible  mutant alleles of , a gene known to be essential for the control of immune response in other insects. In addition to the expected higher level of susceptibility to bacteria, the mutant flies presented higher loads of parasites when infected with , showing that the sand fly immune response impacts its vector competence for this pathogen.

CRISPR/Cas9 Mutagenesis in Phlebotomus papatasi: the Immune Deficiency Pathway Impacts Vector Competence for Leishmania major.

Protozoan parasites in the genus Leishmania produce a broad spectrum of diseases in their human hosts. The strain and species-specific genes controlling these diverse clinical outcomes have remained poorly tractable using reverse genetics approaches. A cryptic sexual cycle involving a meiotic-like process has been described in Leishmania but is so far confined to parasites growing in the sand fly vector. Here, we describe the reproducible in vitro generation of hybrid clones using axenic culture forms of Leishmania tropica promastigotes. Analysis of SNPs marker inheritance and whole-genome sequencing data indicate that the progeny clones are full genomic hybrids. The demonstration that mating-competent forms arise in culture should facilitate experimental study of the mating biology of Leishmania and the generation of large numbers of recombinant parasites for positional cloning of important genes.

Article	Year
The midgut microbiota plays an essential role in sand fly vector competence for Leishmania major. Cellular microbiology\| PubMed ID: 28580630	2017
CRISPR/Cas9 Mutagenesis in Phlebotomus papatasi: the Immune Deficiency Pathway Impacts Vector Competence for Leishmania major. mBio\| PubMed ID: 31455654	2019
In Vitro Generation of Leishmania Hybrids. Cell reports\| PubMed ID: 32294444	2020

Isabelle Louradour

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