A Systematic Analysis of Mosquito-Microbiome Biosynthetic Gene Clusters Reveals Antimalarial Siderophores that Reduce Mosquito Reproduction Capacity

Abstract

21 22 23 24 25 26 2 ABSTRACT 27Advances in infectious disease control strategies through genetic manipulation of insect 28 microbiomes have heightened interest in microbially produced small molecules within 29 mosquitoes. Herein, 33 mosquito-associated bacterial genomes were mined and over 700 30 putative biosynthetic gene clusters (BGCs) were identified, 135 of which belong to known 31 classes of BGCs. After an in-depth analysis of the 135 BGCs, iron-binding siderophores 32 were chosen for further investigation due to their high abundance and well-characterized 33 bioactivities. Through various metabolomic strategies, eight siderophore scaffolds were 34 identified in six strains of mosquito-associated bacteria. Among these, serratiochelin A 35 and pyochelin were found to reduce female Anopheles gambiae overall fecundity likely by 36 lowering their blood feeding rate. Serratiochelin A and pyochelin were further found to 37 inhibit the Plasmodium parasite asexual blood and liver stages in vitro. Our work supplies 38 a bioinformatic resource for future mosquito microbiome studies and highlights an 39 understudied source of bioactive small molecules. 40 41 KEYWORDS 42 Mosquito-Microbiome; Biosynthetic Gene Clusters; Siderophores; Anopheles; 43 Plasmodium 44 45 46 100 transmit avian malaria, P. gallinaceum (Alavi et al., 2003), as well as human infectious 101 agents including dengue virus, Zika virus, and others (2016). As a relevant disease vector,102 Aedes-associated bacteria were also included in our data set. Due to the limited amount 103 of genome sequencing data on mosquito-associated bacteria, especially when compared 104 5 to human microbiomes, our sample size is smaller than analogous studies (Aleti et al., 105 2019; Donia et al., 2014; Helfrich et al., 2018), however this enabled an in-depth 106 bioinformatic analysis. Despite this size, our data set well-represents the unique gut 107 microbiome of mosquitoes. Our previous metagenomic study demonstrated that within the 108 gut microbiota of field-caught A. gambiae mosquitoes four days post blood-meal, 5 109 bacterial genera (Serratia, Elizabethkingia, Acinetobacter, Enterobacter, and 110 Pseudomonas) constitute 84 % of the 16S rRNA reads (Wang et al., 2011), all of which 111 are represented within our sample set. 112 A phylogenetic tree comparing the 33 bacterial strains was generated (Figure 113 1). The best-represented bacterial phylum in our sample set was Proteobacteria (16 γ-114 Proteobacteria, 1 β-Proteobacteria, and 3 α-Proteobacteria), consistent with reports 115 showing Proteobacteria are often the overwhelming microbial phylum in the midgut of 116 field-caught Anopheles mosquitoes (Wang et al., 2011). Species in the Bacteroidetes 117 phylum, which include Elizabethkingia spp., were the second most recurrent group (4 118 Elizabethkingia spp. and 1 Sphingobacterium sp.) in our analysis. Elizabethkingia spp. are 119 well-established mosquito symbionts that are the dominant species within midguts 4-and 120 7-days post-blood meal (PBM) as well as 7-day...

Metabolic interactions between disease-transmitting vectors and their microbiota

“…Oral administration of two siderophores, serratiochelin A and pyochelin, to An. gambiae significantly reduces blood-feeding propensity and fecundity [39]. These studies suggest that mosquito symbionts adjust their gene expression to cope with iron-mediated stress during blood digestion.…”

Recovery of Metagenomic Data from theAedes aegyptiMicrobiome using a Reproducible Snakemake Pipeline: MINUUR

“…Users can search specific genes and metabolic profiles of interest from HUMAaN3’s output within MINUUR. For gene profiles, we show an example using the search term ”siderophore” which are of interest given their previous functional characterization in Anopheles gambiae associated bacteria (55). Here, 17 siderophore related genes associated to nine MetaPhlAn3 classified taxa were identified (Figure 5B).…”

“…HUMAaN3's output within MINUUR. For gene profiles, we show an example using the search term "siderophore" which are of interest given their previous functional characterization in Anopheles gambiae associated bacteria (55).…”

Considerations for mosquito microbiome research from the Mosquito Microbiome Consortium

“…The diverse mosquito microbiome provides a microbial repertoire that can be explored for metabolites or enzymes with anti-pathogen and/ or mosquitocidal activity [153], with recent studies identifying antimalarial [154], antibiotic [155], and insecticidal [156,157] compounds from the mosquito microbiota. As seen in microbiome research in other systems such as plants [158] and humans [159], an advancement in this area would include connecting mosquito microbe-derived metabolites to their biosynthetic gene clusters as exemplified by Ganley et al [160]. Expanding research efforts towards harnessing the strengths of bioinformatics in combination with functional 'omics tools such as metatranscriptomics, metaproteomics, and metabolomics would drive this area of mosquito microbiome research forward.…”

“…Growing evidence demonstrates that these non-prokaryotic microbes may play important roles in mosquito biology and vector competence [168][169][170]. Mosquito microbiome studies using different 'omics approaches and those focusing on neglected microbes are still evolving, with currently limited available reference genomes for mosquito-associated microbes [160]. Future efforts should be directed towards augmenting the number of available reference genomes for mosquito-associated microbes and/or identifying currently available orthologs from other systems.…”