Harokopos, V. | Search bioRxiv

bioRxiv2021

Bee foraging preferences, microbiota and pathogens revealed by direct shotgun metagenomics of honey

Honeybees (Apis mellifera) continue to succumb to human and environmental pressures despite their crucial role in providing essential ecosystem services. Owing to their foraging and honey production activities, honeybees form complex relationships with species across all domains, such as plants, viruses, bacteria (symbiotic and pathogenic), and other hive pests, making honey a valuable biomonitoring tool for assessing their ecological niche. Thus, the application of honey shotgun metagenomics (SM) has paved the way for a detailed description of the species honeybees interact with, in order to better assess the multiple factors governing their health. Here, we describe the implementation of optimized honey DNA extraction methodology coupled to direct shotgun metagenomics (Direct-SM) analysis, and to a computationally optimised and validated pipeline for taxonomic classification of species detected in honey. By comparing honey collected across 3 harvesting seasons in a stable apiary, we show that Direct-SM can describe the variability of sampled plant species, revealing honeybee behavioural adaptation. In addition, we reveal that Direct-SM can non-invasively capture the diversity of species comprising the core and non-core bacterial communities of the gut microbiome. Finally, we show that this methodology is applicable for the monitoring of pathogens and particularly for the biomonitoring varroa infestation. These results suggest that Direct-SM can accurately and comprehensively describe honeybee ecological niches and can be deployed to assess bee health in the field.

ecology↗

bioRxiv2021

A two-color haploid genetic screen identifies novel host factors involved in HIV latency

To identify novel host factors as putative targets to reverse HIV latency, we performed an insertional mutagenesis genetic screen in a latently HIV-1-infected pseudo-haploid KBM7 cell line (Hap-Lat). Following mutagenesis, insertions were mapped to the genome and bioinformatic analysis resulted in the identification of 69 candidate host genes involved in maintaining HIV-1 latency. A select set of candidate genes was functionally validated using shRNA mediated depletion in latent HIV-1 infected J-Lat A2 and 11.1 T cell lines. We confirmed ADK, CHD9, CMSS1, EVI2B, EXOSC8, FAM19A, GRIK5, IRF2BP2, NF1, and USP15 as novel host factors involved in the maintenance of HIV latency. Chromatin immunoprecipitation assays indicated that CHD9, a Chromodomain Helicase DNA-binding protein, maintains HIV latency via direct association with the HIV 5LTR, and its depletion results in increased histone acetylation at the HIV-1 promoter, concomitant with HIV-1 latency reversal. FDA-approved inhibitors 5-Iodotubercidin, Trametinib, and Topiramate, targeting ADK, NF1, and GRIK5, respectively were characterized for their latency reversal potential. While 5-Iodotubercidin exhibited significant cytotoxicity in both J-Lat and primary CD4+ T cells, Trametinib reversed latency in J-Lat cells but not in latently HIV-1-infected primary CD4+ T cells. Crucially, Topiramate reversed latency in cell-line models and latently infected primary CD4+ T cells, without inducing T cell activation or significant toxicity. Thus, using an adaptation of a haploid forward genetic screen, we identified novel and druggable host factors contributing to HIV-1 latency. ImportanceA reservoir of latent HIV-1-infected cells persists in the presence of combination antiretroviral therapy (cART), representing a major obstacle for viral eradication. Reactivation of the latent HIV-1 provirus is part of curative strategies which aim to promote clearance of the infected cells. Using a two-color haploid screen, we identified 69 candidate genes as latency maintaining host factors and functionally validated a subset of 10 of those in additional T-cell based cell line models of HIV-1 latency. We further demonstrated that CHD9 is associated with HIV-1s promoter, the 5LTR while this association is lost upon reactivation. Additionally, we characterized the latency reversal potential of FDA compounds targeting ADK, NF1, and GRIK5 and identify the GRIK5 inhibitor Topiramate as a viable latency reversal agent with clinical potential.

molecular biology↗