Search for: All records

Abstract Background: Nematodes of the genus Steinernema and their Xenorhabdus bacterial symbionts are lethal entomopathogens that are useful in the biocontrol of insect pests, as sources of diverse natural products, and as research models for mutualism and parasitism. Xenorhabdus play a central role in all aspects of the Steinernema lifecycle, and a deeper understanding of their genomes therefore has the potential to spur advances in each of these applications. Results: Here, we report a comparative genomics analysis of Xenorhabdus griffiniae, including the symbiont of Steinernema hermaphroditum nematodes, for which genetic and genomic tools are being developed. We sequenced and assembled circularized genomes for three Xenorhabdus strains: HGB2511, ID10 and TH1. We then determined their relationships to other Xenorhabdus and delineated their species via phylogenomic analyses, concluding that HGB2511 and ID10 are Xenorhabdus griffiniae while TH1 is a novel species. These additions to the existing X. griffiniae landscape further allowed for the identification of two subspecies within the clade. Consistent with other Xenorhabdus, the analysed X. griffiniae genomes each encode a wide array of antimicrobials and virulence-related proteins. Comparative genomic analyses, including the creation of a pangenome, revealed that a large amount of the intraspecies variation in X. griffiniae is contained within the mobilome and attributable to prophage loci. In addition, CRISPR arrays, secondary metabolite potential and toxin genes all varied among strains within the X. griffiniae species. Conclusions: Our findings suggest that phage-related genes drive the genomic diversity in closely related Xenorhabdus symbionts, and that these may underlie some of the traits most associated with the lifestyle and survival of entomopathogenic nematodes and their bacteria: virulence and competition. This study establishes a broad knowledge base for further exploration of not only the relationships between X. griffiniae species and their nematode hosts but also the molecular mechanisms that underlie their entomopathogenic lifestyle. 

ABSTRACT Systemic infections byCandidaspp. are associated with high mortality rates, partly due to limitations in current antifungals, highlighting the need for novel drugs and drug targets. The fungal phosphatidylserine synthase, Cho1, fromCandida albicansis a logical antifungal drug target due to its importance in virulence, absence in the host, and conservation among fungal pathogens. Inhibitors of Cho1 could serve as lead compounds for drug development, so we developed a target-based screen for inhibitors of purified Cho1. This enzyme condenses serine and cytidyldiphosphate-diacylglycerol (CDP-DAG) into phosphatidylserine (PS) and releases cytidylmonophosphate (CMP). Accordingly, we developed anin vitronucleotidase-coupled malachite-green-based high throughput assay for purifiedC. albicansCho1 that monitors CMP production as a proxy for PS synthesis. Over 7,300 molecules curated from repurposing chemical libraries were interrogated in primary and dose-responsivity assays using this platform. The screen had a promising averageZ’ score of ~0.8, and seven compounds were identified that inhibit Cho1. Three of these, ebselen, LOC14, and CBR-5884, exhibited antifungal effects againstC. albicanscells, with fungicidal inhibition by ebselen and fungistatic inhibition by LOC14 and CBR-5884. Only CBR-5884 showed evidence of disruptingin vivoCho1 function by inducing phenotypes consistent with thecho1∆∆mutant, including a reduction of cellular PS levels. Kinetics curves and computational docking indicate that CBR-5884 competes with serine for binding to Cho1 with aK_iof 1,550 ± 245.6 nM. Thus, this compound has the potential for development into an antifungal compound. IMPORTANCEFungal phosphatidylserine synthase (Cho1) is a logical antifungal target due to its crucial role in the virulence and viability of various fungal pathogens, and since it is absent in humans, drugs targeted at Cho1 are less likely to cause toxicity in patients. Using fungal Cho1 as a model, there have been two unsuccessful attempts to discover inhibitors for Cho1 homologs in whole-cell screens prior to this study. The compounds identified in these attempts do not act directly on the protein, resulting in the absence of known Cho1 inhibitors. The significance of our research is that we developed a high-throughput target-based assay and identified the first Cho1 inhibitor, CBR-5884, which acts both on the purified protein and its function in the cell. This molecule acts as a competitive inhibitor with aK_ivalue of 1,550 ± 245.6 nM and, thus, has the potential for development into a new class of antifungals targeting PS synthase.