Identification of Resistance Determinants for a Promising Antileishmanial Oxaborole Series

Current treatment options for visceral leishmaniasis have several drawbacks, and clinicians are confronted with an increasing number of treatment failures. To overcome this, the Drugs for Neglected Diseases initiative (DNDi) has invested in the development of novel antileishmanial leads, including a very promising class of oxaboroles. The mode of action/resistance of this series to Leishmania is still unknown and may be important for its further development and implementation. Repeated in vivo drug exposure and an in vitro selection procedure on both extracellular promastigote and intracellular amastigote stages were both unable to select for resistance. The use of specific inhibitors for ABC-transporters could not demonstrate the putative involvement of efflux pumps. Selection experiments and inhibitor studies, therefore, suggest that resistance to oxaboroles may not emerge readily in the field. The selection of a genome-wide cosmid library coupled to next-generation sequencing (Cos-seq) was used to identify resistance determinants and putative targets. This resulted in the identification of a highly enriched cosmid, harboring genes of chromosome 2 that confer a subtly increased resistance to the oxaboroles tested. Moderately enriched cosmids encompassing a region of chromosome 34 contained the cleavage and polyadenylation specificity factor (cpsf) gene, encoding the molecular target of several related benzoxaboroles in other organisms.

A predation assay using amoebae to screen for virulence factors unearthed the first W. chondrophila inclusion membrane protein

Waddlia chondrophila is an intracellular bacterium phylogenetically related to the well-studied human and animal pathogens of the Chlamydiaceae family. In the last decade, W. chondrophila was convincingly demonstrated to be associated with adverse pregnancy outcomes in humans and abortions in animals. All members of the phylum Chlamydiae possess a Type Three Secretion System that they use for delivering virulence proteins into the host cell cytosol to modulate their environment and create optimal conditions to complete their life cycle. To identify W. chondrophila virulence proteins, we used an original screening approach that combines a cosmid library with an assay monitoring resistance to predation by phagocytic amoebae. This technique combined with bioinformatic data allowed the identification of 28 candidate virulence proteins, including Wimp1, the first identified inclusion membrane protein of W. chondrophila.

Pseudomonas aeruginosaRegulated Intramembrane Proteolysis: Protease MucP Can Overcome Mutations in the AlgO Periplasmic Protease To Restore Alginate Production in Nonmucoid Revertants

ABSTRACTThe progression of cystic fibrosis (CF) from an acute to a chronic disease is often associated with the conversion of the opportunistic pathogenPseudomonas aeruginosafrom a nonmucoid form to a mucoid form in the lung. This conversion involves the constitutive synthesis of the exopolysaccharide alginate, whose production is under the control of the AlgT/U sigma factor. This factor is regulated posttranslationally by an extremely unstable process and has been commonly attributed to mutations in thealgT(algU) gene. By exploiting this unstable phenotype, we isolated 34 spontaneous nonmucoid variants arising from the mucoid strain PDO300, a PAO1 derivative containing themucA22allele commonly found in mucoid CF isolates. Complementation analysis using a minimal tiling path cosmid library revealed that most of these mutants mapped to two protease-encoding genes,algO, also known asprcorPA3257, andmucP. Interestingly, ouralgOmutations were complemented by bothmucPandalgO, leading us to delete, clone, and overexpressmucP,algO,mucE, andmucDin both wild-type PAO1 and PDO300 backgrounds to better understand the regulation of this complex regulatory mechanism. Our findings suggest that the regulatory proteases follow two pathways for regulated intramembrane proteolysis (RIP), where both the AlgO/MucP pathway and MucE/AlgW pathway are required in the wild-type strain but where the AlgO/MucP pathway can bypass the MucE/AlgW pathway in mucoid strains with membrane-associated forms of MucA with shortened C termini, such as the MucA22 variant. This work gives us a better understanding of how alginate production is regulated in the clinically important mucoid variants ofPseudomonas aeruginosa.IMPORTANCEInfection by the opportunistic pathogenPseudomonas aeruginosais the leading cause of morbidity and mortality seen in CF patients. Poor patient prognosis correlates with the genotypic and phenotypic change of the bacteria from a typical nonmucoid to a mucoid form in the CF lung, characterized by the overproduction of alginate. The expression of this exopolysaccharide is under the control an alternate sigma factor, AlgT/U, that is regulated posttranslationally by a series of proteases. A better understanding of this regulatory phenomenon will help in the development of therapies targeting alginate production, ultimately leading to an increase in the length and quality of life for those suffering from CF.

Cellulolytic Activities of the Dung Beetle, Euoniticellus Intermedius, Larva Gut Micro-Flora

Background:The life style and biology of dung beetles offer a significant opportunity for innovation in biofuel production. The larvae of the African dung beetle,Euoniticellus intermedius, feed solely on cow dung, eating and digesting the fibre while adults live on juices found in fresh dung. The larval gut system consists of a small, almost unrecognizable foregut and two distinct chambers; the midgut and hindgut. It is clear that these two chambers are the centres in which the dung material whose composition includes cellulose is processed. The goal of this study was to assess the cellulolytic activities of cultured gut micro-flora derived fromE. intermedius, (Coleoptera: Scarabaeida).Method:Late second to third instar stageE. intermediuslarvae were dissected and the isolated gut micro-flora consortia aerobically cultured in media containing cellulose (filter paper) as the sole carbon source. Genomic DNA isolation was done on the gut consortia cultures after 10 days of culturing, using the ZR Fungal/Bacterial DNA MiniPrep kit (Zymo Research, USA). A complete and unbiased primary cosmid library was then constructed from the isolated genomic DNA using a cloning ready, pWEB-TNC™ Cosmid Cloning kit (EPICENTRE Biotechnologies, USA). The primary cosmid library clones were screened for endo-glucanase and cellobiohydrolase activities using Carboxymethyl Cellulose (CMC) and 4-Methylumbelliferyl-β-D-Cellobioside (MUC) plate assays respectively.Results:Results indicate that a total of 7 colonies out of 160 screened colonies showed positive CMC and MUC activities.Conclusion:This proves thatE. intermediusis a potential source of cellulolytic micro-organisms and enzymes that can be used for cellulose derived biofuel production.

Functional metagenomics using Pseudomonas putida expands the known diversity of polyhydroxyalkanoate synthases and enables the production of novel polyhydroxyalkanoate copolymers

Bacterially produced biodegradable polyhydroxyalkanoates with versatile properties can be achieved using different PHA synthase enzymes. This work aims to expand the diversity of known PHA synthases via functional metagenomics, and demonstrates the use of these novel enzymes in PHA production. Complementation of a PHA synthesis deficient Pseudomonas putida strain with a soil metagenomic cosmid library retrieved 27 clones expressing either Class I, Class II or unclassified PHA synthases, and many did not have close sequence matches to known PHA synthases. The composition of PHA produced by these clones was dependent on both the supplied growth substrates and the nature of the PHA synthase, with various combinations of SCL- and MCL-PHA. These data demonstrate the ability to isolate diverse genes for PHA synthesis by functional metagenomics, and their use for the production of a variety of PHA polymer and copolymer mixtures.

Strong spurious transcription likely a cause of DNA insert bias in typical metagenomic clone libraries

Background: Clone libraries provide researchers with a powerful resource with which to study nucleic acid from diverse sources. Metagenomic clone libraries in particular have aided in studies of microbial biodiversity and function, as well as allowed the mining of novel enzymes for specific functions of interest. These libraries are often constructed by cloning large-inserts (~30 kb) into a cosmid or fosmid vector. Recently, there have been reports of GC bias in fosmid metagenomic clone libraries, and it was speculated that the bias may be a result of fragmentation and loss of AT-rich sequences during the cloning process. However, evidence in the literature suggests that transcriptional activity or gene product toxicity may play a role in library bias. Results: To explore the possible mechanisms responsible for sequence bias in clone libraries, and in particular whether fragmentation is involved, we constructed a cosmid clone library from a human microbiome sample, and sequenced DNA from three different steps of the library construction process: crude extract DNA, size-selected DNA, and cosmid library DNA. We confirmed a GC bias in the final constructed cosmid library, and we provide strong evidence that the sequence bias is not due to fragmentation and loss of AT-rich sequences but is likely occurring after the DNA is introduced into E. coli. To investigate the influence of strong constitutive transcription, we searched the sequence data for consensus promoters and found that rpoD/sigma-70 promoter sequences were underrepresented in the cosmid library. Furthermore, when we examined the reference genomes of taxa that were differentially abundant in the cosmid library relative to the original sample, we found that the bias appears to be more closely correlated with the number of rpoD/sigma-70 consensus sequences in the genome than with simple GC content. Conclusions: The GC bias of metagenomic clone libraries does not appear to be due to DNA fragmentation. Rather, analysis of promoter consensus sequences provides support for the hypothesis that strong constitutive transcription from sequences recognized as rpoD/sigma-70 consensus-like in E. coli may lead to plasmid instability or loss of insert DNA. Our results suggest that despite widespread use of E. coli to propagate foreign DNA, the effects of in vivo transcriptional activity may be under-appreciated. Further work is required to tease apart the effects of transcription from those of gene product toxicity.