Biology subjects

Mark, D.

Publications and source records attributed to Mark, D..

4 recordsLinked to original sources

bioRxiv2025

Phage metagenome assembled genomes portraying anti-ESKAPE and anti CRISPR/Cas potential; datasets from sewage clinical settings of Western Uganda, subSaharan Africa

ESKAPE pathogens include Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp., which account for the major causes of mortality linked to the spread of infection and antimicrobial resistance (AMR) globally. Advances in omics approaches have pointed to bacteriophages as a promising alternative source of antibacterial agents. Here we enriched two samples from sewage and amplified them on Staphylococcus culture, followed by whole metagenome shotgun sequencing with Illumina NovaSe X. We performed metagenomic classification of high-quality sequence reads using the Kraken 2 database, to delineate the diversity and abundances of taxa. Thereafter we assembled the sequence reads with MAGAHIT and binned them with default parameters of the Bacterial and Viral Bioinformatics Resource Center (BV-BRC) before annotating each bin with PhageScope. From assembly, we recovered multiple metagenome-assembled genomes (MAGs) including Alistipes phage, Escherichia phage, Vibrio phage, Staphylococcus phage, Klebsiella phage and Acinetobacter phage, to mention the top six best hits. From annotation, while the Acinetobacter phage is virulent, the two Klebsiella phage and Staphylococcus phage are temperate. All the phages possess more than four lysis genes, with the potential to disrupt bacterial membranes. Exceptionally, Vibrio phage, Acinetobacter phage and Alistipes phage possess anti-CRISPR genes, the potential to counteract normal bacterial immune response to phage infection. These findings also inform that MAGs from the sewage have the potential to recover phages with anti-CRISPR/Cas activity, which is one of the desirable attributes for effective phage-bacterial infection to control the growth and multiplication of bacteria. Our datasets can be utilized for genome-guided selection of potent phages through lytic and host-range assays, towards the purification of endolysins (lysozymes) as alternative antibacterial agents. VALUE OF THE DATAO_LIMetagenome-assembled genomes (MAGs) of lytic phages could present a potential model to combat multidrug-resistant Staphylococcus, Klebsiella and Acinetobacter species, which are WHOs high-priority pathogens. C_LIO_LIPhages with bacterial infective potential can be used as model gene vehicles and vectors for gene and genome editing studies as they possess hydrolytic enzymes targeting the bacterial cell walls, chromosomal DNA sequences and anti-CRISPR/Cas proteins. C_LIO_LIComparing raw and processed datasets, MAGs provide an avenue for the pursuit of novel industrial strains from local resources in East Africa. C_LI

genomics↗

bioRxiv2024

Aurodox, a polyketide from Streptomyces goldiniensis, inhibits transcription of the type III secretion system of multiple Gram-negative pathogens

Gram-negative pathogens pose a significant threat due to their propensity for causing various infections, often coupled with formidable resistance to conventional antibiotic treatments. In light of this challenge, the development of antivirulence (AV) compounds emerges as a promising alternative strategy, aiming to disrupt key virulence mechanisms rather than directly targeting bacterial viability. One such compound, aurodox, derived from Streptomyces goldiniensis, has exhibited promising AV properties in our prior studies. Specifically, aurodox caused a marked downregulation in the expression and function of the E. coli type 3 secretion system (T3SS), a needle-like injectosome structure which is deployed to translocate effector proteins from the cytoplasm to the host target cells. However, the broader spectrum of aurodoxs efficacy against T3SS across diverse pathogens remained unanswered, prompting the focus of this work. Using quantitative real-time PCR, we show that aurodox exerts inhibitory effects on selected T3SS in various pathogens, including Salmonella typhimurium, Yersinia pseudotuberculosis, and Vibrio parahaemolyticus. However, aurodox was not a universal blocker of all secretion systems, showing selectivity in its mode-of-action, even within a single strain. This finding was verified using transcriptomics which demonstrated that aurodox selectively blocks the expression of the Salmonella typhimurium SPI-2 type T3SS whilst other pathogenicity islands, including the SPI-1 system were not inhibited. To delve deeper into the mechanisms governing aurodoxs efficacy against these pathogens, we analysed transcriptomic datasets from both E. coli and S. Typhimurium treated with aurodox. By identifying orthologous genes exhibiting differential expression in response to aurodox treatment across these pathogens, our study sheds light on the potential mechanisms underlying the action of this rediscovered antibiotic.

microbiology↗

bioRxiv2019

Tree Lab: Portable genomics for early detection of plant viruses and pests in Sub-Saharan Africa

In this case study we successfully teamed the PDQeX DNA purification technology developed by MicroGEM, New Zealand, with the MinION and MinIT mobile sequencing devices developed by Oxford Nanopore Technologies to produce an effective point-of-need field diagnostic system. The PDQeX extracts DNA using a cocktail of thermophilic proteinases and cell wall degrading enzymes, thermo-responsive extractor cartridges and a temperature control unit. This single-step closed system delivers purified DNA with no cross contamination. The MinIT is a newly released data processing unit that converts MinION raw signal output into base called data locally in real time, removing the need for high specification computers and large file transfers from the field. All three devices are battery powered with an exceptionally small footprint that facilitates transport and set up.\n\nTo evaluate and validate capability of the system for unbiased pathogen identification by realtime sequencing in a farmers field setting, we analysed samples collected from cassava plants grown by subsistence farmers in three sub-Sahara African countries (Tanzania, Uganda and Kenya). A range of viral pathogens, all with similar symptoms, greatly reduce yield or completely destroy cassava crops. 800 million people worldwide depend on cassava for food and yearly income, and viral diseases are a significant constraint on its production (https://cassavavirusactionproject.com). Early pathogen detection at a molecular level has great potential to rescue crops within a single growing season by providing results that inform decisions on disease management, use of appropriate virus resistant or replacement planting.\n\nThis case study presented conditions of working in-field with limited or no access to mains power, laboratory infrastructure, internet connectivity and highly variable ambient temperature. An additional challenge is that, generally, plant material contains inhibitors of downstream molecular processes making effective DNA purification critical. We successfully undertook real-time on-farm genome sequencing of samples collected from cassava plants on three farms, one in each country. Cassava mosaic begomoviruses were detected by sequencing leaf, stem, tuber and insect samples. The entire process, from arrival on farm to diagnosis including sample collection, processing and provisional sequencing results was complete in under 4 hours. The need for accurate, rapid and on-site diagnosis grows as globalized human activity accelerates. This technical breakthrough has applications that are relevant to human and animal health, environmental management and conservation.

genomics↗

bioRxiv2018

Real time portable genome sequencing for global food security

The United Nations has listed Zero Hunger as one of the 17 global sustainable development goals to end extreme poverty by 2030. Plant viruses are a major constraint to crop production globally causing an estimated $30 billion in damage 1 leaving millions of people food insecure 2. In Africa, agriculture employs up to 50% of the workforce, yet only contributes 15% to the GDP on average 3, suggesting that there is low productivity and limited value addition. This can be addressed through continued innovation in the fields of science and technology as suggested in the Science Agenda for Agriculture in Africa (S3A) 4. Sustainable management of plant viruses and their associated vectors must include efficient diagnostics for surveillance, detection and identification to inform disease management, including the development and strategic deployment of virus resistant varieties. To date, researchers have been utilizing conventional methods such as; PCR, qPCR, high throughput sequencing (RNA-Seq, DNA-Seq) and Sanger sequencing for pathogen identification. However, these methods are both costly and time consuming, delaying timely control actions. The emergence of new tools for real-time diagnostics, such as the Oxford Nanopore MinION, have recently proven useful for early detection of Ebola 6 and Zika 7,8, even in low resourced laboratories. For the first time globally, the MinION portable pocket DNA sequencer was used to sequence whole plant virus genomes. We used this technology to identify the begomoviruses causing the devastating CMD which is ravaging smallholder farmers crops in sub-Saharan Africa. Cassava, a carbohydrate crop from which tapioca originates, is a major source of calories for over eight hundred (800) million people worldwide. With this technology, farmers struggling with diseased crops can take immediate, restorative action to improve their livelihoods based on information about the health of their plants, generated using a portable, real-time DNA sequencing device.

plant biology↗