Biology subjects

Costas, A.

Publications and source records attributed to Costas, A..

3 recordsLinked to original sources

bioRxiv2024

A simple ex vivo bladder infection model permits study of host-pathogen interactions in UTI

Urinary tract infections (UTI) are one of the most common infections, worldwide. To understand mechanisms of UTI pathogenesis and find new treatments, researchers often use animal models, such as mice or rats. However, studying certain phenotypes in animals can be difficult. Additionally, using animals in research comes with significant administrative and ethical challenges. To address these challenges, we developed a simple, reproducible, and cost-effective model to study UTI using donated mouse bladder tissue that would otherwise be discarded. This model allows researchers of all experience levels to study interactions between the host and pathogen in a controlled environment. We tested uropathogenic E. coli colonization and invasion of isolated urothelial sheets from 30 minutes to 24 hours, finding that bacterial burden in our ex vivo model was comparable to in vivo UTI mouse models. To optimize reproducibility, we tested multiple variables, including technical parameters, such as incubator conditions, and biological factors, such as biological sex or prior pregnancy in the donor mouse. This method offers several advantages, including assessment of early host-pathogen interactions, immune cell uptake of bacteria, the impact of age and sex of donor animals in infection, and diverse bacterial strains, mutants, or treatments. In addition, in some countries, sharing material recovered from animals sacrificed for other reasons does not require additional ethical approval by the receiving laboratory, providing a resource for labs without access to animals and reducing administrative burden. Given the breadth of the model with respect to sex, age, mouse and bacterial strain, and the ability to test any parameter that can be included in a 96-well plate, we believe this model will be useful to UTI researchers, with potential application beyond infection or even beyond the bladder to other tissues.

immunology↗

bioRxiv2024

E. coli division machinery drives cocci development inside host cells

Escherichia coli is arguably one of the most studied bacterial model systems in modern biology. Under normal laboratory conditions E. coli adopts its characteristic rod-shape. However, during stress conditions E. coli has been shown to undergo conditional morphology changes to inhibit division and grow into highly elongated forms. Here, on the other end of the morphology spectra, using an in-vitro infection model system combined with advanced imaging we show uropathogenic E. coli rods dividing to form and proliferate as cocci inside human bladder epithelial cells. In these intracellular bacterial communities, the frequency of cell division outpaced the rate of cell growth, resulting in smaller cocci cells. This mechanism was guided by an active FtsZ-governed division machinery, directed to midcell by division-site placement systems. These results show how a previously uncharacterised level of morphological plasticity occurs in bacteria with traditionally well-defined rod shape.

microbiology↗

bioRxiv2023

Genetic requirements for uropathogenic E. coli proliferation in the bladder cell infection cycle

Uropathogenic Escherichia coli (UPEC) requires an adaptable physiology to survive the wide range of environments experienced in the host, including gut and urinary tract surfaces. To identify UPEC genes required during intracellular infection, we developed a transposon-directed insertion-site sequencing (TraDIS) approach for cellular infection models and searched for genes in a library of [~]20,000 E. coli UTI89 transposon-insertion mutants that are specifically required for growth in M9-glycerol minimal medium, and at the distinct stages of infection of cultured bladder epithelial cells. Some of the functional requirements apparent for growth in M9-glycerol overlapped with those for the intracellular stage of infection, notably nutrient utilization, polysaccharide and macromolecule precursor biosynthesis, and cell envelope stress tolerance. Two genes implicated in both conditions were confirmed through independent gene deletion studies: neuC (sialic acid capsule biosynthesis) and hisF (histidine biosynthesis). Distinct sets of UPEC genes were also implicated in bacterial dispersal, where UPEC erupt from bladder cells in highly filamentous or motile forms upon exposure to human urine, and during recovery from infection in rich (LB) medium. Genes linked to septal peptidoglycan processes, ytfB and dedD, appeared to play roles in dispersal and may help stabilize cell division or the envelope during envelope stress created during infection. Our findings support a view that the host intracellular environment and infection cycle are multi-nutrient limited and create stress that demand an array of biosynthetic, cell envelope integrity and biofilm-related functions of UPEC. IMPORTANCEUrinary tract infections (UTIs) are one of the most frequent infections worldwide. Uropathogenic Escherichia coli (UPEC), which accounts for [~]80 % of UTIs, must rapidly adapt to highly variable host environments, such as the gut, bladder sub-surface and urine. In this study, we searched for UPEC genes required for bacterial growth and survival throughout the cellular infection cycle. Genes required for de novo synthesis of biomolecules and cell envelope integrity appeared to be important, and other genes were also implicated in bacterial dispersal and recovery from infection of cultured bladder cells. With further studies of individual gene function, their potential as therapeutic targets may be realized. This study expands knowledge of the UTI infection cycle and establishes an approach to genome-wide functional analyses of stage-resolved microbial infections.

microbiology↗