Biology subjects

MacArthur, I.

Publications and source records attributed to MacArthur, I..

6 recordsLinked to original sources

bioRxiv2024

A Novel Adhesin of B. pertussis is Key to Colonisation of Epithelial Cells

Despite effective vaccines to protect against Whooping cough, or pertussis, the disease is resurgent in many countries. A switch from acellular to whole-cell vaccines has resulted in waning protective immunity, likely contributing to increases in infection prevalence, underlining the need to better understand B. pertussis virulence. As a respiratory pathogen, B. pertussis colonises the upper respiratory tract utilising an array of adhesins, four of which (FHA, pertactin, Fim2/3) are included in the acellular vaccine. In this study, we identified two Bvg regulated genes that are upregulated during virulence conditions and thus potentially involved in pathogenesis. bp1251 and bp1252 encode orphan toxin B subunits, with homology to AB toxin B subunits. Mutation of bp1251 and bp1252 reduced the in vitro adherence of B. pertussis to A549 and BEAS-2B alveolar and bronchial epithelial-like cells. In a murine model of infection, bp1251 and bp1252 mutant strains were recovered from the nasal cavity and lungs at lower levels than WT. In vitro no effect of mutation of bp1251 or bp1252 on cell invasion or toxicity was found suggesting that these proteins do not form part of a toxin. Given their homology to B subunits of AB toxins, and their role in colonisation, we propose that BP1251 and BP1252 are novel adhesins. Our data suggests that these proteins play a significant role in Bordetella infection and have the potential to further the understanding of B. pertussis pathogenesis.

microbiology↗

bioRxiv2020

Duplications drive diversity in Bordetella pertussis on an underestimated scale.

Bacterial genetic diversity is often described using solely base pair changes despite a wide variety of other mutation types likely being major contributors. Tandem duplications of genomic loci are thought to be widespread among bacteria but due to their often intractable size and instability, comprehensive studies of the range and genome dynamics of these mutations are rare. We define a methodology to investigate duplications in bacterial genomes based on read depth of genome sequence data as a proxy for copy number. We demonstrate the approach with Bordetella pertussis, whose insertion sequence element-rich genome provides extensive scope for duplications to occur. Analysis of genome sequence data for 2430 B. pertussis isolates identified 272 putative duplications, of which 94% were located at 11 hotspot loci. We demonstrate limited phylogenetic connection for the occurrence of duplications, suggesting unstable and sporadic characteristics. Genome instability was further described in-vitro using long read sequencing via the Nanopore platform. Clonally derived laboratory cultures produced heterogenous populations containing multiple structural variants. Short read data was used to predict 272 duplications, whilst long reads generated on the Nanopore platform enabled the in-depth study of the genome dynamics of tandem duplications in B. pertussis. Our work reveals the unrecognised and dynamic genetic diversity of B. pertussis and, as the complexity of the B. pertussis genome is not unique, highlights the need for a holistic and fundamental understanding of bacterial genetics.

microbiology↗

bioRxiv2019

A qPCR assay for Bordetella pertussis cells that enumerates both live and dead bacteria.

Bordetella pertussis is the causative agent of whooping cough, commonly referred to as pertussis. Although the incidence of pertussis was reduced through vaccination, during the last thirty years it has returned to high levels in a number of countries. This resurgence has been linked to the switch from the use of whole-cell to acellular vaccines. Protection afforded by acellular vaccines appears to be short-lived compared to that afforded by whole cell vaccines. In order to inform future vaccine improvement by identifying immune correlates of protection, a human challenge model of B. pertussis colonisation has been developed. Accurate measurement of colonisation status in this model has required development of a qPCR-based assay to enumerate B. pertussis in samples that distinguishes between viable and dead bacteria. Here we report the development of this assay and its performance in the quantification of B. pertussis from human challenge model samples. This assay has future utility in diagnostic labs and in research where a quantitative measure of both B. pertussis number and viability is required.

microbiology↗

bioRxiv2018

Prohibitin is a prognostic marker of relapse and therapeutic target to block chemotherapy resistance in Wilms tumor

Wilms tumor (WT) is the most common childhood kidney cancer. To improve risk stratification and identify novel therapeutic targets for patients with WT, we used high-resolution mass spectrometry proteomics to identify urine tumor markers associated with WT relapse. We determined urine proteomes at diagnosis of 49 patients with WT, non-WT renal tumors, and age-matched controls, leading to the quantitation of 6,520 urine proteins. Supervised analysis revealed specific urine markers of renal rhabdoid tumors, kidney clear cell sarcomas, renal cell carcinomas, as well as those detected in cured and relapsed WT. In particular, urine prohibitin was significantly elevated at diagnosis in patients with relapsed as compared to cured WT. In a validation cohort of 139 patients, a specific urine prohibitin enzyme-linked immunosorbent assay demonstrated that prohibitin concentrations greater than 998 ng/mL at diagnosis were significantly associated with ultimate WT relapse. Immunohistochemical analysis revealed that prohibitin was highly expressed in primary WT specimens and associated with disease stage. Using functional genetic experiments, we found that prohibitin was required for the growth and survival of WT cells. Overexpression of prohibitin was sufficient to block intrinsic mitochondrial apoptosis and to cause resistance to diverse chemotherapy drugs, at least in part by dysregulating factors that control apoptotic cytochrome c release from mitochondrial cristae. Thus, urine prohibitin may improve therapy stratification, non-invasive monitoring of treatment response and early disease detection. In addition, therapeutic targeting of chemotherapy resistance induced by prohibitin dysregulation may offer improved therapies for patients with Wilms and other relapsed or refractory tumors.

cancer biology↗

bioRxiv2017

Therapeutic targeting of PGBD5-induced DNA repair dependency in pediatric solid tumors

Despite intense efforts, the cure rates of childhood and adult solid tumors are not satisfactory. Resistance to intensive chemotherapy is common, and targets for molecular therapies are largely undefined. We have now found that the majority of childhood solid tumors, including rhabdoid tumors, neuroblastoma, medulloblastoma and Ewing sarcoma, express an active DNA transposase PGBD5 that can promote site-specific genomic rearrangements in human cells. Using functional genetic approaches, we found that mouse and human cells deficient in non-homologous end joining (NHEJ) DNA repair cannot tolerate the expression of PGBD5. In a chemical screen of DNA damage signaling inhibitors, we identified AZD6738 as a specific sensitizer of PGBD5-dependent DNA damage and apoptosis. We found that expression of PGBD5, but not its nuclease activity-deficient mutant, was sufficient to induce hypersensitivity to AZD6738. Depletion of endogenous PGBD5 conferred resistance to AZD6738 in human tumor cells. PGBD5-expressing tumor cells accumulated unrepaired DNA damage in response to AZD6738 treatment, and underwent apoptosis in both dividing and G1 phase cells in the absence of immediate DNA replication stress. Accordingly, AZD6738 exhibited nanomolar potency against the majority of neuroblastoma, medulloblastoma, Ewing sarcoma and rhabdoid tumor cells tested, while sparing non-transformed human and mouse embryonic fibroblasts in vitro. Finally, treatment with AZD6738 induced apoptosis and regression of human neuroblastoma and medulloblastoma tumors engrafted in immunodeficient mice in vivo. This effect was potentiated by combined treatment with cisplatin, including significant anti-tumor activity against patient-derived primary neuroblastoma xenografts. These findings delineate a therapeutically actionable synthetic dependency induced in PGBD5-expressing solid tumors.

cancer biology↗

bioRxiv2017

Human PGBD5 DNA transposase promotes site-specific oncogenic mutations in rhabdoid tumors

Genomic rearrangements are a hallmark of childhood solid tumors, but their mutational causes remain poorly understood. Here, we identify the piggyBac transposable element derived 5 (PGBD5) gene as an enzymatically active human DNA transposase expressed in the majority of rhabdoid tumors, a lethal childhood cancer. Using assembly-based whole-genome DNA sequencing, we observed previously unknown somatic genomic rearrangements in primary human rhabdoid tumors. These rearrangements were characterized by deletions and inversions involving PGBD5-specific signal (PSS) sequences at their breakpoints, with some recurrently targeting tumor suppressor genes, leading to their inactivation. PGBD5 was found to be physically associated with human genomic PSS sequences that were also sufficient to mediate PGBD5-induced DNA rearrangements in rhabdoid tumor cells. We found that ectopic expression of PGBD5 in primary immortalized human cells was sufficient to promote penetrant cell transformation in vitro and in immunodeficient mice in vivo. This activity required specific catalytic residues in the PGBD5 transposase domain, as well as end-joining DNA repair, and induced distinct structural rearrangements, involving PSS-associated breakpoints, similar to those found in primary human rhabdoid tumors. This defines PGBD5 as an oncogenic mutator and provides a plausible mechanism for site-specific DNA rearrangements in childhood and adult solid tumors.

cancer biology↗