Biology subjects

Dorman, M. J.

Publications and source records attributed to Dorman, M. J..

7 recordsLinked to original sources

bioRxiv2025

Genome-wide analysis exploring mechanisms used by Shigella sonnei to survive long-term nutrient starvation

Shigella is a major cause of severe diarrhoea with S. flexneri and S. sonnei accounting for over 90% of infections. As economies grow, S. sonnei replaces S. flexneri as the dominant cause of shigellosis, however the basis of this epidemiological shift remains unclear. Here we investigated whether S. sonnei is better equipped to survive nutrient starvation, a crucial condition for persistence both outside the host and within the colonic lumen. S. sonnei exhibited greater survival under long-term nutrient starvation (LTNS) than S. flexneri, rapidly activating survival mechanisms. We interrogated the genome of S. sonnei using Transposon Directed Insertion-site Sequencing (TraDIS) revealing that metabolic pathways (ATP, nucleotide, and amino acid synthesis), and envelope homeostasis complexes (e.g., Tol-Pal, Bam) are conditionally essential for LTNS. TraDIS findings were validated by non-competitive and competitive survival of wild-type and deletion mutant strains. We compared the homology of conditionally essential genes between S. sonnei and S. flexneri to identify putative genetic markers of differential interspecies LTNS survival. Analysis of ldcA (a peptidoglycan carboxypeptidase) and rseA (the anti-sigma factor regulator) indicated a major role in sustaining survival in LTNS in S. sonnei; however, allele-swap with S. flexneri alleles restored wild-type survival in S. sonnei suggesting that monogenic changes may not explain the divergent survival of these two species. Together, these data define the molecular adaptations of starvation resistance in S. sonnei and provide insights into its epidemiological dominance in high-income countries. Author summaryUnderstanding why S. sonnei has a higher prevalence over S. flexneri as a country undergoes economic growth is one of the most important challenges in Shigella research. The investigation of their biological and genetic differences is key to tackle the impact of the disease. We discovered that S. sonnei resists nutrient deprivation better than S. flexneri, suggesting a better adaptation to an extracellular lifestyle and a greater preservation of metabolic capabilities. Using a genome-wide transposon sequencing approach we uncovered the key pathways behind the survival of S. sonnei facing nutrient starvation, which include ATP, nucleotide, and amino acid synthesis as well as maintenance of cell envelope integrity. Comparative analysis between S. sonnei and S. flexneri did not identify a single gene responsible for the differing survival and we suggest that the differing survival may stem from the coordination of multigenic differences. Our data provides a genome-wide basis for understanding how S. sonnei adapted to nutrient-deprived settings, which may be advantageous in the gut lumen and also in hostile environments, potentially contributing to its dominance in high-income countries.

microbiology↗

bioRxiv2025

Aeromonas: Genomic Insights into an Environmental Pathogen and Reservoir of Antimicrobial Resistance

Aeromonads are an ecologically versatile group of bacteria that cause infection in aquatic animals and are recognised as an emerging human pathogen. Despite this, our understanding of Aeromonas diversity, especially the relationship between clinical and environmental strains, remains limited. Here, we present a complete view of the Aeromonas genus, comprising 1,853 genomes, and a detailed comparison of clinical and environmental strains from South Asia, including 996 newly sequenced genomes from Bangladesh and India. Phylogenetic analyses revealed that Aeromonas is a highly diverse genus, with no distinct clade separating clinical and environmental isolates. We identified 28 Aeromonas species and 905 novel sequence types, comprising 72.5% of the genomes. Notably, we show a high incidence of AMR genes across all isolates, including against front and last-line antibiotics. Finally, we highlight frequent misidentification of Aeromonas as Vibrio cholerae, key to cholera-endemic regions where both genera co-exist and are associated with diarrhoeal disease. Our study underscores Aeromonas as an important environmental AMR reservoir and emerging multi-species pathogen capable of spilling over into human populations.

genomics↗

bioRxiv2024

De novo pyrimidine biosynthesis inhibition synergizes with BCL-XL targeting in pancreatic cancer

Oncogenic KRAS, the genetic driver of 90% of pancreatic adenocarcinoma (PDAC), induces a metabolic rewiring characterized, in part, by dependency on de novo pyrimidine biosynthesis. Pharmacologic inhibition of dihydroorotate dehydrogenase (DHODH), an enzyme in the de novo pyrimidine synthesis pathway, delays pancreatic tumor growth in vivo; however, limited monotherapy efficacy suggests compensatory pathways and that combinatorial strategies are required for enhanced efficacy. Here, we use an integrated metabolomic, quantitative temporal proteomic and in vitro and in vivo DHODH inhibitor anchored CRISPR/Cas9 genetic screening approach to identify compensatory pathways to DHODH inhibition (DHODHi) and targets for combination strategies. We demonstrate that DHODHi alters the apoptotic regulatory proteome thereby enhancing sensitivity to inhibitors of the anti-apoptotic BCL2L1 (BCL-XL) protein. Combinatorial regimens with DHODH and BCL-XL inhibition synergistically induce apoptosis in PDAC cell lines and patient-derived PDAC organoids. In vivo DHODH inhibition with Brequinar and BCL-XL degradation with DT2216, a proteolysis targeting chimera (PROTAC), significantly inhibits the growth of PDAC tumors. Our data defines mechanisms of adaptation to DHODH inhibition and identifies a combination therapy strategy in PDAC.

cancer biology↗

bioRxiv2022

Genomic epidemiology of the cholera outbreak in Yemen reveals the spread of a multi-drug resistance plasmid between diverse lineages of Vibrio cholerae

The humanitarian crisis in Yemen led in 2016 to the biggest cholera outbreak documented in modern history, with more than 2.5 million suspected cases to date. In late 2018, epidemiological surveillance showed that V. cholerae isolated from cholera patients had turned multi-drug resistant (MDR). We generated genomes from 260 isolates sampled in Yemen between 2018 and 2019 to identify a possible shift in circulating genotypes. 84% of V. cholerae isolates were serogroup O1 belonging to the seventh pandemic El Tor (7PET) lineage, sublineage T13 - same as in 2016 and 2017 - while the remaining 16% of strains were non-toxigenic and belonged to divergent V. cholerae lineages, likely reflecting sporadic gut colonisation by endemic strains. Phylogenomic analysis reveals a succession of T13 clones, with 2019 dominated by a clone that carried an IncC-type plasmid harbouring an MDR pseudo-compound transposon (PCT). Identical copies of these mobile elements were found independently in several unrelated lineages, suggesting exchange and recombination between endemic and epidemic strains. Treatment of severe cholera patients with macrolides in Yemen from 2016 to early 2019 coincides with the emergence of the plasmid-carrying T13 clone. The unprecedented success of this genotype where an SXT-family integrative and conjugative element (SXT/ICE) and an IncC plasmid coinhabit show the stability of this MDR plasmid in the 7PET background, which may durably reduce options for epidemic cholera case management. We advocate a heightened genomic epidemiology surveillance of cholera to help control the spread of this highly-transmissible, MDR clone.

microbiology↗

bioRxiv2021

gbpA and chiA genes are not uniformly distributed amongst diverse Vibrio cholerae

Members of the bacterial genus Vibrio utilise chitin both as a metabolic substrate and a signal to activate natural competence. Vibrio cholerae is a bacterial enteric pathogen, sub-lineages of which can cause pandemic cholera. However, the chitin metabolic pathway in V. cholerae has been dissected using only a limited number of laboratory strains of this species. Here, we survey the complement of key chitin metabolism genes amongst 195 diverse V. cholerae. We show that the gene encoding GbpA, known to be an important colonisation and virulence factor in pandemic isolates, is not ubiquitous amongst V. cholerae. We also identify a putatively novel chitinase, and present experimental evidence in support of its functionality. Our data indicate that the chitin metabolic pathway within the V. cholerae species is more complex than previously thought, and emphasise the importance of considering genes and functions in the context of a species in its entirety, rather than simply relying on traditional reference strains. Impact statementIt is thought that the ability to metabolise chitin is ubiquitous amongst Vibrio spp., and that this enables these species to survive in aqueous and estuarine environmental contexts. Although chitin metabolism pathways have been detailed in several members of this genus, little is known about how these processes vary within a single Vibrio species. Here, we present the distribution of genes encoding key chitinase and chitin-binding proteins across diverse Vibrio cholerae, and show that our canonical understanding of this pathway in this species is challenged when isolates from non-pandemic V. cholerae lineages are considered alongside those linked to pandemics. Furthermore, we show that genes previously thought to be species core genes are not in fact ubiquitous, and we identify novel components of the chitin metabolic cascade in this species, and present functional validation for these observations. Data summaryThe authors confirm that all supporting data, code, and protocols have been provided within the article or through supplementary data files. O_LINo whole-genome sequencing data were generated in this study. Accession numbers for the publicly-available sequences used for these analyses are listed in Supplementary Table 1, Table 2, and the Methods. C_LIO_LIAll other data which underpin the figures in this manuscript, including pangenome data matrices, modified and unmodified sequence alignments and phylogenetic trees, original images of gels and immunoblots, raw fluorescence data, amplicon sequencing reads, and the R code used to generate Figure 7, are available in Figshare: https://dx.doi.org/10.6084/m9.figshare.13169189 (Note for peer-review: Figshare DOI is inactive but will be activated upon publication, please use temporary URL https://figshare.com/s/7795a2d80c13f694f8fa for review). C_LI O_FIG O_LINKSMALLFIG WIDTH=183 HEIGHT=200 SRC="FIGDIR/small/430729v1_fig7.gif" ALT="Figure 7"> View larger version (38K): org.highwire.dtl.DTLVardef@100c246org.highwire.dtl.DTLVardef@d295aborg.highwire.dtl.DTLVardef@1601f69org.highwire.dtl.DTLVardef@1ae3ecb_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 7.C_FLOATNO ChiA-3-6xHis displays chitobiosidase and endochitinase activities, but not -N-acetylglucosaminidase activity. Lysates and supernatants included in Figures 6b and 6c were assayed for chitinase enzyme activity using a fluorometric chitinase assay kit (see Methods for details). Lysed cells from E. coli cultures harbouring pMJD157 and cultured in the presence of arabinose were the only samples which produced detectable and statistically significant signals on triacetylchitotriose and chitobiose substrates (a, b). No signal was detected in the presence of glucosaminide substrate (c). All plots are scaled equivalently. P = pellet; S = supernatant; EV = empty vector (pBAD33). Parametric t-tests performed where indicated: ns = not significant; *** = p<0.001; **** = p<0.0001. C_FIG

microbiology↗

bioRxiv2018

The capsule regulatory network of Klebsiella pneumoniae defined by density-TraDISort

Klebsiella pneumoniae infections affect infants and the immunocompromised, and the recent emergence of hypervirulent and multi-drug resistant K. pneumoniae lineages is a critical healthcare concern. Hypervirulence in K. pneumoniae is mediated by several factors, including the overproduction of extracellular capsule. However, the full details of how K. pneumoniae capsule biosynthesis is achieved or regulated are not known. We have developed a robust and sensitive procedure to identify genes influencing capsule production, density-TraDISort, which combines density gradient centrifugation with transposon-insertion sequencing. We have used this method to explore capsule regulation in two clinically-relevant Klebsiella strains; K. pneumoniae NTUH-K2044 (capsule type K1), and K. pneumoniae ATCC43816 (capsule type K2). We identified multiple genes required for full capsule production in K. pneumoniae, as well as putative suppressors of capsule in NTUH-K2044, and have validated the results of our screen with targeted knockout mutants. Further investigation of several of the K. pneumoniae capsule regulators identified - ArgR, MprA/KvrB, SlyA/KvrA and the Sap ABC transporter - revealed effects on capsule amount and architecture, serum resistance and virulence. We show that capsule production in K. pneumoniae is at the centre of a complex regulatory network involving multiple global regulators and environmental cues, and that the majority of capsule regulatory genes are located in the core genome. Overall our findings expand our understanding of how capsule is regulated in this medically-important pathogen, and provide a technology that can be easily implemented to study capsule regulation in other bacterial species.\n\nImportanceCapsule production is essential for K. pneumoniae to cause infections, but its regulation and mechanism of synthesis are not fully understood in this organism. We have developed and applied a new method for genome-wide identification of capsule regulators. Using this method, many genes that positively or negatively affect capsule production in K. pneumoniae were identified, and we use these data to propose an integrated model for capsule regulation in this species. Several of the genes and biological processes identified have not previously been linked to capsule synthesis. We also show that the methods presented here can be applied to other species of capsulated bacteria, providing the opportunity to explore and compare capsule regulatory networks in other bacterial strains and species.

microbiology↗

bioRxiv2018

KRASG12D and TP53R167H Cooperate to Induce Pancreatic Ductal Adenocarcinoma in Sus Scrofa Pigs

Although survival has improved in recent years, the prognosis of patients with advanced pancreatic ductal adenocarcinoma (PDAC) remains poor. Despite substantial differences in anatomy, physiology, genetics, and metabolism, the overwhelming majority of preclinical testing relies on transgenic mice. Hence, while mice have allowed for tremendous advances in cancer biology, they have been a poor predictor of drug performance/toxicity in the clinic. Given the greater similarity of sus scrofa pigs to humans, we engineered transgenic sus scrofa expressing a LSL-KRASG12D-TP53R167H cassette. By applying Adeno-Cre to pancreatic duct cells in vitro, cells self-immortalized and established tumors in immunocompromised mice. When Adeno-Cre was administered to the main pancreaticduct in vivo, pigs developed extensive PDAC at the injection site hallmarked by excessive proliferation and desmoplastic stroma. This serves as the first large animal model of pancreatic carcinogenesis, and may allow for insight into new avenues of translational research not before possible in rodents.

cancer biology↗