Biology subjects

Bradshaw, J.

Publications and source records attributed to Bradshaw, J..

4 recordsLinked to original sources

bioRxiv2025

citrOgen: a synthesis-free polysaccharide and protein antigen-presentation to antibody-induction platform

Existing technologies employed to generate antibodies against bacterial polysaccharides and proteins rely on the availability of purified or synthetic antigens. Here we present a genetics-based platform that utilises Citrobacter rodentium (CR), an enteric mouse pathogen, to both produce and present complex heterologous polysaccharides and protein antigen complexes during natural infection. As proof of concept, we use lipopolysaccharides (O), capsular polysaccharides (K) and type 3 fimbrial (T3F) antigens expressed by the WHO critical priority pathogens Klebsiella pneumoniae (KP) and Escherichia coli (EC). Following one infection cycle (28 days) CR induces specific IgG antibodies against KPO1, ECO25b, KPK2 and KPT3F. We demonstrate that the antibodies are functional in downstream applications including protection against pathogenic KP challenge, KP capsular serotyping and KP biofilm inhibition. Whilst KP and EC antigens were used as prototypical examples, this modular platform is now readily adaptable to generate antibodies against diverse polysaccharide and protein antigens, with basic science, public health and therapeutic applications.

microbiology↗

bioRxiv2024

The exopolysaccharide Poly-N-Acetyl-Glucosamine (PNAG) coats Klebsiella pneumoniae in vivo

The conserved bacterial polysaccharide Poly-N-Acetyl-Glucosamine (PNAG) is a potential broad-spectrum vaccine candidate. While the immunogenicity of PNAG-based vaccine candidates has been established, characterisation of PNAG production across clinically relevant bacteria remains largely unknown. In particular, PNAG production in the Gram-negative pathogen Klebsiella pneumoniae (KP) is not well understood. Here, we demonstrate that PNAG production is prevalent in clinical KP isolates, where it is secreted as extracellular networks during adherent growth conditions. However, during severe KP pulmonary infection, KP PNAG production undergoes a switch to a cell-associated phenotype, coating the bacterial cell surface. By screening a panel of isogenic KP mutants in prominent cell surface components ({Delta}wcaJ,{Delta} rmpADC,{Delta} rfb,{Delta} ompA and{Delta} ompk36), we identified KP capsular polysaccharide as a key determinant underpinning the phenotype. Deleting genes involved in capsule synthesis ({Delta}wcaJ) and regulation ({Delta}rmpADC) resulted in cell-associated PNAG during adherent growth and infection of alveolar epithelial cells in vitro. Taken together, we describe a novel interaction between KP surface polysaccharides and detect for the first time, cell-associated PNAG in KP during lung infection, highlighting PNAG as an attractive KP vaccine antigen. Author summaryThe Gram-negative pathogen Klebsiella pneumoniae (KP) is a leading cause of hospital-associated lung and bloodstream infections worldwide. As KP exhibits resistance to most frontline antibiotics, there is a growing demand for immune-based strategies to treat KP infections. Poly-N-Acetyl-Glucosamine (PNAG) is a surface sugar produced by most clinically relevant bacteria, including KP. However, relatively little is known about PNAG production in KP. Therefore, we set out to characterise PNAG production in KP during in vitro growth and following lung infection in a pulmonary mouse model. During in vitro growth, KP produces extracellular PNAG networks. In contrast, during an in vivo severe lung infection, PNAG is found cell-associated, coating the bacterial surface. We propose that the visible change in KP PNAG between in vitro and in vivo environments is due to crosstalk with capsule, another polysaccharide on the KP surface. Together, this supports PNAG as an attractive KP antigen.

microbiology↗

bioRxiv2024

Cryo-EM structure and evolutionary history of the conjugation surface exclusion protein TraT

Conjugation plays a major role in dissemination of antimicrobial resistance genes. Following transfer of IncF-like plasmids, recipients become refractory to a second wave of conjugation with the same plasmid via entry (TraS) and surface (TraT) exclusion mechanisms. Here, we show that TraT from the pKpQIL and F plasmids (TraTpKpQIL and TraTF) exhibits plasmid surface exclusion specificity. The cryo-EM structures of TraTpKpQIL and TraTF revealed that they oligomerise into decameric champagne bottle cork-like structures, which are anchored to the outer membrane via a diacylglycerol modified -helical barrel domain. Unexpectedly, we identified chromosomal TraT homologues from multiple Gram-negative phyla which formed numerous deep-branching lineages in a phylogenetic tree of TraT sequences. Plasmid-associated TraT sequences largely cluster into two separate lineages that have more recently evolved, incorporating TraT from Enterobacterales IncF and Legionellaceae F-like plasmids. These findings suggest that different plasmid backbones have acquired and co-opted TraT on independent occasions.

microbiology↗

bioRxiv2023

Aminoglycoside heteroresistance in Enterobacter cloacae is driven by the cell envelope stress response.

Enterobacter cloacae is a Gram-negative nosocomial pathogen of the ESKAPE priority group with increasing multi-drug resistance via the acquisition of resistance plasmids. However, E. cloacae can also display phenotypic antimicrobial resistance, such as heteroresistance or persistence. Here we report that E. cloacae ATCC 13047 and six strains isolated from patients with blood infections display heteroresistance or persistence to aminoglycosides. E. cloacae heteroresistance is transient, accompanied with formation of petite colonies and increased MIC against gentamicin and other aminoglycosides used in the clinic, but not other antibiotic classes. To explore the underlying mechanisms, we performed RNA sequencing of heteroresistant bacteria, which revealed global gene-expression changes and a signature of the CpxRA cell envelope stress response. Deletion of the cpxRA two-component system abrogated aminoglycoside heteroresistance and petite colony formation, pointing to its indispensable role in phenotypic resistance. The introduction of a constitutively active allele of cpxA led to high aminoglycoside MICs, consistent with cell envelope stress driving these behaviours in E. cloacae. Cell envelope stress can be caused by environmental cues, including heavy metals. Indeed, bacterial exposure to copper increased gentamicin MIC in the wild type, but not the {Delta}cpxRA mutant. Moreover, copper exposure also elevated the gentamicin MICs of bloodstream isolates, suggesting that CpxRA- and copper-dependent aminoglycoside resistance is broadly conserved in E. cloacae strains. Altogether, we establish that E. cloacae relies on transcriptional reprogramming via the envelope stress response pathway for transient resistance to a major class of frontline antibiotic. ImportanceEnterobacter cloacae is a bacterium that belongs to the ESKAPE priority group and an increasing threat worldwide due its multidrug resistance. E. cloacae can also display phenotypic resistance to antibiotics, leading to treatment failure. We report that sensitive strains of E. cloacae, including six strains isolated from patients with bloodstream infections, show heteroresistance or persistence to aminoglycoside antibiotics. These are important frontline microbicidal drugs used against Gram-negative bacterial infections, therefore understanding how resistance develops in sensitive strains is important. We show that aminoglycoside resistance is driven by the activation of the cell envelope stress response and transcriptional reprogramming via the CpxRA two-component system. Further, heterologous activation of envelope stress via copper, typically a heavy metal with antimicrobial actions, also increased aminoglycoside MICs in all tested strains of E. cloacae. Our study suggests phenotypic aminoglycoside resistance in E. cloacae could be broadly conserved and cautions against the undesirable effects of copper.

microbiology↗