Biology subjects

Boyce, J. D.

Publications and source records attributed to Boyce, J. D..

3 recordsLinked to original sources

bioRxiv2025

Comparative analysis of shared and unique mechanisms important for diverse strains of Pasteurella multocida to cause systemic infection in mice

Pasteurella multocida is a Gram-negative bacterium that causes a range of distinct diseases in livestock animals. Different P. multocida diseases are associated with different capsule and lipopolysaccharide (LPS) types, but little else is known about what underpins this disease specificity. In this study, we utilised transposon-directed insertion site sequencing (TraDIS) to identify genes in two diverse P. multocida strains that are required for growth in rich media, and genes important for survival during systemic infections in BALB/c mice. Analysis of growth in heart infusion broth showed that the fowl cholera isolate VP161 (capsule type A and LPS type L1) and the bovine haemorrhagic septicaemia isolate M1404 (capsule type B and LPS type L2) shared 461 genes essential for growth in rich media, with 95% of these present in all publicly available closed P. multocida genomes. In vivo fitness analysis identified 63 and 94 genes important for VP161 and M1404 survival in BALB/c mice, respectively. Only 35 homologs were identified as important for survival in both strains, showing that conserved biological systems can be differentially important for different P. multocida strains. Investigation of proteins involved in the catabolite response showed that an active cyclic-adenosine monophosphate (cAMP) receptor protein (CRP) was required for maximal fitness in M1404. Furthermore, disrupting CRP or cAMP production also reduced capsule production in M1404, but increased capsule production in VP161, demonstrating that these P. multocida strains have different regulatory systems for crucial virulence factors. Author SummaryPasteurella multocida is an important livestock pathogen, causing several distinct severe diseases in many different livestock animals. P. multocida can spread rapidly throughout animal populations, with peracute infections causing death within 48 h, resulting in large outbreaks with high mortality. Host predilection and disease presentation often correlate with the capsule and lipopolysaccharide type produced by the causative strain. However, the processes that allow certain strains to cause a particular disease are not well understood. In this study, we have comprehensively identified genes required for two P. multocida strains to cause systemic infection in mice, showing that these diverse strains have differential requirements for survival during a systemic infection. This information is crucial for understanding P. multocida diseases and for the development of new strategies to combat infection.

microbiology↗

bioRxiv2024

Delivery determinants of an Acinetobacter baumannii type VI secretion system bifunctional peptidoglycan hydrolase.

Acinetobacter baumannii is a Gram-negative opportunistic pathogen that is a common cause of nosocomial infections. The increasing development of antibiotic resistance in this organism is a global health concern. The A. baumannii clinical isolate AB307-0294 produces a type VI secretion system (T6SS) that delivers three antibacterial cargo effector proteins (Tse15, Tde16 and Tae17) that give this strain a competitive advantage against other bacteria in polymicrobial environments. These effectors are delivered via specific non-covalent interactions with the T6SS needle tip proteins VgrG15, VgrG16 and VgrG17, respectively. Here we determine the molecular function of the Tae17 effector protein and define the regions of Tae17 and VgrG17 essential for its delivery. Specifically, we show that Tae17 is a multidomain, bifunctional peptidoglycan-degrading enzyme. Tae17 has both lytic transglycosylase activity, which targets the peptidoglycan sugar backbone, and amidase activity, which targets the sugar-peptide bonds. Moreover, we show that the transglycosylase activity was more important for killing Escherichia coli. Using deletion constructs and bacterial two-hybrid analyses, we identify that amino acids 1051-1085 of the VgrG17 needle tip protein and amino acids 1-162 of the Tae17 effector protein are necessary for the Tae17:VgrG17 interaction. Furthermore, we identify the VgrG17 amino acids G1069 and W1075 as crucial for the delivery of Tae17; the first time such specific delivery determinants of T6SS cargo effectors have been defined. This study provides molecular insight into how the T6SS allows A. baumannii strains to gain dominance in polymicrobial communities and thus improve their chances of survival and transmission. IMPORTANCEWe have shown that the Acinetobacter baumannii T6SS effector Tae17 is a modular, bifunctional, peptidoglycan-degrading enzyme that has both lytic transglycosylase and amidase activity. Both activities contribute to the ability to degrade peptidoglycan, but the glycosyltransferase activity was more important for the interbacterial killing of Escherichia coli. We have defined the specific regions of Tae17 and its cognate delivery protein VgrG17 that are necessary for the non-covalent interactions and, for the first time, identified specific amino acids essential for delivery. This work contributes to our molecular understanding of bacterial competition strategies in polymicrobial environments and may provide a window to the design of new therapeutic approaches for combating infection by A. baumannii.

microbiology↗

bioRxiv2023

Structure of a Rhs effector clade domain identifies new mechanistic insights into type VI secretion system toxin delivery.

The type VI secretion system (T6SS) is a molecular machine utilised by many Gram-negative bacteria to deliver antibacterial toxins directly into adjacent bacteria, often providing a competitive advantage. Rearrangement hotspot (Rhs) effectors are known T6SS cargo effectors, but the precise delivery and activation of these toxins is poorly defined. We present the structure of a novel T6SS Rhs effector (Tse15) from the multidrug resistant nosocomial pathogen Acinetobacter baumannii. Tse15 forms a triple layered y-cocoon Rhs domain with an N-terminal -helical clade domain and an unfolded C-terminal toxin domain located entirely inside the Rhs cage. We identify that Tse15 is cleaved into three domains, through two independent auto-cleavage events involving aspartyl protease activity for toxin self-cleavage and a nucleophilic glutamic acid for the cleavage of the N-terminal clade domain. Proteomic analyses showed that the N-terminal clade and toxin domains, but not the y-strand rich Rhs cage, are delivered outside of the cell, suggesting a novel mechanism for Rhs toxin delivery and activation. Our findings suggest that this delivery mechanism requires an interaction between the N-terminal clade and toxin domains, with the clade domain acting as the internal chaperone to mediate tethering of the toxin to the T6SS machinery. Conservation of the clade domain in other Gram-negative bacteria suggest this may be a common mechanism for T6SS toxin delivery.

microbiology↗