Biology subjects

Waterfield, N. R.

Publications and source records attributed to Waterfield, N. R..

7 recordsLinked to original sources

bioRxiv2023

Insight into the emerging insect to human pathogen Photorhabdus revealing geographic differences in immune cell tropism.

Photorhabdus asymbiotica is a species of the insect pathogenic Photorhabdus genus that has been isolated as an etiological agent in human infections. Since then, multiple isolates have been identified worldwide, however actual clinical infections have so far only been identified in North America, Australia and Nepal. Previous research on the clinical isolates had shown that the strains differed in their behaviour when infecting cultured human cells, based on the geographical site of isolation. In this study we investigate the differences between the pathogenic activities of P. asymbiotica isolates from different geographic locations. We present findings from the clinical isolates from Australia (Kingscliff) and North America (ATCC43949), and soil borne nematode isolates from Thailand (PB68) and Northern Europe non-clinical P. asymbiotica genospecies HIT and JUN. We also show the first findings from a new clinical isolate of P. luminescens (Texas), the first non-asymbiotica species to cause a human infection, confirming its ability to infect and survive inside human immune cells. Infection assays were done using both cultured cell lines (THP-1, CHO and HEK cells) and also primary immune cells, Peripheral Blood Mononuclear cells (PBMCs) isolated from human blood. Here for the first time, we show how P. asymbiotica, selectively infects certain immune cells while avoiding others, and that infectivity varies depending on growth temperature. We also show that the infected immune cells vary depending on the geographical location a strain is isolated from, and that the European HIT and JUN strains lack the ability to survive within mammalian cells in tissue culture.

microbiology↗

bioRxiv2023

The Pvc15 D2-Pnf SP Interaction Mediates AAA ATPase Activity, Payload Stability, and Translocation into the PVC Tube Lumen.

The Photorhabdus virulence cassette (PVC) is an elegant, multi-protein, contractile nanostructure which injects functionally bioactive polypeptides into the eukaryotic cytosol. Signal peptides (SPs) are N-terminal amino acid motifs from native payload proteins which can also associate wide range of heterologous proteins to the PVC tube lumen. In addition, Pvc15 is a classic AAA ATPase encoded within the PVC operon which encodes an N-domain and tandem AAA domains D1 and D2. This work finds that Pvc15s ATPase activity is mediated by the E555 residue situated in the Walker B motif of the sole functional ATPase domain, D2. Pvc15 multimerisation may be required to translocate payload into the PVC tube lumen, which is facilitated by intact N and D1 domains. In addition, ATPase activity requires the presence of other PVC operon components which hints at a PVC-regulated mode of action for loading. Furthermore, D2 is a chaperone for the native Pnf SP, though C-terminal truncations of the Pnf SP confers Pvc15-independent stability to the payload. These findings provide insight into the nuanced roles of the Pvc15-SP interaction for payload-PVC association and the effects that SP length and type has on payload stability and PVC-loading ability.

microbiology↗

bioRxiv2022

The influence of extrachromosomal elements in the anthrax "cross-over" strain Bacillus cereus G9241

Bacillus cereus G9241 was isolated from a welder who survived a pulmonary anthrax-like disease. Strain G9241 carries two virulence plasmids, pBCX01 and pBC210, as well as an extrachromosomal prophage, pBFH_1. pBCX01 has 99.6% sequence identity to pXO1 carried by Bacillus anthracis and encodes the tripartite anthrax toxin genes and atxA, a mammalian virulence transcriptional regulator. This work looks at how the presence of pBCX01 and temperature may affect the lifestyle of B. cereus G9241 using a transcriptomic analysis and by studying spore formation, an important part of the B. anthracis lifecycle. Here we report that pBCX01 has a stronger effect on gene transcription at the mammalian infection relevant temperature of 37{degrees}C in comparison to 25{degrees}C. At 37{degrees}C, the presence of pBCX01 appears to have a negative effect on genes involved in cell metabolism, including biosynthesis of amino acids, whilst positively affecting the transcription of many transmembrane proteins. The study of spore formation showed B. cereus G9241 sporulated rapidly in comparison to the B. cereus sensu stricto type strain ATCC 14579, particularly at 37{degrees}C. The carriage of pBCX01 did not affect this phenotype suggesting that other genetic elements were driving rapid sporulation. An unexpected finding of this study was that pBFH_1 is highly expressed at 37{degrees}C in comparison to 25{degrees}C and pBFH_1 expression leads to the production of Siphoviridae-like phage particles in the supernatant of B. cereus G9241. This study provides an insight on how the extrachromosomal genetic elements in B. cereus G9241 has an influence in bacterial phenotypes.

microbiology↗

bioRxiv2022

From cereus to anthrax and back again: The role of the PlcR regulator in the cross-over strain Bacillus cereus G9241

Bacillus cereus G9241 was isolated from a Louisiana welder suffering from an anthrax-like infection. The organism carries two transcriptional regulators that have previously been proposed to be incompatible with each other: the pleiotropic transcriptional regulator PlcR found in most members of the Bacillus cereus group but truncated in all Bacillus anthracis isolates, and the anthrax toxin regulator AtxA found in all B. anthracis strains and a few B. cereus sensu stricto strains. Here we report cytotoxic and haemolytic activity of cell free B. cereus G9241 culture supernatants cultured at 25 {degrees}C to various eukaryotic cells. However, this is not observed at the mammalian infection relevant temperature 37 {degrees}C, behaving much like the supernatants generated by B. anthracis. Using a combination of genetic and proteomic approaches to understand this unique phenotype, we identified several PlcR-regulated toxins to be secreted highly at 25 {degrees}C compared to 37 {degrees}C. Furthermore, we demonstrate that differential expression of the protease involved in processing the PlcR quorum sensing activator molecule PapR appears to be the limiting step for the production of PlcR-regulated toxins at 37 {degrees}C, giving rise to the temperature-dependent haemolytic and cytotoxic activity of the culture supernatants. This study provides an insight on how B. cereus G9241 is able to switch between B. cereus and B. anthracis-like phenotypes in a temperature-dependent manner, potentially accommodating the activities of both PlcR and AtxA.

microbiology↗

bioRxiv2020

Temperature restriction in entomopathogenic bacteria

Temperature plays an important role in bacteria-host interactions and can be a determining factor for host switching. In this study we sought to investigate the reasons behind growth temperature restriction in the entomopathogenic enterobacterium Photorhabdus. Photorhabdus has a complex dual symbiotic and pathogenic life cycle. The genus consists of 19 species but only one subgroup, previously all classed together as P. asymbiotica, have been shown to cause human disease. These clinical isolates necessarily need to be able to grow at 37 {degrees}C, whilst the remaining species are largely restricted to growth temperatures below 34 {degrees}C and are therefore unable to infect mammalian hosts. Here, we have isolated spontaneous mutant lines of P. laumondii DJC that were able to grow up to 36 {degrees}C-37 {degrees}C. Following whole genome sequencing of 29 of these mutants we identified a single gene, encoding a protein with a RecG-like helicase domain, that for the majority of isolates contained single nucleotide polymorphisms. Importantly, provision of the wild-type allele of this gene in trans restored the temperature restriction, confirming the mutations are recessive, and the dominant effect of the protein product of this gene. The gene appears to be part of a short three cistron operon, which we have termed the Temperature Restricting Locus (TRL). Transcription reporter strains revealed that this operon is induced upon the switch from 30 {degrees}C to 37 {degrees}C, leading to replication arrest of the bacteria. TRL is absent from all of the human pathogenic species so far examined, although its presence is not uniform in different strains of the P. luminescens subgroup. In a wider context, the presence of this gene is not limited to Photorhabdus, being found in phylogenetically diverse proteobacteria. We therefore suggest that this system may play a more fundamental role in temperature restriction in diverse species, relating to as yet cryptic aspects of their ecological niches and life cycle requirements.

microbiology↗

bioRxiv2019

Photorhabdus Virulence Cassettes: extracellular multi-protein needle complexes for delivery of small protein effectors into host cells.

Photorhabdus is a highly effective insect pathogen and symbiont of insecticidal nematodes. To exert its potent insecticidal effects, it elaborates a myriad of toxins and small molecule effectors. Among these, the Photorhabdus Virulence Cassettes (PVCs) represent an elegant self-contained delivery mechanism for diverse protein toxins. Importantly, these self-contained nanosyringes overcome host cell membrane barriers, and act independently, at a distance from the bacteria itself. In this study, we demonstrate that Pnf, a PVC needle complex associated toxin, is a Rho-GTPase, which acts via deamidation and transglutamination to disrupt the cytoskeleton. TEM and Western blots have shown a physical association between Pnf and its cognate PVC delivery mechanism. We demonstrate that for Pnf to exert its effect, translocation across the cell membrane is absolutely essential. SIGNIFICANCE STATEMENTHere we provide an up to date analysis of the nano-scale syringe-like molecular devices that Photorhabdus use to manipulate invertebrate hosts, the PVC system. They are related to the Serratia Anti-Feeding Prophage and the Psuedoalteromonas MAC system. All these systems are in turn more distantly related to the well characterized Type VI secretion system currently receiving a great deal of attention. We demonstrate for the first time that the PVC nanosyringes are physically "loaded" with an effector protein payload before being freely released. The PVCs therefore represent bacterial molecular machines that are used as "long-range" protein delivery systems. This widespread class of toxin delivery system will likely prove of great significance in understanding many diverse bacteria/host interactions in future.

microbiology↗

bioRxiv2018

Iso-propyl stilbene: A life-cycle signal?

Members of the Gram-negative bacterial genus Photorhabdus are all highly insect pathogenic and exist in an obligate symbiosis with the entomopathogenic nematode worm Heterorhabditis. All members of the genus produce the small molecule 3,5-dihydroxy-4-isopropyl-trans-stilbene (IPS) as part of their secondary metabolism. IPS is a multi-potent compound that has antimicrobial, antifungal and anticancer activities and also plays an important role in symbiosis with the nematode. In this study we have examined the response of Photorhabdus itself to exogenous ectopic addition of IPS at physiologically relevant concentrations. We observed that the bacteria had a measureable phenotypic response, which included a decrease in bioluminescence and pigment production. This was reflected in changes in its transcriptomic response, in which we reveal a reduction in transcript levels of genes relating to many fundamental cellular processes, such as translation and oxidative phosphorylation. Our observations suggest that IPS plays an important role in the biology of Photorhabdus bacteria, fulfilling roles in quorum sensing, antibiotic-competition advantage and maintenance of the symbiotic developmental cycle.

microbiology↗