Biology subjects

Wren, B. W.

Publications and source records attributed to Wren, B. W..

At least 19 recordsLinked to original sources

bioRxiv2026

Characterisation of novel Campylobacter jejuni Type VI secretion system (T6SS) effectors and exploration of the roles of the C. jejuni T6SS in bacterial antagonism and human host cell interaction

Campylobacter jejuni is a leading global cause of acute foodborne gastroenteritis however, C. jejuni lacks some of the classic virulence determinants associated with other common enteric bacterial pathogens. In recent years an increasing number of C. jejuni isolates have been identified to encode Type Six Secretion System (T6SS), an apparatus utilised by Gram-negative bacteria to secrete toxic bacterial effectors into neighbouring cells. Despite the prevalence of the T6SS and previous investigations, the roles of the C. jejuni T6SS are still not well characterised especially when compared to our knowledge of other clinically relevant T6SS-positive bacterial species. Additionally, as of yet, no C. jejuni T6SS cargo effectors have been characterised. In this study, we show the C. jejuni 488 strain T6SS displays contact-dependent antagonistic behaviour towards T6SS-negative C. jejuni, Campylobacter coli, Escherichia coli and Enterococcus faecium strains suggesting the presence of the T6SS contributes to the competitive capacity of this C. jejuni T6SS-positive strain. Moreover, this antagonistic activity is linked to the functionality of CJ488_0980 and CJ488_0982, two novel putative Tox-REase-7 domain-containing effectors, which were identified through bioinformatical analysis of the C. jejuni 488 strain genome. Additionally, our investigations propose the C. jejuni 488 T6SS contributes to interaction, invasion and intracellular survival in human intestinal epithelial cells (IEC). Collectively, these initial findings are the first examples of in vitro investigation of putative cargo effectors in Campylobacter spp. and provide valuable insights into the roles of C. jejuni T6SS effectors in bacterial competition and pathogenesis. This study highlights the importance of T6SS as an emerging virulence determinant in Campylobacter spp. warranting further investigation.

microbiology↗

bioRxiv2026

Development and evaluation of a dual target glycoconjugate vaccine against Shigella sonnei

BackgroundShigellosis morbidity and mortality, combined with the increase in multidrug-resistant infections make Shigella vaccine development a global imperative. Glycoconjugate vaccines that couple immunogenic O-antigen to protein derived from Shigella may provide broader protection across Shigella species and serogroups. Such an approach also circumvents immunotolerance arising from repeated use of the same carrier. Here we use bioconjugation, exploiting an oligosaccharyltransferase (OST) enzyme to couple O-antigen and carrier protein in vivo, to generate a "double-hit" Shigella glycoconjugate vaccine. MethodGlycoconjugates were synthesised in E. coli SDB1 cells expressing S. sonnei O-antigen, the OST PglS, and one of two Shigella carrier proteins. Recombinant glycoconjugate was purified using anion exchange chromatography and then used to immunise mice. Antibody responses were measured and compared by ELISA. ResultsWhen co-produced in E. coli, PglS was able to transfer the cloned S. sonnei O-antigen onto three carrier proteins, modified to accept glycans from the PglS transferase enzymes- the standard bioconjugate carrier ExoA and two immunogenic Shigella-specific outer membrane proteins, EmrK and MdtA. Production of MdtA or ExoA glycoconjugates for immunisation studies utilised successive rounds of anion exchange chromatography, to remove unglycosylated material and obtain highly purified glycoconjugate proteins for us in vaccination. Analysis of murine sera following immunisation revealed an IgG response was raised against both carrier protein and the S. sonnei O-antigen for each glycoconjugate. ConclusionA novel, conserved Shigella protein can be utilised as an effective carrier for the generation of a "double-hit", immunogenic Shigella glycoconjugate vaccine that elicits IgG responses to both carrier protein and S. sonnei O-antigen.

microbiology↗

bioRxiv2025

Re-evaluating of Chemotaxis in Acanthamoeba castellanii and Acanthamoeba polyphaga: A Modern Perspective Using Advanced Imaging and Tracking Technologies

Chemotaxis, the directed movement of an organism towards nutrients or away from noxious agents is a fundamental process for the survival of many microorganisms. We combined high-resolution imaging, microfluidic gradients, and frame-by-frame tracking to re-evaluate Acanthamoeba chemotaxis to microbial glycans (Mannan, Mannose, N-acetyl-d-glucosamine (GlcNAc), N-acetyl-muramic acid (MurNAc)) and peptides (N-formyl methionyl-leucyl-phenylalanine (fMLP) and Boc-Phe-Leu-Phe-Leu-Phe (BOC_FLFLF)). Our quantitative tracking results on A. castellanii confirm the core patterns in the original studies reported by Schuster and Levandowsky; attraction to fMLP and GlcNAc and lack of response to MurNAc or the peptide antagonist BOC-FLFLF, while revealing previously missed attraction to mannan. In contrast, A. polyphaga demonstrated a more restricted response, with significant chemotaxis observed only toward fMLP, and lack of motility in the presence of MurNAc or BOC-FLFLF. Notably, formyl peptide responses were differentially modulated: BOC-FLFLF reduced fMLP-induced directionality in A. castellanii without impairing motility, while in A. polyphaga, it suppressed both velocity and orientation. These distinct chemoattractant "signatures" are consistent with micro-niche adaptation, and we hypothesise that fine-scale tuning of receptor thresholds to local prey spectra contributes to the observed species differences rather than large-scale habitat segregation. By integrating advanced methodologies with classical paradigms, this study offers new perspectives on Acanthamoeba chemotaxis and supports emerging models of protist pattern recognition paralleling innate immunity.

microbiology↗

bioRxiv2025

Lactate-responsive lysine lactylation in Campylobacter revealed by antibody-based detection of a conserved post-translational modification

Lysine lactylation (Kla) links lactate metabolism to protein regulation yet remains largely unexplored in bacteria. We used a Kla-specific rabbit polyclonal anti-{varepsilon}-N-L-lactyl-lysine antibody to detect for this modification in Campylobacter jejuni (11168H, 81-176) and Campylobacter coli (M8). Immunoblotting of whole-cell lysates resolved discrete, strain-modulated bands under basal conditions and after physiologically relevant L-lactate pulses (4 or 16 mM). A conserved 42-45 kDa species was detected across strains, including in the absence of added lactate. Sample-load titrations revealed additional low-intensity bands, consistent with substoichiometric occupancy typical of a regulatory post-translational modification (PTM). Because Campylobacter, a member of the Epsilonproteobacteria, relies on amino-acid-driven, microaerophilic metabolism, we propose that endogenous and host-derived lactate generates lactyl donors (e.g., lactyl-CoA or lactoyl-glutathione) that enable Kla. We hypothesise that a CobB-like sirtuin mediates reversibility. To our knowledge, these data provide the first antibody-based evidence of Kla in Campylobacter and establish a framework for site-resolved mapping and functional tests that connect redox state and carbon flux to protein function, stress responses, and host interactions. Significance StatementLactate, classically a metabolic end product, can modify lysine residues to regulate proteins, but this process is largely uncharted in bacteria. We examined Campylobacter jejuni and Campylobacter coli and detected lysine lactylation using a polyclonal antibody recognising{varepsilon} -N-L-lactyl-lysine. Immunoblotting of whole-cell lysates revealed discrete, strain-modulated signals under basal conditions and after exposure to physiologically relevant L-lactate. A recurring 42-45 kDa band was present across strains, including in the absence of added lactate, and additional faint bands emerged with greater sample load, consistent with selective, low-occupancy modification. Because Campylobacter relies on amino-acid-driven metabolism under microaerophilic conditions, we suggest that endogenous and host-derived lactate generates activated lactyl donors (e.g., lactyl-CoA or lactoyl-glutathione) that support lysine lactylation, with a CobB-like sirtuin providing reversibility. These findings provide, to our knowledge, the first antibody-based evidence for lysine lactylation in Campylobacter and establish a basis for mapping sites and testing how metabolic state influences protein function, stress responses, and interactions with the hosts.

microbiology↗

bioRxiv2025

Unfolded protein response is a critical mediator in Campylobacter jejuni pathogenesis and host defence

Campylobacter jejuni is the major bacterial cause of foodborne gastroenteritis worldwide. How this pathogen interacts with the host defence machinery of human intestinal epithelial cells (IECs) and is involved in pathogenesis remains elusive. Bacterial pathogens utilise strategies to gain access to the eukaryotic cell machinery that can involve subversion of biological processes in host. Unfolded protein response (UPR) is a highly conserved host cell stress response to the accumulation of misfolded proteins in the endoplasmic reticulum (ER) and is a conserved evolutionary response against invading pathogens. Several bacterial pathogens can induce the UPR for their own survival and thus design a dual scenario where UPR can both protect and facilitate pathogen evasion. Herein, we investigated whether UPR represents a virulence mechanism exploited by C. jejuni during bacterial invasion in human IECs. Our data show that following C. jejuni infection, we observe upregulation of protein kinase R-like ER kinase (PERK), inositol-requiring enzyme 1 (IRE1) and activating transcription factor 6 (ATF6) pathways of the UPR, albeit differentially in human T84 and Caco-2 cells. Chemical induction of UPR by thapsigargin in host cells reduced intracellular survival of C. jejuni while conversely pretreatment with UPR inhibitors increased intracellular survival of C. jejuni and attenuated IL-8 release. Finally, we show that C. jejuni mutant C. jejuni capsular polysaccharide and flagella contribute to UPR activation in IECs. Collectively, these findings provide mechanistic insights into how C. jejuni infection leads to UPR activation and inflammation, potentially contributing to downstream C. jejuni-mediated damage. Author SummaryCampylobacter jejuni is one of the most prevalent bacterial causes of foodborne human gastroenteritis worldwide. The common symptoms of campylobacteriosis include diarrhoea, abdominal pain and fever. C. jejuni-induced diarrhoea is associated with disruption of tight junctions and adherens junctions, downregulation of epithelial sodium channels and disruption of bile reabsorption. In addition, C. jejuni-induced diarrhoea in part result from intestinal inflammation caused by increased C. jejuni colonisation and invasion due to dysfunctional intestinal barrier. C. jejuni was reported to cause an inflammatory signalling cascade via ligand recognition by Toll-like receptor 2 (TLR2), TLR4 and nucleotide-binding oligomerisation domain (NOD) in IECs. However, other potential mechanisms of C. jejuni-induced inflammation, with respect to the IEC responses remains unknown. In this study, we demonstrate UPR activation following C. jejuni infection. The UPR is a conserved eukaryotic regulatory response which is initiated to relieve ER stress and recover ER homeostasis, however several bacteria have evolved to exploit UPR pathways for their own survival and replication, creating a dual scenario where UPR can both protect and facilitate pathogen evasion. This study shows that the activation of the PERK pathway of host UPR is activated in the presence of live C. jejuni or its structural components, flagella and a capsular polysaccharide. Furthermore, we propose that although activation of the UPR is a conserved evolutionary response against invading pathogens, UPR induction induces inflammation which potentially contributes to C. jejuni-mediated damage.

microbiology↗

bioRxiv2024

Acanthamoeba castellanii as a model for unveiling Campylobacter jejuni host-pathogen dynamics

The persistence of the major enteric pathogen Campylobacter jejuni in the natural environment, despite being microaerophilic, remains unsolved. Its survival in the natural atmospheric environment likely stems from several factors, including interactions with amoebae. C. jejuni transiently interacts with Acanthamoebae and this is thought to provide protection against unfavourable atmospheric conditions and subsequently prime the bacteria for interactions with warm-blooded hosts. Acanthamoebae play vital roles in microbial ecosystems by preying on bacterial species, some of which are clinically important. We analysed the whole transcriptome of A. castellanii infected with C. jejuni 11168H. Our findings provide evidence that infection of A. castellanii with C. jejuni triggers distinct and reproducible cellular responses. Upregulated genes were associated with protein synthesis, DNA damage and repair, gluconeogenic pathways, and protein folding and targeting, while downregulated genes were involved in calcium ion transport, osmotic stress response, energy reserve metabolic processes, and protein hydroxylation. From this data we characterized Cj0979c, named here C. jejuni endonuclease (CjeN), which induces DNA damage in A. castellanii. High-resolution microscopy revealed an unexpected association between C. jejuni and host mitochondria, while infected cells show elevated cytosolic calcium levels and metabolic changes favouring "Warburg-like" metabolism. The increased lactate production was subsequently depleted, suggesting that this host metabolic by-product may support C. jejuni survival. These findings identify an unexpected interaction between amoebae and a microaerophilic bacterium and provides a useful model for further research on host-pathogen interactions.

microbiology↗

bioRxiv2024

The Streptococcus pneumoniae transcriptome in patient cerebrospinal fluid identifies novel virulence factors required for meningitis

To better understand Streptococcus pneumoniae pathogenesis we performed RNA sequencing on cerebrospinal fluid (CSF) from meningitis patients to identify bacterial genes expressed during invasion of the central nervous system. Comparison to transcriptome data for serotype 1 S. pneumoniae cultured in ex vivo human CSF defined a subset of 57 genes with high expression during human meningitis. Deletion of two of the most highly expressed genetic loci, bgaA (encodes for a {beta}-galactosidase) or the SP_1801-5 putative stress response operon, resulted in S. pneumoniae strains still able to transmigrate the blood brain barrier but which were more susceptible to complement opsonisation and unable to maintain brain infection in a murine meningitis model. In 1144 meningitis patients, infection with bgaA containing S. pneumoniae strains was associated with a higher mortality (22% versus 14% p=0.02). These data demonstrate that direct bacterial RNAseq from CSF can identify previously undescribed S. pneumoniae virulence factors required for meningitis pathogenesis.

microbiology↗

bioRxiv2024

A platform for the recombinant production of Group A Streptococcus glycoconjugate vaccines

Strep A is a human-exclusive bacterial pathogen killing annually more than 500,000 patients, and no current licensed vaccine exists. Strep A bacteria are highly diverse, but all produce an essential, abundant, and conserved surface carbohydrate, the Group A Carbohydrate, which contains a rhamnose polysaccharide (RhaPS) backbone. RhaPS is a validated universal vaccine candidate in a glycoconjugate prepared by chemical conjugation of the native carbohydrate to a carrier protein. We engineered the Group A Carbohydratte biosynthesis pathway to enable recombinant production using the industry standard route to couple RhaPS to selected carrier proteins within E. coli cells. The structural integrity of the produced recombinant glycoconjugate vaccines was confirmed by NMR spectroscopy and mass spectrometry. Purified RhaPS glycoconjugates elicited carbohydrate-specific antibodies in mice and rabbits and bound to the surface of multiple Strep A strains of diverse M-types, confirming the recombinantly produced RhaPS glycoconjugates as valuable vaccine candidates.

microbiology↗

bioRxiv2023

Understanding Campylobacter coli isolates from the Vietnamese meat production network; a pilot study

Changing farming practices and the associated increase in the use of antibiotics are amongst the main drivers shaping the global increase of Campylobacter infections. The effects farming practices have on Campylobacter species, need to be studied at the global scale, particularly in emerging middle-income countries, where the demand for low-cost poultry meat is rising. While C. jejuni causes the majority of poultry associated diarrhoea, C. coli causes a significant amount of disease but are relatively understudied. In this study we characterised seven C. coli strains isolated from poultry farms and markets in Hanoi, Vietnam. Comprehensive data sets of bacterial Whole-Genome Sequencing; and phenotypic assays, such as, growth, motility, antimicrobial resistant testing along with virulence testing were performed to reveal the genetic relatedness and pathophysiological characteristics of seven C. coli strains. Six isolates were classified as multi-drug resistant, with all isolates resistant to ciprofloxacin, nalidixic acid and tetracycline, but susceptible to phenicols. All isolates had similar growth rates, while five were hyper-motile. Lethality of the isolates towards a tractable host-model system, larvae of the greater wax moth Galleria mellonella, often used to determine Campylobacter virulence was demonstrated for the first time for C. coli. Multilocus sequence typing data correlates with North American, European, and Asian isolates from patients suffering from gastroenteritis, emphasising the global spread of these strains. This work demonstrates that C. coli, with high levels of antimicrobial resistance, is an understudied global threat. Data summaryGenBank database with accession numbers JAKGTW000000000, JAKGTV000000000, JAKGTS000000000, JAKGTU000000000, JAKGTT000000000, JAKGTR000000000 and CP091310 https://www.ncbi.nlm.nih.gov/nuccore/JAKGTW000000000 https://www.ncbi.nlm.nih.gov/nuccore/JAKGTV000000000 https://www.ncbi.nlm.nih.gov/nuccore/JAKGTS000000000 https://www.ncbi.nlm.nih.gov/nuccore/JAKGTU000000000 https://www.ncbi.nlm.nih.gov/nuccore/JAKGTT000000000 https://www.ncbi.nlm.nih.gov/nuccore/JAKGTR000000000 https://www.ncbi.nlm.nih.gov/nuccore/CP091310.1 The authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.

microbiology↗

bioRxiv2022

Survival of Campylobacter jejuni in Acanthamoebae castellanii provides mechanistic insight into host pathogen interactions.

Campylobacter jejuni is the leading cause of bacterial foodborne gastroenteritis world-wide but is rarely transferred between human hosts. Although a recognized microaerophile, C. jejuni is incapable of growing in an aerobic environment. The persistence and transmission of this pathogen outside its warm-blooded avian and mammalian hosts is poorly understood. Acanthamoebae species, are predatory protists and form an important ecological niche with several bacterial species. Here, we investigate the interaction of C. jejuni and Acanthamoebae castellanii at the single-cell level. We observe that a subpopulation of C. jejuni cells can resist killing by A. castellanii and non-digested bacteria are released into the environment where they can persist. In addition, we observe that A. castellanii can harbor C. jejuni even upon encystment. Transcriptome analyses of C. jejuni interactions revealed similar survival mechanisms when infecting both A. castellanii and warm-blooded hosts. In particular, nitrosative stress defense mechanisms and flagellum function are important as confirmed by mutational analyses. This study describes a new host-pathogen interaction for C. jejuni and confirms that amoebae are transient hosts for the persistence, adaptability and potential transmission of C. jejuni.

microbiology↗

bioRxiv2022

Campylobacter jejuni modulates reactive oxygen species production and NADPH oxidase 1 expression in human intestinal epithelial cells

Campylobacter jejuni is the major bacterial cause of foodborne gastroenteritis worldwide. Mechanistically, how this pathogen interacts with intrinsic defence machinery of human intestinal epithelial cells (IECs) remains elusive. To address this, we investigated how C. jejuni counteracts the intracellular and extracellular reactive oxygen species (ROS) in IECs. Our work shows that C. jejuni differentially regulates intracellular and extracellular ROS production in human T84 and Caco-2 cells. C. jejuni downregulates the transcription and translation of Nicotinamide adenine dinucleotide phosphate (NAPDH) oxidase (Nox1), a key ROS-generating enzyme in IECs and antioxidant defence genes cat and sod1. Furthermore, inhibition of Nox1 by diphenylene iodonium (DPI) and siRNA reduced C. jejuni ability to interact, invade and intracellularly survive within T84 and Caco-2 cells. Collectively, these findings provide mechanistic insight into how C. jejuni modulates the IEC defence machinery.

microbiology↗

bioRxiv2021

Survival of Campylobacter jejuni in Amoebae enhances subsequent invasion of mammalian cells

The ubiquitous unicellular eukaryote, Acanthamoeba, is known to play a role in the survival and dissemination of Campylobacter jejuni. C. jejuni is the leading cause of bacterial foodborne gastroenteritis world-wide and is a major public health problem. The ability of C. jejuni to interact and potentially invade epithelial cells is thought to be key for disease development in humans. We examined C. jejuni grown under standard laboratory conditions,11168HCBA with that harvested from within Acanthamoeba castellanii (11168HAC/CBA) or Acanthamoeba polyphaga (11168HAP/CBA), and compared their ability to invade different cell lines. C. jejuni harvested from within amoebae had a [~]3.7-fold increase in invasiveness into T84 human epithelial cells and a striking [~]11-fold increase for re-entry into A. castellanii cells. We also investigated the invasiveness and survivability of six diverse representative C. jejuni strains within Acanthamoeba spp., our results confirm that invasion and survivability is likely host cell dependent. Our survival assay data led us to conclude that Acanthamoeba spp. are a transient host for C. jejuni and that survival within amoebae pre-adapts C. jejuni and enhances subsequent cell invasion. This study provides new insight into C. jejuni interactions with amoebae and its increased invasiveness potential in mammalian hosts.

microbiology↗

bioRxiv2021

Development of a novel glycoengineering platform for the rapid production of conjugate vaccines.

Antimicrobial resistance (AMR) is threatening the lives of millions worldwide. Antibiotics which once saved countless lives, are now failing, ushering in vaccines development as a current global imperative. Conjugate vaccines produced either by chemical synthesis or biologically in Escherichia coli cells, have been demonstrated to be safe and efficacious in protection against several deadly bacterial diseases. However, conjugate vaccines assembly and production have several shortcomings which hinders their wider availability. Here, we developed a tool, Mobile-element Assisted Glycoconjugation by Insertion on Chromosome, MAGIC, a novel method that overcomes the limitations of the current conjugate vaccine design method(s). We demonstrate at least 2-fold increase in glycoconjugate yield via MAGIC when compared to conventional bioconjugate method(s). Furthermore, the modularity of the MAGIC platform also allowed us to perform glycoengineering in genetically intractable bacterial species other than E. coli. The MAGIC system promises a rapid, robust and versatile method to develop vaccines against bacteria, especially AMR pathogens, and could be applied for biopreparedness.

microbiology↗

bioRxiv2021

Probing differences in gene essentiality between the human and animal adapted lineages of the Mycobacterium tuberculosis complex using TnSeq

Members of the Mycobacterium tuberculosis complex (MTBC) show distinct host adaptations, preferences and phenotypes despite being >99% identical at the nucleic acid level. Previous studies have explored gene expression changes between the members, however few studies have probed differences in gene essentiality. To better understand the functional impacts of the nucleic acid differences between Mycobacterium bovis and Mycobacterium tuberculosis we used the Mycomar T7 phagemid delivery system to generate whole genome transposon libraries in laboratory strains of both species and compared the essentiality status of genes during growth under identical in vitro conditions. Libraries contained insertions in 54% of possible TA sites in M. bovis and 40% of those present in M. tuberculosis, achieving similar saturation levels to those previously reported for the MTBC. The distributions of essentiality across the functional categories were similar in both species. 527 genes were found to be essential in M. bovis whereas 477 genes were essential in M. tuberculosis and 370 essential genes were common in both species. CRISPRi was successfully utilised in both species to determine the impacts of silencing genes including wag31, a gene involved in peptidoglycan synthesis and Rv2182c/Mb2204c, a gene involved in glycerophospholipid metabolism. We observed species specific differences in the response to gene silencing, with the inhibition of expression of Mb2204c in M. bovis showing significantly less growth impact than silencing its ortholog (Rv2182c) in M. tuberculosis. Given that glycerophospholipid metabolism is a validated pathway for antimicrobials, our observations suggest that target vulnerability in the animal adapted lineages cannot be assumed to be the same as the human counterpart. This is of relevance for zoonotic tuberculosis as it implies that the development of antimicrobials targeting the human adapted lineage might not necessarily be effective against the animal adapted lineage. The generation of a transposon library and the first reported utilisation of CRISPRi in M. bovis will enable the use of these tools to further probe the genetic basis of survival under disease relevant conditions.

microbiology↗

bioRxiv2020

Impact of industrial production system parameters on chicken microbiomes: mechanisms to improve performance and reduce Campylobacter

BackgroundThe factors affecting host-pathogen ecology in terms of the microbiome remain poorly studied. Chickens are a key source of protein with gut health heavily dependent on the complex microbiome which has key roles in nutrient assimilation and vitamin and amino acid biosynthesis. The chicken gut microbiome may be influenced by extrinsic production system parameters such as Placement Birds/m2 (stocking density), feed type and additives. Such parameters, in addition to on-farm biosecurity may influence performance and also pathogenic bacterial numbers such as Campylobacter. In this study, three different production systems Normal (N), Higher Welfare (HW) and Omega-3 Higher Welfare (O) were investigated "in a natural environment" at day 7 and day 30 with a range of extrinsic parameters assessing performance in correlation with microbial dynamics and Campylobacter presence. ResultsOur data identified production system N as significantly dissimilar from production systems HW and O when comparing the prevalence of genera. An increase in Placement Birds/m2 density led to a decrease in environmental pressure influencing the microbial community structure. Prevalence of genera such as Eisenbergiella within HW and O, and likewise Alistipes within N were representative. These genera have roles directly relating to energy metabolism, amino acid, nucleotide and short chain fatty acid (SCFA) utilisation. Thus, an association exists between consistent and differentiating parameters of the production systems, that affect feed utilisation, advance our knowledge of mechanistic underpinnings, leading to competitive exclusion of genera based on competition for nutrients and other factors. Campylobacter was identified within specific production system and presence was linked with the increased diversity and increased environmental pressure on microbial community structure. Addition of Omega-3 though did alter prevalence of specific genera, in our analysis did not differentiate itself from HW production system. However, Omega-3 was linked with a positive impact on weight gain. ConclusionsOverall, our results show that microbial communities in different industrial production systems are deterministic in elucidating the underlying biological confounders, and these recommendations are transferable to farm practices and diet manipulation leading to improved performance and better intervention strategies against Campylobacter within the food chain. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=125 SRC="FIGDIR/small/084251v2_ufig1.gif" ALT="Figure 1"> View larger version (63K): org.highwire.dtl.DTLVardef@c43951org.highwire.dtl.DTLVardef@8efa5corg.highwire.dtl.DTLVardef@d6db70org.highwire.dtl.DTLVardef@1e09fb9_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2019

Domestication of Campylobacter jejuni NCTC 11168

Reference and type strains of well-known bacteria have been a cornerstone of microbiology research for decades. The sharing of well-characterised isolates among laboratories has parallelised research efforts and enhanced the reproducibility of experiments, leading to a wealth of knowledge about trait variation in different species and the underlying genetics. Campylobacter jejuni strain NCTC 11168, deposited at the National Collection of Type Cultures in 1977, has been adopted widely as a reference strain by researchers worldwide and was the first Campylobacter for which the complete genome was published (in 2000). In this study, we collected 23 C. jejuni NCTC 11168 reference isolates from laboratories across the UK and compared variation in simple laboratory phenotypes with genetic variation in sequenced genomes. Putatively identical isolates identified previously to have aberrant phenotypes varied by up to 281 SNPs (in 15 genes) compared to the most recent reference strain. Isolates also display considerable phenotype variation in motility, morphology, growth at 37{degrees}C, invasion of chicken and human cell lines and susceptibility to ampicillin. This study provides evidence of ongoing evolutionary change among C. jejuni isolates as they are cultured in different laboratories and highlights the need for careful consideration of genetic variation within laboratory reference strains.\n\nImpact statementIn this paper, we comment on the changing role of laboratory reference strains. While the model organism allows basic comparison within and among laboratories, it is important to remember the effect even small differences in isolate genomes can have on the validity and reproducibility of experimental work. We quantify differences in 23 reference Campylobacter genomes and compare them with observable differences in common laboratory phenotypes.\n\nData summaryShort read data are archived on the NCBI SRA associated with BioProject accession PRJNA517467 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA517467).\n\nAll assembled genomes are also available on FigShare (doi: 10.6084/m9.figshare.7849268). Phylogeny visualised on microreact: https://microreact.org/project/NCTC11168.\n\nRepositoriesShort read data are archived on the NCBI SRA repository, associated with BioProject accession PRJNA517467 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA517467; Table S1).\n\nThe authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.

microbiology↗

bioRxiv2019

N-linked glycans confer protein stability and modulate multidrug efflux pump assembly in Campylobacter jejuni

It is now apparent that nearly all bacteria species have at least a single glycosylation system, but the direct effect(s) of these protein post translational modifications are unresolved. In this study, we used the general N-linked glycosylation pathway from Campylobacter jejuni to investigate the biophysical roles of protein modification on the CmeABC multidrug efflux pump complex. The study reveals the multifunctional role of N-linked glycans in enhancing protein thermostability, stabilising protein complexes and the promotion of protein-protein interaction. Our findings demonstrate, for the first time, that regardless of glycan diversification among domains of life, N-linked glycans confer a common evolutionary intrinsic role.

biochemistry↗

bioRxiv2018

Rationally designed mariner vectors to allow functional genomic analysis of Actinobacillus pleuropneumoniae and other bacteria by transposon-directed insertion-site sequencing (TraDIS)

Transposon Directed Insertion Sequencing (TraDIS) is a high-throughput method for mapping insertion sites in large libraries of transposon mutants. The Himar1 (mariner) transposon is ideal for generating near-saturating mutant libraries, especially in AT-rich chromosomes, as the requirement for integration is a TA dinucleotide. In this study, we generated two novel mariner vectors, pTsodCPC9 and pTlacPC9 (differing only in the promoter driving expression of the transposase gene), in order to facilitate TraDIS identification of conditionally essential genes in Actinobacillus pleuropneumoniae and other bacteria. Using the pTlacPC9 vector, we have generated, for the first time, saturating mariner mutant libraries in both A. pleuropneumoniae and Pasteurella multocida that showed a near random distribution of insertions around the respective chromosomes. A preliminary screen of 5000 mutants each identified 8 and 15 genes, respectively, that are required for growth under anaerobic conditions.

microbiology↗