Biology subjects

Jennings, M. P.

Publications and source records attributed to Jennings, M. P..

10 recordsLinked to original sources

bioRxiv2026

Development of a Matrix-Matched Calibration Curve for Multi-Site Quantification of Neu5Gc-Bearing N-Glycans

N-glycolylneuraminic acid (Neu5Gc) has been repeatedly associated with human cancer, but reliable detection has remained elusive, generating controversy regarding its presence in human samples. To address this, matrix-matched calibration curves, which have been pioneered in proteomics and metabolomics for assessing changes in complex mixtures, were measured of released N-glycans at four orders of magnitude dynamic range in defined mixtures, systematically benchmarking Neu5Gc-containing N-glycan detection across multiple LC-MS platforms and sites. Orthogonally, the gold-standard analytical method, consisting of fluorescence detection of labelled monosaccharides separated by LC, was applied to the same samples, yielding absolute concentrations of Neu5Gc. LC-MS demonstrated an extended detection range of three or more orders of magnitude while retaining intact N-glycan measurement, improving assay specificity and enabling detection of the variety of Neu5Gc-bearing N-glycans. By combining orthogonal dimensions of evidence, including chromatographic separation, isotopic distribution matching, and composition-confirming MS/MS, LC-MS confidently resolved Neu5Gc signals from noise, even at low abundance. In comparison, DMB-LC-FLR was limited to two orders of magnitude dynamic range, insufficient for detection of Neu5Gc in commercially available pooled human sera. These findings strongly support that DMB-LC-FLR assay specificity and sensitivity are insufficient for Neu5Gc detection in human samples due to noise overwhelming the Neu5Gc signal. By establishing a reusable benchmarking framework for future glycomic studies, we aim to use LC-MS to improve the measurement of Neu5Gc in clinical samples.

biochemistry↗

bioRxiv2022

Actinobacillus pleuropneumoniae encodes multiple phase-variable DNA methyltransferases that comprise distinct phasevarions

ABSTRACTActinobacillus pleuropneumoniae is the cause of porcine pleuropneumonia, a severe respiratory tract infection that is responsible for major economic losses to the swine industry. Many host-adapted bacterial pathogens encode systems known as phasevarions (phase- variable regulons). Phasevarions result from variable expression of cytoplasmic DNA methyltransferases. Variable expression results in genome-wide methylation differences within a bacterial population, leading to altered expression of multiple genes via epigenetic mechanisms. Our examination of a diverse population of A. pleuropneumoniae strains determined that Type I and Type III DNA methyltransferases with the hallmarks of phase variation were present in this species. We demonstrate that phase variation is occurring in these methyltransferase, and show associations between particular Type III methyltransferase alleles and serovar. Using Pacific BioSciences Single-Molecule, Real-Time (SMRT) sequencing and Oxford Nanopore sequencing, we demonstrate the presence of the first ever characterised phase-variable, cytosine-specific Type III DNA methyltransferase. Phase variation of distinct Type III DNA methyltransferase variants results in the regulation of distinct phasevarions, and in multiple phenotypic differences relevant to pathobiology. Our characterisation of these newly described phasevarions in A. pleuropneumoniae will aid in the selection of stably expressed antigens, and direct and inform development of a rationally designed subunit vaccine against this major veterinary pathogen.

microbiology↗

bioRxiv2022

ModA phasevarions regulate adherence of non-typeable Haemophilus influenzae to the host airway in a tissue-specific manner

Adherence of non-typeable Haemophilus influenzae (NTHi) to the host airway is an essential initial step for asymptomatic colonization of the nasopharynx, as well as development of disease. NTHi relies on strict regulation of multiple adhesins for its pathogenesis. The ModA phasevarion is a bacterial regulatory system important for virulence of NTHi. However, the role of the ModA phasevarion in adherence of NTHi to the host airway is not understood well. This study addressed the role of the ModA phasevarion in the regulation of adherence of NTHi to multiple substrates of the host airway. Assessment of adherence of the modA variants of four clinical isolates of NTHi showed that ModA phasevarions regulated adherence of NTHi to mucus, middle ear epithelial cells, and vitronectin in a substrate-specific manner. The adhesins Protein E and P4 were found to contribute to the ModA-regulated adherence of NTHi to distinct substrates. A better understanding of such tissue-specific regulation of NTHi adherence by the ModA phasevarion will allow identification of virulent NTHi populations at the site of disease within the host airway and facilitate more directed development of vaccines or therapeutics.

microbiology↗

bioRxiv2022

A phasevarion controls multiple virulence traits, including expression of vaccine candidates, in Streptococcus pneumoniae

Streptococcus pneumoniae is the most common cause of bacterial illness worldwide. Current vaccines based on the polysaccharide capsule (PCV-13 and PPSV-23) are only effective against a limited number of the >100 capsular serotypes. A universal vaccine based on conserved protein antigens requires a thorough understanding of gene expression in S. pneumoniae. Restriction-Modification (R-M) systems, classically described as a defence against bacteriophage, are almost ubiquitous in the bacterial domain, and roles other than phage defence. All S. pneumoniae strains encode the SpnIII R-M system. This system contains a phase-variable methyltransferase that randomly switches specificity, and controls expression of multiple genes; a phasevarion. We aimed to determine the role of the SpnIII phasevarion during pneumococcal pathobiology and determine if phase-variation resulted in differences in expression of protein antigens that are being investigated as vaccine candidates. Using locked S. pneumoniae strains that express a single SpnIII methyltransferase specificity, we found significant differences in clinically relevant traits, including survival in blood, and adherence to and invasion of human cells. Crucially, we also observed differences in expression of numerous proteinaceous vaccine candidates, which complicates selection of protein antigens for inclusion in a universal protein-based pneumococcal vaccine. This study will inform future vaccine design against S. pneumoniae by ensuring only stably expressed candidates are included in a rationally designed vaccine. Significance StatementS. pneumoniae is the worlds foremost bacterial pathogen. S. pneumoniae encodes a randomly expressed epigenetic regulator, a phasevarion (phase-variable regulon), that results in random expression of multiple genes. Previous work demonstrated that the pneumococcal SpnIII phasevarion switches between six different expression states, generating six unique phenotypic variants in a pneumococcal population. Here, we show that this phasevarion generates multiple phenotypic differences relevant to pathobiology. Importantly, expression of conserved protein antigens varies with phasevarion switching. As capsule expression, a major pneumococcal virulence factor, is also controlled by the phasevarion, our work will inform the selection of the best candidates to include in a rationally designed, universal pneumococcal vaccine.

microbiology↗

bioRxiv2021

Characterisation of the phase-variable autotransporter Lav reveals a role in host cell adherence and biofilm formation in Non-Typeable Haemophilus influenzae

Lav is an autotransporter protein found in pathogenic Haemophilus and Neisseria species. Lav in non-typeable Haemophilus influenzae (NTHi) is phase-variable: the gene reversibly switches ON-OFF via changes in length of a locus-located GCAA(n) simple DNA sequence repeat tract. The expression status of lav was examined in carriage and invasive collections of NTHi, where it was predominantly not expressed (OFF). Phenotypic study showed lav expression (ON) results in increased adherence to host cells, and denser biofilm formation. A survey of Haemophilus spp. genome sequences showed lav is present in [~]60% of NTHi strains, but lav is not present in most typeable H. influenzae. Sequence analysis revealed a total of five distinct variants of the Lav passenger domain present in Haemophilus spp., with these five variants showing a distinct lineage distribution. Determining the role of Lav in NTHi will help understand the role of this protein during distinct pathologies.

microbiology↗

bioRxiv2021

N-glycolylneuraminic acid serum biomarker levels are elevated in breast cancer patients at all stages of disease.

BackgroundNormal human tissues do not express glycans terminating with the sialic acid N-glycolylneuraminic acid (Neu5Gc), yet Neu5Gc-containing glycans have been consistently found in human tumor tissues, cells and secretions and have been proposed as a cancer biomarker. We engineered a Neu5Gc-specific lectin called SubB2M, and previously reported elevated Neu5Gc biomarkers in serum from ovarian cancer patients using a Surface Plasmon Resonance (SPR)-based assay. Here we report an optimized SubB2M SPR-based assay and use this new assay to analyse sera from breast cancer patients for Neu5Gc levels. MethodsTo enhance specificity of our SPR-based assay, we included a non-sialic acid binding version of SubB, SubBA12, to control for any non-specific binding to SubB2M, which improved discrimination of cancer-free controls from early-stage ovarian cancer. We analysed 96 serum samples from breast cancer patients at all stages of disease compared to 22 cancer-free controls using our optimized SubB2M-A12-SPR assay. We also analysed a collection of serum samples collected at 6 monthly intervals from breast cancer patients at high risk for disease recurrence or spread. ResultsAnalysis of sera from breast cancer cases revealed significantly elevated levels of Neu5Gc biomarkers at all stages of breast cancer. We show that Neu5Gc serum biomarker levels can discriminate breast cancer patients from cancer-free individuals with 98.96% sensitivity and 100% specificity. Analysis of serum collected prospectively, post-diagnosis, from breast cancer patients at high risk for disease recurrence showed a trend for a decrease in Neu5Gc levels immediately following treatment for those in remission. ConclusionsNeu5Gc serum biomarkers are a promising new tool for early detection and disease monitoring for breast cancer that may complement current imaging- and biopsy-based approaches.

cancer biology↗

bioRxiv2021

Streptococcus suis encodes multiple allelic variants of a phase-variable Type III DNA methyltransferase, ModS, that control distinct phasevarions

Streptococcus suis is a significant cause of bacterial meningitis in humans, particularly in S.E. Asia, and is a leading cause of respiratory and invasive disease in pigs. Phase-variable DNA methyltransferases, associated with Restriction-Modification (R-M) systems, are a source of epigenetic gene regulation, controlling the expression of multiple genes. These systems are known as phasevarions (phase-variable regulons), and have been characterised in many host-adapted bacterial pathogens. We recently described the presence of a Type III DNA methyltransferase in S. suis, ModS, which contains a simple sequence repeat (SSR) tract within the open reading frame of the modS gene, and which varied in length between individual strains. We also observed multiple allelic variants of the modS gene were present in a population of S. suis isolates. Here, we demonstrate that a biphasic ON-OFF switching of expression occurs in the two most common ModS alleles, ModS1 and ModS2, and that switching is dependent on SSR tract length. Further, we show that ModS1 and ModS2 are active methyltransferases in S. suis using Single-Molecule, Real Time (SMRT) sequencing. ON-OFF switching of each ModS allele results in the regulation of distinct phasevarions, with the ModS2 phasevarion impacting growth patterns and antibiotic resistance. This is the first demonstration of a phase-variable Type III DNA methyltransferase in a Gram-positive organism that controls a phasevarion. Characterising the phenotypic effects of phasevarions in S. suis is key to understanding pathogenesis and the development of future vaccines. ImportanceStreptococcus suis is a causative agent of meningitis, polyarthritis and polyserositis in swine, and is a major cause of zoonotic meningitis in humans. Here we investigate epigenetic gene regulation in S. suis by multiple phasevarions controlled by the phase-variable Type III DNA methyltransferase ModS. This is the first characterised example of a Type III R-M system regulating a phasevarion in a Gram-positive organism. We demonstrate that biphasic ON-OFF switching of ModS expression results in differences in bacterial growth and antibiotic resistance. Understanding the effects of ModS phase variation is required to determine the stably expressed antigenic repertoire of S. suis, which will direct and inform the development of antimicrobial treatments and vaccines against this important pathogen.

microbiology↗

bioRxiv2020

Systematic analysis of REBASE identifies numerous Type I restriction-modification systems that contain duplicated, variable hsdS specificity genes that randomly switch methyltransferase specificity by recombination.

N6-adenine DNA methyltransferases associated with some Type I and Type III restriction-modification (R-M) systems are able to randomly switch expression by variation in the length of locus-encoded simple sequence repeats (SSRs). SSR tract-length variation causes ON/OFF switching of methyltransferase expression, resulting in genome-wide methylation differences, and global changes in gene expression. These epigenetic regulatory systems are called phasevarions, phase-variable regulons, and are widespread in bacteria. A distinct switching system has also been described in Type I R-M systems, based on recombination-driven changes in hsdS genes, which dictate the DNA target site. In order to determine the prevalence of recombination-driven phasevarions, we generated a program called RecombinationRepeatSearch to interrogate REBASE and identify the presence and number of inverted repeats of hsdS downstream of Type I R-M loci. We report that 5.9% of Type I R-M systems have duplicated variable hsdS genes containing inverted repeats capable of phase-variation. We report the presence of these systems in the major pathogens Enterococcus faecalis and Listeria monocytogenes, which will have important implications for pathogenesis and vaccine development. These data suggest that in addition to SSR-driven phasevarions, many bacteria have independently evolved phase-variable Type I R-M systems via recombination between multiple, variable hsdS genes. ImportanceMany bacterial species contain DNA methyltransferases that have random on/off switching of expression. These systems called phasevarions (phase-variable regulons) control the expression of multiple genes by global methylation changes. In every previously characterised phasevarion, genes involved in pathobiology, antibiotic resistance, and potential vaccine candidates are randomly varied in their expression, commensurate with methyltransferase switching. A systematic study to determine the extent of phasevarions controlled by invertible Type I R-M systems has never before been performed. Understanding how bacteria regulate genes is key to the study of physiology, virulence, and vaccine development; therefore it is critical to identify and characterize phase-variable methyltransferases controlling phasevarions.

microbiology↗

bioRxiv2020

The non-typeable Haemophilus influenzae major adhesin Hia is a dual function lectin that binds to human-specific respiratory tract sialic acid glycan receptors.

NTHi is a human-adapted pathogen that colonises the human respiratory tract. Strains of NTHi express multiple adhesins, however there is a unique, mutually exclusive relationship between the major adhesins Hia and HMW1/2. Approximately 25% of NTHi strains express Hia, a phase-variable autotransporter protein, and which has a critical role in colonisation of the host nasopharynx. The remaining 75% of strains express HMW1/2. Previous work has shown that the HMW1 and HMW2 proteins mediate binding to 2,3- and 2,6-linked sialic acid glycans found in the human respiratory tract. Here we show that that the high affinity binding domain of Hia, binding domain 1 (BD1) is responsible for binding to 2,6-sialyllactosamine glycans. BD1 is highly specific for glycans that incorporate the form of sialic acid expressed by humans, N-acetylneuraminic acid (Neu5Ac). We further show that Hia has lower affinity binding activity for 2,3-linked sialic acid and that this binding activity is mediated via a distinct domain. Thus, Hia with its dual binding activities functionally mimics the combined activities of the HMW1 and 2 adhesins. In addition, we show that Hia has a role in biofilm formation by strains of NTHi that express the adhesin. Knowledge of the binding affinity of a major NTHi adhesin, and putative vaccine candidate, will direct and inform development of future vaccines and therapeutic strategies for this important pathogen. ImportanceHost-adapted bacterial pathogens like NTHi have evolved specific mechanisms to colonize their restricted host niche. Relatively few of the adhesins expressed by NTHi have been characterized as regards their binding affinity at the molecular level. In this work we show that the major NTHi adhesin, Hia, preferentially binds to Neu5Ac-2,6-sialyllactosamine, the form of sialic acid expressed in humans. The receptors targeted by Hia in the human airway mirror those targeted by influenza A virus and indicates the broad importance of sialic acid glycans as receptors for airway pathogens.

microbiology↗

bioRxiv2019

C-type lectins CTL4 and CTLMA2: conserved heterodimeric structure and glycan specificity in Anopheles mosquitoes

AbstractThe C-type lectins CTL4 and CTLMA2 cooperatively influence Plasmodium infection in the malaria vector Anopheles. Here we report the purification and biochemical characterization of CTL4 and CTLMA2 from An. gambiae and An. albimanus. CTL4 and CTLMA2 are known to form a disulfide-bridged heterodimer via an N-terminal tri-cysteine CXCPC motif. We demonstrate in vitro that CTL4 and CTLMA2 intermolecular disulfide formation is promiscuous within this motif. Furthermore, CTL4 and CTLMA2 exhibit charge complementarity that promotes the formation of higher oligomeric states at physiological pH. Both lectins bind specific sugars, with an apparent preference for glycosaminoglycan motifs comprising {beta}1-3/{beta}1-4 linkages between glucose (Glc), galactose (Gal) and their respective hexosamines. Small-angle x-ray scattering data supports a compact heterodimer between the CTL domains. Recombinant CTL4/CTLMA2 is functional in vivo, reversing the enhancement of phenoloxidase activity in dsCTL4-treated mosquitoes. We propose these molecular features underline a common function for CTL4/CTLMA2 in mosquitoes, with species and strain-specific variation in degrees of activity in response to Plasmodium infection. Author SummaryMosquitoes of the genus Anopheles are vectors for the single-celled parasite Plasmodium, the causative agent of malaria. Mosquitoes, like all insects, utilize the process of melanization for both wound healing and defense against pathogens. CTL4 and CTLMA2 are two proteins found in Anopheles mosquitoes that act as inhibitors of melanization, so understanding their molecular function is important to understanding the immune response of Anopheles mosquitoes to Plasmodium infection. We have purified CTL4 and CTLMA2 from two species of Anopheles and studied their molecular properties with a variety of biochemical and biophysical techniques. We also verified that our purified protein is functional by injecting it into mosquitoes. We learned that CTL4 and CTLMA2 are joined together by a disulfide bond between any one of three cysteine residues near the N-terminus of each protein. The CTL4/CTLMA2 complex is compact, but can associate into larger structures in solution, probably because of a loop in each protein that carries an opposite charge. The proteins cooperatively bind calcium and sugars, specifically glycosaminoglycan sugars, which are typically present in the connective tissues of insects. This information will aid in further investigations of the function of CTL4 and CTLMA2.

biochemistry↗