Biology subjects

Holden, E.

Publications and source records attributed to Holden, E..

6 recordsLinked to original sources

bioRxiv2026

An integrated mass spectrometry strategy for quantifying the proteoform diversity of the extensively modified O-glycoprotein Osteopontin

O-glycosylation is among the most abundant and structurally diverse post-translational modifications in eukaryotes, yet its heterogeneity renders O-glycoproteins exceptionally difficult to characterize. To overcome these challenges, we have developed an integrated mass spectrometry (MS) strategy to define the proteoform landscape of O-glycoproteins and applied it to human osteopontin (OPN). OPN is a disease-associated extracellular matrix protein subject to extensive modification. By combining native MS with serial exoglycosidase digestions, we directly resolved truncation, phosphorylation, sulfation, and O-glycosylation of OPN. Matched glycoproteomic analyses, using tailored (glyco)protease combinations, allowed us to quantify glycan heterogeneity inaccessible to conventional trypsin-based approaches or protein-centric methods. We integrated these datasets using forward compositional simulations to infer the intact OPN proteoform distribution and benchmarked the resulting models against an experimental intact-mass distribution obtained by proton-transfer charge-reduction MS. This comparison revealed that bottom-up O-glycoproteomics systematically underestimates the true extent of glycan sialylation, whereas assuming (near-)complete sialylation accurately reproduced the experimental intact OPN mass distribution. Together, these results provide a comprehensive, quantitative view of OPN compositional diversity and demonstrate how intact-protein and peptide-level measurements can be reconciled to resolve highly heterogeneous glycoform populations. The workflow establishes a broadly applicable framework for characterizing extensively O-glycosylated and multiply modified proteins.

biophysics↗

bioRxiv2024

Sex differences in pant-hoot vocalizations in wild Eastern chimpanzees

Chimpanzee pant-hoots are frequently produced long-distance vocalisations that serve a number of social functions, such as to indicate coordinate travelling, food discovery, or social status. Calling often triggers replies by others, offering potential for some form of group-level decision-making through vocal exchanges. However, despite important physiological and social differences between male and female chimpanzees, pant-hoot research has traditionally focused on males, with very little known about female use. To address this gap, we collected all-occurrence behavioural data from wild adult female and male Eastern chimpanzees (Pan troglodytes schweinfurthii) in two communities in the Budongo Forest Reserve, Uganda. We show that females pant-hooted regularly, but less frequently than males and, when they did so, mostly in response to others. Response pant- hoots were more common than initiating pant-hoots in both sexes (females: 90.1%; males: 69.5%), and male and female chimpanzees responded to pant-hoots heard at a similar rate (females: 14.1%, males excluding alpha: 15.8%). Both sexes were more likely to respond to pant-hoots from within their own party, but this effect was stronger for females. The likelihood of male chimpanzees pant-hooting in response was inhibited in larger party sizes, while females response pant-hoots were not. Finally, female response pant-hoots were less common during periods of maximal oestrous as compared to other stages of their reproductive cycle. Overall, our findings demonstrate that response pant-hoots are subject to social factors, such as within-sex social competition and between-sex reproductive strategies, which affect male and female chimpanzees in different ways, a further demonstration of the high degree of audience awareness in this primate species. HighlightsO_LIChimpanzees use pant-hoots in both sex-specific and sex-non-specific ways C_LIO_LIIndividual and social factors impact likelihood of pant-hoot production C_LIO_LIOur findings suggest pant-hoots are important flexible elements of vocal behaviour for male and female chimpanzees. C_LI

animal behavior and cognition↗

bioRxiv2024

pQEB1: a hospital outbreak plasmid lineage carrying blaKPC-2

While conducting genomic surveillance of carbapenemase-producing Enterobacteriaceae (CPEs) from patient colonisation and clinical infections at Birminghams Queen Elizabeth Hospital (QE), we identified an N-type plasmid lineage, pQEB1, carrying several antibiotic resistance genes including the carbapenemase gene blaKPC-2. The pQEB1 lineage is concerning due to its conferral of multi-drug resistance, its host range and apparent transmissibility, and its potential for acquiring further resistance genes. Representatives of pQEB1 were found in three sequence types (STs) of Citrobacter freundii, two STs of Enterobacter cloacae, and three species of Klebsiella. Hosts of pQEB1 were isolated from 11 different patients who stayed in various wards throughout the hospital complex over a 13-month period from January 2023 to February 2024. At present, the only representatives of the pQEB1 lineage in GenBank were carried by an Enterobacter hormaechei isolated from a blood sample at the QE in 2016 and a Klebsiella pneumoniae isolated from a urine sample at University Hospitals Coventry and Warwickshire (UHCW) in May 2023. The UHCW patient had been treated at the QE. Long-read whole-genome sequencing was performed on Oxford Nanopore R10.4.1 flow cells, facilitating comparison of complete plasmid sequences. We identified structural variants of pQEB1 and defined the molecular events responsible for them. These have included IS26-mediated inversions and acquisitions of multiple insertion sequences and transposons, including carriers of mercury or arsenic resistance genes. We found that a particular inversion variant of pQEB1 was strongly associated with the QE Liver speciality after appearing in November 2023, but was found in different specialities and wards in January/February 2024. That variant has so far been seen in five different bacterial hosts from six patients, consistent with recent and ongoing inter-host and inter-patient transmission of pQEB1 in this hospital setting. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=184 SRC="FIGDIR/small/597914v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@17cfa04org.highwire.dtl.DTLVardef@22fd6org.highwire.dtl.DTLVardef@192a339org.highwire.dtl.DTLVardef@1a2434_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2023

Differential development of antibiotic resistance and virulence between Acinetobacter species

The two species that account for most cases of Acinetobacter-associated bacteraemia in the UK are Acinetobacter lwoffii, often a commensal but also an emerging pathogen, and A. baumannii, a well-known antibiotic-resistant species. While these species both cause similar types of human infection and occupy the same niche, A. lwoffii (unlike A. baumannii) has thus far remained susceptible to antibiotics. Comparatively little is known about the biology of A. lwoffii and this is the largest study on it conducted to date, providing valuable insights into its behaviour and potential threat to human health. This study aimed to explain the antibiotic susceptibility, virulence, and fundamental biological differences between these two species. The relative susceptibility of A. lwoffii, was explained as it encoded fewer antibiotic resistance and efflux pump genes than A. baumannii (9 and 30 respectively). While both species had markers of horizontal gene transfer, A. lwoffii encoded more DNA defence systems and harboured a far more restricted range of plasmids. Furthermore, A. lwoffii displayed a reduced ability to select for antibiotic resistance mutations, form biofilm and infect both in vivo and in vitro models of infection. This study suggests that the emerging pathogen A. lwoffii has remained susceptible to antibiotics because mechanisms exist to make it highly selective about the DNA it acquires, and we hypothesise that the fact that it only harbours a single RND system restricts the ability to select for resistance mutations. This provides valuable insights into how development of resistance can be constrained in Gram negative bacteria. ImportanceAcinetobacter lwoffii is often a harmless commensal but is also an emerging pathogen and is the most common cause of Acinetobacter-derived blood stream infections in England and Wales. In contrast to the well-studied, and often highly drug resistant A. baumannii, A. lwoffii has remained susceptible to antibiotics. This study explains why this organism has not evolved resistance to antibiotics. These new insights are important to understand why and how some species develop antibiotic resistance, while others do not and could inform future novel treatment strategies.

microbiology↗

bioRxiv2023

Collateral sensitivity increases the efficacy of a rationally designed bacteriophage combination to control Salmonella enterica

The ability of virulent bacteriophages to lyse bacteria influences bacterial evolution, fitness, and population structure. Knowledge of both host susceptibility and resistance factors is crucial for the successful application of bacteriophages as biological control agents in clinical therapy, food processing and agriculture. In this study, we isolated twelve bacteriophages termed SPLA phage which infect the foodborne pathogen Salmonella enterica. To determine phage host range, a diverse collection of Enterobacteriaceae and Salmonella enterica were used and genes involved in infection by six SPLA phages were identified using Salmonella Typhimurium strain ST4/74. Candidate host receptors included lipopolysaccharide, cellulose and BtuB. Lipopolysaccharide was identified as susceptibility factor for phage SPLA1a and mutations in LPS biosynthesis genes spontaneously emerged during culture with S. Typhimurium. Conversely, LPS was a resistance factor for phage SPLA5b that suggested that emergence of LPS mutations in culture with SPLA1a may represent a case of collateral sensitivity to SPLA5b. We show that combination therapy with SPLA1a and SPLA5b was more successful in limiting the emergence of phage resistance compared to monotherapy. Identification of host susceptibility and resistance genes and understanding infection dynamics is critical step in rationale design of phage cocktails against specific bacterial pathogens.

microbiology↗

bioRxiv2023

The adaptability of the ion binding site by the Ag(I)/Cu(I) periplasmic chaperone SilF.

The periplasmic chaperone SilF has been identified as part of an Ag(I) detoxification system in Gram negative bacteria. Sil proteins also bind Cu(I), but with reported weaker affinity, therefore leading to the designation of a specific detoxification system for Ag(I). Using isothermal titration calorimetry we show that binding of both ions is not only tighter than previously thought, but of very similar affinities. We investigated the structural origins of ion binding using molecular dynamics and QM/MM simulations underpinned by structural and biophysical experiments. The results of this analysis showed that the binding site adapts to accommodate either ion, with key interactions with the solvent in the case of Cu(I). The implications of this are that Gram negative bacteria do not appear to have evolved a specific Ag(I) efflux system but take advantage of the existing Cu(I) detoxification system. Therefore, there are consequences for how we define a particular metal resistance mechanism and understand its evolution in the environment.

biophysics↗