Biology subjects

Karlic, K. I.

Publications and source records attributed to Karlic, K. I..

5 recordsLinked to original sources

bioRxiv2026

MassSpectrum Analyzer: An interactive platform for proteomic searching parameter refinement and peptide modification focused re-scoring

Peptide spectrum annotation is critical for the assignment of peptides and the localisation of modifications. While many existing tools provide spectrum annotation capacities, they often lack the flexibility required to allow bespoke spectral annotation of peptides containing multiple labile modifications or the accurate assignment of peptides in which fragmentation deviates from canonical patterns. In these cases, user-guided annotation is widely used to improve assignment completeness, however it typically does not integrate peptide scoring, making it challenging to assess the empirical improvement of the associated annotation and its impact on downstream false-discovery rate estimations. Here, we introduce an interactive annotation environment, the "MassSpectrum Analyzer", which aims to streamline the exploration and analysis of modified peptides by enabling user-defined customisation with peptide scoring. Using (2-Aminoethyl)trimethylammonium carboxyl-derivatised peptides and glycopeptides as case studies we demonstrate the capacity of the MassSpectrum Analyzer to rapidly explore and allow the assessment of modified peptide datasets. By enabling direct assessment of the impact of user-guided choices on peptide scoring, we show how the detection of highly modified peptides can be improved through post-search integration of modification fragmentation information in a statistically robust manner. Similarly, by permitting comparisons of peptide ion intensities across spectra, we show that global fragmentation patterns can be quantified allowing the interrogation of trends that only become clear when spectra are assessed en masse. Combined, the MassSpectrum Analyzer streamlines the generation of publication-ready spectra and provides a means to assess how the inclusion of annotated features influences assignment scores.

bioinformatics↗

bioRxiv2025

Generation of an optimised Cumate toolkit for tuneable protein expression during in vitro and in vivo studies of Burkholderia cenocepacia

Inducible gene expression is pivotal for dissecting bacterial physiology and virulence mechanisms. Across the Burkholderia genera, a limited range of inducible systems currently exist that show minimal impacts on the proteome and allow tight regulation. In this study, we engineer a set of cumate inducible vectors for use in Burkholderia cenocepacia that offer minimal basal expression and the ability to control B. cenocepacia gene expression within Eukaryotic cells. Through mutagenesis-based studies of cumate circuits and the cumate regulator (CymR), we generate an optimized cumate circuit (PCymRC/CymRGV) which allows the tight and tunable control of protein expression within B. cenocepacia, as assessed by fluorescent and protein O-linked glycosylation analysis. Using comparative proteomics, we demonstrate cumate induction leads to both reduced and orthogonal effects on B. cenocepacia compared to widely used rhamnose based induction systems. Leveraging the cell permeability of cumate and the generation of a CTX-based chromosomal integration vector, we show that inducible control of protein expression is achievable during intracellular replication of B. cenocepacia. Finally, using the ability to control intracellular expression, we demonstrate the requirement of O-linked protein glycosylation for optimal B. cenocepacia intracellular replication. Combined, this work demonstrates that cumate inducible systems allow precise and tuneable gene expression in Burkholderia even within a host-pathogen context. ImportanceThis work establishes optimised cumate-inducible vectors for use in Burkholderia cenocepacia, addressing the need for alternative inducers to available carbohydrate systems. We show cumate-inducible vectors allow precise control of gene expression even within eukaryotic cells, providing a new and orthogonal way to temporally control protein induction. Utilising cumate- based induction, we demonstrate the importance of O-linked protein glycosylation for optimal intracellular replication in B. cenocepacia, highlighting its potential to be used to explore host- pathogen interactions. Combined, this work shows cumate-inducible vectors extend the range of studies which can be undertaken to dissect B. cenocepacia physiology and virulence.

microbiology↗

bioRxiv2025

PAC/SP3 on-bead carboxyl derivatization allows combined C- and N-terminomics

On-bead single-pot solid-phase enhanced sample preparation, SP3, also known as Protein Aggregation Capture (PAC), is a robust, high-throughput, and widely utilized approach for proteomic sample preparation. Recent studies have highlighted PAC/SP3 as an ideal platform for chemoproteomics, allowing chemical labelling by minimizing sample loss and improving recovery of derivatized peptides. In this work, we establish an on-bead PAC/SP3 protein-level amine and carboxyl derivatization approach to facilitate C-terminal focused proteomics. We demonstrate that on-bead protein derivatization of carboxyl groups can be achieved using ethanolamine, (2-aminoethyl)trimethylammonium (AETMA), and (carboxymethyl)trimethylammonium (Girards reagent T, GT) via EDC/HOBt coupling, enabling the labelling of protein C-termini. Using a prokaryotic model system, Acinetobacter baumannii, we demonstrate that AETMA and ethanolamine labelling each enables the identification of unique protein C-terminal peptides, with AETMA improving the identification of C-terminal peptides lacking basic residues. Finally, we apply this approach to interrogate both N- and C-termini in response to etoposide-induced apoptosis within Jurkat cells, demonstrating that combined N- and C-terminomics is achievable using on-bead derivatization, yet provides modest coverage of the C-terminome in its current form. Overall, this work establishes bead-based carboxyl group derivatization as a viable platform to enable future C-terminomics method development.

biochemistry↗

bioRxiv2025

Uncovering bacterial pseudaminylation with pan-specific antibody tools

Pseudaminic acids (Pse) are a family of carbohydrates found within bacterial lipopolysaccharides, capsular polysaccharides and glycoproteins that are critical for the virulence of human pathogens. However, a dearth of effective tools for detecting and enriching Pse has restricted study to only the most abundant Pse-containing glycoconjugates. Here, we devise a synthesis of - and {beta}-O-pseudaminylated glycopeptides to generate pan-specific monoclonal antibodies (mAbs) that recognise - and {beta}-configured Pse and its C8 epimer (8ePse) presented within glycans or directly linked to polypeptide backbones. Structural characterisation reveals the molecular basis of Pse recognition across a range of diverse chemical contexts. Using these mAbs, we establish a glycoproteomic platform to provide unprecedented depth in mapping the Pse glycome of Helicobacter pylori, Campylobacter jejuni, and Acinetobacter baumannii strains. Finally, we demonstrate that the mAbs recognise diverse capsule types in multidrug-resistant Acinetobacter baumannii and enhance phagocytosis to eliminate infections in mice.

biochemistry↗

bioRxiv2024

The Late-Stage Steps of Burkholderia cenocepacia Protein O-Linked Glycan Biosynthesis Are Conditionally Essential.

Periplasmic O-linked protein glycosylation is a highly conserved process observed across the Burkholderia genus. Within Burkholderia, protein glycosylation requires the five gene cluster known as the O-glycosylation cluster (OGC, ogcXABEI) which facilitates the construction of the O-linked trisaccharide attached to periplasmic proteins. Previous studies have reported conflicting results regarding the essentiality of ogcA, predicted to be responsible for the addition of the final carbohydrate of the O-linked trisaccharide and ogcX, the putative O-linked glycan flippase. Within this work, we aimed to dissect the impact of the loss of ogcA and ogcX on Burkholderia cenocepacia viability. We demonstrate that the loss of either ogcA or ogcX are detrimental if glycosylation is initiated leading to marked phenotypic effects. Proteomic analysis supports that the loss of ogcA/ogcX both blocks glycosylation and drives pleotropic effects in the membrane proteome, resulting in the loss of membrane integrity. Consistent with this, strains lacking ogcA and ogcX exhibit increased sensitivity to membrane stressors including antibiotics and demonstrate marked changes in membrane permeability. These effects are consistent with fouling of the undecaprenyl pool due to dead-end O-linked glycan intermediates, and consistent with this, we show that modulation of the undecaprenyl pool through the overexpression of undecaprenyl pyrophosphate synthase (UppS) or the OGC flippase (OgcX) restores viability while expression of early-stage OGC biosynthesis genes (ogcI and ogcB) reduce B. cenocepacia viability. These findings demonstrate disrupting O-linked glycan biosynthesis or transport appears to dramatically impact B. cenocepacia viability, supporting the assignment of ogcA and ogcX as conditionally essential. ImportanceProtein glycosylation, a conserved process in Burkholderia species, utilizes glycans generated by the O-glycosylation cluster (OGC), which is composed of five genes (ogcX, ogcA, ogcB, ogcE, and ogcI). In this study, we demonstrate that the loss of ogcA or ogcX significantly affects the physiology of Burkholderia cenocepacia. Using complementary genetic approaches and proteomic techniques, we show that the loss of ogcA or ogcX blocks glycosylation, alters the cell membrane, and sensitizes cells to stressors such as antibiotics. This increased sensitivity to membrane stress is consistent with the accumulation of dead-end O-linked glycan intermediates, which sequester the limited and essential undecaprenyl pool within B. cenocepacia. These findings highlight that ogcA and ogcX are conditionally essential for B. cenocepacia survival and provides mechanistic insight into how protein glycosylation fidelity--the use of specific glycans for protein glycosylation--is controlled in Burkholderia species.

microbiology↗