Biology subjects

Scott, N. E.

Publications and source records attributed to Scott, N. E..

At least 19 recordsLinked to original sources

bioRxiv2026

Unlocking the secrets of paired terminal genomic repeats utilised by the Burkholderia jumbo phage Matilda

Bacteriophages (phages) are viruses that infect bacteria. Jumbo phages have genomes exceeding 220 kb, which can support greater functional independence and complexity. This work identified vB_BceM_Matilda ({phi}Matilda), a novel Burkholderia-targeting jumbo phage. Before injecting its genome into the host bacterium, {phi}Matilda binds to the bacterial flagellum, the rotation of which is required to drive a productive phage infection. Its linear 267 kb dsDNA genome has a previously undescribed tripartite architecture, with bidirectional terminal elements of 1.3 kb and 25 kb, each flanked by distinct inverted repeats of 1.5 kb and 18 kb, respectively. These architectural elements bookend the genome. Transcriptomic and proteomic analyses tracked the phage infection process and confirmed that genes within these terminal elements are transcribed and translated during infection. The presence of bidirectional terminal sequences in related jumbo phages suggests that the tripartite genome architecture may be conserved and functionally important. This work represents the first description of this genome architecture in any organism, expanding our understanding of genome organisation and raising new questions about the replication, packaging, and evolution of jumbo phage genomes.

microbiology↗

bioRxiv2026

An Expanded Lpt Pathway Couples Surface Lipoprotein Export to Outer Membrane Vesicle Biogenesis in Bacteroides

The ecological dominance of Bacteroidota in the human gut microbiota derives from their exceptional capacity to metabolize diverse dietary and host-derived glycans. This ability relies on surface-exposed lipoproteins (SLPs), which are displayed on the bacterial surface and selectively packaged into outer membrane vesicles (OMVs). Although SLP export and OMV biogenesis are tightly coupled, the mechanism linking these processes has remained unknown. Here, we identify LptZ, a previously uncharacterized protein, as a critical link between SLP surface translocation and OMV biogenesis. Cryo-electron tomography of the lptZ mutant revealed chains of vesicles tethered to the outer membrane, offering a rare glimpse into an arrested stage of OMV formation in which vesicle fission is impaired. Proteomic analysis further showed that, in the absence of LptZ, a subset of SLPs fails to reach the cell surface and instead accumulates at the inner membrane. Unexpectedly, LptZ associates with the lipopolysaccharide transport (Lpt) machinery and repurposes it for SLP translocation. Crosslinking experiments revealed selective associations between multiple SLPs and Lpt proteins, providing mechanistic evidence for the direct involvement of the Lpt machinery in SLP trafficking. We further show that the Bacteroides Lpt machinery contains previously unrecognized components, revealing an unanticipated level of specialization relative to canonical Lpt systems. Together, our findings uncover a previously unknown protein export pathway, expand the functional role of the Lpt machinery, and establish a direct mechanistic link between SLP translocation and OMV biogenesis in Bacteroidota.

microbiology↗

bioRxiv2026

Temporal N-terminomics analysis reveals proteome remodeling following brensocatib-mediated cathepsin C inhibition in promyeloblast cells

Cathepsin C (CatC) is known to activate neutrophil serine proteases (NSPs) involved in innate immune function although its broader impact on cellular proteolytic networks remains poorly defined. Here, we characterized the proteolytic landscape of HL-60 neutrophil progenitor cells following treatment with the CatC inhibitor brensocatib. Activity-based probes confirmed sustained inhibition of CatC by brensocatib, accompanied by progressive suppression of downstream elastase-like protease activation over 16 h, 72 h, and 7 days. An enrichment-free N-terminomics workflow was used to compare control and brensocatib-treated HL-60 cells. Following prolonged CatC inhibition, NSPs were reduced in abundance, while lysosomal cathepsins and endogenous protease inhibitors increased. These findings are consistent with remodeling of the protease-antiprotease network. Cleavage site analysis identified a pronounced, time-dependent NSP-associated P1 cleavage signature enriched for Val, Thr, Ala, Ile, and Cys in control cells that was progressively lost following brensocatib treatment. We also identified established and candidate CatC-dependent cleavage events, together with proteolytic adaptations that emerged in the absence of CatC activity. Collectively, these findings demonstrate that CatC inhibition extends beyond suppression of canonical NSP activation to progressive remodeling of the broader protease-antiprotease network, providing new mechanistic insight into the cellular consequences of therapeutic CatC inhibition.

biochemistry↗

bioRxiv2026

Micafungin exposure drives multidrug resistance in Clavispora lusitaniae

Fungal infections are an escalating global health concern, with rare Candida species posing an urgent threat due to emerging multidrug resistance. Clavispora (Candida) lusitaniae is an uncommon pathogen in which multidrug resistance has been documented during antifungal therapy, yet the selective forces driving this phenotype remain unclear. Here, we show that exposure to the echinocandin micafungin (MCF) alone can select for multidrug resistance in C. lusitaniae. Through controlled evolution experiments we identified individual point mutations in genes encoding ergosterol biosynthesis enzymes (ERGs), sterol trafficking proteins (OSH2), and the echinocandin drug target (FKS1) that confer a significant fitness benefit to one or more classes of antifungals. We find that ERG3 loss-of-function is the primary and independent driver of pan-antifungal resistance to echinocandins, azoles and polyenes. The ERG3 mutants have <1% ergosterol, increased levels of non-toxic sterol intermediates, and increased chitin content, consistent with both cell membrane and cell wall remodeling that enables the fungal pathogen to evade all three drug classes. The convergence of sterol reprogramming and compensatory cell wall remodeling that occurs during adaptation to echinocandin monotherapy can evolve through a single point mutation and parallels our recent case study of acquired multidrug resistance. IMPORTANCEMultidrug resistance in Candida species severely limits treatment options and increases mortality, particularly in immunocompromised patients. Despite increasing reports of multidrug resistance, the molecular mechanisms driving multidrug resistance remain poorly understood. We find that in vitro MCF exposure alone can drive multidrug resistance in C. lusitaniae via acquisition of de novo point mutations in ERG3, an observation that parallels our recent patient case study. By identifying causative mutations and associated physiological changes, we provide mechanistic insight into the emergence of multidrug resistance and highlight the need for surveillance strategies that account for resistance evolution under echinocandin monotherapy.

microbiology↗

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↗

bioRxiv2026

Identification and characterization of the functional LolB ortholog in Bacteroides

Bacteroidota are prolific members of the human gut microbiota, influencing overall health through the degradation of various polysaccharides. To aid in this process, these bacteria deploy cell surface lipoproteins, proteins anchored to the membrane by a lipidated N-terminal cysteine. Despite their importance, lipoprotein synthesis and transport in Bacteroidota is not well defined, particularly how lipoproteins reach the outer membrane and cell surface. In Escherichia coli, lipoproteins are inserted into the outer membrane by LolB, the final step of the Lol pathway for localizing lipoproteins, previously thought to be confined to {gamma}- and {beta}-proteobacteria. Herein, we report a structural ortholog of LolB in Bacteroides that rescues the lethal phenotype associated with LolB depletion in E. coli. We demonstrate this orthologs LolB-like activity through mutagenesis studies and a lipoprotein insertion activity assay. We find that the gene cannot be deleted from Bacteroides, but that depletion does not significantly impact growth on polysaccharides, nor the surface localization of lipoproteins involved in starch degradation. Nonetheless, depletion significantly alters the composition of many proteins in both the inner and outer membranes, while others remain unchanged. Altogether, our findings contribute to elucidating the lipoprotein transport pathway in Bacteroides and how it impacts cell physiology. ImportanceBacteroidota are abundant members of the human gut microbiota that influence health and disease. These bacteria deploy numerous cell surface lipoproteins that mediate their interactions with the host and play key roles in cell physiology. However, their mechanism(s) of lipoprotein transport is understudied. Here, we used a genetic screen to identify orthologs to E. coli LolB, the protein responsible for lipoprotein insertion into the outer membrane. Our screen revealed a structural ortholog to LolB in Bacteroides that performs this function. We show that when LolB is depleted, lipoproteins still reach the cell surface, even though the overall protein composition of the membrane is significantly altered. Our results broaden the understanding of both lipoprotein transport and Bacteroidota physiology.

microbiology↗

bioRxiv2026

Quantitative interactome mapping of skeletal muscle insulin resistance

Protein-protein interactions (PPIs) are dynamic and critical to adaptive homeostasis. While there have been massive efforts to catalogue proteome-wide PPIs, global quantification of changes remains a challenge. Here, we integrate dynamic protein correlation profiling - mass spectrometry (PCP-MS) and quantitative cross linking-mass spectrometry (qXL-MS) using multiplexed stable isotope labelling to characterise global PPI remodelling following the development of chronic skeletal muscle insulin resistance (IR) with or without acute insulin stimulation. We quantify >7,000 unique PPIs amongst 5,346 proteins and show changes in the interactome network dominate the proteome response. Our data show the dysregulation of protein processing in the endoplasmic/sarcoplasmic reticulum involving changes in PPIs with protein chaperones and disulfide isomerases is a major hallmark of skeletal muscle IR. Mechanistically, we show the dysregulation of PPIs with Protein-Disulfide Isomerase 6 (PDIA6) regulates cysteine oxidation and insulin sensitivity. Taken together, we show in vivo quantitative interactome mapping is a powerful approach to understand disease mechanisms and provide new insights into protein network re-organisations with IR.

biochemistry↗

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

Aneuploidy, polyploidy, and loss of heterozygosity distinguish serial bloodstream isolates of Candida albicans

BackgroundThe opportunistic pathogen Candida albicans is the leading species causing invasive Candida infections worldwide. Genomic variation is widespread in clinical isolates and complicates identification of genetic variants underlying antifungal drug resistance and tolerance. Our understanding of genomic and phenotypic diversity during invasive infections is limited and studies of serial isolates from individual patients are uncommon. We performed comparative analyses of 101 C. albicans bloodstream isolates from 55 patients in the Minnesota Minneapolis-Saint Paul region, including serial isolates from 19 patients. We analyzed the phylogenetic relationships of these isolates relative to 199 globally-collected public C. albicans genomes. ResultsThis studys regional isolates span the phylogenetic diversity of C. albicans; 6 isolates represent novel outliers to known clades. Serial isolates from individual patients were separated by limited single nucleotide polymorphisms. Nevertheless, we identified extensive large-scale genomic variation between serial isolates including polyploidy, aneuploidy, copy number variation, loss of heterozygosity, and chromosomal rearrangements. We demonstrated how a heterozygous ERG251 loss of function variant drives azole tolerance in a clinical isolate from a patient with a history of recurrent infections. Using serial isolates, we demonstrated that polyploidy provides an adaptive advantage in the presence of fluconazole despite the absence of overt antifungal drug resistance. ConclusionsOur analysis of serial isolates reveals the genomic plasticity of C. albicans during invasive infections and identifies variation driving antifungal drug tolerance. Our findings reveal limitations in current antifungal susceptibility testing and highlight the need to account for genomic and phenotypic variation during invasive Candida infections.

microbiology↗

bioRxiv2025

Dual Membrane-spanning Anti-Sigma 2 Controls OMV biogenesis and Colonization Fitness in Bacteroides thetaiotaomicron

Bacteroides spp. are Gram-negative, gut commensals that shape the enteric landscape by producing outer membrane vesicles (OMVs) that degrade dietary fibers and traffic immunomodulatory biomolecules. Understanding the mechanism behind OMV biogenesis in Bacteroides spp. is necessary to determine their role in the gut. Recent studies showed that mutation of Dual Membrane-spanning Anti-sigma factor 1 increased OMV production in Bacteroides thetaiotaomicron (Bt) by regulating members of its downstream regulon. Additional members of the Dma family have been identified, but very little is known regarding their roles in Bt. Here, we investigate the role of Dual Membrane-spanning Anti-sigma factor 2 (Dma2) in controlling OMV biogenesis in Bt. We employ biochemical and proteomic analyses to show that mutation of dma2 increases OMV production in a manner that is dependent on the expression of its cognate sigma factor, das2. The precise mechanism by which dma2 increases OMV biogenesis remains elusive. However, transcriptome analyses revealed that{Delta} dma2 has decreased expression of select polysaccharide utilization loci (PULs) that primarily target host-associated glycans. Follow-up comparative proteomics showed that the PUL repertoire was most impacted in the OMV fraction. In vitro growth assessments showed that{Delta} dma2 exhibits delayed growth in the presence of select host-associated glycans. Colonization studies in mice revealed that{Delta} dma2 is outcompeted by the wild-type in the gut, which indicates that dma2 is a key determinant of colonization fitness in Bt. Altogether, these findings expand our knowledge of the Dma familys role in OMV biogenesis and demonstrates their importance in Bacteroides physiology. ImportanceDual membrane-spanning anti-sigma factors (Dma) are a novel class of regulatory system found solely amongst Bacteroidota. Previous studies have demonstrated the role of Dma1 in vesiculation, but the overall role of the Dma family in Bacteroides physiology remains poorly understood. Here, we demonstrate that Dma2 modulates vesiculation and regulates the expression of select polysaccharide utilization loci (PULs) that target host-associated glycans. In vivo studies revealed that Dma2 is an important fitness determinant when competing against kin bacteria. This work begins characterizing the multifaceted involvement of Dma2 in OMV biogenesis, PUL regulation, and colonization fitness.

microbiology↗

bioRxiv2025

Legumain drives processing of cathepsins and nuclear localisation of cathepsin L

Lysosomal proteases such as the cathepsin family and the asparaginyl endopeptidase, legumain, govern vital processes to maintain cellular proteostasis, and their dysregulation contributes to diverse pathologies. Recent studies have reported extra-lysosomal localisation of these proteases, especially in the nucleus, cytoplasm, and extracellularly, yet their function is not completely understood. To examine the relationship between legumain and cathepsins, we assessed the activity and expression of cathepsins in wild-type and legumain-deficient (LGMN-/-) cells using chemical activity-based probes and immunoblots. Processing of cathepsins (CTS) L, V, B, and D from the single-chain to the two-chain form was abrogated in the absence of legumain, with some cell type- and species-specific variation observed. This processing was dependent on legumain activity, although the mechanism remains unclear since recombinant legumain does not appear to directly cleave cathepsins in vitro. In cell types where CTSL exists in the nucleus preferentially in its double chain form, loss of legumain led to a reduction in nuclear CTSL levels. To understand the potential role of these lysosomal proteases in the nucleus, we applied our newly refined chemical N-terminomics pipeline, No-enrichment Identification of Cleavage Events (NICE). This analysis revealed widespread changes in both protein abundance and proteolysis, including putative nuclear substrates of CTSL and legumain, that primarily suggest roles in cell proliferation, cell cycle regulation, inflammation, and ribosomal biogenesis. Overall, this study builds on our understanding of the relationship between legumain and cathepsins and provides the first systematic characterisation of lysosomal protease substrates in the nucleus. Our results offer valuable insight into the potential extra-lysosomal roles of these critical proteases.

biochemistry↗

bioRxiv2025

Phosphorylation by the PknB serine/threonine kinase stimulates dimer formation by WalR

WalKR is a two-component system that regulates cell wall homeostasis and other processes in low-GC Gram-positive bacteria. It is essential for viability in Staphylococcus aureus and regulates genes that encode the autolysins and other critical proteins. The sensor kinase WalK phosphorylates WalR at aspartic acid residue 53 (D53) in the receiver domain, stimulating promoter DNA binding. Unlike other response regulators, WalR is thought to have a second kinase, the serine/threonine kinase PknB, which phosphorylates the receiver domain at threonine residue 101. We previously reported that a walR mutation that changed T101 to a methionine conferred low level resistance to vancomycin and greatly increased susceptibility to tunicamycin along with several other phenotypic traits. In this work we demonstrate similarities between pknB null and the WalRT101M mutants that support a regulatory role for PknB-mediated phosphorylation of WalR. Using a combination of in vitro and in vivo approaches, we confirm the specificity of this posttranslational modification and confirm that phosphorylation at both D53 and T101 is important for dimer formation. Importantly, using an in silico approach, we have discovered that phosphorylation at T101 creates an intermolecular hydrogen bond between the phosphate group and E108, that strengthens contacts along the WalR dimerization interface. This is the first time that a clear molecular role has been assigned to this posttranslational modification and it is the strongest molecular evidence to date for a direct functional relationship between PknB and WalR. We propose that, together with primary activation by WalK, PknB serves as a second potentiating input into the WalKR system, stimulating WalR dimer formation, and target DNA binding and tethering this transcriptional event to important extracellular stimuli.

microbiology↗

bioRxiv2025

Intralysosomal pathogens differentially influence the proteolytic potential of their niche

Avoiding lysosomal degradation is vital to the success of intracellular pathogens. The Gram-negative bacterium Coxiella burnetii and protozoan parasites of the Leishmania genus are unique in being able to replicate within the mature phagolysosomal compartment of host cells, though the exact mechanisms utilised to withstand this hostile environment are not clearly defined. We recently reported that C. burnetii removes the lysosomal protease cathepsin B during infection of mammalian cells. Here, we aimed to determine if this virulence strategy was also employed by the intralysosomal pathogen, Leishmania mexicana. In contrast to C. burnetii, decreases in the activity of specific cathepsins were not detected in L. mexicana-infected host cells as determined using immunoblotting and protease activity-based probes. Co-infection of THP-1 macrophage-like cells with both pathogens resulted in a proteolytic and secretory phenotype consistent with C. burnetii infection, suggesting that C. burnetii-induced remodelling of the lysosome is not influenced by L. mexicana. The host cell proteome and secretome of L. mexicana infected cells was defined using mass spectrometry. This confirmed that, unlike C. burnetii, L. mexicana does not induce increased abundance of lysosomal proteins either intracellularly or in the extracellular milieu. Collectively, this study reveals that although C. burnetii and L. mexicana reside in a phagolysosomal intracellular niche, they employ divergent mechanisms to survive within this hostile compartment.

microbiology↗

bioRxiv2025

Intracellular Acinetobacter baumannii Acts as a Reservoir in Lung Infection via a 'Persist and Resist' Strategy

Although considered primarily extracellular, Acinetobacter baumannii can survive and replicate within macrophages in vitro. Intracellular bacteria are often protected from the host immune system and antibiotic treatment, potentially leading to chronic or recurrent infections. To investigate the role of intracellular A. baumannii during infection, we transferred infected bronchoalveolar lavage fluid (BALF), containing an intracellular bacterial population, to naive immunocompromised mice. This BALF transfer resulted in A. baumannii lung infection, suggesting that intracellular bacteria may serve as a reservoir for recurrent lung infections. Using dual-proteomics, we characterized the A. baumannii-macrophages interactions. Infected macrophages exhibit an inflammatory and Type I interferon response, marked by increased ACOD1 (IRG1) protein levels. Intracellular A. baumannii upregulates proteins involved in evading nutritional immunity, stress response, surface modification, and metabolic adaptation. These findings suggest that A. baumannii employs a multifactorial strategy to survive and replicate within macrophages, which may contribute to recurrent and chronic infections, and compromise treatment efficacy. TeaserIntracellular A. baumannii can act as a reservoir in lung infection

microbiology↗

bioRxiv2025

Heterogeneity of Candida bloodstream isolates in an academic medical center and affiliated hospitals

Invasive Candida bloodstream infections (candidemia) are a deadly global health threat. Rare Candida species are increasingly important causes of candidemia and phenotypic data, including patterns of antifungal drug resistance, is limited. There is geographic variation in the distribution of Candida species and frequency of antifungal drug resistance, which means that collecting and reporting regional data can have significant clinical value. Here, we report the first survey of species distribution, frequency of antifungal drug resistance, and phenotypic variability of Candida bloodstream isolates from an academic medical center and 5 affiliated hospitals in the Minneapolis-Saint Paul region of Minnesota, collected during an 18-month period from 2019 to 2021. We collected 288 isolates spanning 11 species from 119 patients. C. albicans was the most frequently recovered species, followed by C. glabrata and C. parapsilosis, with 10% of cases representing additional, rare species. We performed antifungal drug susceptibility for the three major drug classes and, concerningly, we identified fluconazole, micafungin and multidrug resistance rates in C. glabrata that were [~] 2 times higher than that reported in other regions of the United States. We report some of the first phenotypic data in rare non-albicans Candida species. Through analysis of serial isolates from individual patients, we identified clinically relevant within-patient differences of MIC values in multiple drug classes. Our results provide valuable clinical data relevant to antifungal stewardship efforts and highlight important areas of future research, including within-patient dynamics of infection and the mechanisms of drug resistance in rare Candida species.

microbiology↗

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↗

bioRxiv2023

The mycobacterial glycoside hydrolase LamH enables capsular arabinomannan release and stimulates growth

Mycobacterial glycolipids are important cell envelope structures that drive host-pathogen interactions. Arguably, the most important amongst these are lipoarabinomannan (LAM) and its precursor, lipomannan (LM), which are both trafficked out of the bacterium to the host via unknown mechanisms. An important class of exported LM/LAM is the capsular derivative of these molecules which is devoid of its lipid anchor. Here, we describe the identification of a glycoside hydrolase family 76 enzyme that we term LamH which specifically cleaves -1,6-mannoside linkages within LM and LAM, driving its export to the capsule releasing its phosphatidyl-myo-inositol mannoside lipid anchor. Unexpectedly, we found that the catalytic activity of this enzyme is important for efficient exit from stationary phase cultures where arabinomannan acts as a signal for growth phase transition. Finally, we demonstrate that LamH is important for Mycobacterium tuberculosis survival in macrophages. These data provide a new framework for understanding the biological role of LAM in mycobacteria.

microbiology↗

bioRxiv2023

A patchwork pathway for catabolism of the sulfosugar sulfofucose

The rare sulfosugar sulfofucose (6-deoxy-6-sulfo-D-galactose) is catabolized by Paracoccus wurundjeri sp. nov. strain Merri via a novel tandem pathway that couples sulfofucolytic and sulfolactate sulfolyase reactions. We isolated P. wurundjeri from soil and demonstrated its ability to utilize SFuc as the sole carbon source, leading to complete degradation of sulfofucose and release of sulfite. Proteomics and metabolomics analysis revealed a sulfofucolytic variant of the Entner-Doudoroff (ED) pathway, wherein sulfofucose is converted to sulfolactaldehyde through oxidation, lactone hydrolysis, dehydration and retro-aldol steps. The sulfolactaldehyde is subsequently degraded to pyruvate and sulfite via a downstream biomineralization pathway involving sulfolactate as an intermediate. In vitro reconstitution of the key enzymatic steps validated the pathway, identifying four enzymes: SfcH (dehydrogenase), SfcD (lactonase), SfcF (dehydratase), and SfcE (aldolase), that collectively convert sulfofucose to sulfolactaldehyde and pyruvate. Our findings provide insight into microbial utilization of rare sulfosugars and expand the known metabolic versatility of the genus Paracoccus. ImportanceSulfosugars, such as sulfoquinovose, are integral to the global sulfur cycle, but the metabolism of rarer sulfosugars, like sulfofucose, remains poorly understood. This study identifies a unique bacterial catabolic pathway for sulfofucose degradation, comprising a sulfofucolytic ED pathway followed by biomineralization to sulfite. The identification of this tandem pathway highlights the metabolic flexibility of bacteria to adapt carbohydrate catabolic pathways for processing structurally similar sulfosugars. This work sheds light on the ecological role of sulfofucose metabolism and broadens our understanding of bacterial sulfur metabolism, offering potential implications for biogeochemical sulfur cycling and biotechnological applications.

microbiology↗