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Abstract Mucin-domain glycoproteins are densely O-glycosylated and play critical roles in a host of biological functions. In particular, the T cell immunoglobulin and mucin-domain containing family of proteins (TIM-1, -3, -4) decorate immune cells and act as key regulators in cellular immunity. However, their dense O-glycosylation remains enigmatic, primarily due to the challenges associated with studying mucin domains. Here, we demonstrate that the mucinase SmE has a unique ability to cleave at residues bearing very complex glycans. SmE enables improved mass spectrometric analysis of several mucins, including the entire TIM family. With this information in-hand, we perform molecular dynamics (MD) simulations of TIM-3 and -4 to understand how glycosylation affects structural features of these proteins. Finally, we use these models to investigate the functional relevance of glycosylation for TIM-3 function and ligand binding. Overall, we present a powerful workflow to better understand the detailed molecular structures and functions of the mucinome.

Changes in glycoprotein expression are correlates of disease, but secreted glycoproteins cannot be accurately traced to their cell line of origin. Here, the authors develop a strategy to chemically tag and profile glycoproteins in a cell line-specific manner in co-culture systems and in vivo.

IntroductionOral and/or tongue swabs have demonstrated ability to detect Mycobacterium tuberculosis (Mtb) in adults with pulmonary tuberculosis (TB). Swabs provide useful alternative specimens for diagnosis of TB using molecular assays however, the diagnostic pickup by culture requires further improvement and development. Several studies identified the presence of differentially culturable tubercle bacilli (DCTB) populations in a variety of clinical specimens. These organisms do not grow in routine laboratory media and require growth factors in the form of culture filtrate (CF) from logarithmic phase cultures of Mtb H37Rv.MethodsHerein, we compared the diagnostic performance of sputum and tongue swabs using Mycobacterial Growth Indicator Tube (MGIT) assays, Auramine smear, GeneXpert and DCTB assays supplemented with or without CF.ResultsFrom 89 eligible participants, 83 (93%), 66 (74%) and 79 (89%) were sputum positive by MGIT, smear and GeneXpert, respectively. The corresponding tongue swabs displayed a lower sensitivity with 39 (44%), 2 (2.0%) and 18 (20%) participants respectively for the same tests. We aimed to improve the diagnostic yield by utilizing DCTB assays. Sputum samples were associated with a higher positivity rate for CF-augmented DCTB at 82/89 (92%) relative to tongue swabs at 36/89 (40%). Similarly, sputum samples had a higher positivity rate for DCTB populations that were CF-independent at 64/89 (72%) relative to tongue swabs at 26/89 (29%). DCTB positivity increased significantly, relative to MGIT culture, for tongue swabs taken from HIV-positive participants. We next tested whether the use of an alternative smear stain, DMN-Trehalose, would improve diagnostic yield but noted no substantial increase.DiscussionCollectively, our data show that while tongue swabs yield lower bacterial numbers for diagnostic testing, the use of growth supplementation may improve detection of TB particularly in HIV-positive people but this requires further interrogation in larger studies.

Mucin-domain glycoproteins are densely O-glycosylated proteins with unique secondary structure that imparts a large influence on cellular environments. Here, the authors develop a technique to selectively enrich and characterize mucin-domain glycoproteins from cell lysate and patient biofluids.

MUC1 is a heavily glycosylated protein on the cell surface. Here the authors show that MUC1 prefers negative over positive membrane curvature due to its bulky size, enabling MUC1 to avoid endocytosis and surface removal based on curvature preference.

IntroductionRoutine efficacy assessments of new tuberculosis (TB) treatments include quantitative solid culture or routine liquid culture, which likely miss quantification of drug tolerant bacteria. To improve these assessments, comparative analyses using additional measures such as quantification of differentially culturable tubercle bacteria (DCTB) are required. Essential for enabling this is a comparative measure of TB treatment responses using routine solid and liquid culture with liquid limiting dilutions (LLDs) that detect DCTB in sputum.MethodsWe recruited treatment-naïve TB patients, with and without HIV-infection, and serially quantified their sputum for DCTB over the course of treatment. ResultsSerial sputum sampling in 73 individuals during their first 14 days of treatment demonstrated that clearance of DCTB was slower compared to routine solid culture. Treatment response appeared to be characterized by four patterns: (1) Classic bi-phasic bacterial clearance; (2) early non-responders with slower clearance; (3) paradoxical worsening with an increase in bacterial count upon treatment initiation; and (4) non-responders with no change in bacterial load. During treatment, LLDs displayed greater bacterial yield when compared with quantitative solid culture. Upon treatment completion, 74% [46/62] of specimens displayed residual DCTB and within this group, two recurrences were diagnosed. Residual DCTB upon treatment completion was associated with a higher proportion of MGIT culture, GeneXpert, and smear positivity at two months post treatment. No recurrences occurred in the group without residual DCTB.DiscussionThese data indicate that DCTB assays detect distinct subpopulations of organisms in sputum that are missed by routine solid and liquid culture, and offer important alternatives for efficacy assessments of new TB treatments. The residual DCTB observed upon treatment completion suggests that TB treatment does not always eliminate all bacterial populations, a finding that should be investigated in larger cohorts.

Summary: Background: Despite being highly prevalent in hospitalised patients with severe HIV-associated tuberculosis (TB) and sepsis, little is known about the mycobacteriology of Mycobacterium tuberculosis bloodstream infection (MTBBSI). We developed methods to serially measure bacillary load in blood and used these to characterise MTBBSI response to anti-TB therapy (ATT) and relationship with mortality. Methods: We established a microscopy method for direct visualisation of M. tuberculosis bacilli in blood using a novel lysis-concentration protocol and the fluorescent probe, 4-N,N-dimethylaminonaphthalimide-trehalose (DMN-Tre). We tested blood using GeneXpert® MTB/RIF-Ultra (Xpert-ultra) and Myco/F lytic culture after processing blood through lysis-wash steps to remove PCR inhibitors and anti-microbial drug carry-over. HIV-positive patients predicted to have MTBBSI gave blood samples 0, 4, 24, 48 and 72 h after ATT initiation. Bacillary loads were quantified using microscopy, Xpert-ultra cycle threshold, and culture time-to-positivity. Pharmacodynamics were modelled using these measures combined on an ordinal scale, including association with 12-week mortality. Findings: M. tuberculosis was detected in 27 of 28 recruited participants; 25 (89%) by blood Xpert-ultra, 22 (79%) by DMN-Tre microscopy, and 21 (75%) by Myco/F lytic blood culture. Eight (29%) participants died by 12-week follow-up. In a combined pharmacodynamic model, predicted probabilities of negative DMN-Tre microscopy, blood Xpert-ultra, or blood culture after 72 h treatment were 0·64, 0·27, and 0·94, respectively, in those who survived, compared with 0·23, 0·06, and 0·71 in those who died (posterior probability of slower clearance of MTBBSI in those that died >0·99). DMN-Tre microscopy of blood demonstrated heterogenous bacillary morphologies, including microcolonies and clumps. Bacillary cell-length varied significantly with ATT exposure (mean cell-length increase 0·13 log-µm/day; 95%CrI 0·10–0·16). Interpretation: Pharmacodynamics of MTBBSI treatment can be captured using DMN-Tre microscopy, blood Xpert-ultra and culture. This could facilitate interventional trials in severe HIV-associated TB. Funding: Wellcome Trust, NIH Fogarty International Center, South African MRC, NIHR(UK), National Research Foundation of South Africa.

Summary: Secreted polypeptides represent a fundamental axis of intercellular communication. Here, we present a protocol for the cell type-specific biotinylation, enrichment, and proteomic profiling of secreted plasma proteins directly in mice. This protocol uses conditional “turn-on” adeno-associated viruses expressing an endoplasmic reticulum-targeted biotin ligase to globally biotinylate proteins of the secretory pathway in a cell type-specific manner. Biotinylated secreted proteins can be directly purified from blood plasma and analyzed by SDS-PAGE gel or shotgun proteomics.For complete information on the generation and use of this protocol, please refer to Wei et al. (2021).

Sialidases are glycoside hydrolases that cleave sialosides. Here the authors define the 3-D structure, alone and in complex with products and inhibitors, of the CAZy family GH156 sialidase, EnvSia156, showing it displays a catalytical (β/α) 8-barrel domain distinct from other sialidases and allowing description of its inverting catalytic mechanism.

Summary: Antigen presentation to T cells in major histocompatibility complex class II (MHC class II) requires the conversion of early endo/phagosomes into lysosomes by a process called maturation. Maturation is driven by the phosphoinositide kinase PIKfyve. Blocking PIKfyve activity by small molecule inhibitors caused a delay in the conversion of phagosomes into lysosomes and in phagosomal acidification, whereas production of reactive oxygen species (ROS) increased. Elevated ROS resulted in reduced activity of cathepsin S and B, but not X, causing a proteolytic defect of MHC class II chaperone invariant chain Ii processing. We developed a novel universal MHC class II presentation assay based on a bio-orthogonal “clickable” antigen and showed that MHC class II presentation was disrupted by the inhibition of PIKfyve, which in turn resulted in reduced activation of CD4+ T cells. Our results demonstrate a key role of PIKfyve in the processing and presentation of antigens, which should be taken into consideration when targeting PIKfyve in autoimmune disease and cancer. : Molecular Mechanism of Gene Regulation; Immunology; Immune Response Subject Areas: Molecular Mechanism of Gene Regulation, Immunology, Immune Response

This article contains raw and processed data related to research published in “Role of the polypeptide N-acetylgalactosaminyltransferase 3 in ovarian cancer progression: possible implications in abnormal mucin O-glycosylation” [1]. The data presented here was obtained with the application of a bioorthogonal chemical reporter strategy analyzing differential glycoprotein expression following the knock-down (KD) of the GALNT3 gene in the epithelial ovarian cancer (EOC) cell line A2780s. LC-MS/MS mass spectrometry analysis was then performed and the processed data related to the identified glycoproteins show that several hundred proteins are differentially expressed between control and GALNT3 KD A2780s cells. The obtained data also uncover numerous novel glycoproteins; some of which could represent new potential EOC biomarkers and/or therapeutic targets. Keywords: GALNT3, Glycosylation, Ac4GalNAz labeling, Label free quantification, NetOGlyc and NetNGlyc prediction analysis, Glycoproteomics

Microbial communities are important for the health of mucosal tissues. Traditional culture and gene sequencing have demonstrated bacterial populations on the conjunctiva. However, it remains unclear if the cornea, a transparent tissue critical for vision, also hosts a microbiome. Corneas of wild-type, IL-1R (-/-) and MyD88 (-/-) C57BL/6 mice were imaged after labeling with alkyne-functionalized D-alanine (alkDala), a probe that only incorporates into the peptidoglycan of metabolically active bacteria. Fluorescence in situ hybridization (FISH) was also used to detect viable bacteria. AlkDala labeling was rarely observed on healthy corneas. In contrast, adjacent conjunctivae harbored filamentous alkDala-positive forms, that also labeled with DMN-Tre, a Corynebacterineae-specific probe. FISH confirmed the absence of viable bacteria on healthy corneas, which also cleared deliberately inoculated bacteria within 24 h. Differing from wild-type, both IL-1R (-/-) and MyD88 (-/-) corneas harbored numerous alkDala-labeled bacteria, a result abrogated by topical antibiotics. IL-1R (-/-) corneas were impermeable to fluorescein suggesting that bacterial colonization did not reflect decreased epithelial integrity. Thus, in contrast to the conjunctiva and other mucosal surfaces, healthy murine corneas host very few viable bacteria, and this constitutive state requires the IL-1R and MyD88. While this study cannot exclude the presence of fungi, viruses, or non-viable or dormant bacteria, the data suggest that healthy murine corneas do not host a resident viable bacterial community, or microbiome, the absence of which could have important implications for understanding the homeostasis of this tissue.

Growth and division are two of the most fundamental capabilities of a bacterial cell. While they are well described for model organisms growing in broth culture, very little is known about the cell division cycle of bacteria replicating in more complex environments. Using a D-alanine reporter strategy, we found that intracellular Listeria monocytogenes (Lm) spend a smaller proportion of their cell cycle dividing compared to Lm growing in broth culture. This alteration to the cell division cycle is independent of bacterial doubling time. Instead, polymerization of host-derived actin at the bacterial cell surface extends the non-dividing elongation period and compresses the division period. By decreasing the relative proportion of dividing Lm, actin polymerization biases the population toward cells with the highest propensity to form actin tails. Thus, there is a positive-feedback loop between the Lm cell division cycle and a physical interaction with the host cytoskeleton.

Protein modification by O-linked β-N-acetylglucosamine (O-GlcNAc) is a critical cell signaling modality, but identifying signal-specific O-GlcNAcylation events remains a significant experimental challenge. Here, we describe a method for visualizing and analyzing organelle- and stimulus-specific O-GlcNAcylated proteins and use it to identify the mitochondrial voltage-dependent anion channel 2 (VDAC2) as an O-GlcNAc substrate. VDAC2−/− cells resist the mitochondrial dysfunction and apoptosis caused by global O-GlcNAc perturbation, demonstrating a functional connection between O-GlcNAc signaling and mitochondrial physiology through VDAC2. More broadly, our method will enable the discovery of signal-specific O-GlcNAcylation events in a wide array of experimental contexts.

Progress in understanding the biology of protein fatty acylation has been impeded by the lack of rapid direct detection and identification methods. We first report that a synthetic ω-alkynyl-palmitate analog can be readily and specifically incorporated into GAPDH or mitochondrial 3-hydroxyl-3-methylglutaryl-CoA synthase in vitro and reacted with an azido-biotin probe or the fluorogenic probe 3-azido-7-hydroxycoumarin using click chemistry for rapid detection by Western blotting or flat bed fluorescence scanning. The acylated cysteine residues were confirmed by MS. Second, ω-alkynyl-palmitate is preferentially incorporated into transiently expressed H- or N-Ras proteins (but not nonpalmitoylated K-Ras), compared with ω-alkynyl-myristate or ω-alkynyl-stearate, via an alkali sensitive thioester bond. Third, ω-alkynyl-myristate is specifically incorporated into endogenous co- and posttranslationally myristoylated proteins. The competitive inhibitors 2-bromopalmitate and 2-hydroxymyristate prevented incorporation of ω-alkynyl-palmitate and ω-alkynyl-myristate into palmitoylated and myristoylated proteins, respectively. Labeling cells with ω-alkynyl-palmitate does not affect membrane association of N-Ras. Furthermore, the palmitoylation of endogenous proteins including H- and N-Ras could be easily detected using ω-alkynyl-palmitate as label in cultured HeLa, Jurkat, and COS-7 cells, and, promisingly, in mice. The ω-alkynyl-myristate and -palmitate analogs used with click chemistry and azido-probes will be invaluable to study protein acylation in vitro, in cells, and in vivo.