Mammalian hybrid pre-autophagosomal structure HyPAS generates autophagosomes.

The biogenesis of mammalian autophagosomes remains to be fully defined. Here, we used cellular and in vitro membrane fusion analyses to show that autophagosomes are formed from a hitherto unappreciated hybrid membrane compartment. The autophagic precursors emerge through fusion of FIP200 vesicles, derived from the cis-Golgi, with endosomally derived ATG16L1 membranes to generate a hybrid pre-autophagosomal structure, HyPAS. A previously unrecognized apparatus defined here controls HyPAS biogenesis and mammalian autophagosomal precursor membranes. HyPAS can be modulated by pharmacological agents whereas its formation is inhibited upon severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection or by expression of SARS-CoV-2 nsp6. These findings reveal the origin of mammalian autophagosomal membranes, which emerge via convergence of secretory and endosomal pathways, and show that this process is targeted by microbial factors such as coronaviral membrane-modulating proteins.

Genetic determinants of genus-level glycan diversity in a bacterial protein glycosylation system.

The human pathogens N. gonorrhoeae and N. meningitidis display robust intra- and interstrain glycan diversity associated with their O-linked protein glycosylation (pgl) systems. In an effort to better understand the evolution and function of protein glycosylation operating there, we aimed to determine if other human-restricted, Neisseria species similarly glycosylate proteins and if so, to assess the levels of glycoform diversity. Comparative genomics revealed the conservation of a subset of genes minimally required for O-linked protein glycosylation glycan and established those pgl genes as core genome constituents of the genus. In conjunction with mass spectrometric-based glycan phenotyping, we found that extant glycoform repertoires in N. gonorrhoeae, N. meningitidis and the closely related species N. polysaccharea and N. lactamica reflect the functional replacement of a progenitor glycan biosynthetic pathway. This replacement involved loss of pgl gene components of the primordial pathway coincident with the acquisition of two exogenous glycosyltransferase genes. Critical to this discovery was the identification of a ubiquitous but previously unrecognized glycosyltransferase gene (pglP) that has uniquely undergone parallel but independent pseudogenization in N. gonorrhoeae and N. meningitidis. We suggest that the pseudogenization events are driven by processes of compositional epistasis leading to gene decay. Additionally, we documented instances where inter-species recombination influences pgl gene status and creates discordant genetic interactions due ostensibly to the multi-locus nature of pgl gene networks. In summary, these findings provide a novel perspective on the evolution of protein glycosylation systems and identify phylogenetically informative, genetic differences associated with Neisseria species.

Allelic polymorphisms in a glycosyltransferase gene shape glycan repertoire in the O-linked protein glycosylation system of Neisseria.

Glycosylation of multiple proteins via O-linkage is well documented in bacterial species of Neisseria of import to human disease. Recent studies of protein glycosylation (pgl) gene distribution established that related protein glycosylation systems occur throughout the genus including nonpathogenic species. However, there are inconsistencies between pgl gene status and observed glycan structures. One of these relates to the widespread distribution of pglG, encoding a glycosyltransferase that in Neisseria elongata subsp. glycolytica is responsible for the addition of di-N-acetyl glucuronic acid at the third position of a tetrasaccharide. Despite pglG residing in strains of N. gonorrhoeae, N. meningitidis and N. lactamica, no glycan structures have been correlated with its presence in these backgrounds. Moreover, PglG function in N. elongata subsp. glycolytica minimally requires UDP-glucuronic acid (GlcNAcA), and yet N. gonorrhoeae, N. meningitidis and N. lactamica lack pglJ, the gene whose product is essential for UDP-GlcNAcA synthesis. We examined the functionality of pglG alleles from species spanning the Neisseria genus by genetic complementation in N. elongata subsp. glycolytica. The results indicate that select pglG alleles from N. meningitidis and N. lactamica are associated with incorporation of an N-acetyl-hexosamine at the third position and reveal the potential for an expanded glycan repertoire in those species. Similar experiments using pglG from N. gonorrhoeae failed to find any evidence of function suggesting that those alleles are missense pseudogenes. Taken together, the results are emblematic of how allelic polymorphisms can shape bacterial glycosyltransferase function and demonstrate that such alterations may be constrained to distinct phylogenetic lineages.

Global biochemical and structural analysis of the type IV pilus from the Gram-positive bacterium Streptococcus sanguinis.

Type IV pili (Tfp) are functionally versatile filaments, widespread in prokaryotes, that belong to a large class of filamentous nanomachines known as type IV filaments (Tff). Although Tfp have been extensively studied in several Gram-negative pathogens where they function as key virulence factors, many aspects of their biology remain poorly understood. Here, we performed a global biochemical and structural analysis of Tfp in a recently emerged Gram-positive model, Streptococcus sanguinis In particular, we focused on the five pilins and pilin-like proteins involved in Tfp biology in S. sanguinis We found that the two major pilins, PilE1 and PilE2, (i) follow widely conserved principles for processing by the prepilin peptidase PilD and for assembly into filaments; (ii) display only one of the post-translational modifications frequently found in pilins, i.e. a methylated N terminus; (iii) are found in the same heteropolymeric filaments; and (iv) are not functionally equivalent. The 3D structure of PilE1, solved by NMR, revealed a classical pilin-fold with a highly unusual flexible C terminus. Intriguingly, PilE1 more closely resembles pseudopilins forming shorter Tff than bona fide Tfp-forming major pilins, underlining the evolutionary relatedness among different Tff. Finally, we show that S. sanguinis Tfp contain a low abundance of three additional proteins processed by PilD, the minor pilins PilA, PilB, and PilC. These findings provide the first global biochemical and structural picture of a Gram-positive Tfp and have fundamental implications for our understanding of a widespread class of filamentous nanomachines.

In-depth characterization of Trichoderma reesei cellobiohydrolase TrCel7A produced in Nicotiana benthamiana reveals limitations of cellulase production in plants by host-specific post-translational modifications.

Sustainable production of biofuels from lignocellulose feedstocks depends on cheap enzymes for degradation of such biomass. Plants offer a safe and cost-effective production platform for biopharmaceuticals, vaccines and industrial enzymes boosting biomass conversion to biofuels. Production of intact and functional protein is a prerequisite for large-scale protein production, and extensive host-specific post-translational modifications (PTMs) often affect the catalytic properties and stability of recombinant enzymes. Here we investigated the impact of plant PTMs on enzyme performance and stability of the major cellobiohydrolase TrCel7A from Trichoderma reesei, an industrially relevant enzyme. TrCel7A was produced in Nicotiana benthamiana using a vacuum-based transient expression technology, and this recombinant enzyme (TrCel7Arec ) was compared with the native fungal enzyme (TrCel7Anat ) in terms of PTMs and catalytic activity on commercial and industrial substrates. We show that the N-terminal glutamate of TrCel7Arec was correctly processed by N. benthamiana to a pyroglutamate, critical for protein structure, while the linker region of TrCel7Arec was vulnerable to proteolytic digestion during protein production due to the absence of O-mannosylation in the plant host as compared with the native protein. In general, the purified full-length TrCel7Arec had 25% lower catalytic activity than TrCel7Anat and impaired substrate-binding properties, which can be attributed to larger N-glycans and lack of O-glycans in TrCel7Arec . All in all, our study reveals that the glycosylation machinery of N. benthamiana needs tailoring to optimize the production of efficient cellulases.

Disrupted Synthesis of a Di-N-acetylated Sugar Perturbs Mature Glycoform Structure and Microheterogeneity in the O-Linked Protein Glycosylation System of Neisseria elongata subsp. glycolytica.

The genus Neisseria includes three major species of importance to human health and disease (Neisseria gonorrhoeae, Neisseria meningitidis, and Neisseria lactamica) that express broad-spectrum O-linked protein glycosylation (Pgl) systems. The potential for related Pgl systems in other species in the genus, however, remains to be determined. Using a strain of Neisseria elongata subsp. glycolytica, a unique tetrasaccharide glycoform consisting of di-N-acetylbacillosamine and glucose as the first two sugars followed by a rare sugar whose mass spectrometric fragmentation profile was most consistent with di-N-acetyl hexuronic acid and a N-acetylhexosamine at the nonreducing end has been identified. Based on established mechanisms for UDP-di-N-acetyl hexuronic acid biosynthesis found in other microbes, we searched for genes encoding related pathway components in the N. elongata subsp. glycolytica genome. Here, we detail the identification of such genes and the ensuing glycosylation phenotypes engendered by their inactivation. While the findings extend the conservative nature of microbial UDP-di-N-acetyl hexuronic acid biosynthesis, mutant glycosylation phenotypes reveal unique, relaxed specificities of the glycosyltransferases and oligosaccharyltransferases to incorporate pathway intermediate UDP-sugars into mature glycoforms.IMPORTANCE Broad-spectrum protein glycosylation (Pgl) systems are well recognized in bacteria and archaea. Knowledge of how these systems relate structurally, biochemically, and evolutionarily to one another and to others associated with microbial surface glycoconjugate expression is still incomplete. Here, we detail reverse genetic efforts toward characterization of protein glycosylation mutants of N. elongata subsp. glycolytica that define the biosynthesis of a conserved but relatively rare UDP-sugar precursor. The results show both a significant degree of intra- and transkingdom conservation in the utilization of UDP-di-N-acetyl-glucuronic acid and singular properties related to the relaxed specificities of the N. elongata subsp. glycolytica system.

Characterization of TCDD-inducible poly-ADP-ribose polymerase (TIPARP/ARTD14) catalytic activity.

Here, we report the biochemical characterization of the mono-ADP-ribosyltransferase 2,3,7,8-tetrachlorodibenzo-p-dioxin poly-ADP-ribose polymerase (TIPARP/ARTD14/PARP7), which is known to repress aryl hydrocarbon receptor (AHR)-dependent transcription. We found that the nuclear localization of TIPARP was dependent on a short N-terminal sequence and its zinc finger domain. Deletion and in vitro ADP-ribosylation studies identified amino acids 400-657 as the minimum catalytically active region, which retained its ability to mono-ADP-ribosylate AHR. However, the ability of TIPARP to ADP-ribosylate and repress AHR in cells was dependent on both its catalytic activity and zinc finger domain. The catalytic activity of TIPARP was resistant to meta-iodobenzylguanidine but sensitive to iodoacetamide and hydroxylamine, implicating cysteines and acidic side chains as ADP-ribosylated target residues. Mass spectrometry identified multiple ADP-ribosylated peptides in TIPARP and AHR. Electron transfer dissociation analysis of the TIPARP peptide 33ITPLKTCFK41 revealed cysteine 39 as a site for mono-ADP-ribosylation. Mutation of cysteine 39 to alanine resulted in a small, but significant, reduction in TIPARP autoribosylation activity, suggesting that additional amino acid residues are modified, but loss of cysteine 39 did not prevent its ability to repress AHR. Our findings characterize the subcellular localization and mono-ADP-ribosyltransferase activity of TIPARP, identify cysteine as a mono-ADP-ribosylated residue targeted by this enzyme, and confirm the TIPARP-dependent mono-ADP-ribosylation of other protein targets, such as AHR.

O-GlcNAc site-mapping of liver X receptor-α and O-GlcNAc transferase.

The Liver X Receptor α (LXRα) belongs to the nuclear receptor superfamily and plays an essential role in regulating cholesterol, lipid and glucose metabolism and inflammatory responses. We have previously shown that LXRα is post-translationally modified by O-linked ß-N-acetyl-glucosamine (O-GlcNAc) with increased transcriptional activity. Moreover, we showed that LXRα associates with O-GlcNAc transferase (OGT) in vitro and in vivo in mouse liver. In this study, we report that human LXRα is O-GlcNAc modified in its N-terminal domain (NTD) by identifying a specific O-GlcNAc site S49 and a novel O-GlcNAc modified peptide 20LWKPGAQDASSQAQGGSSCILRE42. However, O-GlcNAc site-mutations did not modulate LXRα transactivation of selected target gene promoters in vitro. Peptide array and co-immunoprecipitation assays demonstrate that LXRα interacts with OGT in its NTD and ligand-binding domain (LBD) in a ligand-independent fashion. Moreover, we map two new O-GlcNAc sites in the longest OGT isoform (ncOGT): S437 in the tetratricopeptide repeat (TPR) 13 domain and T1043 in the far C-terminus, and a new O-GlcNAc modified peptide (amino acids 826-832) in the intervening region (Int-D) within the catalytic domain. We also map four new O-GlcNAc sites in the short isoform sOGT: S391, T393, S399 and S437 in the TPRs 11-13 domain. Future studies will reveal the biological role of identified O-GlcNAc sites in LXRα and OGT.

Structural and genetic analyses of glycan O-acetylation in a bacterial protein glycosylation system: evidence for differential effects on glycan chain length.

O-acetylation is a common modification of bacterial glycoconjugates. By modifying oligosaccharide structure and chemistry, O-acetylation has important consequences for biotic and abiotic recognition events and thus bacterial fitness in general. Previous studies of the broad-spectrum O-linked protein glycosylation in pathogenic Neisseria species (including N. gonorrhoeae and N. meningitidis) have revealed O-acetylation of some of their diverse glycoforms and identified the committed acetylase, PglI. Herein, we extend these observations by using mass spectrometry to examine a complete set of all glycan variants identified to date. Regardless of composition, all glycoforms and all sugars in the oligosaccharide are subject to acetylation in a PglI-dependent fashion with the only exception of di-N-acetyl-bacillosamine. Moreover, multiple sugars in a single oligosaccharide could be simultaneously modified. Interestingly, O-acetylation status was found to be correlated with altered chain lengths of oligosaccharides expressed in otherwise isogenic backgrounds. Models for how this unprecedented phenomenon might arise are discussed with some having potentially important implications for the membrane topology of glycan O-acetylation. Together, the findings provide better insight into how O-acetylation can both directly and indirectly govern glycoform structure and diversity.

Characterization of a Unique Tetrasaccharide and Distinct Glycoproteome in the O-Linked Protein Glycosylation System of Neisseria elongata subsp. glycolytica.

UNLABELLED: Broad-spectrum O-linked protein glycosylation is well characterized in the major Neisseria species of importance to human health and disease. Within strains of Neisseria gonorrhoeae, N. meningitidis, and N. lactamica, protein glycosylation (pgl) gene content and the corresponding oligosaccharide structure are fairly well conserved, although intra- and interstrain variability occurs. The status of such systems in distantly related commensal species, however, remains largely unexplored. Using a strain of deeply branching Neisseria elongata subsp. glycolytica, a heretofore unrecognized tetrasaccharide glycoform consisting of di-N-acetylbacillosamine-glucose-di-N-acetyl hexuronic acid-N-acetylhexosamine (diNAcBac-Glc-diNAcHexA-HexNAc) was identified. Directed mutagenesis, mass spectrometric analysis, and glycan serotyping confirmed that the oligosaccharide is an extended version of the diNAcBac-Glc-based structure seen in N. gonorrhoeae and N. meningitidis generated by the successive actions of PglB, PglC, and PglD and glucosyltransferase PglH orthologues. In addition, a null mutation in the orthologue of the broadly conserved but enigmatic pglG gene precluded expression of the extended glycoform, providing the first evidence that its product is a functional glycosyltransferase. Despite clear evidence for a substantial number of glycoprotein substrates, the major pilin subunit of the endogenous type IV pilus was not glycosylated. The latter finding raises obvious questions as to the relative distribution of pilin glycosylation within the genus, how protein glycosylation substrates are selected, and the overall structure-function relationships of broad-spectrum protein glycosylation. Together, the results of this study provide a foundation upon which to assess neisserial O-linked protein glycosylation diversity at the genus level. IMPORTANCE: Broad-spectrum protein glycosylation systems are well characterized in the pathogenic Neisseria species N. gonorrhoeae and N. meningitidis. A number of lines of evidence indicate that the glycan components in these systems are subject to diversifying selection and suggest that glycan variation may be driven in the context of glycosylation of the abundant and surface-localized pilin protein PilE, the major subunit of type IV pili. Here, we examined protein glycosylation in a distantly related, nonpathogenic neisserial species, Neisseria elongata subsp. glycolytica. This system has clear similarities to the systems found in pathogenic species but makes novel glycoforms utilizing a glycosyltransferase that is widely conserved at the genus level but whose function until now remained unknown. Remarkably, PilE pilin is not glycosylated in this species, a finding that raises important questions about the evolutionary trajectories and overall structure-function relationships of broad-spectrum protein glycosylation systems in bacteria.

The Pediocin PA-1 Accessory Protein Ensures Correct Disulfide Bond Formation in the Antimicrobial Peptide Pediocin PA-1.

Peptides, in contrast to proteins, are generally not large enough to form stable and well-defined three-dimensional structures. However, peptides are still able to form correct disulfide bonds. Using pediocin-like bacteriocins, we have examined how this may be achieved. Some pediocin-like bacteriocins, such as pediocin PA-1 and sakacin P[N24C+44C], have four cysteines. There are three possible ways by which the four cysteines may combine to form two disulfide bonds, and the three variants are expected to be produced in approximately equal amounts if their formation is random. Pediocin PA-1 and sakacin P[N24C+44C] with correct disulfide bonds were the main products when they were secreted by the pediocin PA-1 ABC transporter and accessory protein, but when they were secreted by the corresponding secretion machinery for sakacin A, a pediocin-like bacteriocin with one disulfide bond (two cysteines), peptides with all three possible disulfide bonds were produced in approximately equal amounts. All five cysteines in the pediocin PA-1 ABC transporter and the two cysteines (that form a CxxC motif) in the accessory protein were individually replaced with serines to examine their involvement in disulfide bond formation in pediocin PA-1. The Cys86Ser mutation in the accessory protein caused a 2-fold decrease in the amount of pediocin PA-1 with correct disulfide bonds, while the Cys83Ser mutation nearly abolished the production of pediocin PA-1 and resulted in the production of all three disufide bond variants in equal amounts. The Cys19Ser mutation in the ABC transporter completely abolished secretion of pediocin PA-1, suggesting that Cys19 is in the proteolytic active site and involved in cleaving the prebacteriocin. Replacing the other four cysteines in the ABC transporter with serines caused a slight reduction in the overall amount of secreted pediocin PA-1, but the relative amount with the correct disulfide bonds remained large. These results indicate that the pediocin PA-1 accessory protein has a chaperone-like activity in that it ensures the formation of the correct disulfide bond in pediocin PA-1.

Extended glycan diversity in a bacterial protein glycosylation system linked to allelic polymorphisms and minimal genetic alterations in a glycosyltransferase gene.

Glycans manifest in conjunction with the broad spectrum O-linked protein glycosylation in species within the genus Neisseria display intra- and interstrain diversity. Variability in glycan structure and antigenicity are attributable to differences in the content and expression status of glycan synthesis genes. Given the high degree of standing allelic polymorphisms in these genes, the level of glycan diversity may exceed that currently defined. Here, we identify unique protein-associated disaccharide glycoforms that carry N-acetylglucosamine (GlcNAc) at their non-reducing end. This altered structure was correlated with allelic variants of pglH whose product was previously demonstrated to be responsible for the expression of glucose (Glc)-containing disaccharides. Allele comparisons and site-specific mutagenesis showed that the presence of a single residue, alanine at position 303 in place of a glutamine, was sufficient for GlcNAc versus Glc incorporation. Phylogenetic analyses revealed that GlcNAc-containing disaccharides may be widely distributed within the pgl systems of Neisseria particularly in strains of N. meningitidis. Although analogous minimal structural alterations in glycosyltransferases have been documented in association with lipopolysaccharide and capsular polysaccharide variability, this appears to be the first example in which such changes have been implicated in glycan diversification within a bacterial protein glycosylation system.

Phosphoproteomic changes in response to anoxia are tissue-specific in the anoxia-tolerant crucian carp (Carassius carassius).

Crucian carp (Carassius carassius), a freshwater fish, can survive chronic anoxia for several months at low temperatures. Consequently, anoxia-related physiological and biochemical adaptations in this species have been studied for more than half a century. Still, despite for the well-known role of protein phosphorylation in regulating cellular processes, no studies have comprehensively characterized the phosphoproteome in crucian carp. In this study, we report the global phosphoproteome in crucian carp brain and liver during anoxia and reoxygenation. By applying a bottom-up proteomic approach on enriched phosphopeptides we found that the brain phosphoproteome shows surprisingly few changes during anoxia-reoxygenation exposure with only 109 out of 4200 phosphopeptides being differentially changed compared to normoxic controls. By contrast, in the liver 395 out of 1287 phosphopeptides changed. Although most changes occurred in the liver phosphoproteome, the pattern of changes indicated metabolic depression and decreased translation in both brain and liver. We also found changes in phosphoproteins involved in apoptotic regulation and reactive oxygen species handling in both tissues. In the brain, some of the most changed phosphopeptides belonged to proteins involved in central nervous system development and neuronal activity at the synaptic cleft. Changed phosphoproteins specific for liver tissue were related to glucose metabolism, such as glycolytic flux and glycogenolysis. In conclusion, protein phosphorylation in response to anoxia and reoxygenation showed both common and tissue-specific changes related to the functional differences between brain and liver.

Transcriptomic and proteomic analysis of the virulence inducing effect of ciprofloxacin on enterohemorrhagic Escherichia coli.

Enterohemorrhagic E. coli (EHEC) is considered to be the most dangerous pathotype of E. coli, as it causes severe conditions such as hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS). Antibiotic treatment of EHEC infections is generally not recommended since it may promote the production of the Shiga toxin (Stx) and lead to worsened symptoms. This study explores how exposure to the fluoroquinolone ciprofloxacin reorganizes the transcriptome and proteome of EHEC O157:H7 strain EDL933, with special emphasis on virulence-associated factors. As expected, exposure to ciprofloxacin caused an extensive upregulation of SOS-response- and Stx-phage proteins, including Stx. A range of other virulence-associated factors were also upregulated, including many genes encoded by the LEE-pathogenicity island, the enterohemolysin gene (ehxA), as well as several genes and proteins involved in LPS production. However, a large proportion of the genes and proteins (17 and 8%, respectively) whose expression was upregulated upon ciprofloxacin exposure (17 and 8%, respectively) are not functionally assigned. This indicates a knowledge gap in our understanding of mechanisms involved in EHECs response to antibiotic-induced stress. Altogether, the results contribute to better understanding of how exposure to ciprofloxacin influences the virulome of EHEC and generates a knowledge base for further studies on how EHEC responds to antibiotic-induced stress. A deeper understanding on how EHEC responds to antibiotics will facilitate development of novel and safer treatments for EHEC infections.

Insights into type IV pilus biogenesis and dynamics from genetic analysis of a C-terminally tagged pilin: a role for O-linked glycosylation.

Type IV pili are surface organelles essential for pathogenicity of many Gram-negative bacteria. In Neisseria gonorrhoeae, the major subunit of type IV pili, PilE, is a target of its general O-linked glycosylation system. This system modifies a diverse set of periplasmic and extracellular gonococcal proteins with a variable set of glycans. Here we show that expression of a particular hexa-histidine-tagged PilE was associated with growth arrest. By studying intra- and extragenic suppressors, we found that this phenotype was dependent on pilus assembly and retraction. Based on these results, we developed a sensitive tool to identify factors with subtle effects on pilus dynamics. Using this approach, we found that glycan chain length has differential effects on the growth arrest that appears to be mediated at the level of pilin subunit-subunit interactions and bidirectional remodelling of pilin between its membrane-associated and assembled states. Gonococcal pilin glycosylation thus plays both an intracellular role in pilus dynamics and potential extracellular roles mediated through type IV pili. In addition to demonstrating the effect of glycosylation on pilus dynamics, the study provides a new way of identifying factors with less dramatic effects on processes involved in type IV pilus biogenesis.

Surviving without oxygen involves major tissue specific changes in the proteome of crucian carp (Carassius carassius).

The crucian carp (Carassius carassius) can survive complete oxygen depletion (anoxia) for several months at low temperatures, making it an excellent model for studying molecular adaptations to anoxia. Still, little is known about how its global proteome responds to anoxia and reoxygenation. By applying mass spectrometry-based proteome analyses on brain, heart and liver tissue from crucian carp exposed to normoxia, five days anoxia, and reoxygenation, we found major changes in particularly cardiac and hepatic protein levels in response to anoxia and reoxygenation. These included tissue-specific differences in mitochondrial proteins involved in aerobic respiration and mitochondrial membrane integrity. Enzymes in the electron transport system (ETS) decreased in heart and increased massively in liver during anoxia and reoxygenation but did not change in the brain. Importantly, the data support a special role for the liver in succinate handling upon reoxygenation, as suggested by a drastic increase of components of the ETS and uncoupling protein 2, which could allow for succinate metabolism without excessive formation of reactive oxygen species (ROS). Also during reoxygenation, the levels of proteins involved in the cristae junction organization of the mitochondria changed in the heart, possibly functioning to suppress ROS formation. Furthermore, proteins involved in immune (complement) system activation changed in the anoxic heart compared to normoxic controls. The results emphasize that responses to anoxia are highly tissue-specific and related to organ function.

An extended spectrum of target proteins and modification sites in the general O-linked protein glycosylation system in Neisseria gonorrhoeae.

The bacterial human pathogen Neisseria gonorrhoeae expresses a general O-linked protein glycosylation (Pgl) system known to target at least 12 membrane-associated proteins. To facilitate a better understanding of the mechanisms, significance and function of this glycosylation system, we sought to further delineate the target proteome of the Pgl system. To this end, we employed immunoaffinity enrichment of glycoproteins using a monoclonal antibody against the glycan moiety. Enzymatically generated peptides were subsequently analyzed by MS to identify glycopeptides and glycosylation sites. In this way, we increase the total number of known glycoproteins in N. gonorrhoeae to 19. These new glycoproteins are involved in a wide variety of extracytoplasmic functions. By employing collision fragmentation, we mapped nine new glycosylation sites, all of which were serine. No target sequon was readily apparent, although attachment sites were most often localized with regions of low sequence complexity. Moreover, we found that 5 of the proteins were modified with more than one glycan. This work thus confirms and extends earlier observations on the structural features of Neisseria glycoproteins.

Novel protein substrates of the phospho-form modification system in Neisseria gonorrhoeae and their connection to O-linked protein glycosylation.

The zwitterionic phospho-form moieties phosphoethanolamine (PE) and phosphocholine (PC) are important components of bacterial membranes and cell surfaces. The major type IV pilus subunit protein of Neisseria gonorrhoeae, PilE, undergoes posttranslational modifications with these moieties via the activity of the pilin phospho-form transferase PptA. A number of observations relating to colocalization of phospho-form and O-linked glycan attachment sites in PilE suggested that these modifications might be either functionally or mechanistically linked or interact directly or indirectly. Moreover, it was unknown whether the phenomenon of phospho-form modification was solely dedicated to PilE or if other neisserial protein targets might exist. In light of these concerns, we screened for evidence of phospho-form modification on other membrane glycoproteins targeted by the broad-spectrum O-linked glycosylation system. In this way, two periplasmic lipoproteins, NGO1043 and NGO1237, were identified as substrates for PE addition. As seen previously for PilE, sites of PE modifications were clustered with those of glycan attachment. In the case of NGO1043, evidence for at least six serine phospho-form attachment sites was found, and further analyses revealed that at least two of these serines were also attachment sites for glycan. Finally, mutations altering glycosylation status led to the presence of pptA-dependent PC modifications on both proteins. Together, these results reinforce the associations established in PilE and provide evidence for dynamic interplay between phospho-form modification and O-linked glycosylation. The observations also suggest that phospho-form modifications likely contribute biologically at both intracellular and extracellular levels.

Broad spectrum O-linked protein glycosylation in the human pathogen Neisseria gonorrhoeae.

Protein glycosylation is an important element of biologic systems because of its significant effects on protein properties and functions. Although prominent within all domains of life, O-linked glycosylation systems modifying serine and threonine residues within bacteria and eukaryotes differ substantially in target protein selectivity. In particular, well-characterized bacterial systems have been invariably dedicated to modification of individual proteins or related subsets thereof. Here we characterize a general O-linked glycosylation system that targets structurally and functionally diverse groups of membrane-associated proteins in the gram-negative bacterium Neisseria gonorrhoeae, the etiologic agent of the human disease gonorrhea. The 11 glycoproteins identified here are implicated in activities as varied as protein folding, disulfide bond formation, and solute uptake, as well as both aerobic and anaerobic respiration. Along with their common trafficking within the periplasmic compartment, the protein substrates share quasi-related domains bearing signatures of low complexity that were demonstrated to encompass sites of glycan occupancy. Thus, as in eukaryotes, the broad scope of this system is dictated by the relaxed specificity of the glycan transferase as well as the bulk properties and context of the protein-targeting signal rather than by a strict amino acid consensus sequence. Together, these findings reveal previously unrecognized commonalities linking O-linked protein glycosylation in distantly related life forms.

Sculpting the Bacterial O-Glycoproteome: Functional Analyses of Orthologous Oligosaccharyltransferases with Diverse Targeting Specificities.

Protein glycosylation systems are widely recognized in bacteria, including members of the genus Neisseria. In most bacterial species, the molecular mechanisms and evolutionary contexts underpinning target protein selection and the glycan repertoire remain poorly understood. Broad-spectrum O-linked protein glycosylation occurs in all human-associated species groups within the genus Neisseria, but knowledge of their individual glycoprotein repertoires is limited. Interestingly, PilE, the pilin subunit of the type IV pilus (Tfp) colonization factor, is glycosylated in Neisseria gonorrhoeae and Neisseria meningitidis but not in the deeply branching species N. elongata subsp. glycolytica. To examine this in more detail, we assessed PilE glycosylation status across the genus and found that PilEs of commensal clade species are not modified by the gonococcal PglO oligosaccharyltransferase. Experiments using PglO oligosaccharyltransferases from across the genus expressed in N. gonorrhoeae showed that although all were capable of broad-spectrum protein glycosylation, those from a deep-branching group of commensals were unable to support resident PilE glycosylation. Further glycoproteomic analyses of these strains using immunoblotting and mass spectrometry revealed other proteins differentially targeted by otherwise remarkably similar oligosaccharyltransferases. Finally, we generated pglO allelic chimeras that begin to localize PglO protein domains associated with unique substrate targeting activities. These findings reveal previously unappreciated differences within the protein glycosylation systems of highly related bacterial species. We propose that the natural diversity manifest in the neisserial protein substrates and oligosaccharyltransferases has significant potential to inform the structure-function relationships operating in these and related bacterial protein glycosylation systems. IMPORTANCE Although general protein glycosylation systems have been well recognized in prokaryotes, the processes governing their distribution, function, and evolution remain poorly understood. Here, we have begun to address these gaps in knowledge by comparative analyses of broad-spectrum O-linked protein glycosylation manifest in species within the genus Neisseria that strictly colonize humans. Using N. gonorrhoeae as a well-defined model organism in conjunction with comparative genomics, intraspecies gene complementation, and glycoprotein phenotyping, we discovered clear differences in both glycosylation susceptibilities and enzymatic targeting activities of otherwise largely conserved proteins. These findings reveal previously unappreciated differences within the protein glycosylation systems of highly related bacterial species. We propose that the natural diversity manifest within Neisseria species has significant potential to elucidate the structure-function relationships operating in these and related systems and to inform novel approaches to applied glycoengineering strategies.