Antibodies against Neisseria meningitidis serogroups A, C, W and Y in serum and saliva of Norwegian adolescents.

INTRODUCTION: The incidence of invasive meningococcal disease (IMD) among Norwegian 16-19-year-olds was 1-7/100,000 in the decade before the COVID-19 pandemic, with serogroup Y (MenY) dominance. In contrast to many other European countries, meningococcal vaccines are not part of the national immunisation program (NIP) in Norway. This cross-sectional study aimed to measure the degree of natural immunity against Neisseria meningitidis among adolescents in Norway to evaluate the need for introducing tetravalent meningococcal conjugate vaccine (MCV4) in the NIP. MATERIALS AND METHODS: Serum and saliva samples were collected from students in upper and lower secondary schools in Norway in 2018. Samples were analysed for meningococcal capsular polysaccharide (PS)-specific antibodies using a bead-based multiplex immunoassay. PS-specific antibody levels were linked to data on meningococcal carriage, vaccination status and risk factors for carriage (assessed with questionnaire) and analysed by linear regression of log transformed concentrations. A subset of samples from unvaccinated individuals was analysed for serum bactericidal antibodies (SBA). RESULTS: A total of 1344 participants, median age 16 years (range 12-24), were included in the study. Overall, 60.9% of the participants were female and 1137 (84.6%) were not vaccinated with MCV4. PS-specific antibody concentrations in serum and saliva were low among unvaccinated individuals for all serogroups and only 6.7-20.0% of the subpopulation with high PS-specific antibodies assessed with SBA had protective levels. Unvaccinated MenY carriers had higher levels of MenY anti-PS IgG in serum and IgA in saliva than those not carrying MenY. Use of Swedish snus was associated with lower anti-PS IgG levels in serum and waterpipe use with lower anti-PS IgG levels in saliva. CONCLUSION: Unvaccinated adolescents in Norway have a low degree of natural immunity against the serogroups of N. meningitidis predominating among cases of IMD in this age group. Therefore, introduction of MCV4 for adolescents in the NIP is recommended.

Genetic, Functional, and Immunogenic Analyses of the O-Linked Protein Glycosylation System in Neisseria meningitidis Serogroup A ST-7 Isolates.

Neisseria meningitidis exhibits a general O-linked protein glycosylation system in which pili and other extracytoplasmic proteins are glycosylated. To investigate glycan antigenicity in humans and the significance of high glycan diversity on immune escape mechanisms, we exploited serogroup A meningococcal strains and serum samples obtained from laboratory-confirmed Ethiopian patients with meningococcal disease. The 37 meningococcal isolates were sequenced, and their protein glycosylation (pgl) genotypes and protein glycosylation phenotypes were investigated in detail. An insertion sequence (IS1655) element in pglH reduced glycan variability in the majority of isolates, while phase variation strengthened glycan variability and microheterogeneity. Homologous recombination events within the pgl genes were identified in eight of the 37 isolates, and the phenotypic consequences ranged from none detected to altered glycoforms in two of the isolates in which the whole pgl locus was exchanged. Immunoblotting of sera against a complete panel of glycan-expressing mutant strains demonstrated that most of these patient sera had IgG antibodies against various neisserial protein glycan antigens. Furthermore, using a bactericidal assay comparing a wild-type meningococcal A strain and a glycosylation-null variant strain, we showed that these protein glycan antigens interfere with bactericidal killing by antibodies in patient sera. Altogether, we were largely able to link pgl genotype with glycosylation phenotype. Our study reveals that protein glycans seem to contribute to the ability of N. meningitidis to resist the bactericidal activity of human serum, possibly by masking protein epitopes important for bactericidal killing and thus protection against meningococcal disease. IMPORTANCE Bacterial meningitis is a serious global health problem, and one of the major causative organisms is Neisseria meningitidis. Extensive variability in protein glycan structure and antigenicity is due to phase variation of protein glycosylation genes and polymorphic gene content and function. The exact role(s) of glycosylation in Neisseria remains to be determined, but increasing evidence, supported by this study, suggests that glycan variability can be a strategy to escape the human immune system. The complexity of the O-linked protein glycosylation system requires further studies to fully comprehend how these bacteria utilize variation in pgl genes to produce such high glycoform diversity and to evade the human immune response.

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.

Genotypic and Phenotypic Characterization of the O-Linked Protein Glycosylation System Reveals High Glycan Diversity in Paired Meningococcal Carriage Isolates.

Species within the genus Neisseria display significant glycan diversity associated with the O-linked protein glycosylation (pgl) systems due to phase variation and polymorphic genes and gene content. The aim of this study was to examine in detail the pgl genotype and glycosylation phenotype in meningococcal isolates and the changes occurring during short-term asymptomatic carriage. Paired meningococcal isolates derived from 50 asymptomatic meningococcal carriers, taken about 2 months apart, were analyzed with whole-genome sequencing. The O-linked protein glycosylation genes were characterized in detail using the Genome Comparator tool at the https://pubmlst.org/ database. Immunoblotting with glycan-specific antibodies (Abs) was used to investigate the protein glycosylation phenotype. All major pgl locus polymorphisms identified in Neisseria meningitidis to date were present in our isolate collection, with the variable presence of pglG and pglH, both in combination with either pglB or pglB2 We identified significant changes and diversity in the pgl genotype and/or glycan phenotype in 96% of the paired isolates. There was also a high degree of glycan microheterogeneity, in which different variants of glycan structures were found at a given glycoprotein. The main mechanism responsible for the observed differences was phase-variable expression of the involved glycosyltransferases and the O-acetyltransferase. To our knowledge, this is the first characterization of the pgl genotype and glycosylation phenotype in a larger strain collection. This report thus provides important insight into glycan diversity in N. meningitidis and into the phase variability changes that influence the expressed glycoform repertoire during meningococcal carriage.IMPORTANCE Bacterial meningitis is a serious global health problem, and one of the major causative organisms is Neisseria meningitidis, which is also a common commensal in the upper respiratory tract of healthy humans. In bacteria, numerous loci involved in biosynthesis of surface-exposed antigenic structures that are involved in the interaction between bacteria and host are frequently subjected to homologous recombination and phase variation. These mechanisms are well described in Neisseria, and phase variation provides the ability to change these structures reversibly in response to the environment. Protein glycosylation systems are becoming widely identified in bacteria, and yet little is known about the mechanisms and evolutionary forces influencing glycan composition during carriage and disease.

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.

Whole genome sequencing reveals within-host genetic changes in paired meningococcal carriage isolates from Ethiopia.

BACKGROUND: Meningococcal colonization is a prerequisite for transmission and disease, but the bacterium only very infrequently causes disease while asymptomatic carriage is common. Carriage is highly dynamic, showing a great variety across time and space within and across populations, but also within individuals. The understanding of genetic changes in the meningococcus during carriage, when the bacteria resides in its natural niche, is important for understanding not only the carriage state, but the dynamics of the entire meningococcal population. RESULTS: Paired meningococcal isolates, obtained from 50 asymptomatic carriers about 2 months apart were analyzed with whole genome sequencing (WGS). Phylogenetic analysis revealed that most paired isolates from the same individual were closely related, and the average and median number of allelic differences between paired isolates defined as the same strain was 35. About twice as many differences were seen between isolates from different individuals within the same sequence type (ST). In 8%, different strains were detected at different time points. A difference in ST was observed in 6%, including an individual who was found to carry three different STs over the course of 9 weeks. One individual carried different strains from the same ST. In total, 566 of 1605 cgMLST genes had undergone within-host genetic changes in one or more pairs. The most frequently changed cgMLST gene was relA that was changed in 47% of pairs. Across the whole genome, pilE, differed mostly, in 85% of the pairs. The most frequent mechanisms of genetic difference between paired isolates were phase variation and recombination, including gene conversion. Different STs showed variation with regard to which genes that were most frequently changed, mostly due to absence/presence of phase variation. CONCLUSIONS: This study revealed within-host genetic differences in meningococcal isolates during short-term asymptomatic carriage. The most frequently changed genes were genes belonging to the pilin family, the restriction/modification system, opacity proteins and genes involved in glycosylation. Higher resolution genome-wide sequence typing is necessary to resolve the diversity of isolates and reveals genetic differences not discovered by traditional typing schemes, and would be the preferred choice of technology.

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.

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.

Allelic variation in a simple sequence repeat element of neisserial pglB2 and its consequences for protein expression and protein glycosylation.

Neisseria species express an O-linked glycosylation system in which functionally distinct proteins are elaborated with variable glycans. A major source of glycan diversity in N. meningitidis results from two distinct pglB alleles responsible for the synthesis of either N,N'-diacetylbacillosamine or glyceramido-acetamido trideoxyhexose that occupy the reducing end of the oligosaccharides. Alternative modifications at C-4 of the precursor UDP-4-amino are attributable to distinct C-terminal domains that dictate either acetyltransferase or glyceramidotransferase activity, encoded by pglB and pglB2, respectively. Naturally occurring alleles of pglB2 have homopolymeric tracts of either 7 or 8 adenosines (As) bridging the C-terminal open reading frame (ORF) and the ORF encompassing the conserved N-terminal domain associated with phosphoglycosyltransferase activity. In the work presented here, we explored the consequences of such pglB2 allele variation and found that, although both alleles are functional vis-à-vis glycosylation, the 7A form results in the expression of a single, multidomain protein, while the 8A variant elicits two single-domain proteins. We also found that the glyceramidotransferase activity-encoding domain is essential to protein glycosylation, showing the critical role of the C-4 modification of the precursor UDP-4-amino in the pathway. These findings were further extended and confirmed by examining the phenotypic consequences of extended poly(A) tract length variation. Although ORFs related to those of pglB2 are broadly distributed in eubacteria, they are primarily found as two distinct, juxtaposed ORFs. Thus, the neisserial pglB2 system provides novel insights into the potential influence of hypermutability on modular evolution of proteins by providing a unique snapshot of the progression of ongoing gene fusion.

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.

Hypomorphic glycosyltransferase alleles and recoding at contingency loci influence glycan microheterogeneity in the protein glycosylation system of Neisseria species.

As more bacterial protein glycosylation systems are identified and characterized, a central question that arises is, what governs the prevalence of particular glycans associated with them? In addition, accumulating evidence shows that bacterial protein glycans can be subject to the phenomenon of microheterogeneity, in which variant glycan structures are found at specific attachment sites of a given glycoprotein. Although factors underlying microheterogeneity in reconstituted expression systems have been identified and modeled, those impacting natural systems largely remain enigmatic. On the basis of a sensitive and specific glycan serotyping system, microheterogeneity has been reported for the broad-spectrum, O-linked protein glycosylation system in species within the genus Neisseria. To elucidate the mechanisms involved, a genetic approach was used to identify a hypomorphic allele of pglA (encoding the PglA galactosyltransferase) as a significant contributor to simultaneous expression of multiple glycoforms. Moreover, this phenotype was mapped to a single amino acid polymorphism in PglA. Further analyses revealed that many pglA phase-off variants (containing out-of-frame configurations in simple nucleotide repeats within the open reading frame) were associated with disproportionally high levels of the N,N'-diacetylbacillosamine-Gal disaccharide glycoform generated by PglA. This phenotype is emblematic of nonstandard decoding involving programmed ribosomal frameshifting and/or programmed transcriptional realignment. Together, these findings provide new information regarding the mechanisms of neisserial protein glycan microheterogeneity and the anticipatory nature of contingency loci.

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.

Genetic and molecular analyses reveal an evolutionary trajectory for glycan synthesis in a bacterial protein glycosylation system.

Although protein glycosylation systems are becoming widely recognized in bacteria, little is known about the mechanisms and evolutionary forces shaping glycan composition. Species within the genus Neisseria display remarkable glycoform variability associated with their O-linked protein glycosylation (pgl) systems and provide a well developed model system to study these phenomena. By examining the potential influence of two ORFs linked to the core pgl gene locus, we discovered that one of these, previously designated as pglH, encodes a glucosyltransferase that generates unique disaccharide products by using polyprenyl diphosphate-linked monosaccharide substrates. By defining the function of PglH in the glycosylation pathway, we identified a metabolic conflict related to competition for a shared substrate between the opposing glycosyltransferases PglA and PglH. Accordingly, we propose that the presence of a stereotypic, conserved deletion mutation inactivating pglH in strains of Neisseria gonorrhoeae, Neisseria meningitidis, and related commensals, reflects a resolution of this conflict with the consequence of reduced glycan diversity. This model of genetic détente is supported by the characterization of pglH "missense" alleles encoding proteins devoid of activity or reduced in activity such that they cannot exert their effect in the presence of PglA. Thus, glucose-containing glycans appear to be a trait undergoing regression at the genus level. Together, these findings document a role for intrinsic genetic interactions in shaping glycan evolution in protein glycosylation systems.

Biochemical characterization of the O-linked glycosylation pathway in Neisseria gonorrhoeae responsible for biosynthesis of protein glycans containing N,N'-diacetylbacillosamine.

The O-linked protein glycosylation pathway in Neisseria gonorrhoeae is responsible for the synthesis of a complex oligosaccharide on undecaprenyl diphosphate and subsequent en bloc transfer of the glycan to serine residues of select periplasmic proteins. Protein glycosylation (pgl) genes have been annotated on the basis of bioinformatics and top-down mass spectrometry analysis of protein modifications in pgl-null strains [Aas, F. E., et al. (2007) Mol. Microbiol. 65, 607-624; Vik, A., et al. (2009) Proc. Natl. Acad. Sci. U.S.A. 106, 4447-4452], but relatively little biochemical analysis has been performed to date. In this report, we present the expression, purification, and functional characterization of seven Pgl enzymes. Specifically, the enzymes studied are responsible for synthesis of an uncommon uridine diphosphate (UDP)-sugar (PglD, PglC, and PglB-acetyltransferase domain), glycan assembly (PglB-phospho-glycosyltransferase domain, PglA, PglE, and PglH), and final oligosaccharide transfer (PglO). UDP-2,4-diacetamido-2,4,6-trideoxy-α-d-hexose (DATDH), which is the first sugar in glycan biosynthesis, was produced enzymatically, and the stereochemistry was assigned as uridine diphosphate N'-diacetylbacillosamine (UDP-diNAcBac) by nuclear magnetic resonance characterization. In addition, the substrate specificities of the phospho-glycosyltransferase, glycosyltransferases, and oligosaccharyltransferase (OTase) were analyzed in vitro, and in most cases, these enzymes exhibited strong preferences for the native substrates relative to closely related glycans. In particular, PglO, the O-linked OTase, and PglB(Cj), the N-linked OTase from Campylobacter jejuni, preferred the native N. gonorrhoeae and C. jejuni substrates, respectively. This study represents the first comprehensive biochemical characterization of this important O-linked glycosylation pathway and provides the basis for further investigations of these enzymes as antibacterial targets.

Genetic, structural, and antigenic analyses of glycan diversity in the O-linked protein glycosylation systems of human Neisseria species.

Bacterial capsular polysaccharides and lipopolysaccharides are well-established ligands of innate and adaptive immune effectors and often exhibit structural and antigenic variability. Although many surface-localized glycoproteins have been identified in bacterial pathogens and symbionts, it not clear if and how selection impacts associated glycoform structure. Here, a systematic approach was devised to correlate gene repertoire with protein-associated glycoform structure in Neisseria species important to human health and disease. By manipulating the protein glycosylation (pgl) gene content and assessing the glycan structure by mass spectrometry and reactivity with monoclonal antibodies, it was established that protein-associated glycans are antigenically variable and that at least nine distinct glycoforms can be expressed in vitro. These studies also revealed that in addition to Neisseria gonorrhoeae strain N400, one other gonococcal strain and isolates of Neisseria meningitidis and Neisseria lactamica exhibit broad-spectrum O-linked protein glycosylation. Although a strong correlation between pgl gene content, glycoform expression, and serological profile was observed, there were significant exceptions, particularly with regard to levels of microheterogeneity. This work provides a technological platform for molecular serotyping of neisserial protein glycans and for elucidating pgl gene evolution.

cAMP-dependent protein kinase regulates ubiquitin-proteasome-mediated degradation and subcellular localization of the nuclear receptor coactivator GRIP1.

Nuclear receptors and their coactivators are key regulators of numerous physiological functions. GRIP1 (glucocorticoid receptor-interacting protein) is a member of the steroid receptor coactivator family. Here, we show that GRIP1 is regulated by cAMP-dependent protein kinase (PKA) that induces its degradation through the ubiquitin-proteasome pathway. GRIP1 was down-regulated in transiently transfected COS-1 cells after treatment with 8-para-chlorophenylthio-cAMP or forskolin and 3-isobutyl-1-methylxanthine and in adrenocortical Y1 cells after incubation with adrenocorticotropic hormone. Pulse-chase experiments with transiently transfected COS-1 cells demonstrated that the half-life of GRIP1 was markedly reduced in cells overexpressing the PKA catalytic subunit, suggesting that activation of PKA increases the turnover of GRIP1 protein. The proteasome inhibitors MG132 and lactacystin abolished the PKA-mediated degradation of GRIP1. Using ts20 cells, a temperature-sensitive cell line that contains a thermolabile ubiquitin-activating E1 enzyme, it was confirmed that PKA-mediated degradation of GRIP1 is dependent upon the ubiquitin-proteasome pathway. Coimmunoprecipitation studies of COS-1 cells transfected with expression vectors encoding GRIP1 and ubiquitin using anti-GRIP1 and anti-ubiquitin antibodies showed that the ubiquitination of GRIP1 was increased by overexpression of PKA. Finally, we show that PKA regulates the intracellular distribution pattern of green fluorescent protein-GRIP1 and stimulates recruitment of GRIP1 to subnuclear foci that are colocalized with the proteasome. Taken together, these data demonstrate that GRIP1 is ubiquitinated and degraded through activation of the PKA pathway. This may represent a novel regulatory mechanism whereby hormones down-regulate a nuclear receptor coactivator.

Cloning and characterization of a novel zinc finger protein that modulates the transcriptional activity of nuclear receptors.

The orphan nuclear receptor steroidogenic factor-1 (SF-1) plays pivotal roles in the development and function of steroidogenic organs. It transcriptionally regulates an array of factors required for biosynthesis of steroid hormones and is also necessary for the expression of genes in the pituitary and the male reproductive tract. Here we describe the identification of a novel zinc finger protein that modifies the transcriptional potential of SF-1. This factor, which we call Zip67 (zinc finger protein 67 kDa), was cloned through a two-hybrid screen of a human testis cDNA library using the C-terminal part of SF-1 as the bait. Transient transfection experiments demonstrated that Zip67 represses SF-1-dependent transcription in the context of both multimerized SF-1-binding sites and natural SF-1-inducible promoters. The interaction between Zip67 and SF-1 was dependent on an intact activation function-2 domain of SF-1, and we propose a mechanism whereby Zip67 represses transcription through competition with p160 coactivators for binding to SF-1. Zip67 was detected in SF-1 expressing tissues such as testis, adrenal, ovary and spleen in addition to other tissues. In line with the broader expression pattern, we found that Zip67 also affected transcription mediated by several other nuclear receptors. In conclusion, we have isolated a novel zinc-finger protein that influences gene activation through interaction with the functionally important activation function-2 domain of nuclear receptors.

Characterization of receptor-interacting protein RIP140 in the regulation of SF-1 responsive target genes.

Receptor-interacting protein (RIP) 140 interacts with several nuclear receptors, but its function in regulation of nuclear receptor action has been debated. Here we have examined the role of RIP140 in regulation of Steroidogenic factor-1 (SF-1)-dependent transcription. SF-1 interacts with RIP140 through its activation function-2 (AF-2) domain. Several domains of RIP140 interact directly with SF-1, but the carboxyl-terminal region containing 4 of its 9 LXXLL motifs showed the strongest SF-1 interaction. Coexpression of RIP140 and SF-1 in different cell types demonstrated that RIP140 acts as a potent corepressor of transcription from the SF-1 responsive cAMP regulatory sequence 2 (CRS2) element of the CYP17 gene and a variety of SF-1 responsive promoter genes. RIP140 also counteracted the stimulatory action of p160/SRC coactivators. The inhibitory effect of RIP140 was partially reversed by Trichostatin A, suggesting a role of histone deacetylase (HDAC) activity in RIP140-mediated repression of SF-1. Quantitation of endogenous coregulator mRNA levels revealed cell type specific differences that could affect the repressor action by overexpressed RIP140.

The nuclear receptor coactivators p300/CBP/cointegrator-associated protein (p/CIP) and transcription intermediary factor 2 (TIF2) differentially regulate PKA-stimulated transcriptional activity of steroidogenic factor 1.

Steroidogenic factor-1 (SF-1) is a member of the nuclear receptor superfamily that plays essential roles in the development of endocrine organs. Steroid receptor coactivator 1 and transcription intermediary factor 2 (TIF2) belong to the p160 coactivator family that mediates transcriptional activation by several nuclear receptors, including SF-1. Here, it is reported that another of the p160 coactivators, p/CIP, interacts with SF-1 through the activation function-2 domain. Both p300/CBP/cointegrator-associated protein (p/CIP) and TIF2 potentiated SF-1-mediated transcription from two reporter gene constructs in transfected nonsteroidogenic COS-1 cells and in adrenocortical Y1 cells. PKA was shown to stimulate SF-1 transcriptional activity, and coexpression of p/CIP together with the PKA catalytic subunit stimulated SF-1-mediated transactivation even further. In contrast, PKA catalytic subunit overexpression impaired the ability of TIF2 to potentiate SF-1-dependent transcription. Activation of PKA also inhibited the TIF2-mediated coactivation of other nuclear receptors such as PPAR alpha/-gamma and liver X receptor-alpha. The TIF2 mRNA levels were not affected by PKA, but instead we found that PKA activation led to a decrease in the levels of TIF2 protein. Moreover, the C-terminal activation domain 2 of TIF2 was required for the inhibitory effect of PKA, suggesting that this region is the target for the PKA-mediated down-regulation. Thus, in contrast to the regulation of p/CIP and steroid receptor coactivator 1, we suggest that activation of PKA leads to selective down-regulation of TIF2 and subsequently repression of TIF2 coactivator function.

Differential regulation of SF-1-cofactor interactions.

The orphan nuclear receptor steroidogenic factor-1 (SF-1) plays pivotal roles in the development and function of steroidogenic organs. Here we describe the differential effect of protein kinase A (PKA) on coregulation of SF-1 dependent transcription by two p160 family members, p300/CBP co-integrator-associated protein (p/CIP) and transcription intermediary factor-2 (TIF2). Thus, whereas p/CIP-stimulated SF-1 dependent transcription is further potentiated by PKA, we show that activation of PKA leads to selective downregulation of TIF2 protein and a subsequent repression of TIF2 coactivator function. Using a yeast two-hybrid screen we also identified a novel zinc finger containing protein, which interacts with SF-1 via the AF-2 domain.