Genetic and Functional Analyses of PptA, a Phospho-Form Transferase Targeting Type IV Pili in Neisseria gonorrhoeae

The PilE pilin subunit protein of Neisseria gonorrhoeae undergoes unique covalent modifications with phosphoethanolamine (PE) and phosphocholine (PC). The pilin phospho-form transferase A (PptA) protein, required for these modifications, shows sequence relatedness with and architectural similarities to lipopolysaccharide PE transferases. Here, we used regulated expression and mutagenesis as means to better define the relationships between PptA structure and function, as well as to probe the mechanisms by which other factors impact the system. We show here that pptA expression is coupled at the level of transcription to its distal gene, murF, in a division/cell wall gene operon and that PptA can act in a dose-dependent fashion in PilE phospho-form modification. Molecular modeling and site-directed mutagenesis provided the first direct evidence that PptA is a member of the alkaline phosphatase superfamily of metalloenzymes with similar metal-binding sites and conserved structural folds. Through phylogenetic analyses and sequence alignments, these conclusions were extended to include the lipopolysaccharide PE transferases, including members of the disparate Lpt6 subfamily, and the MdoB family of phosphoglycerol transferases. Each of these enzymes thus likely acts as a phospholipid head group transferase whose catalytic mechanism involves a trans-esterification step generating a protein-phospho-form ester intermediate. Coexpression of PptA with PilE in Pseudomonas aeruginosa resulted in high levels of PE modification but was not sufficient for PC modification. This and other findings show that PptA-associated PC modification is governed by as-yet-undefined ancillary factors unique to N. gonorrhoeae

Identification of surface proteins in Enterococcus faecalis V583

<p>Abstract</p> <p>Background</p> <p>Surface proteins are a key to a deeper understanding of the behaviour of Gram-positive bacteria interacting with the human gastro-intestinal tract. Such proteins contribute to cell wall synthesis and maintenance and are important for interactions between the bacterial cell and the human host. Since they are exposed and may play roles in pathogenicity, surface proteins are interesting targets for drug design.</p> <p>Results</p> <p>Using methods based on proteolytic "shaving" of bacterial cells and subsequent mass spectrometry-based protein identification, we have identified surface-located proteins in <it>Enterococcus faecalis </it>V583. In total 69 unique proteins were identified, few of which have been identified and characterized previously. 33 of these proteins are predicted to be cytoplasmic, whereas the other 36 are predicted to have surface locations (31) or to be secreted (5). Lipid-anchored proteins were the most dominant among the identified surface proteins. The seemingly most abundant surface proteins included a membrane protein with a potentially shedded extracellular sulfatase domain that could act on the sulfate groups in mucin and a lipid-anchored fumarate reductase that could contribute to generation of reactive oxygen species.</p> <p>Conclusions</p> <p>The present proteome analysis gives an experimental impression of the protein landscape on the cell surface of the pathogenic bacterium <it>E. faecalis</it>. The 36 identified secreted (5) and surface (31) proteins included several proteins involved in cell wall synthesis, pheromone-regulated processes, and transport of solutes, as well as proteins with unknown function. These proteins stand out as interesting targets for further investigation of the interaction between <it>E. faecalis </it>and its environment.</p

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

The Major Autolysin Acm2 from Lactobacillus plantarum Undergoes Cytoplasmic O-Glycosylation

The major autolysin Acm2 from the probiotic strain Lactobacillus plantarum WCFS1 contains high proportions of alanine, serine, and threonine in its N-terminal so-called AST domain. It has been suggested that this extracellular protein might be glycosylated, but this has not been experimentally verified. We used high-resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS) to study the possible occurrence of glycans on peptides generated from lactobacillary surface proteins by protease treatment. This approach yielded five glycopeptides in various glycoforms, all derived from the AST domain of Acm2. All five glycopeptides contained the hydroxy-amino acids serine and threonine, suggesting that Acm2 is O-glycosylated. By using lectin blotting with succinylated wheat germ agglutinin, and by comparing the wild-type strain with an Acm2-negative derivative (NZ3557), we found that the attached N-acetylhexosamines are most likely N-acetylglucosamines (GlcNAc). NZ3557 was further used as a genetic background to express an Acm2 variant lacking its secretion signal, resulting in intracellular expression of Acm2. We show that this intracellular version of Acm2 is also glycosylated, indicating that the GlcNAc modification is an intracellular process

-linked pilin glycosylation: functional analyses define both the biosynthetic pathway and glycan structure-9

<p><b>Copyright information:</b></p><p>Taken from "-linked pilin glycosylation: functional analyses define both the biosynthetic pathway and glycan structure"</p><p></p><p>Molecular Microbiology 2007;65(3):607-624.</p><p>Published online 01 Aug 2007</p><p>PMCID:PMC1976384.</p><p>© 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd</p

Detection of acetic acid in the supernatants of an alkaline incubation mixture of Tfp preparations

<p><b>Copyright information:</b></p><p>Taken from "-linked pilin glycosylation: functional analyses define both the biosynthetic pathway and glycan structure"</p><p></p><p>Molecular Microbiology 2007;65(3):607-624.</p><p>Published online 01 Aug 2007</p><p>PMCID:PMC1976384.</p><p>© 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd</p> Total ion chromatograms (TIC) of the supernatant of the alkaline incubation mixtures of PilE at pH 10.5 after 0, 1 and 24 h (insets with selected ion chromatograms (SIC) of 60 for acetic acid). Electron impact ionization (EI) spectrum of the compound eluting at 11.2 min confirms formation of acetic acid over time

-linked pilin glycosylation: functional analyses define both the biosynthetic pathway and glycan structure-10

<p><b>Copyright information:</b></p><p>Taken from "-linked pilin glycosylation: functional analyses define both the biosynthetic pathway and glycan structure"</p><p></p><p>Molecular Microbiology 2007;65(3):607-624.</p><p>Published online 01 Aug 2007</p><p>PMCID:PMC1976384.</p><p>© 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd</p

-linked pilin glycosylation: functional analyses define both the biosynthetic pathway and glycan structure-7

<p><b>Copyright information:</b></p><p>Taken from "-linked pilin glycosylation: functional analyses define both the biosynthetic pathway and glycan structure"</p><p></p><p>Molecular Microbiology 2007;65(3):607-624.</p><p>Published online 01 Aug 2007</p><p>PMCID:PMC1976384.</p><p>© 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd</p