General N-and O-linked glycosylation of lipoproteins in mycoplasmas and role of exogenous oligosaccharide

The lack of a cell wall, flagella, fimbria, and other extracellular appendages and the possession of only a single membrane render the mycoplasmas structurally simplistic and ideal model organisms for the study of glycoconjugates. Most species have genomes of about 800 kb and code for few proteins predicted to have a role in glycobiology. The murine pathogens Mycoplasma arthritidis and Mycoplasma pulmonis have only a single gene annotated as coding for a glycosyltransferase but synthesize glycolipid, polysaccharide and glycoproteins. Previously, it was shown that M. arthritidis glycosylated surface lipoproteins through O-linkage. In the current study, O-linked glycoproteins were similarly found in M. pulmonis and both species of mycoplasma were found to also possess N-linked glycans at residues of asparagine and glutamine. Protein glycosylation occurred at numerous sites on surface-exposed lipoproteins with no apparent amino acid sequence specificity. The lipoproteins of Mycoplasma pneumoniae also are glycosylated. Glycosylation was dependent on the glycosidic linkages from host oligosaccharides. As far as we are aware, N-linked glycoproteins have not been previously described in Gram-positive bacteria, the organisms to which the mycoplasmas are phylogenetically related. The findings indicate that the mycoplasma cell surface is heavily glycosylated with implications for the modulation of mycoplasma-host interactions

Rhamnose Links Moonlighting Proteins to Membrane Phospholipid in Mycoplasmas.

Many proteins that have a primary function as a cytoplasmic protein are known to have the ability to moonlight on the surface of nearly all organisms. An example is the glycolytic enzyme enolase, which can be found on the surface of many types of cells from bacteria to human. Surface enolase is not enzymatic because it is monomeric and oligomerization is required for glycolytic activity. It can bind various molecules and activate plasminogen. Enolase lacks a signal peptide and the mechanism by which it attaches to the surface is unknown. We found that treatment of whole cells of the murine pathogen Mycoplasma pulmonis with phospholipase D released enolase and other common moonlighting proteins. Glycostaining suggested that the released proteins were glycosylated. Cytoplasmic and membrane-bound enolase was isolated by immunoprecipitation. No post-translational modification was detected on cytoplasmic enolase, but membrane enolase was associated with lipid, phosphate and rhamnose. Treatment with phospholipase released the lipid and phosphate from enolase but not the rhamnose. The site of rhamnosylation was identified as a glutamine residue near the C-terminus of the protein. Rhamnose has been found in all species of mycoplasma examined but its function was previously unknown. Mycoplasmas are small bacteria with have no peptidoglycan, and rhamnose in these organisms is also not associated with polysaccharide. We suggest that rhamnose has a central role in anchoring proteins to the membrane by linkage to phospholipid, which may be a general mechanism for the membrane association of moonlighting proteins in mycoplasmas and perhaps other bacteria

Rhamnose Links Moonlighting Proteins to Membrane Phospholipid in Mycoplasmas - Fig 2

<p>GC/MS of a rhamnose standard (panel A), IP-purified membrane enolase isolated from a SDS-PAGE gel (panel B), and the same material as analyzed in panel B after subsequent treatment with phospholipase D (panel C).</p

A Stochastic Mechanism for Biofilm Formation by Mycoplasma pulmonis

Bacterial biofilms are communities of bacteria that are enclosed in an extracellular matrix. Within a biofilm the bacteria are protected from antimicrobials, environmental stresses, and immune responses from the host. Biofilms are often believed to have a highly developed organization that is derived from differential regulation of the genes that direct the synthesis of the extracellular matrix and the attachment to surfaces. The mycoplasmas have the smallest of the prokaryotic genomes and apparently lack complex gene-regulatory systems. We examined biofilm formation by Mycoplasma pulmonis and found it to be dependent on the length of the tandem repeat region of the variable surface antigen (Vsa) protein. Mycoplasmas that produced a short Vsa protein with few tandem repeats formed biofilms that attached to polystyrene and glass. Mycoplasmas that produced a long Vsa protein with many tandem repeats formed microcolonies that floated freely in the medium. The biofilms and the microcolonies contained an extracellular matrix which contained Vsa protein, lipid, DNA, and saccharide. As variation in the number of Vsa tandem repeats occurs by slipped-strand mispairing, the ability of the mycoplasmas to form a biofilm switches stochastically

HPLC plot of the relative abundance of the LLEIEDQLEEAAVFPGK peptide of enolase.

<p>The inset is a plot of another peptide from enolase that lacks lipid modification.</p

SDS-PAGE analysis of <i>M</i>. <i>pulmonis</i>.

<p>Panel A, Coomassie-stained gel of supernatants obtained by treatment of whole cells with phospholipase D (PLD) or heat-killed enzyme (HK-PLD). Proteins were eluted from the gel in wide swaths that were analyzed by ESI-TOF MS for protein identification (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162505#pone.0162505.t001" target="_blank">Table 1</a>). Panel B, membrane proteins were analyzed on gels stained with Coomassie, Pro-Q Emerald (Glycostain), Pro-Q Diamond (Phosphostain), Western blot reacted with antibody to enolase, and a Coomassie-stained gel of IP-purified enolase. Panel C, IP-purified enolase from membrane and cytoplasmic fractions were analyzed on gels stained for total protein (Coomassie), glycoprotein, or phosphoprotein. In all panels numbers refer to the masses of protein standards, and arrows refer to the expected location of enolase.</p

MS/MS of the LLEIEDQLEEAAVFPGK peptide showing modification at Gln437.

<p>Inset A. Model for the 128.05 m/<i>z</i> residue. Inset B. Model of the PTM associated with membrane enolase. The model is based on phosphatidylcholine because this lipid is a main substrate for phospholipase D and is a major lipid for mycoplasmas grown in serum [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162505#pone.0162505.ref042" target="_blank">42</a>].</p

HR-MS of the LLEIEDQLEEAAVFPGK peptide showing the modified and unmodified states for <i>z</i> = 2 and <i>z</i> = 3.

<p>Numbers at the top are the theoretical and experimentally observed masses of the peptide.</p

A Computational Analysis of Central CO2 Chemosensitivity in \u3ci\u3eHelix aspersa\u3c/i\u3e

We created a single-compartment computer model of a CO2 chemosensory neuron using differential equations adapted from the Hodgkin-Huxley model and measurements of currents in CO2 chemosensory neurons from Helix aspersa. We incorporated into the model two inward currents, a sodium current and a calcium current, three outward potassium currents, an A-type current (IKA), a delayed rectifier current (IKDR), a calcium-activated potassium current (IKCa), and a proton conductance found in invertebrate cells. All of the potassium channels were inhibited by reduced pH. We also included the pH regulatory process to mimic the effect of the sodium-hydrogen exchanger (NHE) described in these cells during hypercapnic stimulation. The model displayed chemosensory behavior (increased spike frequency during acid stimulation), and all three potassium channels participated in the chemosensory response and shaped the temporal characteristics of the response to acid stimulation. pH-dependent inhibition ofIKA initiated the response to CO2, but hypercapnic inhibition of IKDR and IKCaaffected the duration of the excitatory response to hypercapnia. The presence or absence of NHE activity altered the chemosensory response over time and demonstrated the inadvisability of effective intracellular pH (pHi) regulation in cells designed to act as chemostats for acid-base regulation. The results of the model indicate that multiple channels contribute to CO2 chemosensitivity, but the primary sensor is probably IKA. pHi may be a sufficient chemosensory stimulus, but it may not be a necessary stimulus: either pHi or extracellular pH can be an effective stimuli if chemosensory neurons express appropriate pH-sensitive channels. The lack of pHi regulation is a key feature determining the neuronal activity of chemosensory cells over time, and the balanced lack of pHi regulation during hypercapnia probably depends on intracellular activation of pHi regulation but extracellular inhibition of pHi regulation. These general principles are applicable to all CO2 chemosensory cells in vertebrate and invertebrate neurons

A Computational Analysis of Central CO2 Chemosensitivity in \u3ci\u3eHelix aspersa\u3c/i\u3e

We created a single-compartment computer model of a CO2 chemosensory neuron using differential equations adapted from the Hodgkin-Huxley model and measurements of currents in CO2 chemosensory neurons from Helix aspersa. We incorporated into the model two inward currents, a sodium current and a calcium current, three outward potassium currents, an A-type current (IKA), a delayed rectifier current (IKDR), a calcium-activated potassium current (IKCa), and a proton conductance found in invertebrate cells. All of the potassium channels were inhibited by reduced pH. We also included the pH regulatory process to mimic the effect of the sodium-hydrogen exchanger (NHE) described in these cells during hypercapnic stimulation. The model displayed chemosensory behavior (increased spike frequency during acid stimulation), and all three potassium channels participated in the chemosensory response and shaped the temporal characteristics of the response to acid stimulation. pH-dependent inhibition ofIKA initiated the response to CO2, but hypercapnic inhibition of IKDR and IKCaaffected the duration of the excitatory response to hypercapnia. The presence or absence of NHE activity altered the chemosensory response over time and demonstrated the inadvisability of effective intracellular pH (pHi) regulation in cells designed to act as chemostats for acid-base regulation. The results of the model indicate that multiple channels contribute to CO2 chemosensitivity, but the primary sensor is probably IKA. pHi may be a sufficient chemosensory stimulus, but it may not be a necessary stimulus: either pHi or extracellular pH can be an effective stimuli if chemosensory neurons express appropriate pH-sensitive channels. The lack of pHi regulation is a key feature determining the neuronal activity of chemosensory cells over time, and the balanced lack of pHi regulation during hypercapnia probably depends on intracellular activation of pHi regulation but extracellular inhibition of pHi regulation. These general principles are applicable to all CO2 chemosensory cells in vertebrate and invertebrate neurons