Listeria monocytogenes tyrosine phosphatases affect wall teichoic acid composition and phage resistance

Tyrosine phosphatase (PTP)-like proteins exist in many bacteria and are segregated into two major groups: low molecular weight and conventional. The latter group also has activity as phosphoinositide phosphatases. These two kinds of PTP are suggested to be involved in many aspects of bacterial physiology including stress response, DNA binding proteins, virulence, and capsule/cell wall production. By annotation, Listeria monocytogenes possesses two potential low molecular weight and two conventional PTPs. Using L. monocytogenes wild-type (WT) strain 10403S, we have created an in-frame deletion mutant lacking all four PTPs, as well as four additional complemented strains harboring each of the PTPs. No major physiological differences were observed between the WT and the mutant lacking all four PTPs. However, the deletion mutant strain was resistant to Listeria phages A511 and P35 and sensitive to other Listeria phages. This was attributed to reduced attachment to the cell wall. The mutant lacking all PTPs was found to lack N-acetylglucosamine in its wall teichoic acid. Phage sensitivity and attachment was rescued in a complemented strain harboring a low molecular weight PTP (LMRG1707

Vertical Transmission of Mycoplasma pneumoniae Infection

Mycoplasma pneumoniae is a significant cause of pneumonia in school-aged children and young adults. We report a case of neonatal M. pneumoniae pneumonia in a preterm child manifesting in the first hours of life. Vertical transmission was demonstrated by the detection of M. pneumoniae in inflamed placental tissue indicating chorioamnionitis

Evolutionarily distinct bacteriophage endolysins featuring conserved peptidoglycan cleavage sites protect mice from MRSA infection

Objectives In the light of increasing drug resistance in Staphylococcus aureus, bacteriophage endolysins [peptidoglycan hydrolases (PGHs)] have been suggested as promising antimicrobial agents. The aim of this study was to determine the antimicrobial activity of nine enzymes representing unique homology groups within a diverse class of staphylococcal PGHs. Methods PGHs were recombinantly expressed, purified and tested for staphylolytic activity in multiple in vitro assays (zymogram, turbidity reduction assay and plate lysis) and against a comprehensive set of strains (S. aureus and CoNS). PGH cut sites in the staphylococcal peptidoglycan were determined by biochemical assays (Park-Johnson and Ghuysen procedures) and MS analysis. The enzymes were tested for their ability to eradicate static S. aureus biofilms and compared for their efficacy against systemic MRSA infection in a mouse model. Results Despite similar modular architectures and unexpectedly conserved cleavage sites in the peptidoglycan (conferred by evolutionarily divergent catalytic domains), the enzymes displayed varying degrees of in vitro lytic activity against numerous staphylococcal strains, including cell surface mutants and drug-resistant strains, and proved effective against static biofilms. In a mouse model of systemic MRSA infection, six PGHs provided 100% protection from death, with animals being free of clinical signs at the end of the experiment. Conclusions Our results corroborate the high potential of PGHs for treatment of S. aureus infections and reveal unique antimicrobial and biochemical properties of the different enzymes, suggesting a high diversity of potential applications despite highly conserved peptidoglycan target site

Rapid Analysis of Listeria monocytogenes Cell Wall Teichoic Acid Carbohydrates by ESI-MS/MS

We report the application of electrospray ionization (ESI) mass spectrometry for compositional characterization of wall teichoic acids (WTA), a major component of Gram-positive bacterial cell walls. Tandem mass spectrometry (ESI-MS/MS) of purified and chemically hydrolyzed monomeric WTA components provided sufficient information to identify WTA monomers and their specific carbohydrate constituents. A lithium matrix was used for ionization of uncharged WTA monomers, and successfully applied to analyze the WTA molecules of four Listeria strains differing in carbohydrate substitution on a conserved polyribitol-phosphate backbone structure. Carbohydrate residues such as N-acetylglucosamine or rhamnose linked to the WTA could directly be identified by ESI-MS/MS, circumventing the need for quantitative analysis by gas chromatography. The presence of a terminal N-acetylglucosamine residue tethered to the ribitol was confirmed using fluorescently labeled wheat-germ agglutinin. In conclusion, the mass spectrometry method described here will greatly facilitate compositional analysis and characterization of teichoic acids and similar macromolecules from diverse bacterial species, and represents a significant advance in the identification of serovar-specific carbohydrates and sugar molecules on bacteria

Non-Standard Errors

In statistics, samples are drawn from a population in a data-generating process (DGP). Standard errors measure the uncertainty in estimates of population parameters. In science, evidence is generated to test hypotheses in an evidence-generating process (EGP). We claim that EGP variation across researchers adds uncertainty: Non-standard errors (NSEs). We study NSEs by letting 164 teams test the same hypotheses on the same data. NSEs turn out to be sizable, but smaller for better reproducible or higher rated research. Adding peer-review stages reduces NSEs. We further find that this type of uncertainty is underestimated by participants

ESI-MS/MS analysis of WTA monomers of <i>L. monocytogenes</i> strains EGDe, WSLC 1485, and WSLC 1442.

<p>Left (<b>A, D, G</b>): Positive ion spectra (ESI-MS) of WTA monomers. Middle (<b>B, E, H</b>): ESI-MS/MS of the [M+H]⁺ ion (B) at <i>m/z</i> 502.22 of WTA monomer EGDe, (E) at <i>m/z</i> 356.15 of WTA monomer 1485, and the [M+Li]⁺ ion (H) at <i>m/z</i> 305.15 of WTA monomer 1442. Right (<b>C, F, I</b>): Schematic structures and side chain fragmentation of corresponding WTA monomers. Peak labels show the measured <i>m/z</i> values. Arrows denote WTA fragmentation.</p

Mass spectrometry data, carbohydrate composition of the WTA repeating units, and WGA binding properties for the bacterial strains used in this study.

1<p>Gal, galactose; Glc, glucose; GlcNAc, <i>N</i>-acetylglucosamine; Rbo, ribitol; Rha, rhamnose; in serovar 4b strain WSLC 1042, GlcNAc is incorporated in the polyribitol-phosphate chain and decorated with Gal and Glc residues.</p>2<p>The calculated masses correspond to the monomeric unit of WTA, i.e., ribitol and attached carbohydrates.</p>3<p>WTAs of strains EGDe and WSLC 1485 were detected as protonated molecules, whereas WTA samples of strains WSLC 1442 and WSLC 1042 were identified as lithiated molecules.</p>4<p>+, binding; −, no binding; WGA: wheat germ agglutinin.</p>5<p><i>L. monocytogenes</i> strain WSLC 1442 is a GlcNAc-negative serovar 1/2a mutant strain <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021500#pone.0021500-Wendlinger1" target="_blank">[27]</a>.</p

Binding of wheat germ agglutinin (WGA) to <i>Listeria</i> cells wall.

<p><i>Listeria</i> cells of strain EGDe (left), WSLC 1485 (middle) and WSLC 1442 (right) were labeled with WGA-Alexa Fluor® 594 conjugate. WGA specifically recognizes GlcNAc residues in WTAs. Top: phase contrast images. Bottom: corresponding fluorescence microscopy (confocal) images.</p

Listeria monocytogenes tyrosine phosphatases affect wall teichoic acid composition and phage resistance

ISSN:0378-1097ISSN:1574-6968ISSN:0168-6496ISSN:0920-8534ISSN:0168-644

The tail-associated depolymerase of Erwinia amylovoraphage L1 mediates host cell adsorption and enzymatic capsule removal, which can enhance infection by other phage

The depolymerase enzyme (DpoL1) encoded by the T7-like phage L1 efficiently degrades amylovoran, an important virulence factor and major component of the extracellular polysaccharide (EPS) of its host, the plant pathogen Erwinia amylovora. Mass spectrometry analysis of hydrolysed EPS revealed that DpoL1 cleaves the galactose-containing backbone of amylovoran. The enzyme is most active at pH 6 and 50°C, and features a modular architecture. Removal of 180 N-terminal amino acids was shown not to affect enzyme activity. The C-terminus harbours the hydrolase activity, while the N-terminal domain links the enzyme to the phage particle. Electron microscopy demonstrated that DpoL1-specific antibodies cross-link phage particles at their tails, either lateral or frontal, and immunogold staining confirmed that DpoL1 is located at the tail spikes. Exposure of high-level EPS-producing Er. amylovora strain CFBP1430 to recombinant DpoL1 dramatically increased sensitivity to the Dpo-negative phage Y2, which was not the case for EPS-negative mutants or low-level EPS-producing Er. amylovora. Our findings indicate that enhanced phage susceptibility is based on enzymatic removal of the EPS capsule, normally a physical barrier to Y2 infection, and that use of DpoL1 together with the broad host range, virulent phage Y2 represents an attractive combination for biocontrol of fire blight