scholarly journals Biosynthesis of the Glycolipid Anchor in Lipoteichoic Acid of Staphylococcus aureus RN4220: Role of YpfP, the Diglucosyldiacylglycerol Synthase

2001 ◽  
Vol 183 (11) ◽  
pp. 3506-3514 ◽  
Author(s):  
Michael Y. Kiriukhin ◽  
Dmitri V. Debabov ◽  
Dean L. Shinabarger ◽  
Francis C. Neuhaus
Keyword(s):  
Electron Microscopy ◽  
Staphylococcus Aureus ◽  
Doubling Time ◽  
Lipoteichoic Acid ◽  
Luria Bertani ◽  
Wild Type ◽  
Glycolipid Anchor ◽  
Triton X ◽  
Pleomorphic Cells

ABSTRACT In Staphylococcus aureus RN4220, lipoteichoic acid (LTA) is anchored in the membrane by a diglucosyldiacylglycerol moiety. The gene (ypfP) which encodes diglucosyldiacylglycerol synthase was recently cloned from Bacillus subtilis and expressed in Escherichia coli (P. Jorasch, F. P. Wolter, U. Zahringer, and E. Heinz, Mol. Microbiol. 29:419–430, 1998). To define the role of ypfP in this strain of S. aureus, a fragment of ypfP truncated from both ends was cloned into the thermosensitive replicon pVE6007 and used to inactivate ypfP. Chloramphenicol-resistant (ypfP::cat) clones did not synthesize the glycolipids monoglucosyldiacylglycerol and diglucosyldiacylglycerol. Thus, YpfP would appear to be the only diglucosyldiacylglycerol synthase in S. aureus providing glycolipid for LTA assembly. In LTA from the mutant, the glycolipid anchor is replaced by diacylglycerol. Although the doubling time of the mutant was identical to that of the wild type in Luria-Bertani (LB) medium, growth of the mutant in LB medium containing 1% glycine was not observed. This inhibition was antagonized by either l- or d-alanine. Moreover, viability of the mutant at 37°C in 0.05 M phosphate (pH 7.2)-saline for 12 h was reduced to <0.1%. Addition of 0.1% d-glucose to the phosphate-saline ensured viability under these conditions. The autolysis of the ypfP::cat mutant in the presence of 0.05% Triton X-100 was 1.8-fold faster than that of the parental strain. Electron microscopy of the mutant revealed not only a small increase in cell size but also the presence of pleomorphic cells. Each of these phenotypes may be correlated with either (or both) a deficiency of free glycolipid in the membrane or the replacement of the usual glycolipid anchor of LTA with diacylglycerol.

scholarly journals Causal Role of Single Nucleotide Polymorphisms within themprFGene of Staphylococcus aureus in Daptomycin Resistance

2013 ◽  
Vol 57 (11) ◽  
pp. 5658-5664 ◽  
Author(s):  
Soo-Jin Yang ◽  
Nagendra N. Mishra ◽  
Aileen Rubio ◽  
Arnold S. Bayer
Keyword(s):  
Staphylococcus Aureus ◽  
Single Nucleotide Polymorphisms ◽  
Point Mutations ◽  
Nucleotide Polymorphisms ◽  
Wild Type ◽  
Single Nucleotide ◽  
Content Type ◽  
Reading Frame ◽  
A Charge

ABSTRACTSingle nucleotide polymorphisms (SNPs) within themprFopen reading frame (ORF) have been commonly observed in daptomycin-resistant (DAPr)Staphylococcus aureusstrains. Such SNPs are usually associated with a gain-in-function phenotype, in terms of either increased synthesis or enhanced translocation (flipping) of lysyl-phosphatidylglycerol (L-PG). However, it is unclear if suchmprFSNPs are causal in DAPrstrains or are merely a biomarker for this phenotype. In this study, we used an isogenic set ofS. aureusstrains: (i) Newman, (ii) its isogenic ΔmprFmutant, and (iii) several intransplasmid complementation constructs, expressing either a wild-type or point-mutated form of themprFORF cloned from two isogenic DAP-susceptible (DAPs)-DAPrstrain pairs (616-701 and MRSA11/11-REF2145). Complementation of the ΔmprFstrain with singly point-mutatedmprFgenes (mprFS295LormprFT345A) revealed that (i) individual and distinct point mutations within themprFORF can recapitulate phenotypes observed in donor strains (i.e., changes in DAP MICs, positive surface charge, and cell membrane phospholipid profiles) and (ii) these gain-in-function SNPs (i.e., enhanced L-PG synthesis) likely promote reduced DAP binding toS. aureusby a charge repulsion mechanism. Thus, for these two DAPrstrains, the definedmprFSNPs appear to be causally related to this phenotype.

scholarly journals Lipoteichoic Acid from Staphylococcus aureus Induces Lung Endothelial Cell Barrier Dysfunction: Role of Reactive Oxygen and Nitrogen Species

PLoS ONE ◽  
2012 ◽  
Vol 7 (11) ◽  
pp. e49209 ◽  
Author(s):  
Amy Barton Pai ◽  
Heena Patel ◽  
Alexander J. Prokopienko ◽  
Hiba Alsaffar ◽  
Nancy Gertzberg ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Endothelial Cell ◽  
Reactive Oxygen ◽  
Lipoteichoic Acid ◽  
Nitrogen Species ◽  
Barrier Dysfunction

scholarly journals Effects of ubiquitin C-terminal hydrolase L1 deficiency on mouse ova

Reproduction ◽  
2012 ◽  
Vol 143 (3) ◽  
pp. 271-279 ◽  
Author(s):  
Sayaka Koyanagi ◽  
Hiroko Hamasaki ◽  
Satoshi Sekiguchi ◽  
Kenshiro Hara ◽  
Yoshiyuki Ishii ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Proteomic Analysis ◽  
Ubiquitin Proteasome System ◽  
Underlying Mechanism ◽  
Wild Type ◽  
Transmission Electron ◽  
Ubiquitin Proteasome

Maternal proteins are rapidly degraded by the ubiquitin–proteasome system during oocyte maturation in mice. Ubiquitin C-terminal hydrolase L1 (UCHL1) is highly and specifically expressed in mouse ova and is involved in the polyspermy block. However, the role of UCHL1 in the underlying mechanism of polyspermy block is poorly understood. To address this issue, we performed a comprehensive proteomic analysis to identify maternal proteins that were relevant to the role of UCHL1 in mouse ova using UCHL1-deficientgad. Furthermore, we assessed morphological features ingadmouse ova using transmission electron microscopy. NACHT, LRR, and PYD domain-containing (NALP) family proteins and endoplasmic reticulum (ER) chaperones were identified by proteomic analysis. We also found that the ‘maternal antigen that embryos require’ (NLRP5 (MATER)) protein level increased significantly ingadmouse ova compared with that in wild-type mice. In an ultrastructural study,gadmouse ova contained less ER in the cortex than in wild-type mice. These results provide new insights into the role of UCHL1 in the mechanism of polyspermy block in mouse ova.

scholarly journals Enhanced production of exopolysaccharide matrix and biofilm by a menadione-auxotrophic Staphylococcus aureus small-colony variant

2010 ◽  
Vol 59 (5) ◽  
pp. 521-527 ◽  
Author(s):  
Rachna Singh ◽  
Pallab Ray ◽  
Anindita Das ◽  
Meera Sharma
Keyword(s):  
Staphylococcus Aureus ◽  
Tricarboxylic Acid Cycle ◽  
Surface Hydrophobicity ◽  
Tricarboxylic Acid ◽  
Polysaccharide Intercellular Adhesin ◽  
Wild Type ◽  
Small Colony Variant ◽  
Enhanced Production ◽  
Small Colony

The role of Staphylococcus aureus small-colony variants (SCVs) in the pathogenesis of biofilm-associated infections remains unclear. This study investigated the mechanism behind increased biofilm-forming potential of a menadione-auxotrophic Staphylococcus aureus SCV compared with the wild-type parental strain, as recently reported by our laboratory. SCVs displayed an autoaggregative phenotype, with a greater amount of polysaccharide intercellular adhesin (PIA), significantly reduced tricarboxylic acid cycle activity and a decreased susceptibility to aminoglycosides and cell-wall inhibitors compared with wild-type. The biofilms formed by the SCV were highly structured, consisting of large microcolonies separated by channels, and contained more biomass as well as significantly more PIA than wild-type biofilms. The surface hydrophobicity of the two phenotypes was similar. Thus, the autoaggregation and increased biofilm-forming capacity of menadione-auxotrophic Staphylococcus aureus SCVs in this study was related to the enhanced production of PIA in these variants.

scholarly journals The β-tail domain (βTD) regulates physiologic ligand binding to integrin CD11b/CD18

Blood ◽  
2006 ◽  
Vol 109 (8) ◽  
pp. 3513-3520 ◽  
Author(s):  
Vineet Gupta ◽  
Annette Gylling ◽  
José Luis Alonso ◽  
Takashi Sugimori ◽  
Petre Ianakiev ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Ligand Binding ◽  
Conformational Changes ◽  
Divalent Cations ◽  
Contact Region ◽  
Allosteric Modulator ◽  
Tail Domain ◽  
Wild Type ◽  
High Affinity

Abstract Crystallographic and electron microscopy studies revealed genuflexed (bent) integrins in both unliganded (inactive) and physiologic ligandbound (active) states, suggesting that local conformational changes are sufficient for activation. Herein we have explored the role of local changes in the contact region between the membrane-proximal β-tail domain (βTD) and the ligand-binding βA domain of the bent conformation in regulating interaction of integrin CD11b/CD18 (αMβ2) with its physiologic ligand iC3b. We replaced the βTD CD loop residues D658GMD of the CD18 (β2) subunit with the equivalent D672SSG of the β3 subunit, with AGAA or with NGTD, expressed the respective heterodimeric receptors either transiently in epithelial HEK293T cells or stably in leukocytes (K562), and measured their ability to bind iC3b and to conformation-sensitive mAbs. In the presence of the physiologic divalent cations Ca2+ plus Mg2+ (at 1 mM each), the modified integrins showed increased (in HEK293) or constitutive (in K562) binding to iC3b compared with wild-type receptors. K562 expressing the βTD-modified integrins bound in Ca2+Mg2+ to the βA-directed high-affinity reporter mAb 24 but not to mAb KIM127, a reporter of the genu-straightened state. These data identify a role for the membrane proximal βTD as an allosteric modulator of integrin activation.

scholarly journals Are B Lymphocytes of Importance in Severe Staphylococcus aureus Infections?

2000 ◽  
Vol 68 (5) ◽  
pp. 2431-2434 ◽  
Author(s):  
Inger Gjertsson ◽  
Olof Hörnquist Hultgren ◽  
Martin Stenson ◽  
Rikard Holmdahl ◽  
Andrzej Tarkowski
Keyword(s):  
Staphylococcus Aureus ◽  
B Cells ◽  
B Cell ◽  
Interleukin 4 ◽  
Wild Type ◽  
Toxic Shock ◽  
Toxic Shock Syndrome Toxin ◽  
Deficient Mice ◽  
Wild Type Control

ABSTRACT To investigate the role of B cells in experimental, superantigen-mediated Staphylococcus aureus arthritis and sepsis, we used gene-targeted B-cell-deficient mice. The mice were inoculated intravenously with a toxic shock syndrome toxin 1 (TSST-1)-producing S. aureus strain. The B-cell-deficient and thus agamma-globulinemic mice showed striking similarities to the wild-type control animals with respect to the development of arthritis, the mortality rate, and the rate of bacterial clearance. Surprisingly, we found that the levels of gamma interferon in serum were significantly lower (P < 0.0001) in B-cell-deficient mice than in the controls, possibly due to impaired superantigen presentation and a diminished expression of costimulatory molecules. In contrast, the levels of interleukin-4 (IL-4), IL-6, and IL-10 in serum were equal in both groups. Our findings demonstrate that neither mature B cells nor their products significantly contribute to the course ofS. aureus-induced septic arthritis.

scholarly journals The length of lipoteichoic acid polymers controls Staphylococcus aureus cell size and envelope integrity

2020 ◽  
Author(s):  
Anthony R. Hesser ◽  
Leigh M. Matano ◽  
Christopher R. Vickery ◽  
B. McKay Wood ◽  
Ace George Santiago ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Growth ◽  
Cell Size ◽  
Cell Envelope ◽  
Lipoteichoic Acid ◽  
Resistant Bacteria ◽  
Beta Lactam ◽  
Beta Lactam Antibiotics ◽  
Outer Leaflet ◽  
Glycolipid Anchor

ABSTRACTThe opportunistic pathogen Staphylococcus aureus is protected by a cell envelope that is crucial for viability. In addition to peptidoglycan, lipoteichoic acid (LTA) is an especially important component of the S. aureus cell envelope. LTA is an anionic polymer anchored to a glycolipid in the outer leaflet of the cell membrane. It was known that deleting the gene for UgtP, the enzyme that makes this glycolipid anchor, causes cell growth and division defects. In Bacillus subtilis, growth abnormalities from the loss of ugtP have been attributed to the absence of the encoded protein, not to loss of its enzymatic activity. Here, we show that growth defects in S. aureus ugtP deletion mutants are due to the long, abnormal LTA polymer that is produced when the glycolipid anchor is missing from the outer leaflet of the membrane. Dysregulated cell growth leads to defective cell division, and these phenotypes are corrected by mutations in the LTA polymerase, ltaS, that reduce polymer length. We also show that S. aureus mutants with long LTA are sensitized to cell wall hydrolases, beta-lactam antibiotics, and compounds that target other cell envelope pathways. We conclude that control of LTA polymer length is important for S. aureus physiology and promotes survival under stressful conditions, including antibiotic stress.IMPORTANCEMethicillin-resistant Staphylococcus aureus (MRSA) is a common cause of community- and hospital-acquired infections and is responsible for a large fraction of deaths caused by antibiotic-resistant bacteria. S. aureus is surrounded by a complex cell envelope that protects it from antimicrobial compounds and other stresses. Here we show that controlling the length of an essential cell envelope polymer, lipoteichoic acid, is critical for controlling S. aureus cell size and cell envelope integrity. We also show that genes involved in LTA length regulation are required for resistance to beta-lactam antibiotics in MRSA. The proteins encoded by these genes may be targets for combination therapy with an appropriate beta-lactam.

scholarly journals The Length of Lipoteichoic Acid Polymers Controls Staphylococcus aureus Cell Size and Envelope Integrity

2020 ◽  
Vol 202 (16) ◽  
Author(s):  
Anthony R. Hesser ◽  
Leigh M. Matano ◽  
Christopher R. Vickery ◽  
B. McKay Wood ◽  
Ace George Santiago ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Growth ◽  
Cell Size ◽  
Cell Envelope ◽  
Lipoteichoic Acid ◽  
Beta Lactam ◽  
Content Type ◽  
Beta Lactam Antibiotics ◽  
Outer Leaflet ◽  
Glycolipid Anchor

ABSTRACT The opportunistic pathogen Staphylococcus aureus is protected by a cell envelope that is crucial for viability. In addition to peptidoglycan, lipoteichoic acid (LTA) is an especially important component of the S. aureus cell envelope. LTA is an anionic polymer anchored to a glycolipid in the outer leaflet of the cell membrane. It was known that deleting the gene for UgtP, the enzyme that makes this glycolipid anchor, causes cell growth and division defects. In Bacillus subtilis, growth abnormalities from the loss of ugtP have been attributed to both the absence of the encoded protein and the loss of its products. Here, we show that growth defects in S. aureus ugtP deletion mutants are due to the long, abnormal LTA polymer that is produced when the glycolipid anchor is missing from the outer leaflet of the membrane. Dysregulated cell growth leads to defective cell division, and these phenotypes are corrected by mutations in the LTA polymerase gene, ltaS, that reduce polymer length. We also show that S. aureus mutants with long LTA are sensitized to cell wall hydrolases, beta-lactam antibiotics, and compounds that target other cell envelope pathways. We conclude that control of LTA polymer length is important for S. aureus physiology and promotes survival under stressful conditions, including antibiotic stress. IMPORTANCE Methicillin-resistant Staphylococcus aureus (MRSA) is a common cause of community- and hospital-acquired infections and is responsible for a large fraction of deaths caused by antibiotic-resistant bacteria. S. aureus is surrounded by a complex cell envelope that protects it from antimicrobial compounds and other stresses. Here, we show that controlling the length of an essential cell envelope polymer, lipoteichoic acid, is critical for controlling S. aureus cell size and cell envelope integrity. We also show that genes involved in LTA length regulation are required for resistance to beta-lactam antibiotics in MRSA. The proteins encoded by these genes may be targets for combination therapy with an appropriate beta-lactam.

NoteThe stringent response genes relA and spoT are important for Escherichia coli biofilms under slow-growth conditions

2002 ◽  
Vol 48 (7) ◽  
pp. 675-680 ◽  
Author(s):  
Grant J Balzer ◽  
Robert J.C McLean
Keyword(s):  
Escherichia Coli ◽  
Cell Density ◽  
Biofilm Formation ◽  
Doubling Time ◽  
Stringent Response ◽  
Growth Conditions ◽  
Luria Bertani ◽  
Confocal Laser ◽  
Wild Type ◽  
Microtiter Plate Assay

In order to see whether the stringent response was involved in biofilm formation, Escherichia coli DS291 (MG1655), and its isogenic relA spoT derivative were grown for 48 h in a chemostat at dilution rates of 0.025 and 0.25 h–1 under serine limitation. The absence of the stringent response genes relA and spoT had little effect on the planktonic cell concentrations. However, a significant (P < 0.001) reduction in biofilm cell density of the relA spoT mutants was seen at a doubling time of 40 h. At a doubling time of 4 h, differences in biofilm cell density were not significant. Scanning confocal laser microscopy demonstrated the cell densities of microcolonies in the relA spoT mutant to be lower than those in the wild type. Using a microtiter plate assay, we found biofilm formation in relA spoT mutants to be similarly reduced in minimal media but to be enhanced in rich media (Luria–Bertani broth). No significant differences in biofilm formation were observed between wild type and isogenic relA mutants under any growth conditions. Overall, these results suggest that both stringent response genes relA and spoT are important in nutrient-limited biofilms. Key words: biofilm, stringent response, nutrient limitation, microcolony, Escherichia coli.

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