Mutation of lipoprotein processing pathway gene lspA or inhibition of LspA activity by globomycin increases MRSA resistance to β-lactam antibiotics

AbstractThe Staphylococcus aureus cell envelope comprises numerous components, including peptidoglycan (PG), wall teichoic acids (WTA), lipoteichoic acids (LTA), targeted by antimicrobial drugs. MRSA resistance to methicillin is mediated by the mecA-encoded β-lactam-resistant transpeptidase, penicillin binding protein 2a (PBP2a). However, PBP2a-dependent β-lactam resistance is also modulated by the activity of pathways involved in the regulation or biosynthesis of PG, WTA or LTA. Here, we report that mutation of the lipoprotein signal peptidase II gene, lspA, from the lipoprotein processing pathway, significantly increased β-lactam resistance in MRSA. Mutation of lgt, which encodes diacylglycerol transferase (Lgt) responsible for synthesis of the LspA substrate did not impact β-lactam susceptibility. Consistent with previous reports, lgt and lspA mutations impaired growth in chemically defined media, but not in complex broth. MRSA exposure to the LspA inhibitor globomycin also increased β-lactam resistance. Mutation of lgt in an lspA background restored β-lactam resistance to wild type. The lspA mutation had no effect on PBP2a expression, PG composition or autolytic activity indicating a potential role for WTA or LTA. The lspA and lgt mutants exhibited marginally increased resistance to the D-alanine pathway inhibitor D-cycloserine. In addition, mutation of lgt and multicopy lspA expression, but not mutation of lspA, significantly increased susceptibility to the lipoteichoic acid synthase inhibitor Congo red revealing complex interplay between lipoprotein processing mutations and the expression/stability of cell surface glycopolymers. These findings indicate that accumulation of the LspA substrate, diacylglyceryl lipoprotein, increases MRSA resistance to β-lactam antibiotics through impacts on cell envelope components other than PG.

Download Full-text

Lipoprotein Processing Is Essential for Resistance of Mycobacterium tuberculosis to Malachite Green

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00647-09 ◽

2009 ◽

Vol 53 (9) ◽

pp. 3799-3802 ◽

Cited By ~ 17

Author(s):

Niaz Banaei ◽

Eleanor Z. Kincaid ◽

S.-Y. Grace Lin ◽

Edward Desmond ◽

William R. Jacobs ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Malachite Green ◽

Cell Envelope ◽

Sodium Dodecyl ◽

Signal Peptidase ◽

Wild Type ◽

First Line ◽

Green Is ◽

Antituberculosis Drugs ◽

Forming Efficiency

ABSTRACT Malachite green, a synthetic antimicrobial dye, has been used for over 50 years in mycobacterial culture medium to inhibit the growth of contaminants. The molecular basis of mycobacterial resistance to malachite green is unknown, although the presence of malachite green-reducing enzymes in the cell envelope has been suggested. The objective of this study was to investigate the role of lipoproteins in resistance of Mycobacterium tuberculosis to malachite green. The replication of an M. tuberculosis lipoprotein signal peptidase II (lspA) mutant (ΔlspA::lspA mut) on Middlebrook agar with and without 1 mg/liter malachite green was investigated. The lspA mutant was also compared with wild-type M. tuberculosis in the decolorization rate of malachite green and sensitivity to sodium dodecyl sulfate (SDS) detergent and first-line antituberculosis drugs. The lspA mutant has a 104-fold reduction in CFU-forming efficiency on Middlebrook agar with malachite green. Malachite green is decolorized faster in the presence of the lspA mutant than wild-type bacteria. The lspA mutant is hypersensitive to SDS detergent and shows increased sensitivity to first-line antituberculosis drugs. In summary, lipoprotein processing by LspA is essential for resistance of M. tuberculosis to malachite green. A cell wall permeability defect is likely responsible for the hypersensitivity of lspA mutant to malachite green.

Download Full-text

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

10.1101/2020.03.23.004671 ◽

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.

Download Full-text

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

Journal of Bacteriology ◽

10.1128/jb.00149-20 ◽

2020 ◽

Vol 202 (16) ◽

Cited By ~ 1

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.

Download Full-text

β-Lactam Resistance in Methicillin-Resistant Staphylococcus aureus USA300 Is Increased by Inactivation of the ClpXP Protease

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02802-14 ◽

2014 ◽

Vol 58 (8) ◽

pp. 4593-4603 ◽

Cited By ~ 34

Author(s):

Kristoffer T. Bæk ◽

Angelika Gründling ◽

René G. Mogensen ◽

Louise Thøgersen ◽

Andreas Petersen ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Cell Envelope ◽

Penicillin Binding Protein ◽

Gene Encoding ◽

Resistance Level ◽

Methicillin Resistant ◽

Content Type ◽

Usa300 Strain ◽

Mrsa Strains ◽

Peptidoglycan Hydrolysis

ABSTRACTMethicillin-resistantStaphylococcus aureus(MRSA) has acquired themecAgene encoding a peptidoglycan transpeptidase, penicillin binding protein 2a (PBP2a), which has decreased affinity for β-lactams. Quickly spreading and highly virulent community-acquired (CA) MRSA strains recently emerged as a frequent cause of infection in individuals without exposure to the health care system. In this study, we found that the inactivation of the components of the ClpXP protease substantially increased the β-lactam resistance level of a CA-MRSA USA300 strain, suggesting that the proteolytic activity of ClpXP controls one or more pathways modulating β-lactam resistance. These pathways do not involve the control ofmecAexpression, as the cellular levels of PBP2a were unaltered in theclpmutants. An analysis of the cell envelope properties of theclpXandclpPmutants revealed a number of distinct phenotypes that may contribute to the enhanced β-lactam tolerance. Both mutants displayed significantly thicker cell walls, increased peptidoglycan cross-linking, and altered composition of monomeric muropeptide species compared to those of the wild types. Moreover, changes in Sle1-mediated peptidoglycan hydrolysis and altered processing of the major autolysin Atl were observed in theclpmutants. In conclusion, the results presented here point to an important role for the ClpXP protease in controlling cell wall metabolism and add novel insights into the molecular factors that determine strain-dependent β-lactam resistance.

Download Full-text

Cation-Induced Transcriptional Regulation of the dlt Operon of Staphylococcus aureus

Journal of Bacteriology ◽

10.1128/jb.188.10.3622-3630.2006 ◽

2006 ◽

Vol 188 (10) ◽

pp. 3622-3630 ◽

Cited By ~ 68

Author(s):

Tomaz Koprivnjak ◽

Vid Mlakar ◽

Lindsey Swanson ◽

Benedicte Fournier ◽

Andreas Peschel ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Cell Envelope ◽

Divalent Cations ◽

Northern Blotting ◽

Start Codon ◽

Two Component System ◽

Reading Frame ◽

Reverse Transcription Pcr ◽

High Concentrations ◽

Wall Teichoic Acids

ABSTRACT Lipoteichoic and wall teichoic acids (TA) are highly anionic cell envelope-associated polymers containing repeating polyglycerol/ribitol phosphate moieties. Substitution of TA with d-alanine is important for modulation of many cell envelope-dependent processes, such as activity of autolytic enzymes, binding of divalent cations, and susceptibility to innate host defenses. d-Alanylation of TA is diminished when bacteria are grown in medium containing increased NaCl concentrations, but the effects of increased salt concentration on expression of the dlt operon encoding proteins mediating d-alanylation of TA are unknown. We demonstrate that Staphylococcus aureus transcriptionally represses dlt expression in response to high concentrations of Na+ and moderate concentrations of Mg2+ and Ca2+ but not sucrose. Changes in dlt mRNA are induced within 15 min and sustained for several generations of growth. Mg2+-induced dlt repression depends on the ArlSR two-component system. Northern blotting, reverse transcription-PCR, and SMART-RACE analyses suggest that the dlt transcript begins 250 bp upstream of the dltA start codon and includes an open reading frame immediately upstream of dltA. Chloramphenicol transacetylase transcriptional fusions indicate that a region encompassing the 171 to 325 bp upstream of dltA is required for expression and Mg2+-induced repression of the dlt operon in S. aureus.

Download Full-text

Salt-Induced Stress Stimulates a Lipoteichoic Acid-Specific Three-Component Glycosylation System inStaphylococcus aureus

Journal of Bacteriology ◽

10.1128/jb.00017-18 ◽

2018 ◽

Vol 200 (12) ◽

Cited By ~ 15

Author(s):

Kelvin Kho ◽

Timothy C. Meredith

Keyword(s):

Staphylococcus Aureus ◽

Sigma Factor ◽

Cell Envelope ◽

Normal Growth ◽

Lipoteichoic Acid ◽

Growth Conditions ◽

Growth Media ◽

Alternative Sigma Factor ◽

Content Type

ABSTRACTLipoteichoic acid (LTA) inStaphylococcus aureusis a poly-glycerophosphate polymer anchored to the outer surface of the cell membrane. LTA has numerous roles in cell envelope physiology, including regulating cell autolysis, coordinating cell division, and adapting to environmental growth conditions. LTA is often further modified with substituents, includingd-alanine and glycosyl groups, to alter cellular function. While the genetic determinants ofd-alanylation have been largely defined, the route of LTA glycosylation and its role in cell envelope physiology have remained unknown, in part due to the low levels of basal LTA glycosylation inS. aureus. We demonstrate here thatS. aureusutilizes a membrane-associated three-component glycosylation system composed of an undecaprenol (Und)N-acetylglucosamine (GlcNAc) charging enzyme (CsbB; SAOUHSC_00713), a putative flippase to transport loaded substrate to the outside surface of the cell (GtcA; SAOUHSC_02722), and finally an LTA-specific glycosyltransferase that adds α-GlcNAc moieties to LTA (YfhO; SAOUHSC_01213). We demonstrate that this system is specific for LTA with no cross recognition of the structurally similar polyribitol phosphate containing wall teichoic acids. We show that while wild-typeS. aureusLTA has only a trace of GlcNAcylated LTA under normal growth conditions, amounts are raised upon either overexpressing CsbB, reducing endogenousd-alanylation activity, expressing the cell envelope stress responsive alternative sigma factor SigB, or by exposure to environmental stress-inducing culture conditions, including growth media containing high levels of sodium chloride.IMPORTANCEThe role of glycosylation in the structure and function ofStaphylococcus aureuslipoteichoic acid (LTA) is largely unknown. By defining key components of the LTA three-component glycosylation pathway and uncovering stress-induced regulation by the alternative sigma factor SigB, the role ofN-acetylglucosamine tailoring during adaptation to environmental stresses can now be elucidated. As thedltand glycosylation pathways compete for the same sites on LTA and induction of glycosylation results in decreasedd-alanylation, the interplay between the two modification systems holds implications for resistance to antibiotics and antimicrobial peptides.

Download Full-text

The Bacillus subtilis Extracytoplasmic-Function σX Factor Regulates Modification of the Cell Envelope and Resistance to Cationic Antimicrobial Peptides

Journal of Bacteriology ◽

10.1128/jb.186.4.1136-1146.2004 ◽

2004 ◽

Vol 186 (4) ◽

pp. 1136-1146 ◽

Cited By ~ 111

Author(s):

Min Cao ◽

John D. Helmann

Keyword(s):

Bacillus Subtilis ◽

Antimicrobial Peptides ◽

Cell Envelope ◽

Resistance Mechanism ◽

Penicillin Binding Protein ◽

Wild Type ◽

Amino Groups ◽

Cationic Antimicrobial Peptides ◽

Teichoic Acids ◽

Positively Charged

ABSTRACT Bacillus subtilis contains seven extracytoplasmic-function σ factors that activate partially overlapping regulons. We here identify four additional members of the σX regulon, pbpX (penicillin-binding protein), ywnJ, the dlt operon (d-alanylation of teichoic acids), and the pss ybfM psd operon (phosphatidylethanolamine biosynthesis). Modification of teichoic acids by esterification with d-alanine and incorporation of phosphatidylethanolamine into the cell membrane have a common consequence: in both cases positively charged amino groups are introduced into the cell envelope. The resulting reduction in the net negative charge of the cell envelope has been previously implicated as a resistance mechanism specific for cationic antimicrobial peptides. Consistent with this notion, we find that both sigX and dltA mutants are more sensitive to nisin than wild-type cells. We conclude that activation of the σX regulon serves to alter cell surface properties to provide protection against antimicrobial peptides.

Download Full-text

The cell envelope of Staphylococcus aureus selectively controls the sorting of virulence factors

Nature Communications ◽

10.1038/s41467-021-26517-z ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Xuhui Zheng ◽

Gerben Marsman ◽

Keenan A. Lacey ◽

Jessica R. Chapman ◽

Christian Goosmann ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Immune Cells ◽

Protein Translocation ◽

Cell Envelope ◽

Lipoteichoic Acid ◽

Membrane Lipid ◽

Secretion System ◽

Bacterial Cells ◽

Genetic Screens ◽

Extracellular Milieu

AbstractStaphylococcus aureus bi-component pore-forming leukocidins are secreted toxins that directly target and lyse immune cells. Intriguingly, one of the leukocidins, Leukocidin AB (LukAB), is found associated with the bacterial cell envelope in addition to secreted into the extracellular milieu. Here, we report that retention of LukAB on the bacterial cells provides S. aureus with a pre-synthesized active toxin that kills immune cells. On the bacteria, LukAB is distributed as discrete foci in two distinct compartments: membrane-proximal and surface-exposed. Through genetic screens, we show that a membrane lipid, lysyl-phosphatidylglycerol (LPG), and lipoteichoic acid (LTA) contribute to LukAB deposition and release. Furthermore, by studying non-covalently surface-bound proteins we discovered that the sorting of additional exoproteins, such as IsaB, Hel, ScaH, and Geh, are also controlled by LPG and LTA. Collectively, our study reveals a multistep secretion system that controls exoprotein storage and protein translocation across the S. aureus cell wall.

Download Full-text

Identification of sequences encoding for ncr-peptides and defensins in the ‘meta-assembly’ of transcriptome of pea (pisum sativum l.) Nitrogen-fixing nodules

Ecological genetics ◽

10.17816/ecogen17339-46 ◽

2019 ◽

Vol 17 (3) ◽

pp. 39-46 ◽

Cited By ~ 1

Author(s):

Evgeny A. Zorin ◽

Marina S. Kliukova ◽

Olga A. Kulaeva ◽

Alexey M. Afonin ◽

Igor A. Tikhonovich ◽

...

Keyword(s):

Antimicrobial Activity ◽

Pisum Sativum ◽

Signal Peptidase ◽

Nitrogen Fixing ◽

Antimicrobial Drugs ◽

Pisum Sativum L ◽

Plant Defensins ◽

New Antibiotics ◽

Emergence Of Resistance

Background. The active and careless applying of antibiotics in medicine and agriculture leads to the emergence of resistance to the existing antimicrobial drugs, which reduces the effectiveness of their use. One of the ways to solve this problem is the development of new antibiotics based on plant peptides with antimicrobial activity, for example plant defensins (which identified in all plants) and NCR peptides that are specifically synthesized in nodules of some leguminous plants. Materials and methods. In the present study, a meta-assembly of a transcriptome was constructed based on publicly available RNA-sequencing transcriptomes of pea nodules (Pisum sativum L.). This meta-assembly was used to search for sequences encoding antimicrobial peptides. Results. As a result, 55 and 908 unique sequences encoding defensins and NCR peptides, respectively, were identified. The recognition site for the signal peptidase was predicted and sequences were divided into the signal and mature part of the peptide. Among mature defensins, 22 peptides possess in silico predicted antimicrobial activity, and for the NCR peptides family their number was 422. Conclusion. Sequences encoding defensins and NCR peptides expressed in nitrogen-fixing pea nodules were identified. They are candidates for testing their antimicrobial activity in vitro.

Download Full-text