Synthetic Effects of secG and secY2 Mutations on Exoproteome Biogenesis in Staphylococcus aureus

ABSTRACT The Gram-positive pathogen Staphylococcus aureus secretes various proteins into its extracellular milieu. Bioinformatics analyses have indicated that most of these proteins are directed to the canonical Sec pathway, which consists of the translocation motor SecA and a membrane-embedded channel composed of the SecY, SecE, and SecG proteins. In addition, S. aureus contains an accessory Sec2 pathway involving the SecA2 and SecY2 proteins. Here, we have addressed the roles of the nonessential channel components SecG and SecY2 in the biogenesis of the extracellular proteome of S. aureus. The results show that SecG is of major importance for protein secretion by S. aureus. Specifically, the extracellular accumulation of nine abundant exoproteins and seven cell wall-bound proteins was significantly affected in an secG mutant. No secretion defects were detected for strains with a secY2 single mutation. However, deletion of secY2 exacerbated the secretion defects of secG mutants, affecting the extracellular accumulation of one additional exoprotein and one cell wall protein. Furthermore, an secG secY2 double mutant displayed a synthetic growth defect. This might relate to a slightly elevated expression of sraP, encoding the only known substrate for the Sec2 pathway, in cells lacking SecG. Additionally, the results suggest that SecY2 can interact with the Sec1 channel, which would be consistent with the presence of a single set of secE and secG genes in S. aureus.

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Defects in Protein Glycosylation Cause SHO1-Dependent Activation of a STE12 Signaling Pathway in Yeast

Genetics ◽

10.1093/genetics/155.3.1005 ◽

2000 ◽

Vol 155 (3) ◽

pp. 1005-1018 ◽

Cited By ~ 6

Author(s):

Paul J Cullen ◽

Janet Schultz ◽

Joe Horecka ◽

Brian J Stevenson ◽

Yoshifumi Jigami ◽

...

Keyword(s):

Cell Wall ◽

Genetic Selection ◽

Protein Glycosylation ◽

Cell Wall Integrity ◽

Growth Defect ◽

Loss Of Function ◽

Pheromone Response ◽

Pheromone Response Pathway ◽

Pathway Activation ◽

Synthetic Growth

Abstract In haploid Saccharomyces cerevisiae, mating occurs by activation of the pheromone response pathway. A genetic selection for mutants that activate this pathway uncovered a class of mutants defective in cell wall integrity. Partial loss-of-function alleles of PGI1, PMI40, PSA1, DPM1, ALG1, MNN10, SPT14, and OCH1, genes required for mannose utilization and protein glycosylation, activated a pheromone-response-pathway-dependent reporter (FUS1) in cells lacking a basal signal (ste4). Pathway activation was suppressed by the addition of mannose to hexose isomerase mutants pgi1-101 and pmi40-101, which bypassed the requirement for mannose biosynthesis in these mutants. Pathway activation was also suppressed in dpm1-101 mutants by plasmids that contained RER2 or PSA1, which produce the substrates for Dpm1. Activation of FUS1 transcription in the mannose utilization/protein glycosylation mutants required some but not all proteins from three different signaling pathways: the pheromone response, invasive growth, and HOG pathways. We specifically suggest that a Sho1 → Ste20/Ste50 → Ste11 → Ste7 → Kss1 → Ste12 pathway is responsible for activation of FUS1 transcription in these mutants. Because loss of pheromone response pathway components leads to a synthetic growth defect in mannose utilization/protein glycosylation mutants, we suggest that the Sho1 → Ste12 pathway contributes to maintenance of cell wall integrity in vegetative cells.

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The Cell Wall Polymer Lipoteichoic Acid Becomes Nonessential inStaphylococcus aureusCells Lacking the ClpX Chaperone

mBio ◽

10.1128/mbio.01228-16 ◽

2016 ◽

Vol 7 (4) ◽

Cited By ~ 21

Author(s):

Kristoffer T. Bæk ◽

Lisa Bowman ◽

Charlotte Millership ◽

Mia Dupont Søgaard ◽

Volkhard Kaever ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Cell Wall ◽

Cell Division ◽

Lipoteichoic Acid ◽

Growth Defect ◽

Gram Positive ◽

Functional Connection ◽

Antibiotic Resistant ◽

Gram Positive Bacteria ◽

Autolytic Activity

ABSTRACTLipoteichoic acid (LTA) is an important cell wall component of Gram-positive bacteria and a promising target for the development of vaccines and antimicrobial compounds againstStaphylococcus aureus. Here we demonstrate that mutations in the conditionally essentialltaS(LTA synthase) gene arise spontaneously in anS. aureusmutant lacking the ClpX chaperone. A wide variety ofltaSmutations were selected, and among these, a substantial portion resulted in premature stop codons and other changes predicted to abolish LtaS synthesis. Consistent with this assumption, theclpX ltaSdouble mutants did not produce LTA, and genetic analyses confirmed that LTA becomes nonessential in the absence of the ClpX chaperone. In fact, inactivation ofltaSalleviated the severe growth defect conferred by theclpXdeletion. Microscopic analyses showed that the absence of ClpX partly alleviates the septum placement defects of an LTA-depleted strain, while other phenotypes typical of LTA-negativeS. aureusmutants, including increased cell size and decreased autolytic activity, are retained. In conclusion, our results indicate that LTA has an essential role in septum placement that can be bypassed by inactivating the ClpX chaperone.IMPORTANCELipoteichoic acid is an essential component of theStaphylococcus aureuscell envelope and an attractive target for the development of vaccines and antimicrobials directed against antibiotic-resistant Gram-positive bacteria such as methicillin-resistantS. aureusand vancomycin-resistant enterococci. In this study, we showed that the lipoteichoic acid polymer is essential for growth ofS. aureusonly as long as the ClpX chaperone is present in the cell. Our results indicate that lipoteichoic acid and ClpX play opposite roles in a pathway that controls two key cell division processes inS. aureus, namely, septum formation and autolytic activity. The discovery of a novel functional connection in the genetic network that controls cell division inS. aureusmay expand the repertoire of possible strategies to identify compounds or compound combinations that kill antibiotic-resistantS. aureus.

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The eIF4E-binding protein Eap1 has similar but independent roles in cell growth and gene expression with the cytoplasmic deadenylase Ccr4

Bioscience Biotechnology and Biochemistry ◽

10.1093/bbb/zbab056 ◽

2021 ◽

Author(s):

Yudai Higuchi ◽

Shiori Fujii ◽

Arvin Lapiz Valderrama ◽

Kaoru Irie ◽

Yasuyuki Suda ◽

...

Keyword(s):

Gene Expression ◽

Cell Wall ◽

Cell Growth ◽

Synthetic Lethality ◽

Growth Defect ◽

Response Gene ◽

Gene Encoding ◽

Cell Wall Integrity Pathway ◽

Synthetic Growth ◽

Stress Response Gene

Abstract eIF4E-binding proteins (4E-BPs) are translational repressors that compete with eIF4G for binding to eIF4E. Here we investigated the roles of yeast 4E-BPs, Eap1 and Caf20, in cell wall integrity pathway and gene expression. We found that eap1∆ mutation, but not caf20∆ mutation, showed synthetic growth defect with mutation in ROM2 gene encoding Rho1 GEF. The eap1∆ mutation also showed synthetic lethality with mutation in CCR4 gene encoding cytoplasmic deadenylase. Ccr4 functions in degradation of LRG1 mRNA encoding Rho1 GAP. Eap1-Y109A L114A, which could not bind to eIF4E, did not suppress the synthetic lethality of eap1∆ ccr4∆ mutant, suggesting that 4E-binding of Eap1 is important for its function. We also found that eap1∆ mutant showed derepression of stress response gene HSP12. 4E-binding of Eap1 was also required for repression of HSP12 expression. Our results indicate that Eap1 has similar but independent roles in cell growth and gene expression with Ccr4.

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Stepwise Decrease in Daptomycin Susceptibility in Clinical Staphylococcus aureus Isolates Associated with an Initial Mutation inrpoBand a Compensatory Inactivation of theclpXGene

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01303-15 ◽

2015 ◽

Vol 59 (11) ◽

pp. 6983-6991 ◽

Cited By ~ 29

Author(s):

Kristoffer T. Bæk ◽

Louise Thøgersen ◽

René G. Mogenssen ◽

Maiken Mellergaard ◽

Line E. Thomsen ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Cell Wall ◽

Wall Stress ◽

Growth Defect ◽

Loss Of Function ◽

Genetic Changes ◽

Content Type ◽

Virulence Traits ◽

Lipopeptide Antibiotic

ABSTRACTDaptomycin is a lipopeptide antibiotic used clinically for the treatment of methicillin-resistantStaphylococcus aureus(MRSA) infections. The emergence of daptomycin-nonsusceptibleS. aureusisolates during therapy is often associated with multiple genetic changes; however, the relative contributions of these changes to resistance and other phenotypic changes usually remain unclear. The present study was undertaken to investigate this issue using a genetically characterized series of four isogenic clinical MRSA strains derived from a patient with bacteremia before and during daptomycin treatment. The first strain obtained after daptomycin therapy carried a single-nucleotide polymorphism (SNP) inrpoB(RpoB A477D) that decreased susceptibility not only to daptomycin but also to vancomycin, β-lactams, and rifampin. Furthermore, therpoBmutant exhibited pleiotropic phenotypes, including increased cell wall thickness, reduced expression of virulence traits, induced expression of the stress-associated transcriptional regulator Spx, and slow growth. A subsequently acquired loss-of-function mutation inclpXpartly alleviated the growth defect conferred by therpoBmutation without changing antibiotic susceptibility. The final isolate acquired three additional mutations, including an SNP inmprF(MprF S295L) known to confer daptomycin nonsusceptibility, and accordingly, this isolate was the only daptomycin-nonsusceptible strain of this series. Interestingly, in this isolate, the cell wall had regained the same thickness as that of the parental strain, while the level of transcription of thevraSR(cell wall stress regulator) was increased. In conclusion, this study illustrates how serial genetic changes selectedin vivocontribute to daptomycin nonsusceptibility, growth fitness, and virulence traits.

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Effect of Staphylococcus aureus Cell-Wall Peptidoglycan on the Rat Myometrial Contractility Regulation by Adenylate Cyclase Signaling System

International Journal of Physiology and Pathophysiology ◽

10.1615/intjphyspathophys.v8.i1.70 ◽

2017 ◽

Vol 8 (1) ◽

pp. 65-76

Author(s):

Lilit S. Nasibyan ◽

Igor B. Philyppov

Keyword(s):

Staphylococcus Aureus ◽

Cell Wall ◽

Adenylate Cyclase ◽

Signaling System ◽

Cell Wall Peptidoglycan ◽

Myometrial Contractility ◽

Adenylate Cyclase Signaling System

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Faculty Opinions recommendation of Transcriptional profiling reveals that daptomycin induces the Staphylococcus aureus cell wall stress stimulon and genes responsive to membrane depolarization.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1098746.558329 ◽

2008 ◽

Author(s):

Lynn Silver

Keyword(s):

Staphylococcus Aureus ◽

Cell Wall ◽

Transcriptional Profiling ◽

Wall Stress ◽

Membrane Depolarization ◽

Cell Wall Stress

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A Metzincin and TIMP-Like Protein Pair of a Phage Origin Sensitize Listeria monocytogenes to Phage Lysins and Other Cell Wall Targeting Agents

Microorganisms ◽

10.3390/microorganisms9061323 ◽

2021 ◽

Vol 9 (6) ◽

pp. 1323

Author(s):

Etai Boichis ◽

Nadejda Sigal ◽

Ilya Borovok ◽

Anat A. Herskovits

Keyword(s):

Cell Wall ◽

Listeria Monocytogenes ◽

Mammalian Cells ◽

Protein Pair ◽

Growth Conditions ◽

Wall Structure ◽

Antibiotic Resistant ◽

Extracellular Milieu ◽

Virion Release ◽

Genes Encoding

Infection of mammalian cells by Listeria monocytogenes (Lm) was shown to be facilitated by its phage elements. In a search for additional phage remnants that play a role in Lm’s lifecycle, we identified a conserved locus containing two XRE regulators and a pair of genes encoding a secreted metzincin protease and a lipoprotein structurally similar to a TIMP-family metzincin inhibitor. We found that the XRE regulators act as a classic CI/Cro regulatory switch that regulates the expression of the metzincin and TIMP-like genes under intracellular growth conditions. We established that when these genes are expressed, their products alter Lm morphology and increase its sensitivity to phage mediated lysis, thereby enhancing virion release. Expression of these proteins also sensitized the bacteria to cell wall targeting compounds, implying that they modulate the cell wall structure. Our data indicate that these effects are mediated by the cleavage of the TIMP-like protein by the metzincin, and its subsequent release to the extracellular milieu. While the importance of this locus to Lm pathogenicity remains unclear, the observation that this phage-associated protein pair act upon the bacterial cell wall may hold promise in the field of antibiotic potentiation to combat antibiotic resistant bacterial pathogens.

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Rhodomyrtone Accumulates in Bacterial Cell Wall and Cell Membrane and Inhibits the Synthesis of Multiple Cellular Macromolecules in Epidemic Methicillin-Resistant Staphylococcus aureus

Antibiotics ◽

10.3390/antibiotics10050543 ◽

2021 ◽

Vol 10 (5) ◽

pp. 543

Author(s):

Ozioma F. Nwabor ◽

Sukanlaya Leejae ◽

Supayang P. Voravuthikunchai

Keyword(s):

Staphylococcus Aureus ◽

Cell Wall ◽

Cell Membrane ◽

Bacterial Cell ◽

Methicillin Resistant Staphylococcus Aureus ◽

Treatment Options ◽

Bacterial Cell Wall ◽

Methicillin Resistant ◽

Gram Positive Bacteria ◽

Inhibitor Compounds

As the burden of antibacterial resistance worsens and treatment options become narrower, rhodomyrtone—a novel natural antibiotic agent with a new antibacterial mechanism—could replace existing antibiotics for the treatment of infections caused by multi-drug resistant Gram-positive bacteria. In this study, rhodomyrtone was detected within the cell by means of an easy an inexpensive method. The antibacterial effects of rhodomyrtone were investigated on epidemic methicillin-resistant Staphylococcus aureus. Thin-layer chromatography demonstrated the entrapment and accumulation of rhodomyrtone within the bacterial cell wall and cell membrane. The incorporation of radiolabelled precursors revealed that rhodomyrtone inhibited the synthesis of macromolecules including DNA, RNA, proteins, the cell wall, and lipids. Following the treatment with rhodomyrtone at MIC (0.5–1 µg/mL), the synthesis of all macromolecules was significantly inhibited (p ≤ 0.05) after 4 h. Inhibition of macromolecule synthesis was demonstrated after 30 min at a higher concentration of rhodomyrtone (4× MIC), comparable to standard inhibitor compounds. In contrast, rhodomyrtone did not affect lipase activity in staphylococci—both epidemic methicillin-resistant S. aureus and S. aureus ATCC 29213. Interfering with the synthesis of multiple macromolecules is thought to be one of the antibacterial mechanisms of rhodomyrtone.

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