Synthetic LPETG-Containing Peptide Incorporation in the Staphylococcus aureus Cell-Wall in a Sortase A- and Growth Phase-Dependent Manner

The housekeeping sortase A (SrtA), a membrane-associated cysteine transpeptidase, is responsible for anchoring surface proteins to the cell wall peptidoglycan in Gram-positive bacteria. This process is essential for the regulation...

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The Nature of Antibacterial Adaptive Immune Responses against Staphylococcus aureus Is Dependent on the Growth Phase and Extracellular Peptidoglycan

Infection and Immunity ◽

10.1128/iai.00733-19 ◽

2019 ◽

Vol 88 (1) ◽

Author(s):

Payal P. Balraadjsing ◽

Lisbeth D. Lund ◽

Yuri Souwer ◽

Sebastian A. J. Zaat ◽

Hanne Frøkiær ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Cell Wall ◽

T Cell ◽

Stationary Phase ◽

Growth Phase ◽

Cell Response ◽

Th17 Cell ◽

Exponential Phase ◽

Th1 Cell ◽

Content Type

ABSTRACT Staphylococcus aureus has evolved different strategies to evade the immune response, which play an important role in its pathogenesis. The bacteria express and shed various cell wall components and toxins during different stages of growth that may affect the protective T cell responses to extracellular and intracellular S. aureus. However, if and how the dendritic cell (DC)-mediated T cell response against S. aureus changes during growth of the bacterium remain elusive. In this study, we show that exponential-phase (EP) S. aureus bacteria were endocytosed very efficiently by human DCs, and these DCs strongly promoted production of the T cell polarizing factor interleukin-12 (IL-12). In contrast, stationary-phase (SP) S. aureus bacteria were endocytosed less efficiently by DCs, and these DCs produced small amounts of IL-12. The high level of IL-12 production induced by EP S. aureus led to the development of a T helper 1 (Th1) cell response, which was inhibited after neutralization of IL-12. Furthermore, preincubation with the staphylococcal cell wall component peptidoglycan (PGN), characteristically shed during the exponential growth phase, modulated the DC response to EP S. aureus. PGN preincubation appeared to inhibit IL-12p35 expression, leading to downregulation of IL-12 and an increase of IL-23 production by DCs, enhancing Th17 cell development. Taken together, our data indicate that exponential-phase S. aureus bacteria induce a stronger IL-12-dependent Th1 cell response than stationary-phase S. aureus and that this Th1 cell response shifted toward a Th17 cell response in the presence of PGN.

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Transcriptional Induction of the Penicillin-Binding Protein 2 Gene in Staphylococcus aureus by Cell Wall-Active Antibiotics Oxacillin and Vancomycin

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.47.3.1028-1036.2003 ◽

2003 ◽

Vol 47 (3) ◽

pp. 1028-1036 ◽

Cited By ~ 27

Author(s):

Susan Boyle-Vavra ◽

Shaohui Yin ◽

Mamatha Challapalli ◽

Robert S. Daum

Keyword(s):

Staphylococcus Aureus ◽

Cell Wall ◽

Growth Phase ◽

Transcript Abundance ◽

Promoter Sequence ◽

Phase Variable ◽

Penicillin Binding Protein ◽

Promoter Regions ◽

Putative Transcription Factor ◽

Transcriptional Induction

ABSTRACT We found an increased abundance of pbpB-specific transcripts in vancomycin intermediate-resistant Staphylococcus aureus (VISA) isolates compared with that found in paired, genetically identical, susceptible isolates. This difference in expression cannot be explained by differences in the pbpB promoter sequence. Since the factors controlling pbpB gene expression have remained largely unexplored, various conditions that might affect pbpB transcript abundance were examined. In both vancomycin-susceptible and VISA strains, pbpB expression varied with the growth phase, with the highest abundance of pbpB-specific transcripts detected during mid-log phase. Interestingly, both vancomycin and oxacillin were able to induce pbpB transcription above a constitutive level. When vancomycin was absent, one of the three pbpB-specific transcripts that were usually faintly detected in non-VISA strains was more readily detected in VISA strains during mid-log but not stationary phase. This transcript was enhanced in non-VISA strains by vancomycin induction. Gel shift assays indicated that an increased amount of the putative transcription factor that binds to both P1 and P1′ promoter regions is present in the cytosol of vancomycin-induced cells. Neither the SigB sigma factor nor the quorum-sensing agr locus was required for growth phase-variable pbpB expression or transcriptional induction of pbpB by vancomycin or oxacillin. Also, MecI, MecR1, BlaI, and BlaR1, regulatory proteins that mediate β-lactam-inducible expression of mecA and β-lactamase, were not required for antibiotic induction of pbpB transcription. These data support the idea that pbpB expression is modulated by a trans-acting factor in response to the presence of the cell wall-active antibiotics vancomycin and oxacillin.

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A PBP 2 Mutant Devoid of the Transpeptidase Domain Abolishes Spermine–β-Lactam Synergy in Staphylococcus aureus Mu50

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.05415-11 ◽

2011 ◽

Vol 56 (1) ◽

pp. 83-91 ◽

Cited By ~ 10

Author(s):

Xiangyu Yao ◽

Chung-Dar Lu

Keyword(s):

Staphylococcus Aureus ◽

Cell Wall ◽

Wall Stress ◽

Enzyme Hydrolysis ◽

Dependent Manner ◽

Content Type ◽

C Terminus ◽

Alkaline Conditions ◽

Exogenous Spermine ◽

Ph Range

ABSTRACTExogenous spermine was reported to enhance the killing of methicillin-resistantStaphylococcus aureus(MRSA) by β-lactams through a strong synergistic effect of unknown nature. Spermine alone also exerts an antimicrobial activity againstS. aureusin a pH-dependent manner. MIC measurements revealed stronger effects of spermine under alkaline conditions, suggesting the nucleophilic property of spermine instead of its positive charge as the cause of adverse effects. A spontaneous suppressor mutant (MuM) of MRSA Mu50 was selected for spermine resistance and conferred complete abolishment of spermine–β-lactam synergy. In comparison to that in Mu50, the spermine MIC in MuM remained constant (64 mM) at pH 6 to 8; however, MuM, a heat-sensitive mutant, also grew in a very narrow pH range. Furthermore, MuM acquired a unique phenotype of vancomycin-spermine synergy. Genome resequencing revealed a 7-bp deletion inpbpB, which results in a truncated penicillin-binding protein 2 (PBP 2) without the transpeptidase domain at the C terminus while the N-terminal transglycosidase domain remains intact. The results of fluorescent Bocillin labeling experiments confirmed the presence of this defective PBP 2 in MuM. All the aforementioned phenotypes of MuM were reverted to those of Mu50 after complementation by the wild-typepbpBcarried on a recombinant plasmid. The anticipated changes in cell wall metabolism and composition in MuM were evidenced by observations that the cell wall of MuM was more susceptible to enzyme hydrolysis and that MuM exhibited a lower level of autolytic activities. Pleiotropic alterations in gene expression were revealed by microarray analysis, suggesting a remarkable flexibility of MuM to circumvent cell wall damage by triggering adaptations that are complex but completely different from that of the cell wall stress stimulon. In summary, these results reveal phenotypic changes and transcriptome adaptations in a uniquepbpBmutant and provide evidence to support the idea that exogenous spermine may perturb normal cell wall formation through its interactions with PBP 2.

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Transcriptome and Functional Analysis of the Eukaryotic-Type Serine/Threonine Kinase PknB in Staphylococcus aureus

Journal of Bacteriology ◽

10.1128/jb.00117-09 ◽

2009 ◽

Vol 191 (13) ◽

pp. 4056-4069 ◽

Cited By ~ 72

Author(s):

Stefanie Donat ◽

Karin Streker ◽

Tanja Schirmeister ◽

Sonja Rakette ◽

Thilo Stehle ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Cell Wall ◽

Biochemical Characterization ◽

Dependent Manner ◽

Serine Threonine Kinase ◽

Expression Of Genes ◽

Biochemical Assays ◽

Genes Encoding ◽

Regulatory Impact ◽

Glutamine Synthesis

ABSTRACT The function of the Staphylococcus aureus eukaryotic-like serine/threonine protein kinase PknB was investigated by performing transcriptome analysis using DNA microarray technology and biochemical assays. The transcriptional profile revealed a strong regulatory impact of PknB on the expression of genes encoding proteins which are involved in purine and pyrimidine biosynthesis, cell wall metabolism, autolysis, and glutamine synthesis. Functional activity of overexpressed and purified PknB kinase was demonstrated using the myelin basic protein as a surrogate substrate. Phosphorylation occurred in a time-dependent manner with Mn2+ as a preferred cofactor. Furthermore, biochemical characterization revealed regulation of adenylosuccinate synthase (PurA) activity by phosphorylation. Phosphorylated PurA showed a 1.8-fold decrease in enzymatic activity compared to unphosphorylated PurA. Loss of PknB led to formation of larger cell clusters, and a pknB deletion strain showed 32-fold-higher sensitivity to the cell wall-active antibiotic tunicamycin. The results of this study strongly indicate that PknB has a role in regulation of purine biosynthesis, autolysis, and central metabolic processes in S. aureus.

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SarA Represses agr Operon Expression in a Purified In Vitro Staphylococcus aureusTranscription System

Journal of Bacteriology ◽

10.1128/jb.182.20.5893-5897.2000 ◽

2000 ◽

Vol 182 (20) ◽

pp. 5893-5897 ◽

Cited By ~ 22

Author(s):

Swarup K. Chakrabarti ◽

Tapan K. Misra

Keyword(s):

Experimental Data ◽

Escherichia Coli ◽

Staphylococcus Aureus ◽

Rna Polymerase ◽

Growth Phase ◽

Surface Protein ◽

Dependent Manner ◽

Operon Expression ◽

Chromosomal Loci

ABSTRACT Mutation and genetic complementation studies suggested that two chromosomal loci, agr and sar, are involved in the upregulation of several exotoxin genes and the downregulation of a number of surface protein genes in a growth phase-dependent manner inStaphylococcus aureus. We purified recombinant T7-tagged SarA from Escherichia coli and determined its effect on transcription from several S. aureus promoters by using purified RNA polymerase reconstituted with either ςA or ςB from S. aureus. Of the seven ςA-dependent promoters that we tested, SarA repressed transcription from agrP2, agrP3,cna, sarP1, and sea promoters and did not affect sec and znt promoters. Furthermore, SarA had no effect on transcription from the ςB-dependent sarP3 promoter. In vitro experimental data presented in this report suggest that SarA expression is autoregulated.

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Growth-phase dependence of susceptibility to antimicrobial peptides in Staphylococcus aureus

Microbiology ◽

10.1099/mic.0.044727-0 ◽

2011 ◽

Vol 157 (6) ◽

pp. 1786-1797 ◽

Cited By ~ 30

Author(s):

Miki Matsuo ◽

Yuichi Oogai ◽

Fuminori Kato ◽

Motoyuki Sugai ◽

Hitoshi Komatsuzawa

Keyword(s):

Staphylococcus Aureus ◽

Cell Surface ◽

Antimicrobial Peptides ◽

Stationary Phase ◽

Surface Charge ◽

Growth Phase ◽

Exponential Phase ◽

Dependent Manner ◽

Phase Dependence ◽

Cell Surface Charge

Bacterial cell surface charge is responsible for susceptibility to cationic antimicrobial peptides. Previously, Staphylococcus aureus dlt and mprF were identified as factors conferring a positive charge upon cell surfaces. In this study, we investigated the regulation of cell surface charge during growth. Using a group of S. aureus MW2 mutants, which are gene-inactivated in 15 types of two-component systems (TCSs), we tested dltC and mprF expression and found that two TCSs, aps and agr, were associated with dltC and mprF expression in a growth phase-dependent manner. The first of these, aps, which had already been identified as a sensor of antimicrobial peptides and a positive regulator of dlt and mprF expression, was expressed strongly in the exponential phase, while its expression was significantly suppressed by agr in the stationary phase, resulting in higher expression of dltC and mprF in the exponential phase and lower expression in the stationary phase. Since both types of expression affected the cell surface charge, the susceptibility to antimicrobial peptides and cationic antibiotics was changed during growth. Furthermore, we found that the ability to sense antimicrobial peptides only functioned in the exponential phase. These results suggest that cell surface charge is tightly regulated during growth in S. aureus.

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Eriodictyol as a Potential Candidate Inhibitor of Sortase A Protects Mice From Methicillin-Resistant Staphylococcus aureus-Induced Pneumonia

Frontiers in Microbiology ◽

10.3389/fmicb.2021.635710 ◽

2021 ◽

Vol 12 ◽

Author(s):

Li Wang ◽

Qianxue Li ◽

Jiaxin Li ◽

Shisong Jing ◽

Yajing Jin ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Cell Wall ◽

Protein A ◽

A549 Cells ◽

Surface Proteins ◽

Reversible Inhibitor ◽

Sortase A ◽

Staphylococcal Protein A ◽

Potential Candidate ◽

Methicillin Resistant

New anti-infective approaches are urgently needed to control multidrug-resistant (MDR) pathogens, such as methicillin-resistant Staphylococcus aureus (MRSA). Sortase A (SrtA) is a membrane-bound cysteine transpeptidase that plays an essential role in the catalysis of covalent anchoring of surface proteins to the cell wall of Staphylococcus aureus (S. aureus). The present study reports identification of a flavonoid, eriodictyol, as a reversible inhibitor of SrtA with an IC50 of 2.229 ± 0.014 μg/mL that can be used as an innovative means to counter both resistance and virulence. The data indicated that eriodictyol inhibited the adhesion of the bacteria to fibrinogen and reduced the formation of biofilms and anchoring of staphylococcal protein A (SpA) on the cell wall. The results of fluorescence quenching experiments demonstrated a strong interaction between eriodictyol and SrtA. Subsequent mechanistic studies revealed that eriodictyol binds to SrtA by interacting with R197 amino acid residue. Importantly, eriodictyol reduced the adhesion-dependent invasion of A549 cells by S. aureus and showed a good therapeutic effect in a model of mouse pneumonia induced by S. aureus. Overall, the results indicated that eriodictyol can attenuate MRSA virulence and prevent the development of resistance by inhibiting SrtA, suggesting that eriodictyol may be a promising lead compound for the control of MRSA infections.

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VraSR Two-Component Regulatory System Contributes tomprF-Mediated Decreased Susceptibility to Daptomycin inIn Vivo-Selected Clinical Strains of Methicillin-Resistant Staphylococcus aureus

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00432-10 ◽

2011 ◽

Vol 56 (1) ◽

pp. 92-102 ◽

Cited By ~ 76

Author(s):

Shrenik Mehta ◽

Arabela X. Cuirolo ◽

Konrad B. Plata ◽

Sarah Riosa ◽

Jared A. Silverman ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Cell Wall ◽

Regulatory System ◽

Dependent Manner ◽

Wild Type ◽

Methicillin Resistant ◽

Content Type ◽

Clinical Strains ◽

Two Component ◽

The Impact

ABSTRACTDaptomycin (DAP) is a new class of cyclic lipopeptide antibiotic highly active against methicillin-resistantStaphylococcus aureus(MRSA) infections. Proposed mechanisms involve disruption of the functional integrity of the bacterial membrane in a Ca-dependent manner. In the present work, we investigated the molecular basis of DAP resistance in a group of isogenic MRSA clinical strains obtained from patients withS. aureusinfections after treatment with DAP. Different point mutations were found in themprFgene in DAP-resistant (DR) strains. Investigation of themprFL826F mutation in DR strains was accomplished by inactivation and transcomplementation of either full-length wild-type or mutatedmprFin DAP-susceptible (DS) strains, revealing that they were mechanistically linked to the DR phenotype. However, our data suggested thatmprFwas not the only factor determining the resistance to DAP. Differential gene expression analysis showed upregulation of the two-component regulatory systemvraSR. Inactivation ofvraSRresulted in increased DAP susceptibility, while complementation ofvraSRmutant strains restored DAP resistance to levels comparable to those observed in the corresponding DR wild-type strain. Electron microscopy analysis showed a thicker cell wall in DR CB5012 than DS CB5011, an effect that was related to the impact ofvraSRandmprFmutations in the cell wall. Moreover, overexpression ofvraSRin DS strains resulted in both increased resistance to DAP and decreased resistance to oxacillin, similar to the phenotype observed in DR strains. These results support the suggestion that, in addition to mutations inmprF,vraSRcontributes to DAP resistance in the present group of clinical strains.

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