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In depth characterization of the Staphylococcus aureus phosphoproteome reveals new targets of Stk1.

ABSTRACT: Staphylococcus aureus is a major cause of infections worldwide and infection results in a variety of diseases. As of no surprise, protein phosphorylation is an important game player in signaling cascades and has been shown to be involved in S. aureus virulence. Albeit long neglected, eukaryotic-type serine/threonine kinases in S. aureus have been implicated in this complex signaling cascades. Due to the sub-stoichiometric nature of protein phosphorylation and a lack of suitable analysis tools, the knowledge of these cascades is however, to date, still limited. Here, were apply an optimized protocol for efficient phosphopeptide enrichment via Fe³⁺-IMAC followed by LC-MS/MS to get a better understanding of the impact of protein phosphorylation on the complex signaling networks involved in pathogenicity. By profiling a serine/threonine kinase and phosphatase mutant from a methicillin-resistant S. aureus mutant library, we generated the most comprehensive phosphoproteome dataset of S. aureus to date, aiding a better understanding of signaling in bacteria. With the identification of 3800 class I p-sites we were able to increase the number of identifications by more than 21 times compared to recent literature. In addition, we were able to identify 74 downstream targets of the only reported eukaryotic-type Ser/Thr kinase of the S. aureus strain USA300, Stk1. This work allowed an extensive analysis of the bacterial phosphoproteome and indicates that Ser/Thr kinase signaling is far more abundant than previously anticipated in S. aureus.

SUBMITTER: Prust N

PROVIDER: S-EPMC7950182 | biostudies-literature |

REPOSITORIES: biostudies-literature

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In depth characterization of the S. aureus phosphoproteome reveals new targets of Stk1

Project description:Staphylococcus aureus is the leading cause of infections worldwide and infection results in a variety of diseases. As of no surprise, phosphorylation is a major game player in signaling cascades and has been shown to be involved in S. aureus virulence. Albeit long neglected, eukaryotic-like serine/threonine kinases have been implicated in these complex signaling cascades. Due to the sub-stoichiometric nature of protein phosphorylation and a lack of suitable analysis tools, the knowledge of these cascades is however, to date, still limited. Here, were apply an optimized protocol for efficient phosphopeptide enrichment via Fe3+-IMAC followed by LC-MS/MS to get a better understanding of the impact of protein phosphorylation on the complex signaling networks involved in pathogenicity. By profiling a serine/threonine kinase and phosphatase mutant from a methicillin-resistant S. aureus mutant library, we generated the most comprehensive phosphoproteome dataset of S. aureus to date, aiding a better understanding of signaling in bacteria.

2020-12-21 | PXD020226 | Pride

Autophosphorylation Mechanism of the Ser/Thr Kinase Stk1 From Staphylococcus aureus.

Project description:The eukaryotic-like Ser/Thr kinase Stk1 is crucial for virulence, cell wall biosynthesis, and drug susceptibility in methicillin-resistant Staphylococcus aureus (S. aureus) (MRSA). Importantly, MRSA lacking Stk1 become sensitive to β-lactam antibiotics, implying that Stk1 could be an alternative target for combination therapy. However, the autophosphorylation mechanism of Stk1 remains elusive. Using a phosphoproteomic study, we identified six in vivo phosphorylated activation loop residues (Ser159, Thr161, Ser162, Thr164, Thr166, and Thr172) of Stk1, which are also phosphorylated in vitro. We further showed that cis autophosphorylation of Thr172 in the GT/S motif is essential for self-activation and kinase activity of Stk1 kinase domain (Stk1-KD), whereas the trans autophosphorylation of other activation loop serines/threonines are required for the optimal kinase activity of Stk1-KD. Moreover, substitution of the activation loop serines/threonines impaired in vivo autophosphorylation activity of kinase variants, while T172A and T172D variants were unable to autophosphorylate in the cellular content, underlining the essential role of Thr172 for Stk1 activity in vivo. This study provides insights into molecular basis for regulation of Stk1 activity from S. aureus.

| S-EPMC5920020 | biostudies-literature

A novel STK1-targeted small-molecule as an "antibiotic resistance breaker" against multidrug-resistant Staphylococcus aureus.

Project description:Ser/Thr protein kinase (STK1) plays a critical role in cell wall biosynthesis of and drug resistance in methicillin-resistant Staphylococcus aureus (MRSA). MRSA strains lacking STK1 become susceptible to failing cephalosporins, such as Ceftriaxone and Cefotaxime. STK1, despite being nonessential protein for MRSA survival, it can serve as an important therapeutic agent for combination therapy. Here, we report a novel small molecule quinazoline compound, Inh2-B1, which specifically inhibits STK1 activity by directly binding to its ATP-binding catalytic domain. Functional analyses encompassing in vitro growth inhibition of MRSA, and in vivo protection studies in mice against the lethal MRSA challenge indicated that at high concentration neither Inh2-B1 nor Ceftriaxone or Cefotaxime alone was able to inhibit the growth of bacteria or protect the challenged mice. However, the growth of MRSA was inhibited, and a significant protection in mice against the bacterial challenge was observed at a micromolar concentration of Ceftriaxone or Cefotaxime in the presence of Inh2-B1. Cell-dependent minimal to no toxicity of Inh2-B1, and its abilities to down-regulate cell wall hydrolase genes and disrupt the biofilm formation of MRSA clearly indicated that Inh2-B1 serves as a therapeutically important "antibiotic-resistance-breaker," which enhances the bactericidal activity of Ceftriaxone/Cefotaxime against highly pathogenic MRSA infection.

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Structural characterization of MepB from Staphylococcus aureus reveals homology to endonucleases.

Project description:The MepRAB operon in Staphylococcus aureus has been identified to play a role in drug resistance. Although the functions of MepA and MepR are known, little information is available on the function of MepB. Here we report the X-ray structure of MepB to 2.1 Å revealing its structural similarity to the PD-(D/E)XK family of endonucleases. We further show that MepB binds DNA and RNA, with a higher affinity towards RNA and single stranded DNA than towards double stranded DNA. Notably, the PD-(D/E)XK catalytic active site residues are not conserved in MepB. MepB's association with a drug resistance operon suggests that it plays a role in responding to antimicrobials. This role is likely carried out through MepB's interactions with nucleic acids.

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Characterization of a new cytotoxin that contributes to Staphylococcus aureus pathogenesis.

Project description:Staphylococcus aureus is an important pathogen that continues to be a significant global health threat because of the prevalence of methicillin-resistant S. aureus strains (MRSA). The pathogenesis of this organism is partly attributed to the production of a large repertoire of cytotoxins that target and kill innate immune cells, which provide the first line of defence against S. aureus infection. Here we demonstrate that leukocidin A/B (LukAB) is required and sufficient for the ability of S. aureus, including MRSA, to kill human neutrophils, macrophages and dendritic cells. LukAB targets the plasma membrane of host cells resulting in cellular swelling and subsequent cell death. We found that S. aureus lacking lukAB are severely impaired in their ability to kill phagocytes during bacteria-phagocyte interaction, which in turn renders the lukAB-negative staphylococci more susceptible to killing by neutrophils. Notably, we show that lukAB is expressed in vivo within abscesses in a murine infection model and that it contributes significantly to pathogenesis of MRSA in an animal host. Collectively, these results extend our understanding of how S. aureus avoids phagocyte-mediated clearance, and underscore LukAB as an important factor that contributes to staphylococcal pathogenesis.

| S-EPMC3312031 | biostudies-literature

Direct targets of CodY in Staphylococcus aureus.

Project description:More than 200 direct CodY target genes in Staphylococcus aureus were identified by genome-wide analysis of in vitro DNA binding. This analysis, which was confirmed for some genes by DNase I footprinting assays, revealed that CodY is a direct regulator of numerous transcription units associated with amino acid biosynthesis, transport of macromolecules, and virulence. The virulence genes regulated by CodY fell into three groups. One group was dependent on the Agr system for its expression; these genes were indirectly regulated by CodY through its repression of the agr locus. A second group was regulated directly by CodY. The third group, which includes genes for alpha-toxin and capsule synthesis, was regulated by CodY in two ways, i.e., by direct repression and by repression of the agr locus. Since S. aureus CodY was activated in vitro by the branched chain amino acids and GTP, CodY appears to link changes in intracellular metabolite pools with the induction of numerous adaptive responses, including virulence.

| S-EPMC2876493 | biostudies-literature

A novel mode of regulation of the Staphylococcus aureus catabolite control protein A (CcpA) mediated by Stk1 protein phosphorylation.

Project description:The Staphylococcus aureus serine/threonine protein kinase Stk1 (also known as PknB) affects different key pathways such as cell wall metabolism, antibiotic susceptibility, and regulation of virulence. Here we report that the catabolite control protein A (CcpA), a highly conserved regulator of carbon catabolite repression and virulence in a number of gram-positive pathogens, was efficiently phosphorylated in vitro and in vivo by Stk1 in S. aureus, whereas the CcpA homologues of Bacillus subtilis and Bacillus anthracis were not affected by the Stk1 orthologue PrkC. Mass spectrometry and mutational analyses identified Thr-18 and Thr-33 as the phosphoacceptors; both are located in the DNA binding domain of this protein. Electrophoretic mobility shift assays demonstrated that the CcpA DNA binding activity was completely abrogated for the phosphorylated CcpA. The physiological relevance of CcpA phosphorylation was assessed by generating CcpA phosphoablative (T18A/T33A) or phosphomimetic (T18D/T33D) mutants. In contrast to the wild-type and phosphoablative ccpA alleles, introduction of the phosphomimetic ccpA allele in a ΔccpA mutant failed to restore the parental biofilm formation profile and the transcription of citZ and hla to levels seen with the wild type. The strong up regulation of ccpA transcripts and CcpA level in the ccpA mutant trans-complemented with the phosphomimetic CcpA variant suggest furthermore that CcpA acts as a negative regulator of its own expression. Together, these findings demonstrate that Stk1-driven phosphorylation of CcpA inhibits its DNA binding activity toward its regulon in S. aureus, representing a novel regulatory mechanism of CcpA activity in addition to the well known regulation via HprKP/Hpr in this clinically important pathogen.

| S-EPMC3527947 | biostudies-literature

Direct targets of CodY in Staphylococcus aureus

Project description:More than 200 direct CodY target genes in Staphylococcus aureus were identified by genome-wide analysis of in vitro DNA binding. This analysis, which was confirmed for some genes by DNase I footprinting assays, revealed that CodY is a direct regulator of numerous transcription units associated with amino acid biosynthesis, transport of macromolecules and virulence. The virulence genes regulated by CodY fell into three groups. One group was dependent on the Agr system for its expression; these genes were indirectly regulated by CodY through its repression of the agr locus. A second group was regulated directly by CodY. The third group, which includes genes for alpha-toxin and capsule synthesis, was regulated by CodY in two ways, i.e., by direct repression and by repression of the agr locus. Since S. aureus CodY was activated in vitro by the branched chain amino acids and GTP, CodY appears to link changes in intracellular metabolite pools with the induction of numerous adaptive responses, including virulence.

2010-04-01 | GSE20973 | GEO

Characterization of a New Staphylococcus aureus Kayvirus Harboring a Lysin Active against Biofilms.

Project description:Staphylococcus aureus is one of the most relevant opportunistic pathogens involved in many biofilm-associated diseases, and is a major cause of nosocomial infections, mainly due to the increasing prevalence of multidrug-resistant strains. Consequently, alternative methods to eradicate the pathogen are urgent. It has been previously shown that polyvalent staphylococcal kayviruses and their derived endolysins are excellent candidates for therapy. Here we present the characterization of a new bacteriophage: vB_SauM-LM12 (LM12). LM12 has a broad host range (>90%; 56 strains tested), and is active against several MRSA strains. The genome of LM12 is composed of a dsDNA molecule with 143,625 bp, with average GC content of 30.25% and codes for 227 Coding Sequences (CDSs). Bioinformatics analysis did not identify any gene encoding virulence factors, toxins, or antibiotic resistance determinants. Antibiofilm assays have shown that this phage significantly reduced the number of viable cells (less than one order of magnitude). Moreover, the encoded endolysin also showed activity against biofilms, with a consistent biomass reduction during prolonged periods of treatment (of about one order of magnitude). Interestingly, the endolysin was shown to be much more active against stationary-phase cells and suspended biofilm cells than against intact and scraped biofilms, suggesting that cellular aggregates protected by the biofilm matrix reduced protein activity. Both phage LM12 and its endolysin seem to have a strong antimicrobial effect and broad host range against S. aureus, suggesting their potential to treat S. aureus biofilm infections.

| S-EPMC5923476 | biostudies-literature

Expression and characterization of the new antimicrobial peptide AP138L-arg26 anti Staphylococcus aureus.

Project description:The low activity and yield of antimicrobial peptides (AMPs) are pressing problems. The improvement of activity and yield through modification and heterologous expression, a potential way to solve the problem, is a research hot-pot. In this work, a new plectasin-derived variant L-type AP138 (AP138L-arg26) was constructed for the study of recombination expression and druggablity. As a result, the total protein concentration of AP138L-arg26 was 3.1 mg/mL in Pichia pastoris X-33 supernatant after 5 days of induction expression in a 5-L fermenter. The recombinant peptide AP138L-arg26 has potential antibacterial activity against selected standard and clinical Gram-positive bacteria (G+, minimum inhibitory concentration (MIC) 2-16 µg/mL) and high stability under different conditions (temperature, pH, ion concentration) and 2 × MIC of AP138L-arg26 could rapidly kill Staphylococcus aureus (S. aureus) (> 99.99%) within 1.5 h. It showed a high safety in vivo and in vivo and a long post-antibiotic effect (PAE, 1.91 h) compared with vancomycin (1.2 h). Furthermore, the bactericidal mechanism was revealed from two dimensions related to its disruption of the cell membrane resulting in intracellular potassium leakage (2.5-fold higher than control), and an increase in intracellular adenosine triphosphate (ATP), and reactive oxygen species (ROS), the decrease of lactate dehydrogenase (LDH) and further intervening metabolism in S. aureus. These results indicate that AP138L-arg26 as a new peptide candidate could be used for more in-depth development in the future. KEY POINTS: • The AP138L-arg26 was expressed in the P. pastoris expression system with high yield • The AP138 L-arg26 showed high stability and safety in vitro and in vivo • The AP138L-arg26 killed S. aureus by affecting cell membranes and metabolism.

| S-EPMC10787893 | biostudies-literature

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