Activated ClpP kills persisters and eradicates a chronic biofilm infection.
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ABSTRACT:
SUBMITTER: Conlon BP
PROVIDER: S-EPMC4031760 | biostudies-literature | 2013 Nov
REPOSITORIES: biostudies-literature
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Project description:ATPases associated with diverse cellular activities (AAA+) proteases power the maintenance of protein homeostasis by coupling ATP hydrolysis to mechanical protein unfolding, translocation, and ultimately degradation. Although ATPase activity drives a large portion of the mechanical work these molecular machines perform, how the peptidase contributes to the forceful denaturation and processive threading of substrates remains unknown. Here, using single-molecule optical trapping, we examine the mechanical activity of the caseinolytic peptidase P (ClpP) from Escherichia coli in the absence of a partner ATPase and in the presence of an activating small-molecule acyldepsipeptide. We demonstrate that ClpP grips protein substrate under mechanical loads exceeding 40 pN, which are greater than those observed for the AAA+ unfoldase ClpX and the AAA+ protease complexes ClpXP and ClpAP. We further characterize substrate-ClpP bond lifetimes and rupture forces under varying loads. We find that the resulting slip bond behavior does not depend on ClpP peptidase activity. In addition, we find that unloaded bond lifetimes between ClpP and protein substrate are on a timescale relevant to unfolding times (up to ∼160 s) for difficult to unfold model substrate proteins. These direct measurements of the substrate-peptidase bond under load define key properties required by AAA+ proteases to mechanically unfold and degrade protein substrates.
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Project description:Staphylococcus aureus has reemerged as an important human pathogen in recent decades. Although many infections caused by this microbial species persist through a biofilm mode of growth, little is known about how the host's adaptive immune system responds to these biofilm infections. In this study, S. aureus cells adhered to pins in culture and were subsequently inserted into the tibiae of C57BL/6 mice, with an infecting dose of 2 × 10? CFU. This model was utilized to determine local cytokine levels, antibody (Ab) function, and T cell populations at multiple time points throughout infection. Like human hosts, S. aureus implant infection was chronic and remained localized in 100% of C57BL/6 mice at a consistent level of approximately 10(7) CFU/gram bone tissue after day 7. This infection persisted locally for >49 days and was recalcitrant to clearance by the host immune response and antimicrobial therapy. Local inflammatory cytokines of the Th1 (interleukin-2 [IL-2], IL-12 p70, tumor necrosis factor alpha [TNF-?], and IL-1?) and Th17 (IL-6 and IL-17) responses were upregulated throughout the infection, except IL-12 p70, which dwindled late in the infection. In addition, Th1 Ab subtypes against a biofilm antigen (SA0486) were upregulated early in the infection, while Th2 Abs and anti-inflammatory regulatory T cells (Tregs) were not upregulated until later. These results indicate that early Th1 and Th17 inflammatory responses and downregulated Th2 and Treg responses occur during the development of a chronic biofilm implant infection. This unrestrained inflammatory response may cause tissue damage, thereby enabling S. aureus to attach and thrive in a biofilm mode of growth.
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Project description:BACKGROUND:ClpP is important for bacterial growth and plays an indispensable role in cellular protein quality control systems by refolding or degrading damaged proteins, but the physiological significance of ClpP in Enterococcus faecalis remains obscure. A clpP deletion mutant (?clpP) was constructed using the E. faecalis OG1RF strain to clarify the effect of ClpP on E. faecalis. The global abundance of proteins was determined by a mass spectrometer with tandem mass tag labeling. RESULTS:The ?clpP mutant strain showed impaired growth at 20?°C or 45?°C at 5% NaCl or 2?mM H2O2. The number of surviving ?clpP mutants decreased after exposure to the high concentration (50× minimal inhibitory concentration) of linezolid or minocycline for 96?h. The ?clpP mutant strain also demonstrated decreased biofilm formation but increased virulence in a Galleria mellonella model. The mass spectrometry proteomics data indicated that the abundances of 135 proteins changed (111 increased, 24 decreased) in the ?clpP mutant strain. Among those, the abundances of stress response or virulence relating proteins: FsrA response regulator, gelatinase GelE, regulatory protein Spx (spxA), heat-inducible transcription repressor HrcA, transcriptional regulator CtsR, ATPase/chaperone ClpC, acetyl esterase/lipase, and chaperonin GroEL increased in the ?clpP mutant strain; however, the abundances of ribosomal protein L4/L1 family protein (rplD), ribosomal protein L7/L12 (rplL2), 50S ribosomal protein L13 (rplM), L18 (rplR), L20 (rplT), 30S ribosomal protein S14 (rpsN2) and S18 (rpsR) all decreased. The abundances of biofilm formation-related adapter protein MecA increased, while the abundances of dihydroorotase (pyrC), orotate phosphoribosyltransferase (pyrE), and orotidine-5'-phosphate decarboxylase (pyrF) all decreased in the ?clpP mutant strain. CONCLUSION:The present study demonstrates that ClpP participates in stress tolerance, biofilm formation, antimicrobial tolerance, and virulence of E. faecalis.
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Project description:The drug resistance rate of Acinetobacter baumannii increases year on year, and the drugs available for the treatment of carbapenem-resistant A. baumannii (CRAB) infection are extremely limited. A. baumannii, which forms biofilms, protects itself by secreting substrates such as exopolysaccharides, allowing it to survive under adverse conditions and increasing drug resistance. Antimicrobial peptides are small molecular peptides with broad-spectrum antibacterial activity and immunomodulatory function. Previous studies have shown that the antimicrobial peptide Cec4 has a strong effect on A. baumannii, but the antibacterial and biofilm inhibition of this antimicrobial peptide on clinical carbapenem resistance A. baumannii is not thoroughly understood. In this study, it was indicated that most of the 200 strains of CRAB were susceptible to Cec4 with a MIC of 4 μg/ml. Cec4 has a strong inhibitory and eradication effect on the CRAB biofilm; the minimum biofilm inhibition concentration (MBIC) was 64-128 μg/ml, and the minimum biofilm eradication concentration (MBEC) was 256-512 μg/ml. It was observed that Cec4 disrupted the structure of the biofilm using scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). A comparative transcriptome analysis of the effects of the antimicrobial peptide Cec4 on CRAB biofilm, identified 185 differentially expressed genes, including membrane proteins, bacterial resistance genes, and pilus-related genes. The results show that multiple metabolic pathways, two-component regulation systems, quorum sensing, and antibiotic synthesis-related pathways in A. baumannii biofilms were affected after Cec4 treatment. In conclusion, Cec4 may represent a new choice for the prevention and treatment of clinical infections, and may also provide a theoretical basis for the development of antimicrobial peptide drugs.
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