| 1. |
- Novak, R, et al.
(författare)
-
Extracellular targeting of choline-binding proteins in Streptococcus pneumoniae by a zinc metalloprotease.
- 2000
-
Ingår i: Molecular Microbiology. - 0950-382X .- 1365-2958. ; 36:2, s. 366-376
-
Tidskriftsartikel (refereegranskat)abstract
- A genetic-based search for surface proteins of Streptococcus pneumoniae involved in adhesion identified a putative zinc metalloprotease (ZmpB). ZmpB shared high amino acid sequence similarities with IgA1 proteases of Gram-positive bacteria, but ZmpB had neither IgA1 nor IgA2 protease activity. Analysis of a family of surface-expressed proteins, the choline-binding proteins (Cbp's), in a zmpB-deficient mutant demonstrated a global loss of surface expression of CbpA, CbpE, CbpF and CbpJ. CbpA was detected within the cytoplasm. The zmpB-deficient mutant also failed to lyse with penicillin, a sign of lack of function of the Cbp LytA. Immunodetection studies revealed that the autolysin (LytA), normally located on the cell wall, was trapped in the cytoplasm colocalized with DNA and the transformation protein CinA. Trafficking of CinA and RecA to the cell membrane during genetic competence was also not observed in the zmpB-deficient mutant. These results suggest a protease dependent regulatory mechanism governing the translocation of CinA and the Cbp's LytA and CbpA of S. pneumoniae.
|
|
| 2. |
- Charpentier, E, et al.
(författare)
-
Mechanisms of antibiotic resistance and tolerance in Streptococcus pneumoniae.
- 2000
-
Ingår i: Microbes and infection. - 1286-4579 .- 1769-714X. ; 2:15, s. 1855-64
-
Tidskriftsartikel (refereegranskat)abstract
- Streptococcus pneumoniae is a major pathogen causing potentially life-threatening community-acquired diseases in both the developed and developing world. Since 1967, there has been a dramatic increase in the incidence of penicillin-resistant and multiply antibiotic-resistant pneumococci worldwide. Prevention of access of the antibiotic to the target, inactivation of the antibiotic and alteration of the target are mechanisms that S. pneumoniae has developed to resist antibiotics. Recent studies on antibiotic-tolerant pneumococcal mutants permitted development of a novel model for the control of bacterial cell death.
|
|
| 3. |
- Charpentier, E, et al.
(författare)
-
Regulation of growth inhibition at high temperature, autolysis, transformation and adherence in Streptococcus pneumoniae by clpC.
- 2000
-
Ingår i: Molecular Microbiology. - 0950-382X .- 1365-2958. ; 37:4, s. 717-26
-
Tidskriftsartikel (refereegranskat)abstract
- The ClpC ATPase is a subfamily of HSP100/Clp molecular chaperones-regulators of proteolysis. By screening a library of loss of function mutants for the ability to survive treatment with penicillin, we identified the gene clpC. The corresponding protein was identified as a ClpC ATPase, sharing strong peptide sequence identity with ClpC of Bacillus subtilis, Listeria monocytogenes and Lactococcus lactis. Northern blot experiments showed that expression of clpC was induced in response to high temperature (40-42 degrees C) versus 37 degrees C, suggesting that ClpC is a heat shock protein. Insertional duplication mutagenesis of clpC resulted in increased tolerance to high temperature; a result in contrast to other bacterial Clp proteases. The clpC-deficient mutant formed long chains and failed to undergo lysis after treatment with penicillin or vancomycin. The effect of the clpC mutation extended to deficiency of adherence to the human type II alveolar cells. Finally, the clpC disruption resulted in decreased genetic transformation. Western blot analysis demonstrated that the mutant failed to express pneumolysin and the choline-binding proteins LytA, CbpA, CbpE, CbpF, CbpJ. These results suggest that the heat shock protein ClpC plays an essential complex pleiotropic role in pneumococcal physiology, including cell growth under heat stress, cell division, autolysis, adherence and transformation.
|
|
| 4. |
|
|
| 5. |
- Novak, R, et al.
(författare)
-
Emergence of vancomycin tolerance in Streptococcus pneumoniae.
- 1999
-
Ingår i: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 399:6736, s. 590-593
-
Tidskriftsartikel (refereegranskat)abstract
- Streptococcus pneumoniae, the pneumococcus, is the most common cause of sepsis and meningitis. Multiple-antibiotic-resistant strains are widespread, and vancomycin is the antibiotic of last resort. Emergence of vancomycin resistance in this community-acquired bacterium would be catastrophic. Antibiotic tolerance, the ability of bacteria to survive but not grow in the presence of antibiotics, is a precursor phenotype to resistance. Here we show that loss of function of the VncS histidine kinase of a two-component sensor-regulator system in S. pneumoniae produced tolerance to vancomycin and other classes of antibiotic. Bacterial two-component systems monitor environmental parameters through a sensor histidine-kinase/phosphatase, which phosphorylates/dephosphorylates a response regulator that in turn mediates changes in gene expression. These results indicate that signal transduction is critical for the bactericidal activity of antibiotics. Experimental meningitis caused by the vncS mutant failed to respond to vancomycin. Clinical isolates tolerant to vancomycin were identified and DNA sequencing revealed nucleotide alterations in vncS. We conclude that broad antibiotic tolerance of S. pneumoniae has emerged in the community by a molecular mechanism that eliminates sensitivity to the current cornerstone of therapy, vancomycin.
|
|
| 6. |
- Novak, R, et al.
(författare)
-
Identification of a Streptococcus pneumoniae gene locus encoding proteins of an ABC phosphate transporter and a two-component regulatory system.
- 1999
-
Ingår i: Journal of Bacteriology. - 0021-9193 .- 1098-5530. ; 181:4, s. 1126-1133
-
Tidskriftsartikel (refereegranskat)abstract
- The Escherichia coli Pst system belongs to the family of ABC transporters. It is part of a phosphate (PHO) regulon which is regulated by extracellular phosphate. Under conditions of phosphate limitation, the response regulator PhoB is phosphorylated by the histidine kinase PhoR and binds to promoters that share a consensus PHO box. Under conditions of phosphate excess, PhoR, Pst, and PhoU downregulate the PHO regulon. Screening of a library of pneumococcal mutants with defects in exported proteins revealed a putative two-component regulatory system, PnpR-PnpS, and a downstream ABC transporter, similar to the Pst system in E. coli including a gene encoding a PhoU protein. Similar to E. coli, mutagenesis of the ATP-binding cassette gene, pstB, resulted in decreased uptake of phosphate. The effects of the loss of the pneumococcal Pst system extended to decreased transformation and lysis. Withdrawal of phosphate led to transformation deficiency in the parent strain R6x but not to penicillin tolerance, suggesting that reduced bacterial death was independent of phosphate. None of these phenotypes was observed in the pneumococcal loss-of-function mutant phoU. By using a lacZ reporter construct, it was demonstrated that expression of the two-component regulatory system PnpR-PnpS was not influenced by different concentrations of phosphate. These results suggest a more complex role of the Pst system in pneumococcal physiology than in that of E. coli.
|
|
| 7. |
- Novak, R, et al.
(författare)
-
Penicillin tolerance genes of Streptococcus pneumoniae : the ABC-type manganese permease complex Psa.
- 1998
-
Ingår i: Molecular Microbiology. - 0950-382X .- 1365-2958. ; 29:5, s. 1285-1296
-
Tidskriftsartikel (refereegranskat)abstract
- Downregulation of the major autolysin in Streptococcus pneumoniae leads to penicillin tolerance, a feature that is characterized by the ability to survive but not grow in the presence of antibiotic. Screening a library of mutants in pneumococcal surface proteins for the ability to survive 10x minimum inhibitory concentration (MIC) of penicillin revealed over 10 candidate tolerance genes. One such mutant contained an insertion in the known gene psaA, which is part of the psa locus. This locus encodes an ABC-type Mn permease complex. Sequence analysis of adjacent DNA extended the known genetic organization of the locus to include two new open reading frames (ORFs), psaB, which encodes an ATP-binding protein, and psaC, which encodes a hydrophobic transmembrane protein. Mutagenesis of psaB, psaC, psaA and downstream psaD resulted in penicillin tolerance. Defective adhesion and reduced transformation efficiency, as reported previously for a psaA- mutant, were phenotypes shared by psaB-, psaC- and psaD- knockout mutants. Western blot analysis demonstrated that the set of mutants expressed RecA, but none of them showed translation of the autolysin gene, which is located downstream of recA. The addition of manganese (Mn) failed to correct the abnormal physiology. These results suggest that this ABC-type Mn permease complex has a pleiotropic effect on pneumococcal physiology including adherence and autolysis. These are the first genes suggested as being involved in triggering autolysin. The results raise the possibility that loss of function of PsaA, by vaccine-induced antibody for instance, may promote penicillin tolerance.
|
|
| 8. |
- Novak, R, et al.
(författare)
-
Signal transduction by a death signal peptide : uncovering the mechanism of bacterial killing by penicillin.
- 2000
-
Ingår i: Molecular Cell. - 1097-2765 .- 1097-4164. ; 5:1, s. 49-57
-
Tidskriftsartikel (refereegranskat)abstract
- The binding of bactericidal antibiotics like penicillins, cephalosporins, and glycopeptides to their bacterial targets stops bacterial growth but does not directly cause cell death. A second process arising from the bacteria itself is necessary to trigger endogenous suicidal enzymes that dissolve the cell wall during autolysis. The signal and the trigger pathway for this event are completely unknown. Using S. pneumoniae as a model, we demonstrate that signal transduction via the two-component system VncR/S triggers multiple death pathways. We show that the signal sensed by VncR/S is a secreted peptide, Pep27, that initiates the cell death program. These data depict a novel model for the control of bacterial cell death.
|
|
| 9. |
|
|
| 10. |
|
|
