Search

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.

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.

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.

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.

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.

The Stilbonemutinue (marine free-living nematodes) arc remarkable for cctosymbiotic bacteria, which cover the greatest part of their body in a highly ordered and species specific pattern. Using SEM we describe the main types of symbiotic cover and give evidence for the role of the bacteria in the nutrition of their host on the basis of stable carbon isotope ratios. In experimental systems the worms migrated repeatedly across a sulfide gradient during 12 h when sulfide concentrations were low, but stayed above the sulfide maximum at high concentrations. The migration across the chemocline exposes the symbionts alternately to reduced sulfur compounds and oxygen; this constitutes an alternative strategy to the ventilation/circulation systems in symbiotic macrofauna from sulfidic habitats