title: Global transcriptional analysis of clpP mutations of type 2 Streptococcus pneumoniae and their effects on physiology and virulence.
abstract: Streptococcus pneumoniae is an important human pathogen that contains single copies of genes encoding the ClpP and FtsH ATP-dependent proteases but lacks the Lon and HslV proteases. We constructed and characterized the phenotypes of clpP, clpC, and clpX deletion replacement mutants, which lack the ClpP protease subunit or the putative ClpC or ClpX ATPase specificity factor. A DeltaclpP mutant, but not a DeltaclpC or DeltaclpX mutant, of the virulent D39 type 2 strain of S. pneumoniae grew poorly at 30 degrees C and failed to grow at 40 degrees C. Despite this temperature sensitivity, transcription of the heat shock regulon determined by microarray analysis was induced in a DeltaclpP mutant, which was also more sensitive to oxidative stress by H2O2 and to puromycin than its clpP+ parent strain. A DeltaclpP mutant, but not a DeltaclpC mutant, was strongly attenuated for virulence in the murine lung and sepsis infection models. All of these phenotypes were complemented in a DeltaclpP/clpP+ merodiploid strain. Consistent with these complementation patterns, clpP was found to be in a monocistronic operon, whose transcription was induced about fivefold by heat shock in S. pneumoniae as determined by Northern and real-time reverse transcription-PCR analyses. Besides clpP, transcription of clpC, clpE, and clpL, but not clpX or ftsH, was induced by heat shock or entry into late exponential growth phase. Microarray analysis of DeltaclpP mutants showed a limited change in transcription pattern (approximately 80 genes) consistent with these phenotypes, including repression of genes involved in oxidative stress, metal ion transport, and virulence. In addition, transcription of the early and late competence regulon was induced in the DeltaclpP mutant, and competence gene expression and DNA uptake seemed to be constitutively induced throughout growth. Together, these results indicate that ClpP-mediated proteolysis plays a complex and central role in numerous pneumococcal stress responses, development of competence, and virulence.
authors.affiliation: Infectious Diseases Research, Lilly Research Laboratories, Indianapolis, Indiana 46285, USA.
authors.forename: Gregory T
authors.initials: GT
authors.lastname: Robertson
authors.forename: Wai-Leung
authors.initials: WL
authors.lastname: Ng
authors.forename: Joseph
authors.initials: J
authors.lastname: Foley
authors.forename: Raymond
authors.initials: R
authors.lastname: Gilmour
authors.forename: Malcolm E
authors.initials: ME
authors.lastname: Winkler
mesh_terms: D000074183:ATPases Associated with Diverse Cellular Activities; D000251:Adenosine Triphosphatases / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D021461:Animals, Outbred Strains; D001329:Autolysis; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D049071:Endopeptidase Clp; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D018869:Heat-Shock Response; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008297:Male; D051379:Mice; D018832:Molecular Chaperones; D008969:Molecular Sequence Data; D009154:Mutation; D020411:Oligonucleotide Array Sequence Analysis; D011008:Pneumococcal Infections / Q000188:drug therapy / Q000382:microbiology; D012697:Serine Endopeptidases / Q000235:genetics* / Q000378:metabolism; D013296:Streptococcus pneumoniae / Q000187:drug effects / Q000472:pathogenicity* / Q000502:physiology*; D014158:Transcription, Genetic; D014774:Virulence / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; C087481:ClpC protein, Bacteria; D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D006861:Hydrogen Peroxide; D012697:Serine Endopeptidases; D049071:Endopeptidase Clp; D000251:Adenosine Triphosphatases; C088072:ClpX protein, E coli; D000074183:ATPases Associated with Diverse Cellular Activities