title: Differences in virulence of pneumolysin and autolysin mutants constructed by insertion duplication mutagenesis and in-frame deletion in Streptococcus pneumoniae.
abstract: BACKGROUND
  Insertion duplication mutagenesis (IDM) and in-frame deletion (IFD) are common techniques for studying gene function, and have been applied to pneumolysin (ply), a virulence gene in Streptococcus pneumoniae (D39). Discrepancies in virulence between the two techniques were observed in both the previous and present studies. This phenomenon was also observed during mutation analysis of autolysin (lytA).
  
  
  RESULTS
  Our data showed that target gene restoration (TGR) occurred in IDM mutants, even in the presence of antibiotics, while the IFD mutants were stable. In PCR result, TGR occurred later in IDM-ply and -lytA mutants cultured in non-supplemented medium (4-5 h) compared with those grown in medium supplemented with erythromycin (erm)/chloramphenicol (cat) (3-4 h), but plateaued faster. Real-time PCR for detecting TGR had been performed. When compared with 8-h culture, TGR detection increased from Day 1 and Day 2 of IDM mutant's culture. erm-sensitive clones from IDM mutant were found. Southern blot hybridization and Western blotting also confirmed the phenomenon of TGR. The median survival of mice following intraperitoneal (IP) injection with a 3-h culture of IDM-mutants was significantly longer than that with an 8-h culture, irrespective of antibiotic usage. The median survival time of mice following IP injection of a 3-h culture versus an 8-h culture of IDM-ply in the absence of antibiotics was 10 days versus 2 days (p = 0.031), respectively, while in the presence of erm, the median survival was 5 days versus 2.5 days (p = 0.037), respectively. For an IDM-lytA mutant, the corresponding values were 8.5 days versus 2 days (p = 0.019), respectively, for non-supplemented medium, and 2.5 versus 2 days (p = 0.021), respectively, in the presence of cat. A comparable survival rate was observed between WT D39 and an 8-h IDM culture.
  
  
  CONCLUSIONS
  TGR in IDM mutants should be monitored to avoid inconsistent results, and misinterpretation of data due to TGR could lead to important biological meaning being overlooked. Therefore, based on these results, IFD is preferable to IDM for disruption of target genes.
authors.forename: Esther Yip-Mei
authors.initials: EY
authors.lastname: Liu
authors.forename: Feng-Yee
authors.initials: FY
authors.lastname: Chang
authors.forename: Jen-Chang
authors.initials: JC
authors.lastname: Chang
authors.affiliation: Institute of Clinical Medicine, School of Medicine, National Yang-Ming University, Taipei 11217, Taiwan. cpfung@vghtpe.gov.tw.
authors.forename: Chang-Phone
authors.initials: CP
authors.lastname: Fung
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D004252:DNA Mutational Analysis; D018390:Gene Targeting / Q000379:methods*; D051379:Mice; D008807:Mice, Inbred BALB C; D016254:Mutagenesis, Insertional; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D017384:Sequence Deletion; D013296:Streptococcus pneumoniae / Q000235:genetics / Q000472:pathogenicity*; D013301:Streptolysins / Q000235:genetics*; D014774:Virulence / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D013301:Streptolysins; C032290:plY protein, Streptococcus pneumoniae; D009238:N-Acetylmuramoyl-L-alanine Amidase