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Open Access Methodology article

Differences in virulence of pneumolysin and autolysin mutants constructed by insertion duplication mutagenesis and in-frame deletion in Streptococcus pneumoniae

Esther Yip-Mei Liu1, Feng-Yee Chang2, Jen-Chang Chang3 and Chang-Phone Fung14*

Author Affiliations

1 Institute of Clinical Medicine, School of Medicine, National Yang-Ming University, Taipei 11217, Taiwan

2 Division of Infectious Diseases, Department of Internal Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei 11490, Taiwan

3 National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Zhunan, Miaoli County 35053, Taiwan

4 Division of Infectious Diseases, Department of Medicine, Taipei Veterans General Hospital and National Yang-Ming University, Taipei 11217, Taiwan

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BMC Biotechnology 2014, 14:16  doi:10.1186/1472-6750-14-16

Published: 21 February 2014

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.

Conclusion

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.

Keywords:
Insertion duplication mutagenesis; In-frame deletion; Pneumolysin; Autolysin; Target gene restoration