A peer-reviewed Formal Comment by Prof. Backert and colleagues discussing results relating to this work has been published as Tegtmeyer N, Lind J, Schmid B, Backert S (2014) Helicobacter pylori CagL Y58/E59 Mutation Turns-Off Type IV Secretion-Dependent Delivery of CagA into Host Cells. PLOS ONE 9(6): e97782. doi:10.1371/journal.pone.0097782
Following discussion among Associate Editor Matt Hodgkinson, the authors, and the handling editor Jun Sun, and in response to the issues raised in the Formal Comment, we are posting a correction to address: 1) confirmation of the strain mutations by PCR, sequencing, and RT-PCR; 2) clarification of the availability of the strains.
The authors have provided DNA sequencing and RT-PCR data in supporting information files:
Figure S1 shows a diagram of the construction of the cagL mutant (A), revertant, and amino acid replacement mutants (B).
Figure S2 confirms the cagL mutant, revertant, and amino acid replacement mutants using PCR.
Figure S3 shows sequencing results of H. pylori clinical strain Hp1033 wild type, cagL insertion mutant, revertant, and amino acid replacement mutants.
Figure S4 shows RT-PCR of cagL expression for Hp1033 wild type, cagL mutant and replacement mutants co-cultured with AGS cells at pH 7.4 for 1 hour.
Table S1 shows the primers used for sequencing and RT-PCR.
The authors have no Anti-CagL antibody, and so they are not able to show protein expression.
Sharing of the strains/isolates of H. pylori reported in the manuscript may be done following a formal request. This application is necessary to comply with the regulatory processes in the authors' institution and the customs/export regulations between Taiwan and other countries.
The diagram of construction of cagL mutant (A), revertant, and amino acid replacement mutants (B).
Confirming cagL mutant, revertant, and amino acid replacement mutants by using PCR. PCR Amplicons from wild type, revertants, and amino acid replacement mutants are 1.1kb (using primer cagL-5 & cagL-6) or 1.4kb (using primer cagIL-1 & cagL-6), from cagL insertion mutants are 2kb. (A) M: marker; w: Hp1033 wild type; lane1-16: Hp1033 cagL::cat. (B) lane1-12: 26695 cagL::cat; lane 13-23: J99 cagL::cat; lane 24: Hp1033 cagL-Y58/E59 revertant. (C) lane 25-29: Hp1033 cagL-Y58D/E59 amino acid replacement mutants; lane 30: Hp1033 cagL::cat ; lane 31: Hp1035 cagL::cat. (D) lane 32-35: Hp1033 cagL-Y58/E59K amino acid replacement mutants. (E) lane 36-39: Hp1033 cagL-Y58D/E59K amino acid replacement mutants ; lane 40: Hp1035 cagL-Y58D/E59K amino acid replacement mutants. Arrows indicate the clones selected in this study.
The sequencing results of H. pylori clinical strain Hp1033 wild type, cagL insertion mutant, revertant, and amino acid replacement mutants. Multiple Alignments were processed by MAFFT L-INS-1 (v6.850b) from EMBL-EBI website. The blue blocks indicate the region of chloramphenicol resistance gene. The yellow block indicates the position of amino acid 58 and 59 residues.
After Hp1033 wild type, cagL mutant and replacement mutants co-cultured with AGS cells at pH 7.4 for 1 hour, cagL expression was examined by RT-PCR. There was no difference in CagL expressions among Hp1033 wild type, revertant, and amino acid replacement mutants. The 3rd lane indicated there was no RNA contamination.
Citation: Yeh Y-C, Cheng H-C, Yang H-B, Chang W-L, Sheu B-S (2014) Correction: H. pylori CagL-Y58/E59 Prime Higher Integrin α5β1 in Adverse pH Condition to Enhance Hypochlorhydria Vicious Cycle for Gastric Carcinogenesis. PLoS ONE 9(6): e101912. https://doi.org/10.1371/journal.pone.0101912
Published: June 27, 2014
Copyright: © 2014 Matthew R. Mason, Haikady N. Nagaraja, Terry Camerlengo, Vinayak Joshi, Purnima S. Kumar. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.