Peptidoglycan hydrolase of an unusual cross-link cleavage specificity contributes to bacterial cell wall synthesis
Authors: Manjula Reddy
Pavan Kumar Chodisetti
Journal: Proceedings of the National Academy of Sciences of the United States of America (2019)
Discipline: Multidisciplinary
Keywords: MepK; YcbK; bacteria; mDAP cross-link; peptidoglycan.
Affiliations: Centre for Cellular and Molecular Biology, Council of Scientific and Industrial Research, Hyderabad, India 500007., Centre for Cellular and Molecular Biology, Council of Scientific and Industrial Research, Hyderabad, India 500007 .

Abstract:  Bacteria are surrounded by a protective exoskeleton, peptidoglycan (PG), a cross-linked mesh-like macromolecule consisting of glycan strands interlinked by short peptides. Because PG completely encases the cytoplasmic membrane, cleavage of peptide cross-links is a prerequisite to make space for incorporation of nascent glycan strands for its successful expansion during cell growth. In most bacteria, the peptides consist of l-alanine, d-glutamate, meso-diaminopimelic acid (mDAP) and d-alanine (d-Ala) with cross-links occurring either between d-Ala and mDAP or two mDAP residues. In Escherichia coli, the d-Ala-mDAP cross-links whose cleavage by specialized endopeptidases is crucial for expansion of PG predominate. However, a small proportion of mDAP-mDAP cross-links also exist, yet their role in the context of PG expansion or the hydrolase(s) capable of catalyzing their cleavage is not known. Here, we identified an ORF of unknown function, YcbK (renamed MepK), as an mDAP-mDAP cross-link cleaving endopeptidase working in conjunction with other elongation-specific endopeptidases to make space for efficient incorporation of nascent PG strands into the sacculus. E. coli mutants lacking mepK and another d-Ala-mDAP-specific endopeptidase (mepS) were synthetic sick, and the defects were abrogated by lack of l,d-transpeptidases, enzymes catalyzing the formation of mDAP cross-links. Purified MepK was able to cleave the mDAP cross-links of soluble muropeptides and of intact PG sacculi. Overall, this study describes a PG hydrolytic enzyme with a hitherto unknown substrate specificity that contributes to expansion of the PG sacculus, emphasizing the fundamental importance of cross-link cleavage in bacterial peptidoglycan synthesis. 

Retracted: No

PubPeer comments:
 This paper is part of a set of 6 papers. According to the authors, all papers are related to the same protocol approved under the **number 0020 2015 SG CNE** by the National Ethics Committee of Gabon. One of the authors is mentioned in the 6 papers. Other authors are mentioned in several papers. This protocol seems to cover different objectives, different types of participants, different type of study designs. According to the protocol approval number (0020 2015 SG CNE) and its format, the protocol has been approved in 2015 by the National Ethics Committee of Gabon. But what does this protocol cover ? **Different therapeutic areas, Different objectives:** -t**Article 1**: Malaria - Aim: to investigate the causes of febrile illnesses among children -t**Article 2**: Malaria - Aim: to investigate the prevalence of mutations in Pfdhfr and Pfdhps associated with sulfadoxine-pyrimethamine resistance. -t**Article 3**: Diarrhea - Aim: To identify and characterize bacterial and parasitic pathogens responsible for childhood diarrhea, and to identify environmental risk factors associated with diarrhea in children under 5 years old -t**Article 4**: Diarrhea - Aim: to assess the prevalence of eighteen IPIs in children with diarrhea -t**Article 5**: Malaria - Aim: adhesion to treatment with Sulfadoxine-Pyrimethamine -t**Article 6**: Head lice - Aim: to investigate the phylogeny of head lice collected in Gabon and their associated bacteria **Different types of samples Different types of participant Different dates of participantsu2019 recruitment** -t**Article 1**: Blood samples Children November 26, 2015 to January 25, 2016 -t**Article 2**: Blood samples Adults and children February 02, 2016 to May 31, 2016, -t**Article 3**: Stools samples Children under 5 years old May 2016 to February 2018 -t**Article 4**: Stools samples Children November 2016 to August 2017 -t**Article 5**: Blood samples Pregnant women February 2018 to May 2018 -t**Article 6**: Head lice Women only March 26th, 2018 to April 11th, 2018 Can the authors explain how a unique protocol cover 6 different types of study design with 6 different objectives and 6 different types of recruitment? -tStudy designs are very different. -tRecruitment periods are different. -tTypes of participants are different -tTypes of sample are different, etcu2026 It seems pretty unusual that a same protocol cover -tblood samples in children to study malaria in 2015 and stool samples one year after, -tthen, head lice in women, 3 years after. Can the authors explain the scope of the protocol 0020 2015 SG CNE and the approval process by the National Ethics Committee of Gabon? How a protocol can cover so many different type of study design? **Article 1: [High Circulation of Malaria and Low Prevalence of Bacteremia in Febrile and Afebrile Children in Northeastern Gabon] ** According to the authors, this study is a retrospective study in children. The aim of the study is to investigate the causes of febrile illnesses among children. Blood sample has been collected between November 26, 2015 and January 25, 2016. These dates raises a serious concern regarding the nature of the study. Indeed, a retrospective study is defined as a study which analyses data already collected. The Ethics approval have been obtained in 2015. However data was collected until 2016. As a consequence, the study collected prospectively the data after the ethical approval. This study should be considered as a prospective study. The authors donu2019t mention the date of the ethical approval. This information should be useful to determine if the ethical approval was obtained before the recruitment of participant or after. It will be in a public interest to disclose the approved protocol by the National Ethics Committee of Gabon in order to understand how a study which have been approved before the end of the data collection has been considered as retrospective by the authors. **Article 2: [High prevalence of genotypes associated with sulfadoxine-pyrimethamine resistance in the rural area of Fougamou, Gabon] ** 204 febrile patients were included, with a mean age of 167.75u00b1138.83 months. Blood samples have been collected. The ethical approval has been obtained in 2015 and the study performed from 2016. Data are collected after the ethical approval. This study is a prospective study. The objective of this study is to investigate the prevalence of mutations in Pfdhfr and Pfdhps associated with sulfadoxine-pyrimethamine resistance in Gabonese rural areas. **Article 3: [Etiology and biodistribution of enterobacteria and parasites, and their associated environmental risk factors among children under 5 years old with diarrhea in East-Central Gabon] ** 132 children under 5 years old were enrolled. Stools samples have been collected. The ethical approval has been obtained in 2015 and the study has been performed in 2016. Data are collected after the ethical approval. This study is a prospective study. The objective of this study is to identify and characterize bacterial and parasitic pathogens responsible for childhood diarrhea, and to identify environmental risk factors associated with diarrhea in children under 5 years old living in Koula-Moutou, East-Central Gabon **Article 4: [Molecular prevalence of intestinal parasites infections in children with diarrhea in Franceville, Southeast of Gabon] ** 100 diarrheic children between 0 and 180 months of age have been recruited from November 2016 to August 2017. Stools samples have been collected. Data are collected after the ethical approval. This study is a prospective study. The objective of this study is to assess the prevalence of eighteen IPIs in children with diarrhea. **Article 5: [Evaluation of the adhesion of pregnant women to intermittent presumptive treatment with Sulfadoxine-Pyrimethamine in a rural area of Gabon] ** 607 pregnant women before their delivery have been recruited from February to May 2018. The aim of the study is the adhesion to treatment with Sulfadoxine-Pyrimethamine. The authors mention that recruitment is prospective. Blood samples and questionnaires have been collected. **Article 6: [Evaluation of the adhesion of pregnant women to intermittent presumptive treatment with Sulfadoxine-Pyrimethamine in a rural area of Gabon] ** From 26 March to 11 April 2018, 691 head lice were collected from 86 women in Franceville, Gabon. Data are collected after the ethical approval. This study is a prospective study. The aim of the study is investigate the phylogeny of head lice collected in Gabon and their associated bacteria. ,