Inhibition of Chlamydial Infection by CRISPR/Cas9-SAM Mediated Enhancement of Human Peptidoglycan Recognition Proteins G
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Inhibition of Chlamydial Infection by CRISPR/Cas9SAM Mediated Enhancement of Human Peptidoglycan Recognition Proteins Gene Expression in HeLa Cells P. A. Bobrovsky1,a*, V. D. Moroz1, V. N. Lavrenova1,2, V. A. Manuvera1, and V. N. Lazarev1 1
Federal Research and Clinical Center of PhysicalChemical Medicine of Federal Medical Biological Agency, 119435 Moscow, Russia 2 Faculty of Biology, Lomonosov Moscow State University, 119991 Moscow, Russia a email: [email protected] Received July 7, 2020 Revised August 22, 2020 Accepted August 23, 2020
Abstract—The global problem of emerging resistance of microorganisms to antibiotics makes the search for new natural sub stances with antibacterial properties relevant. Such substances include peptidoglycan recognition proteins (PGLYRP), which are the components of the innate immunity of many organisms, including humans. These proteins have a unique mechanism of action that allows them to evade the resistance of bacteria to them, as well as to be active against both Gram positive and Gramnegative bacteria. However, the use of antimicrobial recombinant proteins is not always advisable due to the complexity of local delivery of the proteins and their stability; in this regard it seems appropriate to activate the compo nents of the innate immunity. The aim of this study was to increase the expression level of native peptidoglycan recognition protein genes in HeLa cells using genomeediting technology with synergistic activation mediators (CRISPR/Cas9SAM) and evaluate antichlamydial effect of PGLYRP. We demonstrated activation of the chlamydial twocomponent gene system (ctcBctcC), which played a key role in the mechanism of action of the peptidoglycan recognition proteins. We generated the HeLa cell line transduced with lentiviruses encoding CRISPR/Cas9SAM activation system with increased PGLYRP gene expression. It was shown that activation of the own peptidoglycan recognition proteins gene expression in the cell line caused inhibition of the chlamydial infection development. The proposed approach makes it possible to use the capabilities of innate immunity to combat infectious diseases caused by Grampositive and Gramnegative bacteria. DOI: 10.1134/S0006297920110036 Keywords: peptidoglycan, Chlamydia trachomatis, PGLYRP, CRISPR/Cas9
INTRODUCTION At present the increasing number of antibiotic resistant strains represents a global problem. Emergence of the resistant strains results in the decreased efficiency of the therapeutic treatments of the socially significant infectious diseases. Diseases caused by the resistant strains are more difficult to treat and they are often more severe [1]. Serious side effects of some antibiotics have Abbreviations: PGLYRP, peptidoglycan recognition proteins; DMEM, Dulbecco’s modified Eagle’s medium; SAM, syner gistic activation mediator; HSF1, heat shock factor 1; p65, sub unit of NFκB transcription factor; CRISPR, clustered regu larly interspaced short palindromic repeats; dCas9, nuclease dead Cas9, PEI, polyethyleneimine. * To
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