Arch Clin Infect Dis. 2020 June; 15(3):e98625. Published online 2020 September 20. doi: 10.5812/archcid.98625. Research Article Evaluation of Putative Type II Toxin-Antitoxin Systems and Lon Protease Expression in Shigella flexneri Following Infection of Caco-2 Cells Erfan Kheradmand 1 , Shabnam Razavi 2, 3, * , Malihe Talebi 2, 3 and Mahmood Jamshidian 4 1 Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran 2 Department of Microbiology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran 3 Microbial Biotechnology Research Center, Iran University of Medical Sciences, Tehran, Iran 4 Department of Pathobiology, Science and Research Branch, Islamic Azad University, Tehran, Iran * Corresponding author: Microbial Biotechnology Research Center, Iran University of Medical Sciences, Tehran, Iran. Email: razavi.sh@iums.ac.ir Received 2019 October 07; Revised 2020 August 19; Accepted 2020 August 22. Abstract Shigella flexneri causes bacillary dysentery in developing countries. Due to recent reports regarding antimicrobial resistance in hu- man S. flexneri, finding alternative therapeutics is of vital importance. Toxin-antitoxin (TA) systems have recently been introduced as antimicrobial targets owing to their involvement in bacterial survival in stress conditions and “persister” cell formation. In this study, the presence of four TA loci were studied in S. flexneri ATCC 12022. The presence of genes coding for the identified TA loci and Lon protease were confirmed by the PCR method using specific primers. Caco-2 cell lines were then infected with this standard strain, and 8 and 24 h post-infection, expression levels of genes coding for the studied TA loci, and Lon protease were evaluated us- ing a real-time PCR method. Expression of mazF, GNAT (Gcn5-related N-acetyltransferase), yeeU, pfam13975, and Lon genes showed 5.4, 9.8, 2.3, 2.7, and 13.8-fold increase, respectively, 8 h after bacterial invasion of the Caco-2 cell line. In addition, the expression of the aforementioned genes showed 4.8, 10.8, 2.3, 3.7, and 16.8-fold increase after 24 h. The GNAT and lon genes showed significantly higher expression levels compared to the control (P value < 0.05). However, the increase in the expression level of yeeU was the same at 8 h and 24 h post-infection. In addition, mazF expression level showed a slight decrease at 24 h compared to 8h post-infection. Genes coding for GNAT and Lon protease showed a significantly higher expression after invading the Caco-2 cell line. Therefore, target- ing GNAT or Lon protease can be taken into consideration for finding novel antimicrobial drug strategies. The exact functions and mechanisms of TA systems in S. flexneri isolates are suggested to be experimentally determined. Keywords: Shigella flexneri, Toxin-Antitoxin Systems, Lon Protease, Novel Antimicrobial Therapy 1. Background Similar to eukaryotic apoptosis, bacterial cell death can occur due to the overexpression of some bacterial genes, including toxin-antitoxin (TA) systems or addic- tion modules whose activity can also inhibit bacterial cell growth through affecting cellular processes. These in- clude DNA replication, macromolecular synthesis, cell wall synthesis, membrane disruption, phage infection, and cy- toskeletal polymerization (1, 2). Toxin-antitoxin systems are purportedly predisposed to lateral gene transfer and intragenic recombination. They were initially discovered in genes carried by plas- mids, and were subsequently detected in chromosomal genomes where they are involved in the process of stabiliz- ing bacterial chromosomes and conjugative transposons (3, 4). A stable protein toxin and a labile protein or RNA an- titoxin, which are generally encoded on a single operon, constitute TA modules. Based on the basic characteristics and activity of the antitoxins, TA systems are categorized into six types. Among these, type II, IV, V, and VI TA systems have protein antitoxins; whereas, types I and III have sRNA antitoxins (5). Several functions have been attributed to TA systems. It has been corroborated that TA modules enable bacteria to evade antibiotic effects on account of inducing a dormant phase and “persister” cell formation followed by the over- expression of numerous toxins (6, 7). In fact, in type II TA systems, labile protein antitoxins can be degraded by Lon and Clp proteases after bacterial exposure to environmen- tal stress, enabling toxins to inhibit bacterial growth by ei- Copyright © 2020, Author(s). This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (http://creativecommons.org/licenses/by-nc/4.0/) which permits copy and redistribute the material just in noncommercial usages, provided the original work is properly cited.