Abstract
Salmonella enterica serotype Typhimurium is a bacterium that causes gastroenteritis and diarrhea in humans. The genome of S. Typhimurium codes for diverse virulence factors, among which are the toxin-antitoxin (TA) systems. SehAB is a type II TA, where SehA is the toxin and SehB is the antitoxin. It was previously reported that the absence of the SehB antitoxin affects the growth of S. Typhimurium. In addition, the SehB antitoxin can interact directly with the SehA toxin neutralizing its toxic effect as well as repressing its own expression. We identified conserved residues on SehB homologous proteins. Point mutations were introduced at both N- and C-terminal of SehB antitoxin to analyze the effect of these changes on its transcription repressor function, on its ability to form homodimers and on the virulence of S. Typhimurium. All changes in amino acid residues at both the N- and C-terminal affected the repressor function of SehB antitoxin and they were required for DNA-binding activity. Mutations in the amino acid residues at the N-terminal showed a lower capacity for homodimer formation of the SehB protein. However, none of the SehB point mutants were affected in the interaction with the SehA toxin. In terms of virulence, the eight single-amino acid mutations were attenuated for virulence in the mouse model. In agreement with our results, the eight amino acid residues of SehB antitoxin were required for its repressor activity, affecting both homodimerization and DNA-binding activity, supporting the notion that both activities of SehB antitoxin are required to confer virulence to Salmonella enterica.
Highlights
Salmonella enterica comprises Gram-negative bacteria with around 2500 serotypes responsible for severe gastroenteritis and systemic infections in warm-blooded animals, including humans (Haraga et al, 2008; Jajere, 2019)
Uncharacterized SehB homologs are present in serotypes of Salmonella enterica (Heidelberg, Paratyphi, Typhi, and Gallinarum), as well as in in other bacteria species such as Serratia marcescens, Xenorhabdus nematophila, Klebsiella pneumoniae, Pectobacterium carotovora, E. coli, S. flexneri, and Enterobacter cloacae (Figure 1A)
By alignment of the amino acid sequence of this protein with other homologous SehB antitoxins, conserved amino acids were identified throughout the entire protein (Figure 1A)
Summary
Salmonella enterica comprises Gram-negative bacteria with around 2500 serotypes responsible for severe gastroenteritis and systemic infections in warm-blooded animals, including humans (Haraga et al, 2008; Jajere, 2019). S. enterica has evolved to adapt to both extracellular and intracellular conditions, expressing a myriad of virulence factors, such as secretion systems, adhesins, flagella, and toxins, among others (Fabrega and Vila, 2013). In this way, toxin-antitoxin systems have emerged as important elements that affect the pathogenicity of many bacteria, regulating their physiology, persistence and virulence (Lobato-Marquez et al, 2016; Harms et al, 2018; Ronneau and Helaine, 2019). In the type II TA systems, both toxin and antitoxin are proteins and are transcriptionally organized in a bicistronic operon, where mostly the antitoxin gene is upstream of the toxin gene (Yamaguchi and Inouye, 2009)
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