Arginine 58 is indispensable for proper function of the Francisella tularensis subsp. holarctica FSC200 HU protein, and its substitution alters virulence and mediates immunity against wild-type strain

Abstract

HU protein, a member of the nucleoid-associated group of proteins, is an important transcription factor in bacteria, including in the dangerous human pathogen Francisella tularensis . Generally, HU protein acts as a DNA sequence non-specific binding protein and it is responsible for winding of the DNA chain that leads to the separation of transcription units. Here, we identified potential HU protein binding sites using the ChIP-seq method and two possible binding motifs in F. tularensis subsp. holarctica FSC200 depending upon growth conditions. We also confirmed that FSC200 HU protein is able to introduce negative supercoiling of DNA in the presence of topoisomerase I. Next, we showed interaction of the HU protein with a DNA region upstream of the pigR gene and inside the clpB gene, suggesting possible regulation of PigR and ClpB expression. Moreover, we showed that arginine 58 and partially arginine 61 are important for HU protein’s DNA binding capacity, negative supercoiling induction by HU, and the length and winding of FSC200 chromosomal DNA. Finally, in order to verify biological function of the HU protein, we demonstrated that mutations in arginine 58, arginine 61, and serine 74 of the HU protein decrease virulence of FSC200 both in vitro and in vivo and that immunization using these mutant strains is able to protect as many as 100% of mice against wild-type challenge. Taken together, our findings deepen knowledge about the role of the HU protein in tularaemia pathogenesis and suggest that HU protein should be addressed in the context of tularaemia vaccine development.

Bacteriophage SPO1 protein Gp46 suppresses functions of HU protein in Francisella tularensis

“…The bacterial strains and plasmids used in this study are summarized in Supplementary Table S1 . The bacterial strains were cultured as previously described ( Stojkova et al, 2018 ; Pavlik and Spidlova, 2022 ). McLeod agar plate enriched for bovine hemoglobin (BD Diagnostics, 212392) and IsoVitalex (BD Diagnostics, 211876) or Chamberlain medium ( Chamberlain, 1965 ) with shaking 200 rpm at 37°C were used for Francisella cultivation.…”

“…holarctica FSC200 strain HU protein (FTS_0886, AFT92728, CP003862.1) structure was predicted as previously described ( Pavlik and Spidlova, 2022 ). The predicted structure of F. tularensis HU, experimental crystal structure of Gp46 (Bacteriophage SPO1 protein Gp46; RCSB:7BY7) ( Zhang et al, 2022 ), and DNA 16 bp long corresponding to the DNA-binding motif ( Pavlik and Spidlova, 2022 ) of F. tularensis HU found inside the pigR gene were used in molecular docking studies of protein–protein or protein–DNA complexes using the HDOCK server ( Yan et al, 2020 ) with default parameters. The best matching models were visualized using the BIOVIA Discovery Studio Visualizer (Dassault Systèmes BIOVIA, San Diego, CA, USA) and used in further simulation analyses.…”

“…F. tularensis subsp. holarctica FSC200 carrying the HU protein labeled with an HA tag (HU_HA) ( Pavlik and Spidlova, 2022 ) was cultured overnight in Chamberlain’s medium supplemented with kanamycin (20 μg/mL). The pellet was resuspended in Tris-buffered saline (TBS) and lysed using a French press (16,000 psi, three times).…”

“…Several transcription factors such as MglA/SspA ( Lauriano et al, 2004 ; Brotcke et al, 2006 ; Charity et al, 2007 ), PigR ( Brotcke et al, 2006 ; Charity et al, 2007 ; Ramsey et al, 2015 ), and PmrA ( Mohapatra et al, 2007 ; Bell et al, 2010 ; Ramsey and Dove, 2016 ) have been implicated in the regulation of virulence and stress response genes, and various models have been proposed to explain FPI genes expression ( Charity et al, 2007 ; Ramsey and Dove, 2016 ; Cuthbert et al, 2017 ). Our recent research demonstrated that the F. tularensis HU protein is an additional key regulator that should not be overlooked ( Stojkova et al, 2018 ; Pavlik and Spidlova, 2022 ).…”

“…Our latest study showed that arginines 58 and 61 are necessary for the HU protein DNA-binding capacity in F. tularensis . Moreover, we identified a potential DNA-binding motif in the HU protein, suggesting that it can bind DNA in a novel sequence-dependent manner ( Pavlik and Spidlova, 2022 ).…”