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.

KEYWORDS:

• Francisella
• HU protein
• PigR
• virulence
• histone-like protein
• ChIP-seq
• HU regulon
• bacterial pathogenesis
• transcription factor
• nucleoid-associated protein

Acknowledgement

Thanks to Dr Petr Baldrian and Dr Daniel Morais (Institute of Molecular Genetics, Czech Academy of Science in Prague, Czech Republic) for sequencing assistance and Dr Petr Vacha and Dr Stepan Stoces for ChIP data analysis (SEQme s.r.o., Dobris, Czech Republic). Then, the group Dr Jan Pribyl and Dr Radka Oborilova for AFM DNA analysis (CEITEC – Central European Institute of Technology, Brno, Czech Republic).

Disclosure statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Data availability statement

The data that support the findings of this study are openly available in this manuscript and supplementary files.

Ethics statement

All experiments using mice were performed following guidelines of the Animal Care and Use Ethical Committee of the Faculty of Military Health Sciences, University of Defence, Czech Republic. The research protocol was approved by the ethics committee under project no. 121890/2021–1457 (3 May 2021).

Supplementary information

Supplemental data for this article can be accessed online at https://doi.org/10.1080/21505594.2022.2132729

Additional information

Funding

This work was supported by the Ministry of Defence of the Czech Republic - Long-term organization development plan Medical Aspects of Weapons of Mass Destruction of the Faculty of Military Health Sciences, University of Defence (DZRO-ZHN-2017 and DZRO-FVZ-ZHN-II)