Abstract
Francisella tularensis is one of the most virulent pathogenic bacteria causing the acute human respiratory disease tularemia. While the mechanisms underlying F. tularensis pathogenesis are largely unknown, previous studies have shown that a F. novicida transposon mutant with insertions in a gene coding for a putative lysine decarboxylase was attenuated in mouse spleen, suggesting a possible role of its protein product as a virulence factor. Therefore, we set out to structurally and functionally characterize the F. novicida lysine decarboxylase, which we termed LdcF. Here, we investigate the genetic environment of ldcF as well as its evolutionary relationships with other basic AAT-fold amino acid decarboxylase superfamily members, known as key actors in bacterial adaptative stress response and polyamine biosynthesis. We determine the crystal structure of LdcF and compare it with the most thoroughly studied lysine decarboxylase, E. coli LdcI. We analyze the influence of ldcF deletion on bacterial growth under different stress conditions in dedicated growth media, as well as in infected macrophages, and demonstrate its involvement in oxidative stress resistance. Finally, our mass spectrometry-based quantitative proteomic analysis enables identification of 80 proteins with expression levels significantly affected by ldcF deletion, including several DNA repair proteins potentially involved in the diminished capacity of the F. novicida mutant to deal with oxidative stress. Taken together, we uncover an important role of LdcF in F. novicida survival in host cells through participation in oxidative stress response, thereby singling out this previously uncharacterized protein as a potential drug target.
Highlights
Francisella tularensis is one of the most virulent pathogenic bacteria causing the acute human respiratory disease tularemia
The pathogenicity of F. tularensis mainly relies on the Francisella Pathogenicity Island (FPI)[4], a gene cluster encoding 17 proteins comprising a type VI-like secretion system machinery (T6SS)[5,6]
LdcF sequences found in Francisellaceae strains display a high level of sequence identity (83%), and contain four functional regions, corresponding to a wing domain (Pfam ID: PF03709), a pyridoxal 5′-phosphate (PLP)-binding domain and a AAT-like domain, and a C-terminal domain (Pfam ID: PF03711) (Supplementary Fig. S1)
Summary
Francisella tularensis is one of the most virulent pathogenic bacteria causing the acute human respiratory disease tularemia. Due to the ease of culture and the extremely high infectivity by airborne route, F. tularensis is considered as a dangerous bioweapon classified as a category A bioterrorism agent by the Centers for Disease Control and Prevention (CDC)[2] This bacterium, capable of surviving for weeks at low temperature in water, soil, grass or animal carcasses is one of the most infectious pathogens known because inhalation of as few as a dozen of organisms can suffice to cause illness and death. Considering the current knowledge on the other members of the superfamily of AAT-fold PLP-dependent basic amino acid decarboxylases and their recognized involvement in bacterial physiology, stress responses and virulence, we set out to investigate the structure and function of LdcF, using the F. novicida model as a practicable surrogate of F. tularensis for experimental s tudies[3]
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