Dissociation between the critical role of ClpB of Francisella tularensis for the heat shock response and the DnaK interaction and its important role for efficient type VI secretion and bacterial virulence

Abstract

Francisella tularensis, a highly infectious, intracellular bacterium possesses an atypical type VI secretion system (T6SS), which is essential for its virulence. The chaperone ClpB, a member of the Hsp100/Clp family, is involved in Francisella T6SS disassembly and type VI secretion (T6S) is impaired in its absence. We asked if the role of ClpB for T6S was related to its prototypical role for the disaggregation activity. The latter is dependent on its interaction with the DnaK/Hsp70 chaperone system. Key residues of the ClpB-DnaK interaction were identified by molecular dynamic simulation and verified by targeted mutagenesis. Using such targeted mutants, it was found that the F. novicida ClpB-DnaK interaction was dispensable for T6S, intracellular replication, and virulence in a mouse model, although essential for handling of heat shock. Moreover, by mutagenesis of key amino acids of the Walker A, Walker B, and Arginine finger motifs of each of the two Nucleotide-Binding Domains, their critical roles for heat shock, T6S, intracellular replication, and virulence were identified. In contrast, the N-terminus was dispensable for heat shock, but required for T6S, intracellular replication, and virulence. Complementation of the ΔclpB mutant with a chimeric F. novicida ClpB expressing the N-terminal of Escherichia coli, led to reconstitution of the wild-type phenotype. Collectively, the data demonstrate that the ClpB-DnaK interaction does not contribute to T6S, whereas the N-terminal and NBD domains displayed critical roles for T6S and virulence.

The Role of ClpB in Bacterial Stress Responses and Virulence

“…E. coli, F. tularensis , Helicobacter pylori, Pseudomonas putida, Campylobacter coli , and Campylobacter lari are some of the pathogenic bacteria for which the role of ClpB in thermotolerance has been studied ( Squires et al, 1991 ; Allan et al, 1998 ; Meibom et al, 2008 ; Ito et al, 2014 ; Alam et al, 2018 , 2020 ; Riedel et al, 2020 ). F. tularensis , a highly infectious pathogen and a category A bioterrorism agent, is the etiological agent of the zoonotic disease tularemia.…”

“…F. tularensis , a highly infectious pathogen and a category A bioterrorism agent, is the etiological agent of the zoonotic disease tularemia. Deletion of the clpB gene causes a severe defect in survival at elevated temperature ( Meibom et al, 2008 ; Alam et al, 2018 , 2020 ). A similar effect was observed for a clpB mutant of H. pylori , the causative agent of gastric ulcers ( Allan et al, 1998 ).…”

“…Besides promoting stress tolerance, ClpB plays an important role in invasiveness and/or host survival of multiple important bacterial pathogens ( Table 1 ), such as Leptospira interrogans, Yersinia enterocolitica, Francisella noatunensis, F. tularensis, Piscirickettsia salmonis, Mycoplasma pneumoniae, Salmonella typhimurium, Mycobacterium tuberculosis, Porphyromonas gingivalis, Enterococcus faecalis, Listeria monocytogenes , and Staphylococcus aureus ( Badger et al, 2000 ; Chastanet et al, 2004 ; Frees et al, 2004 ; Yuan et al, 2007 ; Capestany et al, 2008 ; Kannan et al, 2008 ; Conlan, 2011 ; de Oliveira et al, 2011 ; Lourdault et al, 2011 ; Alam et al, 2018 , 2020 ; Sangpuii et al, 2018 ; Harnagel et al, 2020 ; Kêdzierska-Mieszkowska and Arent, 2020 ; Tripathi et al, 2020 ). In the case of L. interrogans , the causative agent of the emerging zoonotic disease leptospirosis, a clpB mutant not only showed enhanced susceptibility to high temperature, nutrient-depletion, and oxidative stress, but was also attenuated in a gerbil animal model of acute leptospirosis ( Lourdault et al, 2011 ; Kêdzierska-Mieszkowska and Arent, 2020 ).…”

“…In the absence of ClpB, the resulting mutant showed attenuation in a zebrafish model and also provided efficient protection in zebrafish challenged with wild-type bacteria ( Lampe et al, 2017 ). Moreover, clpB mutants of F. tularensis subspecies holarctica and tularensis were found to be defective for T6S, susceptible to elevated temperature, and completely attenuated in mice ( Alam et al, 2018 , 2020 ). Such mutants also serve as highly efficacious vaccines in animal models of tularemia ( Conlan, 2011 ; Alam et al, 2018 ).…”

Control of Francisella tularensis Virulence at Gene Level: Network of Transcription Factors

“…Most of the RNA-polymerase-regulated genes are required for intracellular growth and full virulence of F. novicida [ 124 ]. Probably the most important of them is ClpB [ 125 ] that directly interacts with DnaK, which is under RpoH control, [ 59 ] and a strain defective for the clpB gene is an auspicious candidate as a live vaccine strain [ 126 , 127 ].…”