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.
Highlights
The zoonotic disease tularemia is caused by the extremely virulent, facultative intracellular Gram-negative coccobacillus Francisella tularensis [1] and the subsp. tularensis and holarctica are important human pathogens
We demonstrate that the ΔclpB mutant of F. novicida is exquisitely susceptible to heat shock, shows defective intracellular growth and markedly decreased virulence in the mouse model, concomitantly with impaired T6S
Our findings reveal that the crucial roles of the F. novicida ClpB for heat shock and regulation of T6S are dependent on distinct regions of the protein and not dependent on the DnaK interaction
Summary
The zoonotic disease tularemia is caused by the extremely virulent, facultative intracellular Gram-negative coccobacillus Francisella tularensis [1] and the subsp. tularensis and holarctica are important human pathogens. The related species F. novicida is a very rare human pathogen, but still highly virulent for mice, and commonly used as a laboratory model for tularemia [2]. The pathogenicity of both Francisella species is linked to the Francisella Pathogenicity Island (FPI), a gene cluster encoding a functional, but atypical type VI secretion system (T6SS) [3, 4]. It has been demonstrated that the FPI of Francisella encodes a functional T6S system, despite that individual components demonstrate low sequence similarity to canonical T6SS proteins, and it lacks the two ATPases, IcmF/TssM and ClpV, both of which are believed to provide the energy for secretion in prototypical T6SS [5,6,7,8,9]. In the absence of ClpV, it is possible that other proteins, such as ClpB, due to its ATPase activity, may contribute to the assembly-disassembly cycle of the T6S apparatus
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