Pullulanase Is Necessary for the Efficient Intracellular Growth of Francisella tularensis

Abstract

Pullulanase, an enzyme that catalyzes the hydrolysis of polysaccharides, has been identified in a broad range of organisms, including bacteria, yeasts, fungi, and animals. The pullulanase (pulB; FTT_0412c) of F. tularensis subspecies tularensis Schu S4 is considered to be a homologue of the type I pullulanase (pulA) of the other Francisella subspecies. The significance of Francisella pullulanase has been obscure until now. In the present study, we characterized a recombinant PulB of F. tularensis SCHU P9, which was expressed as a his-tagged protein in Escherichia coli. The recombinant PulB was confirmed to be a type I pullulanase by its enzymatic activity in vitro. A pulB gene knockout mutant of F. tularensis SCHU P9 (ΔpulB) was constructed using the TargeTron Knockout system and plasmid pKEK1140 to clarify the function of PulB during the growth of F. tularensis in macrophages. The intracellular growth of the ΔpulB mutant in murine macrophage J774.1 cells was significantly reduced compared with that of the parental strain SCHU P9. Expression of PulB in ΔpulB, using an expression plasmid, resulted in the complementation of the reduced growth in macrophages, suggesting that PulB is necessary for the efficient growth of F. tularensis in macrophages. To assess the role of PulB in virulence, the knockout and parent bacterial strains were used to infect C57BL/6J mice. Histopathological analyses showed that tissues from ΔpulB-infected mice showed milder lesions compared to those from SCHU P9-infected mice. However, all mice infected with SCHU P9 and ΔpulB showed the similar levels of bacterial loads in their tissues. The results suggest that PulB plays a significant role in bacterial growth within murine macrophage but does not contribute to bacterial virulence in vivo.

Evaluation of Francisella tularensis ΔpdpC as a candidate live attenuated vaccine against respiratory challenge by a virulent SCHU P9 strain of Francisella tularensis in a C57BL/6J mouse model

“…Bacterial loads were quantified by inoculating 100 μL of homogenates of infected lungs, spleens and livers diluted in RPMI 1640 onto Eugon chocolate agar plates, as previously reported . After cultivation at 37°C for 4 days, CFUs were calculated by multiplying the average number of colonies by 10 to account for the dilution factor.…”

Comparative proteomic analysis reveals novel potential virulence factors of Aeromonas veronii

“…Pullulanase can catalyze the hydrolysis of polysaccharides, and it has been identified in a broad range of organisms, such as bacteria, yeasts, fungi, and animals. 42 However, there are few studies on its function in pathogenic bacteria. Akihiko et al showed that pullulanase is necessary for the efficient growth of Francisella tularensis in macrophages but does not contribute to bacterial virulence in vivo.…”

“…Akihiko et al showed that pullulanase is necessary for the efficient growth of Francisella tularensis in macrophages but does not contribute to bacterial virulence in vivo. 42 Bueren et al 43 considered pullulanase a potential virulence factor for pathogenic streptococci. They found that pullulanase surface anchored by S. pneumoniae and S. pyogenes could bind to alveolar type II cells in mouse lung tissue, leading to a stronger reaction between the pathogen and the host cells.…”

Protective effects of the Francisella tularensis ΔpdpC mutant against its virulent parental strain SCHU P9 in Cynomolgus macaques

“…A previous study reported that the ∆ pdpC mutant strain had the potential to protect mice from lethal intranasal challenge with a virulent strain via subcutaneous immunization 34 . The pdpC gene of the virulent strain SCHU P9 was disrupted by group II intron insertion using the TargeTron gene knockout system 37,38 to generate the Δ pdpC mutant strain 39 . Thus, the genetic stability of strain ∆ pdpC was confirmed by in vivo passages in mice for 10 generations 34 .…”