Differential chitinase activity and production within Francisella species, subspecies, and subpopulations

Abstract

Genotyping of Francisella tularensis (A1a, A1b, A2, and type B) and Francisella novicida has identified multiple differences between species and among F. tularensis subspecies and subpopulations. Variations in virulence, geographic distribution, and ecology are also known to exist among this group of bacteria, despite the >95% nucleotide identity in their genomes. This study expands the description of phenotypic differences by evaluating the ability of F. tularensis and F. novicida to degrade chitin analogs and produce active chitinases. Endochitinase activities were observed to vary among F. tularensis and F. novicida strains. The activity observed for F. tularensis strains was predominantly associated with whole-cell lysates, while the chitinase activity of F. novicida localized to the culture supernatant. In addition, the overall level of chitinase activity differed among the subpopulations of F. tularensis and between the species. Bioinformatic analyses identified two new putative chitinase genes (chiC and chiD), as well as the previously described chiA and chiB. However, the presence of these four open reading frames as intact genes or pseudogenes was found to differ between Francisella species and F. tularensis subspecies and subpopulations. Recombinant production of the putative chitinases and enzymatic evaluations revealed ChiA, ChiB, ChiC, and ChiD possessed dissimilar chitinase activities. These biochemical studies coupled with bioinformatic analyses and the evaluation of chiA and chiC knockouts in F. tularensis A1 and A2 strains, respectively, provided a molecular basis to explain the differential chitinase activities observed among the species and subpopulations of Francisella.

Fig. 1.

In vitro chitinase activities of F. tularensis (A1a, A1b, A2, and type B) and F. novicida strains. A panel of 12 characterized strains (three A1a, two A1b, two A2, three type B, and two F. novicida) were evaluated for endochitinase, chitobiosidase, and N-acetylglucosaminidase activity. (A) endochitinase activities of WCL; (B) endochitinase activities of CS; (C) chitobiosidase activities of CS; (D) N-acetylglucosaminidase activities of CS. The average absorbance at 405 from three biological replicates (two technical replicates) are reported as a dot (●), with the average of all replicates indicated by a bar (—). Little to no chitobiosidase or N-acetylglucosaminidase activities was detected in WCL.

Fig. 2.

Domain features of F. tularensis (A1a, A1b, A2, and type B) and F. novicida chitinases. The relative positions of conserved domains were identified in the chitinase gene products of F. tularensis and F. novicida. Solid black boxes indicate the predicted signal peptide cleavage site, striped boxes indicate the location and completeness of the GH18 domain (truncated GH18 domains appear as a pentagon), open boxes indicate the position of fibronectin type 3 domains, gray boxes indicate carbohydrate binding domains, and dark gray chevrons represent incomplete N-acetylglucosamine-binding protein A domains. (A) chiA gene products; (B) chiB gene products; (C) chiC gene products; (D) chiD gene products. A similar chiC gene product was identified in the genome of F. novicida GA99-3548 and GA99-3549 but not F. novicida GA99-3550.

Fig. 3.

Chitinase activity and substrate specificities of F. tularensis (A1a/A1b, A2, and type B) and F. novicida recombinant chitinases. F. tularensis and F. novicida chitinases with complete or partial GH18 domains were produced as recombinants in a heterologous system and assayed for 9 min to determine their degree and specificity to analogs capable of distinguishing endochitinase (●), chitobiosidase (▵), and N-acetylglucosaminidase (♢) activities. The absorbance at 405 nm was determined for three technical replicates of each enzyme at 37°C. The average of the technical replicates is reported as a bar of the corresponding color and shading.

Fig. 4.

Comparative endochitinase kinetics of F. tularensis (A1a, A1b, A2, and type B) and F. novicida recombinant chitinases. Recombinants that tested positive for endochitinase activity (Fig. 3) were assayed with an endochitinase analog to determine the relative activity of each functional enzyme at 37°C. The average absorbance at 405 nm of three technical replicates is reported for time points at 0, 15, 30, 45, 60, 90, 120, 150, 180, 210, 240, 300, 360, 420, 480, and 540 s, except for ChiC, where activity plateaued at 90 s. The kinetics of ChiA recombinant proteins (A), ChiB recombinant proteins (B), and ChiC recombinant proteins (C) are presented, as well as a relative comparison of all functional chitinases (D).

Fig. 5.

Chitinases produced in vitro by F. tularensis (A1a, A1b, A2, and type B) and F. novicida. Western blots of WCL (15 μg) of each Francisella strain with anti-ChiA antiserum, anti-ChiB antiserum, and anti-ChiC antiserum. Lanes 1 to 3, A1a strains OK01-2528, MO02-4195, and SCHU S4, respectively; lanes 4 and 5, A1b strains MA00-2987 and MD00-2970, respectively; lanes 6 and 7, A2 strains NM99-1823 and WY96-3418, respectively; lanes 8 to 10, type B strains KY99-3387, LVS, and MI00-1730, respectively; lanes 11 and 12, F. novicida strains GA99-3548 and GA99-3550, respectively.

Fig. 6.

Analyses of ChiA and ChiC chitinase knockouts in F. tularensis A1b strain MA00-2987 and F. tularensis A2 strain WY96-3418, respectively. (A) Endochitinase (◆) activity of WCL from WT strain MA00-2987, ΔchiA MA00-2987, and ΔchiA/comp MA00-2987; (B) endochitinase (◆) and chitobiosidase (■) activity of WCL from WT strain WY96-3418, ΔchiC WY96-3418, and ΔchiC/comp WY96-3418. (C) Western blot with anti-ChiA against WCL from WT MA00-2987 (lane 1), ΔchiA MA00-2987 (lane 2), and ΔchiA/comp MA00-2987 (lane 3) strains. (D) Western blot with anti-ChiC against WCL from WT WY96-3418 (lane 1), ΔchiC WY96-3418 (lane 2), and ΔchiC/comp WY96-3418 (lane 3) strains.