Pullulanase Is Necessary for the Efficient Intracellular Growth of <i>Francisella tularensis</i>

<div><p>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 (<i>pulB</i>; <i>FTT_0412c</i>) of <i>F</i>. <i>tularensis</i> subspecies <i>tularensis</i> Schu S4 is considered to be a homologue of the type I pullulanase (<i>pulA</i>) of the other <i>Francisella</i> subspecies. The significance of <i>Francisella</i> pullulanase has been obscure until now. In the present study, we characterized a recombinant PulB of <i>F</i>. <i>tularensis</i> SCHU P9, which was expressed as a his-tagged protein in <i>Escherichia coli</i>. The recombinant PulB was confirmed to be a type I pullulanase by its enzymatic activity <i>in vitro</i>. A <i>pulB</i> gene knockout mutant of <i>F</i>. <i>tularensis</i> SCHU P9 (Δ<i>pulB</i>) was constructed using the TargeTron Knockout system and plasmid pKEK1140 to clarify the function of PulB during the growth of <i>F</i>. <i>tularensis</i> in macrophages. The intracellular growth of the Δ<i>pulB</i> mutant in murine macrophage J774.1 cells was significantly reduced compared with that of the parental strain SCHU P9. Expression of PulB in Δ<i>pulB</i>, using an expression plasmid, resulted in the complementation of the reduced growth in macrophages, suggesting that PulB is necessary for the efficient growth of <i>F</i>. <i>tularensis</i> 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 Δ<i>pulB</i>-infected mice showed milder lesions compared to those from SCHU P9-infected mice. However, all mice infected with SCHU P9 and Δ<i>pulB</i> 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 <i>in vivo</i>.</p></div

Primers used to produce recombinant PulB.

<p>Primers used to produce recombinant PulB.</p

Histopathilogical analyses of mice infected with SCHU9 and Δ<i>pulB</i> mutant bacteria.

<p>Tissue sections obtained from mice infected with SCHU P9 and Δ<i>pulB</i> were examined by hematoxylin and eosin (HE) staining and immunohistochemistry (IHC) using anti-<i>Francisella tularensis</i> LPS monoclonal antibody. (A) Moderate focal necrosis and abscess were observed in lungs from mice infected with SCHU P9. Lungs from mice infected with Δ<i>pulB</i> showed milder pulmonary lesions compared to those from mice infected with SCHU P9. Vacuolar degeneration of hepatocytes, moderate focal necrosis and congestion were observed in livers of mice infected with SCHU P9. Livers from mice infected with Δ<i>pulB</i> showed milder focal necrosis. Marked focal necrosis associated with the accumulation of neutrophils was observed in the white pulp and red pulp of spleens from mice infected with SCHU P9 (3 dpi, original magnification x10). However, the accumulation of neutrophils was more prominent in the white pulp and around the central arteries or arterioles of spleens from mice infected with Δ<i>pulB</i> (right bottom square, original magnification x40). (B) Lung, liver and spleen from mice infected with SCHU P9 and Δ<i>pulB</i> were stained by immunohistochemical stain (IHC) with anti-<i>F</i>. <i>tularensis</i> LPS, N-Histofine Simple Stain MAX PO (M), and visualized by 3,3’-diaminobenzidine (DAB), followed by a hematoxylin counterstain (5 dpi, original magnification x10). LPS antigen positive foci, which are considered to be LPS-positive bacteria, were more prominent in SCHU P9-infected mice at 5 dpi compared to those in Δ<i>pulB</i>-infected mice.</p

The comparisons of virulence of SCHU P9 and <i>ΔpulB</i> mutant bacteria in mice.

<p>Four C57BL/6J mice in each group (seven- to twelve-week-old females; SLC, Inc. Shizuoka, Japan) were intranasally inoculated with 10³ CFU of SCHU P9 and Δ<i>pulB</i>, respectively. Survival rates (A) and body weights (B) of these mice were measured up to 6 dpi. Mice were sacrificed at the indicated dpi, and then the averages ± SD of bacterial CFU in lungs (C), livers (D), and spleens (E) were shown. ND, not detected.</p

Primers used to construct <i>F</i>. <i>tularensis</i> SCHU P9 mutant strains.

<p>Primers used to construct <i>F</i>. <i>tularensis</i> SCHU P9 mutant strains.</p

The intracellular growth of Δ<i>pulB</i> derived from virulent SCHU P9.

<p>(A) The insertion in Δ<i>pulB</i> derived from SCHU P9 was confirmed by PCR. Genomic DNA extracted from SCHU P9 and Δ<i>pulB</i> were subjected to PCR using gene specific sense/antisense (I), gene specific sense/EBS universal (II), and EBS universal/antisense (III) primer pairs. The amplicons and molecular weight marker were electrophoresed on a 0.7% agarose gel. (B) The schematic summary of Δ<i>pulB</i> generated in this study is shown. <i>ltr</i>B introns were inserted into the SCHU P9 <i>pulB</i> gene. The resultant mutant was designated Δ<i>pulB</i>. (C) J774.1 cells inoculated with virulent SCHU P9 (black bar), attenuated SCHU P5 (white bar), and Δ<i>pulB</i> (gray bar) at an MOI of 10, were incubated for 2 h and 26 h. Their intracellular CFUs were measured in triplicate. Mean ± SD of CFU are shown. Statistical significance was determined by using Student’s <i>t</i> test (***<i>P</i> < 0.001). (D) J774.1 cells were infected with Δ<i>pulB</i> complemented with a <i>pulB</i> gene expression plasmid (black bar) or a control plasmid (gray bar). The intracellular CFUs were measured in triplicate. Mean ± SD of CFU are shown. Statistical significance was determined by using Student’s <i>t</i> test (**<i>P</i> < 0.01).</p

Characterization of the recombinant PulB of <i>F</i>. <i>tularensis</i> SCHU P9.

<p>(A) The plasmid pCold TF contains a <i>lac</i> operator, the cold shock protein A (<i>cspA</i>) 5' untranslated region (5' UTR), a translation enhancing element (TEE), a 6x His-tag sequence, the trigger factor sequence, protease cleavage sites, and a multiple cloning site (MCS) downstream of the <i>cspA</i> promoter. This plasmid, which is a cold shock expression vector, can express the target protein fused to a trigger factor under the control of the cold shock protein A (<i>cspA</i>) promoter and <i>lac</i> operator. In this study, the <i>pulB</i> gene fused with an N-terminal TEE sequence and a C-terminal 6x his-tag sequence was amplified from <i>F</i>. <i>tularensis</i> SCHU P9 DNA and then inserted into pCold TF plasmid lacking trigger factor, protease sites, and MCS using the In-Fusion HD Cloning Kit. (B) The expression and purification of the recombinant PulB. PulB expression was induced by IPTG in <i>E</i>. <i>coli</i> BL21(DE3) transformed with the pCold TF-pulB plasmid. Recombinant PulB was purified from the bacterial lysates using an AKTA start system equipped with a HisTrap HP column. Lane M, marker proteins (kDa); lane 1, <i>E</i>. <i>coli</i> BL21(DE3) lysates; lane 2, purified recombinant PulB. (C) The products of pullulan hydrolysis catalyzed by recombinant PulB are shown. Pullulan (P), maltotriose (G3), maltose (G2), and glucose (G1) were incubated in pH 6.2 phosphate buffer with (+) or without (–) the recombinant PulB at 37°C for 24 h. After this incubation, the samples were immediately heat denatured. The samples were subjected to TLC analysis using 2-propanol/acetic acid/water (4:1:1, vol/vol/vol) as the solvent system. (D and E) Effects of pH (D) and temperature (E) on recombinant PulB activity are shown. Recombinant PulB was incubated with 0.25% pullulan at 37°C for 12 h and then immediately heat denatured at 94°C for 15 min. The hydrolyzed products were measured in triplicate using the DNS method. After the background was subtracted from the data, the maximal relative activity was determined at pH 6.2 (D) and 37°C (E). Mean ± SD of relative activity are shown. The optimal pH was determined from a curve fitting (Gaussian) by GraphPad Prism software.</p