Lipoprotein biosynthesis by prolipoprotein diacylglyceryl transferase is required for efficient spore germination and full virulence of Bacillus anthracis

Abstract

Bacterial lipoproteins play a crucial role in virulence in some gram-positive bacteria. However, the role of lipoprotein biosynthesis in Bacillus anthracis is unknown. We created a B. anthracis mutant strain altered in lipoproteins by deleting the lgt gene encoding the enzyme prolipoprotein diacylglyceryl transferase, which attaches the lipid anchor to prolipoproteins. (14)C-palmitate labelling confirmed that the mutant strain lacked lipoproteins, and hydrocarbon partitioning showed it to have decreased surface hydrophobicity. The anthrax toxin proteins were secreted from the mutant strain at nearly the same levels as from the wild-type strain. The TLR2-dependent TNF-α response of macrophages to heat-killed lgt mutant bacteria was reduced. Spores of the lgt mutant germinated inefficiently in vitro and in mouse skin. As a result, in a murine subcutaneous infection model, lgt mutant spores had markedly attenuated virulence. In contrast, vegetative cells of the lgt mutant were as virulent as those of the wild-type strain. Thus, lipoprotein biosynthesis in B. anthracis is required for full virulence in a murine infection model.

Figure 1

Deletion of the lgt gene and in situ complementation. (A) lgt was removed by Cre-loxP system. Underlined region was amplified by PCR to confirm deletion as shown in (B). (B) Ethidium bromide stained agarose gel showing PCR analysis of lgt depletion. Primers were designed to amplify the internal portion between BA5392 and BA5390. W: wild-type B. anthracis, lgt: lgt mutant. (C) For in site complementation, the pSC plasmid containing BA5392-BA5389 was introduced into the lgt knockout mutant, which was then grown at the restrictive temperature. The single-crossover insertion event was selected by the EmR phenotype. Removal of the EmR along with the pSC backbone from the chromosome was achieved by Cre-mediated recombination following transformation with pCrePAS. Growth at 37°C eliminated the pSC and pCrePAS plasmids, resulting in the (re)insertion of BA5391 (lgt gene). Underlined regions were amplified by PCR to analyze the resulting strain as shown in (D). (D) Ethidium bromide stained agarose gel showing PCR analysis of the complementation of lgt gene (BA5391) and the duplication of BA5392. W: wild-type B. anthracis, lgt: lgt mutant, compl: in situ complemented mutant.

Figure 2

Lack of lipidation in lgt mutant. Cells were cultivated in BHI medium with shaking for 4 h at 37°C. Lipoproteins were labeled with [¹⁴C]-palmitic acid and detected by autoradiography. (A) Wild-type strain with empty vector pSW4, lgt mutant with empty vector pSW4, and the complemented mutant with pSW4-lgt. (B) Wild-type strain, lgt mutant, and the in situ complemented mutant.

Figure 3

Lipoproteins of B. anthracis influence bacterial surface hydrophobicity. Bacteria were cultured in BHI medium to an A₆₀₀ of 4.0 and the washed bacterial suspensions were tested for adherence to hexadecane, which results in reduced optical density in the lower aqueous phase. Significant differences (p < 0.0001) between the lgt mutant, wild-type strain, and in situ complemented strain were determined by ANOVA with a Tukey’s multiple comparison post-test and are indicated by asterisks.

Figure 4

Lgt deficiency attenuates germination. (A) Germination of wild-type strain, lgt mutant, and in situ complemented mutant as monitored by the decrease in A₆₀₀ of spore suspensions during incubation in BHI medium. Results are mean values of three independent experiments. Standard deviations are < 10% of the mean. The A₆₀₀ curve of lgt mutant spores differed with statistical significance from the curves of the wild-type and in situ complemented strain spores. (B) Spores of wild-type strain, lgt mutant, and complemented mutant were incubated at in (A) at 37°C for 30 min. At 30 min, samples were heat-treated at 65°C for 30 min. Before and after heat treatment, samples were plated on BHI agar. Plates were incubated overnight and colonies were counted. The fraction of total CFU that were heat sensitive (i.e., germinated) was calculated and are plotted as the mean values ± standard deviation of three independent experiments. Significant differences using one-way ANOVA with Tukey’s post-test (p < 0.001) are indicated by three asterisks (***).

Figure 5

Anthrax toxin protein secretion from wild-type strain, lgt mutant, and in situ complemented mutant. Wild-type strain (W), lgt mutant (G), and in situ complemented mutants (C) were cultivated in NBY medium in air, or NBY supplemented with 0.9% sodium bicarbonate and 9% CO₂ until A₆₀₀ reached 2.0. Supernatants were separated by SDS-PAGE on 4–20% polyacrylamide gels, transferred to nitrocellulose, and blotted with antibody to protective antigen (PA), lethal factor (LF), or edema factor (EF). Intensities of bands were measured with the Odyssey software. Results shown in graph represent the mean ± SD of three independent experiments, and statistical analyses using one-way ANOVA with a Tukey’s post-test for pairwise comparisons revealed no significant differences.

Figure 6

Induction of TNF-α secretion from macrophages by heat-killed B. anthracis and lipoprotein extracts. (A) Bone marrow-derived macrophages from the three types of mice indicated were exposed to heat-killed wild-type strain, lgt mutant, or complemented mutant at 100 μg ml⁻¹. Macrophages were incubated for 18 h and supernatants were analyzed for TNF-α by enzyme-linked immunosorbent assay. Data are plotted as the mean values ± standard deviation of three independent experiments. Significant differences where p < 0.001 are indicated by three asterisks (***) and where p < 0.0001, indicated by four asterisks (****). (B) Coomassie blue staining of proteins in TX-114 fraction of B. anthracis (second lane). First lane contains molecular weight markers. (C) C57BL/6J bone marrow-derived macrophages from the two types of mice indicated were incubated with medium, TX-114 fraction, or lipoprotein lipase digested TX114 fraction for 18 h and supernatants were analyzed for TNF-α by enzyme-linked immunosorbent assay. Data are plotted as the mean values ± standard deviation of three independent experiments. Significant difference (p < 0.05) between lipoprotein lipase treatment and no treatment is indicated by an asterisk (*). All statistical tests used the unpaired two-tailed t-test.

Figure 7

Survival curves of mice challenged subcutaneously (SC) with spores or vegetative bacteria. A/J mice (A, B) or C57BL/6J (C, D) mice were injected SC with 2 × 10³ spores (A, n=10), 20 vegetative cells (B, n=8), 2 × 10⁷ spores (C, n=5) or 1 × 10⁵ vegetative cells (D, n=5). The survival curves following lgt mutant spore challenge differed with statistical significance (by the logrank test) from curves of the wild-type and complemented strains for both A/J challenges (p < 0.0001), as well as C57BL/6J challenges (p< 0.05), while no significant differences were found in vegetative challenges.

Figure 8

Edema in mouse forelegs following infection with lgt mutant or in situ complemented strain. Groups of three A/J mice were injected with lgt mutant or complemented mutant (5 × 10⁷ spores, 50 μl, SC). Percent increases in foot edema dorsal/ventral measurements relative to pre-infection measurements are shown at 3, 6, and 24 h following infection. Each symbol represents measurements of an individual mouse. P-values comparing lgt mutant to in situ complemented strain at 6 and 24 h are < 0.02 by unpaired two-tailed t-test.

Figure 9

Spore germination following subcutaneous (SC) and intravenous (IV) infection. (A) A/J mice (n=3/group) were injected SC in the foreleg with 5 × 10⁷ spores in 50 μl, and (B) C57BL/6J mice (n=2/group) were injected IV with 1 × 10⁶ spores in 200 μl. Skin and underlying tissues were obtained at 6 h from the A/J mice and spleen and liver were obtained at 90 min from the C57BL/6J mice. Tissues were processed as described in Experimental Procedures and total CFUs determined before and after heat treatment. Values plotted are the number of spores (solid bars, “Heat”) and the sum of spores + germinated bacteria (open bars, “No Heat”). Asterisks indicate significant differences (p < 0.05) by 2-way ANOVA test. (C) Representative histology images showing staining of vegetative bacteria (left panels) and spores (right panels) in foreleg skin sections harvested 6 h post-infection from A/J mice injected SC with 1 × 10⁸ spores. Upper panels show lgt mutant and lower panels show complemented strain. The upper and lower pairs of images were prepared from proximal but independent sections.