Importance of srtA and srtB for growth of Bacillus anthracis in macrophages

Abstract

We examined the effect of mutation of two sortase genes of Bacillus anthracis, srtA and srtB, on the ability of the bacterium to grow in J774A.1 cells, a mouse macrophage-like cell line. While disruption of either srtA or srtB had no effect on the ability of the bacteria to grow in rich culture media, mutations in each of these genes dramatically attenuated growth of the bacterium in J774A.1 cells. Complementation of the mutation restored the ability of bacteria to grow in the cells. Since the initial events in inhalation anthrax are believed to be uptake of B. anthracis spores by alveolar macrophages followed by germination of the spores and growth of the bacteria within the macrophages, these results suggest that two sortases of B. anthracis may be critical in the early stages of inhalation anthrax.

FIG. 1.

Insertional mutagenesis of srtA and srtB. The regions of the B. anthracis genome encompassing the srtA (A) and srtB (B) genes are shown. Gene designations are from Read et al. (26). While srtA (BA0688) is located in a region of the chromosome devoid of genes encoding putative LPXTG-containing substrates, srtB (BA4783) is located approximately 6 kb from BA4789, which encodes a potential substrate (see the text). Insertional mutagenesis of the srt genes was accomplished as diagrammed. A portion of the srt gene (gray-shaded box) lacking critical regions at both the 5′ and 3′ ends was inserted into a temperature-sensitive plasmid. At the nonpermissive temperature, plasmid-encoded drug resistance is maintained only if the plasmid recombines with the chromosome. Homologous recombination results in the formation of two incomplete copies of the srt gene, each of which is predicted to encode a nonfunctional protein (see the text). Asterisks indicate the positions of the primers used to generate PCR fragments used in the complementing plasmids (see the text for details).

FIG. 2.

Growth of B. anthracis Sterne 7702 and srtA mutant strains in J774A.1 cells. J774A.1 cells (7.5 × 10⁵ cells/well) were infected at an MOI of 1 with either B. anthracis Sterne 7702 (•), the srtA mutant strain B. anthracis Sterne 7702::pHYZ2(pUTE29) (▪), or the srtA mutant strain complemented with the srtA gene, B. anthracis Sterne 7702::pHYZ2(pEZ31) (▴). At each time point, the number of intracellular (A) and extracellular (B) bacteria were determined as described in the text. Data points and error bars represent mean CFU of the triplicate samples ± standard deviation. The experiment was performed three times, and the results shown are representative of those obtained in each experiment.

FIG. 3.

Micrographs of J774A.1 cells infected with B. anthracis strains. J774A.1 cells were infected at an MOI of 1 with the indicated strains of B. anthracis. After 8 h, the cells were washed, stained with Giemsa, and photographed using an Olympus IX71 inverted microscope. The panels show representative fields (400× magnification) for B. anthracis Sterne 7702 (A), B. anthracis 9131 (a strain lacking both pXO1 and pXO2) (B), the srtA mutant strain B. anthracis Sterne 7702::pHYZ2 (C), and the srtB mutant strain B. anthracis Sterne 7702::pHYZ3 (D). In panels A and B, heavily stained bacteria are readily apparent. In panels C and D, bacteria (some indicated by arrows) are fewer in number.

FIG. 4.

Growth of B. anthracis Sterne 7702 and srtB mutant strains in J774A.1 cells. J774A.1 cells (1 × 10⁶ cells/well) were infected at an MOI of 1 with either B. anthracis Sterne 7702 (•), or the srtB mutant strain B. anthracis Sterne 7702::pHYZ3 (pUTE29) (▪), or the srtB mutant strain complemented with the srtB gene, B. anthracis Sterne 7702::pHYZ3 (pEZ34) (▴). At each time point, the number of intracellular (A) and extracellular (B) bacteria was determined as described in the text. Data points and error bars represent mean CFU of the triplicate samples ± standard deviation. The experiment was performed three times, and the results shown are representative of those obtained in each experiment.