Legionella pneumophila utilizes the same genes to multiply within Acanthamoeba castellanii and human macrophages

Abstract

In previous reports we described a 22-kb Legionella pneumophila chromosomal locus containing 18 genes. Thirteen of these genes (icmT, -R, -Q, -P, -O, -M, -L, -K, -E, -C, -D, -J, and -B) were found to be completely required for intracellular growth and killing of human macrophages. Three genes (icmS, -G, and -F) were found to be partially required, and two genes (lphA and tphA) were found to be dispensable for intracellular growth and killing of human macrophages. Here, we analyzed the requirement of these genes for intracellular growth in the protozoan host Acanthamoeba castellanii, a well-established important environmental host of L. pneumophila. We found that all the genes that are completely required for intracellular growth in human macrophages are also completely required for intracellular growth in A. castellanii. However, the genes that are partially required for intracellular growth in human macrophages are completely required for intracellular growth in A. castellanii. In addition, the lphA gene, which was shown to be dispensable for intracellular growth in human macrophages, is partially required for intracellular growth in A. castellanii. Our results indicate that L. pneumophila utilizes the same genes to grow intracellularly in both human macrophages and amoebae.

FIG. 1

Intracellular growth of an icmP insertion mutant in HL-60-derived macrophages and A. castellanii. (A) Chromosomal arrangement of the region surrounding icmP. The location of the insertion in icmP (GS3002) is shown. Intracellular growth in HL-60-derived macrophages (B) and in A. castellanii (C) was tested as described in Materials and Methods; the experiments were done at least three times, and results similar to those shown were obtained. ■, JR32; ⧫, 25D; ▵, GS3002 containing pMMB207αb-Km-14; ○, GS3002 containing pGS-Lc-34-14.

FIG. 2

Intracellular growth of icmT and icmS insertion mutants in HL-60-derived macrophages and A. castellanii. (A) Chromosomal arrangement of the region surrounding icmS. The locations of the deletion substitutions (GS3001 and GS3011) are shown. Intracellular growth in HL-60-derived macrophages (B) and in A. castellanii (C) was tested as described in Materials and Methods; the experiments were done at least three times, and results similar to those shown were obtained. ■, JR32; ○, GS3011 containing pMMB207αb-Km-14; ▵, GS3011 containing pGS-Lc-37-14; □, GS3001 containing pMMB207αb-Km-14; ◊, GS3001 containing pGS-Lc-37-14.

FIG. 3

Intracellular growth of icmN and icmM insertion mutants in HL-60-derived macrophages and A. castellanii. (A) Chromosomal arrangement of the region surrounding icmN. The chromosomal arrangements of the mutants tested are shown above the genes. Intracellular growth in HL-60-derived macrophages (B) and in A. castellanii (C) was tested as described in Materials and Methods; the experiments were done at least three times, and results similar to those shown were obtained. ■, JR32; ○, GS3005, □, GS3006, ▵, GS3007; ◊, GS3008.

FIG. 4

Intracellular growth of icmG and icmC insertion mutants in HL-60-derived macrophages and A. castellanii. (A) Chromosomal arrangement of the region surrounding icmG. The locations of the deletion substitutions (MW635 and MW645) are shown. Intracellular growth in HL-60-derived macrophages (B) and in A. castellanii (C) was tested as described in Materials and Methods; the experiments were done at least three times, and results similar to those shown were obtained. ■, JR32; ○, MW635 containing pMMB207-Km-14; ▵, MW635 containing pGS-Lc-63-14; □, MW645 containing pMMB207-Km-14; ◊, MW645 containing pGS-Lc-63-14.

FIG. 5

Intracellular growth of icmF and tphA insertion mutants in HL-60-derived macrophages and A. castellanii. (A) Chromosomal arrangement of the region surrounding icmF. The locations of the insertions (LELA1275 and MW627) are shown. Intracellular growth in HL-60-derived macrophages (B) and in A. castellanii (C) was tested as described in Materials and Methods; the experiments were done at least three times, and results similar to those shown were obtained. ■, JR32; ◊, LELA1275 containing pMMB207αb-Km-14; ○, LELA1275 containing pGS-Lc-55-14; ▵, MW627.

FIG. 6

Intracellular growth requirements for genes in region II. The first line under the genes indicates the growth phenotype in HL-60-derived macrophages, and the second line indicates the growth phenotype in A. castellanii. −, no intracellular growth was observed; +/−, partial intracellular growth was observed; +, intracellular growth was similar to that of the wild-type strain.