The Legionella pneumophila icmGCDJBF genes are required for killing of human macrophages

Abstract

Previously, a collection of mutants of Legionella pneumophila that had lost the ability to multiply within and kill human macrophages was generated by Tn903dIIlacZ transposon mutagenesis and classified into DNA hybridization groups. A subset of these mutants was complemented by a plasmid, pMW100, containing a 13.5-kb genomic DNA insert. This plasmid restored the ability to multiply within and produce cytopathic effects on human macrophages to members of DNA hybridization groups II, IV, VI, and XVII. A region of the genomic insert of pMW100 was sequenced, and eight potential genes were identified and named icmE, icmG, icmC, icmD, icmJ, icmB, icmF, and tphA. None of the genes encode potential protein products with significant homology to previously characterized proteins, except for tphA, whose product has significant homology to a family of metabolite/H+ symport proteins from gram-negative bacteria. The positions of the Tn903dIIlacZ insertions within the genes were determined by nucleotide sequencing. No Tn903dIIlacZ insertions mapped to icmG, icmJ, or tphA; therefore, these loci were mutated to test whether they were required for macrophage killing. Complementation analysis was used to evaluate the roles of the potential gene products and provide information on the organization of transcriptional units within the region. The results indicate that all identified open reading frames except tphA are required for killing of human macrophages.

FIG. 1

(A) Intracellular multiplication of strain LELA3896 within HL-60-derived macrophages. Differentiated HL-60 cells (1.5 × 10⁶ per well) were infected with approximately 10⁴ bacteria on day 0. The number of CFU of each strain per milliliter was determined daily for 5 days, in triplicate, on ABCYE plates. ▪, JR32; □, LELA3896; •, LELA3896(pMMB207); ○, LELA3896(pMW100). Error bars indicate the standard error of the mean. (B) Cytotoxicity of strain LELA3896 for HL-60-derived macrophages. Differentiated HL-60 cells (4 × 10⁵ per well) were infected with the indicated strains of bacteria at concentrations ranging from 10 to 10⁶ CFU/ml. After 6 days, the remaining viable macrophages in the monolayer were quantitated by adding the dye MTT and measuring the absorbance at 570 nm (A₅₇₀). ▪, JR32(pMMB207); □, JR32(pMW100); •, LELA3896; ○, LELA3896(pMMB207); ▴, LELA3896(pMW100). Values are the average of three determinations. Error bars indicate the standard error of the mean.

FIG. 2

(A) EcoRI restriction map of the genomic insert on plasmid pMW100. EcoRI restriction sites are labeled R. The approximate size of each EcoRI restriction fragment is shown in kilobases. P_tac of the pMMB207 vector directs transcription to the left into the 5.5-kb EcoRI fragment. The dotted line at the 3′-end of the 5.5-kb EcoRI fragment represents the region not sequenced. Mak⁻ DNA hybridization groups represented on the genomic insert of pMW100 as shown by Southern analysis are shown above the EcoRI fragments. (B) ORFs present on the genomic insert of pMW100 and locations of Tn903dIIlacZ insertions (▹) and deletion substitutions (▪) in the Mak⁻ mutants. Triangles on top of one another indicate that the LELA strains contain a Tn903dIIlacZ insertion at the same nucleotide. Vertical numbers indicate the strain number. ORFs shown were identified by nucleotide sequencing and MacDNAsis analysis. Solid arrows indicate the direction of transcription of each ORF. The location with respect to each EcoRI restriction site and size of each ORF is approximate. icmE is represented as a partial ORF, as indicated by a dotted 5′ end.

FIG. 3

Cytotoxicity of the icmF mutant strains for HL-60-derived macrophages. Differentiated HL-60 cells (4 × 10⁵ cells per well) were infected with the indicated strains of bacteria at concentrations ranging from 10 to 10⁶ CFU/ml. After 6 days, the remaining viable macrophages in the monolayer were quantitated by adding the dye MTT and measuring the A₅₇₀. (A) icmF mutant strain LELA1275. ▪, JR32(pMMB207); □, LELA1275(pMMB207); •, LELA1275(pMW100). (B) icmF mutant strain LELA1718. ▪, JR32(pMMB207); □, LELA1718(pMMB207); •, LELA1718(pMW100). Values are the average of three determinations. Error bars indicate the standard error of the mean.

FIG. 4

Representation of predicted transmembrane domains in the icm gene products. The hydropathic profiles of the predicted Icm proteins are shown according to the Kyte-Doolittle scale with a window size of 20 amino acids. The transmembrane domains as identified by the Psort program are represented as bars above the graphs. The vertical axis represents the hydrophobic value as measured on the Kyte-Doolittle scale, and the horizontal axis represents the number of amino acids in the icm gene products.

FIG. 5

Cytotoxicity of the icmB mutant strains containing different plasmids for HL-60-derived macrophages. Differentiated HL-60 cells (4 × 10⁵ per well) were infected with the indicated strains of bacteria at concentrations ranging from 10 to 10⁶ CFU/ml. After 6 days, the remaining viable macrophages in the monolayer were quantitated by adding the dye MTT and measuring the A₅₇₀. (A) icmB mutant strain LELA3393. ▪, JR32(pMMB207); □, LELA3393(pBC SK+); •, LELA3393(pMW100); ○, LELA3393(pMW560). (B) icmB mutant strain LELA1012. ▪, JR32(pMMB207); □, LELA1012(pBC SK+); •, LELA1012(pMW100); ○, LELA1012(pMW560). (C) icmB mutant strain LELA1223. ▪, JR32(pMMB207); □, LELA1223(pBC SK+); •, LELA1223(pMW100); ○, LELA1223(pMW560). (D) icmB mutant strain LELA3896. ▪, JR32(pMMB207); □, LELA3896(pBC SK+); •, LELA3896(pMW100); ○, LELA3896(pMW560). Values are the average of three determinations. Error bars indicate the standard error of the mean.

FIG. 6

Cytotoxicity assay of L. pneumophila strains containing different plasmids for HL-60-derived macrophages. Differentiated HL-60 cells (4 × 10⁵ cells per well) were infected with the indicated strains of bacteria at concentrations ranging from 10 to 10⁶ CFU/ml. After 6 days, the remaining viable macrophages in the monolayer were quantitated by adding the dye MTT and measuring the A₅₇₀. (A) icmJ allelic exchange strain MW656. ▪, JR32(pMMB207); □, MW656(pMMB207αb); •, MW656(pMW100); ○, MW656(pMW680); , MW656(pMW681); ▵, MW656(pMW560); ▾▴, MW656(pMW560, pMW790). (B) icmD mutant strain LELA1205. ▪, JR32(pMMB207); □, LELA1205(pMMB207αb); •, LELA1205(pMW100); ○, LELA1205(pMW734); , LELA1205(pMW736). (C) icmC allelic exchange strain MW645. ▪, JR32(pMMB207); □, MW645(pMMB207αb); •, MW645(pMW100); ○, MW645(pMW604); , MW645(pMW728); ▵, MW645(pMW730); ▾▴, MW645(pMW734). (D) icmG allelic exchange strain MW635. ▪, JR32(pMMB207); □, MW635(pMMB207αb); •, MW635(pMW100); ○, MW635(pMW741); , MW635(pMW743). Values are the average of three determinations. Error bars indicate the standard error of the mean.