Identification of a hypervariable region containing new Legionella pneumophila Icm/Dot translocated substrates by using the conserved icmQ regulatory signature

Abstract

Legionella pneumophila is an intracellular pathogen that has been shown to utilize the Icm/Dot type IV secretion system for pathogenesis. This system was shown to be composed of Icm/Dot complex components, accessory proteins, and a large number of translocated substrates. In this study, comparison of the icmQ regulatory regions from many Legionella species revealed a conserved regulatory sequence that includes the icmQ -10 promoter element. Mutagenesis of this conserved regulatory element indicated that each of the nucleotides in it affects the level of expression of the icmQ gene but not in a uniform fashion. A genomic analysis discovered that four additional genes in L. pneumophila contain this conserved regulatory sequence, which was found to function similarly in these genes as well. Examination of these four genes indicated that they are dispensable for intracellular growth, but two of them were found to encode new Icm/Dot translocated substrates (IDTS). Comparison of the genomic regions encoding these two IDTS among the four available L. pneumophila genomic sequences indicated that one of these genes is located in a hypervariable genomic region, which was shown before to contain an IDTS-encoding gene. Translocation analysis that was performed for nine proteins encoded from this hypervariable genomic region indicated that six of them are new IDTS which are translocated into host cells in an Icm/Dot-dependent manner. Furthermore, a bioinformatic analysis indicated that additional L. pneumophila genomic regions that contain several neighboring IDTS-encoding genes are hypervariable in gene content.

FIG. 1.

The regulatory regions of the icmQ genes from different Legionella species contain a conserved regulatory element. The regulatory sequences of the icmQ genes from 27 Legionella species were aligned. The regulatory elements are in bold, the L. pneumophila icmQ transcription start site is marked in bold and underlined, and the distances to the first ATG are indicated. The conserved regulatory element is indicated at the bottom (R is A or G, and M is A or C). The Legionella species from which the icmQ regulatory regions were aligned (and their accession numbers) are L. longbeachae (Lng, AY512558), L. gratiana (Gra, AY860642), L. worsleiensis (Wor, AY860646), L. shakespearei (Sha, AY860647), L. moravica (Mor, AY860644), L. quateirensis (Qua, AY860645), L. waltersii (Wal, AY860648), L. pneumophila (Lpn, Y12705), L. hackeliae (Hac, AY753534), L. jamestowniensis (Jam, AY860649), L. brunensis (Bru, AY860650), L. feeleii (Fee, AY753535), L. jordanis (Jor, AY860651), L. fairfieldensis (Fai, AY860653), L. lansingensis (Lan, AY860652), L. micdadei (Mic, AY512559), L. maceachernii (Mac, AY860654), L. nautarum (Nau, AY860655), L. drozanskii (Dro, AY860662), L. birminghamensis (Bir, AY860641), L. quinlivanii (Qui, AY860656), L. erythra (Ery, AY860658), L. rubrilucens (Rub, AY860659), L. spiritensis (Spi, AY860657), L. israelensis (Isr, AY860663), L. londiniensis (Lon, AY860660), and L. adelaidensis (Ade, AY860661).

FIG. 2.

Mutation analysis of the conserved icmQ regulatory signature. (A) Sequence logo of the conserved icmQ signature, representing the most conserved nucleotides in the sequence. The transcription start site (TSS) that was determined for the L. pneumophila icmQ gene is indicated. (B) Each of the nucleotides in the conserved icmQ regulatory signature was mutated (as described in Materials and Methods), and the effects of the different mutations are shown in comparison to the wild-type levels of expression (white bar). The levels of expression were measured at early stationary phase by using a β-galactosidase assay as described in Materials and Methods. The results (Miller units [MU]) are the averages ± standard deviations of at least three independent experiments.

FIG. 3.

The icmQ regulatory signature is found in four additional genes in the L. pneumophila genome. (A) Sequence alignment of the regulatory regions of icmQ, lpg0541, lpg1836 (ceg25), lpg1962, and lpg1983. The putative consensus sequence is in boldface, the transcription start sites were determined for all of the genes and are marked in bold and underlined, the putative PmrA binding site is underlined for lpg1836 (ceg25), and the distances to the first ATG are indicated. (B) The regulatory element identified is functional also in the four new genes found. The expression levels of lacZ fusions containing the wild-type (W.T.) regulatory region as well as two mutations (Mut-1 and Mut-2) (Fig. 2) in the regulatory signatures of lpg0541, lpg1962, and lpg1983 were determined at early stationary phase. (C) Analysis of the expression of lpg1836 (ceg25) in the wild-type strain and the pmrA deletion mutant (ΔpmrA) (left). The effects of the two mutations, indicated in panel B, on the level of expression of lpg1836 in the wild-type strain (middle) and in the pmrA deletion mutant (right) are shown. The levels of expression were determined using the β-galactosidase assay as described in Materials and Methods. Statistical analyses of the expression of the wild-type fusion and each of the mutants (Mut-1 and Mut-2) were performed using a standard t test, and the results were found to be significantly different (P < 0.0001) in all of them. The results (Miller units [MU]) are the averages ± standard deviations of at least three independent experiments.

FIG. 4.

Involvement of the four new genes in L. pneumophila pathogenesis. Null mutants constructed with mutations in the four new genes (lpg0541, filled triangle; lpg1836, open triangle; lpg1962, ×; and lpg1983, open diamond) were examined for intracellular growth in A. castellanii (A) and HL-60-derived human macrophages (B) in comparison to growth in the wild-type strain (filled diamond) and an icmT deletion mutant (filled square). (C) Translocation of CyaA fusions into HL-60-derived human macrophages was examined for the four new genes from the wild-type strain JR32 (gray) and the icmT mutant (white), and the lpg1836 (ceg25) and lpg1962 CyaA fusions were found to be translocated into host cells in an Icm/Dot-dependent manner. cAMP, cyclic AMP.

FIG. 5.

lpg1962 is located in a hypervariable genomic region. (A) Schematic presentation of the genes located between lpg1956 and lpg1967 of L. pneumophila Lens (Lpl), L. pneumophila Corby (Lpc), L. pneumophila Philadelphia-1 (Lpg), and L. pneumophila Paris (Lpp). Homologous genes are indicated by dashed lines, genes that encode IDTS are colored in black (lpg1958 [legL5], lpg1960 [lirA], lpg1962 [lirB], lpg1963 [lirC], lpg1964 [lirD], lpg1965 [lirE], and lpg1966 [lirF]), and genes encoding transposases/integrases are marked in white. A gray background indicates genes that are present in all four strains. (B) All of the genes located in this hypervariable region are expressed. The expression of the genes was determined using RT-PCR; the PCR was carried out for 25 cycles for all of the genes (+RT). Equivalent amounts of RNA were used for RT reactions without reverse transcriptase and then examined by PCR for the same number of cycles (−RT), and no product was obtained.

FIG. 6.

Icm/Dot-dependent translocation of the proteins encoded by genes located at the variable region. Wild-type strain JR32 (gray bars) and icmT mutant GS3011 (white bars) harboring the CyaA fusion proteins (indicated below the bars) were used to infect HL-60-derived human macrophages, and the cyclic AMP (cAMP) levels of the infected cells were determined (as described in Materials and Methods). The data are the means for the amount of cAMP per well obtained, and the error bars indicate standard deviations. A Western blot analysis using anti-CyaA antibody is presented below each bar, indicating that similar levels of the CyaA fusions were present in wild-type JR32 (left) and the icmT mutant (right) used for infection.

FIG. 7.

Examination of the effects of different regulators on the IDTS identified. The levels of expression of the eight IDTS lacZ fusions (lirA, lirB, lirC, lirD, lirE, lirF, legL5, and ceg25) in wild-type strain JR32 (gray), a pmrA deletion mutant (checkers), a cpxR deletion mutant (dots), an rpoS deletion mutant (stripes), and a letA deletion mutant (waves) were examined. The levels of expression were determined using a β-galactosidase assay, as described in Materials and Methods. The results (Miller units [MU]) are the averages ± standard deviations (error bars) of at least three different experiments.

FIG. 8.

Additional regions encoding known IDTS are hypervariable. Schematic presentation of the genomic regions between lpg2398 and lpg2411 (A) and between lpg2824 and lpg2832 (B) in L. pneumophila Lens (Lpl), L. pneumophila Corby (Lpc), L. pneumophila Philadelphia-1 (Lpg), and L. pneumophila Paris (Lpp). Homologous genes are indicated by dashed lines, genes that encode known IDTS are colored in black (lpg2400 [legL7], lpg2407, lpg2409 [ceg29], lpg2410 [vpdA], lpg2829 [sidH], lpg2830 [lubX], and lpg2831 [vipD]), the predicted IDTS-encoding gene lpg2826 (ceg31) is hatched, and genes encoding transposases/integrases are marked in white. A gray background indicates genes that are present in all four strains.