The response regulator CpxR directly regulates expression of several Legionella pneumophila icm/dot components as well as new translocated substrates

Abstract

Legionella pneumophila 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 and accessory proteins, as well as a large number of translocated substrates. Bioinformatic analysis of the regulatory regions of all the genes revealed that several icm/dot genes, as well as two genes encoding icm/dot translocated substrates, contain the conserved CpxR regulatory element, a regulator that has been shown previously to control the expression of the icmR gene. An experimental analysis, which included a comparison of gene expression in a L. pneumophila wild-type strain and gene expression in a cpxR deletion mutant, construction of mutants with mutations in the CpxR conserved regulatory elements, controlled expression studies, and mobility shift assays, demonstrated the direct relationship between the CpxR regulator and the expression of the genes. Furthermore, genomic analysis identified nine additional genes that contain a putative CpxR regulatory element; five of these genes (two legA genes and three ceg genes) were suggested previously to be putative icm/dot translocated substrates. The three ceg genes identified, which were shown previously to contain a putative PmrA regulatory element, were found here to be regulated by both CpxR and PmrA. The other six genes (two legA genes and four new genes products were found to be regulated by CpxR. Moreover, using the CyaA translocation assay, these nine gene products were found to be translocated into host cells in an Icm/Dot-dependent manner. Our results establish that the CpxR regulator is a fundamental regulator of the icm/dot type IV secretion system in L. pneumophila.

FIG. 1.

Analysis of the expression of known pathogenesis-related genes containing the CpxR consensus sequence. (A) Sequence alignment of the regulatory regions of icm/dot genes (icmR, icmV, and icmW), the icm/dot accessory gene lvgA, and two IDTS-encoding genes (sidM and sidH). The putative consensus sequence is indicated by boldface type, and the distances to the first ATG codon are also indicated. The complementary strand is shown for genes in which the consensus regulatory element was identified on the reverse strand (icmV, icmW, and lvgA). (B) Expression of lacZ fusions in wild-type L. pneumophila and the cpxR deletion mutant. The expression of lacZ fusions of three icm/dot genes (icmR, icmV, and icmW), the lvgA gene, two IDTS-encoding genes (sidM and sidH) that contain the CpxR consensus regulatory element, and lepB that does not contain the consensus regulatory element in wild-type strain JR32 (shaded bars) and the cpxR deletion mutant OG2002 (open bars) was examined. β-Galactosidase activity was determined as described in Materials and Methods. The data (in Miller units [MU]) are the averages ± standard deviations (error bars) of at least three different experiments.

FIG. 2.

Analysis of mRNA levels for genes containing the CpxR consensus sequence in the wild type and a cpxR deletion mutant. Total RNA derived from the wild type (wt) and the cpxR deletion mutant (ΔcpxR) was used in RT-PCR to assess the mRNA levels for seven genes containing the CpxR consensus sequence (icmV, sidM, ceg33, ceg07, cegC1, cegC2, and legA11) and two genes that do not contain the CpxR consensus sequence (sidF and sdeB). The PCR was carried out for 19 cycles for icmV, legA11, and sidM, for 21 cycles for ceg07, ceg33, cegC1, sidF, and sdeB, and for 23 cycles for cegC2. Quantification (values are indicated on the right) was performed using the TINA image analysis software, and the values were obtained by dividing the mRNA level in the cpxR mutant by the mRNA level in the wild-type strain.

FIG. 3.

Analysis of the relationship between the cpxR consensus and the CpxR regulator. Expression of icmV, icmW, lvgA, sidM, and sidH lacZ fusions in L. pneumophila was determined. For each of the fusions, data are shown for the wild-type regulatory region (wt) and the mutated regulatory region (mut) in the JR32 wild-type strain (wild-type) and cpxR mutant strain OG2002 (ΔcpxR). β-Galactosidase activity was determined as described in Materials and Methods. The data are the averages ± standard deviations (error bars) of at least three different experiments. MU, Miller units.

FIG. 4.

CpxR directly regulates the genes containing the consensus sequence. The expression of wild-type fusions (grey bars) of sidM::lacZ (A) and lvgA::lacZ (B), as well as the same fusions containing a mutation in the CpxR regulatory element (open bars), was examined in E. coli having a deletion in the cpxR gene (PAD215). The bacteria examined contained a plasmid with the L. pneumophila cpxR gene cloned under control of the P_tac promoter (activated by IPTG), and they were grown in media containing different concentrations of IPTG (indicated below the bars). Bacteria containing the same lacZ fusions without the cpxR gene were used as controls (left columns in both panels). β-Galactosidase activity was determined as described in Materials and Methods. The data are the averages ± standard deviations (error bars) of at least three different experiments. MU, Miller units.

FIG. 5.

L. pneumophila His₆-CpxR protein binds to the sidM, lvgA, and cegC2 regulatory regions. Mobility shift assays were performed with pure His₆-CpxR protein and DIG-labeled sidM (136 pg) (A), lvgA (154 pg) (B), and cegC2 (123 pg) (C) regulatory regions in the presence (plus sign) or absence (minus sign) of unlabeled probe (Specific comp.) (50 ng in panels A and B and 150 ng in panel C) or the same amount of herring sperm DNA (Non-specific). The amount of His₆-CpxR is indicated above each lane.

FIG. 6.

legA and cegC genes are regulated by the CpxR regulator. (A) Sequence alignment of the regulatory regions of the legA and cegC genes (legA10, legA11, cegC1, cegC2, cegC3, and cegC4). The putative consensus sequence is indicated by boldface type, and the distances to the first ATG codon are also indicated. The complementary strand is shown for genes (legA10, cegC1, and cegC2) in which the consensus regulatory element was identified on the reverse strand. (B) Expression of the four cegC genes and the two legA genes in L. pneumophila wild-type strain (dark grey bars) and cpxR mutant strain OG2002 (open bars). For the cegC genes lacZ fusions containing a mutation in the CpxR consensus sequence were also examined in the L. pneumophila wild-type strain (light grey bars). β-Galactosidase activity was determined as described in Materials and Methods. The data are the averages ± standard deviations (error bars) of at least three different experiments. MU, Miller units.

FIG. 7.

ceg genes are regulated by the CpxR and PmrA regulators. (A and B) Sequence alignment of the regulatory regions of the three ceg genes according to the CpxR consensus sequence (A) or the PmrA consensus sequence (B). The putative PmrA consensus sequence is indicated by shading, the putative CpxR consensus sequence is indicated by boldface type, the putative −10 promoter elements are underlined, and the distances to the first ATG codon are also indicated. (C) Expression of the three lacZ fusions in wild-type L. pneumophila (W.T.), cpxR deletion mutant OG2002 (ΔcpxR), pmrA deletion mutant HK-PQ1 (ΔpmrA), and cpxR pmrA double-deletion mutant EA-CRPA (ΔcpxR ΔpmrA). (D) Expression of the wild-type ceg18::lacZ fusion (grey bars), the ceg18::lacZ fusion containing a mutation in the CpxR consensus sequence (open bars), and the ceg18::lacZ fusion containing a mutation in the PmrA consensus sequence (black bars) in wild-type L. pneumophila (Wild-type), cpxR deletion mutant OG2002 (ΔcpxR), pmrA deletion mutant HK-PQ1 (ΔpmrA), and cpxR pmrA double-deletion mutant EA-CRPA (ΔcpxR-ΔpmrA). β-Galactosidase activity was determined as described in the Materials and Methods. The data are the averages ± standard deviations (error bars) of at least three different experiments. MU, Miller units.

FIG. 8.

Icm/Dot-dependent translocation of CpxR-regulated genes. (A) Wild-type strain JR32 (grey bars) and icmT mutant GS3011 (open bars) harboring the CyaA fusion proteins (indicated below the bars) were used to infect HL-60-derived human macrophages, and the cAMP levels of the infected cells were determined as described in Materials and Methods. (B) Effect of the cpxR deletion mutant on the translocation of sidM and cegC4 cyaA fusions in wild-type strain JR32 (grey bars), cpxR mutant OG2002 (black bars), and icmT mutant GS3011 (open bars). The fusions were cloned under control of P_tac or under control of their natural promoter. The data are the means for the amount of cAMP per well obtained in three independent experiments, and the error bars indicate standard deviations.

FIG. 9.

Schematic diagrams of CpxR-regulated promoters. The CpxR boxes, PmrA boxes, −10 promoter elements, and transcription start sites (arrow) are indicated along with their orientations with respect to the first ATG codon of the gene (black region). For icmV and icmW (upper bar) the relevant regulatory elements for each of the two genes are indicated. The genes that were found to have a certain type of regulation are indicated on the right; the genes that were found to be repressed by CpxR are underlined.

FIG. 10.

Model of regulation of the L. pneumophila Icm/Dot pathogenesis system. The three two-component systems (CpxRA, PmrAB, and LetAS) which were found to be involved in the regulation of pathogenesis-related genes and/or phenotypes are shown. The environmental signals sensed by CpxA and PmrB are currently not known, and the phosphorylation of these components is expected to activate by transfer of the phosphate group to the cognate response regulators CpxR and PmrA, respectively, which then activate or repress the transcription of their target genes. The target genes of CpxR and PmrA overlap in some cases, but only CpxR was found to control the expression of icm/dot genes; both regulators control the expression of IDTS-encoding genes. No target genes are currently known for the LetA-CsrA regulatory cascade. The dotted line indicates connections that might be indirect.