Essential role for the response regulator PmrA in Coxiella burnetii type 4B secretion and colonization of mammalian host cells

Abstract

Successful host cell colonization by the Q fever pathogen, Coxiella burnetii, requires translocation of effector proteins into the host cytosol by a Dot/Icm type 4B secretion system (T4BSS). In Legionella pneumophila, the two-component system (TCS) PmrAB regulates the Dot/Icm T4BSS and several additional physiological processes associated with pathogenesis. Because PmrA consensus regulatory elements are associated with some dot/icm and substrate genes, a similar role for PmrA in regulation of the C. burnetii T4BSS has been proposed. Here, we constructed a C. burnetii pmrA deletion mutant to directly probe PmrA-mediated gene regulation. Compared to wild-type bacteria, C. burnetii ΔpmrA exhibited severe intracellular growth defects that coincided with failed secretion of effector proteins. Luciferase gene reporter assays demonstrated PmrA-dependent expression of 5 of 7 dot/icm operons and 9 of 11 effector-encoding genes with a predicted upstream PmrA regulatory element. Mutational analysis verified consensus sequence nucleotides required for PmrA-directed transcription. RNA sequencing and whole bacterial cell mass spectrometry of wild-type C. burnetii and the ΔpmrA mutant uncovered new components of the PmrA regulon, including several genes lacking PmrA motifs that encoded Dot/Icm substrates. Collectively, our results indicate that the PmrAB TCS is a critical virulence factor that regulates C. burnetii Dot/Icm secretion. The presence of PmrA-responsive genes lacking PmrA regulatory elements also suggests that the PmrAB TCS controls expression of regulatory systems associated with the production of additional C. burnetii proteins involved in host cell parasitism.

FIG 1

C. burnetii ΔpmrA has severe intracellular growth defects. Replication of wild-type C. burnetii, the ΔpmrA mutant, and the complemented mutant (comp) in ACCM-2 (A) and Vero cells (B). Fold increases in C. burnetii genome equivalents (GE) after 6 days of growth are depicted. Results are expressed as the means of results from two biological replicates from four independent experiments. Error bars indicate the standard deviations from the means, and asterisks indicate a statistically significant difference (P < 0.0001) compared to values for wild-type C. burnetii. (C) Confocal fluorescence micrographs of Vero cells infected for 4 days with wild-type C. burnetii, the ΔpmrA mutant, or the complemented mutant. LAMP3 (green) and C. burnetii (red) are stained by indirect immunofluorescence, and DNA (blue) is stained with DAPI. Bar, 5 μm.

FIG 2

Transcription of pmrA declines following entry of C. burnetii into stationary phase. Luciferase assays were conducted after 2, 3, 4, 6, 10, and 14 days of growth in axenic medium of wild-type C. burnetii carrying lux fused to the pmrA promoter region. Bioluminescent readings are expressed as relative light units (RLU). Results are expressed as the means of results from two biological replicates from three independent experiments. Error bars indicate the standard deviations from the means, and asterisks indicate a statistically significant difference (*, P < 0.05; ****, P < 0.0001).

FIG 3

The C. burnetii dot/icm locus is regulated by PmrA. (A) Linkage and predicted operon structures of C. burnetii dot/icm genes. Operons with upstream PmrA regulatory elements are colored. Operon-specific promoters are indicated with an arrow below the first gene of each predicted operon. icmF is truncated due to a frameshift (icmF^FS). Antibodies specific for proteins encoded by boldface genes were used in immunoblotting, described below. (B) Luciferase activities of the lux operon transcriptionally fused to the promoter regions of control genes (left) and dot/icm genes (right). Assays were conducted after 4 days of growth in axenic medium of wild-type C. burnetii and the ΔpmrA mutant expressing lux fusions. Bioluminescent readings are expressed as relative light units (RLU). Results are expressed as the means of results from two biological replicates from three independent experiments. Error bars indicate the standard deviations from the means, and asterisks indicate a statistically significant difference (P < 0.0001) between wild-type C. burnetii and the ΔpmrA mutant. (C) Immunoblots of lysates of wild-type C. burnetii (lane 1), the ΔpmrA mutant (lane 2), and the complemented mutant (lane 3) probed with anti-IcmD (αIcmD), -IcmK, and -DotA antibodies. Probing for EF-Ts was conducted as a loading control.

FIG 4

Expression of C. burnetii Dot/Icm substrates is regulated by PmrA. Luciferase activities of the lux operon transcriptionally fused to the promoter regions of Dot/Icm substrate-coding genes with predicted pmrA regulatory elements. Assays were conducted after 4 days of growth in axenic medium of wild-type C. burnetii and the ΔpmrA mutant expressing lux fusions. Bioluminescent readings are expressed as relative light units (RLU). Results are expressed as the means of results from two biological replicates from three independent experiments. Error bars indicate the standard deviations from the means, and asterisks indicate a statistically significant difference between wild-type C. burnetii and the ΔpmrA mutant (*, P < 0.05; **, P < 0.005; ***, P < 0.0005; ****, P < 0.0001).

FIG 5

PmrA is required for secretion by the Dot/Icm T4BSS. Cytosolic levels of cAMP were measured following infection of THP-1 macrophages for 2 days with wild-type C. burnetii or the ΔpmrA mutant expressing CyaA alone or CyaA fused to the previously defined Dot/Icm substrates CpeD and CpeE. Elevated levels of cAMP indicating secretion were observed only with wild-type C. burnetii expressing CyaA-CpeD or -CpeE fusion proteins. Results shown are from one experiment conducted in duplicate and are representative of three independent experiments. Error bars indicate the standard deviations from the means.

FIG 6

Mutational analysis defines regulatory element nucleotides required for PmrA-driven expression. (A) Regions upstream of pmrA-regulated cbu0021 and the CoxigA gene. Boldface nucleotides show mutational changes of the two TTAA regions within the predicted PmrA regulatory element (MUT1 and MUT2), while underlined nucleotides show mutational changes within the −10 promoter region (MUT3). (B) Luciferase activity of wild-type (WT) C. burnetii expressing transcriptional proteins of the lux operon fused to nonmutated promoter regions or regions containing the MUT1, MUT2, or MUT3 mutation. Luciferase activity was also assessed for the ΔpmrA mutant expressing lux fused to nonmutated promoters and wild-type C. burnetii and the ΔpmrA mutant expressing lux alone. Assays were conducted after 4 days of growth in axenic medium. Bioluminescent readings are expressed as relative light units (RLU). Results are expressed as the means of results from two biological replicates from three independent experiments. Error bars indicate the standard deviations from the means, and asterisks indicate a statistically significant difference (P < 0.0001) with respect to wild-type C. burnetii expressing a nonmutated promoter lux fusion.

FIG 7

RNA-seq reveals new Dot/Icm substrates. Cytosolic levels of cAMP were measured following infection of THP-1 macrophages for 2 days with wild-type C. burnetii or the ΔdotA mutant expressing CyaA alone or CyaA fused to possible Dot/Icm substrates. Elevated levels of cAMP indicating secretion were observed for CBU0122, CBU1530, CBU1614, CBU1685, CBU1686, and CBU1752. Results shown are from one experiment conducted in duplicate and are representative of three independent experiments. Error bars indicate the standard deviations from the means.