Dot/Icm type IVB secretion system requirements for Coxiella burnetii growth in human macrophages - PubMed (original) (raw)
Dot/Icm type IVB secretion system requirements for Coxiella burnetii growth in human macrophages
Paul A Beare et al. mBio. 2011.
Abstract
Central to Q fever pathogenesis is replication of the causative agent, Coxiella burnetii, within a phagolysosome-like parasitophorous vacuole (PV) in mononuclear phagocytes. C. burnetii modulates PV biogenesis and other host cell functions, such as apoptotic signaling, presumably via the activity of proteins delivered to the host cytosol by a Dot/Icm type IVB secretion system (T4BSS). In this study, we utilized a C. burnetii strain carrying IcmD inactivated by the Himar1 transposon to investigate the requirements for Dot/Icm function in C. burnetii parasitism of human THP-1 macrophage-like cells. The icmD::Tn mutant failed to secrete characterized T4BSS substrates, a defect that correlated with deficient replication, PV development, and apoptosis protection. Restoration of type IVB secretion and intracellular growth of the icmD::Tn mutant required complementation with icmD, -J, and -B, indicating a polar effect of the transposon insertion on downstream dot/icm genes. Induction of icmDJB expression at 1 day postinfection resulted in C. burnetii replication and PV generation. Collectively, these data prove that T4BSS function is required for productive infection of human macrophages by C. burnetii. However, illustrating the metabolic flexibility of C. burnetti, the icmD::Tn mutant could replicate intracellularly when sequestered in a PV generated by wild-type bacteria, where Dot/Icm function is provided in trans, and within a phenotypically similar PV generated by the protozoan parasite Leishmania amazonensis, where host cells are devoid of Dot/Icm T4BSS effector proteins.
Figures
FIG 1
A Himar1 transposon insertion inactivates C. burnetii icmD. (A) Schematic showing the location of a Himar1 transposon insertion in icmD. The transposon inserted at base 75 of the 414-bp gene. ITR, inverted terminal repeat; H, HindIII site. (B) Southern blot assay of HindIII-digested genomic DNA from NMII and the icmD::Tn mutant hybridized with a probe specific to icmD (black bar in panel A). A 1.62-kb _icmD_-containing HindIII fragment was disrupted in the icmD mutant. (C) Immunoblotting of lysates from NMII and the icmD::Tn mutant grown in ACCM-2 for 6 days. IcmD-specific antiserum detected the 14.3-kDa IcmD protein in lysates from NMII but not in those of the icmD::Tn mutant.
FIG 2
icmD is required for intracellular growth of C. burnetii. (A) One-step growth curves of NMII and the icmD::Tn mutant grown in ACCM-2 (left panel) or THP-1 macrophages (right panel). Growth was measured by enumerating GE. ACCM-2 results are expressed as the means of three biological replicates from one experiment and are representative of two independent experiments. Error bars indicate the standard deviations of the means. THP-1 macrophage results are expressed as the means of two biological replicates from one experiment and are representative of three independent experiments. Error bars indicate the standard deviations of the means. (B) Fluorescence micrographs of THP-1 macrophages infected for 6 days with NMII or the icmD::Tn mutant. C. burnetii (red) is stained by indirect immunofluorescence, and DNA (blue) is stained with DAPI. Cells contained multiplying NMII harbored within 1 or 2 large and spacious PVs per cell, while the icmD::Tn mutant was typically observed as a single organism in dispersed, tight-fitting PVs. Bar, 20 µm.
FIG 3
icmD is required for cytosolic delivery of T4BSS substrates. Cytosolic levels of cAMP following infection of THP-1 macrophages for 2 days with NMII or the icmD::Tn mutant expressing CyaA alone or CyaA fusions to the previously defined Dot/Icm substrates CpeD and CpeE. Elevated levels of cAMP, indicating secretion, were observed only with NMII expressing CyaA-CpeD or -CpeE fusion protein. Results shown are from one experiment conducted in duplicate and are representative of two independent experiments. Error bars indicate the standard errors of the means. Immunoblotting signals of CyaA and CyaA-effector fusion proteins, depicted above respective histogram bars, show comparable levels of protein expression by NMII and the icmD::Tn mutant after 6 days of growth in ACCM-2. Blots were probed with anti-CyaA antibody.
FIG 4
icmD is required for inhibition of apoptosis. THP-1 macrophages were infected with NMII or the icmD::Tn mutant for 2 days and then treated with staurosporine for 4 h to induce apoptosis. Uninfected cells were used as a control. (A) Detection of cleaved PARP-positive nuclei. C. burnetii (red) and cleaved PARP (green) were labeled by indirect immunofluorescence. DNA (blue) was stained with DAPI. Bar, 20 µm. (B) Enumeration of cleaved, PARP-positive (apoptotic) nuclei. The results shown are from one experiment conducted in triplicate and are representative of two independent experiments. A total of 750 cells were counted for each condition. Error bars indicate the standard deviations of the means, and asterisks indicate a statistically significant difference (P < 0.0005) from cells infected with NMII.
FIG 5
Coinfection with NMII or L. amazonensis rescues intracellular growth of the icmD::Tn mutant. (Top) THP-1 macrophages were coinfected for 6 days with NMII expressing mCherry red fluorescent protein (24) and the icmD::Tn mutant. Both C. burnetii strains (green) and LAMP-3 (blue) were stained by indirect immunofluorescence. Confocal fluorescence micrographs show coinhabited PVs with replicating NMII (yellow due to red and green overlay) and icmD::Tn mutant bacteria (green only). Bar, 5 µm. (Bottom) Vero cells were infected for 1 day with L. amazonensis promasitgotes expressing GFP and then superinfected for 4 days with the icmD::Tn mutant. The icmD::Tn mutant (red) and LAMP-3 (blue) were stained by indirect immunofluorescence. Confocal fluorescence micrographs showed coinhabited PVs with replicating L. amazonensis and icmD::Tn mutant bacteria. Bar, 5 µm.
FIG 6
Intracellular growth of the icmD::Tn mutant requires the expression of icmD, icmJ, and icmB. (A) THP-1 macrophages infected for 6 days with the icmD::Tn mutant transformed with a Tn_7_ construct containing icmD, icmDJ, or icmDJB under the control of a native C. burnetii promoter. C. burnetii (red) was stained by indirect immunofluorescence, and DNA (blue) was stained with DAPI. Fluorescence micrographs show large PVs harboring replicating C. burnetii only within cells infected with the icmD::Tn mutant transformed with Tn_7_::icmDJB. Bar, 20 µm. (B) Increases in GE of NMII or the icmD::Tn mutant transformed with Tn_7_::icmD, Tn_7_::icmDJ, or Tn_7_::icmDJB after 6 days of growth in THP-1 macrophages. The asterisk indicates a statistically significant difference (P < 0.05) between the icmD::Tn mutant and the mutant transformed with Tn_7_::icmDJB. (C) icmD, icmJ, icmB, and CBU1169 transcript levels in NMII, the icmD mutant, and the icmD mutant complemented with Tn_7_::icmDJB (icmD::Tn comp) after 4 days of growth in ACCM-2. Expression is shown as relative light units (RLU). (D) Immunoblotting of lysates of C. burnetii grown in ACCM-2 for 6 days showing production of IcmD by the complemented mutant and NMII. The experiments depicted in panels B and C were performed in triplicate, and error bars indicate the standard errors of the means.
FIG 7
Induction of icmDJB expression after infection rescues the growth of the icmD::Tn mutant. (A) aTc-inducible expression of icmD. The icmD::Tn mutant transformed with a Tn_7_ construct containing aTc-inducible icmDJB was cultivated in ACCM-2 for 3 days and then induced with aTc for 1 day. Immunoblotting showed aTc-induced synthesis of IcmD. (B and C) aTc induction of icmDJB concurrent with or after infection results in PV production and significant replication by the icmD::Tn mutant at 6 days postinduction. THP-1 macrophages were infected with the icmD::Tn mutant transformed with Tn_7_::TetRA-icmDJB, where aTc was added to the culture medium at 0 h (CI) or 1 day (DI) p.i. Transformant infections were also conducted without aTc (NI) and with NMII. (B) One asterisk (P < 0.05) and three (P < 0.0005) asterisks indicate significant differences in GE. The experiment was performed three times in duplicate, and error bars indicate the standard deviations of the means. (C) The icmD::Tn mutant grown under CI, but not NI, conditions forms typical large, LAMP-3-positive PVs at 6 days p.i. C. burnetii and LAMP-3 were stained by indirect immunofluorescence and appear red and green, respectively, in the merged image. Bar, 10 µm. (D) Induced expression of icmDJB restores T4BSS function by the icmD::Tn mutant. THP-1 macrophages were infected with the icmD::Tn mutant transformed with a Tn_7_ construct encoding aTc-inducible icmJBD and a CyaA fusion to CpeD expressed from the constitutive CBU1169 promoter. C. burnetii expressing CyaA alone was used as a control. For induction of icmDJB expression, aTc was added at 1 day p.i. CyaA assays were conducted at 2 days p.i. Elevated levels of cAMP, indicating secretion, were observed only with the complemented mutant expressing CyaA-CpeD under inducing conditions. The results shown are from one experiment conducted in duplicate and are representative of two independent experiments. Error bars indicate the standard errors of the means.
References
- Voth DE, Heinzen RA. 2007. Lounging in a lysosome: the intracellular lifestyle of Coxiella burnetii. Cell. Microbiol. 9:829–840 - PubMed
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