CtpV: a putative copper exporter required for full virulence of Mycobacterium tuberculosis

Abstract

Copper is a required micronutrient that is also toxic at excess concentrations. Currently, little is known about the role of copper in interactions between bacterial pathogens and their human hosts. In this study, we elucidate a mechanism for copper homeostasis in the human pathogen Mycobacterium tuberculosis via characterization of a putative copper exporter, CtpV. CtpV was shown to be required by M. tuberculosis to maintain resistance to copper toxicity. Furthermore, the deletion of ctpV resulted in a 98-gene transcriptional response, which elucidates the increased stress experienced by the bacteria in the absence of this detoxification mechanism. Interestingly, although the ΔctpV mutant survives close to the wild-type levels in both murine and guinea pig models of tuberculosis, animals infected with the ΔctpV mutant displayed decreased lung damage, and mutant-infected mice had a reduced immune response to the bacteria as well as a significant increase in survival time relative to mice infected with wild-type M. tuberculosis. Overall, our study provides the first evidence for a connection between bacterial copper response and the virulence of M. tuberculosis, supporting the hypothesis that copper response could be important to intracellular pathogens, in general.

Figure 1. Construction of ΔctpV mutant using the M. tuberculosis H37Rv strain

A. The ΔctpV mutant was constructed with homologous recombination via pML21, a derivative of pPR27, which resulted in the deletion of 2.1 kB of the ctpV coding region (represented in black) and the insertion of a 3.5 kB region encoding a hygromycin resistance cassette. B. The mutant was confirmed with Southern blots BamHI-digested genomic DNA (5 μg) from H37Rv or ΔctpV. Incubation with a P32 labeled probe for the remaining ctpV region (left, gray arrows) revealed the increased size of the band in ΔctpV (7.5 kB) relative to H37Rv (4.7 kB) resulting from the loss of two BamHI restriction enzyme sites within the ctpV coding region (see 1A) when it was replaced with HygR, which contains no BamHI sites. Additionally, a probe for the hygromycin resistance cassette (right, black arrows) hybridized only to the BamHI-digested gDNA from ΔctpV. C. The polarity of the ctpV knockout mutant was addressed using RT-PCR to check for transcription of its downstream gene. In the wild-type strain (left), positive bands show that ctpV and the downstream gene rv0970 are both encoded in the genome and transcribed (able to be amplified from cDNA), with negative amplification from RNA shown as a negative control. In the isogenic mutant ΔctpV (right), the ctpV coding region is not present in the genome or as cDNA, but transcript for the downstream gene rv0970 is detectable.

Figure 2. Growth experiments of wild-type Mtb (H37Rv), ΔctpV, and ΔctpV::ctpV

A. Growth experiments of H37Rv, its isogenic mutant ΔctpV, and the complemented strain ΔctpV:ctpV in 7H9+ADC liquid media. Cultures were seeded from stock to OD₆₀₀ 0.10 and allowed to grow for 14 days, with CFUs taken at 0, 4, 8, and 14 days via plating on 7H10+ADC solid media. The average of two biological replicates is shown. Limit of detection = 10 CFUs. B. Cultures (30 ml) were grown in copper-free Sauton’s liquid media, with defined amounts of CuCl₂ added. Colony forming units (CFUs) were determined at 0, 4, 8, and 14 days post-exposure via plating on 7H10+ADC solid media, plus hygromycin in the case of the mutant and complemented strains. The average of two biological replicates is shown. Growth at 0 and 50 μM were identical between the three strains, so wild type data is shown for figure clarity (full data available in Fig. S2). Using a t-test, the difference between WT and ΔctpV at d8 is statistically significant (p=0.001), as is the difference between WT and ΔctpV:ctpV at d14 (p=0.02). Limit of detection is 10 CFUs. No CFUs were detected for ΔctpV at day 8 or for WT and ΔctpV at day 14, as determined after plating 100 uL of the culture in triplicate and incubating for 6 weeks.

Figure 2. Growth experiments of wild-type Mtb (H37Rv), ΔctpV, and ΔctpV::ctpV

A. Growth experiments of H37Rv, its isogenic mutant ΔctpV, and the complemented strain ΔctpV:ctpV in 7H9+ADC liquid media. Cultures were seeded from stock to OD₆₀₀ 0.10 and allowed to grow for 14 days, with CFUs taken at 0, 4, 8, and 14 days via plating on 7H10+ADC solid media. The average of two biological replicates is shown. Limit of detection = 10 CFUs. B. Cultures (30 ml) were grown in copper-free Sauton’s liquid media, with defined amounts of CuCl₂ added. Colony forming units (CFUs) were determined at 0, 4, 8, and 14 days post-exposure via plating on 7H10+ADC solid media, plus hygromycin in the case of the mutant and complemented strains. The average of two biological replicates is shown. Growth at 0 and 50 μM were identical between the three strains, so wild type data is shown for figure clarity (full data available in Fig. S2). Using a t-test, the difference between WT and ΔctpV at d8 is statistically significant (p=0.001), as is the difference between WT and ΔctpV:ctpV at d14 (p=0.02). Limit of detection is 10 CFUs. No CFUs were detected for ΔctpV at day 8 or for WT and ΔctpV at day 14, as determined after plating 100 uL of the culture in triplicate and incubating for 6 weeks.

Figure 3

Bulk analysis of copper present in cell pellets of M. smegmatis containing only the empty pSE100 vector as a control, or M. smegmatis expressing the cso operon, as determined by neutron activation analysis. Values are normalized to cell mass and expressed as PPM (microgram copper per gram of biomass) copper. The experiment was repeated twice and each experiment was done in triplicate. Values for M. smegmatis + pSE100 are significantly higher than those for M. smegmatis::cso within each replicate as determined by a T-test (replicate 1 p=0.02, replicate 2 p=0.002). Lines represent average value for each replicate.

Figure 4

Transcriptional responses to the deletion of ctpV under toxic levels of copper. Transcript levels of all genes within the Mtb genome predicated to encode metal-transporting P-type ATPases at 500 μM vs. 0 μM copper, in both H37Rv and ΔctpV, were measured using qRT-PCR. Data are displayed as fold-change relative to expression at 0 μM Cu, and are normalized to expression of 16S rRNA. Averaged data from two biological replicates are shown.

Figure 5

Guinea pigs infection with M. tuberculosis and its isogenic mutant ΔctpV. A) Bacterial colonization of guinea pig lungs after aerosol infection with either wild-type H37Rv, ΔctpV, or the complemented strain ΔctpV::ctpV. CFUs were determined following homogenization of lungs from infected guinea pigs. Colonization levels between H37Rv and ΔctpV are statistically different at 21 days post-infection (p=0.04) but not at 42 days post-infection. The experiment was performed once with 3–4 animals per strain per time point. B) Pathology of infected guinea pigs. a) Necropsy of lungs of guinea pigs infected with the H37Rv, ΔctpV, and ΔctpV::ctpV at 42 days post-infection. b) Hematoxylin and eosin (H&E) stained guinea pig tissue (40× magnification, scale bar=500 μm) at 42 days post-infection are shown.

Figure 5

Guinea pigs infection with M. tuberculosis and its isogenic mutant ΔctpV. A) Bacterial colonization of guinea pig lungs after aerosol infection with either wild-type H37Rv, ΔctpV, or the complemented strain ΔctpV::ctpV. CFUs were determined following homogenization of lungs from infected guinea pigs. Colonization levels between H37Rv and ΔctpV are statistically different at 21 days post-infection (p=0.04) but not at 42 days post-infection. The experiment was performed once with 3–4 animals per strain per time point. B) Pathology of infected guinea pigs. a) Necropsy of lungs of guinea pigs infected with the H37Rv, ΔctpV, and ΔctpV::ctpV at 42 days post-infection. b) Hematoxylin and eosin (H&E) stained guinea pig tissue (40× magnification, scale bar=500 μm) at 42 days post-infection are shown.

Figure 6

Murine infection with ΔctpV. A) Bacterial colonization of mouse lungs after aerosol infection with either wild-type H37Rv, its isogenic mutant ΔctpV, or the complemented strain ΔctpV::ctpV. CFUs were determined via homogenization of lungs from infected mice (N=3–5 per time point) in PBS and plating on 7H10+ADC, with hygromycin added in the case of the mutant and complemented strain. Colonization levels between the three strains did not display a statistically significant difference over the course of the infection. The experiment was performed twicefor early time points and once for late time points. B) Survival of mouse groups after aerosol infection with H37Rv, ΔctpV, or ΔctpV::ctpV. Survival is displayed as the time from infection (week 0) until the time declared morbid by animal care staff. Morbid mice were subsequently euthanized, with tuberculosis determined as the cause of illness via necropsy. A log-rank statistical test was used to analyze survival of mice groups. Survival of mice infected with H37Rv was significantly different from those infection with ΔctpV (p-value =0.002), and those infected with the ΔctpV::ctpV strain (p-value=0.02). Experiment was performed once with 5–10 mice/group.

Figure 6

Murine infection with ΔctpV. A) Bacterial colonization of mouse lungs after aerosol infection with either wild-type H37Rv, its isogenic mutant ΔctpV, or the complemented strain ΔctpV::ctpV. CFUs were determined via homogenization of lungs from infected mice (N=3–5 per time point) in PBS and plating on 7H10+ADC, with hygromycin added in the case of the mutant and complemented strain. Colonization levels between the three strains did not display a statistically significant difference over the course of the infection. The experiment was performed twicefor early time points and once for late time points. B) Survival of mouse groups after aerosol infection with H37Rv, ΔctpV, or ΔctpV::ctpV. Survival is displayed as the time from infection (week 0) until the time declared morbid by animal care staff. Morbid mice were subsequently euthanized, with tuberculosis determined as the cause of illness via necropsy. A log-rank statistical test was used to analyze survival of mice groups. Survival of mice infected with H37Rv was significantly different from those infection with ΔctpV (p-value =0.002), and those infected with the ΔctpV::ctpV strain (p-value=0.02). Experiment was performed once with 5–10 mice/group.

Figure 7. Pathology of murine infection with H37Rv, ΔctpV, and ΔctpV::ctpV

A) H&E stained mouse lung tissue (40× magnification, scale bar = 500 μm) at 8 weeks post-infection. Inset images (1000× magnification, scale bar = 20 μm) show the Mtb bacilli (purple) which were visible in lung tissue starting from 8 weeks forward. B) H&E stained mouse lung tissue (40× magnification) at 38 weeks post-infection. C) Immunohistochemisty of infected murine lung tissue at 8 weeks post-infection. Murine lung tissue was sectioned and stained with antibody for mouse IFN-γ, which appears brown in the images (40× magnification). Inset images (200× magnification) of lesions displaying IFN- γ expression. D) Immunohistochemisty of infected murine lung tissue at 38 weeks post-infection. Inset images (200× magnification) of lesions displaying IFN- γ expression.