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. 2006 Jun;74(6):3125-33.
doi: 10.1128/IAI.02061-05.

Mycobacterium marinum Erp is a virulence determinant required for cell wall integrity and intracellular survival

Christine L Cosma  1 , Kathryn Klein, Rosa Kim, Dana Beery, Lalita Ramakrishnan
Affiliations

Mycobacterium marinum Erp is a virulence determinant required for cell wall integrity and intracellular survival

Christine L Cosma et al. Infect Immun. 2006 Jun.

Abstract

The Mycobacterium tuberculosis exported repetitive protein (Erp) is a virulence determinant required for growth in cultured macrophages and in vivo. To better understand the role of Erp in Mycobacterium pathogenesis, we generated a mutation in the erp homologue of Mycobacterium marinum, a close genetic relative of M. tuberculosis. erp-deficient M. marinum was growth attenuated in cultured macrophage monolayers and during chronic granulomatous infection of leopard frogs, suggesting that Erp function is similarly required for the virulence of both M. tuberculosis and M. marinum. To pinpoint the step in infection at which Erp is required, we utilized a zebrafish embryo infection model that allows M. marinum infections to be visualized in real-time, comparing the erp-deficient strain to a DeltaRD1 mutant whose stage of attenuation was previously characterized in zebrafish embryos. A detailed microscopic examination of infected embryos revealed that bacteria lacking Erp were compromised very early in infection, failing to grow and/or survive upon phagocytosis by host macrophages. In contrast, DeltaRD1 mutant bacteria grow normally in macrophages but fail to induce host macrophage aggregation and subsequent cell-to-cell spread. Consistent with these in vivo findings, erp-deficient but not RD1-deficient bacteria exhibited permeability defects in vitro, which may be responsible for their specific failure to survive in host macrophages.

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Figures

FIG. 1.
FIG. 1.
The M. marinum erp::aph mutant is growth attenuated in both murine macrophages and frogs. (A) J774A.1 macrophages were infected as described in Materials and Methods with strains M (○, 8.8 × 103 CFU), KK33 (▴, 7.7 × 103 CFU), KK60 (×, 4.4 × 103 CFU), or RD1-6 (▪, 1.6 × 104 CFU). All datum points were obtained in triplicate, and the means and standard deviations are shown. The assay was performed two additional times with similar results. (B) Leopard frogs (n = 5) were infected as described in Materials and Methods with strain M (○, 6.7 × 105 CFU), KK33 (▴, 2.9 × 105 CFU), KK60 (×, 2.5 × 105 CFU), or RD1-6 (▪, 2.7 × 105 CFU). Mean log CFU per liver are shown with the standard deviation, and the Students unpaired t test was used to assess significance. For the 2-week time point, P < 0.05 (KK33 versus KK60). For the 12-week time point, P < 0.05 (M versus RD1-6 and M versus KK33) and P < 0.01 (KK33 versus KK60). Similar results were obtained from the spleen (data not shown).
FIG. 2.
FIG. 2.
erp::aph and ΔRD1 mutants exhibit reduced virulence and growth in zebrafish embryos. (A) Kaplan-Meier curves for zebrafish embryos infected with the following doses (CFU): strain M (20 CFU), KK33 (38 CFU), RD1-6 (22 CFU), and KK60 (53 CFU). The data are shown as percent surviving each day postinfection. (B) Embryos were infected with the following doses (CFU): KK11 (161 CFU), CC59 (200 CFU), and KK47 (226 CFU). Embryos were imaged at both 1 h and at 5 days postinfection by using a fluorescence microscope at ×40 total magnification. Images shown are 10-s exposures of green fluorescence. Some autofluorescence is visible along the dorsal side due to the presence of melanophores.
FIG. 3.
FIG. 3.
erp::aph mutant bacteria fail to grow or survive in zebrafish embryo macrophages. Ten embryos were infected per strain and were examined by fluorescence and DIC microscopy at 5 days postinfection at ×600 magnification. Mean infection doses (CFU) were as follows: KK11 (158 CFU), CC59 (214 CFU), and KK47 (279 CFU). Shown are representative images of infected macrophages for KK11 (A), CC59 (B), and KK47 (C). For each panel, upper images are overlays of DIC (grayscale) and bacterial fluorescence (green), while bottom images are fluorescence only. Scale bar, 25 μm. Arrows indicate heavily infected macrophages, arrowheads indicate lightly infected macrophages, and the asterisk indicates an aggregate of infected macrophages. For the bottom panels of panel C, the brightness and contrast were adjusted to allow better visualization of KK47 bacteria, which were dimmer than the other strains. (D) Schematic diagram of the zebrafish embryo. (E) Average proportion of macrophages containing <5, 5 to 10, or >10 bacteria, when embryos were infected with wild-type, ΔRD1, or erp::aph bacteria. Significance was assessed by ANOVA, comparing the proportion in each category across the three strains. The mean number of individual infected macrophages did not differ between strains. See the text for additional details.
FIG. 4.
FIG. 4.
Proposed pathway for the early stages of Mycobacterium pathogenesis, highlighting the steps impacted by the Erp and RD1 virulence determinants. See the text for details.
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