Essential Role of the ESX-5 Secretion System in Outer Membrane Permeability of Pathogenic Mycobacteria

Abstract

Mycobacteria possess different type VII secretion (T7S) systems to secrete proteins across their unusual cell envelope. One of these systems, ESX-5, is only present in slow-growing mycobacteria and responsible for the secretion of multiple substrates. However, the role of ESX-5 substrates in growth and/or virulence is largely unknown. In this study, we show that esx-5 is essential for growth of both Mycobacterium marinum and Mycobacterium bovis. Remarkably, this essentiality can be rescued by increasing the permeability of the outer membrane, either by altering its lipid composition or by the introduction of the heterologous porin MspA. Mutagenesis of the first nucleotide-binding domain of the membrane ATPase EccC5 prevented both ESX-5-dependent secretion and bacterial growth, but did not affect ESX-5 complex assembly. This suggests that the rescuing effect is not due to pores formed by the ESX-5 membrane complex, but caused by ESX-5 activity. Subsequent proteomic analysis to identify crucial ESX-5 substrates confirmed that all detectable PE and PPE proteins in the cell surface and cell envelope fractions were routed through ESX-5. Additionally, saturated transposon-directed insertion-site sequencing (TraDIS) was applied to both wild-type M. marinum cells and cells expressing mspA to identify genes that are not essential anymore in the presence of MspA. This analysis confirmed the importance of esx-5, but we could not identify essential ESX-5 substrates, indicating that multiple of these substrates are together responsible for the essentiality. Finally, examination of phenotypes on defined carbon sources revealed that an esx-5 mutant is strongly impaired in the uptake and utilization of hydrophobic carbon sources. Based on these data, we propose a model in which the ESX-5 system is responsible for the transport of cell envelope proteins that are required for nutrient uptake. These proteins might in this way compensate for the lack of MspA-like porins in slow-growing mycobacteria.

Fig 1. Expression of MycP5 is essential for growth of M. bovis BCG.

A, B) The BCG-Pasteur c-mycP5-tet-on (A) and c-mycP5-tet-off (B) mutants were grown for 21 days on Middlebrook 7H10 agar plates containing the indicated ATc concentrations. Full growth of c-mycP5-tet-on was only observed at 10 μg/ml ATc, whereas this concentration of ATc did not completely abolish colony growth of c-mycP5-tet-off. C, D) Resazurin reduction is dependent on ATc-induced expression/repression of mycP5. Cells of the BCG-Pasteur c-mycP5-tet-on (C), or c-mycP5-tet-off (D) mutants were grown as liquid cultures in 96-well microtiter plates for 6 days at 37°C at the indicated ATc concentrations, after which 10% Alamar Blue was added and fluorescence (585 nm) was measured after 16 h incubation to determine metabolic activity as a correlate of growth. Values are means of triplicates; error bars represent the standard deviation.

Fig 2. mas mutation or introduction of mspA lead to increased outer membrane permeability.

A, B) Sensitivity to a combination of ampicillin and clavulanic acid of different M. marinum strains was measured by performing a disc diffusion assay on the indicated strains and measuring the surface of the growth inhibition zone. The mas transposon (mas::tn) mutants exhibit increased sensitivity, independent of the presence of an intact copy of eccC5 (A). Similarly, introduction of pSMT3::mspA also leads to an increase in sensitivity independent on the presence of mycP5 (B). Values are the means of triplicates; error bars indicate the standard deviation. C) Uptake of EtBr, measured by flow cytometric analysis. M. marinum wild-type (dark grey) and M. marinum::mspA (black) were incubated with 20 μM EtBr for 60 min and 20.000 events were analyzed for their fluorescence intensity at 585/540 nm. Light-grey lines indicate unstained samples. All measurements are depicted in duplicates and are representatives of three independent experiments. ΔeccC5-C and ΔmycP5-C refer to the complementation strains of the M. marinum ΔeccC5 or the ΔmycP5 mutants complemented with pMV::eccBC5 or pMV::mycP5 respectively.

Fig 3. Secretion analysis of ESX-5 mutant strains.

A) A schematic representation of the ESX-5 region of M. marinum with the different ESX-5 mutations used in this study. Bars above the gene cluster indicate regions deleted by targeted knock-out mutagenesis. Arrows below indicate position and orientation of transposons (named LA1 to LA12) in mutants of the parental strain M. marinum::mspA defective in ESX-5 dependent secretion. B) Secretion analysis of M.marinum::mspA (WT::mspA), a mycP5 transposon mutant (mycP5::tn, corresponding to LA9 in (A)) and the complemented version of this strain (mycP5::tn-C). Secreted proteins (S) were separated from bacterial cells (P) by centrifugation. In addition, surface-associated proteins were enriched from the bacterial cells by extraction with 0.5% Genapol X-080 (GS) and separated from non-solubilized proteins (GP) by centrifugation. All fractions were analyzed for the presence of PE_PGRS proteins by immunoblotting. GroEL2 staining was used as a loading and lysis control. C) Expression of EccB₅ and EspG₅ was analyzed by immunoblotting of total cell lysates of wild-type M. marinum (WT), the Δesx-5::mspA mutant and the complemented Δesx-5::esx-5tub strain. D) The same strains as under (C) were analyzed for their ability to express and secrete PE_PGRS proteins following the same procedure as under (B).

Fig 4. Role of NBDs domains of EccC5 in ESX-5 dependent secretion and membrane complex assembly.

A) Predicted transmembrane domains (dark grey), and NBD (light grey) of EccC₅ are indicated. The positions of relevant residues are depicted with a black bar. The numbers represent the position in amino acids. B) Secretion of PE_PGRS proteins in the different EccC₅ mutant strains was analyzed by immunoblot of supernatants and cell pellets of wild-type (WT) M. marinum and the eccC5 deletion strain (ΔeccC5) complemented with various eccC5 mutated genes. GroEL2 staining was used as a control for lysis and equal loading. C) Immunoblot analysis of EccC₅ expression in isolated membranes of indicated strains. D) Blue native PAGE and immunoblot analysis using an anti-EccD₅ antibody of the ESX-5 membrane of M. marinum ΔeccC5::mspA, complemented either with an empty vector (-) or with various eccC5 mutated genes. For all samples that contained EccC₅ variants the characteristic pattern of ESX-5 membrane complexes was observed [28], consisting of the largest ~1.5 MDa complex and two additional smaller subcomplexes (indicated by the three arrowheads).

Fig 5. ESX-5 is involved in fatty acid uptake.

A) Growth of indicated M. marinum strains on Tween-80 as a sole carbon source was assessed by measuring optical density at different time points. Depicted is the average of three biological replicates. Error bars indicate standard deviations. B) Uptake of a fluorescently labeled fatty acid after 72 hours of hypoxic growth was measured by FACS analysis. 20.000 events gated for similar size were acquired for WT::mspA (black), ΔmycP5::mspA (light grey) or ΔmycP5-C::mspA (dark grey). C) Quantification of FACS analysis. Mean fluorescent intensity of three experiments per strain was acquired by FACS. Background staining, quantified by adding the fluorescent fatty acid to an unstained culture one hour before washing the cells, was deducted from the measured values. Error bars indicate the standard deviations and One-way ANOVA showed a statistical difference between the samples of p = 0.010. D) Uptake of the fluorescently labeled fatty acid and formation of lipid bodies was confirmed by confocal microscopy.

Fig 6. Model for the essentiality of ESX-5.

A) Presented is the working hypothesis, in which the ESX-5 system is responsible for the insertion of several channel- or pore-forming protein (indicated by the question marks) that mediates the uptake of essential nutrients and/or other metabolites. B) The lack of PDIMs (left) or the presence of MspA-like porins (right) increases the permeability of the outer membrane, allowing the passive diffusion of the hypothesized essential nutrient(s), thereby circumventing the essentiality of ESX-5.