Fig 1.
Mutants affected in the ESX-1 accessory proteins EspG1, EspH and EccA1 differently affect the ESX-1 secretome.
A. Genetic organization of espG1-espH-eccA1 in the esx-1 locus. Genes are color-coded according to the localization of their proteins—see key. B and C. Secretion analysis of EsxA and EspE substrates reveals that single deletion of espG1, espH and eccA1 affects secretion at different levels. Immunoblot analysis using protein preparations of wild-type M. marinum and the indicated mutants. In B we analyzed cell pellets not treated with detergent Genapol X-080 and culture supernatant fractions. In C we analyzed cell pellets treated with Genapol X-080 and the concomitant supernatant fractions. D and E. Complementation of the mutant strains fully restores ESX-1 secretion. In D the secretion of EsxA was analyzed and in E the secretion of EspE. In both experiments, GroEL2 was used as loading control and PE_PGRS as cell-surface control fraction. Equivalent OD units were loaded; 0.2 OD for pellet or Genapol pellet and 0.5 OD for supernatant or Genapol supernatant fractions.
Fig 2.
Secretion of EsxA by the eccA1 mutant is growth-medium dependent.
Secretion analysis of the WT M. marinum MUSA, the eccCb1 mutant and the knockout strains espG1, espH and eccA1 grown in Sauton’s defined medium. Immunoblot analysis with anti-EsxA confirmed a requirement of EccA1 for a full secretion of EsxA when cells were grown in this medium. Anti-GroEL2 was used as a loading and lysis control for all samples. Anti-PGRS antibodies, staining the ESX-5 dependent substrates PE_PGRS proteins, were used as a supernatant control for all samples. Equivalent OD units were loaded; 0.3 OD for pellet and 0.6 OD for supernatant or supernatant fractions.
Fig 3.
Quantitative proteomics analysis of the Genapol-enriched fractions of different M. marinum ESX-1 mutant strains.
Volcano plots representing the statistical significance of changes of cell-surface enriched proteins between the WT M. marinum and each ESX-1 mutant. The vertical lines depict p value on the–log base 10 scale. The horizontal lines denote fold change on the log base 2 scale. Only proteins with an accumulative number of more than 10 spectral counts are shown. Each dot corresponds to a single identified protein and the size of the dots correlates to the accumulative spectral counts of the protein of the WT and the corresponding mutant. Proteins with a spectral count difference of more than eight folds were set to eight. In blue: proteins that showed more than 4 folds of change, otherwise in red. Only putative ESX-1 substrates, SecA2 and Mak are annotated. A. WT versus the eccCb1 mutant. B. WT versus the ΔespG1 mutant. C. WT versus the ΔespH mutant. D. WT versus the ΔeccA1 mutant.
Fig 4.
Quantitative proteomics analysis of the supernatant of different M. marinum ESX-1 mutant strains.
Volcano plots representing the statistical significance of changes of the secreted proteins in the supernatant between the WT M. marinum and each ESX-1 mutant. The same quantitative method was used as in Fig 3 for the Genapol-enriched fractions. A. WT versus the eccCb1 mutant. B. WT versus the ΔespG1 mutant. C. WT versus the ΔespH mutant. D. WT versus the ΔeccA1 mutant.
Fig 5.
EspH specifically interacts with EspE in M. marinum.
A. Immunoblots of pulldown assays using Strep-tactin agarose. EspE-Strep was purified from soluble lysates of the eccCb1 mutant expressing only EspE-Strep/EspF or EspE-Strep/EspF together with EspH-His. A strain containing empty plasmids was included as negative control. Total input material (I), unbound proteins (FT), the final washing step (W), three fractions of eluted proteins (E1, E2, E3) and boiled beads fractions were separated by SDS-PAGE and further immunoblotted using antisera directed against the Strep- or His-tag. The elution fractions were loaded 10 times more compared to the other fractions. Endogenous EspE, PPE68 and EsxA substrates were detected using anti-EspE, anti-PPE68 and anti-EsxA, respectively. B. Immunoblots of pulldown assays using Ni-NTA beads. EspH-His proteins were purified from soluble lysates of the eccCb1 strain carrying a plasmid expressing EspH-His or the corresponding empty plasmid. Total input material (I), unbound proteins (FT), the last washing step (W), proteins eluted with 50 mM (E1), 100 mM (E2), and 200 mM (E3) imidazole and boiled bead fraction were separated by SDS-PAGE and probed with His-specific antiserum. The elution fractions were loaded 10 times more compared to the other fractions. Endogenous EspE, PPE68 and EsxA proteins were detected using anti-EspE, anti-PPE68 and anti-EsxA, respectively.
Fig 6.
ESX-1 mutant strains have lost hemolytic activity.
Contact-dependent hemolysis of red blood cells (RBCs) by various M. marinum strains grown in the presence of Tween-80. Hemolysis was quantified by determining the OD405 absorption of the released hemoglobin. The data shown here is generated from two independent experiments, each time in triplicates. In A, the ESX-1 mutants and in B the complemented strains with restored hemolytic activity are shown. Significance is indicated, **** < 0.0001. Ctrl = control sample with PBS.
Fig 7.
Intracellular growth of ΔeccA1, ΔespG1 and ΔespH in different hosts.
A. Flow cytometry experiment showing percentage of infected A. castellanii at 4 hours post infection (hpi) versus 24 hpi, graph shows pooled data from two independent experiments. B. Graph shows fold change in percentage infected A. castellanii presented in A. C. Similar flow cytometry experiment with infected RAW macrophages when comparing percentage infected cells at 3hpi and 24 hpi, graph shows representative data of 1 out of 3 biological replicates.,. D. Graph shows fold change in percentage infected RAW macrophages presented in C. **** = p<0.001, ns = non-significant.
Fig 8.
In vivo effect of ΔeccA1, ΔespG1 and ΔespH in zebrafish larvae.
Graphs A-C show relative levels of infection as determined by automated pixel count software for infection of zebrafish larvae. The larvae were infected with ~75–150 CFU red fluorescent M. marinum mutant strains and analyzed at 4 dpi. Graphs show combined data of three independent biological replicates per mutant strain, each dot represents one larva. Bars represent mean and standard error of the mean. A. Systemic infection of zebrafish larvae with M. marinum ΔeccA1, B. M. marinum ΔespG1 and C. M. marinum ΔespH, * = <0.05, **** <0.001. Representative bright field and corresponding fluorescent images are depicted in: D. WT infection, F. eccCb1 mutant infection, H. M. marinum ΔeccA1, J. M. marinum ΔespG1, L. M. marinum ΔespH. Confocal imaging of a single cluster of infected L-plastin labeled phagocytic cells (cyan) in the tail of infected larvae confirmed the phenotype seen in fluorescent imaging: E. WT infection, G. eccCb1 mutant infection, I. M. marinum ΔeccA1, K. M. marinum ΔespG1, M. M. marinum ΔespH, depicting a cording phenotype (closed arrows) and intense fluorescent spots suggestive for phagocytic cell debris (open arrows). Scale bar E, G, I, K, M = 50 μm.
Fig 9.
EspH-mutant strain is hypervirulent in zebrafish larvae.
A-C. Systemic M. marinum WT infection (red) of Tg(fli:GFP) larvae with green fluorescent blood vessels was followed over time, representative images are shown in A. 1dpi, B. 2dpi, C. 4dpi. Larvae were stained with anti-L-plastin to label phagocytic cells (cyan). D-F. Representative images of systemic infection with M. marinum ΔespH over time in D. 1dpi, E. 2dpi, F. 4dpi. Scale bar = 50 μm.