Modification of a PE/PPE substrate pair reroutes an Esx substrate pair from the mycobacterial ESX-1 type VII secretion system to the ESX-5 system

Bacterial type VII secretion systems secrete a wide range of extracellular proteins that play important roles in bacterial viability and in interactions of pathogenic mycobacteria with their hosts. Mycobacterial type VII secretion systems consist of five subtypes, ESX-1-5, and have four substrate classes, namely, Esx, PE, PPE, and Esp proteins. At least some of these substrates are secreted as heterodimers. Each ESX system mediates the secretion of a specific set of Esx, PE, and PPE proteins, raising the question of how these substrates are recognized in a system-specific fashion. For the PE/PPE heterodimers, it has been shown that they interact with their cognate EspG chaperone and that this chaperone determines the designated secretion pathway. However, both structural and pulldown analyses have suggested that EspG cannot interact with the Esx proteins. Therefore, the determining factor for system specificity of the Esx proteins remains unknown. Here, we investigated the secretion specificity of the ESX-1 substrate pair EsxB_1/EsxA_1 in Mycobacterium marinum Although this substrate pair was hardly secreted when homologously expressed, it was secreted when co-expressed together with the PE35/PPE68_1 pair, indicating that this pair could stimulate secretion of the EsxB_1/EsxA_1 pair. Surprisingly, co-expression of EsxB_1/EsxA_1 with a modified PE35/PPE68_1 version that carried the EspG5 chaperone-binding domain, previously shown to redirect this substrate pair to the ESX-5 system, also resulted in redirection and co-secretion of the Esx pair via ESX-5. Our results suggest a secretion model in which PE35/PPE68_1 determines the system-specific secretion of EsxB_1/EsxA_1

The ESX-1 Substrate PPE68 Has a Key Function in ESX-1-Mediated Secretion in Mycobacterium marinum

Mycobacteria use specialized type VII secretion systems (T7SSs) to secrete proteins across their diderm cell envelope. One of the T7SS subtypes, named ESX-1, is a major virulence determinant in pathogenic species such as Mycobacterium tuberculosis and the fish pathogen Mycobacterium marinum. ESX-1 secretes a variety of substrates, called Esx, PE, PPE, and Esp proteins, at least some of which are folded heterodimers. Investigation into the functions of these substrates is problematic, because of the intricate network of codependent secretion between several ESX-1 substrates. Here, we describe the ESX-1 substrate PPE68 as essential for secretion of the highly immunogenic substrates EsxA and EspE via the ESX-1 system in M. marinum. While secreted PPE68 is processed on the cell surface, the majority of cell-associated PPE68 of M. marinum and M. tuberculosis is present in a cytosolic complex with its PE partner and the EspG1 chaperone. Interfering with the binding of EspG1 to PPE68 blocked its export and the secretion of EsxA and EspE. In contrast, esxA was not required for the secretion of PPE68, revealing a hierarchy in codependent secretion. Remarkably, the final 10 residues of PPE68, a negatively charged domain, seem essential for EspE secretion, but not for the secretion of EsxA and of PPE68 itself. This indicates that distinctive domains of PPE68 are involved in secretion of the different ESX-1 substrates. Based on these findings, we propose a mechanistic model for the central role of PPE68 in ESX-1-mediated secretion and substrate codependence. IMPORTANCE Pathogenic mycobacteria, such Mycobacterium tuberculosis and Mycobacterium marinum, use a type VII secretion system (T7SS) subtype, called ESX-1, to mediate intracellular survival via phagosomal rupture and subsequent translocation of the mycobacterium to the host cytosol. Identifying the ESX-1 substrate that is responsible for this process is problematic because of the intricate network of codependent secretion between ESX-1 substrates. Here, we show the central role of the ESX-1 substrate PPE68 for the secretion of ESX-1 substrates in Mycobacterium marinum. Unravelling the mechanism of codependent secretion will aid the functional understanding of T7SSs and will allow the analysis of the individual roles of ESX-1 substrates in the virulence caused by the significant human pathogen Mycobacterium tuberculosis

The ESX-1 Substrate PPE68 Has a Key Function in ESX-1-Mediated Secretion in Mycobacterium marinum

Mycobacteria use specialized type VII secretion systems (T7SSs) to secrete proteins across their diderm cell envelope. One of the T7SS subtypes, named ESX-1, is a major virulence determinant in pathogenic species such as Mycobacterium tuberculosis and the fish pathogen Mycobacterium marinum. ESX-1 secretes a variety of substrates, called Esx, PE, PPE, and Esp proteins, at least some of which are folded heterodimers. Investigation into the functions of these substrates is problematic, because of the intricate network of codependent secretion between several ESX-1 substrates. Here, we describe the ESX-1 substrate PPE68 as essential for secretion of the highly immunogenic substrates EsxA and EspE via the ESX-1 system in M. marinum. While secreted PPE68 is processed on the cell surface, the majority of cell-associated PPE68 of M. marinum and M. tuberculosis is present in a cytosolic complex with its PE partner and the EspG1 chaperone. Interfering with the binding of EspG1 to PPE68 blocked its export and the secretion of EsxA and EspE. In contrast, esxA was not required for the secretion of PPE68, revealing a hierarchy in codependent secretion. Remarkably, the final 10 residues of PPE68, a negatively charged domain, seem essential for EspE secretion, but not for the secretion of EsxA and of PPE68 itself. This indicates that distinctive domains of PPE68 are involved in secretion of the different ESX-1 substrates. Based on these findings, we propose a mechanistic model for the central role of PPE68 in ESX-1-mediated secretion and substrate codependence. IMPORTANCE Pathogenic mycobacteria, such Mycobacterium tuberculosis and Mycobacterium marinum, use a type VII secretion system (T7SS) subtype, called ESX-1, to mediate intracellular survival via phagosomal rupture and subsequent translocation of the mycobacterium to the host cytosol. Identifying the ESX-1 substrate that is responsible for this process is problematic because of the intricate network of codependent secretion between ESX-1 substrates. Here, we show the central role of the ESX-1 substrate PPE68 for the secretion of ESX-1 substrates in Mycobacterium marinum. Unravelling the mechanism of codependent secretion will aid the functional understanding of T7SSs and will allow the analysis of the individual roles of ESX-1 substrates in the virulence caused by the significant human pathogen Mycobacterium tuberculosis

Boosting PPE10 specific immune responses does not increase protection against <i>M</i>. <i>tuberculosis</i>.

<p>A) Graphical representation of the prime-boost vaccination protocol. Mice were immunized with either BCG or BCG38 (Green). 60 days post-infection (d.p.i.) C57BL/6 x CBA F1 mice were injected s.c. with a booster consisting of adjuvant CpG(DOTAP), alone or in combination with a mix of PPE10_221-235 and PPE10_381-395 peptides (blue). The same formulation was intranasally administered four weeks later. Nine days after the intranasal boost, mice were exposed to <i>M</i>. <i>tuberculosis</i> H37Rv aerosol infection (220 CFU/lung 1 d.p.i). Bacterial lung (B) and Spleen (C) burdens were assessed by dilution and counting 4 weeks post-infection (experimental end-point) after being photographed for macroscopic investigation (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007139#ppat.1007139.s002" target="_blank">S2C and S2D Fig</a>). Each data point represents the CFU value of one organ from a single mouse, error bars depict the standard deviation. No significant differences between the vaccination conditions were detected by ordinary one-way ANOVA followed by Tukey’s test of multiple comparisons. All vaccination conditions resulted in a significant (<i>p</i><0.01) reduction in lung burden compared to unimmunized controls (Ordinary one way ANOVA; Dunnett’s test of multiple comparisons against a single control). For simplicity, this latter information is not depicted in the figure. Reduction in spleen CFUs was not significant for any of the vaccination conditions. Statistical analyses were performed using PRISM software.</p

Restoring PPE38-dependent protein secretion of BCG does not increase protection against <i>M</i>. <i>tuberculosis</i> in C57BL/6 mice.

<p>Lung (A) or spleen (B) bacterial burdens of C57BL/6 mice infected with <i>M</i>. <i>tuberculosis</i> H37Rv via aerosol administration. Mice were vaccinated s.c. four weeks before the challenge, with 1 x 10⁶ CFU/mouse of either BCG or BCG38 (indicated in green). Both strains were prepared, either in standard culture conditions in medium containing 0.025% Tween-80 considered as no capsule (indicated with (-)), or in culture allowing capsule formation/retention in detergent free condition (indicated with (+)). Photographs of the assessed organs are depicted in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007139#ppat.1007139.s002" target="_blank">S2A and S2B Fig</a>. Each data point represents the CFU/organ of one single mouse counted and averaged from two technical duplicates. Error bars depict the standard deviation. Differences between different vaccination conditions were non-significant (<i>p</i>>0.05), but all vaccination conditions were statistically different from the unimmunized control group (<i>p</i><0.01). For simplicity, this latter information is not depicted in the figure. Significance was calculated with Prism software using ordinary one-way ANOVA followed by Tukey’s test for multiple comparisons.</p

Epitope mapping of PPE10 identifies two novel immunogenic T-cell epitopes.

<p>C57BL/6 H-2^b (black) or C57BL/6 x CBA (H-2^b/k) F1 mice (B6CBAF1, blue) were immunized s.c. with 1 x 10⁶ CFU/mouse of <i>M</i>. <i>tuberculosis</i> H37Rv (<i>Mtb</i>, filled bars), or were left non-immunized (N.I. empty bars). Three weeks post-immunization, splenocytes were stimulated with control peptides or a library of 15-mers spanning PPE10 excluding the PPE domain. T-cell mediated IFN-γ responses were quantified buy ELISA as a measure of immunogenicity. Two immunogenic PPE10-peptides were identified (PPE10_221-235 & PPE10_381-395) in B6CBAF1 mice. Error bars depict standard deviation over two technical replicates. This figure depicts only newly identified epitopes and controls. Full results of the pep-scan epitope mapping can be found in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007139#ppat.1007139.s002" target="_blank">S2 Fig</a>.</p

RD5-like genetic deletions in the <i>M</i>. <i>tuberculosis</i> complex and their effect on PE_PGRS secretion.

<p>A) The genetic organization of the RD5 locus in <i>M</i>. <i>tuberculosis</i> strains CDC1551 and H37Rv is depicted in colored arrows. Bars below the genes indicate the size and location of different RD5-like and <i>ppe38</i>-deletions examined in this work. Arrows above the genes indicate primers used in this study to verify the presence of RD5 associated genes, sequences can be found in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007139#ppat.1007139.s011" target="_blank">S4 Table</a>. Functional PE_PGRS secretion is indicated by shading of the strain name in green, while red shading represents strains in which PE_PGRS secretion is not functional (based on immunoblot analysis). Figure adapted from Mc Evoy et al. 2009 with permission [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007139#ppat.1007139.ref038" target="_blank">38</a>]. B) Immunoblot secretion analysis of animal-adapted MTBC strains verifies that strains with RD5 deletions do not secrete PE_PGRS proteins. Samples were prepared as described in materials and methods section. C) Immunoblot secretion analysis reveals PE_PGRS secretion defect in BCG, comparable to the <i>M</i>. <i>tuberculosis ppe38-</i>71-deletion strain or a general ESX-5 secretion mutant (<i>eccC</i>_<i>5</i>::<i>tn</i>). SigA was used as a loading and lysis control. Some lysis could be found in both BCG and BCG38, but was not markedly different between strains. Please note that these immunoblots correspond to the same pre-cultures as those that were used in the immunogenicity experiment depicted in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007139#ppat.1007139.g005" target="_blank">Fig 5</a> and therefore also include the Δ<i>ppe10</i> and Δ<i>ppe25-pe19</i> isolates. Full western blots corresponding to panels depicted in B-C are depicted in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007139#ppat.1007139.s005" target="_blank">S5 Fig</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007139#ppat.1007139.s007" target="_blank">S7 Fig</a>, respectively.</p