MMARE11_28540 is required for the processing of LipY. (A) Secretion of LipY-OVA₂ by M. marinum wild-type E11, mmarE11_28540::tr, and complemented strains was detected by double filter assay. (B) Alignment between model of MMARE11_28540 (red, amino acids 269 to 551 out of 554) and crystal structure of PE_PGRS16 (blue, 4EHC). Aspartic acids within the putative active site DTG/DSG motifs are depicted as spheres. (C) Immunoblot analysis of whole-cell lysate from M. marinum M wild-type and Δmmar_2933 strains expressing C-terminally HA-labeled LipY_tub or LipY_A150D. LipY was detected with anti-HA antibody. (D) The cleavage sites of MMAR_2933 in the linker domain of LipY_tub as determined by N-terminal Edman sequencing; the N-terminal cleavage site (aa 136A to 137A) and the previously identified C-terminal cleavage (aa 149G to 150A) (23) in LipY_tub are indicated. (E) Immunoblot analysis of whole-cell lysate from M. marinum M wild-type and Δmmar_2933 strains expressing C-terminally HA-labeled LipY_Δ98-201. Proteins were detected with anti-HA antibody.

PecA does not affect LipY surface localization and lipase activity. (A) Surface localization of LipY was measured on whole cells by flow cytometry for M. marinum M wild-type and ΔpecA strains expressing LipY_tub. LipY_tub was detected via its C-terminal HA tag by anti-HA antibody (10,000 bacteria were counted per condition). (B) Lipase activity was measured by the fluorescently based DGGR assay. Standard deviation values are shown (n = 3). a.u., arbitrary units; n.s., not significant.

PecA is subjected to self-cleavage. (A) Expression of PecA_myc and PecA_D293Gmyc in M. marinumΔpecA was detected by immunoblotting of whole-cell lysate. (B) Subcellular localization and secretion analysis of M. marinum wild type (indicated with “-”) or wild type expressing PecA_myc or PecA_D293Gmyc. WCL, whole-cell lysate; GP, Genapol-treated pellet (membrane associated and intracellular); GS, Genapol supernatant (membrane associated); CF, culture filtrate. GroEL2 was probed as a cytosolic control. (C) Immunoblot analysis of PecA_myc and PecA_D293Gmyc expressed in E. coli (whole-cell lysate). (D) Immunoblot analysis of in vitro translation of PecA_myc and PecA_D293Gmyc. (E) Immunoblot analysis of M. marinum wild type; wild type expressing PecA_myc, PecA_A84Dmyc, or PecA_AA88EDmyc; and ΔpecA mutant expressing PecA_D293Gmyc (whole-cell lysate). The arrow indicates the fully cleaved form of PecA. Proteins were detected by their C-terminal Myc tag with an anti-Myc antibody.

PecA is required for the processing of PE_PGRS proteins. Immunoblotting of whole-cell lysate from M. marinum wild-type M strain, ΔpecA strain, and ΔpecA strain complemented with M. marinumpecA_myc and pecA_D293Gmyc (A) or M. tuberculosispecA_tub (B) for endogenous PE_PGRS proteins. PE_PGRS proteins were detected with monoclonal antibodies recognizing the PGRS domain.

Deletion of PecA causes attenuation of virulence in an in vivo zebrafish infection model. (A) A total of 273 zebrafish larvae were infected 1 day postfertilization (dpf) with wild-type (75 larvae), ΔpecA (71 larvae), ΔpecA plus pecA_myc (62 larvae), or ΔpecA plus ΔpecA_D293Gmyc (65 larvae) M. marinum M strains (see Table S2 for input CFU). Graph shows relative infection from 4 independent experiments as calculated by normalized integrated fluorescent intensity per larva at 4 days postinfection, with mean intensity and standard deviation for the entire population. Similar effects were obtained for CFU counts at 4 dpi with wild-type, ΔpecA, and ΔpecA plus pecA_myc strains (Fig. S6). (B) Representative images of infected larvae are shown for each strain.

Model: PecA processes PE-PGRS proteins and is important for virulence. (A) PecA removes the PE domain of PE-PGRS proteins, including LipY_tub and itself, at the surface of Mycobacterium marinum. The processed PE_PGRS proteins might be of importance during infection, although their exact role, as well as the fate of the PE domains, remains unknown. (B) Proteolytic activity of PecA is suggested to play a role in virulence of M. marinum, as was observed during infection in zebrafish larvae.