We show that T. gondii
cathepsin L-like protease, TgCPL, is associated with endocytic organelles, including most prominently a VAC that is distinct from other well known apical organelles of T. gondii
. Given is location proximal to the LE marked by TgRab7 and TgVP1, it is reasonable to consider that the VAC is the T. gondii
equivalent of a lysosome or lytic vacuole. Few studies have attempted to identify lysosome-like organelles in this parasite, and the findings are varied. Norrby et. al. (Norrby et al., 1968
) showed that acridine orange, a fluorescent dye that accumulates in acidic structures, labels small vesicular structures within extracellular and intracellular parasites. The same study showed the presence of multiple black spots in intracellular tachyzoites stained with the Gomori technique for lysosomes, and that these structures increased with time after invasion. However, using the acidotropic agent DAMP and electron microscopy, Shaw et. al
. (Shaw et al., 1998
) failed to detect a lysosomal-like compartment. This study did, however, suggest that pre-rhoptries and mature rhoptries are acidic and this, along with a series of studies by Keith Joiner's group (Hoppe et al.
, 2000a; Hoppe et al., 2000b
; Ngo et al., 2003
), led to the idea that rhoptries are secretory lysosomes (reviewed in Ngo et al., 2003
). Our findings along with those of Miranda et al (accompanying article) strongly suggest that T. gondii
has an endolysosomal system that includes at least one typical lysosomal protease, TgCPL.
We also showed that the VAC is a dynamic organelle that undergoes a cell cycle dependent fragmentation process. This phenomenon is reminiscent of the inheritance mechanism used bySaccharomyces cerevisiae
to actively distribute organelles into the budding cell. Early in the yeast cell cycle a portion of the mother cell organelles, including the vacuole, mitochondria, the endoplasmic reticulum (ER), late-Golgi elements and peroxisomes are actively transported to the budding daughter cell (reviewed in (Weisman, 2006
)). We show that tubulovesicular projections from the VAC appear immediately before daughter cell formation. With the current view it is not possible to distinguish whether the small TgCPL positive structures are migrating into the new daughters or are instead moving toward the posterior end of the mother cell to possibly function in catabolism of proteins in the residual body. It is also not clear if the organelle fragmentation and movement is part of a regulated mechanism to modulate and coordinate the function of the VAC with the cell cycle of the parasite. Gaining further insight into the dynamics of VAC fragmentation will require new markers of the VAC suitable for fluorescent tagging and live cell visualization during intracellular replication.
Huang et. al. also partially characterized TgCPL (Huang et al., 2009
). Our findings extend this work by showing that TgCPL occupies the internal periphery of the VAC, and that it contributes to the maturation of proMICs during trafficking to the micronemes. Whereas the previous report found that recombinant TgCPL is most active at pH 6.0-6.5 (Huang et al., 2009
), we our findings indicate its activity is maximal at pH 5.5. This may reflect the use of different substrates since Huang et. al. used a synthetic peptide substrate while we used proteinaceous substrates including rTgCPL itself (autoactivation) and rproTgM2AP. Huang et. al. also found that rTgCPL cleaves a substrate with P2 Leu approximately two times better than Phe, and they suggested this was due to Asp216 creating a relatively shallow S2 binding pocket based on a structural homology model of TgCPL (Huang et al., 2009
). However, the recent reported crystal structure of TgCPL (Larson et al., 2009
) showed that Asp216 does not impinge the S2 binding pocket, which is consistent with our finding that substrates with a P2 Leu or Phe are cleaved approximately equally well, albeit with some distinctions due to preferences a the P3 position. The source of the recombinant enzyme, P. pastoris
(Huang et al., 2009
) or E. coli
(current study), might also influence the catalytic properties of rTgCPL.
Proteolytic maturation is a common feature of regulated secretory pathways. We provide multiples lines of evidence for TgCPL acting as a maturase for at least two microneme precursor proteins, proTgM2AP and proTgMIC3, within a regulated secretory pathway of T. gondii
. This notion is further supported by the preference of TgCPL for P2 Leu and other hydrophobic residues, which is consistent with the propeptide cleavage sites of several proMICs, and the correct processing of rproTgM2AP by rTgCPL. TgCPL is expressed in bradyzoites along with TgM2AP and TgMIC3 (Fig. S2B-C), thus it also has the potential to act as a maturase in encysted, chronic stage of the parasite. However, maturation of TgM2AP and TgMIC3 was not completely abrogated in the RHΔcpl
strain, indicating that at least one additional maturase is capable of processing these substrates. Accordingly, we found that treatment of RHΔsub1
parasites with PMSF or subtilisin inhibitor III impair processing of proTgMIC3 (Fig. S8), implying the involvement of an additional subtilisin-like enzyme. Moreover, that TgCPB has very similar substrate preferences to TgCPL could hint to it being responsible for the residual maturation of TgM2AP in RHΔcpl
parasites. TgCPB has been reported to occupy the rhoptries and act as a maturase for rhoptry proteins (Que et al., 2002
), hence it probably also traffics through the endolysosomal system where it could encounter microneme substrates. However, treatment of RHΔcpl
parasites with the cathepsin B inhibitor CA074Me did not affect the maturation of proTgM2AP or proTgMIC3 (data not shown), thus additional experiments will be necessary to clarify the involvement of TgCPB. Maturation of TgMIC6, TgMIC5, TgAMA1, and was not affected in RHΔcpl
parasites, providing further evidence of distinct maturases.
To correctly process proMIC substrates without unwanted degradation, TgCPL zymogen activation and protease activity are probably regulated at several levels. We show that the autocatalytic processing of proTgCPL and the maturation of at least two proMICs (proTgM2AP and proTgMIC3) are both pH dependent, albeit in a distinguishable manner. The differential sensitivity to pH antagonists could indicate that TgCPL zymogen activation occurs in a distinct compartment from that of proMIC maturation, thereby segregating these events as a first level of regulation. Moreover, pH modulation by vacuolar H+-ATPases or other types of proton pumps could be an important secondary factor governing proMIC maturation by TgCPL as well as the activity of alternative maturases. Whether the vacuolar H+-ATPase-positive structures associated with the VAC (Miranda et. al., accompanying article) are involved in proMIC processing will require further investigation. TgCPL activity might be controlled at a third level by segregation of the protease within intralumenal vesicles of endosomes. It is well established in several eukaryotic systems that multivesicular endosomes like the VAC are dynamic organelles that use intralumenal vesicles to partition storage, recycling, and sorting functions from degradative activities (Woodman et al., 2008). Finally, regulation could be governed by allowing only limited amounts of TgCPL access to proMICs in the processing compartment. The low levels of TgCPL in the LE during microneme biogenesis are consistent with this notion. Stringent regulation would help avoid inappropriate processing or degradation and favor limited proteolysis of proMICs and possibly other substrates.
Our findings imply that the endocytic and exocytic pathways converge in common intermediate compartments for the biogenesis of the micronemes, and possibly other apical organelles. The hypothetical model depicted in illustrates that after being synthesized in the ER microneme proteins are routed through the Golgi apparatus and transported to micronemes via a series of post-Golgi endosomal compartments including the EE, LE, and possibly the VAC. Rather than the TGN being the site of regulated secretory organelle biogenesis as in many eukaryotic cells, our model predicts that micronemes are derived from the endocytic system, specifically the LE or VAC. The use of endocytic organelles for exocytic trafficking is not without precedent since recent studies have shown that recycling endosomes are used as sorting stations for the exocytosis of, for example, E-cadherin in epithelial cells (Desclozeaux et al., 2008
) and the cytokines IL-6 and TNFα in macrophages (Manderson et al., 2007
). Our model further predicts that proteolytic maturation of proMICs mediated by TgCPL occurs within the LE and/or VAC. The observation of TgCPL within micronemes proximal to an endosome also raises the possibility of cleavage occurring within nascent micronemes prior to recycling of the protease back to its cognate compartment. In any case, since microneme propeptides contain targeting information (Harper et al., 2006
; Brydges et al., 2008
; El Hajj et al., 2008
), it is expected that these cleavable elements are removed only after they have fulfilled their role in targeting. Precisely where and how they perform this function remains to be determined. Future studies aimed at disabling vesicular transport at specific points in the secretory pathway may help further elucidate the principal sites of proTgCPL and proMIC maturation.
Hypothetical model of trafficking and maturation in the endo/exocytic pathway