PfClpC Is an Essential Clp Chaperone Required for Plastid Integrity and Clp Protease Stability in Plasmodium falciparum

Abstract

Summary The deadly malaria parasite, Plasmodium falciparum, contains a non-photosynthetic plastid known as the apicoplast, that functions to produce essential metabolites and drugs that target the apicoplast are clinically effective. Several prokaryotic caseinolytic protease (Clp) genes have been identified in the Plasmodium genome. Using phylogenetic analysis, we focused on the Clp members that may form a regulated proteolytic complex in the apicoplast. We genetically targeted members of this complex and generated conditional mutants of the apicoplast-localized PfClpC chaperone and PfClpP protease. Conditional inhibition of the PfClpC chaperone resulted in growth arrest and apicoplast loss, and was rescued by addition of the essential apicoplast-derived metabolite, IPP. Using a double conditional-mutant parasite line, we discovered that the chaperone activity is required to stabilize the mature protease, revealing functional interactions. These data demonstrate the essential function of PfClpC in maintaining apicoplast integrity and its role in regulating the proteolytic activity of the Clp complex.

The endoplasmic reticulum chaperone PfGRP170 is essential for asexual development and is linked to stress response in malaria parasites

“…A growth defect was seen within 24 hr after the removal of TMP, resulting in parasite death ( Figure 2a). Consistent with data from other chaperones tagged with the DDD (Beck et al, 2014;Florentin et al, 2017;Muralidharan et al, 2012), TMP removal did not result in degradation of PfGRP170-GFP-DDD (Figure 2c). Consistent with data from other chaperones tagged with the DDD (Beck et al, 2014;Florentin et al, 2017;Muralidharan et al, 2012), TMP removal did not result in degradation of PfGRP170-GFP-DDD (Figure 2c).…”

“…We have generated conditional mutants that allow us to probe the role of this protein using the DDD conditional auto-inhibition system Florentin et al, 2017;Ganter et al, 2017;Ito et al, 2017;Muralidharan et al, 2011;Muralidharan et al, 2012). We have generated conditional mutants that allow us to probe the role of this protein using the DDD conditional auto-inhibition system Florentin et al, 2017;Ganter et al, 2017;Ito et al, 2017;Muralidharan et al, 2011;Muralidharan et al, 2012).…”

“…The endogenous PfGRP170 gene encodes a C-terminus SDEL sequence, a potential ER retention signal, and therefore, we added an SDEL sequence after the DDD domain in order to avoid mislocalisation of the tagged protein. Intramolecular binding of the chaperone to the unfolded domain inhibits normal chaperone function (Figure 1b; Beck et al, 2014;Florentin et al, 2017;Muralidharan et al, 2012). However, if TMP is removed from the culture medium, the DDD unfolds and becomes unstable (Beck et al, 2014;Cobb et al, 2017;Florentin et al, 2017;Muralidharan et al, 2012;Muralidharan, Oksman, Iwamoto, Wandless, & Goldberg, 2011).…”

“…The resulting product was then digested with Nhe1 and BglII and inserted into pCEN-DHFR (Iwanaga et al, 2012) that was digested with Nhe1 and BglII (New England Biolabs) using a T4 ligase and transformed into bacteria as described previously Florentin et al, 2017). The PfGRP170 transit peptide PCR was digested with Nhe1 and AatII (New England Biolabs), and the GFP PCR was digested with AatII and BglII (New England Biolabs).…”

“…Southern blot analysis was performed on DNA isolated from PfGRP170-GFP-DDD parasites (1B2 and 1B11) as described previously Florentin et al, 2017). The assay was also performed on PM1KO parental DNA and the pGRP170-DDD plasmid as a control.…”