Selected publications

CRISPR/Cas9 gene editing to make conditional mutants of the human malaria parasite, P. falciparum

Kudyba M.K.*, Cobb D.W.*, Florentin A., Krakowiak M., and Muralidharan v. (2018) J. vis. exp. (*– Equal Contributions)

PfClpC is an essential Clp chaperone required for plastid integrity and Clp protease stability in Plasmodium falciparum

Florentin A., Cobb D.W., Fishburn J.D., Cipriano M.J., Kim P.S., Fierro M.A., Striepen B., and Muralidharan V. (2017) Cell Reports

The deadly malaria parasite Plasmodium falciparum contains a nonphotosynthetic 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.

Plasmodium-specific exported chaperone, PfHsp70x is dispensable for parasite growth

Cobb D.W.*, Florentin A.*, Fierro M.A., Krakowiak M., Moore J.M., and Muralidharan V. (2017) mSphere,  (*– Equal Contributions)

An outstanding question in the field is how Plasmodium falciparum undertakes the essential process of trafficking its proteins within the host cell.In most organisms, chaperones such as Hsp70 are employed in protein trafficking. Of the Plasmodium species causing human disease, the chaperone PfHsp70x is unique to P. falciparum, and it is the only parasite protein of its kind exported to the host . This has placed PfHsp70x as an ideal target to inhibit protein trafficking and kill the parasite. However, we show that PfHsp70x is not required for export of parasite effectors and it is not essential for parasite survival inside the RBC.

The PTEX chaperone, PfHSP101,  is required for protein export

Beck J.R.*, Muralidharan V.*, Oksman A., and Goldberg D.E. (2014) Nature (*– equal contributions)

The Plasmodium translocon of exported proteins or PTEX is present at the cellular interface between P. falciparum and human red blood cells.   We show that all classes of exported proteins use the PTEX to get across the vacuolar membrane encasing the parasite. Our data demonstrates that a component of this translocon, PfHSP101, is essential for growth within RBCs and conditional inhibition of this chaperone leads to a complete block in protein export to the host cell.  This chaperone is required for sexual development of the parasite, a process that is essential for transmission of the parasite. Therefore, drugs targeting this crucial bottleneck for parasite-induced remodeling of the host cell will be an ideal transmission blocking antimalarial.

Asparagine repeats in Plasmodium falciparum

Muralidharan V., and Goldberg d.e. (2013) PLoS Pathogens

Most sequenced genomes have a low abundance of amino acid repeats as these are usually disfavored due to their propensity to form disordered structures. However, the proteome of P. falciparum is unusually rich polyasparagine runs (found in more than 2000 proteins). Such repeats have severe consequences but our work has shown that P. falciparum has figured out how to deal with the aggregate-forming tendencies of asparagine repeats. In this review, we propose that these repeats may have an evolutionary advantage, because parasite chaperones can act as capacitors for positive evolutionary change. They allow the propagation of asparagine repeats in the genome that can go on to evolve into protein domains with novel functions over evolutionary time scales. At the same time, under periods of high stress, they can allow some proteins to aggregate causing novel phenotypes that may help in dealing with environmental stresses.

The asparagine-rich Plasmodium falciparum proteome is stabilized by the PfHsp110c chaperone

Muralidharan V., Oksman A., Pal P., Lindquist S., and Goldberg D.E. (2012) Nature Communications

Nearly one in three proteins in the genome of the deadly human parasite, P. falciparum, possess large repeats of the amino acid, asparagine. Such repeats have been shown to cause proteins to aggregate especially at elevated temperatures, such as those encountered by malaria parasites during fever or when going from humans to mosquitos or vice versa. We show that the P. falciparum chaperone, PfHsp110c, is exceptionally good at preventing protein aggregation, in both parasites and in mammalian cells. It is even able to prevent aggregation of glutamine-rich repeats such as those seen in neurodegenerative diseases such as Huntingtons'. Our data show that the parasite chaperone, PfHsp110c, vital for maintaining a stable proteome especially during malarial fevers. It also raises the intriguing possibility of using parasite chaperones to prevent protein misfolding diseases. 

A regulatable DHFR degradation domain to assess asparagine repeats in P. falciparum

Muralidharan V., Oksman A., Iwamoto M., Wandless T.J., and Goldberg D.E. (2011) PNAS

One in four proteins in Plasmodium falciparum contains asparagine repeats. We probed the function of one such 28-residue asparagine repeat present in the P. falciparum proteasome lid subunit 6, Rpn6. To aid our efforts, we developed a regulatable, fluorescent affinity (RFA) tag that allows cellular localization, manipulation of cellular levels, and affinity isolation of a chosen protein in P. falciparum. The tag comprises a degradation domain derived from Escherichia coli dihydrofolate reductase together with GFP. The expression of RFA-tagged proteins is regulated by the simple folate analog trimethoprim (TMP). Parasite lines were generated in which full-length Rpn6 and an asparagine repeat-deletion mutant of Rpn6 were fused to the RFA tag. The knockdown of Rpn6 upon removal of TMP revealed that this protein is essential for ubiquitinated protein degradation and for parasite survival, but the asparagine repeat is dispensable for protein expression, stability, and function. The data point to a genomic mechanism for repeat perpetuation rather than a positive cellular role. The RFA tag should facilitate study of the role of essential genes in parasite biology.

Plasmepsin V licenses Plasmodium proteins for export into the host RBC

Russo I., Babbit S.*, Muralidharan V.*, Butler T., Oksman A., and Goldberg D.E. (2010) Nature (*– equal contributions)

During their intraerythrocytic development, malaria parasites export hundreds of proteins to remodel their host cell. Nutrient acquisition, cytoadherence and antigenic variation are among the key virulence functions effected by this erythrocyte takeover. Proteins destined for export are synthesized in the endoplasmic reticulum (ER) and cleaved at a conserved (PEXEL) motif, which allows translocation into the host cell via an ATP-driven translocon called the PTEX complex. We report that plasmepsin V, an ER aspartic protease with distant homology to the mammalian processing enzyme BACE, recognizes the PEXEL motif and cleaves it at the correct site. This enzyme is essential for parasite viability and ER residence is essential for its function. We propose that plasmepsin V is the PEXEL protease and is an attractive enzyme for antimalarial drug development.