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Summary The pathogenicity of the most deadly human malaria parasite, Plasmodium falciparum, relies on the export of virulence factors to the surface of infected erythrocytes. A novel membrane compartment, referred to as Maurer's clefts, is transposed to the host erythrocyte, acting as a marshal platform in the red blood cell cytoplasm, for exported parasite proteins addressed to the host cell plasma membrane. We report here the characterization of three new P. falciparum multigene families organized in 9 highly conserved clusters with the Pfmc-2tm genes in the subtelomeric regions of parasite's chromosomes and expressed at early trophozoite stages. Like the PfMC-2TM proteins, the PfEPF1, 3 and 4 proteins encoded by these families are exported to the Maurer's clefts, as peripheral or integral proteins of the Maurer's cleft membrane and largely exposed to the red cell cytosolic face of this membrane. A promoter titration approach was used to question the biological roles of these P. falciparum-specific exported proteins. Using the Pfepf1 family promoter, we observed the specific downregulation of all four families, correlating with the inefficient release of merozoites while the parasite intra-erythrocytic maturation and Maurer's clefts morphology were not impacted.

The export of numerous proteins to the plasma membrane of its host erythrocyte is essential for the virulence and survival of the malaria parasite Plasmodium falciparum. The Maurer's clefts, membrane structures transposed by the parasite in the cytoplasm of its host erythrocyte, play the role of a marshal platform for such exported parasite proteins. We identify here the export pathway of three resident proteins of the Maurer's clefts membrane: the proteins are exported as soluble forms in the red cell cytoplasm to the Maurer's clefts membrane in association with the parasite group II chaperonin (PfTRIC), a chaperone complex known to bind and address a large spectrum of unfolded proteins to their final location. We have also located the domain of interaction with PfTRiC within the amino-terminal domain of one of these Maurer's cleft proteins, PfSBP1. Because several Maurer's cleft membrane proteins with different export motifs seem to follow the same route, we propose a general role for PfTRiC in the trafficking of malarial parasite proteins to the host erythrocyte.

The pathogenicity of the most deadly human malaria parasite, Plasmodium falciparum, relies on the export of virulence factors to the surface of infected erythrocytes. A novel membrane compartment, referred to as Maurer's clefts, is transposed to the host erythrocyte, acting as a marshal platform in the red blood cell cytoplasm, for exported parasite proteins addressed to the host cell plasma membrane. We report here the characterization of three new P. falciparum multigene families organized in 9 highly conserved clusters with the Pfmc-2tm genes in the subtelomeric regions of parasite's chromosomes and expressed at early trophozoite stages. Like the PfMC-2TM proteins, the PfEPF1, 3 and 4 proteins encoded by these families are exported to the Maurer's clefts, as peripheral or integral proteins of the Maurer's cleft membrane and largely exposed to the red cell cytosolic face of this membrane. A promoter titration approach was used to question the biological roles of these P. falciparum-specific exported proteins. Using the Pfepf1 family promoter, we observed the specific downregulation of all four families, correlating with the inefficient release of merozoites while the parasite intra-erythrocytic maturation and Maurer's clefts morphology were not impacted.

Defining the pharmacological target(s) of currently used drugs and developing new analogues with greater potency are both important aspects of the search for agents that are effective against drug-sensitive and drug-resistant Mycobacterium tuberculosis. Thiacetazone (TAC) is an anti-tubercular drug that was formerly used in conjunction with isoniazid, but removed from the antitubercular chemotherapeutic arsenal due to toxic side effects. However, several recent studies have linked the mechanisms of action of TAC to mycolic acid metabolism and TAC-derived analogues have shown increased potency against M. tuberculosis. To obtain new insights into the molecular mechanisms of TAC resistance, we isolated and analyzed 10 mutants of M. tuberculosis that were highly resistant to TAC. One strain was found to be mutated in the methyltransferase MmaA4 at Gly101, consistent with its lack of oxygenated mycolic acids. All remaining strains harbored missense mutations in either HadA (at Cys61) or HadC (at Val85, Lys157 or Thr123), which are components of the β-hydroxyacyl-ACP dehydratase complex that participates in the mycolic acid elongation step. Separately, a library of 31 new TAC analogues was synthesized and evaluated against M. tuberculosis. Two of these compounds, 15 and 16, exhibited minimal inhibitory concentrations 10-fold lower than the parental molecule, and inhibited mycolic acid biosynthesis in a dose-dependent manner. Moreover, overexpression of HadAB HadBC or HadABC in M. tuberculosis led to high level resistance to these compounds, demonstrating that their mode of action is similar to that of TAC. In summary, this study uncovered new mutations associated with TAC resistance and also demonstrated that simple structural optimization of the TAC scaffold was possible and may lead to a new generation of TAC-derived drug candidates for the potential treatment of tuberculosis as mycolic acid inhibitors.