Cytosolic eukaryotic 2-Cys-peroxiredoxins have been widely reported to act as dual-function proteins, either detoxifying reactive oxygen species or acting as chaperones to prevent protein aggregation. Several stimuli, including peroxide-mediated sulfinic acid formation at the active site cysteine, have been proposed to trigger the chaperone activity. However, the mechanism underlying this activation and the extent to which the chaperone function is crucial under physiological conditions in vivo remained unknown. Here we demonstrate that in the vector-borne protozoan parasite Leishmania infantum, mitochondrial peroxiredoxin (Prx) exerts intrinsic ATP-independent chaperone activity, protecting a wide variety of different proteins against heat stress-mediated unfolding in vitro and in vivo. Activation of the chaperone function appears to be induced by temperature-mediated restructuring of the reduced decamers, promoting binding of unfolding client proteins in the center of Prx's ringlike structure. Client proteins are maintained in a folding-competent conformation until restoration of nonstress conditions, upon which they are released and transferred to ATP-dependent chaperones for refolding. Interference with client binding impairs parasite infectivity, providing compelling evidence for the in vivo importance of Prx's chaperone function. Our results suggest that reduced Prx provides a mitochondrial chaperone reservoir, which allows L. infantum to deal successfully with protein unfolding conditions during the transition from insect to the mammalian hosts and to generate viable parasites capable of perpetuating infection.chaperone | Leishmania | peroxiredoxin

Two-cysteine peroxiredoxins are ubiquitous peroxidases that play various functions in cells. In Leishmania and related trypanosomatids, which lack catalase and selenium-glutathione peroxidases, the discovery of this family of enzymes provided the molecular basis for peroxide removal in these organisms. In this report the functional relevance of one of such enzymes, the mitochondrial 2-Cys peroxiredoxin (mTXNPx), was investigated along the Leishmania infantum life cycle. mTXNPx null mutants (mtxnpx−) produced by a gene replacement strategy, while indistinguishable from wild type promastigotes, were found unable to thrive in a murine model of infection. Unexpectedly, however, the avirulent phenotype of mtxnpx− was not due to lack of the peroxidase activity of mTXNPx as these behaved like controls when exposed to oxidants added exogenously or generated by macrophages during phagocytosis ex vivo. In line with this, mtxnpx− were also avirulent when inoculated into murine hosts unable to mount an effective oxidative phagocyte response (B6.p47phox−/− and B6.RAG2−/− IFN-γ−/− mice). Definitive conclusion that the peroxidase activity of mTXNPx is not required for parasite survival in mice was obtained by showing that a peroxidase-inactive version of this protein was competent in rescuing the non-infective phenotype of mtxnpx−. A novel function is thus proposed for mTXNPx, that of a molecular chaperone, which may explain the impaired infectivity of the null mutants. This premise is based on the observation that the enzyme is able to suppress the thermal aggregation of citrate synthase in vitro. Also, mtxnpx− were more sensitive than controls to a temperature shift from 25°C to 37°C, a phenotype reminiscent of organisms lacking specific chaperone genes. Collectively, the findings reported here change the paradigm which regards all trypanosomatid 2-Cys peroxiredoxins as peroxide-eliminating devices. Moreover, they demonstrate, for the first time, that these 2-Cys peroxiredoxins can be determinant for pathogenicity independently of their peroxidase activity.

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Highlights d An early PSE misspecification promotes identity hybridism between PSE and CC d Meristematic CC and MSE retain plastic identity to safeguard phloem functionality d RPK2 excludes PSE identity from PSE-surrounding cells within the root meristem d CLE45 maintains PSE and PSE-surrounding cells in a plastic identity stage