Cellular remodeling during differentiation is essential for lifecycle progression of many unicellular eukaryotic pathogens such as Leishmania, but the mechanisms involved are largely uncharacterized. The role of endosomal sorting in differentiation was analyzed in Leishmania major by overexpression of a dominant-negative ATPase, VPS4. VPS4 E235Q accumulated in vesicles from the endocytic pathway, and the mutant L. major was deficient in endosome sorting. Mutant parasites failed to differentiate to the obligate infective metacyclic promastigote form. Furthermore, the autophagy pathway, monitored via the expression of autophagosome marker GFP-ATG8, and shown to normally peak during initiation of metacyclogenesis, was disrupted in the mutants. The defect in late endosome-autophagosome function in the VPS4 E235Q parasites made them less able to withstand starvation than wild-type L. major. In addition, a L. major ATG4-deficient mutant was found also to be defective in the ability to differentiate. This finding, that transformation to the infective metacyclic form is dependent on late endosome function and, more directly, autophagy, makes L. major a good model for studying the roles of these processes in differentiation.

SummaryEcotin is a potent inhibitor of family S1A serine peptidases, enzymes lacking in the protozoan parasite Leishmania major. Nevertheless, L. major has three ecotin-like genes, termed inhibitor of serine peptidase (ISP). ISP1 is expressed in vectorborne procyclic and metacyclic promastigotes, whereas ISP2 is also expressed in the mammalian amastigote stage. Recombinant ISP2 inhibited neutrophil elastase, trypsin and chymotrypsin with Kis between 7.7 and 83 nM. L. major ISP2-ISP3 double null mutants (Disp2/3) were created. These grew normally as promastigotes, but were internalized by macrophages more efficiently than wild-type parasites due to the upregulation of phagocytosis by a mechanism dependent on serine peptidase activity. Disp2/3 promastigotes transformed to amastigotes, but failed to divide for 48 h. Intracellular multiplication of Disp2/3 was similar to wild-type parasites when serine peptidase inhibitors were present, suggesting that defective intracellular growth results from the lack of serine peptidase inhibition during promastigote uptake. Disp2/3 mutants were more infective than wild-type parasites to BALB/c mice at the early stages of infection, but became equivalent as the infection progressed. These data support the hypothesis that ISPs of L. major target host serine peptidases and influence the early stages of infection of the mammalian host.

Leishmania major is a protozoan parasite that causes skin ulcerations in cutaneous leishmaniasis. In the mammalian host, the parasite resides in professional phagocytes and has evolved to avoid killing by macrophages. We identified L. major genes encoding inhibitors of serine peptidases, ISPs, which are orthologues of bacterial ecotins and found that ISP2 inhibits trypsin-fold S1A family peptidases. Here we show that L. major mutants deficient in ISP2 and ISP3 (Δisp2/3) trigger higher phagocytosis by macrophages through a combined action of the complement type-3 receptor (CR3), toll-like receptor 4 (TLR4) and unregulated activity of neutrophil elastase (NE), leading to parasite killing. While all three components are required to mediate enhanced parasite uptake, only TLR4 and NE are necessary to promote parasite killing after infection. We found that the production of superoxide by macrophages in the absence of ISP2 is the main mechanism controlling the intracellular infection. Furthermore, we show that NE modulates macrophage infection in vivo, and that the lack of ISP leads to reduced parasite burdens at later stages of the infection. Our findings support the hypothesis that ISPs function to prevent the activation of TLR4 by NE during the Leishmania-macrophage interaction in order to promote parasite survival and growth.

Leishmania major, an intracellular parasitic protozoon that infects, differentiates and replicates within macrophages, encodes two closely related MIF-like proteins, which have only ~20% amino acid identity with mammalian MIF. Recombinant L. major MIF1 and MIF2 have been expressed and the structures, resolved by X-ray crystallography, show a trimeric ring architecture similar to mammalian MIF but with some structurally distinct features. LmjMIF1, but not LmjMIF2, has tautomerase activity, indicating that the LmjMIFs have evolved potentially different biological roles. This is further demonstrated by the differential life cycle expression of the proteins. LmjMIF2 is found in all life cycle stages whereas LmjMIF1 is found exclusively in amastigotes, the intracellular stage responsible for mammalian disease. The findings are consistent with parasite MIFs modulating or circumventing the host macrophage response and thereby promoting parasite survival, however analysis of the L. braziliensis genome showed that this species lacks intact MIF genes -highlighting that MIF is not a virulence factor in all species of Leishmania.Parasites have adopted many strategies to avoid or subvert their hosts' immune responses including antigenic variation (1), signalling subversion (2) and immune evasion genes (3). Leishmania parasites are obligate intracellular pathogens and the causative agents of leishmaniases. The form and severity of the disease ranges from relatively mild dermal lesions to often fatal visceral infection depending on the infecting Leishmania species and the immune status of the host (4). Leishmania enter macrophages by receptor-mediated endocytosis (5) where they differentiate into amastigotes. A number of strategies are employed by the amistagotes to survive within parasitophorous vacuoles in host macrophages, including inhibition of NO production, hydrolytic enzymes and calcium chelation (6).During the annotation of the L. major genome (7), two genes in tandem (LmjMIF1 and LmjMIF2) were identified that encode proteins related to mammalian Macrophage Migration Inhibitory Factor (MIF), which is a major mediator of inflammatory processes (8). Increased serum levels of MIF contribute to a plethora of auto-immune diseases There are now over 30 X-ray structures of MIF available from 8 species including, human (23), rat (24), mouse (25), frog (26) and the parasites Brugia malayi (27) and L. major (28) and this study). The trimeric architecture is maintained in all structures. Sequence identities among mammalian MIF sequences are high (above 85%) however this drops to 20-27% identity between the human and protozoan Leishmania and Plasmodium sequences. All of the structurally characterised MIF sequences show keto-enol tautomerase activity with model substrates like p-hydroxyphenylpyruvate and D-dopachrome, though the biological relevance of this well characterised activity is still unknown. Small molecule tautomerase inhibitors of mammalian MIF (29) are being developed for treatment of inflammatory diseases suc...