Unveiling Leishmania invasion of fibroblasts: calcium signaling, lysosome recruitment and exocytosis culminate with actin-independent invasion

Intracellular parasites of the genus Leishmania are the causative agents of human leishmaniasis, a widespread emergent tropical disease. The parasite is transmitted by the bite of a hematophagous sandfly vector that inoculates motile flagellated promastigote forms into the dermis of the mammalian host. After inoculation, parasites are ultimately captured by macrophages and multiply as round-shaped amastigote forms. Macrophages seem not to be the first infected cells since parasites were observed invading neutrophils first whose leishmania-containing apoptotic bodies were latter captured by macrophages, thereby becoming infected. The fact that Leishmania spp are able to live and replicate inside immune phagocytic cells and that macrophages are the main cell type found infected in chronicity created the perception that Leishmania spp are passive players waiting to be captured by phagocytes. However, several groups have described the infection of non-phagocytic cells in vivo and in vitro. The objective of this work was to study the cellular mechanisms involved in the invasion of non-professional phagocytes by Leishmania. We show that promastigotes of L.amazonensis actively induces invasion in fibroblasts without cytoskeleton activity, thus by a mechanism that is distinct from phagocytosis. Inside fibroblasts parasites transformed in amastigotes, remained viable for at least two weeks and re-transformed in promastigotes when returned to insect vector conditions. Similarly to what was observed for T. cruzi, infection involves calcium signaling, recruitment and exocytosis of lysosomes involved in plasma membrane repair and lysosome-triggered endocytosis. Conditions that alter lysosomal function such as cytochalasin-D and brefeldin-A treatment or the knockout of host cell lysosomal proteins LAMP-1 and 2 dramatically affected invasion. Likewise, triggering of lysosomal exocytosis and lysosome-dependent plasma membrane repair by low doses of streptolysin-O dramatically increased parasite entry. Together our results show that L.amazonensis promastigotes are able to take advantage of calcium-dependent lysosomal exocytosis and lysosome-induced endocytosis to invade and persist in non-phagocytic cells. AUTHOR SUMMARY Intracellular parasites of the genus Leishmania are the causative agents of leishmaniasis. The disease is transmitted by the bite of a sand fly vector which inoculates the parasite into the skin of mammalian hosts, including humans. During chronic infection the parasite lives and replicates inside phagocytic cells, notably the macrophages. An interesting but overlooked finding on Leishmania infection is that non-phagocytic cells have also been found infected by amastigotes. Nevertheless, the mechanisms by which Leishmania invades non-phagocytic cells were not studied to date. Here we show that L. amazonensis can actively induce their own entry into fibroblasts independently of actin cytoskeleton activity, thus by a mechanism that is distinct from phagocytosis. Invasion involves subversion of host cell functions such as calcium signaling and recruitment and exocytosis of host cell lysosomes involved in plasma membrane repair and whose positioning and content interfere in invasion. Parasites were able to replicate and remained viable in fibroblasts, suggesting that cell invasion trough the mechanism demonstrated here could serve as a parasite hideout and reservoir, facilitating infection amplification and persistence.


INTRODUCTION
The genus Leishmania comprises several species of intracellular parasites that cause a  [12]. 117 Despite its potential importance, the mechanism by which Leishmania spp. invade such cells 118 remains elusive. Therefore, we sought to investigate how the parasite invades cells unable to   Figure 1C shows that as early as 15 142 min about 18% of cells were RFP-positive. From 30 min to 4 h there were no substantial 143 changes, but after 24 h, 50% of the cells were infected. Since external parasites can be easily 144 removed by trypsin treatment, we can assume that RFP-positive cells are the infected cells.

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To verify whether host cell actin polymerization participates in the process of invasion, 146 MEFs were pre-treated with cytochalasin D to inhibit actin polymerization, and infection was 147 assessed. The result (Fig. 1D) shows not only that host cell actin polymerization is dispensable 148 for cell invasion, but that actin filament disassembly facilitates parasite entry, leading to 149 almost four-fold increase in the infection rate. In order to determine whether invasion of MEFs 150 is a unique property of metacyclic promastigotes, cells were incubated with either procyclic 151 or metacyclic LLa-RFP promastigotes (Fig. 1E). We observed that, unlike metacyclic forms, 152 procyclic promastigotes were not able to infect cells, indicating that the ability to invade MEFs 153 7 is acquired during metacyclogenesis. To determine whether cell entry depended on the 154 viability of parasites, MEFs were incubated with PFA-fixed or heat-treated LLa. We observed 155 that, while the infection rate by living parasites reached 18% (4 h) and 56% (24 h), no PFA-156 fixed or heat-treated promastigotes were internalized by MEFs, apart from a negligible amount 157 of heat-treated parasites at 24 h ( Fig. 1F and G). This result showed that only living metacyclic 158 promastigotes are able to enter MEFs.

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In order to determine whether lysosomes fused with parasite-containing intracellular 160 compartments, we stained cells with antibodies against the lysosomal protein LAMP-2 and 161 analyzed cells by fluorescence microscopy. Figure 1H shows a single focal plane of an    3A) was performed in calcium-free medium. As observed, parasites were able to trigger 219 calcium signaling even when calcium was absent in the extracellular media (Figs. 3 F and G).

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Together these results demonstrate that both intracellular calcium signaling and extracellular 221 calcium influx occur during contact of L. amazonensis promastigotes and host fibroblasts.   for example, leads to calcium-dependent exocytosis of lysosomes, which is followed by a 297 massive compensatory endocytosis that removes the damaged membrane from cell surface 298 [19]. Since we observed that parasites were inducing all these processes during cell entry, we LAMP-1-labeled infected cells were analyzed by fluorescence microscopy (Fig 7C to E). The 310 results showed multi-infected cells (Fig 7D) in which parasites also subsequently transformed 311 into the replicating amastigote forms (Fig. 7E).

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The remarkable ability of Leishmania spp. to survive and replicate inside phagocytes,  [12] [26] (reviewed by Rittig & Bogdan, 2000). In spite 318 of the importance of such observations, almost no effort has been made to understand how 319 these parasites succeed in infecting cells that are unable to perform classical phagocytosis.

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Here, using MEFs as a model, we show that entry of L. amazonensis into fibroblasts is a 321 process that involves the ability of these parasites to actively induce a cell invasion mechanism   (Figs. 1A, 1B and 1C), it became evident that these cells could be invaded by 329 the parasites, and that these parasites were found inside lysosome-derived vacuoles (Fig. 1H) 330 as observed for macrophages. However, unlike the phagocytosis-mediated entry that occurs  In the model described here, contact with live L. amazonensis promastigotes also induced 341 strong intracellular calcium transients in MEFs (Fig. 3A-B and 3F-G). Calcium seems to be 342 an important requirement for cell invasion, since its increase in the extracellular medium 343 positively modulated parasite entry (Fig. 3D). 344 We reasoned that one mechanism for the parasites to trigger calcium elevation in the 345 cytoplasm might be the generation of host cell PM wounds during invasion. Indeed, we 346 showed that contact with live L. amazonensis promastigotes wounds the PM of host cells, and 347 the lesions are promptly repaired in the presence of calcium ( Fig. 3C and 3D). In fact, when    Interestingly, in the experiments described here the exocytosis of the lysosomal enzyme beta-371 Hex peaked at 15 min of infection (Fig. 4D), matching the early triggering of calcium 372 transients (Fig. 3A) and the appearance of infected cells as early as 15 min after parasite 373 inoculation (Fig. 1C). It is known that after exocytosis from lysosomes, ASM cleaves 374 sphingomyelin on cell surface producing ceramide, a lipid that promotes negative curvature 375 of the PM enabling endocytosis [19]. A ceramide-rich vacuole, as opposed to actin-rich 376 vacuole, is precisely what is observed in endosomes derived from the extracellular action of 377 16 ASM during T. cruzi internalization [5]. Also similar to earlier observations, we found that 378 recently internalized Leishmania parasites are surrounded by a tight PV (Fig. 2E-insert), 379 which is intensely stained by anti-LAMP-1 (Fig. 4G) and anti-ceramide antibodies (Fig. 4H). 380 This indicates that invasion actually takes advantage of exocytosis of lysosomes, which 381 provide the membrane that allows parasite entry, in a mechanism that is markedly distinct 382 from classical parasite internalization by phagocytosis in macrophages. This is corroborated 383 by the facts that L. amazonensis parasites can still invade MEFs pre-treated with cytochalasin-384 D (Fig 1D), and that recently internalized parasites do not co-localize with actin filaments  (Fig. 6H and 6I) when compared to wild type cells 398 (Fig. 6G), similar to what was observed for T. cruzi infection with the same cell lines [25]. 399 Additionally, our results indicate that Leishmania promastigotes are able to trigger calcium 400 signaling in host cells from intracellular stores (Fig. 3F and G) since signaling also occurs in 401 the absence of extracellular calcium. Further investigation will be needed to identify the 402 molecules involved in this signaling. However, regardless of the origin of the calcium, from 403 extracellular influx or intracellular reservoirs, the downstream effects important for cell 404 invasion such as lysosomal exocytosis and its derived endocytosis would be triggered. 405 We still do not know how parasites induce PM injury in MEFs (Fig. 3C-D). However, Leishmania spp. infection, notably at the early stages. Since these parasites are able to 451 replicate inside fibroblasts, as we report here (Fig 2A) and as described by others (reviewed 452 by Rittig and Bogdan, 2000), it is possible that a first round of replication inside these cells 453 could be an important step leading to infection amplification, prior to macrophage invasion.

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The ability to actively induce cell invasion characterized here is a neglected feature of 455 Leishmania spp, probably due to the fact that these parasites have been largely perceived as