Higher expression of proline dehydrogenase altered mitochondrial function and 1 increased Trypanosoma cruzi differentiation in vitro and in the insect vector 2 3 4

The pathogenic protist Trypanosoma cruzi uses kissing bugs as intermediate hosts that vectorize the infection among mammals. This parasite oxidizes proline to glutamate through two enzymatic steps and one nonenzymatic step. In insect vectors, T. cruzi differentiates from a noninfective replicating form to nonproliferative infective forms. Proline sustains this differentiation, but to date, a link between proline metabolism and differentiation has not been established. In T. cruzi, the enzymatic steps of the proline-glutamate oxidation pathway are catalysed exclusively by the mitochondrial enzymes proline dehydrogenase [TcPRODH, EC: 1.5.5.2] and D1-pyrroline-5-carboxylate dehydrogenase [TcP5CDH, EC: 1.2.1.88]. Both enzymatic steps produce reducing equivalents that are able to directly feed the mitochondrial electron transport chain (ETC) and thus produce ATP. In this study, we demonstrate the contribution of each enzyme of the proline-glutamate pathway to ATP production. In addition, we show that parasites overexpressing these enzymes produce increased levels of H2O2, but only those overexpressing TcP5CDH produce increased levels of superoxide anion. We show that parasites overexpressing TcPRODH, but not parasites overexpressing TcP5CDH, exhibit a higher rate of differentiation into metacyclic trypomastigotes in vitro. Finally, insect hosts infected with parasites overexpressing TcPRODH showed a diminished parasitic load but a higher percent of metacyclic trypomastigotes, when compared with controls. Our data show that parasites overexpressing both, PRODH and P5CDH had increased mitochondrial functions that orchestrated different oxygen signalling, resulting in different outcomes in relation to the efficiency of parasitic differentiation in the invertebrate host.


4
[19], oxidative imbalance [19][20][21] and thermal stress [19,22]. In addition, proline 76 oxidation is able to fulfil the energy demands of the parasite [10,20,23,24]. 77 Early studies of proline metabolism in T. cruzi epimastigotes showed that this 78 amino acid triggers O 2 consumption and is converted into glutamate and further into 79 several tricarboxylic acid (TCA) cycle intermediates [10]. Later, it was shown that T. (mediated by PRODH) had higher oxygen consumption rates and ATP synthesis, and 99 stimulated hydrogen peroxide production. Notably, this increased mitochondrial 100 Downloaded from http://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20210428/922042/bcj-2021-0428.pdf by guest on 22 October 2021 Biochemical Journal. This is an Accepted Manuscript. You are encouraged to use the Version of Record that, when published, will replace this version. The most up-to-date-version is available at https://doi.org/10.1042/BCJ20210428 7 exchange chromatography using resin DOWEX 50 WX8-400 (Sigma  ) according to 152 the method of [31] with small modifications as described in Mantilla et al. (2015). 153

Western blot analysis 154
Total protein extracts from epimastigotes (pTEX, pTEX-TcPRODH and pTEX-155 TcP5CDH) were prepared for western blot analysis. The cells were harvested, washed 156 twice with PBS, and resuspended in lysis buffer (50 mM Tris-HCl, pH 7.4, 50 mM 157 NaCl, 2 mM EGTA, 2 mM EDTA, 20% glycerol (v/v), 0.1% Triton X-100 (v/v), 1 158 mM PMSF and protease inhibitor mixture (Sigma ® ). The lysates were clarified by 159 centrifugation (16,000 g for 15 min at 4°C), and the protein concentrations of the 160 supernatants were determined by the Bradford method using bovine serum albumin 161 (BSA) as a standard (Bradford, 1976 seeded onto poly-L-lysine-coated cover slips. The cells were fixed (20 min) with 2% 181 phosphomolybdate was then precipitated with 800 μL t-butyl acetate. The resulting 241 precipitate was washed twice with the same reagent, and the remaining aqueous phase 242 containing glucose-6-32 P (900 L) was spotted on filter paper for radioactivity counts. 243 The relative net synthesis of ATP was expressed in relation to the number of 244 scintillations per minute (CPM) obtained under basal conditions. 245

3.1.Obtaining TcPRODH-and TcP5CDH-overexpressing (OE) parasites. 288
To better understand the role of the enzymes involved in the catabolism of 289 proline in T. cruzi (Fig. 1A), we obtained parasite lineages that overexpressed the 290 TcPRODH and TcP5CDH genes in the epimastigote insect stage. The detection of 291 TcPRODH and TcP5CDH by immunoblotting showed augmented protein levels in 292 the transfected lineages compared to controls ( Fig. 1B and 1C). Both overexpression 293 lines had PRODH and P5CDH enzymatic activities that were increased 2.5-and 3-294 fold, respectively, compared to those of the WT and pTEX (control) cell lines (Fig.  295   1D and 1E). We also ensured that their intracellular locations were preserved via 296 immunofluorescence microscopy. This confirmed the mitochondrial distribution of 297 the overexpressed proteins which were visualized by MitoTracker costaining for both 298 TcPRODH-OE and TcP5CDH-OE cell lines ( Fig. 2A, B). After characterization of 299 the mutants, we performed a first phenotypic analysis by assaying possible changes in 300 the cell proliferation profile. Our results showed that proliferation rates, as well as cell 301 density at the stationary phase, did not change by overexpressing any of the enzymes 302 involved in proline catabolism when compared to controls ( Figure S1). 303

3.2.Effects of TcPRODH and TcP5CDH overexpression on mitochondrial 304
physiology 305 As mentioned earlier, epimastigotes of T. cruzi use proline for ATP 306 production, and both the PRODH and P5CDH enzymes involved in catabolizing 307 proline to glutamate are able to energize oxidative phosphorylation [20, 23]. Thus, we 308 were interested in measuring the overexpression effects of each enzyme involved in 309 proline catabolism on proline-stimulated parasite respiration. We observed that 310 parasites overexpressing TcPRODH increased their basal and maximal respiratory 311 capacities by approximately 1.5-and 2-fold compared to WT and pTEX controls, 312 respectively (Fig. 3A). When comparing TcP5CDH cells to both controls, we found 313 that their basal and maximal respiratory capacities were increased by approximately 314 1.2-and 1.5-fold, relative to WT and pTEX controls, respectively ( Fig. S2 A and Fig.  315 3A). The stimulation with succinate (as a control) did not show significant changes in 316 the respiratory parameters, showing that both overexpressing lineages did not have a 317 spurious increase in their mitochondrial respiration (Fig. S2 B): differences in O 2 318 consumption between pTEX and pTEX-TcPRODH, or pTEX and pTEX-TcP5CDH 319 are not significant (p = 0.3864 and 0.4027, respectively). Since TcPRODH and 320 TcP5CDH had their activities increased by approximately 2.5X and 3X, respectively, 321 it seems that both enzymes contributed similarly to the increase in the delivery of 322 electrons into the ETC (Fig. 3A). As expected, lineages overexpressing TcPRODH or 323 TcP5CDH showed significantly higher proline-dependent ATP synthesis than 324 controls, and this effect was fully abolished upon antimycin addition (Fig. 3B). 325 Similarly, P5C-dependent ATP synthesis was increased in TcP5CDH mutants (Fig.  326   S3). This finding reinforces the idea that in proline-stimulated parasites, reduced 327 equivalents (FADH 2 and NADH) contribute to cellular respiration, and this process is 328 coupled to ATP synthesis, as previously shown [20,23]. To this end, we used the fluorescent probe Amplex Red, which allowed us to detect 341 increased amounts of H 2 O 2 in digitonin-permeabilized cells overexpressing each 342 enzyme in relation to the controls (Fig. 4A). Both overexpressing parasites had an 343 increased H 2 O 2 production, however, it was more pronounced in TcP5CDH-OE 344 parasites (Fig. 4A, 2.5-fold increase). Interestingly, the superoxide generation 345 (observed using the Mito-SOX probe, Fig. 4B), was only increased in TcP5CDH-OE 346 parasites, which reached similar levels to those observed with Antimycin A. .No 347 significant difference was detected for the production of these species by TcPRODH-348 OE cells (Fig 4B, right). 349

3.3.TcPRODH and TcP5CDH overexpression in T. cruzi increases 350 metacyclogenesis in the vector host. 351
It is well-known that apart from contributing to the cellular energy supply, 352 proline is involved in T. cruzi cellular differentiation from epimastigotes to metacyclic 353 trypomastigotes [12,13]. Therefore, we decided to investigate the possible 354 involvement of the enzymes of the proline catabolism pathway in metacyclogenesis in 355 vitro and in the insect vector. To this end, we initially submitted the parasites to in 356 vitro metacyclogenesis. Parasites overexpressing TcPRODH differentiated earlier, 357 and 1.8-fold more than those overexpressing TcP5CDH or controls (Fig. 5A). As we 358 observed that only TcPRODH affected metacyclogenesis, we investigated whether 359 this lineage showed increased metacyclogenesis in vivo. To this end, we infected 360 reduviid insects (Rhodnius prolixus) with the TcPRODH-OE cell line or with control 361 parasites. We initially measured the parasite loads in each infected insect by qPCR. 362 Surprisingly, insects with the TcPRODH cell line had lower parasitic loads than 363 insects with the controls (Fig. 5B). However, we observed that the percentage of 364 developed metacyclic trypomastigotes in kissing bugs infected with parasites 365 overexpressing TcPRODH almost doubled in relation to the controls (Fig. 5C). The 366 increased proportion of metacyclic trypomastigotes in the insects that had been 367 infected with parasites overexpressing TcPRODH remained until 12 days post 368 infection of R. prolixus (Fig. 5C). Typical microscopic images used for counting 369 metacyclic trypomastigotes from vector excreta content are shown in Figure S4. (alone or in different combinations), such as glucose, proline, aspartate or glutamine, 399 is necessary for the parasite to complete the differentiation process [12,13,45]. In 400 fact, previous studies focused on the ability of proline alone to trigger 401 metacyclogenesis [13,19]. On this basis, we investigated whether an increase in the 402 expression of the enzymes involved in proline metabolism would affect the 403 differentiation process. Our data revealed that parasites with increased PRODH 404 activity, and thus a higher proline oxidation capacity, had increased ATP production 405 and mildly increased H 2 O 2 production, and were less efficient in establishing the 406 infection but more efficient in differentiating into metacyclic trypomastigotes in R. 407 prolixus. This effect was specific to PRODH since it was not observed when the 408 P5CDH activity was increased to a similar extent. In this case, excess P5C produced 409 by increased PRODH activity could be exported into the cytoplasm and reduced back