Binding of Leishmania infantum LPG to the midgut is not sufficient to define vector competence in Lutzomyia longipalpis sand flies

The major surface lipophosphoglycan (LPG) of Leishmania parasites is critical to vector competence in restrictive sand fly vectors by mediating Leishmania attachment to the midgut epithelium, considered essential to parasite survival and development. However, the relevance of LPG for sand flies that harbor multiple species of Leishmania remains elusive. We tested binding of Leishmania infantum wild type (WT), LPG-defective (Δlpg1 mutants) and add-back lines (Δlpg1 + LPG1) to sand fly midguts in vitro and their survival in Lutzomyia longipalpis sand flies in vivo. Le. infantum WT parasites attached to the Lu. longipalpis midgut in vitro with late-stage parasites binding to midguts in significantly higher numbers compared to early-stage stage promastigotes. Δlpg1 mutants did not bind to Lu. longipalpis midguts, and this was rescued in the Δlpg1 + LPG1 lines, indicating that midgut binding is mediated by LPG. When Lu. longipalpis sand flies were infected with either Le. infantum WT, Δlpg1, or Δlpg1 + LPG1 of the BH46 or BA262 strains, the BH46 Δlpg1 mutant, but not the BA262 Δlpg1 mutant, survived and grew to similar numbers as the WT and Δlpg1 + LPG1 lines. Exposure of BH46 and BA262 Δlpg1 mutants to blood engorged midgut extracts led to the mortality of the BA262 Δlpg1 but not the BH46 Δlpg1 parasites. These findings suggest that Le. infantum LPG protects parasites on a strain-specific basis early in infection, likely against toxic components of blood digestion, however, it is not necessary to prevent Le. infantum evacuation along with the feces in the permissive vector Lu. longipalpis. IMPORTANCE It is well established that LPG is sufficient to define the vector competence of restrictive sand fly vectors to Leishmania parasites. However, the permissiveness of other sand flies to multiple Leishmania species suggests that other factors might define vector competence for these vectors. In this study, we investigated the underpinnings of Leishmania infantum survival and development in its natural vector Lutzomyia longipalpis. We found out that LPG-mediated midgut binding persists in late-stage parasites. This observation is paradigm-changing and suggests that only a subset of infective metacyclics lose their ability to attach to the midgut with implications for parasite transmission dynamics. However, our data also demonstrate that LPG is not a determining factor in Leishmania infantum retention in the midgut of Lutzomyia longipalpis, a permissive vector. Rather, LPG appears to be more important in protecting some parasite strains from the toxic environment generated during blood meal digestion in the insect gut. Thus, the relevance of LPG in parasite development in permissive vectors appears to be complex and should be investigated on a strain-specific basis.


Le. infantum binding to the midgut epithelium of Lu. longipalpis is stage-dependent 147
To validate and extend our observation, we used a different strain of Le. infantum, 148 MCAN/BR/09/52 (LAB), and assessed its binding to sand fly midguts. Parasites harvested on 149 day 3 attached to the midgut epithelium of Lu. longipalpis at a median of 5,250 parasites/midgut 150 ( Fig. 2A). As the LAB parasite cultures aged, the number of parasites binding the sand fly 151 midgut increased proportionally, peaking with day 6 parasites at a median of 8,500 152 parasites/midgut ( Fig. 2A). A greater proportion of parasite binding to the midgut epithelium as 153 the culture aged was also observed for the Le. infantum BH46 strain ( Fig. S1A-D), ranging from 154 a median of 4,200 parasites/midgut for day 4 old culture parasites (Fig. S1A) to a median of 155 26,000 parasites/midgut with day 6 old culture parasites (Fig. S1C). As the culture got older (Fig. 156 2B; Fig. S2A), the parasites began to differentiate to the infective form, the metacyclic 157 promastigotes, increasing from 10-30% on day 4 to about 80% on day 7 of culture, respectively 8 ( Fig. 2C; Fig. S2B). As we have observed that many, if not most, of the bound parasites were at 159 late stages of differentiation (leptomonads and metacyclic), we used a Ficoll gradient to separate 160 early-stage parasites (ESP) from late-stage parasites (LSP) and tested if they could bind to sand 161 fly midgut epithelium. Surprisingly, we observed that significantly more LSP bound to the sand 162 fly midgut epithelium compared to ESP, for both Le. infantum LAB ( Fig. 2D and E) and BH46 163 ( Fig. 2F and G) strains, harvested on both day 4 and day 5. 164 In order to assess the abundance of LPG on the surface of BH46 WT parasites, we 165 stained LSP and ESP Ficoll-purified parasites with a LPG backbone-specific monoclonal 166 antibody (CA7AE). Using flow cytometry, we show that LSP parasites exhibited a 2-to 20-fold 167 higher antibody binding than ESP samples for 4 and 5-day old cultures, respectively (

Le. infantum binding to the midgut epithelium of Lu. longipalpis is LPG-dependent 176
In order to assess whether or not the major surface glycoconjugate of Le. infantum (LPG) 177 was the parasite ligand to the midgut epithelium of Lu. longipalpis, we carried out midgut 178 binding assays with the Le. infantum BH46 wild type strain (BH46 WT) as well as both the LPG-179 deficient (Dlpg1) mutant and the add-back (Dlpg1 + LPG1) lines. Similar to what was observed 180 for unfed midguts (Fig. S1), aging parasites from the BH46 WT line bound to the epithelium of 9 midguts dissected 5 days after blood feeding with increasing efficiency, whereas parasite binding 182 was limited in unopened midguts (Fig. 4). For both fed and unfed Lu. longipalpis midguts, 183 binding of the BH46 Dlpg1 + LPG1 was intermediate and significantly higher than that of the 184 BH46 Dlpg1 mutant that failed to bind, exhibiting less than 1,500 parasite/midgut regardless the 185 age of the harvested parasites or the feeding status of the midguts ( Fig. 4 and Fig. S1). Similar 186 results were obtained with the Le. infantum BA262 Dlpg1 mutant, which also failed to bind to 187 unfed midguts (Fig. S3). Comparatively, the BA262 WT bound with increased efficiency as the 188 parasites aged ( Fig. S3  WT or BH46 Dlpg1 + LPG1 (Fig. 5C-J). When the infection was started with 2 million 203 parasites/mL, the BH46 Dlpg1 mutant displayed a lower, yet not significant, parasite load on day 204 3 (Fig. S5A), but higher parasite loads than either the WT strain or the Dlpg1 + LPG1 from day 6 205 onwards ( Fig. S5B-E). 206 207 Le. infantum BA262 Dlpg1 mutants fail to grow in the midguts of Lu. longipalpis 208 As the LPG side chain decorations are polymorphic amongst Le. infantum strains (32), 209 we also assessed the ability of BA262 WT strain, along with the BA262 Dlpg1 and BA262 Dlpg1 210 + LPG1 lines (33), to develop in Lu. longipalpis (Fig. 6). Different from the BH46 strain, which 211 exhibits side chains with 1 to 3 b-glucose residues, the LPG of BA262 is devoid of side chains, 212 like most of the Le. infantum strains (32). Seeded with 5 million parasites/mL, the BA262 Dlpg1 213 mutant displayed lower parasite loads on days 2 and 3 (median 0-500 parasites/midgut) as 214 compared to the WT (median 3,000-5,000 parasites/midgut) and Dlpg1 + LPG1 (median 1,600-215 13,750 parasites/midgut) lines, and a lower infection prevalence before the blood was passed 216 To investigate the differences in sand fly survival of the BH46 and BA262 Dlpg1 225 mutants, we tested if resistance to by-products blood meal digestion could be a factor that 226 explains these differences. For this, we incubated in vitro parasites in exponential phase of 227 growth with extracts of midguts, collected at 24 and 48 hours after blood feeding, for 4 hours at 228 26°C, and compared them to WT and Dlpg1 + LPG1 lines (Fig. 7). BH46 Dlpg1 mutants were 229 not affected by the components of the digested sand fly blood meal (Fig. 7A). In contrast, the 230 BA262 Dlpg1 mutants were severely affected by incubation with midguts collected 24h and 48h 231 post-blood feeding (Fig. 7B). The WT and Dlpg1 + LPG1 lines of both strains were not affected 232 by incubation with midgut extracts (Fig. 7A-B). Many studies have demonstrated that binding by Le. major to the midguts of the 236 restrictive vectors is mediated by LPG (6,10,11,[23][24][25]34). Incubation of P. papatasi midguts 237 with purified PGs from procyclic Le. major parasites prevented the binding of procyclic Le. 238 major parasites (6), and Le. major Dlpg1 parasites cannot develop in P. papatasi (23, 34) or P. 239 duboscqi (24, 25) sand flies. On the other hand, the Le. major Dlpg1 binds to midguts and 240 develops well in permissive vectors, such as Lu. longipalpis (23, 24), as well as Phlebotomus 241 arabicus (23), P. argentipes (25), and P. perniciosus (25). Based on such findings, an LPG-242 independent mechanism for midgut binding was proposed for the permissive vectors (27). 243 Differences in midgut glycosylation between restrictive and permissive vectors were observed 244 that could possibly account for non-specific binding of parasites in the latter. It was hypothesized 245 that a lectin on the Leishmania surface might bind to the O-linked glycans of the midgut 246 microvilli of permissive vectors allowing parasite binding (25,27). Nonetheless, such studies 247 were carried out using Le. major and unnatural permissive vectors (23)(24)(25)27); thereby, the LPG-248 independent mechanism could be restricted to Le. major development in unnatural permissive 249 vectors. Therefore, we decided to revisit the role of LPG in parasite development in natural 250 permissive vectors, focusing on two Le. infantum strains bearing intraspecies polymorphisms in 251 their LPGs (25). The LPG of one strain, BA262, is devoid of side chains (type I) whereas that of 252 the BH46 strain has b-glucose sugars branching-off the repeat unit backbone (type III; (20)). 253 Here, we demonstrate that Le. infantum (BH46) wild type and Dlpg1 + LPG1 lines bound to both 254 unfed and 5 days post-feeding midgut epithelia of Lu. longipalpis in vitro. In contrast, the LPG-255 deficient Dlpg1 failed to bind to Lu. longipalpis unfed and fed midguts. Similar results were 256 reported for Dlpg1 mutants of Le. mexicana (MPRO/BR/72/M1845) and Le. infantum 257 (MHOM/BR/76/M4192) that exhibited poor binding to midguts of Lu. longipalpis and P. 258 perniciosus (26), respectively. Altogether, these data indicate that LPG mediates Leishmania 259 binding to midguts of natural permissive vectors, and suggest that the previously described LPG-260 independent midgut binding mechanism may be limited to Le. major binding to midguts of 261 permissive vectors. 262 Similar to conclusions for Le. major binding to the midgut of its natural restrictive vector, 263 previous reports claimed that Le. infantum and Le. donovani binding to the midgut of a natural 264 permissive vector was also restricted to early-stage parasites (19,20). Nonetheless, the fact that 265 PNA was used to sort procyclic and metacyclic Le. infantum (20)  Knowing whether stronger LPG labelling is related to an increase in the number and/or length of 284 LPG molecules during metacyclogenesis will shed light on the binding mechanism of the Le. 285 infantum LPG to the receptor on the midgut microvilli. 286 Electron microscopy images of Le. infantum developing in Lu. longipalpis show that 287 parasites, then described as long and short nectomonads, and now termed nectomonads and 288 leptomonads, respectively, were observed attached to the posterior and anterior midguts 289 throughout the parasite life cycle in the sand fly (37). In our experiments, early-stage parasites 290 were not able to attach to midguts in vitro, which might be explained by a lack of bona fide 291 nectomonad parasites in cultures expressing LPG on their surface. On the other hand, we 292 observed strong Lu. longipalpis midgut binding by late-stage Le. infantum parasites, enriched in 293 leptomonads and metacyclics, confirming early observations by electron microscopy that Le. 294 infantum late-stage parasites also bind to the Lu. longipalpis midgut epithelium (37). Whether 295 midgut binding by late stage parasites is necessary for parasite migration to the anterior midgut 296 (38), genetic exchange (39, 40), and/or preventing metacyclic parasites to be pushed to posterior 297 midgut upon sequential blood meals (41), has yet to be determined. 298 Our proposed model of LPG-mediated Le. infantum attachment to the midgut of a 299 permissive vector complements and expands upon previous findings demonstrating that changes 300 in LPG during metacyclogenesis mediates parasite detachment from the midgut epithelium to be 301 transmitted. Here, we propose that metacyclics encompass two subpopulations: one that binds to 302 the midgut epithelium, as was observed in this study and also earlier (37), and one free-303 swimming that is transmitted by a sand fly bite (19,20). The existence of such subpopulations is 304 also supported by polymorphisms in the LPG cap of metacyclic Le. infantum (20)  Dlpg1 mutant struggled to survive at the time of blood digestion on day 3, yet it thrived after the 316 blood was passed, reaching similar parasite loads as the WT and add-back lines at later time 317 points. In contrast, the BA262 Dlpg1 mutant not only struggled to survive up to day 3 but 318 succumbed afterwards, revealing important inter-strain differences in Le. infantum in vivo 319 development. These data indicate that LPG-mediated epithelium binding is not a determinant of 320 parasite survival in the midgut for this parasite-vector pair. However, the presence of LPG does 321 confer protection to some Le. infantum strains such as BA262. 322 Toxicity of blood components can affect the survival of Leishmania parasites in the sand 323 fly midgut during digestion (9). In the present study, incubation of the Le. infantum strains, 324 BH46 and BA262, harvested from 3 days old cultures with extracts of engorged midguts 325 produced different outcomes. Whereas the BH46 and BA262 WT and Dlpg1 + LPG1 lines, as 326 well as the BH46 Dlpg1 mutant, survived exposure to the toxic components of the digested 327 blood, the BA262 Dlpg1 mutant was highly affected by midgut extracts. These results correlated 328 well with the survival of the BH46 Dlpg1 mutant and the lack of further development of the 329 BA262 lpg1 KO line in sand flies. Together, these results indicate that neither LPG type I nor III 330 is needed to prevent Le. infantum from being eliminated along with the digested blood bolus in a 331 permissive vector, but it is likely important for early survival during the digestive period for 332 some but not all parasite strains. The survival of the BH46 Dlpg1 mutant after exposure to 333 engorged midgut extracts is intriguing; the nature of the glycosylation of the galactose-mannose 334 backbone of other surface glycoconjugates may provide protection for this strain and needs to be 335 further explored. 336 As stated by Sacks and Kamhawi (15), for permissive species such as Lu. longipalpis, 337 persistence of parasites in the midgut after blood is passed is possibly due to factors other than 338 attachment, such as a slower peristaltic movement. These observations are supported by our 339 findings which indicate that LPG-docking of parasites does occur but appears not to define 340 vector competence for Le. infantum in Lu. longipalpis. Rather, vector competence in permissive 341 vectors seems to be more complex, affected by strain-specific differences and involving multiple 342 stages of parasite development in the midgut. BH46 Add-back, Hygromycin (50ug/mL) and Geneticin (G418, 5ug/mL) were added to the 358 medium. In addition, Zeocin (75ug/mL) was added to BH46 Add-back cultures. For both BA262 359 Dlpg1 and BA262 Dlpg1 + LPG1, Hygromycin (50ug/mL) and Geneticin (G418, 70ug/mL) were 360 added to the medium. In addition, Zeocin (100ug/mL) was added to BA262 Dlpg1 + LPG1 361 cultures. 362 363

Sorting of early-stage and late-stage parasites in Ficoll gradient 364
Procedure was performed as described elsewhere (44), with slight modifications. Briefly, 365 cultures were spun down, and parasites were washed twice in PBS and resuspended in 2mL PBS. 366 Then, parasites were overlaid with 40% Ficoll in 2mL PBS, followed by addition of 10% Ficoll 367 in 2mL M199 medium, and spun at 365 x g for 10 min at room temperature. Metacyclic-enriched 368 parasites were collected from the layer in the interface between 10% Ficoll and PBS whereas 369 procyclic-enriched parasites were collected after removing supernatant and resuspending the 370 pellet. Thereafter, both parasite samples were washed twice in PBS for further experimentation. 371 372

Parasite Growth Curves 373
Parasite cultures were seeded with 1 x 10 5 parasites/mL as described above, and parasites 374 were counted daily direct from the medium or diluted in PBS using Neubauer improved 375 chambers (Incyto). 376 377

Midgut binding assays 378
Parasite cultures were spun down once and washed with PBS twice at 3,500 RPMs for 15 379 minutes. Parasites were counted and diluted to 5 x 10 7 parasites/mL in PBS. The midguts of P. 380 papatasi and Lu. longipalpis unfed as well as Lu. longipalpis 5 days after blood feeding were 381 dissected in a drop of PBS, opened up transversally in Y shape with fine entomological pins 382 when needed, and groups of 10 midguts were exposed to 2 x 10 6 parasites in 40uL of PBS in a 383 well of an electron microscopy 9 cavity Pyrex pressed plate (Fisher Scientific). Chambers were 384 transferred to a humidified chamber, and incubation was performed for 45 min at room 385 temperature. Afterwards, midguts were passed through fresh PBS twice and transferred 386 individually to 1.7mL Eppendorf tubes (Denville Scientific) with 30uL of PBS. 387

Sand fly infections 389
Defibrinated naïve rabbit blood (Noble Life Sciences, Gaithersburg, MD), was spun 390 down at 2,000 RPMs for 10 min, and plasma was collected and transferred to a fresh vial. RBCs 391 were washed at least twice with PBS (until most of the free heme was removed) whereas plasma 392 was heat-inactivated at 56°C for 1 hour. Parasite cultures were spun down and washed twice with 393 PBS as described above. Then, RBCs were reconstituted with heat-inactivated plasma and 394 seeded with either 5 x 10 6 (BH46 and BA262) or 2 x 10 6 parasites/mL (BH46). Infectious blood 395 was loaded into a custom-made glass feeder (Chemglass Life Sciences, CG183570), capped with 396 a chick skin and heated by a circulating water bath set for 37°C. Sand flies were allowed to feed 397 for 3 hours. Afterwards, midguts were dissected and transferred individually to 1.7mL Eppendorf 398 tubes (Denville Scientific) in 50uL PBS. 399 400

Midgut Leishmania load assessment 401
Midguts were homogenized with a cordless motor and disposable pellet mixers (Kimble). 402 In order to assess metacyclic proportions, formalin was added to the vials at a 0.005% final 403 concentration. Samples were diluted as necessary and 10uL was loaded onto Neubauer improved 404 chambers (Incyto). 405 406

Leishmania incubation with extracts of blood engorged midguts 407
For sand fly feeding on naïve rabbit blood (Noble Life Sciences, Gaithersburg, MD), 408 RBCs and plasma were processed as described above. Sand flies were fed on the naïve (heat-409 inactivated) blood, and midguts were dissected at 24 and 48 hours after feeding. Midguts were 410 individually transferred to 0.2mL tubes, frozen in dry ice, and stored at -80°C. Two batches of 411 midguts were obtained from sand flies fed on at two different days. Before incubation with 412 parasites, midguts underwent 10 cycles of freeze-thaw (dry ice/room temperature; 5 minutes 413 each). Parasites were harvested from 3 days old culture, washed twice in 1X PBS, and diluted to 414 5 x 10 6 parasites/mL in complete Grace medium. One microliter (5,000 parasites) was incubated 415 with either the extract of a single midgut or PBS (1ul) for 4 hours at 26°C. Afterwards, 20uL of 416 PBS were added to each sample, and parasites were counted with Neubauer improved chambers. 417 418