Calcium homeostasis plays important roles in the internalisation and activities of the small synthetic antifungal peptide PAF26

Fungal diseases are responsible for the deaths of over 1.5 million people worldwide annually. Antifungal peptides represent a useful source of antifungals with novel mechanisms-of-action, and potentially provide new methods of overcoming resistance. Here we investigate the mode-of-action of the small, rationally designed synthetic antifungal peptide PAF26 using the model fungus Neurospora crassa. Here we show that the cell killing activity of PAF26 is dependent on extracellular Ca2+ and the presence of fully functioning fungal Ca2+ homeostatic/signalling machinery. In a screen of mutants with deletions in Ca2+-signalling machinery, we identified three mutants more tolerant to PAF26. The Ca2+ ATPase NCA-2 was found to be involved in the initial interaction of PAF26 with the cell envelope. The vacuolar Ca2+ channel YVC-1 was shown to be essential for its accumulation and concentration within the vacuolar system. The Ca2+ channel CCH-1 was found to be required to prevent the translocation of PAF26 across the plasma membrane. In the wild type, Ca2+ removal from the medium resulted in the peptide remaining trapped in small vesicles as in the Δyvc-1 mutant. It is therefore apparent that cell killing by PAF26 is complex and unusually dependent on extracellular Ca2+ and components of the Ca2+-regulatory machinery. AUTHOR SUMMARY Life threatening diseases can be caused when fungi invade human tissues. These invasions often occur when a person’s immune defences are down, often due to treatments for cancer or transplantation. These infections are commonly buried deep within the body and as such are difficult to access and treat. Current medications are often highly toxic to the patient. There is also a worrying rise in drug resistance seen in fungi sampled from patients, with infections effectively untreatable – a death sentence. Antifungal peptides such as PAF26 provide a possible solution by offering a cheap and rapidly produced alternative to conventional drugs. However, unlike antibacterial peptides, little is known about how these small molecules mostly exert their effects and cause death. Using live-cell imaging and deletion mutants, this study provides an analysis of the important roles that Ca2+-homeostasis and Ca2+-signalling, and possible accompanying vacuolar fusion, play during the dynamic internalization and interaction with and within the fungal cell following PAF26 treatment.

162 Zasloff et al. 1991, Antebi and Fink 1992, Iida, Nakamura et al. 1994, Lapinskas, Cunningham et al. 163 1995, Levina, Lew et al. 1995, Paidhungat and Garrett 1997, Erdman, Lin et al. 1998, Kanzaki 1999 164 Benito, Garciadeblas et al. 2000, Locke, Bonilla et al. 2000, Muller, Locke et al. 2001, Palmer, Zhou 165 et al. 2001, Courchesne 2002, Courchesne and Ozturk 2002, Denis and Cyert 2002, Gupta, Ton et 166 al. 2003, Kaiserer, Oberparleiter et al. 2003, Muller, Mackin et al. 2003, Zhou, Batiza et al. 2003 Zelter, Bencina et al. 2004, Brand, Shanks et al. 2007, Hallen and Trail 2008 168 2008, Benito, Garciadeblás et al. 2009, Bormann and Tudzynski 2009 169 2009, Binder, Chu et al. 2010, Bowman, Abreu et al. 2011, Cavinder, Hamam et al. 2011 170 171  In order to gain more insight into which stages of the PAF26 interaction, uptake and 211 distribution process the CCH-1, YVC-1 and NCA-2 proteins influence, the time-dependent 212 staining by TMR-PAF26 in each of the Δ cch-1, Δ yvc-1, and Δ nca-2 mutants were compared 213 with that in the wild type. As a baseline for the comparison, four staining patterns were 214 identified in the wild type macroconidia that could be readily quantified at 30 min intervals 215 following treatment with 3.5 µM TMR-PAF26. These staining patterns related to different 216 events in the time-dependent uptake and distribution to different organelles which precedes 217  Accumulation of the peptide in vacuoles was much slower and reached a maximum of 6% of 254 the cells with this localization pattern after 120 min. Virtually no cells were observed with 255 peptide exclusively in the cytoplasm at the three time points of measurement over the 120 256 min by which time only 5% of cells were internally permeabilised. After 120 min, 79% of the 257 ∆ yvc-1 cells had taken up TMR-PAF26 compared with 100% in the wild type. Thus, less 258 TMR-PAF26 was taken up by ∆ yvc-1 cells, most accumulated in small vesicles and few cells 259 had internal membrane permeabilisation after 120 min (5% compared with 57% in the wild 260 type). These results are consistent with YVC-1 being required for the transport of PAF26 261 from vesicles to vacuoles. 262 NCA-2 is required for the interaction of PAF26 and the cell envelope 263 When the ∆ nca-2 mutant was treated with TMR-PAF26 ( Fig. 2B and 3C) the cell 264 envelopes of the macroconidia were visibly less fluorescent than that of the wild type. Thus, 265 the affinity of TMR-PAF26 for the cell envelope of this mutant seemed to be reduced and its 266 staining was delayed compared with that of the wild type. The ∆ nca-2 macroconidia also 267 internalized TMR-PAF26 at a far reduced rate compared with the other mutants and the 268 peptide mostly became trapped within the small vesicles, resulting in virtually no TMR-269 PAF26 being taken up by vacuoles and a correspondingly extremely small percentage (~ 270 2%) of the cells having permeabilised internal membranes after 120 min (Fig. 2B). 271 CCH-1 plays a key role in the energy-and Ca 2+ -dependent 272 internalisation of PAF26 by fungal cells 273 The pattern of TMR-PAF26 internalization and intracellular transport within 274 macroconidia of the Δ cch-1 mutant appeared to be opposite to that in the wild type (Fig. 3D). 275 After treatment for 30 min the peptide was primarily localized in the cytoplasm (in 67% of 276 cells) and to a much lesser extent in small vesicles (~ 18% of cells) and to a very low level in 277 large vacuoles (~ 5% of cells). Between 30 and 90 min, TMR-PAF26 was removed from the 278 cytoplasm and increased in amount in the small vesicles and large vacuoles. Between 90 279 and 120 min the large vacuoles containing TMR-PAF26 are fragmenting into smaller 280 vesicles. This was reflected by a dramatic increase in the number of stained small vesicles 281 (Fig. 2B). 282 The overall rate of internalisation of TMR-PAF26 by Δ cch-1 macroconidia was faster 283 than in the wild type because in the former it was initially taken up directly into the cytoplasm 284 whilst in the latter it first appeared intracellularly in small vesicles that are presumed to be 285 mostly endosomes. It seems unlikely that TMR-PAF26 is taken up by means of non-specific 286 permeabilization of the plasma membrane because this would likely have resulted in rapid 287 cell death. Furthermore, 30 min after the addition of TMR-PAF26 it was clear that the 288 vacuolar membrane had not become permeabilized because the peptide was excluded from 289 the vacuoles of cells in which the cytoplasm was fluorescent. 290 In order to clarify whether the passage of PAF26 across the plasma membrane into 291 the cytoplasm of the Δ cch-1 mutant was a result of passive translocation or active uptake, 292 macroconidia were pre-treated with the metabolic inhibitor NaN 3 (Muñoz, Marcos et al. 2012) 293 at a concentration of 5 µM for 15 min before the addition of TMR-PAF26 and subsequent 294 imaging and quantification of localization over 120 min (Figs. 2C and 3F). 295 In the presence of NaN 3 , over the whole 120 min period of incubation with TMR-296 PAF26, the peptide remained bound to the cell envelope and was not internalized by most of 297 the macroconidia. Clearly the metabolic inhibitor NaN 3 had almost completely abolished the 298 movement of TMR-PAF26 across the plasma membrane indicating that the uptake of the 299 peptide into the cells of the Δ cch-1 mutant is an ATP-dependent process. The results also 300 showed that the rate of internal membrane permeabilisation, whilst faster over the first 30 301 min, remained similar to that of the non-azide treated  These results are consistent with the normal endocytic internalization of PAF26 being 306 dependent on the Ca 2+ channel protein, CCH-1. As CCH-1 appears to initiate PAF26 reported to be immunolocalized to the plasma membrane (Locke et al., 2000). Furthermore, 310 we had previously shown that removal of Ca 2+ from VM made macroconidia more resistant 311 to being killed by PAF26. To attempt to mimic the effect of cch-1 deletion in the wild type, 312 Ca 2+ was removed from the external medium by the addition of the Ca 2+ chelator BAPTA ( towards the end of this period (Fig. 4B). When the PAF26 added was at a final concentration 331 of 2.5 µM, which is close to its IC 50 value, the initial increase in [Ca 2+ ] cyt was slightly greater 332 (to 0.22 ± 0.03 µM) (Fig. 4B). Again, this was followed by a period of sustained [Ca 2+ ] cyt 333 increase, but there was also a more pronounced exponential increase in [Ca 2+ ] cyt which 334 began at ~ 900 sec after treatment. With the higher dose, there was also an increase in the 335 standard deviations of the measurements with time. This is due to aequorin consumption 336 during the course of the experiment, resulting in less sensitivity after prolonged exposure to 337 high [Ca 2+ ] (note that luminescence measurements from 6 wells are averaged per time 338 point). When the wild type was treated with 5 µM PAF26, which was above its IC 50 value, the 339 [Ca 2+ ] cyt response followed the same general pattern but with a much larger initial [Ca 2+ ] cyt 340 increase to 0.32 ± 0.02 µM and a shorter period of sustained increase before the [Ca 2+ ] cyt 341 increase became exponential at ~ 430 sec (Fig. 4B)

401
In order to investigate whether PAF26 was indeed causing a dose dependent rise in 402 [Ca 2+ ] cyt , conidia expressing GCaMP6s were imaged during PAF26 treatment using widefield 403 fluorescence microscopy. Individual macroconidia were then isolated from the background in 404 FIJI and fluorescence measured over time. As Figure 5A shows, the individual responses 405 are far more varied than the measurements using aequorin suggested. In the middle of the 406 timecourse, the moment the PAF26 reaches the field of view and interacts with the cells can 407 be clearly seen in a sudden increase in Ca 2+ spiking in all the individuals. Given that the 408 aequorin measurements are across a whole population, it is not hard to see how the 409 cumulative effects of an increase in Ca 2+ spikes and waves could be interpreted as a dose 410 dependent rise in [Ca 2+ ] cyt .In order to determine whether there was a predictable response to 411 PAF26 addition, several repeated experiments were run in which the peptide was added and 412 fluorescence intensity recorded. No distinct pattern or regularity was found within any of the 413 data sets, except for an increase in Ca 2+ signalling after PAF26 addition. As the only 414 noticeable response, the amplitude and frequency of these Ca 2+ spikes was quantified. 415 Conidia were treated either with ddH 2 O or ddH 2 O containing 3.5 µM PAF26. A second set 416 were pre-treated with 5 mM BAPTA before ddH 2 O or PAF26 treatment. The conidia treated 417 with ddH 2 O have a relatively low frequency of Ca 2+ spiking with the mean being 4 spikes 418 over the course of the measurement. This is consistent with our findings as to the rate of 419 Ca 2+ spiking in germinating and fusing conidia (Read, unpublished). When the cells are 420 treated with PAF26 however, there is a marked increase in both frequency and amplitude. 421 The mean frequency of the Ca 2+ spikes increases to 19 and the amplitude increases to 422 around 6 times the resting level from 4 ( Figure 5B). Both of these results are significant at P 423 < 0.01. When the conidia are pre-treated with BAPTA however, all Ca 2+ spiking ceases 424 completely. Whilst there were no apparent recurring patterns in the spiking events, 425 observations appeared to show that they correspond to vesicles and vacuoles coming into 426 close proximity. A Ca 2+ trace is shown in Fig has previously been localised to the vacuole and plasma membrane in mature hyphae 448 (Bowman, Draskovic et al. 2009). We conducted experiments to localize NCA-2 tagged with 449 obvious signal at the plasma membrane (Fig. 6).). 451 In order to localise the CCH-1 and YVC-1 proteins, plasmids were constructed based 452 on FJ457002 for N-terminal tagging and FJ457006 for C-terminal tagging (Honda and Selker 453   2009). 454 Both the C-and N-terminal YVC-1:GFP fusion proteins fluoresced, although the N-455 tagged protein showed a stronger more stable fluorescence. Fluorescence mainly appeared 456 within vacuoles in conidia, germ tubes and mature hyphae (Fig. 6). The CCH-1:GFP fusion 457 products produced very little fluorescence, both from N and C tagged versions. This 458 appeared to be localised to vacuoles within conidia and intracellular membranes within older 459 hyphae. Single punctate spots of GFP fluorescence could however be seen at the plasma 460 membrane of mature hyphae, Fig.6. 461 macroconida re shown for this RFP expressing strain (Bowman, Draskovic et al. 2009). CCH-1 466 appears to be localised in the vacuolar system in macroconidia and germlings, but also appears as 467 distinct points in the plasma membrane of mature hyphae. YVC-1 appears to localise to the vacuolar 468 system in all cell types. RFP tagged NCA-2 localises to the vacuole in macroconidia. Bar = 5 μm. Deletion of nca-2 appears to influence the binding of PAF26 to the cell envelope, the Δ nca-2 493 mutant has been shown to accumulate up to 10 fold more Ca 2+ than wild type cells, 494 suggesting that NCA-2 serves to remove Ca 2+ from the cell (Bowman, Abreu et al. 2011). 495 We found no significant difference in the resting level of [Ca 2+ [ cyt from the wild type however 496 under unstimulated conditions. Interestingly however, the Δ nca-2 mutant has a membrane 497 potential reversed from the wild type due to a lack of cell surface H + ATPase function 498 (Hamam and Lew 2012). In S. cerevisiae the H + ATPase is one of the most abundant cell 499 surface proteins (Bagnat, Chang et al. 2001) and makes up a significant amount (up to 10%) cause a rapid depolarization of the membrane in wild type cells in an energy independent 502 manner (Muñoz, Marcos et al. 2012). Given that PAF26 has a net positive charge, the 503 membrane potential reversed Δ nca-2 should technically not be inhibited in PAF26 504 membrane binding from an electrostatic view. It is therefore possible to propose that PAF26 505 directly inhibits H + -ATPase action, possibly by direct binding; misfunctioning H + --ATPase at 506 the plasma membrane is sent to the vacuole for degradation in yeast, a possible mechanistic 507 for the accumulation of PAF26 (Bagnat, Chang et al. 2001, Liu, Sitaraman et al. 2006. 508

DISCUSSION
When the peptide is internalised in the Δ yvc-1 mutant, it remains in small vesicles with very 509 little sign of the peptide entering the vacuolar system; adding support for the hypothesis that 510 YVC-1 and a threshold amount of PAF26 is required to initiate vacuolar fusion. Vacuolar 511 fusion occurs through conformational change of the docking SNARE proteins, triggered by 512 the release of Ca 2+ from the vacuole in S. cerevisiae (Bayer, Reese et al. 2003, Merz and 513 Wickner 2004, Coonrod, Graham et al. 2013. In S. cerevisiae, isolated vacuoles are able to 514 catalyse their own fusion through the release of Ca 2+ from yvc1p present in the vacuolar 515 membrane (Peters and Mayer 1998). This does not appear to be the case in N. crassa 516 however, as both deletion of YVC-1 and removal of extracellular Ca 2+ resulted in the trapping 517 of the peptide in small vesicles. Therefore extracellular Ca 2+ is required to initiate the release 518 of Ca 2+ from the vacuoles and trigger fusion. This raises questions as to why internal 519 membrane fusion is reliant on external stimuli. In the Δ cch-1 mutant the peptide was directly 520 translocated across the plasma membrane into the cytoplasm in an energy dependent 521 manner, before accumulation in vacuoles, meaning PAF26 does not kill by being present in 522 the cytosolic space. CCH-1 is therefore required to initiate the endocytic pathway. Energy 523 dependent peptide uptake into the vacuolar system is also seen in the Penicillium 524  In order to create pTef1AeqS, aequorin was amplified from pAB19 using the primers 578 AeqS-F and AeqS-R. The vector pCC019 was digested with PacI and EcoRI and the vector 579 gel purified. Aequorin was then amplified with AeqS-IF-Fw and AeqS-IF-Rv to add overlaps 580 homologous to the backbone whilst maintaining digestion sites and the vector was 581 assembled using Gibson assembly. To generate pYVC1CGFP and pYVC1NGFP, the yvc-1 582 ORF was amplified from gDNA using the primers NC-YVC1-AMP-FW and NC-YVC-1-AMP-583 RV. The purified product was then amplified using NC-YVC1-N-FW & NC-YVC1-N-RV for N 584 terminal tagging and NC-YVC1-C-FW & NC-YVC1-C-RV for C terminal tagging. The 585 FJ457002 vector was digested using PacI and XbaI and the FJ457006 vector digested with 586 AscI and NotI. Following gel extraction both vectors were assembled using Gibson 587 assembly. 588 Tagging of CCH-1 was not straightforward due to the size of the ORF -6.5kb. The 589 CCH-1 ORF was amplified first using the primer sets NC-CCH1 AMP-FW and NC-CCH1-590 3.5Rv and NC-CCH1-3.5Fw and NC-CCH1-AMP-RV to amplify the gene in two segments, 5' 591 and 3'. The complete constructs were built using fusion PCR; primer NC-CCH1-C-FW, 5' 592 segment, 3' segment and primer NC-CCH1-C-RV, the N terminal tagging vector used the 593 primers NC-CCH1-N-FW and NC-CCH1-N-RV. Following successful fusion the vectors were concentration was of 5 X 10 5 in 10% VM. Absorbance measurements were obtained at 610 603 nm. This wavelength is close to the 595nm at which the relationship between optical density 604 and fungal biomass is linear (Broekaert, Terras et al. 1990). 605 Conidia were diluted to a concentration of 1 X 10 6 in 10% VM, coelenterazine was 611 added to a final concentration of 2.5 µM. 100 µl of conidial suspension was used per well of 612 a white microtitre plate and the plates incubated at 25°C in the dark for 6 hours. The light 613 output of aequorin was measured by counting the photons emitted by a single well over one 614 second, each well in a row of six being measured once every cycle of 7 seconds. After 615 recording baseline luminescence, a single row was treated with PAF26 and the light output 616 measured over time. Following this the second row was run with 100 µl 3M

638
All microscopy was carried out on a Nikon Eclipse ® TE2000-E inverted microscope, 639 using a Nikon Plan Apo 60 X 1.2 N.A. DIC H water immersion objective and Nikon G-2A and 640 B-2A filter sets, excitation was provided by a CoolLED illumination system set at 550 nm for 641 use with the G-2A filter or 470 nm for use with the B2-A filter. 642 Confocal microscopy was carried out using a Leica TCS SP8 equipped with two 643 hybrid GaAsP detectors (HyD) and two photomultiplier tubes (PMT). Excitation was provided 644 using either the Leica tuneable white light laser (450 -750 nm), an argon laser (458nm, 645 476nm, 488nm and 496nm) or UV laser (405nm). Images were captured using LAS X 646 software and the Leica 63 X water immersion objective. All image handling and analysis was 647 carried out using Fiji (fiji.sc/Fiji). 648

Quantification of Ca 2+ signalling
649 Fluorescence values were exported from Fiji and analysed in Excel. Data was 650 normalised using feature scaling. This scales the data to removes irregularities in GCaMP6 651 expression and photon yield. The function used was: x I = MIN + (x-min x )(MAX-MIN)/(max x -normalised value. min x is the minimum value for that data set and max x is the maximum, 654 max x was defined as the maximum value from all experiments plus 10 to avoid artificial 655 amplification of noise. The noise was removed using an IF function: =IF(x>y,x,0) where x is 656 the data point and y is the noise threshold limit. Finally, an IF(AND(x>x -1 , x>x +1 ),1,0) 657 function, where x -1 and x +1 are the surrounding measurements, quantified each peak in 658 ( :  t  h  e  m  e  c  h  a  n  i  s  m  o  f  a  c  t  i  o  n  o  f  c  e  l  l  -p  e  n  e  t  r  a  t  i  n  g  a  n  t  i  f  u  n  g  a  l  p  e  p  t  i  d  e  s  u  s  i  n  g  t  h  e  r  a  t  i  o  n  a  l  l  y  d  e  s  i  g  n  e t  h  e  m  e  c  h  a  n  i  s  m  o  f  a  c  t  i  o  n  o  f  c  e  l  l  -p  e  n  e  t  r  a  t  i  n  g  a  n  t  i  f  u  n  g  a  l  p  e  p  t  i  d  e  s  u  s  i  n  g  t  h  e  r  a  t  i  o  n  a  l  l  y  d  e  s  i  g  n  e