Draft - University of Toronto T-Space · Draft 3 48 Introduction 49 Scedosporium aurantiacum is an...

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Effect of proteases secreted by the opportunistic pathogen Scedosporium aurantiacum on human epithelial cells

Journal: Canadian Journal of Microbiology

Manuscript ID cjm-2019-0212.R1

Manuscript Type: Article

Date Submitted by the Author: 14-Jun-2019

Complete List of Authors: Han, Zhiping; Macquarie University, Department of Molecular Sciences; Lingnan Normal University, School of Chemistry and Chemical EngineeringKautto, Liisa; Macquarie University, Department of Molecular Sciences; Macquarie University, Biomolecular Discovery and Design Research CentreMeyer, Wieland; The University of Sydney, Centre for Infectious Diseases and MicrobiologyChen, Sharon ; The University of Sydney, Centre for Infectious Diseases and Microbiology; Westmead Hospital, Centre for Infectious Diseases and Microbiology Laboratory ServicesNevalainen, Helena; Macquarie University, Department of Molecular Sciences; Macquarie University, Biomolecular Discovery and Design Research Centre

Keyword: Scedosporium aurantiacum, human alveolar epithelial cells, cell attachment, cell viability, peptidases

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Canadian Journal of Microbiology

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1 Effect of peptidases secreted by the opportunistic pathogen Scedosporium

2 aurantiacum on human epithelial cells

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4 Zhiping Han 1,5*, Liisa Kautto1,2, Wieland Meyer3, Sharon C-A. Chen3,4 and Helena Nevalainen1,2

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6 1Department of Molecular Sciences, Macquarie University, Sydney, Australia

7 2 Biomolecular Discovery and Design Research Centre, Macquarie University, Sydney, Australia

8 3Molecular Mycology Research Laboratory, Centre for Infectious Diseases and Microbiology,

9 Marie Bashir Institute for Infectious Diseases and Biosecurity, Sydney Medical School –

10 Westmead Hospital, The University of Sydney, Westmead Institute for Medical Research, Sydney,

11 Australia

12 4Centre for Infectious Diseases and Microbiology Laboratory Services, ICPMR, New South Wales

13 Health Pathology, Westmead Hospital, Westmead, NSW, Australia

14 5Current address: School of Chemistry and Chemical Engineering, Lingnan Normal University,

15 Zhanjiang, China

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17 *Corresponding author: Zhiping Han, Tel.: +86 759 3174023, Email: [email protected]

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24 Abstract

25 Peptidases secreted by a clinical high-virulence Scedosporium aurantiacum isolate (strain WM

26 06.482; CBS 136046) under normoxic as well as hypoxic conditions were separated using size

27 exclusion chromatography, and peptidase activities present in each fraction were determined using

28 class-specific substrates. Effects of the fractions demonstrating peptidase activity on the

29 attachment and viability of A549 human lung epithelial cells were assessed in vitro. From the

30 peptidases detected in the exclusion chromatography fractions, the elastase-like peptidase reduced

31 cell viability, chymotrypsin-like peptidase was associated with cell detachment and cysteine

32 peptidases were able to abolish both cell attachment and viability. The loss of cell viability and

33 attachment became more prominent with an increase in the peptidase activity and could also be

34 specifically prevented by addition of class-specific peptidase inhibitors. Our findings indicate

35 that peptidases secreted by S. aurantiacum can breach the human alveolar epithelial cell barrier

36 and thus may have a role in the pathobiology of the organism.

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38 Keywords Scedosporium aurantiacum; peptidases; human alveolar epithelial cells; cell

39 attachment; cell viability

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48 Introduction49 Scedosporium aurantiacum is an emerging opportunistic fungal pathogen especially in

50 immunocompromised patients (Harun et al. 2010), and can colonise lungs previously damaged by

51 chronic inflammation, with potential to cause invasive disease with time (Nevalainen et al. 2018;

52 Pihet et al. 2009). Symptoms caused by S. aurantiacum infection are similar to those caused by

53 Aspergillus fumigatus (Cortez et al. 2008; Kleinschmidt-DeMasters 2002). Scedosporium

54 infection including that due to S. aurantiacum is initiated by either inhalation of air-borne conidia

55 or by penetration of the host tissue at the site of injury (Cortez et al. 2008). Infection sites include

56 the eye, ear, central nervous system, internal organs and more commonly the lungs, especially the

57 lungs of cystic fibrosis patients (Steinbach and Perfect 2003). Mortality from S. aurantiacum is

58 high in the mouse model (60-100%) (Harun et al. 2010), however, very little is known about the

59 infection mechanism of this pathogen.

60 Peptidases secreted by fungi have been demonstrated to play a role in fungal pathogenicity by

61 degrading and breaching the extracellular matrix barrier in the host cells (Latgé 2001). For

62 example, secreted serine and cysteine peptidases from A. fumigatus were able to breach the A549

63 alveolar epithelial cell barrier by disruption of the actin cytoskeleton and sites of focal attachment

64 in human lung cancer cells (Kogan et al. 2004). Elastase secreted by A. fumigatus and A. flavus is

65 capable of breaking down the major structural component (elastin) of lungs to enable fungal

66 germination and penetration into mice lungs (Kolattukudy et al. 1993). Aspartic peptidases

67 secreted by Candida albicans were found to aid fungal attachment, colonization and penetration

68 of host tissue by degradation of molecules that protect mucosal surfaces such as mucin and

69 secretory immunoglobulin A (sIgA) (Naglik et al. 2003). Scedosporium spp. produce secrete

70 various types of peptidases including subtilisin-like, chymotrpsin-like, elastase-like, trypsin-like,

71 aspartic and metallopeptidases (Han et al. 2017; Larcher et al. 1996; Santos et al. 2009; Ramirez-

72 Garcia et al. 2017). Amongst the most studied are peptidases secreted by S. apiospermum of which

73 the subtilisin-like peptidases were found to be able to degrade human inflammatory response

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74 mediator protein (Larcher et al. 1996). Another study on S. apiospermum showed that this fungus

75 secreted six metallopeptidases (28-94 kDa), which may digest human serum proteins (Santos et

76 al. 2009). A recent proteome-based study on S. boydii identified an aspartic peptidase although its

77 relationship to fungal virulence has not been established as yet (Ramirez-Garcia et al. 2017).

78 In previous work, we profiled the types of peptidases secreted by S. aurantiacum grown under

79 both normoxic and hypoxic conditions (Han et al. 2017; Han et al. 2018). It was found that

80 subtilisin- and trypsin-like serine peptidases and aspartic peptidases were secreted by the clinical

81 and environmental strains under both conditions. Cysteine peptidases were secreted only under

82 hypoxia. The clinical strain did not produce chymotrypsin-like serine peptidase while the

83 environmental strain did not produce elastase-like serine peptidase under normoxia two days post

84 inoculation (Han et al. 2017; Han et al. 2018). Overall, serine peptidases were responsible for the

85 majority of proteolytic activity under normoxia while aspartic peptidases dominated under

86 hypoxia. However, the roles of these secreted peptidases in the pathogenicity of S. aurantiacum

87 are so far unclear.

88 Lungs are the primary organs to receive the air from the external environment. Accordingly,

89 lung epithelium cells are the initial point of contact with the inhaled fungi, and are the very first

90 cells affected by fungal growth (Filler and Sheppard 2006; Vareille et al. 2011). Cell attachment

91 and viability are two important indices in cell proliferation, which involves assembly of individual

92 cells into tissues and the ability of a cell or a tissue to maintain its function respectively (Alberts

93 et al. 2008; Aplin et al. 1999; Gumbiner 1996). Reduction of cell viability can result in necrosis

94 or cell death caused by toxic effects of foreign compounds or a decrease in cellular proliferation

95 (Alberts et al. 2008).

96 The A549 cell line has a high degree of metabolic and morphological similarity with lung

97 alveolar epithelial cells, and thus has been widely used in in vitro studies. For example, A549 cells

98 were used to study the effects of A. fumigatus culture filtrate on human lung epithelium cells; the

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99 results showed that cell death was initiated by necrosis but factors responsible for the necrosis

100 have not yet been identified (Daly and Kavanagh 2002; Daly et al. 1999). Thakur et al. used the

101 A549 cell line to investigate the effects of Paecilomyces hepiali on human lung adenocarcinoma

102 in vitro and found that the extract from this fungus limited cell proliferation, induced apoptosis,

103 and caused cell cycle arrest (Thakur et al. 2011). So far, there are two reports describing the

104 interaction between Scedosporium spp. and A549 cells. Both found that the fungal conidia

105 germinate and penetrate into the A549 cell membrane shortly after their attachment to the cells,

106 and the infection process resembles that of A. fumigatus (Pinto et al. 2004; Kaur et al. 2015).

107 In the current work, we examined the effects of peptidases secreted by a clinical high-

108 virulence S. aurantiacum isolate on the viability and attachment of A549 with a view of

109 investigating a potential link between particular peptidases and fungal pathogenicity.

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111 Materials and methods

112 Cell culture

113 The model human cell line used in this work was A549 (ATCC CCL 185, derived from a

114 human lung carcinoma). RPMI 1640 medium (Life Technologies) supplemented with 10 % v/v

115 FBS (Foetal bovine serum, Life Technologies, Australia) and 1 mM glutamine (Life Technologies)

116 was used for cell culture at 37 °C under 5 % CO2. The cells were sub-cultured when 80 % confluent

117 as described previously (Phelan and May 2015). Detection of a potential Mycoplasma

118 contamination was performed according to the protocol obtained from the Garvan Institute of

119 Medical Research using a PCR-based method for the analysis of the 16S-23S rRNA

120 (https://www.garvan.org.au/research/capabilities/molecular-genetics).

121 Fungal strain and liquid culture medium

122 S. aurantiacum strain WM 06.482 (CBS 136046; originally isolated from broncho-alveolar

123 lavage of a cystic fibrosis patient in Australia), was obtained from the culture collection of the

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124 Molecular Mycology Research Laboratory, Centre for Infectious Diseases and Microbiology,

125 Westmead Hospital, Sydney, Australia.

126 Liquid cultures of this strain were performed in a synthetic cystic fibrosis sputum medium

127 (SCFM+M) supplemented with 1 % (w/v) glucose and 1 % (w/v) mucin (from porcine stomach,

128 type III; Sigma-Aldrich), prepared as described in (Han et al. 2017; Palmer et al. 2007).

129 Normoxic and hypoxic culture of S. aurantiacum

130 Normoxic and hypoxic cultures were conducted as described previously (Han et al. 2017; Han

131 et al. 2018). Incubation was carried out at 37 °C on an orbital shaker at 200 rpm for 4 d under

132 normoxia and 3 d under hypoxia with three individual flasks dedicated to each condition. Culture

133 times were selected based on time-dependent general peptidase activity measured from the culture

134 supernatants as per our earlier study (Han et al. 2017; Han et al. 2018). The supernatants were

135 collected by centrifugation at 4500 g for 30 min and filtered through a 0.22 μm membrane

136 (Millipore) at 4 °C. Cleared supernatants were then dialyzed against sterilized Milli-Q H2O

137 overnight at 4 °C using 10 kDa cut-off dialysis tubing (Amico Ultra-15, Millipore). Dialyzed

138 supernatants were freeze-dried and stored at -80 °C until use.

139 Fractionation of proteins in the fungal culture supernatant

140 Freeze-dried culture supernatants were resuspended in PBS buffer (pH 7.4) to achieve a final

141 protein concentration of around 2 mg·ml-1 measured by the Bradford assay (Bradford 1976).

142 Aliquots of 0.5 ml (1 mg protein) were separated at 4 °C by size exclusion chromatography (SEC)

143 using a 24 ml Superdex S-200 column (GE Healthcare Life Sciences) in filter sterilized PBS (pH

144 7.4) at 0.5 ml·min-1. One 1 ml fractions were collected and absorbance at 280 nm was measured

145 using an ÄKTAPrime protein purification system (GE Healthcare Life Sciences). Fractionation

146 was performed twice for two sets of freeze-dried fungal culture supernatants and fractions

147 containing similar type of peptidase activity (see below) were pooled together.

148

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149 Classification of peptidases

150 Types and activities of peptidases present in each SEC fraction were tested using class-

151 specific substrates featuring synthetic peptides coupled with either 7-Amino-4-methylcoumarin

152 (MCA) or ρ-nitroanilide (ρ-NA) (Peptide Institute Inc, Japan) (Han et al. 2017). All peptidase

153 activity assays were performed under sub-saturating conditions. Peptidase activities were

154 calculated based on fluorescence of the released MCA or absorbance of released ρ-NA. All assays

155 were performed in triplicates, and statistical analysis was conducted using Excel.

156 Peptidase inhibitors were added into each SEC fraction depending on the type of peptidase

157 activity detected (PF + inh). Inhibitors used included 50 µM elastatinal (elastase peptidase

158 inhibitor; Santa Cruz Biotechnology), 100 µM Boc-VF-NHO-Bz-Pcl (subtilisin peptidase

159 inhibitor; Santa Cruz Biotechnology), 5 mg ml-1 chymostatin (chymotrypsin peptidase inhibitor;

160 Sigma), 100 nM aprotinin (trypsin peptidase inhibitor; Sigma), 10 μM E-64 (cysteine peptidase

161 inhibitor; Sigma), 100 μM pepstatin A (aspartic peptidase inhibitor; inhibitor; Sigma), 13 mM

162 galardin (metallopeptidases inhibitor; Sigma), 1 mM PMSF (serine peptidase inhibitor; Sigma-

163 Aldrich), 5 mM EDTA (metallopeptidase inhibitor; Sigma) and 0.05% v/v peptidase inhibitor

164 cocktail (Sigma). Concentration of peptidase inhibitors was determined according to the published

165 literature and prior testing in our laboratory.

166 Each SEC fraction was pre-incubated with a selected peptidase inhibitor for 30 min at room

167 temperature prior to performing a class-specific peptidase activity assay or adding to A549 cell

168 cultures to assay cell viability and attachment. SEC fractions without inhibitors were also included

169 in the assay for comparison. All assays were performed in triplicate.

170 Cell attachment assay

171 A wash assay was carried out to assess changes in cell attachment in a static culture, following

172 the method of Kogan et al. (Kogan et al. 2004). Briefly, 1×105 A549 cells per well containing 3

173 ml of medium were incubated in a 6-well-culture plate (Corning Costar, Sigma-Aldrich) for 24 h.

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174 After incubation, the medium was discarded and cells were washed three times with 1 x PBS to

175 remove detached cells. Fresh medium consisting of 50 % (v/v) of supplemented RPMI 1640, and

176 10 %, 20 % or 50 % (v/v) of a SEC fraction, made to 3 ml with 1 x PBS was added. After 4, 8, 12,

177 24, or 48 h incubation, the medium was discarded and cells were washed three times with 1 x PBS

178 to remove the floating cells. The remaining cells were detached by trypsinization, stained with

179 trypsin blue (Sigma-Aldrich,) and counted using an automated cell counter (TC20, Bio-Rad). Cells

180 grown in supplemented RPMI 1640 plus 1 x PBS (1:1) were included in the assay as a negative

181 control for each time point independently. The assay with peptidase inhibitors was conducted in a

182 similar fashion. All assays were performed in biological triplicates.

183 Cell viability assay

184 MTS assay based on the reduction of MTS tetrazolium compound by viable cells to generate

185 a colored formazan soluble in cell culture medim was conducted to measure cell viability (Bernas

186 and Dobrucki 2002). A sample of 5×103 A549 cells per well containing 100 µl medium were

187 grown in a 96-well-culture plate (Corning Costar, Sigma-Aldrich). The culturing was performed

188 following the same procedures as above in the cell attachment assay. At 4, 8, 12, 24 or 48 h post

189 inoculation, MTS Reagent (Promega) 20 µl/well was added to the cultures, followed by 2 h

190 incubation at 37 °C under 5% CO2. Absorbance of each well was read at 490 nm. The number of

191 viable cells was calculated using a standard curve prepared by plotting the absorbance at 490 nm

192 versus known concentration of viable cells. Cells grown in RPMI 1640 plus PBS (1:1 volume)

193 were included in the assay as a negative control for each time point independently. The assay with

194 peptidase inhibitors was conducted in a similar fashion. All assays were performed in biological

195 triplicates.

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199 Light microscopy

200 Cell cultures in the multi-well plates were observed under optical Leica microscope (Leica

201 DMIL). Images were acquired and processed with Leica Application Suite Version 4.3 software

202 (Las v4.3).

203

204 Results

205 Separation and classification of peptidases

206 In order to separate peptidases secreted by the S. aurantiacum isolate WM 06.482 (CBS

207 136046), proteins concentrated from the day-4 supernatant of the normoxic culture and from day-3

208 supernatant of the hypoxic culture were fractionated by SEC. These supernatants were selected

209 because they had the highest general peptidase activity at the respective condition. Fractionation

210 was performed at 4 °C to retain peptidase activity, and protein content in each fraction was

211 monitored by reading the absorbance at 280 nm (Fig 1).

212

213 - Fig 1 here -

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215 Each fraction was assayed using class-specific substrates to identify fractions containing

216 peptidase activity. Subtilisin-, elastase- and trypsin-like peptidase activities were detected from

217 normoxic cultures, while chymotrypsin-like, cysteine and aspartic peptidase activities were

218 detected from hypoxic cultures (Table 1). Subtilisin-, elastase- and trypsin-like peptidases secreted

219 by S. aurantiacum were not separated well and were present in more than one fraction. This may

220 be due to their fairly similar molecular size (20-45 kDa), determined in previous studies into fungal

221 extracellular serine peptidases (Biaggio et al. 2016; Savitha et al. 2011). The normoxic fractions

222 15, 17 and 19 (N15, N17, N19) showing elastase-, trypsin- and subtilisin-like peptidase activity,

223 and hypoxic fractions 12, 16 and 20 (H12, H16, H20) representing aspartic, cysteine and

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224 chymotrypsin-like peptidase activity were selected for subsequent studies so that the overlap of

225 peptidase classes in the selected fractions was minimal.

226

227 -Table 1 here-

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229 Peptidases involved in cell detachment

230 The six selected SEC fractions with peptidase activity were separately co-incubated with

231 A549 cells, and detachment of cells from the culture wells was monitored over 48 h (not shown).

232 It turned out that only the chymotrypsin-like and cysteine peptidases present in the hypoxic

233 fractions were able to cause detachment of A549 cells by rounding the cells up (Fig 2A), and the

234 rate of detachment increased with an increase of the volumetric concentration of the SEC fraction

235 added into the cell culture. Addition of 10 %, 20 % and 50 % (v/v) of the SEC fraction with

236 chymotrypsin-like peptidase activity (H20) into the cell culture medium caused detachment of

237 around 20 %, 50 % and 80 % of cells respectively within 24 h. Addition of the above volumes of

238 cysteine peptidase fraction (H16) into the cell culture lead to approximately 10 %, 20 % and 50 %

239 detachment (Fig 2B). In addition, A549 cells began to detach from the surface of the culture wells

240 within 30 minutes of addition of chymotrypsin-like peptidase fraction (H20, data not shown);

241 approximately 60 % of cells detached after 4 h of exposure, after which the rate of detachment

242 slowed (Fig 2C). The detachment of A549 cells caused by peptidases was decreased by class-

243 specific peptidase inhibitors (chymostatin for chymotrypsin like peptidase and E-64 for cysteine

244 peptidase), suggesting that the chymotrypsin-like and cysteine peptidase activity measured from

245 the SEC fractions tested was the main factor for cell detachment. The relative abundance of

246 individual peptidases within the SEC fractions was not examined.

247

248 - Fig 2 here -

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250 Peptidases associated with a decrease in cell viability

251 Cell viability as determined using the MTS assay yielded results that corresponded to those

252 obtained by cell counting (Fig S1), indicating accuracy of the method. The limitation of MTS

253 assay here was investigated to be 0-60 000 cells per well (Fig S2).

254 From the six SEC fractions showing peptidase activity only H16 containing cysteine peptidase

255 activity and N15 with elastase-like peptidase activity could cause a decrease in viability of the

256 A549 cells (Fig 3A). The rate of decrease increased with an increase in the volumetric percentage

257 amount of the peptidase fraction added into the cultures. Addition of 10 %, 20 % and 50 % (v/v)

258 of N15 with elastase-like peptidase activity resulted in the loss of viability of approximately 20 %,

259 45 % and 80 % of cells. The same volumetric additions of the SEC fraction with cysteine peptidase

260 activity (H16) led to approximately 2 %, 11 % and 20 % decrease in cell viability (Fig 3B). The

261 A549 cells exposed to elastase-like peptidase primarily began to lose their viability after 4 h, after

262 which the number of viable cells decreased gradually; about 12 h later, 50 % of cells had lost their

263 viability (Fig 3C). Cell viability assay was also conducted in the presence of trypsin-like peptidase

264 inhibitor, because both trypsin- and elastase-like peptidases were detected in the same SEC

265 fraction. Since cell viability could not be reduced by addition of a trypsin-specific inhibitor,

266 trypsin-like peptidases were not associated with cell death.

267

268 - Fig 3 here -

269

270 Microscopic features of the cells exposed to elastase-like peptidase (N15) and cysteine

271 peptidases (H16) were different. A549 cells exposed to elastase-like peptidase were still attached

272 to the surface of the culture well and looked different to untreated cells. Cysteine peptidase

273 rounded up A549 cells and cell content seemed to become released into environment (Fig 3A).

274 This suggested that the mechanism by which the elastase-like peptidase and cysteine peptidases

275 affected cell viability may be different.

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276

277 Discussion

278 Studies on the effects of secreted fungal peptidases on lung epithelium cells have primarily

279 focused on major fungal pathogens, such as A. fumigatus and Candida spp. (Borger et al. 1999;

280 Daly et al. 1999; Kogan et al. 2004; Naglik et al. 2003). In spite of clinical relevance of

281 Scedosporium spp., there are very limited data available on the interaction between Scedosporium

282 peptidases and lung cells. Most of the studies have investigated the structures and components of

283 conidial and hyphal cell walls, as well as their link to fungal attachment to epithelial cells and the

284 secreted enzymes (Cortez et al, 2008; Proskuryakov et al, 2003; Lackner et al, 2013). There are a few

285 studies investigating the virulence factors of S. apiospermum, which report that the fungus mainly

286 secretes polysaccharides, peptidases and phosphatases in the development of infection. However,

287 the contribution of these secreta on Scedosporium pathogenesis is still poorly understood (Lackner

288 et al, 2013).

289 In our previous studies, we found that the peptidases produced by clinical and environmental

290 S. aurantiacum strains were different, and trypsin-like and elastase-like peptidases were secreted

291 by the clinical strain only (Han et al, 2017). The aim of this study was to explore the effects of

292 peptidases secreted by the emerging pathogen S. aurantiacum on the attachment and viability of

293 human lung epithelium cells. The results will provide insights into roles of secreted proteases in

294 fungal invasion, and may assist in designing a strategy to prevent the establishment of S.

295 aurantiacum infection. S. aurantiacum has been shown to invade human alveolar epithelial cells

296 (A549 cell line) by initial attachment to the pneumocytes and then penetration of the cells (Kaur

297 2015). Our study demonstrated, for the first time, that secreted S. aurantiacum peptidases can

298 impair the attachment and viability of A549 cells, and that the impact of chymotrypsin-, elastase-

299 like and cysteine peptidases was different.

300 Cysteine peptidases secreted by S. aurantiacum under hypoxia caused cell detachment by

301 rounding them up (Fig 2A); also, viability of a portion of the floating cells was reduced seemingly

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302 by release of cell content into environment (Fig 3A). A cysteine peptidase produced by A.

303 fumigatus has reported to have a similar effect on A549 cells (Daly et al. 1999). Release of the

304 cell content may result in further damage to the surrounding cells and an inflammatory response

305 as indicated in the study of A. fumigatus using a mouse model (Proskuryakov et al. 2003).

306 In our study, secreted S. aurantiacum chymotrypsin-like peptidase also rounded up the A549 cells,

307 leading to detachment of cells (Fig 2A), however, these floating cells were still viable (Fig 3B).

308 After 4 h exposure to the 50 % (v/v) chymotrypsin-like peptidase fraction, 60 % of A549 cells

309 detached from the bottom of the culture well (Fig 2C). As seen from Fig 1 and Table 1, the overall

310 protein concentration of the chymotrypsin-like peptidase fraction (H20) was low compared to

311 other peptidase fractions, suggesting that the secreted chymotrypsin-like peptidase had high

312 specific activity exerting a strong effect on cell attachment.

313 Disruption of cell attachment may result in deficiency in the normal cell function, such as

314 failure in provision of a physical barrier and less effective cell-to-cell connection and

315 communication. In turn, a fungal pathogen may benefit from cell detachment enabling the fungus

316 to invade deeper into the tissue (Ivanov et al. 2010; Kogan et al. 2004). Exact comparison of the

317 effect of chymotrypsin-like and cysteine peptidases on cell detachment would require purification

318 of the enzyme proteins.

319 The observation by Kaur et al. that S. aurantaicum WM 06.482 required at least 4 h for the

320 conidia to attach to the A549 cells (Kaur 2015), supports our findings that peptidases secreted by

321 S. aurantaicum destroy the physical barrier and cell-to-cell connection of A549 cells within 4 h to

322 facilitate attachment of conidia to the cells to start the invasion. The drops of cell attachment at

323 the subsequent time points were small. This might be due to the loss of chymotrypsin-like

324 peptidase activity, as some fungal peptidases were subjected to autolysis even at 4 °C (Tunlid et

325 al. 1994).

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326 Secreted elastase-like peptidase(s) has been considered a virulence factor of A. fumigatus. The

327 enzyme has been linked to germination and penetration of this fungus into mice lungs

328 (Kolattukudy et al. 1993), deterioration of respiratory function (Robinson et al. 1990), and lung

329 injury (Ladarola et al. 1998). In the current study, we demonstrated that the S. aurantiacum

330 elastase-like peptidase decreased the viability of A549 cells (Fig 3B), which points to the

331 possibility that the enzyme has a similar role to that of the A. fumigatus elastase. These effects

332 require evaluation in animal models. Addition of 50 % (v/v) fraction of elastase-like peptidase

333 activity could cause around 80 % of the cells to lose their variability within 24 h (Fig 3B). This is

334 similar to the findings of Kaur et al. who also observed that infection of A549 cells by the isolate

335 S. aurantaicum WM 06.482 caused cell death after 24 h incubation (Kaur 2015). Together, these

336 studies suggest that elastase-like peptidase secreted by this fungal species was the main reason for

337 cell death. Elastase peptidase secreted by the bacterium Pseudomonas aeruginosa also caused

338 death of A549 cells (Lee et al. 2011).

339 There was a visible difference in the morphology of A549 cells exposed to elastase-like

340 peptidases and cysteine peptidases. Elastase-like peptidase failed to round cells up, yet some

341 morphological changes were observed (Fig 3A). Cells exposed to cysteine peptidase were rounded

342 up, lost their membrane integrity and released inner contents to the environment (Fig 2A). This is

343 consistent to the study of Kaur et al., who found that the A549 cell line infected by the clinical S.

344 aurantaicum isolate (WM 06.482) lost the membrane integrity (indicated by membrane blebbing

345 and cell rounding) (Kaur 2015).

346 In summary, our study is the first exploring the effect of peptidases secreted by the clinical

347 S.aurantiacum isolate (WM 06.482) on cell attachment and viability in vitro using human alveolar

348 epithelial cells (A549 cell line). The chymotrypsin-like peptidase caused detachment of cells

349 elastase-like peptidase was associated with loss of cell viability. Similar effect caused by elastase

350 has been previously described for other fungi. Cysteine peptidases were involved in destroying

351 both the cell attachment and viability; however, the mechanism seemed different to that of

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352 elastase-like peptidases. This study contributes to the understanding of the role of peptidases in

353 invasion of lung cells by S. aurantiacum paving the way for future studies aimed at assessing the

354 effects of these peptidases in vivo.

355

356 Acknowledgements. Z. Han was supported by International Macquarie University Research

357 Excellence Scholarship (iMQRES).

358

359 Compliance with Ethical Standards

360 Conflict of interest The authors declare that they have no conflict of interest.

361 Ethical Approval The work was conducted under the MQ Biosafety approval NRLD Ref

362 5201200092

363 Informed Consent NA (no patients participating)

364

365 References

366 Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., and Walter, P. 2008. Chapter 17: Cell Cycle; 367 Chapter 18: Apoptosis; Chapter 19: Cell Junctions, Cell Adhesion, and the Extracellular Matrix. 368 In Molecular biology of the cell. 5th ed. Garland Science, New York. pp. 1053-1149.

369 Aplin, A.E., Howe, A.K., and Juliano, R.L. 1999. Cell adhesion molecules, signal transduction and cell 370 growth. Curr Opin Cell Biol. 11(6): 737-744.

371 Bernas, T., and Dobrucki, J. 2002. Mitochondrial and nonmitochondrial reduction of MTT: interaction of 372 MTT with TMRE, JC‐1, and NAO mitochondrial fluorescent probes. Cytometry. 47(4): 236-242.

373 Biaggio, R.T., Silva, R.R., Rosa, N.G., Leite, R.S., Arantes, E.C., Cabral, T.P., Juliano, M.A., Juliano, L., 374 and Cabral, H. 2016. Purification and biochemical characterization of an extracellular serine 375 peptidase from Aspergillus terreus. Prep Biochem Biotechnol. 46(3): 298-304.

376 Borger, P., Koeter, G.H., Timmerman, J.A.B., Vellenga, E., Tomee, J.F.C., and Kauffman, H.F. 1999. 377 Proteases from Aspergillus fumigatus induce interleukin (IL)-6 and IL-8 production in airway 378 epithelial cell lines by transcriptional mechanisms. J Infect Dis. 180(4): 1267-1274.

379 Bradford, M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein 380 utilizing the principle of protein-dye binding. Analyt Biochem. 72(1): 248-254.

381 Cortez, K.J., Roilides, E., Quiroz-Telles, F., Meletiadis, J., Antachopoulos, C., Knudsen, T., Buchanan, 382 W., Milanovich, J., Sutton, D.A., and Fothergill, A. 2008. Infections caused by Scedosporium spp. 383 Clin Microbiol Rev. 21(1): 157-197.

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384 Daly, P., and Kavanagh, K. 2002. Immobilization of Aspergillus fumigatus colonies in a soft agar matrix 385 allows visualization of A549 cell detachment and death. Med Mycol. 40(1): 27-33.

386 Daly, P., Verhaegen, S., Clynes, M., and Kavanagh, K. 1999. Culture filtrates of Aspergillus fumigatus 387 induce different modes of cell death in human cancer cell lines. Mycopathologia. 146(2): 67-74.

388 Filler, S.G., and Sheppard, D.C. 2006. Fungal invasion of normally non-phagocytic host cells. PLoS Pathog. 389 2(12): e129.

390 Gumbiner, B.M. 1996. Cell adhesion: the molecular basis of tissue architecture and morphogenesis. Cell. 391 84(3): 345-357.

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394 Han, Z., Kautto, L., Meyer, W., Chen, S.C.A., and Nevalainen, H. 2018. Growth and protease secretion of 395 Scedosporium aurantiacum under conditions of hypoxia. Microbiol Res. 216: 23-29.

396 Harun, A., Serena, C., Gilgado, F., Chen, S.C., and Meyer, W. 2010. Scedosporium aurantiacum is as 397 virulent as S. prolificans, and shows strain-specific virulence differences, in a mouse model. Med 398 Mycol. 48: S45-51.

399 Ivanov, V.A., Gewolb, I.H., and Uhal, B.D. 2010. A new look at the pathogenesis of the Meconium 400 aspiration syndrome: a role for fetal pancreatic proteolytic enzymes in epithelial cell detachment. 401 Pediatr Res. 68(3): 221-224.

402 Kaur, J. 2015. Biological studies into Scedosporium aurantiacum, an opportunistic pathogen colonising 403 human lungs. Ph.D. thesis, Macquarie University, Sydney, Australia.

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464 Table 1. Peptidase activity present in each fraction, tested using class-specific peptidase substrates

Peptidases Source Fraction No. Activity

Subtilisin 18,19 625 µM ρ-NA·ml-1·min-1

Elastase 15-18 32410 nM MCA·ml-1·min-1

Trypsin

Normoxic

16-19 18930 nM MCA·ml-1·min-1

Chymotrypsin 19,20 566 µM ρ-NA·ml-1·min-1

Cysteine 15,16 6109 nM MCA·ml-1·min-1

Aspartic

Hypoxic

12 147500 nM MCA·ml-1·min-1

465

466

467

468

469

470

471

472

473

474

475

476

477

478

479

480

481

482

483

484

485

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486

487 Figure legends

488

489 Fig 1. Fractionation of proteins secreted by Scedosporium. aurantiacum under normoxia and

490 hypoxia. One mg of protein was subjected to size exclusion chromatography and protein levels

491 were measured continuously by UV absorbance at 280 nm. One ml fractions were collected at

492 4 °C.

493

494 Fig 2. Peptidases involved in cell detachment. (A) Microscopic images of detached A549 cells

495 after 24-hour exposure to 50 % (v/v) chymotrypsin-like and cysteine peptidase fractions. Bars =

496 20 µm. (B) Effect of chymotrypsin-like and cysteine peptidase fractions (% v/v) on A549 cell

497 detachment after 24 h exposure. (C) Time-dependent effect of 50 % (v/v) chymotrypsin-like

498 peptidase fraction on A549 cell attachment. Ctrl: cells in RPMI plus PBS (1:1 v/v) at each

499 indicated time point; PF+inh: SEC peptidase fractions with addition of class-specific peptidase

500 inhibitor. Chymostatin with a final concentration of 5 mg·ml-1 was used as chymotrypsin-like

501 peptidase inhibitor, and E-64 with a final concentration of 10 µM was used for cysteine peptidases

502 inhibitor; Ctrl+inh: control with addition of class-specific peptidase inhibitor; 10 %, 20 %, 50 %

503 (v/v) of a SEC fraction in the cell culture medium. Chym: H20 with chymotrypsin-like peptidase

504 activity, Cyst: H16 with cysteine peptidase activity. Data represent a mean ± SD (three replicates).

505 * means significant difference between the samples and Ctrl (p < 0.05), and ** means highly

506 significant difference between the samples and Ctrl (p < 0.01). P-values were calculated using

507 Student’s t test.

508

509 Fig 3. Peptidases associated with loss of cell viability. (A) Microscopic images of A549 cells

510 with decreased viability after 24-hour exposure to 50 % (v/v) elastase-like and cysteine peptidase

511 fractions. Bars = 20 µm. (B) Effects of elastase-like and cysteine peptidase fractions (% v/v) on

512 A549 cell viability after 24 h exposure. (C) Time-dependent effect of 50 % (v/v) elastase-like

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513 peptidase fraction on A549 cell viability. Ctrl: cells in RPMI plus PBS (1:1 v/v) at each indicated

514 time point; PF+inh: SEC peptidase fractions with addition of class-specific peptidase inhibitor.

515 Elastatinal with a final concentration of 50 µM was used as elastase-like peptidase inhibitor, and

516 E-64 with a final concentration of 10 µM was used for cysteine peptidases inhibitor; Ctrl+inh:

517 control with addition of class-specific peptidase inhibitor; 10 %, 20 %, 50 %: volumetric

518 percentage of a SEC fraction in cell culture medium (v/v). Elas: N15 with elastase-like peptidase

519 activity, Cyst: H16 with cysteine peptidase activity. Trypsin-like peptidase had no effect on cell

520 viability (data not shown). Data represent a mean ± SD (three replicates). * means significant

521 difference between the samples and Ctrl (p < 0.05), and ** means highly significant difference

522 between the samples and Ctrl (p < 0.01). P-values were calculated using Student’s t test.

523

524 Fig S1. The number of cells measured by MTS assay and counting using automated cell

525 counter. Various numbers of A549 cells were added to the wells of a 96-well plate in RPMI

526 supplemented with 10 % (v/v) FBS and 1 mM glutamine. The medium was allowed to equilibrate

527 for 1 hour; then MTS assay or counting using automated cell counter was performed. Each point

528 represents the mean ± SD of 3 replicates.

529

530 Fig S2. Effect of cell number on absorbance at 490nm measured by MTS Assay. Various

531 numbers of A549 cells were added to the wells of a 96-well plate in RPMI supplemented with

532 10 % (v/v) FBS and 1 mM glutamine. The medium was allowed to equilibrate for 1 hour; then 20

533 µl/well of MTS Reagent was added. After 2 h at 37°C in a humidified 5 % CO2 atmosphere, the

534 absorbance at 490 nm was recorded using a Fluostar plate reader. Each point represents the mean

535 ± SD of 3 replicates. The correlation coefficient of the line labelled with asterisk () was 0.9974,

536 indicating a linear response between cell number (≤60000 cells per well) and absorbance at 490

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537 nm. When cell number was bigger than 60000 cells per well (line labelled with dot ()), the

538 correlation coefficient lowered, suggesting that there was not a linear relationship between cell

539 number and absorbance. The background absorbance shown at zero cells/well was not subtracted

540 from these data.

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Fig 1. Fractionation of proteins secreted by Scedosporium. aurantiacum under normoxia and hypoxia. One mg of protein was subjected to size exclusion chromatography and protein levels were measured

continuously by UV absorbance at 280 nm. One ml fractions were collected at 4 °C.

126x79mm (300 x 300 DPI)

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Fig 2. Peptidases involved in cell detachment. (A) Microscopic images of detached A549 cells after 24-hour exposure to 50 % (v/v) chymotrypsin-like and cysteine peptidase fractions. Bars = 20 µm. (B) Effect of

chymotrypsin-like and cysteine peptidase fractions (% v/v) on A549 cell detachment after 24 h exposure. (C) Time-dependent effect of 50 % (v/v) chymotrypsin-like peptidase fraction on A549 cell attachment. Ctrl: cells in RPMI plus PBS (1:1 v/v) at each indicated time point; PF+inh: SEC peptidase fractions with addition

of class-specific peptidase inhibitor. Chymostatin with a final concentration of 5 mg•ml-1 was used as chymotrypsin-like peptidase inhibitor, and E-64 with a final concentration of 10 µM was used for cysteine

peptidases inhibitor; Ctrl+inh: control with addition of class-specific peptidase inhibitor; 10 %, 20 %, 50 % (v/v) of a SEC fraction in the cell culture medium. Chym: H20 with chymotrypsin-like peptidase activity,

Cyst: H16 with cysteine peptidase activity. Data represent a mean ± SD (three replicates). * means significant difference between the samples and Ctrl (p < 0.05), and ** means highly significant difference

between the samples and Ctrl (p < 0.01). P-values were calculated using Student’s t test.

128x100mm (300 x 300 DPI)

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Fig 3. Peptidases associated with loss of cell viability. (A) Microscopic images of A549 cells with decreased viability after 24-hour exposure to 50 % (v/v) elastase-like and cysteine peptidase fractions. Bars = 20 µm.

(B) Effects of elastase-like and cysteine peptidase fractions (% v/v) on A549 cell viability after 24 h exposure. (C) Time-dependent effect of 50 % (v/v) elastase-like peptidase fraction on A549 cell viability. Ctrl: cells in RPMI plus PBS (1:1 v/v) at each indicated time point; PF+inh: SEC peptidase fractions with addition of class-specific peptidase inhibitor. Elastatinal with a final concentration of 50 µM was used as

elastase-like peptidase inhibitor, and E-64 with a final concentration of 10 µM was used for cysteine peptidases inhibitor; Ctrl+inh: control with addition of class-specific peptidase inhibitor; 10 %, 20 %, 50 %: volumetric percentage of a SEC fraction in cell culture medium (v/v). Elas: N15 with elastase-like peptidase

activity, Cyst: H16 with cysteine peptidase activity. Trypsin-like peptidase had no effect on cell viability (data not shown). Data represent a mean ± SD (three replicates). * means significant difference between

the samples and Ctrl (p < 0.05), and ** means highly significant difference between the samples and Ctrl (p < 0.01). P-values were calculated using Student’s t test.

129x100mm (300 x 300 DPI)

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