The Antimicrobial Peptide TAT-Rasgap317-326 Inhibits the Formation and the Expansion

bioRxiv preprint doi: https://doi.org/10.1101/2020.09.29.318378; this version posted September 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

1 The antimicrobial peptide TAT-RasGAP317-326 inhibits the formation and the expansion

2 of bacterial biofilms in vitro

3 Tytti Heinonen1, Simone Hargraves1, Maria Georgieva2, Christian Widmann2, Nicolas

4 Jacquier1.*

5 1Department of Laboratories, Institute of Microbiology, Lausanne University Hospital and

6 University of Lausanne, Lausanne, Switzerland

7 2Department of Physiology, University of Lausanne, Lausanne, Switzerland

9 Short title: Effect of TAT-RasGAP317-326 on biofilm formation and expansion

10 *Corresponding author: Nicolas Jacquier, Department of Laboratories, Institute of

11 Microbiology, Lausanne University Hospital and University of Lausanne, Rue du Bugnon 48,

12 CH-1011 Lausanne, Switzerland

13 Tel: +41 21 314 85 39

14 E-mail: [email protected] ; ORCID: 0000-0002-1974-8161

16 Number of figures: 3

17 Number of tables: 1

19 Abstract

20 Biofilms are structured aggregates of bacteria embedded in a self-produced matrix. Pathogenic

21 bacteria can form biofilms on surfaces and in tissues leading to nosocomial and chronic

22 infections. While antibiotics are largely inefficient in limiting biofilm formation and expansion,

23 antimicrobial peptides (AMPs) are emerging as alternative anti-biofilm treatments. In this study,

24 we explore the effect of the newly described AMP TAT-RasGAP317-326 on Acinetobacter

25 baumannii, Pseudomonas aeruginosa and Staphylococcus aureus biofilms. We observe that

26 TAT-RasGAP317-326 inhibits the formation of biofilms at concentrations equivalent or two times

27 superior to the minimal inhibitory concentration (MIC) of the corresponding planktonic bacteria.

28 Moreover, TAT-RasGAP317-326 limits the expansion of A. baumannii and P. aeruginosa

29 established biofilms at concentrations 2-4 times superior to the MIC. These results further

30 confirm the potential of AMPs against biofilms, expand the antimicrobial potential of TAT-

31 RasGAP317-326 and support further development of this peptide as an alternative antimicrobial

32 treatment.

34 Keywords: biofilms, antimicrobial peptide, antibiotic resistance, TAT-RasGAP317-326

36 Introduction

37 The emergence of antibiotic resistance is a major threat to public health. Infections

38 caused by multidrug resistant bacteria are an increasing concern, leading to disabilities and

39 possibly death [1]. One limitation in the development of novel antibiotics is the use of free-living

40 (also called planktonic) bacteria in axenic medium as model system. This model is not

41 representative of infections, as the majority of them are caused by multicellular aggregates of

42 bacteria in a self-produced matrix, called biofilms. [2]. The formation of biofilms is dynamic and

43 triggered by signals such as nutrient limitation, antibiotic exposure and oxygen availability [3].

44 The matrix embedding the biofilm is composed of polysaccharides, DNA and proteins, and

45 forms a scaffold for bacterial attachment, protects bacteria from external insults, and enables

46 a compartmentalisation of the biofilms with distinct bacterial subpopulations [2, 4].

47 Pathogenic bacteria, including Acinetobacter baumannii, Pseudomonas aeruginosa

48 and Staphylococcus aureus, can form biofilms on medical devices implanted in humans,

49 develop in some tissues, e.g. lungs, teeth and skin, and may persist on healthcare surface [5,

50 6]. Moreover, bacteria present in biofilms can be 10 to1000 times more resistant to antibiotics

51 than planktonic bacteria [7]. Bacteria contained in biofilms strongly differ from planktonic

52 bacteria regarding both their gene expression profile and their functional properties. Moreover,

53 bacterial subpopulations composing biofilms are heterogeneous, further complicating biofilm

54 treatment. Current biofilm treatment is tedious and resembles cancer therapy starting with the

55 surgical removal of the biofilm followed by administration of antimicrobials [8]. This strategy is

56 not optimal since antimicrobials are often unable to eradicate all bacterial subpopulations

57 embedded in the biofilm. Moreover, this treatment may not completely disrupt the biofilm

58 scaffold, potentially allowing its colonization by other microorganisms. The combination of

59 intrinsic antibiotic resistance with resistance properties provided by the biofilm organisation

60 renders these nosocomial infections highly challenging to treat. We thus need alternatives to

61 classical antibiotics to treat biofilms formed by resistant pathogens.

62 Antimicrobial peptides (AMPs) could be such an alternative. AMPs are short peptides

63 (up to 100 amino acids) produced by all living organisms as a defence mechanism that usually

64 target a broad range of pathogens [9]. While the ability of bacteria to develop resistance

65 towards AMPs is debated [10], pathogens carrying resistance genes to one or multiple

66 antibiotics often show increased sensitivity towards AMPs [11, 12]. Some AMPs, alone or

67 combined with antibiotics, act both on the inhibition of biofilm formation and on the disruption

68 of mature biofilm [13].

69 TAT-RasGAP317-326 is a chimeric peptide consisting of the cell-permeable HIV peptide

70 TAT48-57 linked to a 10-amino acid sequence of the Src Homology 3 Domain (SH3 domain) of

71 p120 RasGAP [14]. TAT-RasGAP317-326 was first described for its anticancer properties, being

72 able to sensitise cancer cells to anticancer therapies [15-17], directly kill cancer cells [18] and

73 display anti-metastatic effect [19]. More recently, we showed that TAT-RasGAP317-326 exerts a

74 broad antimicrobial activity against both Gram-positive and Gram-negative human pathogens

75 including A. baumannii, P. aeruginosa and S. aureus [20]. Like other AMPs, TAT-RasGAP317-

76 326 is positively charged and contains several arginine and tryptophan residues. Arginine

77 residues may interact with the negatively charged bacterial membrane while tryptophan may

78 insert in the bacterial membrane [21, 22]. These interactions may enable AMPs to translocate

79 in the bacteria without disrupting membrane and to target intracellular components. While the

80 tryptophan at position 317 of RasGAP domain is essential for the antimicrobial activity of TAT-

81 RasGAP317-326, the mode of action of this peptide remains unknown.

82 In this report, we questioned the potential effect of TAT-RasGAP317-326 on biofilm

83 formation and on mature biofilm expansion. We show that TAT-RasGAP317-326 inhibits the

84 formation of A. baumannii, P. aeruginosa and S. aureus biofilms in vitro at concentrations equal

85 or 2 times the MIC of planktonic bacteria. Moreover, treatment of mature biofilms with TAT-

86 RasGAP317-326 reduces the expansion of A. baumannii and P. aeruginosa biofilms, but, similarly

87 to other tested antibiotics and AMPs, cannot completely eradicate the biofilm scaffold. These

88 results highlight the potential of TAT-RasGAP317-326 in the prevention and treatment of biofilms

89 and encourage further development of AMPs as alternative antimicrobial compounds.

91 Material and methods

92 Bacterial strains and growth conditions

93 Acinetobacter baumannii ATCC 19606 (A. baumannii) and Staphylococcus aureus

94 ATCC 29213 (S. aureus) were from American Type Culture Collection (ATCC; Manassas, VA).

95 Pseudomonas aeruginosa PA14 (P. aeruginosa) was obtained from Professor Leo Eberl

96 (Department of Plant and Microbial Biology, University of Zürich, Switzerland). Bacteria were

97 routinely grown in Mueller-Hinton broth (A. baumannii), tryptic soy broth (TSB; S. aureus) and

98 Luria-Bertani broth (LB; P. aeruginosa).

99 Peptides and antibiotics

100 TAT-RasGAP317-326 is a retro-inverso peptide (i.e. reversed direction compared to

101 natural sequence including D-amino acids) with an antimicrobial activity [20] composed of

102 amino acids 48-57 of HIV TAT protein (RRRQRRKKRG) and 317-326 of human RasGAP

103 protein (DTRLNTVWMW) linked with two glycines. TAT-RasGAP317-326 was synthesized by

104 SBS Genetech (Beijing, China). Ciprofloxacin, tetracycline and gentamicin were from

105 Applichem (Darmstadt, Germany), rifampicin and polymyxin B from Sigma-Aldrich (Saint-

106 Louis, MO), and melittin from Enzo Life Sciences (Farmingdale, NY).

107 MIC measurement

108 The minimal inhibitory concentrations (MICs) of antibiotics and AMPs on bacterial

109 strains were determined as described [23]. Briefly, overnight cultures were diluted to 0.1 OD600,

110 grown for 1h at 37°C with 200 rpm shaking and diluted 1:200 in 96-well plates containing

111 increasing amount of antibiotics or AMPs. Plates were incubated statically for 18h at 37°C. The

112 lowest concentration at which no turbidity could be observed was determined as MIC.

113 Biofilm formation assay and treatment

114 Overnight cultures of bacteria were diluted 1:50 and grown to exponential phase at

7 9 115 37°C. Cultures were washed with PBS and adjusted to 0.1 OD600 (10 -10 CFU/ml) in BM2

116 medium (62 mM potassium phosphate buffer, 7 mM ammonium sulfate, 10 µM iron sulfate,

117 0.4% (w/v) glucose, 0.5% (w/v) casaminoacids, 2 mM magnesium sulfate) with or without

118 antibiotics or AMPs. Hundred microliters of culture were plated in polypropylene plates

119 (Greiner, Kremsmünster, Austria) and biofilms were allowed to form for 24h (A. baumannii and

120 P. aeruginosa) and 48h (S. aureus) at room temperature (RT). For biofilm eradication, mature

121 biofilms were washed twice with PBS and incubate with antibiotics or AMPs in BM2 medium

122 for 24h at RT.

123 Measurement of biofilm biomass and bacterial viability

124 To assess total biofilm biomass, biofilms were washed with water and stained with 0.1%

125 crystal violet (Sigma-Aldrich) as described [24]. Stained biofilms were dried overnight at RT

126 and dissolved in 30% acetic acid. Absorbance was measured at 590 nm. To assess bacterial

127 viability, biofilms were washed twice with PBS and incubated with 4 µg/ml resazurin (Sigma-

128 Aldrich) in BM2. Plates were incubated 90 minutes at 37°C. Fluorescence was measured with

129 a FLUOstar Omega plate reader (BMG Labtech, Ortenberg, Germany) with Ex/Em = 540/580

130 and gain = 500.

131 Calculations

132 The antibiotic or AMP concentration added at biofilm induction that reduced the

133 formation of biofilm (viability and biomass) by ≥ 90% was set as BPC90 (biofilm prevention

134 concentration). For mature biofilms, the MBIC (minimal biofilm inhibitory concentration) was

135 set as the concentration of antibiotic or AMP that resulted in no expansion of an existing biofilm

136 (viability and biomass equal or lower than the value at treatment). The MBEC90 (minimal biofilm

137 eradication concentration) was defined as the concentration of antibiotic or AMP that reduced

138 the biofilm initial biomass by ≥ 90% during treatment. All calculations were adapted from [25].

139

140 Results

141 Classical antibiotics have a moderate effect on A. baumannii and P. aeruginosa biofilms

142 A. baumannii and P. aeruginosa form biofilms that show increased resistance to

143 antibiotics compared to planktonic bacteria [25, 26]. We first confirmed this observation in our

144 settings by determining the minimal inhibitory concentration of antibiotics on planktonic

145 bacteria (minimal inhibitory concentration, MIC) and on biofilm formation (biofilm prevention

146 concentration, BPC90). For A. baumannii, we measured a 128 to over 512 times higher BPC90

147 than the corresponding MIC for all tested antibiotics (Table 1). The effect was less striking for

148 P. aeruginosa with a BPC90/MIC ratio of 2-4 (Table 1).

149 In a further step, we tested whether these antibiotics could limit biofilm expansion

150 (measuring the minimal biofilm inhibitory concentration, MBIC) and eradicate established

151 biofilms (measuring the minimal biofilm eradication concentration, MBEC90). We used the

152 reduction of resazurin to resofurin as a surrogate of bacterial viability and measured total

153 biomass with crystal violet staining. All the tested antibiotics were ineffective at both inhibiting

154 expansion and eradicating A. baumannii biofilms (MBIC and MBEC90 > 256 µg/ml, Table 1).

155 While ciprofloxacin, tetracycline and gentamicin inhibited the proliferation of viable bacteria in

156 P. aeruginosa biofilms at concentrations corresponding to 2-4 times the BPC90, we observed

157 no effect on biomass (Table 1). These results confirm the low efficiency of classical antibiotics

158 on biofilms.

159 TAT-RasGAP317-326 potently inhibits the formation and expansion of A. baumannii and P.

160 aeruginosa biofilms

161 Well-described AMPs such as melittin and polymyxin B efficiently reduce bacterial

162 biofilm formation and the biomass of established biofilms [27, 28]. In line with published results,

163 we observed a consistent inhibitory effect of these AMPs on A. baumannii and P. aeruginosa

164 biofilm formation. The BPC90 of melittin was equal to the MIC for A. baumannii biofilms and 2-

165 4 times higher than the MIC for P. aeruginosa biofilms (Table 1). For polymyxin B, the

166 BPC90/MIC ratios were 2-4 and 1-2 for A. baumannii and P. aeruginosa respectively (Table 1).

167 As TAT-RasGAP317-326 is efficient against planktonic A. baumannii and P. aeruginosa, we

168 hypothesized that it would also potently inhibit the formation of biofilms. TAT-RasGAP317-326

169 inhibited A. baumannii biofilm formation at 8-16 µg/ml corresponding to 1-2 times the MIC (Fig.

170 1A-B and Table 1). The BPC90 of TAT-RasGAP317-326 on P. aeruginosa biofilms was equal to

171 its MIC (32 µg/ml; Fig. 2A-B and Table 1).

172 Since classical antibiotics had little to no effect on biofilm expansion and eradication

173 (Table 1), we tested the effect of AMPs on established biofilms. TAT-RasGAP317-326 limited the

174 expansion of A. baumannii biofilms with a MBIC/MIC ratio of 4 (Fig. 1C-D and Table 1). Melittin

175 was effective at 8 times the MIC, while Polymyxin B had a MBIC/MIC ratio of 8 for viability and

176 32 for biomass (Table 1). TAT-RasGAP317-326 efficiently inhibited P. aeruginosa biofilm

177 expansion with a MBIC/MIC ratio of 2 (Fig. 2C-D and Table 1). In contrast, polymyxin B had a

178 higher MBIC/MIC ratio of 16 while the MBIC of melittin could not be determined, being higher

179 than 256 µg/ml (Table 1). However, none of the tested AMPs had the potency to eradicate

180 more than 90% of A. baumannii or P. aeruginosa biofilms at the maximal tested concentration

181 of 256 µg/ml.

182 TAT-RasGAP317-326 inhibits the formation of S. aureus biofilms

183 TAT-RasGAP317-326 has an effect on both Gram-negative and Gram-positive bacteria in

184 planktonic cultures [20]. We thus tested its impact on S. aureus biofilms. Despite the high MIC

185 of TAT-RasGAP317-326 on planktonic S. aureus (128 µg/ml), we could measure a BPC90/MIC

186 ratio of 2 (Table 1). In comparison, the BPC90/MIC ratio of ciprofloxacin was 320-640 (Table

187 1). However, as expected due to the high MIC and BPC90 of TAT-RasGAP317-326, the MBIC and

188 MBEC90 on S. aureus biofilm could not be estimated being higher than 256 µg/ml.

189

190 Discussion

191 Bacterial biofilms cause nosocomial infections and underlie several chronic infections.

192 The complex structure of biofilms renders them refractory to treatments with classical

193 antibiotics. Here we show that the AMP TAT-RasGAP317-326 has a potent inhibitory effect on A.

194 baumannii, P. aeruginosa and, to a lesser extent, S. aureus biofilm formation and expansion.

195 These observations support the potential of AMPs as alternative to classical antibiotics in anti-

196 biofilm treatment.

197 In order to test the quality of the biofilm we produced in vitro, we measured its

198 resistance to classical antibiotics. As reported in literature, we observed increased resistance

199 of A. baumannii and P. aeruginosa biofilms to classical antibiotics. A. baumannii biofilms

200 formed in the presence of concentrations of antibiotics equivalent to 128 to more than 512

201 times the MIC on planktonic bacteria (Table 1), while P. aeruginosa biofilm formation was

202 abolished with concentrations equivalent to 2-4 times the MIC. This difference in biofilm

203 resistance to antibiotics is possibly caused by a faster resistance development during A.

204 baumannii biofilm formation, partly triggered by the antibiotics themselves [29]. While

205 antibiotics showed some effect on biofilm formation at moderate to high concentrations, they

206 were not efficient in inhibiting proliferation or disrupting mature biofilms (Table 1, [30]). This

207 may be due to the low permeability of the extracellular matrix to classical antibiotics, and

208 highlights the need of alternative strategies to treat infections involving biofilms.

209 In contrast to antibiotics, TAT-RasGAP317-326, polymyxin B and melittin limited both A.

210 baumannii and P. aeruginosa biofilm formation and expansion. Similar promising results have

211 been observed with other AMPs including the human AMP LL-37 and the bacterial bacteriocins

212 [31, 32]. Moreover and despite its high MIC, TAT-RasGAP317-326 also reduced S. aureus biofilm

213 formation. These results are encouraging and suggest that combinations of TAT-RasGAP317-

214 326 with other drugs might be helpful in the treatment of biofilm-associated infections. As biofilm

215 are composed of heterogeneous bacterial subpopulations, the combination of AMPs with

216 antibiotics is a valuable way to avoid the survival of persister cells. The combination of melittin

217 and LL-37 with classical antibiotics reduced the corresponding MBEC several folds [33]. We

218 need to test in future studies the anti-biofilm effect of combinations of TAT-RasGAP317-326 with

219 classical antibiotics. Indeed, TAT-RasGAP317-326 might have advantages compared to other

220 AMPs: its quite low toxicity to mammalian cells [20] and its chimeric nature against which

221 bacteria should not have developed resistance. Moreover, since biofilms are often composed

222 of multiple bacterial species, the broad-spectrum antimicrobial activity of TAT-RasGAP317-326 is

223 interesting for future clinical developments. The current limitation of TAT-RasGAP317-326 for

224 clinical use is its high excretion rate and subsequent low bioavailability [20]. We thus need to

225 develop strategies to increase the distribution of this peptide in vivo, which could be achieved

226 by chemical modifications or specific delivery methods.

227 In summary, we show in this report that the AMP TAT-RasGAP317-326 has potent anti-

228 biofilm activity in vitro against A. baumannii and P. aeruginosa biofilms and is relatively active

229 on S. aureus biofilms. This makes TAT-RasGAP317-326 a promising tool in the treatment of

230 biofilm-associated infections and might lead to the development of new strategies to prevent

231 biofilm formation.

232

233 Authorship

234 TH and SH performed experiments. TH, MG, CW and NJ were involved in the planning of the

235 project and discussed the results. TH analysed the results. TH and NJ wrote the manuscript.

236 Acknowledgements

237 NJ and CW received an interdisciplinary grant from the University of Lausanne. We thank Prof.

238 Gilbert Greub for providing laboratory equipment. We thank the Prof. Nibbering group (Leiden,

239 Netherlands) for their assistance in the implementation of the protocol of in vitro formation of

240 biofilms and Prof. Eberl (Zurich, Switzerland) for providing strains.

241 Conflicts of Interest Disclosure

242 The authors declare no competing financial interests.

243

244 Table 1

MBIC (µg/ml) Antibiotic/AMP MIC (μg/ml) BPC (μg/ml) MBEC (μg/ml) 90 Resazurin / CV 90

A. baumannii ATCC 19606

Ciprofloxacin 1 > 256 > 256 > 256

Tetracycline 1 128-256 > 256 > 256

Gentamicin 32 > 256 > 256 > 256

Levofloxacin 0.5 > 256 > 256 > 256

TAT-RasGAP 317-326 8 8-16 32 > 256

Polymyxin B 4 8-16 32 / 128 > 256

Melittin 16 16 128 > 256

P. aeruginosa PA14

Ciprofloxacin 0.2 0.4 < 2 / > 256 > 256

Tetracycline 16 8-32 32-64 / > 256 > 256

Gentamicin 4 8-16 32 / > 256 > 256

TAT-RasGAP317-326 32 32 64 > 256

Polymyxin B 2 2-4 32 > 256

Melittin 64 128 > 256 > 256

S. aureus ATCC 29213

Ciprofloxacin 0.4 128-256 > 256 > 256

TAT-RasGAP317-326 128 128-256 > 256 > 256 245 MIC: minimal inhibitory concentration, BPC: biofilm prevention concentration, MBIC: minimal 246 biofilm inhibitory concentration, MBEC: minimal biofilm eradication concentration, CV: crystal 247 violet. For MBIC, the indication of a single value means that the result was the same with 248 resazurin and CV assays. 249 250

251

252 Figure 1: TAT-RasGAP317-326 inhibits A. baumannii biofilm formation and expansion. The

253 effect of TAT-RasGAP317-326 on biofilm formation (A,B) and on mature biofilm expansion (C,D)

254 was assessed with resazurin reduction (A,C) and crystal violet (B,D) assays. Dotted lines

255 indicate 10% of initial signal. Dashed lines indicate values for mature biofilms before treatment.

256 Data are means ± SD of three experiments performed in quadruplicate.

257 258

259

260 Figure 2: TAT-RasGAP317-326 inhibits P. aeruginosa biofilms formation and expansion.

261 TAT-RasGAP317-326 was added simultaneously with bacteria (A-B) to measure inhibition of

262 biofilm formation, or on mature biofilm (C-D) to assess biofilm expansion limitation. Bacterial

263 viability was approximated with resazurin reduction assay (A,C) and biofilm biomass with

264 crystal violet staining (B,D). Dotted lines indicate 10% of initial signal. Dashed lines indicate

265 values for mature biofilms before treatment. Data are means ± SD of three experiments

266 performed in quadruplicate.

267 268

269

270 Figure 3: TAT-RasGAP317-326 reduces S. aureus biofilm formation. (A-B) Addition of TAT-

271 RasGAP317-326 at times of biofilm induction reduced the viability (A) and the biomass (B) of S.

272 aureus. (C-D) Treatment of mature biofilms with TAT-RasGAP317-326 had no effect on bacterial

273 viability (C) and biomass (D). Viability was measured with resazurin (A,C) and biomass with

274 crystal violet (B,D) assays. Dotted lines indicate 10% of initial signal. Dashed lines indicate

275 values for mature biofilms before treatment. Data are means ± SD of three experiments

276 performed in quadruplicate.

277 278

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