Unraveling the Biotechnological Potential of the Secretome of Burkholderia Cepacia Complex, with Focus on Its Antimicrobial Activity

Unraveling the biotechnological potential of the secretome of Burkholderia cepacia complex, with focus on its antimicrobial activity

Carina Andreia Ribeiro Galhofa

Thesis to obtain the Master of Science Degree in

Biotechnology

Supervisors:

Prof. Doctor Isabel Maria de Sá Correia Leite de Almeida; Doctor Patrick de Oliveira Freire

Examination Committee

Chairperson: Doctor Arsénio do Carmo Sales Mendes Fialho Supervisor: Prof. Doctor Isabel Maria de Sá Correia Leite de Almeida Members of the Committee: Doctor Inês Batista Guinote

December 2017

Acknowledgements

The wish to become a future microbiologist and to study the “little bugs” that were able to cause diseases in individuals way bigger than them has been present since I was a child. As such, I would like to acknowledge those that helped me from the beginning to the end of this work, making this possible. To Prof. Dr.Isabel Sá Correia, I would like to express my gratitude not only for advising, but also for accepting me in this project which I enjoyed to do so much. To Dr. Patrick Freire, my advisor within BioMimetx, not only for all the guidance and patience but, most of all, for the motivation and believing in my work and Dr. Carla Coutinho for the support, for bearing my inexperience and for all the tips given that prevented me from exploding the laboratory…and the centrifuge. To the funding of IBB (Institute for Bioengineering and Biosciences) and BioMimetx, that gave me all the resources and conditions that enabled the conduction of my work. To Amir Hassan, for staying up late at the lab waiting for me to finish my work and for the “Goooooodddd woooorkkkkk” motivational screams. Words cannot describe how grateful I am for all your help and how a good part of this thesis wouldn’t be done without your unconditional patience, motivation and incomprehensible wake up calls, usually in arabic. Special thanks go to Inês Leonardo, for the whole work performed that guided this project and for all the support and time spent with the lessons and tips that enabled me to start my work. To the BioMimetx team for the warm welcome. In particular, to Dr. Inês Guinote, Raquel Marques, Sílvia Ribeiro, Susana Saraiva and Tânia Chança for the cheering, help and good times, making my work so much easier and delightful. To João Nascimento, José Teles Reis, Kamran Azmaliyev, Kcénia Bougrova, Lauren Lopes, Renato Dimas and Sónia Santos, for always being there for me when it seemed to get harder and to Emilia Wójcik for all the polish good vibes, the wonderful friendship and for believing in me from the absolute beginning. Without her this couldn’t be possible, ever! To my lab colleagues at BSRG (Biological Sciences Research Group) for the nice times spent at the laboratory and the library. It was a great pleasure to share all the thesis adventures with you. At last, the most special thanks to my family, in particular, to my parents and my sister for all the love and support. Thank you for feeding my love for science and for bearing my enthusiasm for my work even though you may not have found it that interesting.

To my teachers and professors, from my first grade to my masters, for the inspiration and for the enlightenment that enabled me to get this far… ...My most sincere thank you.

“What better microbial challenge to unite agricultural and medical microbiologists than an organism that reduces an onion to a macerated pulp, protects other crops from bacterial and fungal disease, devastates the health and social life of cystic fibrosis patients, and not only is resistant to the most famous of antibiotics, penicillin, but can use it as a nutrient!" -J. R. W. Govan, 1998 ii

Abstract

In this project, developed in collaboration with BioMimetx, a start-up company dedicated to the development of innovative biological biocides, the secretomes of B. cenocepacia IST01 were explored for potential biotechnological applications. Culture supernatants of IST01 were harvested in mid exponential, transition to the stationary, early and late stationary phases and tested against Escherichia coli, Enterococcus faecalis, Listeria monocytogenes and Staphylococcus aureus. The highest bactericidal effectiveness was exhibited by the culture supernatants of the late and the early stationary phases, that were also effective against the multidrug resistant isolates Burkholderia cenocepacia, Pseudomonas aeruginosa and Methicillin-resistant Staphylococcus aureus. These two culture supernatant samples presented a distinct protein migration pattern when compared with the samples of the mid exponential and transition to the stationary phase, in analyses by SDS-PAGE, and the highest proteolytic activity values among the tested samples. Taking these results into account, early stationary phase supernatants were selected for further fractionation, by centrifugal ultrafiltration and separation of the molecules in the samples according to their different molecular weights for the characterization of the mix that is of value to activity. However accounted for over 78% of the total secretome and was the only one which exhibited antimicrobial activity, probably consisting in a mixture of secondary metabolites and small molecular proteins or peptides.

The results obtained in this project are encouraging as they support a possible future use of the Bcc secretomes as an antimicrobial solution cannot be lifted.

Key words: Burkholderia cepacia complex, bacterial secretomes, antimicrobial activity, lipolytic activity, proteolytic activity

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Resumo

Neste projecto, desenvolvido em colaboração com a BioMimetx, uma start-up que se dedica ao desenvolvimento de biocidas biológicos, os secretomas de B. cenocepacia IST01 foram explorados e caracterizados para potenciais aplicações biotecnológicas. Sobrenadantes de cultura do isolado foram recolhidos a meio da fase exponencial, transição para a fase estacionária, no início da fase estacionária e na fase estacionária tardia e testados contra Escherichia coli, Enterococcus faecalis, Listeria monocytogenes e Staphylococcus aureus. As amostras da fase estacionária tardia e do início da fase estacionária demonstraram ser as mais eficazes, de forma bactericida, contra os alvos testados, sendo as últimas capazes ainda de inibir o crescimento de isolados conhecidos pela sua multiresistência, nomeadamente, Burkholderia cenocepacia, Pseudomonas aeruginosa e Staphylococcus aureus resistente à meticilina. Estas duas amostras apresentaram um perfil de proteínas, obtido por SDS-PAGE, distinto das restantes amostras recolhidas e os valores de actividade proteolítica mais elevados.

Assim sendo sobrenadantes recolhidos no início da fase estacionária foram seleccionados para fraccionamento, por ultrafiltração, permitindo o isolamento de uma fracção do secretoma, composta por moléculas de peso molecular abaixo dos 3 kDa, que correspondeu a 78% do secretoma total e a única capaz de inibir o crescimento dos alvos testados, podendo ser composta por metabolitos produzidos durante o crescimento e pequenas proteínas.

Os resultados obtidos neste projecto encorajam, assim, a possibilidade do futuro uso dos secretomas do Bcc para aplicações biotecnológicas, em particular como uma solução antibacteriana.

Palavras chave: Complexo de Burkholderia cepacia, secretoma bacteriano, actividade antibacteriana, actividade proteolítica, actividade lipolítica

Abbreviations

 AHL- Acyl homoserine lactones  AMP- Antimicrobial peptides  ATCC- American type culture collection  Bcc- Burkholderia cepacia complex  B. cenocepacia- Burkholderia cenocepacia  BCL- Burkholderia cepacia lipase  BHI- Brain heart infusion  BSA- Bovine serum albumin  BSRG/IBB- Biological sciences research group /Institute for bioengineering and biosciences  CF- Cystic fibrosis  CFU- Colony forming units  CNCM- Collection nationale de cultures de microorganismes  DSM- Deutsche sammlung von mikroorganismem  E. coli- Escherichia coli  E. faecalis- Enterococcus faecalis  FITC- Fluorecein Isothiocyanide  HGH- Horizontal gene transfer  IST- Instituto superior técnico  LB- Luria-Bertani broth  LES- Liverpool epidemic strain  L. monocytogenes- Listeria monocytogenes  MGE- Mobile genetic elements  MBC- Minimal bactericidal concentration  MIC- Minimal inhibitory concentration  MRSA- Methicillin resistant Staphylococcus aureus  MWCO- Molecular weight cut-off  NCTC- National collection of type culture  P. aeruginosa- Pseudomonas aeruginosa  OD- Optical density  QS- Quorum sensing  QQ- Quorum quenching  S. aureus- Staphylococcus aureus  SDS- Sodium dodecyl sulfate  TBS- Tris-buffered saline  TCE- Trichloroethylene

Index of figures

Figure 1- Beneficial and harmful effects of Burkholderia cepacia complex bacteria ...... 11

Figure 2- Scheme of the fractionation process, by centrifugal ultrafiltration used for the recovery of the fractions of the IST01 culture medium, according to the molecular weights...... 22

Figure 3- B. cenocepacia IST01 growth curve obtained in LB media, at 37ºC. The arrows indicate the times, in hours, chosen to collect IST01 culture supernatants. The values represent the average of two independent bacterial cultivations...... 23

Figure 4- Protein quantification, in g/L of culture medium, of lyophilized B. cenocepacia IST01 culture supernatants, collected at different phases of the growth curve, by the Bradford method. The data represents the average of three independent experiments performed with supernatants harvested from three independent bacterial cultivations...... 26

Figure 5- Protein profile of B. cenocepacia IST01 culture supernatants obtained at the mid exponential phase (3h), transition to the stationary phase (7h), early stationary phase (20h) and late stationary phase (30h) and separated by SDS-PAGE (Resolving gel with 12,5% acrylamide). The samples were subjected to dialysis and 20 μL was loaded into the gel with 5 μL of loading buffer. MW- Molecular weight marker, QC- Sample of the culture supernatant of a reference strain used as an internal control, C- (LB)- Negative control of the medium, that consisted on sterilized liquid LB media resuspended with water and subjected to dialysis, as performed with the IST01 culture supernatant samples ...... 27

CNCM-I 4031, Purple- MIC values for S. aureus NCTC 8325. The data represents the average of the proteolytic activity values obtained for supernatants of three independent bacterial cultivations...... 28

Figure 7- Proteolytic activity of the IST01 culture medium samples collected at different phases of the bacterial growth, expressed in fluorescence units/g of protein. The data represents the average of the proteolytic activity values obtained for supernatants of three independent bacterial cultivations...... 29

Figure 8- Composition, according to the molecular weight of the molecules, of IST01 culture supernatant samples harvested in the early stationary phase, in % (weight, in g, of each freeze dried fraction/weight, in g, of total freeze dried culture supernatant sample) collected at the early stationary phase), recovered after fractionation, obtained by weighing each fraction after freeze-drying and comparing to the weight of total non-fractioned lyophilized culture supernatant used for the fractionation. The assays were performed using fractions obtained from two independent fractionations of the same total IST01 sample from the early stationary phase...... 30

Figure 9- Protein separation, by SDS PAGE (resolving gel with 18% acrylamide) of the fractions recovered after fractionation of the samples of B. cenocepacia IST01 culture medium harvested in the early stationary phase. QC-Sample of the culture supernatant of a reference strain used as an internal control. Non fractioned- Non-fractioned total culture supernatant sample collected in the early stationary phase. >50- Fraction with molecules above 50 kDa, 50-30 kDa- Fraction with molecules between 50 and 30 kDa, 30-10 kDa- Fraction with molecules between 30 and 10 kDa, 10-3 kDa- Fraction with molecules between 10 and 3 kDa, <3- Fraction with molecules below 3 kDa...... 31

Figure 10- Protein concentration, in g/L of culture medium, of the fractions recovered from the total IST01 culture supernatant collected in the early stationary phase. The assays were performed using fractions obtained from two independent fractionations of the same total IST01 sample from the early stationary phase...... 32

Figure 11-Susceptibility of A-E. coli ATCC 25922 and B- S. aureus NCTC 8325, to the total sample of IST01 culture supernatant of the early exponential phase and recovered reconstituted fractions after fractionation. The results are expressed in percentage (%) of growth, representing the increase/decrease of OD595, comparing with the OD595 of the positive controls (water and liquid culture of the bacterial target, in black). Grey- Non-fractioned IST01 culture supernatant collected in the early stationary phase, Blue- Fraction with molecules above 50 kDa, Orange- Fraction with molecules between 50 and 30 kDa, Green-Fraction with molecules between 30 and 10 kDa; Purple- Fraction with molecules between 10 and 3 kDa, Pink- Fraction with molecules below 3 kDa. The assays were performed using fractions from two independent fractionations, reconstituted to the original concentration of the total IST01 supernatant from the early stationary phase...... 33

Figure 12- Susceptibility, in % growth, of A- E. coli ATCC 25922, B- B. cenocepacia IST05, C- P. aeruginosa LES400, D-E. faecalis DSM 20478, E- L. monocytogenes CNCM-I 4031, F- S. aureus vii

NCTC 8325, G- S. aureus (MRSA) ATCC 33591 for the IST01 culture supernatants harvested along bacterial cultivation determined by comparision between the OD values obtained and those of the positive controls. The data represents the average of three independent experiments performed with supernatants harvested from three independent bacterial cultivations...... 52

Figure 13- Protein profile of the IST01 culture medium samples obtained at the mid exponential phase (3h), transition to the stationary phase (7h), early stationary phase (20h) and late stationary phase (30h) and separated by SDS-PAGE (Resolving gel with 12,5% acrylamide). On A, 20 μL of the sample was directed loaded into the gel wells, with 5 μL of loading buffer), without any treatment. On B, the sample was pretreated with acetone 100% (v/v) for protein precipitation and loaded into the gel wells. MW- Molecular weight marker, QC- Sample of the culture supernatant of a reference strain used as an internal control...... 58

Figure 14- Trypsin standard curve used as Quality Control for the proteolytic activity assessment of the IST01 culture supernatants...... 59

Figure 15- Glycerol standard curves used for the quantification of the lipase activity. A- Standard curve obtained after 2 minutes of incubation, at 37ºC. B- Standard curve obtained after 27 minutes of incubation at 37ºC ...... 60

Figure 16- Lipase activity, in Units/L of solution, of the culture supernatant samples of B. cenocepacia IST01, harvested at different phases of the growth curve. The IST01 culture medium samples, previously diluted to 600 mg/mL, were diluted to a fixed amount of protein (6 μg/mL), with lipase assay buffer and according to the previous quantification by the Bradford assay. The A595 values at Tinitial (after 2 minutes of incubation at 37ºC) and Tfinal (after 27 minutes of incubation) of each sample were compared with the glycerol standard curves obtained at these time points for determining the amount of glycerol formed due to lipase activity. 1 unit of lipase corresponds to the amount of enzyme that will form 1 μmole of glycerol from triglycerides, per minute, at 37ºC. The data represents the average of one experiment performed with supernatants harvested from two independent bacterial cultivations. 60

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Index of tables

Table 1- Times of cultivation and corresponding growth phases for the secretome extractions of IST01...... 14

Table 2- Conditions used for the freeze-drying of the supernatants...... 14

Table 3- Target bacterial strains used in the antimicrobial assays with the IST01 supernatants. In bold are the multidrug resistant strains in which only the supernatant of the early stationary phase, collected after 20h of cultivation, was tested...... 15

Table 4- Bovine Serum Albumin (BSA) standard curve solutions used, in μg/mL, for the Bradford assay...... 16

Table 5- Composition of the Running gel (12.5% acrylamide) used for the analysis of the IST01 culture supernatants...... 18

Table 6- Running gel solution composition (18% acrylamide) used for analysing the fractions of the Bcc culture supernatant...... 18

Table 7- Composition of the stacking gel (4%) used for SDS-PAGE...... 18

Table 8- Composition fof the running buffer 10 x (pH= 8.3) stock solution used for SDS-PAGE...... 19

Table 9- Trypsin standard curve solutions used for the proteolytic activity assay, in ng/μL...... 19

Table 10- Preparation of the reaction mixes used for the lipolytic activity assay...... 20

Table 11- Glycerol standard curve solutions prepared for for the lipolytic activity assay...... 20

Table 12- Minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC), in mg/mL, obtained for the culture supernatants collected at the four different phases of the growth curve of IST01, against E. coli ATCC 25922, B. cenocepacia IST05, P. aeruginosa LES400, E. faecalis DSM 20478, L. monocytogenes CNCM-I 4031, S. aureus NCTC 8325 and S. aureus (MRSA) ATCC 33591. Minimum inhibitory concentration was considered to be the concentration leading to a growth reduction of, at least, 90% (in OD595), comparing to the positive controls (bacterial culture without the tested sample). Minimum bactericidal concentration was considered to be the concentration leading to a viability reduction of, at least, 99,9% (in viability), comparing to the positive controls (bacterial culture previously incubated without the tested sample). The data represents the average of three independent experiments performed with supernatants harvested from three independent bacterial cultivations...... 25

Table 13- Yields obtained for the IST01 culture supernatants harvested along bacterial cultivation after freeze drying, in g of freeze dried product/mL of harvested liquid supernatant. Data represents the average of supernatants obtained from three independent bacterial cultivations ...... 49

Table 14- Viability , in % viability, of E. coli ATCC 25922 previously incubated with IST01 culture supernatants harvested along the growth curve, determined by subculture of the bacterial targets previously incubated with test concentrations of the samples and comparision between the CFU/mL obtained with those of the subcultured positive controls. The data represents the average of three independent experiments performed with supernatants harvested from three independent bacterial cultivations...... 53

Table 15- Viability of E. coli ATCC 25922 previously incubated with IST01 culture supernatants harvested along the growth curve, determined by subculture of the bacterial targets previously incubated with test concentrations of the samples and comparision between the CFU/mL obtained with those of the subcultured positive controls. The data represents the average of three independent experiments performed with supernatants harvested from three independent bacterial cultivations. .. 54

Table 16- Viability of L. monocytogenes CNCM-I 4031previously incubated with IST01 culture supernatants harvested along the growth curve, determined by subculture of the bacterial targets previously incubated with test concentrations of the samples and comparision between the CFU/mL obtained with those of the subcultured positive controls. The data represents the average of three independent experiments performed with supernatants harvested from three independent bacterial cultivations...... 55

Table 17- Viability of S. aureus NCTC 8325 previously incubated with IST01 culture supernatants harvested along the growth curve, determined by subculture of the bacterial targets previously incubated with test concentrations of the samples and comparision between the CFU/mL obtained with those of the subcultured positive controls. The data represents the average of three independent experiments performed with supernatants harvested from three independent bacterial cultivations. .. 56

Table 18- Viability of B. cenocepacia IST05, P. aeruginosa LES 400, S. aureus (MRSA) ATCC 33591 previously incubated with IST01 culture supernatants harvested at early stationary phase, determined by subculture of the bacterial targets previously incubated with test concentrations of the samples and comparision between the CFU/mL obtained with those of the subcultured positive controls. The data represents the average of three independent experiments performed with supernatants harvested from three independent bacterial cultivations...... 57

supernatant sample used for fractionation). The assays were performed using fractions from two independent fractionations ...... 59

Table of contents

Acknowledgements ...... i

Abstract...... iii

Resumo ...... iv

Abbreviations ...... v

Index of figures ...... vi

Index of tables ...... ix

Table of contents ...... xii

1. Introduction ...... 1

1.1. Thesis outline ...... 1

1.2. How to fight bacterial infections? ...... 3

1.3. Bacteria as a source of new antimicrobial molecules ...... 5

1.3.1. Quorum quenching molecules ...... 5

1.3.2. Antimicrobial peptides and bacteriocins ...... 6

1.4. Biotechnological potential of bacterial proteases and lipases ...... 7

1.5. Burkholderia cenocepacia ...... 7

1.5.1. Characteristics and virulence ...... 7

1.5.2. Biotechnological potential of Burkholderia ...... 10

2. Materials and Methods ...... 14

2.1. Bacterial isolates and culture conditions...... 14

2.2. Harvest of culture supernatant samples along bacterial cultivation ...... 14

2.3. Antibacterial activity of IST01 culture supernatant samples ...... 15

xii

2.4.Protein quantification B. cenocepacia IST01 culture supernatant samples ...... 16

2.5. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) ...... 16

2.5.1. Sample pretreatment ...... 16

2.5.2. Sample, gel preparation and electrophoresis procedure ...... 17

2.6. Proteolytic activity assessement of B. cenocepacia IST01 culture supernatant samples ...... 19

2.7. Lipolytic activity assessement B. cenocepacia IST01 culture supernatant samples ...... 20

2.8. Fractionation of B. cenocepacia IST01 culture supernatant samples ...... 20

3. Results ...... 23

3.1. Antimicrobial activity of the Burkholderia cenocepacia IST01 culture supernatants harvested during cultivation ...... 23

3.2. Protein quantification and profile of B. cenocepacia IST01 culture supernatants harvested during cultivation ...... 25

3.3. Quantification of the proteolytic activity of IST01 culture supernatants harvested along cultivation ...... 27

3.4. Characterization of the fractions of the B. cenocepacia IST01 supernatant collected on the early stationary phase ...... 29

3.4.1. Characterization of the composition, according to the different molecular weights, of the B. cenocepacia IST01 supernatant collected on the early stationary phase ...... 29

3.4.2. Characterization of the protein profile of the fractions of the B. cenocepacia IST01 supernatants sample collected on the early stationary phase ...... 31

3.4.3. Characterization of the protein concentration of the B. cenocepacia IST01 supernatants collected on the early stationary phase ...... 32

3.4.4. Characterization of the antimicrobial activity of the B. cenocepacia IST01 secretome fractions collected on the early stationary phase ...... 32

4. Discussion ...... 34

5. Future works ...... 37 xiii

References ...... 38

Annexes ...... 49

Annex I ...... 49

Annex II ...... 50

Annex III ...... 53

Annex IV...... 58

Annex V...... 59

Annex VI...... 59

Annex VII...... 60

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1. Introduction

1.1. Thesis outline

The Burkholderia cepacia complex group (Bcc) is known to include several pathogenic bacteria for plants, animals and vulnerable humans, such as cystic fibrosis (CF) patients. However, the Burkholderia genus has been gaining an increasing relevance also due to their biotechnological potential, with some members inclusively registered for commercial use.

The BSRG/IBB of IST, in collaboration with the major portuguese Cystic Fibrosis Center of Hospital de Santa Maria, has been carrying out, for the past 20 years, systematic epidemiological surveys of Bcc bacteria in respiratory infections of CF patients. Additionally, several phenotypic, genomics, transcriptomics and metabolomics studies have been performed with sequential Bcc isolates retrieved during chronic infections, giving an important contribution to further understand the adaptative strategies acquired by Bcc bacteria during long-term chronic infections.

In a previous study, secretomes from liquid cultures of Burkholderia cenocepacia clinical isolates, recovered from respiratory secretions of the same CF patient and widely studied by the IBB/BSRG group and two environmental B. cenocepacia and B. dolosa strains from the LMG culture collection (Laboratory of Microbiology, Ghent University) were harvested, processed, by freeze-drying, followed by resuspension in water and tested, using broth microdilution assays, against reference Gram- negative (Escherichia. coli ATCC 25922) and Gram-positive (Staphylococcus aureus ATCC 33591 and Enterococcus faecalis DSM 20478) bacterial strains. Most secretomes were able to inhibit the bacterial growth of all the bacterial strains tested leading to a decrease of the specific growth rate and final biomass obtained in a dose dependent manner. Particularly, the secretomes obtained from B. cenocepacia IST01 stood out as the most effective with the strongest antibacterial activity. Acorrelation between such AM activity and N- acyl homoserine lactones (AHL) production could not be established. Moreover, it was not possible to define whether the secretomes had a bactericidal or bacteriostatic activity on the bacterial strains used as targets for the assays.

Therefore, the goals for this thesis were to:

1- Evaluate the potential of B. cenocepacia IST01 as producer of molecules with antibacterial, lipolytic and proteolytic activities, relevant in industrial settings.

2- Characterization of IST01 cell culture supernatants by fractionation and analysis of protein profile and antibacterial activity of the subfractions

In the present study, B. cenocepacia IST01 culture supernatants were harvested at different phases of the growth curve, grown in LB medium at 37ºC, processed by freeze-drying and tested for antimicrobial activity against Escherichia coli ATCC 25922, Enterococcus faecalis DSM 20478, 1

Staphylococcus aureus NCTC 8325 and Listeria monocytogenes CNCM-I 4031, by broth microdilution assays followed by subculture in agar plates without the test supernatant, according to the CLSI guidelines, in order to calculate the minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) were calculated, unveiling which are the most effective secretomes to be eventually used as antibacterial agents. The secretomes obtained under the optimal conditions were tested, following the same methodology, against multidrug resistant bacteria: Burkholderia cenocepacia IST05, Pseudomonas aeruginosa LES400 and Methicillin-resistant Staphylococcus aureus ATCC 33591 attesting an antibacterial effect on those.

The secretomes’ protein content was determined using the Bradford method in 96-well microtiter plates, but no correlation was observed between protein levels and antimicrobial activity. Furthermore, SDS-PAGE after sample dialysis was performed and protein profile of the secretomes was successfully obtained discriminating secretome samples according to the bacterial growth phase in which the samples were collected.

Considering the high biotechnological interest in lipases and proteases, such as for food and detergent industries and the fact that the expression of enzymes with lipase and protease activitie are part of the multiple virulence traits exhibited by Bcc bacteria, that could additionally be contributing for the antibacterial activity of the samples, the IST01 secretomes were also assessed for protease and lipase activities using commercial kits (Pierce™ fluorescent protease kit, from Thermo Scientific and lipase assay kit, from Sigma Aldrich) in 96-well-microtiter plates. Protease activity could be detected but results for lipase activity were not conclusive and are therefore not shown. Additionally, in order to identify in which fraction(s) of the secretome the active compunds was present, the fractionation of the IST01 cell culture supernatant, produced in optimal bacterial growth conditions, was performed. Molecular weight cut-offs (MWCO) of 50 kDa, 30 kDa, 10 kDa and 3 kDa were selected, allowing the recovery of five fractions, with molecules above 50 kDa, between 50 and 30 kDa, between 30 and 10 kDa, between 10 and 3 kDa and below 3 kDa, that accounted for over 78% of the total secretome. Afterwards, the individual fractions obtained were tested against a gram-negative and a Gram-positive bacterial target (Escherichia coli ATCC 25922 and Staphylococcus aureus NCTC 8325) for antibacterial activity, using broth microdilution assays and analysed for protein quantification, using the same approach and protein profile analysis, by SDS-PAGE, with a resolving gel with 18% acrylamide. The method used allowed to successfully isolate a fraction below 3 kDa responsible for the antimicrobial activity and thought to be composed of peptides, secondary metabolites and small molecular weight molecules produced and secreted by IST01 during bacterial growth.

To conclude, the results obtained are encouraging for the pursuit of further studies on the characterization of the IST01 cell culture supernatants, in particular, the fraction below 3 kDa and for optimization envisaging the production of a more active secretome combination, as antibacterial and proteolytic compounds.

1.2. How to fight bacterial infections?

Bacteria predate humans for millions of years, which enabled them to develop complex mechanisms that have allowed their survival under harsh conditions, where toxic metabolites could be present. Additionally, these mechanisms can be responsible for their virulence and ability to cause infectious diseases, which, in order to be controlled, require antimicrobial agents.

These affect the growth of microorganisms either by inhibiting their growth, without killing them (bacteriostatic) or by killing them (bactericidal and lytic)1. Antibiotics can be synthetically or naturally produced, and even be further modified artificially to enhance their activity. In spite of the majority of known antibiotics, due to high toxicity and deficit uptake by host cells, not being used clinically, the few useful ones effective in clinical settings have had a major impact on the treatment of infectious diseases.

Concerning their mechanisms of action, they can target several cellular processes, structures and enzymes crucial for bacterial survival, namely the nucleic acid synthesis, by either affecting topoisomerase (quinolones)2, the DNA-dependent transcription (rifampicins)3, the cell wall synthesis (β-lactams and glycopeptides)4,5, the folic acid metabolism (sulfonamides and trimethroprim) required for the synthesis of several important molecules such as aminoacids or nucleic acids6or RNA polymerases (rifamycins)7 and the protein synthesis, by interacting with the major and minor ribosomal subunits8–12.

Furthermore, depending on the group of microorganisms susceptible to these agents, each antimicrobial drug can in addition be classfied as broad-spectrum, intermediate-spectrum, or narrow- spectrum, affecting a limited and well defined group of microorganisms. For example, isoniazid, is an antimicrobial drug specifically active against Mycobacterium but ineffective against other microorganisms13. It should be noted that the spectra of activity may also vary due to the aquisition of resistance genes by the target bacteria. The great majority of the antibiotics discovered until today are naturally produced by bacteria as secondary metabolites, and in general ensure survival functions of the producing cell, as they may be used, for example, as competitive weapons against susceptible competitive neighbours14.

1.2.1. The problem of antibiotic resistance: Mechanistic and genetic basis

When antibiotics were introduced in 1911, the evolution of resistance towards them was thought to be unlikely, as it was assumed then that the frequency of mutations that could lead to resistant bacteria was negligible. However, time has proved the opposite, and the heavy use of antibiotics in the last decades to control infections in medicine and agriculture, has created the perfect conditions for the mobilization of resistance elements within bacterial populations and, as a consequence, their capture by previously antibiotic susceptible pathogens and selection of antimicrobial resistant bacteria 15.

1.2.1.1 Genetic basis

From an evolutionary point of view, two genetic mechanisms can be used by bacteria to adapt to the presence of an antibiotic. The most frequent occurs through horizontal gene transfer (HGT), in which there is an uptake of exogenous DNA, that can occur from one similar organism to another, but also between different species, consisting in a way to circumvent the usual parent-to-progeny vertical route of genetic flow16. Besides chromosomal genes, mobile genetic elements (MGE) can also act as vectors where antibiotic resistance genes are most frequently found17. Alternatively, antibiotic resistance can arise spontaneously from sequential mutations in the chromosomes, as observed for the acquisition of resistance to fluoroquinolones in some E. coli strains18,19.

1.2.1.2. Mechanistic basis

Bacteria may reflect resistance to antibiotics through several mechanisms that typically fall into three categories:

Efflux/changes in permeability

The antimicrobial access to the target may be reduced by the overexpression of membrane proteins, that can act as export or efflux pumps, as observed for P. aeruginosa, in which multidrug resistance is for the most part due to the acquisition of genes encoding for multidrug resistance, such as MexAB- OmpM. Another mechanism, well establish in gram negative bacteria, consists in the reduction of permeability, that can be achieved for example, by downregulating the expression of porins, limiting the entry of the antibacterial agent into the bacterial cell20–22.

Destroying the antibiotic

By modifying the permeability of the cell, even though the internal accumulation of the antibiotic is prevented, its integrity remains unchanged. Thus, another resistance strategy involves the destruction of the antibiotic core structure itself which is known, for example, to be responsible for the resistance to β-lactamic drugs. 23 On the other hand, if the antibiotic core structure is not that easily hydrolysed, it can be alternatively altered with chemical substituents leading to disruption of its binding affinity 23 .

Changing the structure of the target

Moreover, resistance can also be achieved by a change or camouflage of its target. Such mechanism is widely known to occur in Staphylococcus aureus, that is able to express genes encoding for alternative targets that reduce the impact of the antibiotic, determining, for instance, resistance to methicilin, and creating the widely spreaded phenotype of methicillin-resistant Staphylococcus aureus (MRSA)24.

1.3. Bacteria as a source of new antimicrobial molecules

The fact that the problem of drug resistance has been increasing among important bacterial pathogens has been putting a lot of pressure and recurrent need towards the development of new molecules with antibiotic properties25.

On one hand, the production and development of synthetic molecules, such as fluoroquinolones26, sulfonamides27 and oxazolidinones28 has been very important and should be pursued, in order to design new structures that could aim at different bacterial targets, besides the ones already identified, not only by changing its shape and linkers with different configurations, lenghts and polarities, but also modifying surface functional groups that allow its rapid elaboration. However, the optimization process of turning the molecules into drugs that could work inside the host is very difficult, costly and long.

On the other hand, throughout history, microorganisms themselves have been the richest source of antibiotics, as seen for example, for penicillins29, cephalosporins30, vancomycin31, tetracyclin8 and aminoglycosides.32. Nowadays, renewed increasing attention has been paid to natural strategies used by prokaryotes against neighbour microorganisms, that are going to be addressed below.

1.3.1. Quorum quenching molecules

Quorum sensing (QS) consists of a cell-to-cell comunication system, based on the synthesis and secretion of diffusible signal molecules, such as N-acyl homoserine lactones, that once sensed above a certain threshold, eventually lead to the induction or repression of target genes, depending on the concentration of the signaling molecules and being, therefore, dependent on cell density33,34. For pathogens, this system is widely known to occur for sensing its population density and synchronize the expression of virulence genes35. The rapid increase of knowledge on this field has also led to the discovery of naturally occuring mechanisms able to interfere effectively with quorum sensing, generally known as quorum-quenching (QQ). These are known to play an important role for microbe- microbe and pathogen-host interactions and have been identified in many gram-negative and Gram- positive microorganisms36. The inhibition of quorum sensing can be accomplished in several ways, including enzymatic degradation of signaling molecules, signal generation blockage and signal reception blockage. Considering the type of inhibitors, they can be divided in two groups, depending on their functions and structures. The first consists on molecules that are structurally similar to quorum sensing signaling molecules, such as halogenated furanones and synthetic autoinducing peptides37. The other group comprises the so-called quorum quenching enzymes, already identified in quorum sensing and non-quorum sensing organisms, with several purposes such as metabolization of QS molecules for cell growth, as already reported for Pseudomonas aeruginosa strains, or blocking unnecessary gene expression and pathogenic phenotypes, acting as a defense mechanism37–40.

Currently, the quorum quenching has been proposed as a promising growth control therapy gaining interest for both biotechnology and research, as several tests using quorum quenching therapies have 5

shown encouraging results41. Even though, in a number of cases, the use of QQ molecules was not enough to decrease completely the QS activity, some studies have shown that a combination of QS strategies with other antibiotic treatments have led to a significant reduction of bacterial susceptibility to the antibiotic42.

Considering that it is more limited in terms of selective pressure than antibiotics, it could represent a solution for virulence reduction of quorum sensing microbes without resulting in antimicrobial resistance33.

1.3.2. Antimicrobial peptides and bacteriocins

Antimicrobial peptides (AMPs) are oligopeptides, composed by a varying number of aminoacids, that can range from 5 to over 100, and with a broad spectrum antimicrobial activity against fungi, viruses and bacteria43. Since the identification of the first AMP from the fractionation of an extract of a Bacillus strain, initially reported to have antimicrobial activity, thousands of AMPs have been discovered so far44.

Natural AMPs are known to occur in both eukaryotes and prokaryotes, suggesting that their role has been long standing and must have contributed to an organism’s fitness.

The most studied AMPs to date are the antibacterial AMP which target the bacterial cell membrane causing the disruption of the lipid bilayer structure45. However, some studies also reported, at lower concentrations, the ability of AMPs to kill bacteria by inhibiting important cell pathways such as DNA replication and protein synthesis46.

A special interest has been focused in the study of antimicrobial peptides produced by bacteria, categorized as bacteriocins. These AMPs are known to be remarkably diverse and can have a narrower spectrum, confining its antibacterial activity to closely related species, or a broad spectrum of activity, including many different bacterial species, functioning as a strategy to maintain population and reduce competitors in the natural environment47,48. Currently, bacteriocins produced by lactic-acid bacteria are recognized as non toxic and have been used for food science in order to extend the duration of food preservation49. However, other potential applications in pharmaceuticals have been recognised due to their potent bactericidal activity against important animal and plant pathogens such as Staphylococcus aureus (MRSA) and P.aeruginosa, which are of a major concern nowadays due to their known multidrug resistance50,51 .

These studies, combined with their successful use as food preservative, showcase the potential of bacteriocins already studied and others, still undiscovered, that may hold promise for their use as antibiotics.

1.4. Biotechnological potential of bacterial proteases and lipases

Nowadays, proteases and lipases are known to constitute an important and relevant group of enzymes for biotechnology and commercial purposes, being used in several industries, namely food, detergent and pharmaceutical industries.

Even though enzymes with proteolytic and lipolytic activities are mostly produced by animals and plants, microorganisms are considered much more attractive as producers of these enzymes due to their biochemical diversity, scientific and economic advantages52.

Microbial proteases can be either intracellular or extracellular and it is widely known that their production is strongly influenced by nutricional and physicochemical conditions such as pH, nitrogen and carbon sources53,54. Most commercial proteases are neutral or alkaline and produced by Bacillus strains. Regarding the neutral proteases, due to their low thermotolerance and ease of reactivity control, they are used for the production of food hydrolysates with a low degree of hydrolysis while alkaline proteases, due to their stability in alkaline pHs, are more suitable for use in the detergent industry55. In addition to proteases, enzymes with lipase activity have been also attracting more attention being now considered the most important group of biocatalysts for biotechnological applications. Lipases can, not only hydrolyze carboxylic ester bonds, but also catalyse esterification, transesterification and interesterification reactions giving them a high versatility for a multitude of industrial applications such as in the detergent, pharmaceutical, leather, textile, paper and food industries56–58. For food applications, lipases from yeasts or other fungi are prefered due to their GRAS-status (Generally Regarded as Safe)56. However, extracellular lipases from species of Pseudomonas are currently available for detergent applications56. Taking into account the promising and already successful use in industry, the continuous study and commercialization of bacterial proteases and lipases is encouraging.

1.5. Burkholderia cenocepacia

1.5.1. Characteristics and virulence

Burkholderia cenocepacia is a member of a bacterial group refered to as the Burkholderia cepacia complex (Bcc). Bcc bacteria are incredibly diverse, residing in soil, plant rhizosphere, water and can also be a plant and human pathogen, well known for causing chronic infections in patients with underlying vulnerability, being particularly problematic for cystic fibrosis (CF) patients.59 There are several Bcc species that can be transmissible from one patient to another, leading to epidemic

outbrakes. However, B. cenocepacia and B. multivorans are the most predominant, being found in a higher percentage, accounting for 85% to 97% of Bcc infections60.

Usually, these bacteria colonize humans for a long period of time and lead to a more rapid decline in lung function or, in some cases, to the development of a fatal necrotizing pneumonia accompanied by septicemia, the so-called “cepacia-syndrome”61.

Even though the impact can be minimized by therapeutic options, these bacteria possess a remarkable genome plasticity and a multitude of mechanisms that allow them, not only to become resistant to the most clinically used antibiotics, making virtually impossible the irradication from the CF lung, but also to adapt to the highly stressful environment that is the respiratory tract62,60,63. Some mechanisms of virulence and adaptation are described herein:

1.5.1.1. Genomic islands

Generally, Burkholderia species are known to have some of the most complex bacterial genomes. A complete genome analysis of a B. cenocepacia strain, J2315, isolated from a CF patient, reported 14 genomic islands that were absent from other B. cenocepacia strains such as the B. cenocepacia island (cci), that encodes for multiple genes, being the majority associated to accessory functions in quorum sensing, lipid biosynthesis, transcription regulation and aminoacid transport. Additionally, the Island may play a role in virulance enhancement in B. cenocepacia64.

1.5.1.2. Quorum sensing

In Bcc bacteria, the quorum sensing system is composed of an acyl homoserine lactone (AHL) synthase and an AHL receptor responsive to acyl homoserine lactones. Two complete AHL QS systems have been already reported in B. cenocepacia J2315: The CepIR, present in all Burkholderia strains, and the CciR, only present in pathogenic strains, with the cci64. These global regulators are known for controlling the expression of genes involved in siderophore biosynthesis, biofilm formation, production of extracelular enzymes, among others65,66.

Additionally, the presence of a gene encoding for a regulator but not paired with a synthase (CepR2) and the Burkholderia Diffusible Signal Factor-Based system (RpfFBC) were found in B. cenocepacia strains67,68.

1.5.1.3. Secreted proteins

In general, bacteria are able to produce a great variety of enzymes, among which proteases and lipases are largely represented and some have shown to play an important role in pathogenesis in several bacterial species69,70,71.

Considering proteases, in B. cenocepacia, the capability to secrete the metalloproteases ZmpA and ZmpB has been described 72,73. Previous studies have reported that both these enzymes can cleave fibronectin and collagen, causing tissue damage. Additionally, their ability to specifically cleave proteinase inhibitors known to play a role in the modulation of host defences was also recognized 72. Moreover, the capacity to acquire iron from heme and ferrin by the secretion of the siderophores ornibactin and pyochelin is widely known in B. cenocepacia. However, alternatively, proteolytic degradation in order to acquire sequestered iron can occur, additionally contributing for the colonization and persistence in the CF lung74.

Additionally, secreted lipases have also been reported to be produced by B. cenocepacia and to play a role in invasion. In a study carried out by Mullen et al, pretreatment of epithelial cells with lipases resulted in an increase of invasion while a decrease was observed after pretreatment of a B. cenocepacia strain with a lipase inhibitor75, 76.

1.5.1.4. Phenotypic variation

Chronic lung infection is the leading cause of early death due to tissue deterioration and rapid decline in lung function. During long-term colonization, Bcc bacteria experience a highly fluctuating and stressful environment within the respiratory tract resulting from the immune system of the host, antimicrobial therapy and reduced availability of nutrients77–79.

With the aim to further understand the adaptive strategies acquired by Bcc bacteria during long-term chronic infections, in collaboration with the CF center of the Hospital of Santa Maria, in Lisbon, several systematic and active studies involving not only epidemiological studies but also phenotypic, genotypic and genome-wide expression analysis of sequencial Bcc isolates retrieved during chronic infection, have been carried out by the BSRG/IBB of IST and giving an important contribution to this field.

In this context, a case-study widely studied by our group was the collection of 11 isolates, retrieved from the respiratory secretions of Patient J, chronically colonized with the same B. cenocepacia strain for over 3.5 years until death from “cepacia syndrome”.

Characterization studies of these isolates, considering the phenotypic traits, including antimicrobial susceptibility, cell motility and hydrophobicity, colony and cell morphology, growth under iron limitation/load, exopolysaccharide production, among others, were performed and a significant variance was observed between the isolates believed to have initiated the infection and those collected during later stages of the course of infection. Among other differences, IST4113, retrieved over 3 years after exacerbated infection and intravenous therapy, reported to be much more resistant than IST439, the first isolate collected and thought to have initiated the infection, also reported for other Bcc isolates collected from other patients, pointing out to the role of the antibiotic stress to the emergence of resistant populations80,81. In the same study, other mechanisms of adapted strategy were reported for the first time, including the alteration of membrane fatty acid composition81. 9

Moreover, transcriptomics studies also performed in this context and comparing the two same isolates, reported over 1000 genes that were differently expressed evidencing a pronounced reprogramming of genomic expression during chronic infection. Among them were genes involved in translation, iron uptake, central carbon metabolism and energy production79. These differences were also observed in metabolomics and proteomics studies, that also reported the higher virulence potential of the late isolates, comparing with IST439 as they exhibited a higher ability to invade epithelial cells and affect epithelial monolayer 78,82.

Lastly, another important virulence determinant in gram negative bacteria is the lipopolysaccharide (LPS). Particularly, in Bcc bacteria, the LPS has a potent endotoxic activity eliciting high levels of pro inflammatory cytokines such as tumour necrosis factor, inteleukin-6, interleukin-8, among others83. In studies performed using dendritic cells, that aimed to disclose how Bcc bacteria subvert the immune system observed that the late variants IST4113 and IST4134, retrieved right before the patient’s death with cepacia syndrome and that, in recent studies, reveiled to lack the LPS O-antigen, were much more internalized than IST439, in which the OAg was present, pointing out to the idea that the loss of the OAg consists in an additional mechanism of adaptative strategy during long-term respiratory infections84,85.

To conclude, these studies emphasize the need of adaptation to the host environment in order to develop chronic lung infections.

1.5.2. Biotechnological potential of Burkholderia

In spite of the known pathogenicity of some members of the genus Burkholderia, several strains have been used for many biotechnological applications, summarized on figure 1.

Figure 1- Beneficial and harmful effects of Burkholderia cepacia complex bacteria

1.5.2.1. Antimicrobial agents

Antimicrobial agents recovered from bacteria are usually secondary metabolites and its production is controlled by several factors, such as pH, composition of the growth media and temperature86.

Several Burkholderia species have been reported to be able to synthesize a great variety of antimicrobial agents from antifungals to antibiotics, with reported activity against multiresistant pathogens87.

Among known metabolites produced by Burkholderia with antifungal properties are pyrrolnitrin, xylocandins, cepafungins/glidobactins, altericidins, cepacines, occidiofungins, cepaciamides, phenazines and quinolone derivatives 87.

Although mostly the antifungal properties in Burkholderia have been widely explored throughout history, the ability of producing antibacterial agents by these bacteria has been reported. A study performed, that involved 268 Bcc isolates, observed that B. ambifaria isolates were able to inhibit several bacterial strains, such as B. multivorans Glasgow CF strain, A. Baumanii OXA23 clone 2 and B. dolosa Boston CF strain. The authors also performed gene expression analysis which revealed the 11

presence,within the genome of B. ambifaria AMMD, of a cluster of several genes for the synthesis of the polyene enacycloxin IIa, that targets elongation factor Tu. Additionally, it was found, within this cluster, the presence of two orphan luxR-type homologues suggesting the important role of QS in regulation of the biosynthesis of this antibiotic88.

Moreover, for agricultural purposes, certain Bcc strains are registered for commercial use due to their potencial for crop protection against many fungal diseases, such as root rot, caused A. Euteiches or the known “damping off” seed-damaging fungal infection due to colonization by Rhizoctonia solani and Phytum species89,90.

Pyrrolnitrin, a well known molecule with potent antifungal and antibacterial activities against Candida species and Gram-positive bacteria, has been reported to be under control of the CepI/R QS system and mutations in CepI /CepR led to the loss of antimicrobial activity. However, this activity was able to be restored with the addition of exogenous AHL’s in CepI mutants91.

Similarly, mutant studies with B. ambifaria HSJ1 allowed the identification of over 20 QS controlled genes, mainly related to the production of metabolites with antifungal and antimicrobial properties such as burkholdines and pyrrolnitrin92.

Additionally, in a recent study performed in our group, Bcc secretomes, collected from cultures of environmental and clinical isolates were explored as producers of secretomes with antimicrobial activity using Escherichia coli ATCC 25922, Enterococcus faecalis DSM 20478 and Staphylococcus aureus ATCC 33591 as target bacteria and observed that the majority of the secretomes were able to inhibit the bacterial growth of both Gram-positive and gram-negative bacteria decreasing the maximum specific growth rate and final biomass obtained in a dose specific manner. In particular, B. cenocepacia IST01 secretomes were the ones in which the strongest antibacterial activity was reported but was not identical for all the species tested providing the idea that Bcc bacteria could be a potential producer of antibacterials93.

1.5.2.2. Other applications

In the Burkholderia genus, the capability to degrade complex carbon sources, typically found in pesticides and herbicides, such as chlorinated aromatic compounds and groundwater pollutants has been shown. One example is the Burkholderia vietnamensis strain G4, which is able to co- metabolically degrade TCE, in the presence of inducers, such as toluene or phenol94,95.

Additionally, several species of Burkholderia are known to be phytopathogenic. Nevertheless, others, such as B. unamae, can carry out beneficial interactions with plants, existing in plant rhizosphere, functioning as plant endophytes or even as microsymbionts of legume root nodules, becoming attractive replacements of pesticides, herbicides and fertilizers, in which health hazards are already recognized96,97,98 .

Considering Burkholderia is able to produce lipases, with high stability to heat and organic solvents, an interest in these bacteria for biofuel production has been increasing. A study that aimed to test the stability of Burkholderia cepacia lipase (BCL) towards methanol, revealed the capability of this enzyme to maintain its transterification activity at high methanol concentrations99. In order to make them more stable and attractive for industrial use, several methods for BCL immobilization have been developed, which emphasize the potential of BCL for biofuel industry100,101.

2. Materials and Methods

2.1. Bacterial isolates and culture conditions

The Bcc isolate used, Burkholderia cenocepacia IST01, was obtained at the Hospital of Santa Maria, in January 1999, from the respiratory secretions of a chronically infected CF patient, patient J, that was infected with the same B. cenocepacia (recA lineage III-A) for over 3.5 years until death by “cepacia syndrome” and belongs to a collection of 11 sequential isolates widely studied and characterized by the IBB research group of Instituto Superior Técnico. Bacterial cultures were stored in -80ºC in 1:1(vol/vol) glycerol and, when needed, bacteria were grown in pseudomonas isolation agar(Difco) plates. IST01 growth was performed in shake flasks, with Luria-Bertani (LB) liquid medium, at 37ºC, with orbital agitation (250 rpm), at an initial OD640 of 0.05.

2.2. Harvest of culture supernatant samples along bacterial cultivation

Several extractions were performed, along the growth curve of IST01, in order to assess the best phase of the growth curve of IST01 to obtain secretomes more efficient as antibacterials (table 1).

Table 1- Times of cultivation and corresponding growth phases for the secretome extractions of IST01.

Time of growth for the extraction Growth phase

3h Mid exponential

7h Transition to the stationary phase

20h Early stationary

30h Late stationary

At the time-points chosen for the extraction, the culture was centrifuged at 4ºC, for 22 minutes and the culture free supernatant obtained was filtered and preserved at -80ºC, in 400 mL plastic cups, until freeze-drying.

For the lyophilization of the samples, two cycles were employed with a total duration of 120h (4 days) each, in the conditions described at table 2.

Table 2- Conditions used for the freeze-drying of the supernatants.

Temperature: -25ºC 96h Shelves Temperature: 25ºC 24h

Pressure: 0.01 hPa Chamber Temperature: -50ºC

The lyophilized culture supernatants, in powder, were preserved at -20ºC and resupended to a given final concentration for the antimicrobial assays.

2.3. Antibacterial activity of IST01 culture supernatant samples

For the minimum inhibitory concentrations (MIC) determination, broth microdilution assays were performed. Liquid cultures of the bacterial species, described on table 3, previously kept at -80ºC, in 3:1 glycerol (vol/vol), were grown in liquid media, at 37ºC, with orbital agitation, until mid exponential phase and added to a polystyrene 96-well microtiter plate, with 50 μL of secretome solution previously resuspended in water, to give a final inocculum of 106 colony forming units (CFU) in each well. The assay was performed according to the CLSI guidelines102.

Species Isolate Origin Culture media Reference

Clinical Burkholderia Luria Bertani 93 IST05 isolate of a cenocepacia (LB) CF patient

Escherichia coli ATCC 25922 Clinical isolate Luria Bertani (LB) 103

Enterococcus faecalis DSM 20478 - Luria Bertani (LB) 104

Brain Heart Listeria CNCM-I 4031 - Infusion Broth 105 monocytogenes (BHI) Clinical Pseudomonas Luria Bertani 106 LES400 isolate of a aeruginosa (LB) CF patient

Staphylococcus Luria Bertani 107 ATCC 33591 - aureus (LB) Clinical isolate Staphylococcus NCTC 8325 of a sepsis Luria Bertani (LB) 108 aureus patient

Serial dilutions of the test supernatant samples were performed in water, as well as positive controls, prepared with water and strain, and negative controls, with water or the diluted supernatant in the tested concentration, with media. The microplates were incubated for 16h, at 37ºC and the OD595 values of the cultures in the wells were measured in a FLUOstar omega microplate reader (BMG LABTECH). The MIC values are the minimum concentrations in which a growth reduction of at least

90% (in OD595) was observed, comparing with the positive controls.

For determining the minimum bactericidal concentrations (MBC), 20 μL of each culture previously cultivated with inhibitory concentrations and half the minimum inhibitory concentration of the tested

supernatant, as well as positive and negative controls, were diluted in 180 μL of liquid media. Serial dilutions were performed and 5μL of each well were placed on agar plates, that were incubated at 37ºC, for 16h. The MBC was considered the minimum concentrations in which a reduction of cell viability, in CFU, of at least 99,9% was observed comparing with the positive controls.

2.4.Protein quantification B. cenocepacia IST01 culture supernatant samples

Protein quantification in the several secretomes was determined through the Bradford method, using bovine serum albumin (BSA) as standard curve. The standard curve solutions were prepared from an initial stock solution of BSA (Albumin fraction, Merck) at 500 μg/mL, according to the table 4. IST01 culture supernatants were resuspended with water to a final concentration of 300 g/L. In order for the samples to be within the BSA standard curve range, 70 μL of each sample was further diluted in 70 μL of water and 20 μL of each was added to each well on a 96-well microtiter plate with 180 μL of Bradford reagent (VWR AMRESCO, proteomics grade). or the standard curve, 20 μL of each BSA solution was added with 180 μL of Bradford reagent. After agitation at 200 rpm, for 30s, followed by incubation in the dark at room temperature, for 30 minutes, the absorbance was read at 595nm, in a FLUOstar omega microplate reader (BMG LABTECH)

Table 4- Bovine Serum Albumin (BSA) standard curve solutions used, in μg/mL, for the Bradford assay.

BSA (µg/mL) 0 15 30 45 60 75 90 BSA solution 1x at 500 0 60 120 180 240 300 360 µg/mL Water (µL) 2000 1940 1880 1820 1760 1700 1640

2.5. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

In order to analyse the protein profile of the supernatant samples, SDS-PAGE was performed, according to the following steps:

2.5.1. Sample pretreatment

For the preparation of the supernatant solutions to be analysed, samples of lyophilized supernatants, along with a sample of freeze dried LB, were previously diluted in water to 300 g/L and, in some cases, further subjected to pre-treatment (protein precipitation or dialysis) as described below.

2.5.1.1. Protein precipitation

In order to remove impurities and to concentrate the proteins in the IST01 culture supernatant samples, protein precipitation was performed with acetone. A volume of 2 mL of acetone 100% (v/v) 16

was added to each aliquote of IST01 supernatant sample with a volume contaning a fixed amount of protein (4 μg), according to a previous quantification by the Bradford assay and conserved at -20ºC overnight. After centrifugation (10000 rpm, 10 min, 4ºC) the acetone was carefully removed and discarded whereas the pellet was let to dry for 20 minutes.

2.5.1.2. Dialysis

For the removal of salts and other molecules that may interfere with the SDS-PAGE analysis, dialysis was performed, using D-Tube TM dialysers (Novagen®).

Each tube was firstly filled with 1 mL of H2O and let to rest for 10 min for membrane equilibration.

IST01 lyophilized supernatants, along with a sample of lyophilized LB broth, were diluted in H2O to a final concentration of 300 g/L and let to rest for one hour. Afterwards, each dialyser was filled with 800

µL of sample and put on an individual floating rack inside a beaker with 2 L of ultrapure H2O, that was placed at 4ºC, for one hour. Then, the content was mixed, for 5 minutes, in a magnetic stirrer, and the beakers were placed again at 4ºC for one more hour. Later, 400 μL of sample was removed and stored at -20ºC, the water was discarded and the beakers were refilled with H2O, for additional 2h of dialysis, which ended with the removal of 400 μL of each sample and conserved at -20ºC for the SDS- PAGE analysis.

2.5.2. Sample, gel preparation and electrophoresis procedure

Different conditions were chosen for sample preparation depending if the samples were loaded in the gel with a fixed volume or with a volume fulfilling a fixed amount of protein (4 μg). For the SDS-PAGE gels performed with a fixed amount of volume, 20 μL of each sample was mixed with 5 μL of loading buffer (NZYtech) followed by incubation at 95ºC, without shaking, for 5 minutes and loaded in each well. Furthermore, for the SDS-PAGE gels performed with a fixed amount of protein, the samples were mixed with 20 μL of loading buffer and then incubated at 95ºC, for 10 minutes, with shaking and added to each well.

The running and stacking gels used were prepared according to the table 5 and 7. After the addition of the reagents, the running gel solution (12.5% acrylamide) was added into the gel cast, followed by distilled H2O, to allow polymerization, for 20 minutes. Afterwards, the H2O was removed and the stacking gel solution (4% acrylamide) was added, along with the comb caster. After 20 minutes of polymerization, the samples were added to each well, along with an internal control sample (culture supernatant of a reference strain) and 5 μL of molecular weight marker (NZYtech MB17602). Afterwards, the electrophoresis was performed at 80 V until completion, with 1x running buffer, previously prepared from a 10 x stock solution, with composition at table 8.

After finishing, the gels were placed in a container with 20 mL of BlueSafe staining solution (NZYtech MB15201), for 1h, and the photos were taken after destaining with distilled water.

Table 5- Composition of the Running gel (12.5% acrylamide) used for the analysis of the IST01 culture supernatants.

Reagent Volume per gel

H2O 2.108 mL

Tris HCl 1.5 M pH=8.8 1.25 mL

40% (w/v) Bis Acrylamide (NZYtech MB15601) 1.57 mL

10% (w/v) SDS 50 µL

10% (w/v) APS 25 µL

TEMED (NZYtech) 10 µL

Table 6- Running gel solution composition (18% acrylamide) used for analysing the fractions of the Bcc culture supernatant.

Reagent Volume per gel

H2O 1.415 mL

Tris HCl 1.5 M pH=8.8 1.25 mL

40% Bis Acrylamide (NZYtech MB15601) 2.25 mL

10% SDS 50 µL

10% APS 25 µL

TEMED (NZYtech) 10 µL

Table 7- Composition of the stacking gel (4%) used for SDS-PAGE.

Reagent Volume per gel

H2O 1.585 mL

Tris HCl 0.5 M pH=6.8 625 µL

40% (w/v) Bis Acrylamide (NZYtech MB15601) 250 µL

10% (w/v) SDS 25 µL

10% (w/v) APS 12.5 µL

TEMED (NZYtech) 10 µL

Table 8- Composition fof the running buffer 10 x (pH= 8.3) stock solution used for SDS-PAGE.

Reagent g/L

Tris base (NZYtech) 30.285

Glycine 144.1344

SDS 10% (w/v) 10

2.6. Proteolytic activity assessement of B. cenocepacia IST01 culture supernatant samples

The determination of proteolytic activity in the Bcc bacterial secretomes was performed using the Pierce™ fluorescent protease assay kit (ThermoScientific), that takes advantage of the increase of fluorescence that occurs after digestion of FITC (fluorescein isothiocyanate)-labelled casein, which is a general substrate for proteases.

Briefly, FITC-casein, previously kept in aliquotes at -80ºC was diluted to 0.01 mg/mL with tris buffered saline (TBS) buffer and 100 μL was added to each well of a 96-well microtiter plate along with 100 μL of each IST01 culture supernatant sample diluted to 0.3 ng/μL according to the previous quantification by the Bradford assay. Additionally, a trypsin standard curve was used as quality control of the assay (Annex V) For the preparation of the standard curve solutions, frozen aliquotes with 5 μL of trypsin (at 200 ng/μL), previously kept at -80ºC, were diluted in TBS buffer to make a stock solution of 0.5 ng/μL

(S0.5) from which the other solutions were prepared according to the table 9. A volume of 100 μL of each standard curve solution of trypsin was added to each well with 100 μL of the FITC-casein solution at 0.01 mg/mL, prepared as described. Blanks, that consisted on TBS buffer and FITC casein were also added to the assay. After the addition of the casein, the microplate was immediately mixed, for 10s, at 300 rpm and incubated, for 60 min, in the dark. In the end, the fluorescence was measured at 485 nm excitation/520 nm emission in a FLUOstar omega microplate reader (BMG LABTECH).

Table 9- Trypsin standard curve solutions used for the proteolytic activity assay, in ng/μL.

Trypsin standard (ng/μL) Trypsin volume TBS assay buffer(μL)

0.5 5 μL 0f 200 ng/ μL 1995

0.4 1060 μL of 0.5 ng/ μL 265

0.3 825 μL of 0.4 ng/ μL 275

0.2 600 μL of 0.3 ng/ μL 300

0.1 500 μL of 0.2 ng/ μL 500

0.05 500 μL of 0.1 ng/ μL 500

0.01 100 μL of 0.05 ng/ μL 400

0 0 μL of 0.5 ng/ μL 500

2.7. Lipolytic activity assessement B. cenocepacia IST01 culture supernatant samples

For the determination of the lipolytic activity of the supernatants of IST01, a lipase assay kit was used (Sigma-Aldrich ® MAK046) that relies on the hydrolysis of a triglyceride substrate, leading to the formation of glycerol, which is coupled with a change of the peroxidase substrate absorbance giving a colorimetric product (Abs=570nm) which is proportional to the enzymatic activity of the sample.

Each supernatant sample (50 μL), further diluted to 6 ng/μL, was added to each well of a 96-well microtiter plate, along with 100 µL of reaction mix A, prepared according to the table 10. In order to discard the effect of the presence of alcohols in the samples that generate a background signal, sample blanks were also added with 100 μL of reaction mix B (table 10). For the standard curve, 50 μL of previously prepared glycerol solutions (table 11) with different concentrations were added to each well along with the reaction mix A. Afterwards, the plates were mixed well, incubated at 37ºC and the absorbance, at 595 nm, was measured every 5 minutes, until the most active outreached the value of the highest value of the standard curve, being the Tfinal the penultimate reading.

Table 10- Preparation of the reaction mixes used for the lipolytic activity assay.

Reagent Reaction mix A Reaction mix B

Lipase Assay Buffer 93 μL 96 μL

Peroxidase substrate 2 μL 2 μL

Enzyme mix 2 μL 2 μL

Lipase substrate 3 μL -

Table 11- Glycerol standard curve solutions prepared for for the lipolytic activity assay.

nmol/well 0 (0 mM) 2 (0.04 mM) 4 (0.08 mM) 6 (0.12 mM) 8 (0.16 mM) 10 (0.2 mM)

Glycerol 500 μL of 333,5 μL of 250 μL of 200 μL of 800μL of 1 0 volume 0.08 mM 0.12 mM 0.16 mM 0.2 mM mM Lipase assay buffer 1000 μL 500 μL 666.5 μL 750 μL 800 μL 200 μL volume

2.8. Fractionation of B. cenocepacia IST01 culture supernatant samples

The fractionation of the Bcc culture supernatant of the stationary phase (time 20h of growth) was performed using VivaspinTM 20 ultrafiltration devices (GE Healthcare) that allow the separation according to the molecular weights of the solution. The separation was performed with four different ultrafiltration devices with the molecular weight cut-offs (MWCO) of 50 kDa, 30 kDa, 10 kDa and 3 kDa, according to the figure 2. A solution was prepared by resuspension of lyophilized IST01 culture 20

supernatant with H2O, at 300 mg/mL. Then, the solution was filtered with 0.2 μm and 20 mL were put on each concentrator and centrifuged at 4ºC, 4400 rpm. Afterwards, four washing steps were employed with a volume of H2O for a dilution of 1:10 of the retentate which was, at the end, further diluted to the starting volume (20 mL), to reconstitute the original solution. The filtrate was poured into the concentrator with the lower MWCO. Finally, the several fractions recovered were preserved at - 80ºC until freeze-drying (conditions described at table 2) or aliquoted and used for protein (SDS PAGE, Bradford assay) and antimicrobial assays, on E. coli ATCC 25922 and S. aureus NCTC 8325. All assays used for the analysis of the fractions were performed as already described, with exception of the SDS-PAGE in which a running gel with 18% acrylamide was used instead of the 12.5%, with composition described on table 6.

Figure 2- Scheme of the fractionation process, by centrifugal ultrafiltration used for the recovery of the fractions of the IST01 culture medium, according to the molecular weights.

3. Results

3.1. Antimicrobial activity of the Burkholderia cenocepacia IST01 culture supernatants harvested during cultivation

B cenocepacia IST01 secretomes were assessed to determine the best condition to obtain the most effective ones as antimicrobial agents. Samples of culture supernatant were collected at the mid- exponential phase (after 3h of cultivation), transition to the stationary phase (after 7h of cultivation), early stationary phase (after 20h of cultivation) and late stationary phase (after 30h of cultivation) (Figure 3) and freeze dried. The weight of freeze dried IST01 culture supernatants obtained from the harvested liquid samples are represented on annex I

Antimicrobial activity was assessed against Escherichia coli ATCC 25922, Enterococcus faecalis DSM 20478, Listeria monocytogenes CNCM-I 4031 and Staphylococcus aureus NCTC 8325 by broth microdilution assays (annex II) Bacterial viabilities were evaluated by culturing the bacterial targets, previously incubated with the inhibitory concentrations of the tested samples, in agar plates to assess whether the samples had a bactericidal or a bacteriostatic effect (annex III). In all the samples a minimum inhibitory concentration (MIC), defined as the lowest concentration of antimicrobial agent that completely inhibits the growth of organisms in microdilution wells, and a minimum bactericidal concentration (MBC) defined as the minimum concentration of antimicrobial agent needed to kill most (≥99.9%)102 of the viable organisms after incubation, were possible to be determined (table 12). The similarity of MIC and MBC values obtained for all the culture supernatants tested indicate that all the 23

samples collected were able to inhibit, with a bactericidal effect, the growth of the four bacterial targets being S. aureus NCTC 8325 the most susceptible to the tested samples.

Considering that the IST01 bacterial cultivation was performed in LB media and that after freeze- drying and resuspension for the antimicrobial assays, these samples are much more concentrated than when they are harvested from the culture, the salt concentration present in LB could, at least partially, be responsible for the inhibition of the growth of the target bacteria, in addition to the antimicrobial molecules eventually secreted by IST01 and present in the culture supernatant. Therefore, an additional antimicrobial assay was performed, as a negative control. This assay consisted in liquid LB subjected to the same treatment by freeze-drying and resuspension, as performed with the tested IST01 culture supernatant samples, using E. coli ATCC 25922 and S. aureus NCTC 8325 as bacterial targets. The results show a similarity between the values obtained for this control and the ones obtained for the samples collected at mid exponential phase, for both bacterial targets, evidencing that the high salt concentration present in this culture supernatant could largely be responsible for the inhibitory effect registered for the IST01 samples obtained after 3h of cultivation (mid exponential phase).

Taking into consideration that the most significant inhibitory effect was obtained for the culture supernatant samples of the early stationary phase, three multidrug resistant isolates were chosen as targets for this sample, namely the Gram-positive methicillin resistant Staphylococcus aureus ATCC 33591 and the gram-negative strains Pseudomonas aeruginosa LES400 and Burkholderia cenocepacia IST05, that represent a major clinical threat nowadays. The results show that the samples of the early stationary phase were able to inhibit, with a bactericidal effect, the growth of the three isolates under study. Interestingly, the most susceptible bacterial target, for which the lowest minimum inhibitory concentration/minimum bactericidal concentration (MIC/MBC) values were obtained, was B. cenocepacia IST05, that was retrieved from the same patient as IST01 but after 3 years, approximately, of chronic infection and a period of agressive antibiotic treatment.

Culture Transition to Mid Early Late samples Control the exponential stationary stationary (LB) stationary phase phase phase Bacterial phase strain MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC 150- 150- E. coli ATCC 25922 300 300 300 300 150 300 75 75 75 75 B. cenocepacia 18,75 - - - - - 9,38 - - IST05 -9,38 P. aeruginosa 75------75 - - LES400 37,5 E. faecalis DSM 600- 300- 300- 300- 300- - 300 300 300 20478 300 150 150 150 150 L. monocytogenes 150- 150- 75- 75- 75- 75- - 300 300 CNCM-I 4031 75 75 37,5 37,5 37,5 37,5 S. aureus NCTC 600- 600- 150- 150- 75- 75- 150- 150- 300 600 8325 300 300 75 75 37,5 37.5 75 75 S. aureus (MRSA) - - - - 75 75 - - ATCC 33591

3.2. Protein quantification and profile of B. cenocepacia IST01 culture supernatants harvested during cultivation

In order to evaluate a correlation between the amount of protein and the antimicrobial activity of the culture supernatant samples, the protein quantification was performed by the Bradford method, using bovine serum albumin (BSA) as a standard curve (figure 4). The results obtained show that proteins/peptides are present in all the samples collected being the highest protein concentration found in the late stationary phase.

The protein profile of the B. cenocepacia IST01 culture supernatant samples was assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE), using a resolving gel with 12.5% acrylamide, a stacking gel with 4% acrylamide and stained with BlueSafe. In a first attempt, the samples were analysed without any treatment. The IST01 lyophilized culture supernatant samples were firstly resuspended with water to a concentration of 300 g/L and directly loaded into each well (20 μL of sample with 5 μL of loading buffer). Additionally, a sample of culture supernatant of a reference strain was used as an internal control (Figure 13 A, annex IV). This experimental condition reveiled unsuccessful to get the protein profile of the samples since the protein bands are not defined, presumably due to the high concentration of salts present that may have affected the migration of the proteins. As an alternative, proteins were precipitated with acetone 100% (v/v) and the volume loaded in the gel wells fulfilled a fixed amount of protein of 4 μg (with 20 μL of loading buffer), according to the previous quantification by the Bradford assay (Figure 13 B, annex I). Again, this treatment was not enough to remove some of the salts present in the sample, causing the same phenomenon as observed without any treatment. Taking these results into account, as a third attempt, the dialysis of the samples was performed with D-TubeTM dialysers and a fixed volume of the dialysed samples (20 μL with 5μL of loading buffer) was loaded into the gel wells (Figure 5). The results show the detection of much more defined protein bands, being this experimental condition successful for adequate protein separation. Moreover, for the samples collected at the transition to the stationary phase, a protein band with an estimated size of 36 kDa starts to emerge. Other proteins with higher molecular weights are also observed for the samples collected in the transition to the stationary phase. These bands are more defined on extracts harvested in the early stationary and late stationary phase, where, in addition, bands with lower molecular weights can be observed. These bands may correspond to 26

proteins secreted by B. cenocepacia IST01, or be products of cell lysis and degradation of other proteins.

kDa 180 140 100 72

3.3. Quantification of the proteolytic activity of IST01 culture supernatants harvested along A cultivation

Taking into account the previous results that pointed out to the appearance of a distinct protein, after separation by SDS-PAGE, in the samples where antimicrobial activity was registered and considering that Bcc bacteria are known to excrete proteins with proteolytic activity with a role in virulence78,109, the next step was to quantify the proteolytic activity of the IST01 culture supernatant samples along the growth curve, to compare the antimicrobial and the proteolytic activities of the culture samples. For this, a protease assay kit (Pierce, ThermoScientific) was used to detect the presence of proteases by a fluorescence increase, that occurs after the hydrolysis of casein which is labelled with fluorecein isothiocyanide (figure 6). The results obtained indicate a strong increase of protease activity, in Fluorescence units/L of culture medium, along the growth curve until the early and late stationary 27

phases. These results are in the line with those obtained for the antimicrobial activities. When expressed in fluorescence units/g of protein (figure 7) a decrease is observed between the samples of the early and late stationary phase, due to the secretion of other proteins into the extracellular media, which is consistent to the different protein profiles obtained by the SDS-PAGE for these samples, where several proteins bands start to emerge on the late stationary phase ones (figure 5).The pattern observed for the protease activity, reflects the increase of secretion of either higher amount of extracellular proteases or more active proteases, that occurs during stationary phase, essential for the virulence of B. cenocepacia and adaptation in the CF lung.

Figure 6-Proteolytic activity of the IST01 supernatants, in Fluorescence units/L of culture medium (above) and minimum inhibitory concentration values (MIC), in mg/mL, obtained for the samples collected at different phases of the bacterial growth curve (below). Blue- MIC values for E. coli ATCC 25922, Orange- MIC values for E. faecalis DSM 20478, Green- MIC values for L. monocytogenes CNCM-I 4031, Purple- MIC values for S. aureus NCTC 8325. The data represents the average of the proteolytic activity values obtained for supernatants of three independent bacterial cultivations.

3.4. Characterization of the fractions of the B. cenocepacia IST01 supernatant collected on the early stationary phase

3.4.1. Characterization of the composition, according to the different molecular weights, of the B. cenocepacia IST01 supernatant collected on the early stationary phase

According to the results obtained for the SDS-PAGE analysis, protein bands with different molecular weights were observed namely on the culture supernatants of the early and late stationary phases, where the highest antimicrobial efficacy was also reported. Besides proteins, within the IST01 secretomes, it is expected to find several other molecules excreted by this isolate during cultivation, among them secondary metabolites that, alternatively, could attribute the antibacterial properties of the tested supernatant samples. Therefore, the separation and characterization of the IST01 culture supernatant of the early stationary phase was performed in order to find out in which fraction of the complex mixture the antibacterial activity of interest could be contained. For the fractionation, centrifugal ultrafiltration devices were used with four different molecular weight cut-offs (MWCO) allowing the recovery of five fractions composed of molecules above 50 kDa, from 50 to 30 kDa, from 30 to 10 kDa, from 10 to 3 kDa and below 3 kDa. After freeze-drying, the proportion of each fraction within the total secretome, in % (weight (g) of freeze dried fraction/weight (g) of total freeze dried culture supernatant) was obtained by weighing each lyophilized fraction and comparing with the weight of the total non-fractioned lyophilized culture supernatant sample used for the fractionation

(annex VI). The results obtained show that the largest part, accounting for over 78% of the total culture supernatant sample, was recovered in the fraction with molecules below 3 kDa, in contrast with the ones above 50 kDa, from 50 to 30 kDa and from 30 to10 kDa, in which only 1% was possible to be recovered (figure 8).

In the case of the fractions composed by molecules above 50 kDa, between 50 and 30 kDa, from 30 to10 kDa and from 10 to 3 kDa, after fractionation, the retentate was resuspended in water to the initial volume of unprocessed total culture supernatant sample loaded in each ultracentrifugation device. With this, it was possible to reconstitute their original solution within the unprocessed samples of IST01 supernatants, for all the assays envisaging the characterization of the fractions performed afterwards. However, after fractionation, the fraction with molecules of molecular weight below 3 kDa, which was the last one to be recovered, is highly diluted and therefore, the protein quantification, protein separation and antimicrobial assays, for the characterization of this fraction, were performed with the freeze-dried fraction sample resuspended in water.

3.4.2. Characterization of the protein profile of the fractions of the B. cenocepacia IST01 supernatants sample collected on the early stationary phase

In order to characterize the protein profile of each fraction, protein separation of the fractions was performed using SDS-PAGE, using a resolving gel with 18% acrylamide, in order to detect the presence of small molecular proteins (figure 9). The results indicate that the majority of the proteins of the IST01 supernatants sample analysed got retained in the fraction with molecules above 50 kDa. Additionally, on the fraction with molecules below 3 kDa, small molecular weight compounds can be seen that are probably small peptides or proteins produced by IST01 or products of degradation of other proteins.

kDa 180 140 100 72 60 46

20 16

Figure 9- Protein separation, by SDS PAGE (resolving gel with 18% acrylamide) of the fractions recovered after fractionation of the samples of B. cenocepacia IST01 culture medium harvested in the early stationary phase. QC- Sample of the culture supernatant of a reference strain used as an internal control. Non fractioned- Non-fractioned total culture supernatant sample collected in the early stationary phase. >50- Fraction with molecules above 50 kDa, 50-30 kDa- Fraction with molecules between 50 and 30 kDa, 30-10 kDa- Fraction with molecules between 30 and 10 kDa, 10-3 kDa- Fraction with molecules between 10 and 3 kDa, <3- Fraction with molecules below 3 kDa.

3.4.3. Characterization of the protein concentration of the B. cenocepacia IST01 supernatants collected on the early stationary phase

To determine the protein concentration of each fraction, the protein quantification with Bradford assay was carried out (figure 10). The results indicate that the fraction with the highest protein concentration was obtained in the fraction with molecules below 3 kDa. Considering that Bradford method relies on the interactions between Coomassie Brilliant Blue with peptides/proteins by reacting with basic (arginine, lysine and hystidine) and aromatic aminoacid residues (phenylalanine and tryptophan) in peptidic chains.110 Therefore, it does not seem surprising the ablity to detect the presence of smaller peptides and proteins contained in the IST01 secretome of the early stationary phase.

3.4.4. Characterization of the antimicrobial activity of the B. cenocepacia IST01 secretome fractions collected on the early stationary phase

In order to further identify which could be the most interesting fraction of molecules in the complex mixture that is the secretome of B. cenocepacia IST01, responsible for its antimicrobial activity, broth microdilution assays were performed with the fractions with molecules above 50 kDa, between 50 and 30 kDa, from 30 to10 kDa, from 10 to 3 kDa and below 3 kDa using E. coli ATCC 25922 and S. aureus NCTC 8325 as bacterial targets (figure 11). The results obtained show that the fraction with molecules below 3 kDa was able to inhibit the growth of E. coli ATCC 25922 and S. aureus NCTC

8325. On the other hand, on the fraction with molecules above 50 kDa, from 50 to 30 kDa, from 30 to 10 kDa and from 10 to 3 kDa no inhibition of growth was observed. Interestingly, in both target strains, a growth increase in percentage, comparing with the positive controls, was observed in test concentrations. This behaviour may indicate the ability of E. coli ATCC 25922 and S. aureus NCTC 8325 to use, in non-inhibitory concentrations, the components in the IST01 secretome as nutrients.

4. Discussion

With the increase of drug resistance in several bacterial pathogens, Bcc bacteria have been considered a highly problematic pathogen for vulnerable individuals such as cystic fibrosis and immunocompromised patients, as well as causing diseases in plants, with significant impact in agriculture. The increasing societal pressure and need for the development of new antimicrobial agents, in spite of the detrimental effects of Bcc, renews the biotechnological interest for the applications of such bacteria in industry, that has been increasing in the past years, with some species already registered for agriculture as plant growth promotors, due to their ability to carry out beneficial interactions with plants, and biocontrol strains as antimicrobial agent producers111, 112. Most recently, a study performed in a context of a thesis project, in 2016, by Leonardo, in collaboration with BioMimetxthat aimed to evaluate the antibacterial potential of culture supernatants from several clinical and environmental Bcc isolates against reference gram negative bacteria, observed that most Bcc supernatants can reduce the growth of the bacterial targets. One of the most interesting Bcc isolates tested was IST01, collected from the sputa of a chronically infected CF patient, which showed to be an efficient producer of secretomes as antibacterial, being chosen as primary source for production of bio-compounds for this project93.

The cultivation of bacteria leads to the excretion and accumulation of a complex mixture of molecules into the extracellular media. The number and types of molecules produced have been reported to be strongly dependent not only of the cultivation properties but also the phase of growth of bacteria113.

In this study, the evaluation of the most effective secretomes of Burkholderia cenocepacia IST01 as antibacterials was performed, according to the phase of growth they are produced. This was achieved by harvesting supernatant samples of IST01 at different time points along the growth curve, evaluating the antimicrobial activity of the freeze dried and sterile supernatants, by broth microdilution assays. Reference Gram-positive and gram negative bacteria were used as bacterial targets, followed by subculturing in agar plates to count viable cells to assess whether the supernatants had a bacteriostatic or bactericidal effect. All the samples tested could inhibit, as a bactericidal product, the growth of all the bacterial targets but, in general, the supernatants of the early and late stationary phases were the most effective ones, being chosen for deeper antimicrobial assays against multidrug resistant bacteria. Interestingly, the most susceptible target was the clinical isolate B. cenocepacia IST05, recovered from the same patient as IST01, and established as its clonal derivative, but three years after the start of infection. Several studies at transcriptome, proteome and metabolome levels aiming to compare IST439, the first isolate collected from a chronically infected CF patient, with IST4113, collected in a more advanced state of chronic lung infection, have reported marked differences between these two isolates, pointing that these genomic, transcriptomic, proteomic and metabolomic differences are crucial for successful adaptation in the CF lung during long-term infections78,82,79. Additionally, in the study performed by Leonardo already mentioned, IST05 culture supernatants were generally much less efficient, as antibacterials, than IST0193. Taking these studies 34

into account, it is likely that IST05 naturally produces and excretes to the extracellular milieu molecules like IST01, however in a less amount or with more specialized functions due to the distinct setting in which the bacteria has adapted, as a way to cope with the CF lung, during the course of the infection. The concentration of the antibacterial molecules produced by IST01 supernatants is too high to possibilitate the growth of IST05, indiciating both a loss of the productive capacity, as well as the loss of any protective mechanism that was initially present.

For Bcc bacteria, it is widely known their capability to produce several secreted proteins such as proteases, lipases, cytotoxins, haemolysins among others, that play an important role in pathogenesis73,75,114,115. Additionally, a previous study observed a difference in virulence efficacy of Bcc bacteria according to the phases of growth in which bacterial cells were harvested and used for invasion studies. The same authors carried out the profilling of the cell culture supernatants, harvested at the early stationary phase and mid logarithmic phase, using a gel based proteomics approach leading to the identification of proteins only released at the early stationary phase109. In this project, an initial quick evaluation of the protein composition was achieved by the protein separation with SDS- PAGE, using dialysis for the pretreatment of the samples and staining with BlueSafe, and a difference of protein profile was observed according to the length of bacterial cultivation. As an example, a distinct band with an estimated size of 36 kDa was seen in the samples retrieved from the early and late stationary phase, where protein bands with higher and lower molecular weights were also observed. However, it should be noted that, with the experimental conditions used, only proteins with estimated molecular weights ranging from 180 kDa to 16 kDa could be detected and therefore, the data obtained will not cover all the proteins present on the analysed IST01 culture supernatant samples. Among the proteins known to be excreted by Bcc bacteria, are included proteases and lipases. One example of protease, known to act extracellularly and play a crucial role in virulence in chronic lung infections is the zinc metalloprotease ZmpA, that is usually expressed in a preproenzyme form, that suffers autoproteolytic cleavage into a 36 kDa mature enzyme. Moreover, this enzyme is known to play a role in the degradation of several important biological constituents such as neutrophil inhibitors, globulins, collagen and fibronectin being essential for causing tissue damage and affecting the immune system. However, other proteases can be produced by Bcc bacteria and can even take over the role of ZmpA in a case of a defect in its production116,72. Taking into account the important role of proteases for Bcc bacteria, it is not surprising that proteolytic activity was found in the supernatant samples of IST01, with potential contribution to its antibacterial properties. On the other hand, even though lipases were expected to be present in the cell culture supernatants, the results obtained for the quantification of lipase activity were not conclusive (Figure 13, annex VII) as the signal obtained in the samples of the early and late stationary phases can be due to the presence of lipases or external factors, such as the composition of the culture media, that may have interfered with the A595 values obtained. Besides proteins, in the Burkholderia genus, the capacity to secrete several metabolites is known, with some already reported antibacterial and antifungal activities87. In this project, the fractionation of the cell culture supernatant of B. cenocepacia IST01 harvested on the early stationary phase was carried out, enabling the recovery of the fractions above 50 kDa, from 50- 35

30 kDa, from 30-10 kDa, from 10-3 kDa and below 3 kDa, and the determination of which exhibited antibacterial activity. Taking into consideration the protein quantification and the antimicrobial activity results, that clearly shows that the antibacterial activity is concentrated in molecules with low molecular weight (below 3 kDa), the antimicrobial activity may be essentially due to secondary metabolites and/or small proteins or peptides present in this mixture. Indeed, the ability of Bcc bacteria to produce and secrete a wide range of antimicrobial secondary metabolites, such as cepacines, xylocandines and quinolones, and peptides, such as the AFC-BC11 is well known117. Additionally, genome-wide expression analysis have allowed the discovery of more biosynthetic pathways for antimicrobials production, such as enacycloxins suggesting the potential of Bcc bacteria as an underexploited resource for undiscovered new antimicrobials88. The results reported in this work, are of major importance for supporting the hypothesis that Bcc bacteria can contribute decisively for a solution for the problem of antimicrobial resistance, but also be applied in other industrial fields, and deserves to be further explored. For companies like BioMimetx, developing antimicrobial solutions, Bcc culture supernatants or subfractions can be the object of significant interest for diverse biotechnological applications, even though their known pathogenicity should be taken into account and might cause barriers to some commercial applications.

5. Future works

Considering the results obtained with this project, regarding the characterization of the Bcc culture supernatants and their potential use as antimicrobial solution, they support the interest to proceed with additional work. Firstly, taking into consideration that the antimicrobial activity was exhibited mainly by the subfraction composed by molecules below 3 kDa, the identification and purification of the antimicrobial molecules, along with the characterization of its inhibitory effects and the mode of action of each agent present should be taken into consideration. Additionally, the identification of the genes encoding for key elements on the possible pathways for antimicrobial and protease biosynthesis, as well as further investigating the expression and characterization of the antimicrobials and proteases encoded should be performed, in different growth conditions for production optimization. Lastly, the screening of Bcc bacteria cell culture supernatants of more clinical isolates, for potential antimicrobial agents, would be interesting to be performed, focusing on the first isolate of other patients.

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Annexes

Annex I

Supernatant sample yield (g/L) SD

Mid exponential phase (3h) 28.90 0.95 Transition to the stationary phase (7h) 25.94 0.66 Early stationary phase (20h) 22.53 0.69 Late stationary phase (30h) 23.20 0.89

Annex II

Figure 12- Susceptibility, in % growth, of A- E. coli ATCC 25922, B- B. cenocepacia IST05, C- P. aeruginosa LES400, D-E. faecalis DSM 20478, E- L. monocytogenes CNCM-I 4031, F- S. aureus NCTC 8325, G- S. aureus (MRSA) ATCC 33591 for the IST01 culture supernatants harvested along bacterial cultivation, namely mid exponential phase (black), transition to the stationary phase (yellow), early stationary phase (green) and late stationary phase (blue) determined by comparision between the OD values obtained and those of the positive controls. In grey, are represented the negative controls performed with lyophilized LB on E. coli ATCC 25922 and S. aureus NCTC 8325. The data represents the average of three independent experiments performed with supernatants harvested from three independent bacterial cultivations.

Annex III

Table 14- Viability, in % viability, of E. coli ATCC 25922 previously incubated with IST01 culture supernatants harvested along the growth curve, determined by subculture of the bacterial targets previously incubated with test concentrations of the samples and comparision between the CFU/mL obtained with those of the subcultured positive controls. The data represents the average of three independent experiments performed with supernatants harvested from three independent bacterial cultivations.

E. coli ATCC 25922 Mid exponential phase Transition to the Early stationary phase Late stationary phase Control (LB) Concentration (3h) stationary phase (7h) (20h) (30h) (mg/mL) % viability SD % viability SD % viability SD % viability SD % viability SD

Control 100 0.00 100.00 0.00 100.00 0.00 100.00 0.00 100.00 0.00

600 0.00 0.00 0.00 0.00 ------

300 0.00 0.00 0.10 0.18 0.16 0.22 - - - -

150 - - 4654.80 3286.38 12.63 1.38 - - - -

75 ------0.00 0.00 0.10 0.04

37,5 ------621.59 499.09 18.82 0.07

E. faecalis DSM 20478

Mid exponential phase Transition to the Early stationary phase Late stationary phase Concentration (3h) stationary phase (7h) (20h) (30h) (mg/mL) % viability SD % viability SD % viability SD % viability SD

Control 100.00 0.00 100.00 0.00 100.00 0.00 100.00 0.00 600 ------300 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

150 1894.58 394.58 22.69 6.02 13.70 19.37 132.08 62.26

75 60.24 31.07 97.15 67.00 - - - -

L. monocytogenes CNCM-I 4031

Control 100.00 0.00 100.00 0.00 100.00 0.00 100.00 0.00

300 0.00 0.00 ------

150 686.48 63.52 0.00 0.00 - - - -

75 - - 23.09 17.46 - - - -

37,5 - - 8.99 12.72 1.24 1.24 1.27 0.63

18.75 - - - - 18.20 7.52 17.22 11.78

S. aureus NCTC 8325

Mid exponential phase Transition to the Early stationary phase Late stationary phase Control (LB) Concentration (3h) stationary phase (7h) (20h) (30h) (mg/mL) % viability SD % viability SD % viability SD % viability SD % viability SD

Control 100.00 0.00 100.00 0.00 100.00 0.00 100.00 0.00 100.00 0.00

600 0.00 0.00 0.00 0.00 ------

300 16.46 10.21 10.43 9.57 ------

150 - - 25988.51 18344.83 0.00 0.00 - - - -

75 - - - - 0.14 0.14 0.00 0.00 18.67 12.64

37,5 - - - - 140.00 0.00 27.56 27.37 6.38 0.77

Early stationary phase (20h) S. aureus (MRSA) ATCC B. cenocepacia IST05 P. aeruginosa LES400 Concentration 33591 (mg/mL) % viability SD % viability SD % viability SD

Control 100.00 0.00 100.00 0.00 100.00 0.00

75 - - 0.00 0.00 0.00 0.00

37.5 - - 23.14 22.57 18.07 2.28

18.75 0.01 0.01 - - - -

9.38 3.00 2.93 - - - -

Annex IV

kDa A 180 140 100 72

kDa B 180 140 100 72

Annex V

Figure 14- Trypsin standard curve used as Quality Control for the proteolytic activity assessment of the IST01 culture supernatants.

Annex VI

Table 19- Weight in g obtained after freeze drying of the fractions obtained after fractionation of the IST01 culture supernatants and their corresponding proportions in the total IST01 supernatant of early stationary phase, in % (weight, in g, of each freeze dried fraction/ weight, in g, of total culture supernatant sample used for fractionation). The assays were performed using fractions from two independent fractionations % (in g obtained/g of g obtained after Samples weight used for SD freeze drying fractionation) Above 50 kDa 0.18 1.49 0.24 Between 50 and 30 0.16 1.29 0.15 kDa Between 30 and 10 0.14 1.17 0.25 kDa Between 10 and 3 kDa 0.54 4.52 1.62

Below 3 kDa 9.36 78.03 0.70 Total IST01 supernatant recovered 10.38 86.49 1.68 after fractionation Total weight of IST01 12 g used for fractionation Losses 1.62 13.51 1.68

Annex VII

A B

previous quantification by the Bradford assay. The A595 values at Tinitial (after 2 minutes of incubation at 37ºC) and Tfinal (after 27 minutes of incubation) of each sample were compared with the glycerol standard curves obtained at these time points for determining the amount of glycerol formed due to lipase activity. 1 unit of lipase corresponds to the amount of enzyme that will form 1 μmole of glycerol from triglycerides, per minute, at 37ºC. The data represents the average of one experiment performed with supernatants harvested from two independent bacterial cultivations.