Human Lysozyme Peptidase Resistance Is Perturbed by the Anionic

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Human lysozyme peptidase resistance is perturbed by the anionic glycolipid biosurfactant rhamnolipid produced by the opportunistic pathogen Pseudomonas aeruginosa Kell K. Andersen, Brian Stougaard Vad, Carsten Scavenius, Jan Johannes Enghild, and Daniel Erik Otzen Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b01009 • Publication Date (Web): 08 Dec 2016 Downloaded from http://pubs.acs.org on December 10, 2016

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Biochemistry

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Human lysozyme peptidase resistance is perturbed by the anionic glycolipid

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biosurfactant

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Pseudomonas aeruginosa

rhamnolipid

produced

by

the

opportunistic

pathogen

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Kell K. Andersen§*, Brian S. Vad, Carsten Scavenius, Jan J. Enghild and Daniel E. Otzen*

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Interdisciplinary Nanoscience Center (iNANO), Department of Molecular Biology and Genetics,

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Aarhus University, Gustav Wieds Vej 14, DK – 8000 Aarhus C

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§

Present address: Agro Business Park A/S, Niels Pedersens Allé 2, DK-8830 Tjele.

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* Corresponding authors

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Email: [email protected] (K.K.A.) or [email protected] (D.E.O.)

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Key words: rhamnolipid; lysozyme; virulence factors; proteolysis; degradation

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FUNDING STATEMENT:

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K.K.A., B.S.V. and D.E.O. are supported by grants from the Danish Research Council | Technology

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and Production (grant nr. 12-126 186 to K.K.A. and grant nr. 6111-00241B to B.S.V. and D.E.O.).

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D.E.O. is supported by grant no. 11283 from The Novo Nordisk Foundation (Biotechnology-based

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Synthesis and Production).

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1 ACS Paragon Plus Environment

Biochemistry

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Abbreviations:

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CD, circular dichroism; cmc, critical micelle concentration; DDM, dodecyl maltoside; EDTA,

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Ethylenediaminetetraacetic acid; HLZ, human lysozyme; ITC, isothermal titration calorimetry;

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MBTACT, Methylumbelliferyl β-D-N,N′,N′-triacetylchitotrioside hydrate; PA, Pseudomonas

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aeruginosa; PAE, Pseudomonas aeruginosa Elastase; PMSF, Phenylmethylsulfonyl fluoride; QS,

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quorum sensing; RL, rhamnolipid; SDS, sodium dodecyl sulfate; SPITC, 4-sulfophenyl isothiocyanate


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Biochemistry

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ABSTRACT

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Infection by the opportunistic pathogen Pseudomonas aeruginosa (PA) is accompanied by the secretion

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of virulence factors such as the secondary metabolite rhamnolipid (RL) as well as an array of bacterial

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enzymes, including the peptidase elastase. The human immune system tries to counter this via

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defensive proteins such as human lysozyme (HLZ). HLZ targets the bacterial cell wall but may also

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have other antimicrobial activities. The enzyme contains four disulfide bonds and shows high

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thermodynamic stability and resistance to proteolytic attack. Here we show that RL promotes HLZ

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degradation by several unrelated peptidases, including the PA elastase and human peptidases. This

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occurs although RL does not by itself denature HLZ. Nevertheless, RL binds in a sufficiently high

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stoichiometry (8 RL:1 HLZ) to neutralize the highly cationic surface of HLZ. The initial cleavage sites

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agree well with the domain boundaries of HLZ. Thus, RL binding to native HLZ may be sufficient to

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allow proteolytic attack at slightly exposed sites on the protein, leading to subsequent degradation.

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Furthermore, biofilms of RL-producing strains of PA are protected better against high concentrations of

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HLZ than RL-free PA strains. We conclude that pathogen-produced RL may weaken host defenses by

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facilitating degradation of key host proteins.

42 43


Biochemistry

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The gram-negative bacterium Pseudomonas aeruginosa (PA) is an opportunistic pathogen that can

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cause severe chronic infection, especially in the airways of cystic fibrosis (CF) patients. CF is the most

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common inherited disease causing mortality in Caucasians and leads to an average life expectancy of

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37-40 years (1). The respiratory tract is the most severely affected organ, and PA infections are the

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primary cause of death in patients with cystic fibrosis (2). PA can also adhere to damaged corneal

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epithelium, causing keratinitis (3).

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The pathogenesis of PA follows a multipronged strategy. PA produces exopolysaccharides (EPS),

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functional amyloid and extracellular DNA that together form a biofilm, making it difficult for the

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human immune system to combat microbial invasion. In addition, PA also secretes an array of enzymes

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including peptidases that protect the bacterium, assist in substrate acquisition and attack host defensive

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proteins. These attacks may play a key role in PA pathogenesis. Thus the extracellular endopeptidase

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Pseudomonas aeruginosa Elastase (PAE) (Uniprot: P14756, also known as Pseudolysin and Elastase,

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gene name: LasB) is reported to cleave numerous proteins including human lysozyme (HLZ) (4),

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lactoferrin (LF) (5) and immunoglobulins (Ig) (6, 7). The name elastase refers to its ability to degrade

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elastin, first observed by Moiriha et al. who were able to isolate, crystallize and characterize the

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enzyme (8). Mature PAE is 33 kDa with a pI of 5.9 and a pH optimum between 7-9 (8). PAE is

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stabilized by two disulfide bonds, one Ca2+ and one Zn2+ atom (9). PAE favors cleavage at sites that

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contain hydrophobic or aromatic amino acids residues at the P1′ position (the position immediately

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after the cleavage site) (10).

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During a bacterial infection in the airways, a number of human defensive proteins and enzymes are

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mobilized. These include HLZ, LF, secretory IgA (the major immunoglobulin of the respiratory tract)

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and peptidases, which are all abundant in airway fluids and mucus secretions (11). Of particular interest

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in the current study is the 14.3 kDa glycosyl hydrolase HLZ that has anti-bacterial activity. HLZ is

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secreted at high concentrations into human airway secretions from surface epithelium, submucosal

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tracheal glands and pulmonary alveolar macrophages (12). HLZ contains 4 disulfide bonds and has a

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high positive net charge (~+8) at physiological pH (7.4). Together with a high thermodynamic stability

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(13), this makes it highly peptidase resistant, although PAE has been reported to digest HLZ in vitro in

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0.1 M Tris pH 8, 2 mM CaCl2 (4). It is widely acknowledged that HLZ’s primary substrate is the

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bacterial cell-wall component peptidoglycan, allowing HLZ to kill both gram-positive and gram-


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Biochemistry

negative bacteria, including PA (14). Remarkably, the catalytic activity (muramidase activity) of HLZ is not required to kill either gram-negative or gram-positive bacteria (14, 15). This indicates that other factors than the muramidase activity contributes to the antibacterial properties of HLZ. Such factors could include a conserved bactericidal C-terminal cationic domain with a helix-loop-helix motif that has a potent bactericidal effect on both gram-negative and gram-positive bacteria (16, 17). In addition to enzymes, PA also secretes several secondary metabolites including rhamnolipids (RLs) (Fig. 1 insert). RLs are glycolipid biosurfactants that are composed of either a mono- (RL1) or dirhamnose (RL2) head group coupled to modified fatty acids, making RL anionic. Expression of genes responsible for the synthesis of RL is regulated by the quorum-sensing (QS) system that allows for intercellular communication and serves as a method for assessing local population densities and regulation of expressing of individual genes. PA has three QS circuits; Las, Rhl and Pqr (18). These circuits are interwoven in a hierarchical order. Thus the LasR gene product from the Las circuit is responsible for the transcription of rhlR from the rhl circuit, while the gene product of rhlR is the transcription factor RhlR responsible for the transcription of the genes required for rhamnolipid synthesis. Interestingly, translation of mRNA from the rhlR gene is highly elevated at body temperature compared to 30oC. This is due to a so-called RNA-thermometer situated in the 5` untranslated region (19). At 37oC, this region is unstructured, enabling translation, while at 30oC the region is highly structured and prevents efficient translation. In contrast, to the thermoregulated production of RL, no effect of temperature is observed for the production of PAE. RL is involved in motility (20), biofilm development, modification of the bacterial cell surface (21), transport of proteins across the human stratum corneum (22), protection against and lysis of monocytederived macrophages and polymorphonuclear leukocytes of the human immune system (23). Presence of rhamnolipid has been verified in vivo in cystic fibrosis patients infected with PA. Reported RL concentrations range from 8 µg/ml in sputum (24) to 65 µg/ml in lung secretions (25). RL concentrations > 100 g/L have been reported under optimized fermentation in bioreactors (26). RL can both stabilize and destabilize proteins. RL denatures globular proteins such as α-lactalbumin and myoglobin (27), while bovine serum albumin (BSA) only binds 1-2 RL without undergoing denaturation (28). Remarkably, RL can also fold and stabilize outer membrane proteins from gramnegative bacteria (29). To our knowledge, however, there are no studies on how biosurfactants from


Biochemistry

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pathogenic microorganisms may contribute to the pathogenesis by interacting with host defensive proteins. Peptidase attack is often linked to the folding behavior of a protein; peptidases typically attack the most dynamic regions of a protein which may also constitute the first site of unfolding, and thus cleavage sites can be used to identify domain boundaries and transitions from folded to unfolded regions (30, 31). We hypothesized that RL may sensitize defense proteins to peptidase attack and thus promote bacterial infection. Here we show that RL from PA interacts with HLZ without denaturing the enzyme; thus, HLZ maintains activity at concentration well above RL’s critical micelle concentration (cmc). Nevertheless, the binding of anionic RL, which neutralizes HLZ’ positive surface potential, makes HLZ highly susceptible to PAE and leads to accelerated proteolysis. RL also makes HLZ susceptible to human peptidases which usually do not digest HLZ (31) and protects PA biofilm against HLZ attack. RL may thus be involved in PA pathogenesis by charge-neutralizing - rather than directly destabilizing or inactivating - host defensive proteins.


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Biochemistry

MATERIALS AND METHODS Materials: Pseudomonas aeruginosa Elastase (PAE), Human Leucocyte Elastase and Human Leucocyte Cathepsin G were from Elastin Products Company, Inc (Owensville, Missouri, USA). 4Methylumbelliferyl β-D-N,N′,N′-triacetylchitotrioside hydrate (MBTACT), Ethylenediaminetetraacetic acid (EDTA), Phenylmethylsulfonyl fluoride (PMSF), Sodium dodecyl sulfate (SDS), Trypsin from Bovine pancreas, Human Lactoferrin, Human Lysozyme and Human IgA, Proteinase K from Tritirachium album were from Sigma-Aldrich (St. Louis, MO, USA). Dodecyl maltoside (DDM) was from Anatrace (Maumee, OH). JBR515 rhamnolipid (RL) was provided by Jeneil Biosurfactant Company (Saukville, WI, USA) as a liquid solution consisting of 15 % RL of the highest grade (>99% pure). JBR515 is a 1:0.35 mixture of mono-rhamnolipid (RL1) and di-rhamnolipid (RL2) with molecular masses of 504 and 650 Da, respectively. All reactions were carried out in 20 mM sodium phosphate pH 7. Determination of the critical micelle concentration and surfactant clustering by pyrene fluorescence: The cmc of RL in PBS buffer was determined by pyrene fluorescence as described (32). Pyrene’s fluorescence is sensitive to the environment and the ratio of the intensities of two emission peaks at 372.5 (I1) and 383.5 nm (I3) changes as pyrene partitions into surfactant micelles, making I1/I3 a good probe for the polarity of pyrene’s environment (33). Briefly, different concentrations of RL in buffer were prepared. After equilibration for 30 min, pyrene was added from a 100 µM stock in ethanol to a final concentration of 1 µM. The low concentratration of ethanol does not have any measurable effect on cmc measurements (34). Fluorescence scans were performed on an LS-55 luminescence spectrometer (Perkin-Elmer Instruments, UK) at 25oC, using an excitation wavelength of 335 nm, emission from 360 to 410 nm and excitation/emission slits of 5/2.5 nm. Possible complexes formed between RL and HLZ at concentrations below the cmc were investigated by incubation of 2 µM HLZ with RL for 60 minutes before pyrene addition. Circular dichroism: CD Spectra were recorded on a JASCO J-810 spectropolarimeter (Jasco Spectroscopic Co. Ltd., Japan) equipped with a Jasco PTC-423S temperature control unit. Far-UV CD scans were recorded in the wavelength range 200–250 nm, with a bandwidth of 2 nm, a scanning speed of 50 nm/min and a response of 2 seconds. Measurements were conducted in a 0.1 cm quartz cuvette. Twelve accumulations were averaged and buffer background contributions were subtracted. Final HLZ


Biochemistry

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concentration was 0.1 mg/mL (7 µM). Near-UV CD scans were recorded in the wavelength range 250350 nm with a bandwidth of 2 nm, a scanning speed of 50 nm/min and a response of 2 seconds. Measurements were conducted in a 1 cm quartz cuvette. Six accumulations were averaged and buffer background contributions were subtracted. Final HLZ concentration was 0.5 mg/mL (35 µM). Lysozyme activity assays: Lysozyme activity at increasing RL concentrations was determined using 4Methylumbelliferyl β-D-N,N′,N′-triacetylchitotrioside hydrate as substrate (35) at 37 °C in a Varioskan Flash Multimode Reader (Thermo Scientific, Waltham, MA) in a 96 well plate. 5 µM of HLZ was mixed with 0 to 8 mM of RL and left to equilibrate for 1 hr. 2.5 mM substrate in DMSO was added to a final concentration of 50 µM, and fluorescence of the cleaved product (4-methylumbelliferone) was followed at 455 nm, using an excitation wavelength of 360 nm and slit widths of 12 nm. Due to temperature equilibration of the plate taking place at the beginning of the reaction, the HLZ activity was based on data measured from 30 to 90 minutes. Activity of HLZ in the presence of RL was normalized to the activity in the absence of RL. All measurements were conducted in triplicate. Isothermal titration calorimetry: Calorimetric measurements were conducted on a VP-ITC calorimeter (MicroCal, Northampton, MA). The reference cell was filled with water. In a typical experiment, the sample cell was loaded with a solution of 0 – 4 mg/mL (0-280 µM) HLZ in PBS buffer. The cell solution was titrated with aliquots of 4 µl of 25 mM RL in PBS buffer. All titrations were performed at 25 °C, where RL demicellization gives a very low enthalpic signal. Therefore, enthalpic contributions from the demicellization of RL upon injection can be neglected in data analysis. The obtained heat signals from the ITC were integrated using the Origin software supplied by MicroCal. For the calculation of the binding stoichiometry, protein dilution during ITC analysis was taken into account. Visualization of HLZ Electrostatic Potential: The electrostatic potential was calculated at pH 7.4 using the program ABPS (36) and pdb file 1LZ1 (37) and visualized using PyMOL (DeLano Scientific, San Carlos, CA). Analysis of HLZ Proteolysis by SDS-PAGE: All peptidases except Trypsin were dissolved in PBS buffer on ice and immediately flash frozen using liquid nitrogen and stored at -80 °C. Trypsin was dissolved in 1 mM HCl pH 3 and stored at -20 °C. In a typical experiment, 0.2-0.4 mg/mL (14-28 µM) of HLZ in PBS was equilibrated with increasing concentrations of RL in PBS for 30 minutes. After equilibration, 0.04 mg/ml peptidase (i.e. 1.7 µM trypsin, 1.4 µM proteinase K, 1.1 µM cathepsin or 1.5 8 ACS Paragon Plus Environment

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Biochemistry

µM elastase) was added to the mixture. Proteolysis was stopped by addition of PMSF and EDTA to final concentrations of 2 mM and 5 mM respectively. Proteolysis was estimated by SDS-PAGE. Samples were mixed 5:1 with 6x reducing sample buffer, after which 9 µL was analyzed by SDS– PAGE. Band intensities were quantified by ImageJ. Identification of initial proteolytic cleavage site by mass spectrometry: Limited proteolysis was performed by incubating 0.8 mg/mL (56 µM) HLZ in buffer with 2 mM RL. After one hr of equilibration, samples was alliquoted and mixed with different concentrations of peptidase. During proteolysis final concentrations were 0.4 mg/mL (28 µM) HLZ and 1 mM RL. After one hr, proteolysis was stopped by addition of PMSF and EDTA to final concentrations of 2 mM and 5 mM respectively. After proteolytic treatment of HLZ, newly formed N-termini were modified with 4-sulfophenyl isothiocyanate (SPITC) as previously described (38). The samples were denatured, reduced and alkylated in 100 mM ammonium bicarbonate, 6 M Urea, pH 8 containing 10 mM DTT followed by the addition of iodoacetamide to a concentration of 30 mM. The reduced and alkylated samples were micro-purified using custom-made micro-columns packed with Poros R2 (50 µm, Applied Biosystems, Framingham, MA) (39). The lyophilized samples were dissolved in 100 mM ammonium bicarbonate and digested with trypsin (1:25 w/w) at 37°C for 16 hrs. Prior to analysis by mass spectrometry the tryptic peptides were micro-purified (39). Mass spectrometric analysis: NanoESI-MS/MS analyses were performed on an EASY-nLC II system (ThermoScientific) connected to a TripleTOF 5600+ mass spectrometer (AB Sciex). The trypsin digested samples were suspended in 0.1% formic acid, injected, trapped and desalted on a RP ReproSilPur C18-AQ 3 µm column (2 cm x 100 µm I.D). The peptides were eluted from the trap column and separated on a 15-cm analytical column (75 µm i.d.) RP ReproSil-Pur C18-AQ 3 µm resin (Dr. Marisch GmbH, Ammerbuch-Entringen, Germany). Peptides were eluted using 250 nl/min and a 20 min gradient from 5% to 35% phase B (0.1% formic acid and 90% acetonitrile) and sprayed directly into the mass spectrometer. The collected MS files were converted to Mascot generic format (MGF) using the AB SCIEX MS Data Converter beta 1.1 (AB SCIEX) and the “proteinpilot MGF” parameters. The generated peak lists were searched against the Swiss-prot database using an in-house Mascot search engine (Matrix Science). Search parameters allowed semi-tryptic peptides, as well as one, missed cleavage site. Carbamidomethyl was set as a fixed modification and SPITC as variable modification


Biochemistry

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with peptide tolerance and MS/MS tolerance set to 10 ppm and 0.2 Da respectively. All peptides with SPITC assignment were manually inspected. Crystal Violet biofilm assay: Two strains of P. aeruginosa were used, one with and one without the ability to produce rhamnolipid (40), i.e. the wild type strain PAO1 and an isogenic rhlA mutant. These strains were kindly donated by Dr. Tim Tolker-Nielsen. Biofilm formation was quantified using crystal violet as follows: A culture was set up with a single P. aeruginosa colony transferred to 20 ml LB medium and incubated overnight at 25oC. The medium was diluted to OD600 0.5 and transferred to a 96 well flat bottomed plate (Nunc, Roskilde Denmark) and a peg lid (Nunc-Tsp, Roskilde Denmark) was placed on the plate for 30 min to inoculate the pegs. The peg lid was then transferred to a 96 well plate with fresh medium and 0-100 µg/ml HLZ (using a stock solution dissolved and diluted in medium) and grown for 24 h at 25oC. The peg lid was removed and dried for 1 hour at room temperature and transferred to a 0,5 % solution of crystal violet and incubated for 15 min. The peg lid was gently washed in milli-Q water before transferring to a 96 well plate with 96% ethanol to release the bound crystal violet. The peg lid was removed and the amount of crystal violet in each well was measured by absorption at 585 nm. All assays were performed in 6-fold replicates.


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Biochemistry

RESULTS RL does not form clusters on HLZ below the cmc Proteins may interact with surfactants both in the monomeric and micellar state (41), and it is therefore important to know RL’s critical micelle concentration (cmc) where micelles are formed. We did so using the hydrophobic probe pyrene, whose fluorescence is sensitive to the environment. Incubation of pyrene with RL reveals a systematic development in the pyrene I3/I1 fluorescence ratio with increasing RL concentration. The I3/I1 ratio is stable at ~0.6 between 0 and 0.1 mM RL; between 0.1 and 1 mM RL it increases to reach a plateau value of 1.1 (Fig. 1). This indicates that pyrene partitions into RL micelles that are formed in the concentration range 0.1-1 mM RL, leading to an estimated cmc value of 0.1 mM. A possible interaction between HLZ and monomeric RL was also investigated using pyrene. Other proteins such as ACBP (42) can bind clusters of surfactants on their surface below the cmc into which pyrene can partition, leading to altered pyrene fluorescence profiles. However, incubation of 2 µM HLZ with RL followed by addition of pyrene did not have any such effect. The absence of an effect cannot be ascribed to the low HLZ concentration used; other proteins such as ACBP can induce SDS clusters at 2 µM (42). We conclude that HLZ does not form sub-cmc RL clusters on the HLZ surface into which pyrene can partition. HLZ maintains native structure and activity in the presence of RL The influence of RL on the secondary and tertiary structure of HLZ was investigated using far-UV and Near-UV circular dichroism (CD), respectively (Fig. 2). Even at concentrations well above the cmc (1.5-2.0 mM RL), there is no change in either far-UV or near-UV CD spectra, indicating that HLZ is not denatured by either monomeric or micellar RL. Far-UV CD thermal scans also revealed that the thermal stability of HLZ is not reduced by RL (in both cases the midpoint temperature is around 74oC, data not shown). The possible influence of RL on HLZ hydrolytic activity was investigated using the substrate 4Methylumbelliferyl β-D-N,N′,N′-triacetylchitotrioside hydrate (MBTACT), which fluoresces upon cleavage. While increasing concentrations of RL led to a monotonic decline in the activity of HLZ (Fig. 3), >50% activity remained up to 8 mM (ca. 5 mg/ml or 350 µM) RL. Thus HLZ maintains high levels


Biochemistry

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of residual activity well above RL’s cmc and above reported in vivo RL concentrations (8 to 65 µg/mL, i.e. 0.012-0.1 mM) (24, 25). Isothermal titration calorimetry reveals a low level of RL binding to HLZ The decrease in activity by HLZ could be caused by RL interactions with HLZ or MBTACT. Possible RL-HLZ interactions were investigated using isothermal titration calorimetry (ITC), which is a wellestablished approach to provide information on the stoichiometry of surfactant binding to proteins (27, 43-47). Solutions with different concentrations (0-4 mg/mL or 0-280 µM) of HLZ were each titrated with increasing concentrations of RL and the recorded heat flow at each injection point was integrated, leading to a series of enthalpograms (Fig 4A). RL titrations into buffer alone resulted in a small endothermic signal at low RL concentrations, resulting from demicellization of RL micelles. The signal levels out slightly above 0.5 mM RL, indicating that no further demicellization occurs because the cmc has been reached. This is consistent with pyrene data (Fig 1). The HLZ/RL enthalpograms were characterized by a large exothermic signal that increased with increasing protein concentration. The point where the endothermic signal converges with the signal for titration into buffer (indicated by an arrow in Fig. 4A for the highest HLZ concentration) increased with increasing HLZ concentration. This clearly demonstrates interactions between RL and HLZ. To determine the binding stoichiometry, the concentration of HLZ is plotted as a function of the RL concentration where the HLZ titration merges with the buffer titration (Fig 4B). The binding stoichiometry is derived using the following mass balance. [ܴ‫் ]ܮ‬௢௧௔௟ = [ܴ‫]ܮ‬ி௥௘௘ + ܰ[ܲ‫]݊݅݁ݐ݋ݎ‬

(1)

where N is the number of RL molecules bound per protein and [RL]free is the concentration of surfactant that is not bound to protein (48). (Note that this mass balance does not provide information about the affinity between RL and HLZ, but formally assumes that all binding sites at the given inflexion point are filled by surfactant molecules). The linear fit shows that HLZ binds ~8 RL molecules per HLZ or 1 RL per ~16.25 amino acid residue. For proteins undergoing denaturation, the binding number is ~1 RL per 4 amino acid residue (49). The small binding number is consistent with our spectroscopic data that show HLZ not to be denatured by RL. Interestingly, HLZ has a calculated net charge of approximately +8 at pH 7.4, thanks to 19 basic Lys/Arg residues and 11 acidic Asp/Glu residues. This may be a coincidence, since only a few of these residues engage in internal salt bridges. 12 ACS Paragon Plus Environment

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Biochemistry

Nevertheless, the magnitude of the binding stoichiometry suggests that RL via electrostatic interactions may bind to and neutralize a significant proportion of exposed cationic amino acid residues on the surface of HLZ without denaturing the enzyme. The synthetic anionic model surfactant SDS also binds 8 SDS molecules to HLZ at low SDS concentrations without denaturing HLZ (50). However, unlike RL, high SDS concentrations lead to further binding and enzyme denaturation (50). HLZ has a highly positive surface potential at pH 7.4 (Fig. 5). One exception is however, the groove of the active site that shows a highly negative potential. Electrostatic interactions between HLZ and RL are therefore less likely around the active site and this may also be a reason why HLZ maintains significant activity upon binding of RL. HLZ undergoes accelerated proteolysis by both bacterial and human peptidases in the presence of RL We hypothesized that the neutralization of HLZ’s highly cationic surface by RL could increase HLZ’ susceptibility to both bacterial and human peptidases encountered in vivo. PA secretes an array of different peptidases and HLZ has been shown to be susceptible to PAE (4). We incubated HLZ with increasing concentrations of RL, after which PAE was added and the mixture was incubated for 4 hrs at 37oC. Proteolysis was stopped by addition of Phenylmethylsulfonyl fluoride (PMSF) and Ethylenediaminetetraacetic acid (EDTA) and evaluated by SDS-PAGE (Fig 6A). While RL-free HLZ remained intact, increasing RL concentrations clearly had an effect: the overall band intensity declined and there was a small but significant shift in the band to lower molecular mass. This indicates that even low concentrations of RL make HLZ more susceptible to proteolysis. When we followed the time course of HLZ proteolysis by PAE in the absence and presence of 1 mM RL (Fig 6B) over 6 hrs at 37 o

C, we observed a steady decline in the concentration of intact HLZ. Jacqout et al report that RL-free

PAE can degrade HLZ, (4), but they performed proteolysis in buffer with 2 mM CaCl2. Since Ca2+ is a PAE cofactor (9), addition of surplus Ca2+ may increase the activity. In our hands, the absence of surplus Ca2+ avoids proteolysis of RL-free HLZ and thus provides a cleaner background control. In vivo, HLZ in airway secretions encounters both bacterial as well as human peptidases. Neutrophils in particular, secrete a number of peptidases into the airway surface liquid. We therefore investigated the susceptibility of HLZ to human neutrophil elastase as well as Cathepsin G, both of which occur in elevated concentrations in the airway surface liquid in CF patients. In addition, we also investigated two model peptidases, namely bovine trypsin (highly specific for Lys and Arg residues) and T. album 13 ACS Paragon Plus Environment

Biochemistry

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Proteinase K (a highly aggressive broad-specificity peptidase). Under the applied buffer conditions, both proteinase K and Cathepsin G show a minor degree of degradation of RL-free HLZ and formation of degradation products, while trypsin and Human elastase have no effect as reported before for Human elastase (4). Importantly, HLZ’s susceptibility to all four peptidases increases markedly in the presence of 1 mM RL (Fig 7A) as also observed with PAE. To investigate if RL’s ability to increase HLZ’s peptidase susceptibility was a general feature of surfactants, we tested the non-ionic surfactant dodecyl maltoside (DDM), which solubilizes membrane proteins in the native state (51) but only interacts weakly if at all with globular proteins (52, 53). As a positive control we used SDS that both binds and denature HLZ (50) and indeed led to almost complete degradation of HLZ (Fig 7B). In contrast, DDM failed to promote proteolytic degradation of HLZ (Fig 7B). This indicates that elimination of electrostatic repulsion is important to initiate surfactant-promoted HLZ proteolysis (although this may be underpinned by additional interactions via the hydrophobic surfactant moiety). To pursue the impact of electrostatic effects further, we investigated the effect of RL on the degradation of the two defensive proteins lactoferrin and IgA. These proteins have pI values of 8.7 (54) and 5-6.5 (55). However, in both cases, the proteins are susceptible to peptidase digestion even in the absence of RL (data not shown), preventing any firm conclusions from being made. RL had no effect on the rate of trypsin autoproteolysis (data not shown), indicating that that it is the interaction between RL and HLZ (rather than between RL and peptidase) that is determining for digestion/inactivation of HLZ. Limited proteolysis reveals peptidase sites of attack on HLZ We used limited proteolysis with different peptidases to identify regions where peptidases cleave HLZ in the presence of 1 mM RL. Samples that had undergone limited proteolysis were labeled with 4sulfophenyl isothiocyanate (SPITC) prior to trypsin treatment to identify N-termini generated during the limited proteolysis. The samples were analyzed by nLC-MS/MS and SPITC modifications identified by the mascot search engine. The SPITC modification allows us to differentiate between the N-termini generated during limited proteolysis and those produced by trypsin treatment prior to LCMS/MS analysis. The identified cleavage sites were all located in three regions around residues 32-33, 59-62 and 116-118 (Table 1). We suspect that additional peptidase-specific cleavage sites identified at residue 69 (Trypsin and HNE), 51 (proteinase K) and 140 (HNE) arise from further degradation after the initial cleavage. The shared cleavage sites highlight a region that is exposed and cleaved after RL


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Biochemistry

binding to HLZ. Summed spectral counts of all MSMS spectra assigned to the three different positions indicated that the order of cleavage was 116-118 > 59-62 >> 32-33. Both region 59-62 and region 116118 are located on the exterior of the protein (Fig 8). Therefore, the RL-promoted proteolysis of HLZ most likely occurs by an initial cleavage in region 116-118 or 59-62 that leads to further destabilization of the structure and secondary degradation. Rhamnolipids protects Pseudomonas aeruginosa biofilm against HLZ Our in vitro data suggest that RL could protect P. aeruginosa against attack by HLZ by enlisting the help of peptidases. We decided to investigate the consequences for the growth of biofilm using two P. aeruginosa strains, of which the wildtype produces RL naturally at concentrations of around 50 µg/ml (0.08 mM, i.e. very close to its cmc), while the isogenic rhlA strain does not (40). Attempts to grown biofilm of these strains in the presence of different concentrations of exogenously added HLZ and peptidases failed to yield conclusive results because of high standard deviations (data not shown). However, when we grew biofilm in the presence of 0-100 µg/ml HLZ, there was a modest but significant protective effect by RL at high HLZ concentrations (Fig. 9) (p = 0.05). While the rhlA strain suffered a reduction in biofilm production at 100 µg/ml HLZ to 54±6% of the HLZ-free amount, the wt RL-producing strain only decreased to 69±7%, suggesting that RL can indeed to some extent mitigate the bacteriocidal effect of HLZ.


Biochemistry

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DISCUSSION RL is a multifunctional biomolecule that has been reported to be involved in a number of processes important for PA pathogenicity. The role of microbial surfactants such as RL in the interactions with human defensive proteins has until now remained unexplored. Synthetic surfactants and microbial surfactant share numerous properties, e.g. the ability to lower surface tension and form macromolecular assemblies such as micelles. However, their obvious differences in molecular structure lead to different physicochemical properties and functionalities, influencing how surfactants interact with proteins (56). A mild and non-denaturing surfactant may still promote proteolytic degradation The present study shows that HLZ-RL interactions are much simpler and have much more modest structural repercussions than those involving SDS (50). Interactions with both RL and SDS at low concentrations give rise to an overall endothermic signal that can be explained by the binding of ~8 surfactant molecules. However, this specific binding does not lead to protein structural changes that can be monitored by CD (for RL) or Fourier Transform Infrared Spectroscopy (for SDS (50)). After the exothermic peak, the HLZ/RL enthalpogram does not reveal further interactions between RL and HLZ. In contrast, the HLZ/SDS enthalpogram reveals both endothermic and exothermic processes with increasing SDS concentration (50). These processes involve further SDS binding and are also accompanied by structural changes (50). The fact that RL does not denature HLZ may be explained by the major molecular differences between the two anionic surfactants. RL is almost twice the mass of SDS and occupies twice the space, giving a much lower charge to size ratio for RL than for SDS. The hydrophobic moiety, which is derived from the fusion of two carboxylic groups, is branched as opposed to SDS’ linear alkyl tail, and will therefore most likely not pack as effectively against HLZ. Furthermore the carboxylic acid group is not so strongly ionized as SDS, having a much higher pKa (ca. 5 for –COOH versus < 2 for SDS). Surfactant charge is important for protein denaturation, and non-ionic surfactants only denature highly unstable proteins such as apo-α-lactalbumin (52). Therefore, the charge/size ratio of RL may not be high enough to perturb the native structure of HLZ. We have previously shown that while both SDS and RL can denature the two proteins apo α-lactalbumnin and myoglobin, RL is a much less potent denaturant than SDS, illustrated by the slow denaturation kinetics which are around an order of magnitude slower with RL (27). It is therefore not surprising that an extracellular enzyme like HLZ, designed to preserve native structure and activity in a hostile


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Biochemistry

environment where it can encounter peptidases, is resistant to denaturation and inactivation by a mild anionic surfactant such as RL. Nevertheless, RL still interacts with the enzyme. ITC investigations demonstrated that HLZ binds RL at very low surfactant concentrations and at saturation HLZ binds 8 RL molecules. Although RL does not destabilize HLZ according to structural and thermal denaturation studies, it slightly reduces HLZ activity, possibly by interfering with substrate access to the active site. Crucially, binding and progressive neutralization of the positive surface potential with increasing RL concentration had a profound effect on proteolytic resistance. While HLZ was not digested by peptidases in the absence of RL, it is surprising that even small in vivo relevant concentrations of RL (< 0.1 mM RL; RL is reported to accumulate at around 0.08 mM in vivo (40)) resulted in HLZ digestion. The fact that the effect of RL on HLZ susceptibility to proteolysis was observed with many different peptidases from different origins, microbial as well as human, show that the effect was clearly induced by RL binding to HLZ and not a peptidase specific effect. The degradation pattern of HLZ agrees well with previous studies on the proteolytic susceptibility of HLZ and its structural homologues (57, 58). HLZ consists of a large discontinuous α-helical domain (residues 1-33 and residues 85-123) interrupted by a smaller β-sheet subdomain (residues 34-57). The β-subdomain is the least stable part of lysozyme and the first to unfold under denaturing conditions (59-61). Thus, the identified cleavage sites around 59-62 (and to a smaller extent around residue 33) agree well with domain boundaries. Position 116-118 is close in space to the N-terminal part of the βdomain and may therefore become more accessible to cleavage once the β-domain has become nicked by peptidases. Thus, binding of RL does not appear to lead to a structural transition to a non-native state that would expose hitherto unknown binding sites; rather, we suggest that RL reduces the electrostatic barrier to binding of peptidases sufficiently to allow them to access the most flexible sites in the native state. RL likely bind to cationic side chains, which could interfere with the activity of peptidases such as trypsin which cleave after Arg and Lys; however, given HLZ degradation by trypsin at low RL concentrations, it appears that this potential obstacle is outweighed by the additional benefits of reducing overall electrostatic repulsion. Biological role of RL-induced peptidase susceptibitility


Biochemistry

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It is remarkable that HLZ becomes not only more susceptible to PAE proteolysis but also becomes sensitive to human defensive peptidases that normally do not target HLZ. Secretion of small secondary metabolites such as RL is energetically much less expensive and only requires simple resources, compared to e.g. production and secretion of the much larger peptidases. Secretion of anionic RL may therefore be a simple and inexpensive mechanism for PA to destabilize and inactivate host proteins and utilize host peptidases for its own benefit. Inactivation of the rhlA gene that prevents rhamnolipid synthesis reduces the tolerance of established PA biofilm towards monocyte-derived macrophages and polymorphonuclear leukocytes (PMNs) (23). Wild-type PA which produces RL caused lysis and necrosis of the host’s innate immune cells that arrive at infected sites while the PA rhlA mutant does not. Based on this observation the authors state that RL acts as a “protective shield” (23). The current study shows that RL also protect PA not only on the cellular level but also at the molecular level where RL can interact with host defensive proteins. We could also extend this to the cellular level by demonstrating increased protection of biofilm against HLZ attack by P. aeruginosa strain producing RL compared to a RL-free strain. Expression of PAE also influences RL production (62). A PAE deletion mutant showed reduced RL production that resulted in significantly decreased bacterial attachment, microcolony formation, and extracellular matrix linkage in biofilm. Supplementation with exogenous rhamnolipids however restored biofilm formation. This indicates that RL is important for biofilm formation and that RL expression is correlated to expression of PAE, highlighting an intricate and multifaceted colonization and invasion mechanism that will probably have more surprises in store for future investigations.


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AUTHOR CONTRIBUTIONS: K.K.A. and D.E.O. designed experiments, K.K.A., B.S.V. and C.S. performed experiments, D.E.O. and J.E. supervised experiments, K.K.A. and D.E.O. analyzed data and wrote the manuscript.

ACKNOWLEDGEMENTS We are grateful to Dr. Tim Tolker-Nielsen for providing P. aeruginosa strains with and without rhamnolipid production.


Biochemistry

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Biochemistry

Otzen, D. E., Sehgal, P., and Westh, P. (2009) α-lactalbumin is unfolded by all classes of detergents but with different mechanisms, J. Coll. Int. Sci. 329, 273-283. Giehm, L., Oliveira, C. L. P., Christiansen, G., Pedersen, J. S., and Otzen, D. E. (2010) SDS-induced fibrillation of α-synuclein: An alternative fibrillation pathway, Journal of Molecular Biology 401, 115133. Furmanski, P., Li, Z. P., Fortuna, M. B., Swamy, C. V., and Das, M. R. (1989) Multiple molecular forms of human lactoferrin. Identification of a class of lactoferrins that possess ribonuclease activity and lack iron-binding capacity, The Journal of experimental medicine 170, 415-429. Mairs, R. J., and Beeley, J. A. (1987) Isoelectric focusing of human salivary secretory-IgA, Archives of oral biology 32, 873-877. Otzen, D. E. (2016) Biosurfactants and surfactants interacting with membranes and proteins: same but different?, Biochim. Biophys. Acta In press. Tsai, C.-J., De Laureto, P. P., Fontana, A., and Nussinov, R. (2002) Comparison of protein fragments identified by limited proteolysis and by computational cutting of proteins, Prot. Sci. 11, 1753-1770. Polverino de Laureto, P., Frare, E., Gottardo, R., Van Dael, H., and Fontana, A. (2002) Partly folded states of members of the lysozyme/lactalbumin superfamily: a comparative study by circular dichroism spectroscopy and limited proteolysis, Protein Sci 11, 2932-2946. Radford, S. E., Dobson, C. M., and Evans, P. A. (1992) The Folding of Hen Lysozyme Involves Partially Structured Intermediates and Multiple Pathways., Nature 358, 302-307. Dobson, C. M., Evans, P. A., and Radford, S. E. (1994) Understanding how proteins fold: the lysozyme story so far, Trends Biochem. Sci. 19, 31-37. Matagne, A., Radford, S. E., and Dobson, C. M. (1997) Fast and slow tracks in lysozyme folding: insight into the role of domains in the folding process, Journal of Molecular Biology 267, 1068-1074. Yu, H., He, X., Xie, W., Xiong, J., Sheng, H., Guo, S., Huang, C., Zhang, D., and Zhang, K. (2014) Elastase LasB of Pseudomonas aeruginosa promotes biofilm formation partly through rhamnolipid-mediated regulation, Canadian journal of microbiology 60, 227-235.


Biochemistry

1 2 3 4 5 599 6 7 600 8 9 601 10 11 602 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 37

TABLES

Table 1. Proteolysis products obtained from lysozyme upon exposure to different peptidases and identified by mass spectrometry. Precursor

Mass precision

New N-

Peptidase

(m/z)

(ppm)

Score

terminal *

Peptide identified

Human

591.7275

0.91

42

32

K.RLGMDGYR.G

627.7881

2.27

48

117

R.VVRDPQGIR.A

513.6795

3.87

40

33

R.LGMDGYR.G

676.7589

0.03

55

59

T.RATNYNAGDR.S

563.1939

7.24

37

61

A.TNYNAGDR.S

512.6654

1.13

41

62

T.NYNAGDR.S

808.3301

0.89

67

69

R.STDYGIFQINSR.Y

627.7884

1.86

58

117

R.VVRDPQGIR.A

578.2556

0.51

52

118

V.VRDPQGIR.A

528.7207

0.86

41

119

V.RDPQGIR.A

641.2432

1.34

57

140

V.RQYVQGCGV.-

513.6764

2.16

35

33

R.LGMDGYR.G

457.1361

1.37

38

34

L.GMDGYR.G

Cathepsin

Human Neutrophil elastase

Proteinase K


Page 25 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 603 41 42 604 43 44 45 605 46 47 606 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Trypsin

678.259

2.63

40

51

A.KWESGYNTR.A

598.7094

1.6

55

60

R.ATNYNAGDR.S

627.7882

2.11

59

117

R.VVRDPQGIR.A

578.2534

3.4

47

118

V.VRDPQGIR.A

591.7287

1.04

38

32

K.RLGMDGYR.G

513.678

0.91

31

33

R.LGMDGYR.G

598.7089

0.8

71

60

R.ATNYNAGDR.S

860.0273

1.91

43

60

R.ATNYNAGDRSTDYGI FQINSR.Y

P.aeruginosa

808.3288

0.73

74

69

R.STDYGIFQINSR.Y

627.7904

1.4

61

117

R.VVRDPQGIR.A

455.6441

0.94

14

63

N.YNAGDR.S

493.1968

0.31

7

117

R.VVRDPQG.I

Elastase

*Uniport numbering


Biochemistry

1 2 3 4 5 607 6 7 608 8 9 609 10 610 11 12 611 13 14 612 15 16 613 17 18 614 19 20 615 21 22 616 23 24 617 25 26 618 27 28 619 29 30 620 31 621 32 33 622 34 35 623 36 37 624 38 39 625 40 41 626 42 43 627 44 45 628 46 47 629 48 630 49 50 631 51 52 632 53 54 633 55 56 634 57 58 59 60

Page 26 of 37

FIGURE LEGENDS Figure 1. Determination of the RL critical micelle concentration in PBS buffer using pyrene. The emission ratio (I3/I1) of pyrene changes in the concentration range 0.1-1 mM RL, and this is not affected by the presence of 2 µM HLZ, indicating that RL does not form clusters on the surface of HLZ. Insert shows chemical structure of rhamnolipid. Figure 2. Influence of RL on the secondary- and tertiary structure of HLZ. (A) far-UV CD and (B) near-UV CD spectra in PBS buffer and in the presence of 2 mM RL (>cmc). Figure 3. Influence of RL on HLZ activity. (A) Hydrolysis of 4-methylumbelliferyl β-D-N,N′,N′triacetylchitotrioside hydrate (structure shown in graph) over time followed by fluorescence. (B) Normalized activity of HLZ with increasing concentration of RL, based on the slope of data in figure A. Figure 4. Binding stoichiometry of RL to HLZ. (A) ITC enthalpograms for the titration of 25 mM RL into different concentrations of HLZ. Inflexion point (where the different enthalpograms for titration of RL into HLZ solutions merges with the enthalpogram for titration into buffer) is indicated with an arrow for the enthalpogram carried out with 4.0 mg/ml (280 µM) HLZ. (B) The RL concentration at each inflexion point plotted as a function of HLZ concentration. Binding numbers are derived from fitting data to Eq. (1). See text for details. Figure 5. Visualization of the electrostatic surface potential of HLZ at pH 7.4 (PDB code 1LZ1). The surface is dominated by a positive potential except around the active site where a negative potential is observed. Figure 6. Proteolysis of HLZ with PAE. (A) In the absence of RL, incubation of HLZ with PAE for 4 hrs at 37°C does not lead to detectable proteolysis. HLZ is however digested by PAE with increasing concentrations of RL; the band shifts to a slightly lower molecular mass and the overall intensity (right panel) declines. (B) Time course of HLZ digestion by PAE in the absence and presence of 1 mM RL. In the absence of RL no proteolysis is observed while HLZ incubated with 1 mM RL reveals progressive digestion of HLZ over time. Figure 7. HLZ proteolysis by model and human peptidases in the presence and absence of chemical and microbial surfactants. (A) In the absence of RL slight proteolysis of HLZ is observed when


Page 27 of 37

1 2 3 4 5 635 6 636 7 8 637 9 10 638 11 12 639 13 14 640 15 16 641 17 18 642 19 20 643 21 22 644 23 24 645 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

incubated with proteinase K, or Human Cathepsin G but not with Trypsin or Human elastase. In the presence of 1 mM RL, all peptidases digest HLZ. (B) Effect of surfactant type on proteolysis of HLZ. HLZ incubated with nonionic DDM is not digested but is completely digested in the presence of RL or SDS. Figure 8. Structure of HLZ highlighting residue 60 (red) and 116 (green) as the first sites of attack by RL-promoted proteolytic degradation. Fig. 9. Effect of RL production on biofilm growth. Two strains of Pseudomonas aeruginosa, namely the wt RL-producing variant and the deletion variant rhlA lacking RL biosynthesis, were made to form biofilm in a peg lid assay in the presence of 0-100 µg/ml HLZ.


Biochemistry

Figure 1.

1.2

1.1

1

Pyrene I3/I1

1 2 3 4 5 646 6 7 647 8 9 648 10 11 649 12 13 14 650 15 16 651 17 18 652 19 20 21 653 22 23 654 24 25 655 26 27 656 28 29 30 657 31 32 658 33 34 659 35 36 37 660 38 39 661 40 41 662 42 43 663 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 37

0.9

Rhamnolipid 0.8

0.7

0.6

I3/I1 - No HLZ I3/I1 - 2µM HLZ 0.5

0

0.5

1

1.5

2

2.5

3

[Rhamnolipid] (mM)


3.5

4

Page 29 of 37

Figure 2 4

1.5 10

A 4

Molar elepticity

1 10

5000

0

-5000

4

-1 10

0 mM RL 2 mM RL

4

-1.5 10

200

210

220

230

240

250

Wavelength (nm) 0

B -100

-200

Molar elepticity

1 2 3 4 5 664 6 7 665 8 9 666 10 11 667 12 13 14 668 15 16 669 17 18 670 19 20 21 671 22 23 672 24 25 673 26 27 674 28 29 30 675 31 32 676 33 34 677 35 36 37 678 38 39 679 40 41 680 42 43 681 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

-300

-400

-500

-600 0 mM RL 2 mM RL

-700 260

280

300

320

340

Wavelength (nm)


Biochemistry

Figure 3

∆Fluorescence at 450nm (A.U.)

0.5

0mM RL - No HLZ 0mM RL - HLZ 8mM RL - HLZ 0.4

0.3

0.2

0.1

0

-0.1

A 0

10

20

30

40

50

60

Time (min) 120 Normalized activity 100

Normalized activity (%)

1 2 3 4 5 682 6 7 683 8 9 684 10 11 685 12 13 14 686 15 16 687 17 18 688 19 20 21 689 22 23 690 24 25 691 26 27 692 28 29 30 693 31 32 694 33 34 695 35 36 37 696 38 39 697 40 41 698 42 43 699 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 37

80

60

40

20

B

0 0

2

4

6

8

[RL] (mM)


10

Page 31 of 37

Figure 4

0

A ∆H (kcal/mol Rhamnolipid)

-200

-400

-600

-800 0.0mg/mL LZ 0.5mg/mL LZ 1.0mg/mL LZ 1.5mg/mL LZ 2.0mg/mL LZ 2.5mg/mL LZ 3.0mg/mL LZ 3.5mg/mL LZ 4.0mg/mL LZ

-1000

-1200

-1400 0

1

2

3

4

5

RL (mM) 2.5

B 2

Rhamnolipid (mM)

1 2 3 4 5 700 6 7 701 8 9 702 10 11 703 12 13 14 704 15 16 705 17 18 706 19 20 21 707 22 23 708 24 25 709 26 27 710 28 29 30 711 31 32 712 33 34 713 35 36 37 714 38 39 715 40 41 716 42 43 717 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

1.5

1

0.5 2

y = 0.27568 + 8.4293x R = 0.99938 Saturation 0 0

0.05

0.1

0.15

0.2

0.25

LZ (mM) 31 ACS Paragon Plus Environment

0.3

Biochemistry

1 2 3 4 5 718 6 7 719 8 9 720 10 11 721 12 13 14 722 15 16 723 17 18 724 19 20 21 725 22 23 726 24 25 727 26 27 728 28 29 30 729 31 32 730 33 34 731 35 36 37 732 38 39 733 40 41 734 42 43 735 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 5


Page 32 of 37

Page 33 of 37

4.0 mM RL

1.2 Residual fraction

1

Fraction of HLZ

2.0 mM RL

1.0mM RL

0.5 mM RL

0.25 mM RL

0.125mM RL

0.063mM RL

0.031mM RL

0mM RL

0mM RL

A

0.016 mM RL

Figure 6

15 kDa

0.8 0.6 0.4 0.2

10 kDa

PAE

-

+

+

+

+

+

+

+

+

+

0

+

0

1

2

3

4

[RL] (mM)

360min

1.2 0 m M RL 1 m M RL

1 Fraction of intact HLZ

240min

180min

120min

60min

0min

360min

240min

60min

120min

180min

B 0min

1 2 3 4 5 736 6 7 737 8 9 738 10 11 739 12 13 14 740 15 16 741 17 18 742 19 20 21 743 22 23 744 24 25 745 26 27 746 28 29 30 747 31 32 748 33 34 749 35 36 37 750 38 39 751 40 41 752 42 43 753 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

15 kDa

10 kDa

0.8 0.6 0.4 0.2 0 0

1mM RL -

-

-

-

-

-

+

+

+

+

+

+


1

2

3

Time (h )

4

5

6

Biochemistry

Human Elastase

Proteinase K

Trypsin

No peptidase Trypsin

No peptidase

A

Human Elastase

Proteinase K Human Caphepsin G Human Caphepsin G

Figure 7

15 kDa

10 kDa

Trypsin:

-

+

+

-

+

-

+

-

+

1 mM DDM

1 mM RL

B

+ 1 mM SDS

1mM RL -

Buffer

1 2 3 4 5 754 6 7 755 8 9 756 10 11 757 12 13 14 758 15 16 759 17 18 760 19 20 21 761 22 23 762 24 25 763 26 27 764 28 29 30 765 31 32 766 33 34 767 35 36 37 768 38 39 769 40 41 770 42 43 771 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 37

+


-

+

Page 35 of 37

1 2 3 4 5 772 6 7 773 8 9 774 10 11 775 12 13 14 776 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 777 43 44 778 45 46 779 47 48 780 49 50 781 51 52 53 54 55 56 57 58 59 60

Biochemistry

Figure 8


Biochemistry

Figure 9

100

Relative amount of biofilm [%]

1 2 3 4 5 782 6 7 783 8 9 784 10 11 785 12 13 14 786 15 16 787 17 18 788 19 20 21 789 22 23 790 24 25 791 26 27 792 28 29 30 793 31 32 794 33 34 795 35 36 37 796 38 39 797 40 41 798 42 43 799 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 36 of 37

RL negative RL positive

80

60

40

20

0 0

0,001

0,01

0,1

1

10

[Human lysozyme] (µg/ml)


100

1000

Page 37 of 37

Proteases

0.016 mM RL

0.031mM RL

0.063mM RL

0.125mM RL

0.25 mM RL

0.5 mM RL

1.0mM RL

2.0 mM RL

4.0 mM RL

Protease

0mM RL

cmc 0mM RL

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

Biochemistry

-

+

+

+

+

+

+

+

+

+

+

ACS Paragon Plus Environment

Human Lysozyme Peptidase Resistance Is Perturbed by the Anionic

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