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Incorporation of a VLK-Containing Substrate in the Bacterial Cell Wall Silvie Hansenová-Mañásková, Floris J. Bikker, Kamran Nazmi, Rianne van Zuidam, Johan A Slotman, Wiggert A van Cappellen, Adriaan B Houtsmuller, Enno C.I. Veerman, and Wendy E Kaman Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00381 • Publication Date (Web): 09 Sep 2016 Downloaded from http://pubs.acs.org on September 10, 2016

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Bioconjugate Chemistry

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Incorporation of a VLK – containing

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substrate in the bacterial cell wall

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Silvie Hansenová Maňásková 1,2, Floris J. Bikker 2, Kamran Nazmi 2, Rianne van Zuidam 1,

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Johan A. Slotman 3, Wiggert A. van Cappellen 3, Adriaan B. Houtsmuller 3, Enno C. I.

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Veerman 2, Wendy E. Kaman 1,2*

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1

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Department of Medical Microbiology and Infectious Diseases, Erasmus MC, Wytemaweg 80, 3015 CE Rotterdam, the Netherlands

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9

2

Department of Oral Biochemistry, Academic Centre for Dentistry Amsterdam, University of

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Amsterdam and VU University Amsterdam, Gustav Mahlerlaan 3004, 1081 LA Amsterdam,

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the Netherlands

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3

Optical Imaging Center, Department of Pathology, Josephine Nefkens Institute, Erasmus MC, Dr Molewaterplein 50, Rotterdam 3015 GE, the Netherlands

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* Mailing address: Department of Medical Microbiology and Infectious Diseases, Erasmus

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MC, Wytemaweg 80, 3015 CE Rotterdam, the Netherlands. Phone: (+31) 10 703 2177. E-

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mail: [email protected]

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ACS Paragon Plus Environment

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Abstract

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The emergence of antibiotic resistant bacteria is a major public health threat and therefore

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novel antimicrobial targets and strategies are urgently needed. In this regard, cell wall

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associated proteases are envisaged as interesting antimicrobial targets due to their role in cell

25

wall re-modeling. Here, we describe the discovery and characteristics of a protease substrate

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which is processed by a bacterial cell wall associated protease. Stationary phase grown Gram-

27

positive bacteria were incubated with fluorogenic protease substrates, and their cleavage and

28

covalent incorporation into the cell wall was analyzed. Of all substrates used only one

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substrate, containing a valine/leucine/lysine (VLK-) motif, was covalently incorporated into

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the bacterial cell wall. Linkage of the VLK- peptide substrate appeared unrelated to sortase A

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and B activity as both wild-type and sortase A and B knock out Staphylococcus aureus strains

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incorporated this substrate into their cell wall with comparable efficiency. Additionally, the

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VLK-peptide substrate showed significantly higher incorporation in the cell wall of VanA

34

positive Enterococcus faecium strains than in VanB, and vancomycin susceptible isolates.

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In conclusion, the VLK-substrate identified in this study shows promise as vehicle e.g. for

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targeting antimicrobial compounds and diagnostic contrast agents to the bacterial cell wall.

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Introduction

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The growing prevalence of antibiotic resistant bacteria underscores the need to discover

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treatment strategies that will enable the development of novel antimicrobial agents. A group

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of bacterial proteins current under investigation as a tool in novel antimicrobial strategies is

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the group of cell wall associated proteases 1, 2. These proteases, e.g. the bacterial sortases, are

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of particular interest due to their ability to covalently link proteins to the peptidoglycan layer

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of Gram-positive bacteria

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cleavage of specific sorting recognition motifs of endogenous cell wall proteins 4, 5. Cell wall

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modeling may thus be influenced by incorporation of exogenous synthetic compounds

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containing such a recognition motif. For example, recombinant sortase activity has been used

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to display antibodies and antigens to the cell wall of lactic acid bacteria 6. Coupling of these

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proteins to non-pathogenic bacteria protects them from proteolytic degradation and increases

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their on-site concentration. In line, it is envisaged that cell wall associated proteases can be

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exploited to target antimicrobial compounds or antibiotic loaded vesicles to the bacterial cell

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wall. A recent study from our group demonstrated the feasibility of this strategy by showing

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the successful incorporation of a synthetic sortase A specific substrate into the cell wall of

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Gram-positive bacteria 7-9.

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Here, we studied the suitability of a general protease substrate, and its analogs, to target the

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bacterial cell wall. We examined incorporation efficiency of the substrates using Gram-

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positive bacteria including vancomycin resistant isolates and compared their linkage velocity

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with a sortase A synthetic substrate. Additionally, the relationship between substrate

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incorporation and sortase activity was studied.

3, 4

. A crucial step in protein incorporation is the recognition and

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Results

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Proteolytic activity of S. aureus on substrate PEK-054 and its analogs

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The protease-specific substrates (Table 1) were incubated with whole S. aureus cells and

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culture supernatant of an overnight S. aureus culture, potentially loaded with proteases. Next

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increase in fluorescence, representing protease activity, was determined for 1 hr. All protease

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substrates tested were cleaved by an overnight culture of S. aureus with the parent substrate

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PEK-054 being degraded with the highest efficiency (Figure 1A). Similar results were

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obtained using S. aureus supernatant. However substrate PEK-054 was hydrolyzed by the

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supernatant at a lower rate than by the overnight culture. No differences could be detected

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between degradation by culture supernatant and cell culture of the PEK-054 analogs (Figure

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1A). To confirm that degradation of the protease substrates was indeed related to protease

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activity peptide cleavage was monitored in the presence of leupeptin, EDTA, and a protease

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inhibitor cocktail (PI). Leupeptin had no effect on degradation of substrate #1, containing the

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unique valine/leucine/lysine (VLK-) motif (Table 1, underlined), though EDTA and the PI did

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inhibit substrate degradation (Figure 1B). When the assay was performed with heat-

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inactivated S. aureus overnight culture (HI) no cleavage was observed as well (Figure 1B).

79 80

Table 1. Sequences of protease substrate PEK-054 and its analogs Peptide

Sequence

Hydrophobicity

PEK-054

FITC-

NleKKKKVLPIQLNAATDK

-KDbc

44%

#1

FITC-

NleKKKKVL

-KDbc

38%

#2

FITC-

-KDbc

43%

#3

FITC-

-KDbc

63%

#4

FITC-

-KDbc

50%

#5

FITC-

-KDbc

38%

KKKVLP VLPIQLN IQLNAAT LNAATDK

81


4

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Figure 1. Cleavage and incorporation of protease-specific peptide substrates in the cell

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wall of S. aureus. (A) Degradation efficiency of PEK-054 and its analogs by S. aureus

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overnight culture and filtered culture supernatant was measured for 1 hr at 37 oC using 16 µM

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of each protease substrate. Proteolytic activity was defined in ∆F/min. (B) The effect of

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protease inhibitors leupeptin, EDTA and protease inhibitor cocktail (PI) or heat-inactivation

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(HI) of S. aureus culture supernatant on VLK-peptide substrate (#1) degradation was

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monitored with 250 µM protease substrate. Proteolytic activity was measured for 1 hr at 37 oC

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and defined in ∆F/min. Results are expressed as mean ± SEM (n = 3).

91 92

Incorporation of a VLK-peptide substrate to the cell wall of S. aureus

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In order to locate the protease involved the protease substrates were incubated with S. aureus

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bacteria followed by an SDS heat treatment to select for covalent binding. Next, incorporation

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was measured using FACS analysis and the binding location was visualized with a Structured

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Illumination Microscope (SIM) which has a two times higher resolution compared to


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conventional optical microscopes. Although all tested protease substrates were cleaved by S.

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aureus only the protease substrate #1 (VLK-peptide substrate) was covalently linked to the

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bacterial cell wall upon cleavage (Figure 2A). SIM analysis showed incorporation of the

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FITC-labelled VLK-peptide fragment in the bacterial cell wall (Figure 2B, incl. insert a and

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b). No fluorescence, i.e. VLK-peptide incorporation, could be detected in the septa of dividing

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S. aureus cells (Figure 2B, insert c). No direct relation was observed between bacterial

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binding and hydrophobicity of the protease substrates (Table 2). Incorporation of the VLK-

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peptide substrate was further characterized by varying the protease substrate concentration

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and incubation time. It was found that linkage of this substrate to the S. aureus cell wall

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occurs in a dose-dependent manner (Figure 2C) with a plateau reached after 30 min (Figure

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2D).

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Figure 2. Characterization of VLK-peptide substrate incorporation in the S. aureus cell

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wall. (A) Linkage of the protease substrates to the bacterial cell wall was monitored by

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incubating 250 µM protease substrate with stationary phase S. aureus cells in PBS. After SDS

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heat-treatment bacteria were analyzed for peptide linkage using flow cytometry. (B)

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Incorporation position of protease substrate #1 (VLK-peptide substrate) was monitored using

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SIM. Scale bar represents 1 µm. (C) Dose dependency of VLK-peptide substrate

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incorporation was analyzed using flow cytometry by incubating stationary phase S. aureus

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cells in PBS with varying concentrations substrate. (D) Time dependency of VLK-peptide

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incorporation was analyzed using flow cytometry by incubating stationary phase S. aureus

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cells in PBS with varying incubation times (250 µM). Results are expressed as mean ± SEM

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(n = 3).

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Table 2. Enterococcus faecium clinical isolates used in this study 15 MIC (µg/mL)

VanA

VanB

Sensitive

123

a

Isolate no

Type a

Vancomycin

Teicoplanin

26

AF3

> 256

32

29

AF3

> 256

32

30

AF3

> 256

32

37

AA1

> 256

1

39

AA1

> 256

1

10

B

8

0.5

11

B

8

0.5

12

B

8

0.5

22

B

16

0.5

42

B

8

0.5

44

B

8

0.5

S1

n/a

< 0.5

< 0.5

S2

n/a

1

< 0.5

S3

n/a

1

< 0.5

S4

n/a

< 0.5

< 0.5

n/a: not applicable

124


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VLK-peptide substrate cell wall linkage is not related to sortase A or B activity

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To study the role of sortase A and B in VLK-peptide linkage, incorporation efficiency of the

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VLK-peptide substrate was monitored using S. aureus sortase A and B knock-out strains.

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Protease substrate #5, without VLK-motif, was used as a negative control. Both the sortase A

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and B knock-out strains were able to link the VLK-peptide substrate to their cell wall with

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similar efficiency as the wild-type strain (Figure 3A). No incorporation was observed using

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the control substrate without VLK-motif. Additionally, the effectiveness of cell wall linkage

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of the cleaved VLK-peptide substrate was compared to linkage of a substrate specific for

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sortase A (LPETG) using the S. aureus wild-type strain 7, 8. It was found that the VLK-peptide

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substrate was incorporated with a significant higher efficiency than the sortase A substrate

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(Figure 3B). Using the scrambled version of the sortase A substrate (EGLTP) no

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incorporation was observed.

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Figure 3. The role of sortase A and B in VLK-peptide substrate incorporation. (A)

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Stationary phase S. aureus wild-type (WT), sortase A knock-out (srtA KO) and sortase B

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knock-out (srtB KO) cells in PBS were incubated with 1 mM VLK-peptide substrate (#1) or

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its negative control (#5). (B) Additionally incorporation efficiency of the VLK-peptide

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substrate was compared with cell wall linkage of a sortase A specific substrate (LPETG). S.

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aureus WT cells were incubated with 1 mM LPETG, its scrambled analog EGLTP (negative

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control), VLK-peptide substrate (#1) or its negative control (#5). After 1 hr incubation at 37

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o

C the bacteria were 10 min heat-treated with 1% SDS, washed and analyzed for peptide

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linkage using flow cytometry. Results are expressed as mean ± SEM (n = 3). P-values were

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calculated using an unpaired, two-tailed students t-test (* P < 0.05).

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Bacterial incorporation spectrum of the VLK-peptide substrate

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To obtain more information on the specificity of the VLK-peptide substrate we studied

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incorporation of this substrate into the cell walls of S. epidermidis, E. faecium and E. faecalis.

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In all bacteria the VLK-peptide became associated to the bacterial cell wall to the same extent

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(Figure 4A). SIM analysis showed clear cell wall staining for the Staphylococci. In the

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Enterococci however cell wall staining was less pronounced probably due to the fact that

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these bacteria are smaller and reached beyond SIM resolution (Figure 4B). To explore the

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ability of the VLK-peptide substrate to covalently bind to the cell wall of antibiotic resistant

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bacteria we screened the incorporation efficiency of the protease substrate with high (VanA)

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and low (VanB) vancomycin resistant and vancomycin susceptible E. faecium isolates. We

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determined that the peptide was incorporated in the cell walls of the vancomycin resistant

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isolates with at least comparable linkage efficiency as the susceptible isolates (Figure 4C).

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The VanA E. faecium isolates even showed a significant higher incorporation efficiency

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compared to the VanB E. faecium isolates which is related to the higher proteolytic activity

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measured in culture supernatants of these isolates (Figure 4D).

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Figure 4. Incorporation spectrum of the VLK-peptide substrate. Stationary phase S.

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aureus, S. epidermidis, E. faecium and E. faecalis were incubated with 250 µM VLK-peptide

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substrate. After 1 hr incubation at 37 oC the bacteria were SDS heat-treated and analyzed for

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peptide linkage using (A) flow cytometry and (B) Structured Illumination Microscopy (SIM).

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Scale bar represents 1 µm. (C) VLK-peptide substrate incorporation efficiency was analyzed

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using E. faecium VanA, VanB and vancomycin sensitive strains. (D) Degradation of the

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VLK-peptide substrate by E. faecium VanA, VanB and vancomycin sensitive overnight

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culture was measured for 1 hr at 37 oC using 16 µM peptide. Proteolytic activity was defined

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in ∆F/min. Results are expressed as mean ± SEM (n = 3). P-values were calculated using an

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one-way ANOVA with Bonferroni correction (* P < 0.05).

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180

Discussion

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S. aureus, E. faecium, E. faecalis and S. epidermidis are able to cleave a VLK-containing


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substrate and link it to their cell wall. The observation that the VLK-peptide substrate remains

183

incorporated in the bacterial cell wall after SDS heat-treatment suggests a covalent binding of

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the substrate to the bacterial cell wall. A group of bacterial proteases known to cleave and

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subsequently link proteins to this part of the bacterial cell wall are the cell wall associated

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sortases 3. These sortases might thus play a role in VLK-peptide substrate cleavage and

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linkage. However experiments using sortase A and B knock-out strains showed that

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incorporation of the VLK-peptide substrate is unrelated to sortase A and B activity suggesting

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that another bacterial protease is involved. Since the VLK-peptide substrate was also

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degraded by S. aureus culture supernatant the responsible protease might be secreted, or

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released, by the bacterium. Activity of the protease was inhibited in the presence of EDTA

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which suggests that this protease belongs to the family of metallo proteases. Therefore it is

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postulated that proteases involved in cleavage of the VLK-peptide substrate are aureolysin

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from S. aureus and coccolysin from E. faecalis. Both proteases belong to the same metallo

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protease family (M04) and recognize substrate sequences wherein valine, leucine and lysine

196

are present 10.

197

Although all the protease substrates tested in this study were cleaved by S. aureus, only the

198

VLK-containing substrate was incorporated in the bacterial cell wall. This might be related to

199

the fact that as well valine as leucine belong to the group of Branched Chain Amino Acids

200

(BCAAs). S. aureus needs BCAA transport for growth whereas it exhibits an auxotrophic

201

phenotype for leucine and valine. Genes for the synthesis of these amino acids are present in

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S. aureus, though the bacterium prefers to take up these BCAAs from its environment 11. The

203

hypothesis that the presence of BCAAs in the VLK-peptide substrate is important for cell wall

204

linkage is strengthened by the fact that S. aureus is known to take up BCAAs from its

205

environment with a high efficiency

206

which the VLK-peptide substrate was incorporated.

12

. This might relate to the observed high velocity with


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From our results it may be concluded that BCAA-containing motifs could also be used by

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Gram-positive bacteria to couple (host-) proteins to their peptidoglycan layer. A BLAST

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search of the protease substrate revealed that the amino acid sequence of the VLK-peptide

210

substrate (KKVLK) is present in the human protein CXCL-9. In this view it is interesting to

211

note that CXCL-9 is linked to the cell wall of S. aureus by a yet unknown mechanism

212

Possibly S. aureus links CXCL-9 to its cell wall using the same mechanism by which it

213

cleaves and incorporates the VLK-peptide substrate. Based on the results described in this

214

study we suggest that BCAA-containing proteins which are present in the surroundings of the

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bacterium are recognized and cleaved by secreted bacterial proteases. As a result these

216

BCAAs become available for the synthesis of bacterial proteins and/ or modelling of the

217

bacterial cell wall.

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Taken together, this study has revealed the presence of a highly active cell wall associated

219

protease in several clinically relevant Gram-positive bacterial pathogens. Cell wall linkage of

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the discovered protease-specific peptide substrate appeared to be more effective compared to

221

sortase-related linkage and has high potential for use as a bacterial linker. The knowledge

222

obtained within this study could be exploited for the design of novel antimicrobial treatments

223

and/or diagnostic strategies. For example via the anchoring of (novel) antimicrobial

224

compounds or antibiotic containing liposomes to the bacterial cell wall. Additionally the

225

VLK- peptide substrate described could be used for diagnostic purposes; the recognition motif

226

for the novel protease could be used as a tracer for molecular imaging techniques e.g.

227

ultrasound microbubbles 14.

13

.

228 229

Experimental Procedures

230

Bacteria


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231

The bacteria used in this study were Staphylococcus aureus 8325-4 wild-type (WT) and its

232

isogenic sortase A (srtA KO) and B (srtB KO) knock-out strains. Besides, Enterococcus

233

faecium (clinical isolates, Table 2,

234

Staphylococcus epidermidis ATCC12228 were used. For the preparation of culture

235

supernatants, bacteria were grown overnight in 5 mL Brain Heart Infusion (BHI) medium

236

(BioTrading, Mijdrecht, the Netherlands) at 37 oC (200 rpm). Bacteria were then pelleted by

237

centrifugation for 5 min at 10,000 rpm. Both the overnight culture and culture supernatant

238

were used to characterize proteolytic activity.

15

), Enterococcus faecalis ATCC29212 and

239 240

Substrate synthesis

241

The fluorogenic protease substrates used in this study were synthesized by solid-phase peptide

242

synthesis using Fmoc chemistry with a MilliGen 9050 peptide synthesizer (Milligen-

243

Biosearch, Bedford, MA, USA) (Table 2). The protease substrates were C-terminally flanked

244

with a fluorescent probe; FITC, N-terminally flanked with a lysine coupled quencher; Dabcyl

245

(Dbc), and purified by reverse-phase high-performance liquid chromatography (RP-HPLC).

246

Authenticity and purity of the peptides was confirmed by mass spectrometry conducted as

247

described previously 16. Stock solutions of the substrates were prepared in DMSO (800 µM,

248

protease assay) or 0.01% acetic acid in PBS, pH 6.3 (2 mM, incorporation experiments) and

249

stored at – 20 oC. The sortase A specific peptide substrate, FITC-LPETG-amide, and its

250

scrambled analog FITC-EGTLP-amide (negative control) were synthesized as described

251

previously 8.

252 253

Protease activity assay

254

Proteolytic activity was determined by incubating 16 µM substrate with 50 µL overnight

255

culture or bacterial culture supernatant at 37 oC in blackwell 96-well plates (Corning, Lowell,


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256

USA) as described earlier

. BHI medium was used as a negative control. Increase in

257

fluorescence, representing substrate degradation, was monitored for 60 min with 2 min

258

intervals using a fluorescence microplate reader (FLUOstar Galaxy, BMG Laboratories,

259

Offenburg, Germany) at an excitation wavelength of 485 nm and an emission wavelength of

260

530 nm. Additionally, the assay was performed with S. aureus overnight culture in which the

261

present proteases were heat-inactivated by boiling for 15 min at 95 oC. VLK-peptide

262

degradation was also monitored with S. aureus overnight culture in the presence of 1 mM

263

EDTA (Sigma Aldrich, Zwijndrecht, the Netherlands), 1 mM leupeptin (Sigma Aldrich,

264

Zwijndrecht, the Netherlands) or 1x cOmplete mini protease inhibitor cocktail (Roche, Sigma

265

Aldrich, Zwijndrecht, the Netherlands). Fluorescence values were corrected for background

266

fluorescence using the negative control (BHI medium). Protease activity was expressed as ∆F

267

per minute (∆F/min).

268 269

FACS analysis

270

Bacteria were grown in 5 mL BHI at 37 oC and the next day 500 µL culture was centrifuged

271

for 5 min at 14,000 rpm. The bacterial pellet was washed twice with 1 mL PBS, and then

272

suspended in 500 µL PBS. Subsequently 25 µL of the bacterial suspension was incubated

273

with varying concentrations protease substrate in 0.01% acetic acid in PBS (pH 6.3) in a

274

blackwell, 96-well plate (Corning, Lowell, USA) under shaking conditions (200 rpm). After

275

the indicated time periods (0 - 60 min) the samples were transferred to 1.5 mL tubes and

276

washed three times with PBS. Subsequently the bacteria were incubated with 1% SDS at 60

277

o

C for 10 min to remove non-covalently attached substrates. The bacteria were washed three

278

times with PBS and incorporation was measured using an Accuri C6 Flow Cytometer (BD

279

Biosciences, Breda, the Netherlands) equipped with a 488 nm argon laser and a 533/30 nm

280

band pass filter (FL1). Fluorescence intensity was corrected for the negative control (bacteria


16

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incubated with 0.01% acetic acid in PBS (pH 6.3)) and expressed as median arbitrary

282

fluorescence units (a.u.) of the 10,000 events that were counted for each data set. The data

283

was analyzed using the BD Accuri C6 Software (version 1.0.264.21).

284 285

Structured Illumination Microscopy

286

Bacteria were incubated with 250 µM protease substrate in 0.01% acetic acid in PBS (pH

287

6.3). After 30 min, bacteria were centrifuged for 5 min at 14,000 rpm and the obtained pellet

288

was washed three times with PBS. Subsequently the bacteria were incubated with 1% SDS at

289

60 oC for 10 min to remove non-covalently attached substrates. The bacteria were washed

290

three times with PBS, fixed with 4% formaldehyde in PBS and analyzed using Structured

291

Illumination Microscopy (SIM). 3D-SIM data was acquired using a 63x 1.4NA oil objective.

292

A 488 nm diode laser, together with a BP 495-575 + LP 750 emission filter, was used to

293

excite the fluorophores. For 3D-SIM imaging a 28 µm grating was present in the light path.

294

The grating was modulated in 5 phases and 5 rotations, and multiple z-slices with an interval

295

of 110 nm were recorded on an Andor iXon DU 885, 1002x1004 EMCCD camera. Raw

296

images were reconstructed using the Zeiss Zen 2012 software and transmission images from

297

the same field of view were recorded.

298 299

Acknowledgments

300

We would like to thank Dr. J.P. Hays for providing the E. faecium VanA and VanB clinical

301

isolates and Dr. W.J.B. van Wamel for the S. aureus 8325-4 wild-type strain and the isogenic

302

sortase A and B knock-out strains.

303 304

Abbreviations

305

VLK

: Valine/leucine/lysine


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306

BCAAs : Branched Chain Amino Acids

307

SIM

: Structured Illumination Microscopy

308

FITC

: Fluorescein isothiocyanate

309

Dbc

: Dabcyl

310

BHI

: Brain Heart Infusion

Page 18 of 22

311


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