Supporting Information Bacterial Capture Efficiency and Antimicrobial ...
Supporting Information
Bacterial Capture Efficiency and Antimicrobial Activity of Phage Functionalized Model Surfaces
ZEINAB HOSSEINIDOUST1, THEO G.M. VAN DE VEN2, and NATHALIE TUFENKJI*,1
1
Department of Chemical Engineering, McGill University, Montreal, Quebec H3A 2B2, Canada
2
*
Department of Chemistry, McGill University, Montreal, Quebec H3A 2K6, Canada
Corresponding Author. Phone: (514) 398-2999;
Fax: (514) 398-6678;
E-mail:
[email protected] 1
Theoretical background Phages P22, PRD1, and PR772 infect S. typhimurium. P22 is an icosahedral shaped virus from the Podoviridae family (Table 1 and Figure 1a). P22 is approximately 52–60 nm in length and its genetic material comprises of 43,400 bp of double-stranded (ds) DNA 1. P22 consists of an icosahedral capsid and a short tail; its receptors are located on its tail spikes, therefore its orientation is an issue for this phage when immobilized. PRD1 and PR772 are cubic bacteriophages from the Tectiviridae family and have linear ds DNA genome; PR772 bacteriophage is 97.2% identical to PRD1 at the genetic level 2. The family Tectiviridae includes phages with a lipid membrane beneath the icosahedral shell. PRD1 is a well studied phage; while less is known about the structure of PR772, it can be surmised to be highly similar on the basis of the overall similarity of Tectiviridae family phages2. PRD-1 is a somatic phage, meaning it attaches to the bacterial cell surface. Upon attachment to the bacterial surface, the lipoprotein vesicle becomes a tail-like tube about 60 nm in length, thus a nucleic acid ejection device . A single phage structural protein located at the vertices is responsible for PRD1 attachment to its host 3. As this protein is located symmetrically on all the vertices of the capsid (Figure 1b), orientation is not an issue for either PRD1 or PR772. PR772 also infects E. coli, however, its mode of infection is different. PR772 is an F-specific phage and it adsorbs by one of its vertices to tips of F-pili (mating organelle) in E. coli 4. MS2 is a single strand (ss) RNA phage that also infects E.coli. Single-stranded RNA coliphages are found wherever E. coli lives, for example in the intestinal tract of humans 5. MS2 is also an F-specific phage but attaches laterally along the pili of the host (as most ssRNA 2
phage). Both PR772 and MS2 attach their RNA/DNA to the pili which will then have to transfer the nucleic acids into the cell. MS2, although symmetrical in shape, is not symmetrical in terms of attachment proteins; it has only one attachment protein called a “maturation protein” 5 on one of the vertices. T4 phage is another phage infecting E. coli, and consists of a long contractile tail and an elongated icosahedral capsid which encapsulates a ds-DNA genome (Figure 1c). The T4 phages are typically 200 nm long and perform lytic cycles inside the host bacterium E. coli 6.T4 is one of the most well studied phages and this explains why many of the published studies on the use of immobilized phage are performed with T4 despite its highly asymmetrical structure and specific requirements for infectivity.
Methods Surface characterization XPS (X-ray photoelectron spectroscopy) was performed at the LASM (École Polytechnique Surface Analysis Laboratory, Montreal, Quebec) on model surfaces at various stages of functionalization. XPS studies were performed on a VG ESCALAB 3 MKII (VG, Thermo Electron Corporation, UK). Samples were irradiated using an MgKα source at a take-off angle of 0o (i.e., perpendicular); analyzed surface was 2 mm × 3 mm and the depth sampled was ~50-100 Å. Both low resolution and N1s, C1s and O1s high-resolution analyses, with a scan width of 20 eV, were performed. The quality of APTES-coated surfaces before and after functionalization was examined using atomic force microscopy (AFM). The surfaces were used as prepared without any further 3
modifications. Imaging was performed using a NanoScope IIIa scanning probe microscope (Veeko/Digital Instruments, USA) in air under tapping mode using a commercial n+-silicone cantilever (Nanoscience Instruments, Phoenix, USA) 240 μm long and 35 um wide, with a resonant frequency of 50-130 kHz and spring constant of 9.0 N/m. The scanning rate was 1.0 Hz, at 0 angle. Image processing was performed using Research NanoScope III software version 6.11r1. All images were filtered using the flattening built-in tool and roughness values were obtained by utilizing the built-in tool for cross-sectional analysis. The streaming potential of APTES-coated surfaces was measured using an asymmetric clamping cell using an electrokinetic analyzer (Anton Paar, Graz, Austria) 7. The measurements were performed at room temperature with 10 mM KCl buffered with KHCO3 to pH 7.5. For streaming potential measurements, rectangular cover slides (No.2, 24×60 mm, VWR) were cleaned and coated as described previously. Measurements were performed in triplicate and the zeta potential was calculated from the measured streaming potential as per 8. Contact angle goniometry was used as a measure of the degree of hydrophobicity of the substrate. Prior to measurement of contact angles, the slides were rinsed in DI water and dried under a stream of high purity N2. Equilibrium contact angle measurements were performed using an OCA-30 goniometer (Future Digital Scientific Corp., NJ, USA) on sessile drops (1 μL droplets) by measuring the tangent to the drop at its intersection with the surface. All measurements were repeated at least three times.
4
Bacteria and phage characterization Phages were examined by transmission electron microscopy (TEM). A glow-discharged carbon coated copper grid was placed on a 5 μL drop of purified phage stock mixed with a 5 μL 4% uranyl acetate for 1 min and washed 3 times by placing on drops of water for 10 sec. Samples were examined on a Philips Tecnai 12 120KV TEM and images were captured with a Gatan 792 Bioscan 1k × 1k Wide Angle Multiscan CCD Camera. Scanning electron microscopy (SEM) was used to image bacteria and selected discs with immobilized phage and captured bacteria. Suspended bacteria were washed twice, pelleted, and resuspended in 2.5% gluteraldehyde in SM-buffer and kept at 4
for 1 hr for fixation. The fixed samples were
subsequently washed with SM-buffer. A drop of fixed sample was placed on poly-L-lysine coated slides for 15 min and the slide was blot dried. The slides were subsequently dehydrated with a graded series of 10-min ethanol immersions (30 to 100%) and a graded series of amyl acetate in ethanol solutions (25 to 100%). The slides were air dried and coated with 300 of AuPd coating (Hummer VI Au-Pd sputter coater). The discs with captured bacteria were immediately fixed with gluteraldehyde and the dehydration and coating steps were carried out as described above. Samples were examined with a Hitachi S-4700 Field Emission-STEM (FESTEM, Tokyo, Japan) and images were recorded using Quartz PCI v. 6.0 software. Dynamic light scattering (DLS) was used to evaluate the hydrodynamic diameter of bacteria and phage particles using the Malvern Instruments Zetasizer Nano-ZS. Bacteria were cultured as previously described, washed three times to remove growth media, and finally resuspended in SM buffer. Phage was propagated and purified as previously described and was 5
also in SM buffer. Each measurement was repeated five times at room temperature and standard quartz cuvettes were used for all measurements. The electrophoretic mobility of the phage and bacteria was measured using the same equipment. The zeta potential of the phage and bacteria was calculated from the electrophoretic mobility measurements using the Henry equation 8. Samples were prepared as described for DLS measurements and the standard universal dip cell™ was used for the measurements.
Figure S1. Schematic representation of different phages used in this study (a) PRD1 and PR772, (b) MS2, (c) T4, and (d) P22. Reproduced from ref.
9
with permission from Oxford University
Press, USA. Images are not to scale.
6
Figure S2. Scanning electron micrographs of (a) PRD1 bacteriophage immobilized on the surface of APTES coated discs functionalized with EDC-NHS, (b) higher magnification image of area enclosed by black square in (a), showing the shape and size of PRD1.
7
Figure S3. Immobilized phage is infective as indicated by the lysis ring around the disc (result presented for PRD1 and S. typhimurium).
Figure S4. (a) S. typhimurium attached to phage functionalized surface, (b) higher magnification image of the same sample as (a) showing lysed S. typhimurium on the phage-functionalized discs.
8
Figure S5. Growth curve for bacteria with and without the presence of phage (a) S. typhimurium LT2, (b) E. coli 15597. A 25 µL aliquot of overnight culture of host bacteria (diluted in 100 uL of fresh TSB) was infected with 25 µL of phage and monitored in a 96 well plate for 12 hours at 37C. As no effort was made to optimize the multiplicity of infection (MOI), phages that are known to be very sensitive to MOI (namely, MS2 and PR772) do not exhibit significant host growth inhibition. References 1. Ackermann, H. W., Advances in virus research 1998, 51, 135-201. 2. Lute, S.; Aranha, H.; Tremblay, D.; Liang, D.; Ackermann, H. W.; Chu, B.; Moineau, S.; Brorson, K., Appl. Environ. Microbiol. 2004, 70, (8), 4864-4871. 3. Grahn, A. M.; Butcher, S. J.; Bamford, J. K. H.; Bamford, D. H., PRD1: Dissecting the Genome, Structure, and Entry. In The Bacteriophages, Calendar, R. L., Ed. Oxford university press: 2005. 4. Coetzee, J. N.; Lecatsas, G.; Coetzee, W. F.; Hedges, R. W., Journal of General Microbiology 1979, 110, (2), 263-273. 5. Van Duin, J.; Tsareva, N., Single-Stranded RNA Phages. In The bacteriophages, Calendar, R. L., Ed. Oxford University Press: 2005. 6. Mosig, G.; Eiserling, F., T4 and Related Phages: Structure and Development. In The bacteriophages, Calendar, R. L., Ed. 2005. 7. Walker, S. L.; Bhattacharjee, S.; Hoek, E. M. V.; Elimelech, M., Langmuir 2002, 18, (6), 2193-2198. 8. Levich, V. G., Physicochemical Hydrodynamics. Prentice-Hall: Englewood Cliffs: NJ, 1962. 9. Calendar, R. L., The Bacteriophages. Oxford University Press, USA: 2005.
9
Bacterial Capture Efficiency and Antimicrobial Activity of Phage Functionalized Model Surfaces
ZEINAB HOSSEINIDOUST1, THEO G.M. VAN DE VEN2, and NATHALIE TUFENKJI*,1
1
Department of Chemical Engineering, McGill University, Montreal, Quebec H3A 2B2, Canada
2
*
Department of Chemistry, McGill University, Montreal, Quebec H3A 2K6, Canada
Corresponding Author. Phone: (514) 398-2999;
Fax: (514) 398-6678;
E-mail:
[email protected] 1
Theoretical background Phages P22, PRD1, and PR772 infect S. typhimurium. P22 is an icosahedral shaped virus from the Podoviridae family (Table 1 and Figure 1a). P22 is approximately 52–60 nm in length and its genetic material comprises of 43,400 bp of double-stranded (ds) DNA 1. P22 consists of an icosahedral capsid and a short tail; its receptors are located on its tail spikes, therefore its orientation is an issue for this phage when immobilized. PRD1 and PR772 are cubic bacteriophages from the Tectiviridae family and have linear ds DNA genome; PR772 bacteriophage is 97.2% identical to PRD1 at the genetic level 2. The family Tectiviridae includes phages with a lipid membrane beneath the icosahedral shell. PRD1 is a well studied phage; while less is known about the structure of PR772, it can be surmised to be highly similar on the basis of the overall similarity of Tectiviridae family phages2. PRD-1 is a somatic phage, meaning it attaches to the bacterial cell surface. Upon attachment to the bacterial surface, the lipoprotein vesicle becomes a tail-like tube about 60 nm in length, thus a nucleic acid ejection device . A single phage structural protein located at the vertices is responsible for PRD1 attachment to its host 3. As this protein is located symmetrically on all the vertices of the capsid (Figure 1b), orientation is not an issue for either PRD1 or PR772. PR772 also infects E. coli, however, its mode of infection is different. PR772 is an F-specific phage and it adsorbs by one of its vertices to tips of F-pili (mating organelle) in E. coli 4. MS2 is a single strand (ss) RNA phage that also infects E.coli. Single-stranded RNA coliphages are found wherever E. coli lives, for example in the intestinal tract of humans 5. MS2 is also an F-specific phage but attaches laterally along the pili of the host (as most ssRNA 2
phage). Both PR772 and MS2 attach their RNA/DNA to the pili which will then have to transfer the nucleic acids into the cell. MS2, although symmetrical in shape, is not symmetrical in terms of attachment proteins; it has only one attachment protein called a “maturation protein” 5 on one of the vertices. T4 phage is another phage infecting E. coli, and consists of a long contractile tail and an elongated icosahedral capsid which encapsulates a ds-DNA genome (Figure 1c). The T4 phages are typically 200 nm long and perform lytic cycles inside the host bacterium E. coli 6.T4 is one of the most well studied phages and this explains why many of the published studies on the use of immobilized phage are performed with T4 despite its highly asymmetrical structure and specific requirements for infectivity.
Methods Surface characterization XPS (X-ray photoelectron spectroscopy) was performed at the LASM (École Polytechnique Surface Analysis Laboratory, Montreal, Quebec) on model surfaces at various stages of functionalization. XPS studies were performed on a VG ESCALAB 3 MKII (VG, Thermo Electron Corporation, UK). Samples were irradiated using an MgKα source at a take-off angle of 0o (i.e., perpendicular); analyzed surface was 2 mm × 3 mm and the depth sampled was ~50-100 Å. Both low resolution and N1s, C1s and O1s high-resolution analyses, with a scan width of 20 eV, were performed. The quality of APTES-coated surfaces before and after functionalization was examined using atomic force microscopy (AFM). The surfaces were used as prepared without any further 3
modifications. Imaging was performed using a NanoScope IIIa scanning probe microscope (Veeko/Digital Instruments, USA) in air under tapping mode using a commercial n+-silicone cantilever (Nanoscience Instruments, Phoenix, USA) 240 μm long and 35 um wide, with a resonant frequency of 50-130 kHz and spring constant of 9.0 N/m. The scanning rate was 1.0 Hz, at 0 angle. Image processing was performed using Research NanoScope III software version 6.11r1. All images were filtered using the flattening built-in tool and roughness values were obtained by utilizing the built-in tool for cross-sectional analysis. The streaming potential of APTES-coated surfaces was measured using an asymmetric clamping cell using an electrokinetic analyzer (Anton Paar, Graz, Austria) 7. The measurements were performed at room temperature with 10 mM KCl buffered with KHCO3 to pH 7.5. For streaming potential measurements, rectangular cover slides (No.2, 24×60 mm, VWR) were cleaned and coated as described previously. Measurements were performed in triplicate and the zeta potential was calculated from the measured streaming potential as per 8. Contact angle goniometry was used as a measure of the degree of hydrophobicity of the substrate. Prior to measurement of contact angles, the slides were rinsed in DI water and dried under a stream of high purity N2. Equilibrium contact angle measurements were performed using an OCA-30 goniometer (Future Digital Scientific Corp., NJ, USA) on sessile drops (1 μL droplets) by measuring the tangent to the drop at its intersection with the surface. All measurements were repeated at least three times.
4
Bacteria and phage characterization Phages were examined by transmission electron microscopy (TEM). A glow-discharged carbon coated copper grid was placed on a 5 μL drop of purified phage stock mixed with a 5 μL 4% uranyl acetate for 1 min and washed 3 times by placing on drops of water for 10 sec. Samples were examined on a Philips Tecnai 12 120KV TEM and images were captured with a Gatan 792 Bioscan 1k × 1k Wide Angle Multiscan CCD Camera. Scanning electron microscopy (SEM) was used to image bacteria and selected discs with immobilized phage and captured bacteria. Suspended bacteria were washed twice, pelleted, and resuspended in 2.5% gluteraldehyde in SM-buffer and kept at 4
for 1 hr for fixation. The fixed samples were
subsequently washed with SM-buffer. A drop of fixed sample was placed on poly-L-lysine coated slides for 15 min and the slide was blot dried. The slides were subsequently dehydrated with a graded series of 10-min ethanol immersions (30 to 100%) and a graded series of amyl acetate in ethanol solutions (25 to 100%). The slides were air dried and coated with 300 of AuPd coating (Hummer VI Au-Pd sputter coater). The discs with captured bacteria were immediately fixed with gluteraldehyde and the dehydration and coating steps were carried out as described above. Samples were examined with a Hitachi S-4700 Field Emission-STEM (FESTEM, Tokyo, Japan) and images were recorded using Quartz PCI v. 6.0 software. Dynamic light scattering (DLS) was used to evaluate the hydrodynamic diameter of bacteria and phage particles using the Malvern Instruments Zetasizer Nano-ZS. Bacteria were cultured as previously described, washed three times to remove growth media, and finally resuspended in SM buffer. Phage was propagated and purified as previously described and was 5
also in SM buffer. Each measurement was repeated five times at room temperature and standard quartz cuvettes were used for all measurements. The electrophoretic mobility of the phage and bacteria was measured using the same equipment. The zeta potential of the phage and bacteria was calculated from the electrophoretic mobility measurements using the Henry equation 8. Samples were prepared as described for DLS measurements and the standard universal dip cell™ was used for the measurements.
Figure S1. Schematic representation of different phages used in this study (a) PRD1 and PR772, (b) MS2, (c) T4, and (d) P22. Reproduced from ref.
9
with permission from Oxford University
Press, USA. Images are not to scale.
6
Figure S2. Scanning electron micrographs of (a) PRD1 bacteriophage immobilized on the surface of APTES coated discs functionalized with EDC-NHS, (b) higher magnification image of area enclosed by black square in (a), showing the shape and size of PRD1.
7
Figure S3. Immobilized phage is infective as indicated by the lysis ring around the disc (result presented for PRD1 and S. typhimurium).
Figure S4. (a) S. typhimurium attached to phage functionalized surface, (b) higher magnification image of the same sample as (a) showing lysed S. typhimurium on the phage-functionalized discs.
8
Figure S5. Growth curve for bacteria with and without the presence of phage (a) S. typhimurium LT2, (b) E. coli 15597. A 25 µL aliquot of overnight culture of host bacteria (diluted in 100 uL of fresh TSB) was infected with 25 µL of phage and monitored in a 96 well plate for 12 hours at 37C. As no effort was made to optimize the multiplicity of infection (MOI), phages that are known to be very sensitive to MOI (namely, MS2 and PR772) do not exhibit significant host growth inhibition. References 1. Ackermann, H. W., Advances in virus research 1998, 51, 135-201. 2. Lute, S.; Aranha, H.; Tremblay, D.; Liang, D.; Ackermann, H. W.; Chu, B.; Moineau, S.; Brorson, K., Appl. Environ. Microbiol. 2004, 70, (8), 4864-4871. 3. Grahn, A. M.; Butcher, S. J.; Bamford, J. K. H.; Bamford, D. H., PRD1: Dissecting the Genome, Structure, and Entry. In The Bacteriophages, Calendar, R. L., Ed. Oxford university press: 2005. 4. Coetzee, J. N.; Lecatsas, G.; Coetzee, W. F.; Hedges, R. W., Journal of General Microbiology 1979, 110, (2), 263-273. 5. Van Duin, J.; Tsareva, N., Single-Stranded RNA Phages. In The bacteriophages, Calendar, R. L., Ed. Oxford University Press: 2005. 6. Mosig, G.; Eiserling, F., T4 and Related Phages: Structure and Development. In The bacteriophages, Calendar, R. L., Ed. 2005. 7. Walker, S. L.; Bhattacharjee, S.; Hoek, E. M. V.; Elimelech, M., Langmuir 2002, 18, (6), 2193-2198. 8. Levich, V. G., Physicochemical Hydrodynamics. Prentice-Hall: Englewood Cliffs: NJ, 1962. 9. Calendar, R. L., The Bacteriophages. Oxford University Press, USA: 2005.
9
