CHEMBIOCHEM COMMUNICATIONS DOI: 10.1002/cbic.201300410
A Predicted Immunity Protein Confers Resistance to Pyocin S5 in a Sensitive Strain of Pseudomonas aeruginosa Bahareh Haji Rasouliha, Hua Ling, Chun Loong Ho, and Matthew Wook Chang*[a] Pseudomonas aeruginosa is one of the leading etiological agents that cause nosocomial infections among patients with cystic fibrosis or compromised immunity.[1] Because of its inherent resistance towards many antibiotics, there is a demand for an effective strategy to specifically target and treat P. aeruginosa infections. In a competitive environment, P. aeruginosa secretes bacteriocins (“pyocins”) to limit the growth of other related species.[2] Three types of pyocins (F, R, and S) are secreted by P. aeruginosa strains,[3] and, in theory, the immunity proteins of P. aeruginosa render pyocin-producing strains insensitive to their own pyocins.[4] Amongst the S-type pyocins, S5 is an important bacteriocin; it has a pore-forming activity similar to that of colicin Ia, with which it shares high homology of protein sequence and secondary structure.[5] Likewise, a putative pyocin S5 immunity protein (S5I) shows considerable homology to the colicin Ia and Ib immunity proteins,[6] but the role of this protein has yet to be experimentally described. Here, we confirmed the identity of the predicted pyocin S5 immunity gene (S5I) in P. aeruginosa PAO1 (ATCC 15692), and elucidated its protective role in confering resistance to pyocin S5 in the S5-sensitive strain P. aureginosa DWW3. Furthermore, we demonstrated that expression of S5I in S5-sensitive strain conferred pyocin S5 resistance through prevention of membrane damage. This study provides potentially useful insights for exploiting pyocins as specific treatment against Pseudomonas strains. BlastP (BlastP, http://blast.ncbi.nlm.nih.gov) was used to identify similarities between S5I (PA0984, 108 aa) encoded by P. aeruginosa (http://www.pseudomonas.com/) and colicin immunity proteins, and DNAMAN software was used for the alignment (Lynnon Corp., Quebec, Canada). The results indicated multiple regions with high degrees of amino acid sequence conservation between S5I and colicin Ia and E1 immunity proteins (Figure 1 A). Furthermore, the secondary structures and transmembrane domains of these immunity proteins were analyzed by using the Hierarchical Neural Network (HNN)[7] and SOSUI programs.[8] The HNN analysis predicted nine random coils in S5I, and nine and seven random coils in colicin Ia and E1 immunity proteins, respectively (Figure 1 B). By the SOSUI analysis, putative transmembrane domains (VAWKGSVFPSLASVNPLVVAGLS and GLYAVFYLSCYLFSIPLGMVFLF) were [a] B. H. Rasouliha, Dr. H. Ling, C. L. Ho, Prof. Dr. M. W. Chang School of Chemical and Biomedical Engineering Nanyang Technological University (NTU) 62 Nanyang Drive, 637459 (Singapore) E-mail: [email protected] Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cbic.201300410.
2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 1. Pyocin S5I protein and homologous proteins. A) Multiple sequence alignment in DNAMAN of P. aeruginosa pyocin S5I (NP_249675.1) against Escherichia coli immunity proteins colicin Ia (YP_003502719.1) and E1 (ZP_ 12077771.1). Conserved and similar residues are highlighted in black and blue, respectively. B) Secondary structure prediction for pyocin S5I, colicin Ia, and colicin E1 immunity proteins (HNN program). The long, middle, and short bars represent alpha helix, sheet, and random coil forms, respectively.
found at the C and N termini of S5I. Colicin Ia and E1 immunity proteins have similar C- and N-terminal transmembrane domains; thus, we hypothesized that S5I is a transmembrane protein with an immunizing role against pyocin S5. To test our hypothesis, we cloned S5I into the pUCP18 plasmid,[9] and the recombinant plasmid was transformed into a pyocin S5-sensitive P. aeruginosa strain, DWW3, by electroporation as previously described.[10] Expression of S5I from DWW3S5I was confirmed by semi-quantitative and quantitative PCR assays (Figure S1 in the Supporting Information). To examine changes in sensitivity of the DWW3 strain to pyocin S5, we conducted an overlay assay with purified pyocin S5 as described previously (Figure S2).[5] Consistent with the reported sensitivity of DWW3 to pyocin S5,[5] growth of DWW3 was inhibited after exposure to a range of concentrations of pyocin followed by overnight incubation at 30 8C (Figure 2 A). In contrast, DWW3-S5I showed unimpeded growth after pyocin S5 treatment (Figure 2 B), thus demonstrating that expression of S5I in DWW3 provides resistance towards pyocin S5. This finding was supported by quantitative cell viability tests, such as ChemBioChem 2013, 14, 2444 – 2446
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www.chembiochem.org content leakage, including nucleic acids. We investigated pyocin S5-mediated membrane damage by using the nucleicacid-labeling fluorescent dye, SYTO9, to assess membrane permeability. DWW3 and DWW3-S5I cells were incubated with pyocin S5 (198.4 mg mL 1) for 1 h, stained, and viewed by using a fluorescent microscope. Most of the pyocin S5-treated DWW3 cells fluoresced red, thus indicating a high proportion of dead cells (62.23 1.5 %; Figure 3 A). Most of the remaining S5-treated DWW3 cells fluoresced green, but weaker than treated DWW3-S5I cells, thus indicating low concentrations of nucleic acid, possibly a result of membrane damage (Figure 3 A and B). On the other hand, most of the S5-treated DWW3-S5I cells (99.2 1.4 %) showed high green fluorescence, thus indicating that they are viable with intact membrane (Figure 3 B). Similarly, without pyocin S5 treatment, both DWW3 and DWW3-S5I cells showed high green fluorescence intensity (Figure S3). On the basis of our previous study,[5] we postulate that cell death of S5-treated DWW3 is attributable to cell surface damage. To further validate that cell death is attributable to cell surface damage by pyocin S5, we performed field emission scanning electron microscopy (FESEM) on S5-treated DWW3 and DWW3-S5I cells. To investigate cell morphology, DWW3 and DWW3- S5I cells were treated with pyocin S5 (198 mg mL 1),
Figure 2. Overlay assay and IC50 quantification of S5-treated cells. Pyocin S5 sensitivity was tested by overnight incubation of A) DWW3 and B) DWW3-S5I with various concentrations of pyocin S5 (overlay assay). Inhibitory zones represent the sensitivity of DWW3 cells to pyocin S5. Growth curve and halfmaximal inhibitory concentration (IC50) of pyocin S5 at exponential growth phase (3–6 h) against C) DWW3 and D) DWW3-S5I were examined (IC50 = 0.5337 and 3.2674 mg mL 1 for DWW3 and DWW3-S5I, respectively). v0 (initial velocity) is cellular division rate with various pyocin concentrations. E) CFU count of DWW3 and DWW3-S5I treated with pyocin S5 for 1 h; incubation at 37 8C.
half-maximal inhibitory concentration (IC50) and colony forming unit (CFU) count. To determine IC50, cultured cells (100 mL) of each P. aureginosa strains were initially grown to OD600 = 0.1, followed by incubation with various concentrations of pyocin S5 for 6 h. OD600 was measured every hour (Figure 2 C and D), and IC50 was evaluated by plotting exponential growth rate (3– 6 h, initial velocity, v0) against various pyocin concentrations. DWW3-S5I cells showed sixfold higher resistance against pyocin S5 (IC50 values of 0.5337 and 3.2674 mg mL 1 for DWW3 and DWW3-S5I , respectively, with pyocin S5; Figure 2 C and D). To validate the IC50 values, we performed CFU counting for DWW3 and DWW3-S5I cells treated with various concentrations of pyocin S5. DWW3 was not viable after exposure to pyocin S5, even at the lowest concentration (3.1 mg mL 1), whereas DWW3-S5I was viable at the highest concentration examined (198.4 mg mL 1; Figure 2 E). To investigate the role of the S5I protein in protecting the cellular membrane, we stained DWW3 and DWW3-S5I cells with a LIVE/DEAD BacLight Bacterial Viability Kit (Invitrogen) after exposure to pyocin S5. Consistent with our previous study,[5] pyocin S5-mediated cell damage resulted in cellular 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 3. Fluorescence and FESEM cell imaging of S5-treated DWW3 and DWW3-S5I. SYTO9 (green) and propidium iodide (PI; red) staining of A) DWW3 and B) DWW3-S5I after 1 h pyocin S5 treatment. DWW3 cells have high population of cells that fluoresce red (cell sensitivity towards pyocin S5), whereas most DWW3-S5I cells fluoresce green (cellular survival) resulting from pyocin S5 resistance, upon pyocin treatment. FESEM microscopy of C) DWW3 and D) DWW3-S5I cells, without pyocin S5 treatment, and E) DWW3 and F) DWW3-S5I after 1 h pyocin S5 treatment. Scale bars: 10 mm (A and B), 1 mm (C–F).
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CHEMBIOCHEM COMMUNICATIONS fixed and dried as described in the Methodology in the Supporting Information. Cells were coated with platinum (20 mA, 80 s), and cell morphology was examined by FESEM at 5.0 kV.[5] There was no damage to the cell envelope of DWW3 without S5 treatment (Figure 3 C and D), whereas most of the S5-treated DWW3 cells had shrunk and showed visible protuberances and concave areas on the cell surface (Figure 3 E), thus suggesting cell surface damage upon exposure to pyocin S5, in support of the aforementioned membrane damage as proved by the LIVE/DEAD viability staining assay. In contrast, S5-treated DWW3-S5I showed normal cell morphology (Figure 3 F). The differences in cell morphology indicate that expression of S5I in DWW3 prevented cell surface damage by pyocin S5, consistent with our observation by fluorescence microscopy. In conclusion, we identified the putative S5I gene in P. aeruginosa PAO1. Furthermore, we proved that S5I confers pyocin S5 resistance through prevention of membrane damage. Future efforts might include screening for S5I inhibitors, for an S5 combinatorial therapeutic strategy to target S5-resistant strains.[11]
Acknowledgements We thank Dr. Peter Greenberg from University of Washington for the provision of plasmid pUCP18 and Dr. Richard Waite from Queen Mary, University of London for the provision of the DWW3
2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org Pseudomonas aeruginosa clinical isolate. This research was supported by the National Medical Research Council of Singapore (CBRG11nov109) and Singapore International Graduate Award. Keywords: antibiotics · immunity proteins · membrane proteins · pseudomonas aeruginosa · pyocin S5 [1] J. R. Govan, V. Deretic, Microbiol. Rev. 1996, 60, 539 – 574. [2] F. Jacob, A. Lwoff, A. Siminovitch, E. Wollman, Ann. Inst. Pasteur 1954, 84, 222 – 224. [3] Y. Michel-Briand, C. Baysse, Biochimie 2002, 84, 499 – 510. [4] a) Y. Sano, H. Matsui, M. Kobayashi, M. Kageyama, J. Bacteriol. 1993, 175, 2907 – 2916; b) Y. Sano, M. Kageyama, Mol. Gen. Genet. 1993, 237, 161 – 170. [5] H. Ling, N. Saeidi, B. H. Rasouliha, M. W. Chang, FEBS Lett. 2010, 584, 3354 – 3358. [6] A. Parret, R. De Mot, Mol. Microbiol. 2000, 35, 472 – 473. [7] C. Combet, C. Blanchet, C. Geourjon, G. Delage, Trends Biochem. Sci. 2000, 25, 147 – 150. [8] T. Hirokawa, S. Boon-Chieng, S. Mitaku, Bioinformatics 1998, 14, 378 – 379. [9] D. G. Davies, M. R. Parsek, J. P. Pearson, B. H. Iglewski, J. W. Costerton, E. P. Greenberg, Science 1998, 280, 295 – 298. [10] K.-H. Choi, A. Kumar, H. P. Schweizer, J. Microbiol. Methods 2006, 64, 391 – 397. [11] N. Saeidi, C. K. Wong, T.-M. Lo, H. X. Nguyen, H. Ling, S. S. J. Leong, C. L. Poh, M. W. Chang, Mol. Syst. Biol. 2011, 7, 521. Received: June 24, 2013 Published online on November 12, 2013
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A Predicted Immunity Protein Confers Resistance to Pyocin S5 in a Sensitive Strain of Pseudomonas aeruginosa Bahareh Haji Rasouliha, Hua Ling, Chun Loong Ho, and Matthew Wook Chang*[a] Pseudomonas aeruginosa is one of the leading etiological agents that cause nosocomial infections among patients with cystic fibrosis or compromised immunity.[1] Because of its inherent resistance towards many antibiotics, there is a demand for an effective strategy to specifically target and treat P. aeruginosa infections. In a competitive environment, P. aeruginosa secretes bacteriocins (“pyocins”) to limit the growth of other related species.[2] Three types of pyocins (F, R, and S) are secreted by P. aeruginosa strains,[3] and, in theory, the immunity proteins of P. aeruginosa render pyocin-producing strains insensitive to their own pyocins.[4] Amongst the S-type pyocins, S5 is an important bacteriocin; it has a pore-forming activity similar to that of colicin Ia, with which it shares high homology of protein sequence and secondary structure.[5] Likewise, a putative pyocin S5 immunity protein (S5I) shows considerable homology to the colicin Ia and Ib immunity proteins,[6] but the role of this protein has yet to be experimentally described. Here, we confirmed the identity of the predicted pyocin S5 immunity gene (S5I) in P. aeruginosa PAO1 (ATCC 15692), and elucidated its protective role in confering resistance to pyocin S5 in the S5-sensitive strain P. aureginosa DWW3. Furthermore, we demonstrated that expression of S5I in S5-sensitive strain conferred pyocin S5 resistance through prevention of membrane damage. This study provides potentially useful insights for exploiting pyocins as specific treatment against Pseudomonas strains. BlastP (BlastP, http://blast.ncbi.nlm.nih.gov) was used to identify similarities between S5I (PA0984, 108 aa) encoded by P. aeruginosa (http://www.pseudomonas.com/) and colicin immunity proteins, and DNAMAN software was used for the alignment (Lynnon Corp., Quebec, Canada). The results indicated multiple regions with high degrees of amino acid sequence conservation between S5I and colicin Ia and E1 immunity proteins (Figure 1 A). Furthermore, the secondary structures and transmembrane domains of these immunity proteins were analyzed by using the Hierarchical Neural Network (HNN)[7] and SOSUI programs.[8] The HNN analysis predicted nine random coils in S5I, and nine and seven random coils in colicin Ia and E1 immunity proteins, respectively (Figure 1 B). By the SOSUI analysis, putative transmembrane domains (VAWKGSVFPSLASVNPLVVAGLS and GLYAVFYLSCYLFSIPLGMVFLF) were [a] B. H. Rasouliha, Dr. H. Ling, C. L. Ho, Prof. Dr. M. W. Chang School of Chemical and Biomedical Engineering Nanyang Technological University (NTU) 62 Nanyang Drive, 637459 (Singapore) E-mail: [email protected] Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cbic.201300410.
2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 1. Pyocin S5I protein and homologous proteins. A) Multiple sequence alignment in DNAMAN of P. aeruginosa pyocin S5I (NP_249675.1) against Escherichia coli immunity proteins colicin Ia (YP_003502719.1) and E1 (ZP_ 12077771.1). Conserved and similar residues are highlighted in black and blue, respectively. B) Secondary structure prediction for pyocin S5I, colicin Ia, and colicin E1 immunity proteins (HNN program). The long, middle, and short bars represent alpha helix, sheet, and random coil forms, respectively.
found at the C and N termini of S5I. Colicin Ia and E1 immunity proteins have similar C- and N-terminal transmembrane domains; thus, we hypothesized that S5I is a transmembrane protein with an immunizing role against pyocin S5. To test our hypothesis, we cloned S5I into the pUCP18 plasmid,[9] and the recombinant plasmid was transformed into a pyocin S5-sensitive P. aeruginosa strain, DWW3, by electroporation as previously described.[10] Expression of S5I from DWW3S5I was confirmed by semi-quantitative and quantitative PCR assays (Figure S1 in the Supporting Information). To examine changes in sensitivity of the DWW3 strain to pyocin S5, we conducted an overlay assay with purified pyocin S5 as described previously (Figure S2).[5] Consistent with the reported sensitivity of DWW3 to pyocin S5,[5] growth of DWW3 was inhibited after exposure to a range of concentrations of pyocin followed by overnight incubation at 30 8C (Figure 2 A). In contrast, DWW3-S5I showed unimpeded growth after pyocin S5 treatment (Figure 2 B), thus demonstrating that expression of S5I in DWW3 provides resistance towards pyocin S5. This finding was supported by quantitative cell viability tests, such as ChemBioChem 2013, 14, 2444 – 2446
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www.chembiochem.org content leakage, including nucleic acids. We investigated pyocin S5-mediated membrane damage by using the nucleicacid-labeling fluorescent dye, SYTO9, to assess membrane permeability. DWW3 and DWW3-S5I cells were incubated with pyocin S5 (198.4 mg mL 1) for 1 h, stained, and viewed by using a fluorescent microscope. Most of the pyocin S5-treated DWW3 cells fluoresced red, thus indicating a high proportion of dead cells (62.23 1.5 %; Figure 3 A). Most of the remaining S5-treated DWW3 cells fluoresced green, but weaker than treated DWW3-S5I cells, thus indicating low concentrations of nucleic acid, possibly a result of membrane damage (Figure 3 A and B). On the other hand, most of the S5-treated DWW3-S5I cells (99.2 1.4 %) showed high green fluorescence, thus indicating that they are viable with intact membrane (Figure 3 B). Similarly, without pyocin S5 treatment, both DWW3 and DWW3-S5I cells showed high green fluorescence intensity (Figure S3). On the basis of our previous study,[5] we postulate that cell death of S5-treated DWW3 is attributable to cell surface damage. To further validate that cell death is attributable to cell surface damage by pyocin S5, we performed field emission scanning electron microscopy (FESEM) on S5-treated DWW3 and DWW3-S5I cells. To investigate cell morphology, DWW3 and DWW3- S5I cells were treated with pyocin S5 (198 mg mL 1),
Figure 2. Overlay assay and IC50 quantification of S5-treated cells. Pyocin S5 sensitivity was tested by overnight incubation of A) DWW3 and B) DWW3-S5I with various concentrations of pyocin S5 (overlay assay). Inhibitory zones represent the sensitivity of DWW3 cells to pyocin S5. Growth curve and halfmaximal inhibitory concentration (IC50) of pyocin S5 at exponential growth phase (3–6 h) against C) DWW3 and D) DWW3-S5I were examined (IC50 = 0.5337 and 3.2674 mg mL 1 for DWW3 and DWW3-S5I, respectively). v0 (initial velocity) is cellular division rate with various pyocin concentrations. E) CFU count of DWW3 and DWW3-S5I treated with pyocin S5 for 1 h; incubation at 37 8C.
half-maximal inhibitory concentration (IC50) and colony forming unit (CFU) count. To determine IC50, cultured cells (100 mL) of each P. aureginosa strains were initially grown to OD600 = 0.1, followed by incubation with various concentrations of pyocin S5 for 6 h. OD600 was measured every hour (Figure 2 C and D), and IC50 was evaluated by plotting exponential growth rate (3– 6 h, initial velocity, v0) against various pyocin concentrations. DWW3-S5I cells showed sixfold higher resistance against pyocin S5 (IC50 values of 0.5337 and 3.2674 mg mL 1 for DWW3 and DWW3-S5I , respectively, with pyocin S5; Figure 2 C and D). To validate the IC50 values, we performed CFU counting for DWW3 and DWW3-S5I cells treated with various concentrations of pyocin S5. DWW3 was not viable after exposure to pyocin S5, even at the lowest concentration (3.1 mg mL 1), whereas DWW3-S5I was viable at the highest concentration examined (198.4 mg mL 1; Figure 2 E). To investigate the role of the S5I protein in protecting the cellular membrane, we stained DWW3 and DWW3-S5I cells with a LIVE/DEAD BacLight Bacterial Viability Kit (Invitrogen) after exposure to pyocin S5. Consistent with our previous study,[5] pyocin S5-mediated cell damage resulted in cellular 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 3. Fluorescence and FESEM cell imaging of S5-treated DWW3 and DWW3-S5I. SYTO9 (green) and propidium iodide (PI; red) staining of A) DWW3 and B) DWW3-S5I after 1 h pyocin S5 treatment. DWW3 cells have high population of cells that fluoresce red (cell sensitivity towards pyocin S5), whereas most DWW3-S5I cells fluoresce green (cellular survival) resulting from pyocin S5 resistance, upon pyocin treatment. FESEM microscopy of C) DWW3 and D) DWW3-S5I cells, without pyocin S5 treatment, and E) DWW3 and F) DWW3-S5I after 1 h pyocin S5 treatment. Scale bars: 10 mm (A and B), 1 mm (C–F).
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CHEMBIOCHEM COMMUNICATIONS fixed and dried as described in the Methodology in the Supporting Information. Cells were coated with platinum (20 mA, 80 s), and cell morphology was examined by FESEM at 5.0 kV.[5] There was no damage to the cell envelope of DWW3 without S5 treatment (Figure 3 C and D), whereas most of the S5-treated DWW3 cells had shrunk and showed visible protuberances and concave areas on the cell surface (Figure 3 E), thus suggesting cell surface damage upon exposure to pyocin S5, in support of the aforementioned membrane damage as proved by the LIVE/DEAD viability staining assay. In contrast, S5-treated DWW3-S5I showed normal cell morphology (Figure 3 F). The differences in cell morphology indicate that expression of S5I in DWW3 prevented cell surface damage by pyocin S5, consistent with our observation by fluorescence microscopy. In conclusion, we identified the putative S5I gene in P. aeruginosa PAO1. Furthermore, we proved that S5I confers pyocin S5 resistance through prevention of membrane damage. Future efforts might include screening for S5I inhibitors, for an S5 combinatorial therapeutic strategy to target S5-resistant strains.[11]
Acknowledgements We thank Dr. Peter Greenberg from University of Washington for the provision of plasmid pUCP18 and Dr. Richard Waite from Queen Mary, University of London for the provision of the DWW3
2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org Pseudomonas aeruginosa clinical isolate. This research was supported by the National Medical Research Council of Singapore (CBRG11nov109) and Singapore International Graduate Award. Keywords: antibiotics · immunity proteins · membrane proteins · pseudomonas aeruginosa · pyocin S5 [1] J. R. Govan, V. Deretic, Microbiol. Rev. 1996, 60, 539 – 574. [2] F. Jacob, A. Lwoff, A. Siminovitch, E. Wollman, Ann. Inst. Pasteur 1954, 84, 222 – 224. [3] Y. Michel-Briand, C. Baysse, Biochimie 2002, 84, 499 – 510. [4] a) Y. Sano, H. Matsui, M. Kobayashi, M. Kageyama, J. Bacteriol. 1993, 175, 2907 – 2916; b) Y. Sano, M. Kageyama, Mol. Gen. Genet. 1993, 237, 161 – 170. [5] H. Ling, N. Saeidi, B. H. Rasouliha, M. W. Chang, FEBS Lett. 2010, 584, 3354 – 3358. [6] A. Parret, R. De Mot, Mol. Microbiol. 2000, 35, 472 – 473. [7] C. Combet, C. Blanchet, C. Geourjon, G. Delage, Trends Biochem. Sci. 2000, 25, 147 – 150. [8] T. Hirokawa, S. Boon-Chieng, S. Mitaku, Bioinformatics 1998, 14, 378 – 379. [9] D. G. Davies, M. R. Parsek, J. P. Pearson, B. H. Iglewski, J. W. Costerton, E. P. Greenberg, Science 1998, 280, 295 – 298. [10] K.-H. Choi, A. Kumar, H. P. Schweizer, J. Microbiol. Methods 2006, 64, 391 – 397. [11] N. Saeidi, C. K. Wong, T.-M. Lo, H. X. Nguyen, H. Ling, S. S. J. Leong, C. L. Poh, M. W. Chang, Mol. Syst. Biol. 2011, 7, 521. Received: June 24, 2013 Published online on November 12, 2013
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