Liposomal Delivery of Newly Identified Prophage Lysins in a Pseudomonas aeruginosa Model
Abstract
:1. Introduction
2. Results
2.1. Complete Prophages Are Common in P. aeruginosa Genomes
2.2. Pseudomonas Phage Lysins Are Functionally and Genetically Diverse
2.3. Expression and Antibacterial Activity of Phage Lysins: Putative Lysins Are Lysins de Facto
2.4. Pa7 and Pa119 Have Peptidoglycan Hydrolase Activity
2.5. Liposome Deliver Phage Lysins to the Peptidoglycan of P. aeruginosa
3. Discussion
4. Materials and Methods
4.1. P. aeruginosa Genomes
4.2. Prophage Identification and Classification
4.3. Endolysin Identification
4.4. Lysin Genes Cloning
4.5. Protein Expression and Purification
4.6. Zymogram Analysis
4.7. Antimicrobial Activity of Endolysins
4.8. Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration Determination (MBC)
4.9. Encapsulation of Selected Endolysins in Liposomes
4.10. Antimicrobial Activity of Encapsulated Endolysins
4.11. Cocktail Assays
5. Conclusions
Author Contributions
Funding
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Prophage | GC(%) | Size (kb) | CDS | Coding Region (%) | tRNA | Family | Reference |
---|---|---|---|---|---|---|---|
41Z | 60.60 | 40.94 | 70 | 92.57 | 0 | Siphoviridae | [27] |
42argF | 61.80 | 41.59 | 67 | 92.92 | 0 | Siphoviridae | [27] |
44G | 63.00 | 43.67 | 54 | 90.81 | 0 | Siphoviridae | [27] |
L1378_17 | 64.20 | 28.58 | 36 | 91.26 | 0 | Myoviridae | This study |
L1378_56 | 57.30 | 38.16 | 50 | 86.99 | 3 | Siphoviridae | This study |
L1379_42 | 62.00 | 37.57 | 51 | 90.39 | 0 | Myoviridae | This study |
L1395_55 | 58.70 | 35.27 | 60 | 81.40 | 2 | ND | This study |
L1395_78 | 60.00 | 21.74 | 25 | 84.85 | 0 | Siphoviridae | This study |
L3941_30 | 62.50 | 31.45 | 38 | 89.85 | 0 | Myoviridae | This study |
L3942_9 | 61.10 | 62.09 | 84 | 82.46 | 0 | Siphoviridae | This study |
L3944_13 | 60.80 | 34.57 | 50 | 91.42 | 0 | Siphoviridae | This study |
L3944_48 | 58.80 | 56.82 | 101 | 77.15 | 0 | ND | This study |
L3944_59 | 59.90 | 45.34 | 68 | 88.79 | 0 | Siphoviridae | This study |
L3945_7 | 58.90 | 33.76 | 42 | 77.92 | 0 | Siphoviridae | This study |
L3946_2 | 64.40 | 37.21 | 57 | 95.86 | 0 | Siphoviridae | This study |
L3946_67 | 57.30 | 30.85 | 42 | 89.90 | 3 | ND | This study |
L3955_1 | 60.20 | 37.76 | 48 | 87.82 | 0 | Myoviridae | This study |
L3955_10 | 62.70 | 36.34 | 45 | 88.47 | 0 | Myoviridae | This study |
L3955_30 | 58.90 | 53.78 | 87 | 86.32 | 1 | Siphoviridae | This study |
L4112_2 | 57.80 | 49.05 | 72 | 85.93 | 2 | Siphoviridae | This study |
L4112_49 | 62.70 | 42.67 | 53 | 87.04 | 0 | Siphoviridae | This study |
L4137_3 | 62.00 | 66.16 | 65 | 90.81 | 0 | Podoviridae | This study |
L4139_13 | 62.70 | 39.72 | 55 | 95.00 | 0 | Siphoviridae | This study |
L4140_27 | 58.70 | 28.55 | 53 | 82.15 | 1 | ND | This study |
L4140_48 | 63.90 | 29.22 | 44 | 88.65 | 0 | ND | This study |
L4441_19 | 60.80 | 74.64 | 77 | 84.36 | 0 | ND | This study |
L4441_52 | 62.20 | 38.28 | 46 | 87.18 | 0 | Siphoviridae | This study |
O4_56090 | 64.20 | 37.18 | 55 | 95.37 | 0 | Siphoviridae | [28] |
O5_46060 | 59.80 | 58.78 | 45 | 86.48 | 0 | ND | [28] |
O5_46403 | 55.40 | 35.8 | 34 | 88.88 | 0 | ND | [28] |
O8_56090 | 59.00 | 60.54 | 99 | 85.63 | 2 | Siphoviridae | [28] |
O1_56090 | 61.70 | 40.52 | 62 | 93.48 | 0 | Siphoviridae | [28] |
O4_16214 | 60.30 | 48.74 | 53 | 74.68 | 1 | Siphoviridae | [28] |
O3_43328 | 63.40 | 38.77 | 53 | 95.02 | 0 | Siphoviridae | [28] |
O5_61780 | 62.30 | 39.78 | 60 | 93.77 | 0 | Siphoviridae | [28] |
O7_12585 | 61.90 | 72.9 | 69 | 93.46 | 0 | Siphoviridae | [28] |
O8_43328 | 64.60 | 57.23 | 66 | 92.92 | 0 | Podoviridae | [28] |
O8_61780 | 62.10 | 53.39 | 72 | 85.91 | 0 | Siphoviridae | [28] |
Putative Lysin | InterPro [34] | Phyre [35] | |
---|---|---|---|
Family | Description | Description | |
41Z_15 | Repressor Cro | Helix-turn-helix | Chitinase |
41Z_58 | - | - | Endolysin |
41Z_63 | - | - | Endolysin |
42argF_542 * | Phage lysis | Phage lytic protein Rz | Endopeptidase |
L4112_2_7 * | Phage lysozyme | Lysozyme | Lysozyme |
L3944_13 * | Glycosidic hydrolase family 19 | Chitinase class I | Endolysin |
L4139_13_15 * | Transglycosylase | Transglycosylase | Transglycosylase |
L3955_10_119 * | Muramidase | N-acetylmuramidase | Peptidoglycan binding domain |
L1378_17 | Phage lysozyme | Lysozyme | Endolysin |
L1395_55 | - | - | Endolysin |
L3941_30 | Phage lysozyme | Lysozyme | Endolysin |
L3944_48 | - | - | Endolysin |
L3944_482 | - | - | Endolysin |
L3946_2 | - | - | Endolysin |
L3946_22 | - | - | Endolysin |
L4112_2 | Endopeptidase | Endopeptidase | Lysozyme |
L4137_3 | Phage lysozyme | Lysozyme | Lysozyme |
L4140_27 | - | - | Transglycosylase |
O4_16214 | Peptidase | Endopeptidase | Hydrolase |
Lysin | BLASTp [37] | Molecular Weight (kDa) | Theoretical pI [38] | Number of Aminoacids | Zymogram (Peptidoglycan Hydrolyzing Activity) | |||||
---|---|---|---|---|---|---|---|---|---|---|
Classification | Query Cover (%) | Identity (%) | E-Value | Predicted Protparam [38] | Experimental SDS-PAGE | M. luteus | P. aeruginosa | |||
Pa7 | Lysozyme | 100 | 99.31 | 1.00 × 10−101 | 16.20 | 16.5 | 9.69 | 144 | yes | yes |
Pa13 | Chitinase | 100 | 100 | 3.00 × 10−151 | 22.86 | 25.0 | 9.41 | 205 | yes | yes |
Pa15 | Transglycosylase | 100 | 98.56 | 6.00 × 10−150 | 23.50 | 18.5 | 9.54 | 209 | yes | yes |
Pa119 | N-acetylmuramidase | 99 | 99.25 | 0.00 | 29.30 | 30.0 | 7.13 | 268 | yes | yes |
Pa542 | Rz lytic protein | 99 | 95.97 | 8.00 × 10−99 | 16.06 | ND | 9.23 | 150 | ND | ND |
Lipid Composition (Molar Ratio) | Lysin | (Prot/Lip)i (μg/μmol) | (Prot/Lip)f (μg/μmol) | E.E. (%) | Mean Size (nm) | P. I. | Zeta Pot (mV) |
---|---|---|---|---|---|---|---|
DMPC:DOPE:CHEMS (4:4:2) | Pa7 | 16 ± 5 | 5 ± 2 | 33 ± 7 | 151 ± 6 | 0.111 ± 0.011 | −21 ± 2 |
DMPC:DOPE:CHEMS (4:4:2) | Pa119 | 10 ± 3 | 3 ± 1 | 32 ±9 | 149 ± 4 | 0.106 ± 0.012 | −22 ± 2 |
DMPC:DOPE:CHEMS (4:4:2) | EL | NA | NA | NA | 142 ± 2 | 0.125 ± 0.015 | −22 ± 1 |
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Morais, D.; Tanoeiro, L.; Marques, A.T.; Gonçalves, T.; Duarte, A.; Matos, A.P.A.; Vital, J.S.; Cruz, M.E.M.; Carvalheiro, M.C.; Anes, E.; et al. Liposomal Delivery of Newly Identified Prophage Lysins in a Pseudomonas aeruginosa Model. Int. J. Mol. Sci. 2022, 23, 10143. https://doi.org/10.3390/ijms231710143
Morais D, Tanoeiro L, Marques AT, Gonçalves T, Duarte A, Matos APA, Vital JS, Cruz MEM, Carvalheiro MC, Anes E, et al. Liposomal Delivery of Newly Identified Prophage Lysins in a Pseudomonas aeruginosa Model. International Journal of Molecular Sciences. 2022; 23(17):10143. https://doi.org/10.3390/ijms231710143
Chicago/Turabian StyleMorais, Diana, Luís Tanoeiro, Andreia T. Marques, Tiago Gonçalves, Aida Duarte, António Pedro Alves Matos, Joana S. Vital, Maria Eugénia Meirinhos Cruz, Manuela Colla Carvalheiro, Elsa Anes, and et al. 2022. "Liposomal Delivery of Newly Identified Prophage Lysins in a Pseudomonas aeruginosa Model" International Journal of Molecular Sciences 23, no. 17: 10143. https://doi.org/10.3390/ijms231710143