Phage-encoded cationic antimicrobial peptide required for lysis
Most phages of Gram-negative hosts encode spanins for disruption of the outer membrane, the last step in host lysis. However, bioinformatic analysis indicates that ∼15% of these phages lack a spanin gene, suggesting they have an alternate way of disrupting the OM. Here, we show that the T7-like coli...
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| Vydáno v: | Journal of bacteriology Ročník 204; číslo 1; s. JB0021421 |
|---|---|
| Hlavní autoři: | , , , , , , , , , , , |
| Médium: | Journal Article |
| Jazyk: | angličtina |
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United States
18.01.2022
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| ISSN: | 1098-5530, 1098-5530 |
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| Abstract | Most phages of Gram-negative hosts encode spanins for disruption of the outer membrane, the last step in host lysis. However, bioinformatic analysis indicates that ∼15% of these phages lack a spanin gene, suggesting they have an alternate way of disrupting the OM. Here, we show that the T7-like coliphage phiKT causes the explosive cell lysis associated with spanin activity despite not encoding spanins. A putative lysis cassette cloned from the phiKT late gene region includes the hypothetical novel gene
located between the holin and endolysin genes and supports inducible lysis in
K-12. Moreover, induction of an isogenic construct lacking gene
resulted in divalent cation-stabilized spherical cells rather than lysis, implicating gp
in OM disruption. Additionally, gp
was shown to complement the lysis defect of a spanin-null λ lysogen. Gene
encodes a 56-amino acid cationic protein with predicted amphipathic helical structure and is membrane-associated after lysis. Urea and KCl washes did not release gp
from the particulate, suggesting a strong hydrophobic membrane interaction. Fluorescence microscopy supports membrane localization of the gp
protein prior to lysis. Gp
is similar in size, charge, predicted fold, and membrane association to the human cathelicidin antimicrobial peptide LL-37. Synthesized gp
behaved similar to LL-37 in standard assays mixing peptide and cells to measure bactericidal and inhibitory effects. Taken together, these results indicate that phiKT gp
is a phage-encoded cationic antimicrobial peptide that disrupts bacterial outer membranes during host lysis and thus establishes a new class of phage lysis proteins, the disruptins.
We provide evidence that phiKT produces an antimicrobial peptide for outer membrane disruption during lysis. This protein, designated as a disruptin, is a new paradigm for phage lysis and has no similarities to other known lysis genes. Although many mechanisms have been proposed for the function of antimicrobial peptides, there is no consensus on the molecular basis of membrane disruption. Additionally, there is no established genetic system to support such studies. Therefore, the phiKT disruptin may represent the first genetically tractable antimicrobial peptide, facilitating mechanistic analyses. |
|---|---|
| AbstractList | Most phages of Gram-negative hosts encode spanins for disruption of the outer membrane, the last step in host lysis. However, bioinformatic analysis indicates that ∼15% of these phages lack a spanin gene, suggesting they have an alternate way of disrupting the OM. Here, we show that the T7-like coliphage phiKT causes the explosive cell lysis associated with spanin activity despite not encoding spanins. A putative lysis cassette cloned from the phiKT late gene region includes the hypothetical novel gene
located between the holin and endolysin genes and supports inducible lysis in
K-12. Moreover, induction of an isogenic construct lacking gene
resulted in divalent cation-stabilized spherical cells rather than lysis, implicating gp
in OM disruption. Additionally, gp
was shown to complement the lysis defect of a spanin-null λ lysogen. Gene
encodes a 56-amino acid cationic protein with predicted amphipathic helical structure and is membrane-associated after lysis. Urea and KCl washes did not release gp
from the particulate, suggesting a strong hydrophobic membrane interaction. Fluorescence microscopy supports membrane localization of the gp
protein prior to lysis. Gp
is similar in size, charge, predicted fold, and membrane association to the human cathelicidin antimicrobial peptide LL-37. Synthesized gp
behaved similar to LL-37 in standard assays mixing peptide and cells to measure bactericidal and inhibitory effects. Taken together, these results indicate that phiKT gp
is a phage-encoded cationic antimicrobial peptide that disrupts bacterial outer membranes during host lysis and thus establishes a new class of phage lysis proteins, the disruptins.
We provide evidence that phiKT produces an antimicrobial peptide for outer membrane disruption during lysis. This protein, designated as a disruptin, is a new paradigm for phage lysis and has no similarities to other known lysis genes. Although many mechanisms have been proposed for the function of antimicrobial peptides, there is no consensus on the molecular basis of membrane disruption. Additionally, there is no established genetic system to support such studies. Therefore, the phiKT disruptin may represent the first genetically tractable antimicrobial peptide, facilitating mechanistic analyses. Most phages of Gram-negative hosts encode spanins for disruption of the outer membrane, the last step in host lysis. However, bioinformatic analysis indicates that ∼15% of these phages lack a spanin gene, suggesting they have an alternate way of disrupting the OM. Here, we show that the T7-like coliphage phiKT causes the explosive cell lysis associated with spanin activity despite not encoding spanins. A putative lysis cassette cloned from the phiKT late gene region includes the hypothetical novel gene 28 located between the holin and endolysin genes and supports inducible lysis in E. coli K-12. Moreover, induction of an isogenic construct lacking gene 28 resulted in divalent cation-stabilized spherical cells rather than lysis, implicating gp28 in OM disruption. Additionally, gp28 was shown to complement the lysis defect of a spanin-null λ lysogen. Gene 28 encodes a 56-amino acid cationic protein with predicted amphipathic helical structure and is membrane-associated after lysis. Urea and KCl washes did not release gp28 from the particulate, suggesting a strong hydrophobic membrane interaction. Fluorescence microscopy supports membrane localization of the gp28 protein prior to lysis. Gp28 is similar in size, charge, predicted fold, and membrane association to the human cathelicidin antimicrobial peptide LL-37. Synthesized gp28 behaved similar to LL-37 in standard assays mixing peptide and cells to measure bactericidal and inhibitory effects. Taken together, these results indicate that phiKT gp28 is a phage-encoded cationic antimicrobial peptide that disrupts bacterial outer membranes during host lysis and thus establishes a new class of phage lysis proteins, the disruptins. Significance We provide evidence that phiKT produces an antimicrobial peptide for outer membrane disruption during lysis. This protein, designated as a disruptin, is a new paradigm for phage lysis and has no similarities to other known lysis genes. Although many mechanisms have been proposed for the function of antimicrobial peptides, there is no consensus on the molecular basis of membrane disruption. Additionally, there is no established genetic system to support such studies. Therefore, the phiKT disruptin may represent the first genetically tractable antimicrobial peptide, facilitating mechanistic analyses.Most phages of Gram-negative hosts encode spanins for disruption of the outer membrane, the last step in host lysis. However, bioinformatic analysis indicates that ∼15% of these phages lack a spanin gene, suggesting they have an alternate way of disrupting the OM. Here, we show that the T7-like coliphage phiKT causes the explosive cell lysis associated with spanin activity despite not encoding spanins. A putative lysis cassette cloned from the phiKT late gene region includes the hypothetical novel gene 28 located between the holin and endolysin genes and supports inducible lysis in E. coli K-12. Moreover, induction of an isogenic construct lacking gene 28 resulted in divalent cation-stabilized spherical cells rather than lysis, implicating gp28 in OM disruption. Additionally, gp28 was shown to complement the lysis defect of a spanin-null λ lysogen. Gene 28 encodes a 56-amino acid cationic protein with predicted amphipathic helical structure and is membrane-associated after lysis. Urea and KCl washes did not release gp28 from the particulate, suggesting a strong hydrophobic membrane interaction. Fluorescence microscopy supports membrane localization of the gp28 protein prior to lysis. Gp28 is similar in size, charge, predicted fold, and membrane association to the human cathelicidin antimicrobial peptide LL-37. Synthesized gp28 behaved similar to LL-37 in standard assays mixing peptide and cells to measure bactericidal and inhibitory effects. Taken together, these results indicate that phiKT gp28 is a phage-encoded cationic antimicrobial peptide that disrupts bacterial outer membranes during host lysis and thus establishes a new class of phage lysis proteins, the disruptins. Significance We provide evidence that phiKT produces an antimicrobial peptide for outer membrane disruption during lysis. This protein, designated as a disruptin, is a new paradigm for phage lysis and has no similarities to other known lysis genes. Although many mechanisms have been proposed for the function of antimicrobial peptides, there is no consensus on the molecular basis of membrane disruption. Additionally, there is no established genetic system to support such studies. Therefore, the phiKT disruptin may represent the first genetically tractable antimicrobial peptide, facilitating mechanistic analyses. |
| Author | Ramsey, Jolene Maddox, Lori T Holt, Ashley Galbadage, Thushara Cahill, Jesse Sule, Preeti Young, Ry O'Leary, Chandler Martin, Cody Sharan, Riti Cirillo, Jeffrey D Moreland, Russell |
| Author_xml | – sequence: 1 givenname: Ashley surname: Holt fullname: Holt, Ashley organization: Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas, USA – sequence: 2 givenname: Jesse surname: Cahill fullname: Cahill, Jesse organization: Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas, USA – sequence: 3 givenname: Jolene surname: Ramsey fullname: Ramsey, Jolene organization: Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas, USA – sequence: 4 givenname: Cody surname: Martin fullname: Martin, Cody organization: Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas, USA – sequence: 5 givenname: Chandler surname: O'Leary fullname: O'Leary, Chandler organization: Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas, USA – sequence: 6 givenname: Russell surname: Moreland fullname: Moreland, Russell organization: Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas, USA – sequence: 7 givenname: Lori T surname: Maddox fullname: Maddox, Lori T organization: Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas, USA – sequence: 8 givenname: Thushara surname: Galbadage fullname: Galbadage, Thushara organization: Department of Microbial Pathogenesis and Immunology, Texas A&M University Health, Bryan, Texas, USA – sequence: 9 givenname: Riti surname: Sharan fullname: Sharan, Riti organization: Department of Microbial Pathogenesis and Immunology, Texas A&M University Health, Bryan, Texas, USA – sequence: 10 givenname: Preeti surname: Sule fullname: Sule, Preeti organization: Department of Microbial Pathogenesis and Immunology, Texas A&M University Health, Bryan, Texas, USA – sequence: 11 givenname: Jeffrey D surname: Cirillo fullname: Cirillo, Jeffrey D organization: Department of Microbial Pathogenesis and Immunology, Texas A&M University Health, Bryan, Texas, USA – sequence: 12 givenname: Ry surname: Young fullname: Young, Ry organization: Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas, USA |
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