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Species-selective killing of bacteria by antimicrobial peptide-PNAs.

Mondhe M, Chessher A, Goh S, Good L, Stach JE - PLoS ONE (2014)

Bottom Line: An S.Typhimurium-specific PNA targeting ftsZ resulted in elongated cells that were not observed in E. coli, providing phenotypic evidence of the selectivity of PNA-based antimicrobials.This work provides a basis for the development of a new class of antimicrobial with a tuneable spectrum of activity.

View Article: PubMed Central - PubMed

Affiliation: School of Biology, Newcastle University, Newcastle upon Tyne, United Kingdom.

ABSTRACT
Broad-spectrum antimicrobials kill indiscriminately, a property that can lead to negative clinical consequences and an increase in the incidence of resistance. Species-specific antimicrobials that could selectively kill pathogenic bacteria without targeting other species in the microbiome could limit these problems. The pathogen genome presents an excellent target for the development of such antimicrobials. In this study we report the design and evaluation of species-selective peptide nucleic acid (PNA) antibacterials. Selective growth inhibition of B. subtilis, E. coli, K. pnuemoniae and S. enterica serovar Typhimurium in axenic or mixed culture could be achieved with PNAs that exploit species differences in the translation initiation region of essential genes. An S. Typhimurium-specific PNA targeting ftsZ resulted in elongated cells that were not observed in E. coli, providing phenotypic evidence of the selectivity of PNA-based antimicrobials. Analysis of the genomes of E. coli and S. Typhimurium gave a conservative estimate of >150 PNA targets that could potentially discriminate between these two closely related species. This work provides a basis for the development of a new class of antimicrobial with a tuneable spectrum of activity.

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Species-selective antibacterial peptide-PNAs in axenic and two-species mixed culture.E. coli (dark grey), K. pneumoniae (white) and S. Typhimurium (light grey). All cultures were incubated for 16 hrs. A) axenic cultures of the species were treated with E. coli- specific Ec1000 at 3.2 µM, K. pneumoniae-specific Kp0001 at 3.2 µM and S. Typhimurium-specific Se0001 at 2.0 µM. Asterisks indicate species-selective growth inhibition of E. coli, K. pneumoniae and S. Typhimurium respectively. B) Two-species mixed cultures treated with peptide-PNAs as above. The control cultures show the relative proportion of the two species without treatment, the two treatments to the left of the control represent the same mixed culture treated with a peptide-PNA. Black arrows indicate non species-selective growth inhibition of S. Typhimurium by Ec1000. Error bars are standard error for biological replicates (n = 3).
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pone-0089082-g001: Species-selective antibacterial peptide-PNAs in axenic and two-species mixed culture.E. coli (dark grey), K. pneumoniae (white) and S. Typhimurium (light grey). All cultures were incubated for 16 hrs. A) axenic cultures of the species were treated with E. coli- specific Ec1000 at 3.2 µM, K. pneumoniae-specific Kp0001 at 3.2 µM and S. Typhimurium-specific Se0001 at 2.0 µM. Asterisks indicate species-selective growth inhibition of E. coli, K. pneumoniae and S. Typhimurium respectively. B) Two-species mixed cultures treated with peptide-PNAs as above. The control cultures show the relative proportion of the two species without treatment, the two treatments to the left of the control represent the same mixed culture treated with a peptide-PNA. Black arrows indicate non species-selective growth inhibition of S. Typhimurium by Ec1000. Error bars are standard error for biological replicates (n = 3).

Mentions: The peptide-PNAs designed in this study were assayed for their antibacterial activity against both target and non-target species. The previously reported En108 peptide-PNA (called Ec108 in [17]) was used as a control to test the feasibility of species-selectivity at a broad taxonomic level; E. coli, K. pneumoniae and S. Typhimurium (Gram-negative, Enterobacteriaceae) have identical acpP TIRs and thus all three species should be susceptible to En108, while the acpP TIR of Bacillus subtilis (Gram-positive, Bacillaceae) has six base-pair mismatches and should be resistant to En108. Antibacterial assays with En108 proved this to be the case; En108 had an MIC of 1.2, 0.4 and 0.3 µM for E. coli, K. pneumoniae and S. Typhimurium respectively (Table 1), and had no detectable antibacterial activity against B. subtilis at concentrations of up to 20 µM (data not shown). Similarly, the species-selective PNAs for B. subtilis, K. pneumoniae and S. Typhimurium were only antibacterial to the intended species (Figure 1A). The E. coli-selective Ec1000 was unexpectedly cross-reactive with S. Typhimurium (discussed below). In all cases treatment with non-specific PNAs resulted in increased, but statistically insignificant (standard error P>0.05), growth. Table 2 shows the analysis of potential binding sites within the genomes of the target species. Of note is the difference in MIC between E. coli, K. pneumoniae and S. Typhimurium when treated with the acpP-targeting En108; the MIC of this PNA was 3 and 4 fold less in K. pneumoniae and S. Typhimurium, respectively (Table 1). Analysis of the binding sites of En108, Kp0001 and Se0001 in the genomes of these species revealed that En108 likely binds in the TIR region of other essential genes in K. pneumoniae (mukF and ribH) and S. Typhimurium (yhhM) each with a 1 bp mismatch. This could account for the decreased MIC in these species, however the relationship is not straightforward as Se0001 is predicted to bind in the TIR of at least three other genes determined to be essential in E. coli and S. Typhimurium (hemK, lnt and rluA) and has an MIC equivalent to that of En108 in E. coli (Table 1). Furthermore, there is no obvious relationship between the MIC of a peptide-PNA and the number of off-targets in the genome of the target species, including those that bind in the TIRs of both essential and non-essential genes (Table 2). Reasons for the possible differences between the MICs of the different peptide-PNAs are discussed below.


Species-selective killing of bacteria by antimicrobial peptide-PNAs.

Mondhe M, Chessher A, Goh S, Good L, Stach JE - PLoS ONE (2014)

Species-selective antibacterial peptide-PNAs in axenic and two-species mixed culture.E. coli (dark grey), K. pneumoniae (white) and S. Typhimurium (light grey). All cultures were incubated for 16 hrs. A) axenic cultures of the species were treated with E. coli- specific Ec1000 at 3.2 µM, K. pneumoniae-specific Kp0001 at 3.2 µM and S. Typhimurium-specific Se0001 at 2.0 µM. Asterisks indicate species-selective growth inhibition of E. coli, K. pneumoniae and S. Typhimurium respectively. B) Two-species mixed cultures treated with peptide-PNAs as above. The control cultures show the relative proportion of the two species without treatment, the two treatments to the left of the control represent the same mixed culture treated with a peptide-PNA. Black arrows indicate non species-selective growth inhibition of S. Typhimurium by Ec1000. Error bars are standard error for biological replicates (n = 3).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3928365&req=5

pone-0089082-g001: Species-selective antibacterial peptide-PNAs in axenic and two-species mixed culture.E. coli (dark grey), K. pneumoniae (white) and S. Typhimurium (light grey). All cultures were incubated for 16 hrs. A) axenic cultures of the species were treated with E. coli- specific Ec1000 at 3.2 µM, K. pneumoniae-specific Kp0001 at 3.2 µM and S. Typhimurium-specific Se0001 at 2.0 µM. Asterisks indicate species-selective growth inhibition of E. coli, K. pneumoniae and S. Typhimurium respectively. B) Two-species mixed cultures treated with peptide-PNAs as above. The control cultures show the relative proportion of the two species without treatment, the two treatments to the left of the control represent the same mixed culture treated with a peptide-PNA. Black arrows indicate non species-selective growth inhibition of S. Typhimurium by Ec1000. Error bars are standard error for biological replicates (n = 3).
Mentions: The peptide-PNAs designed in this study were assayed for their antibacterial activity against both target and non-target species. The previously reported En108 peptide-PNA (called Ec108 in [17]) was used as a control to test the feasibility of species-selectivity at a broad taxonomic level; E. coli, K. pneumoniae and S. Typhimurium (Gram-negative, Enterobacteriaceae) have identical acpP TIRs and thus all three species should be susceptible to En108, while the acpP TIR of Bacillus subtilis (Gram-positive, Bacillaceae) has six base-pair mismatches and should be resistant to En108. Antibacterial assays with En108 proved this to be the case; En108 had an MIC of 1.2, 0.4 and 0.3 µM for E. coli, K. pneumoniae and S. Typhimurium respectively (Table 1), and had no detectable antibacterial activity against B. subtilis at concentrations of up to 20 µM (data not shown). Similarly, the species-selective PNAs for B. subtilis, K. pneumoniae and S. Typhimurium were only antibacterial to the intended species (Figure 1A). The E. coli-selective Ec1000 was unexpectedly cross-reactive with S. Typhimurium (discussed below). In all cases treatment with non-specific PNAs resulted in increased, but statistically insignificant (standard error P>0.05), growth. Table 2 shows the analysis of potential binding sites within the genomes of the target species. Of note is the difference in MIC between E. coli, K. pneumoniae and S. Typhimurium when treated with the acpP-targeting En108; the MIC of this PNA was 3 and 4 fold less in K. pneumoniae and S. Typhimurium, respectively (Table 1). Analysis of the binding sites of En108, Kp0001 and Se0001 in the genomes of these species revealed that En108 likely binds in the TIR region of other essential genes in K. pneumoniae (mukF and ribH) and S. Typhimurium (yhhM) each with a 1 bp mismatch. This could account for the decreased MIC in these species, however the relationship is not straightforward as Se0001 is predicted to bind in the TIR of at least three other genes determined to be essential in E. coli and S. Typhimurium (hemK, lnt and rluA) and has an MIC equivalent to that of En108 in E. coli (Table 1). Furthermore, there is no obvious relationship between the MIC of a peptide-PNA and the number of off-targets in the genome of the target species, including those that bind in the TIRs of both essential and non-essential genes (Table 2). Reasons for the possible differences between the MICs of the different peptide-PNAs are discussed below.

Bottom Line: An S.Typhimurium-specific PNA targeting ftsZ resulted in elongated cells that were not observed in E. coli, providing phenotypic evidence of the selectivity of PNA-based antimicrobials.This work provides a basis for the development of a new class of antimicrobial with a tuneable spectrum of activity.

View Article: PubMed Central - PubMed

Affiliation: School of Biology, Newcastle University, Newcastle upon Tyne, United Kingdom.

ABSTRACT
Broad-spectrum antimicrobials kill indiscriminately, a property that can lead to negative clinical consequences and an increase in the incidence of resistance. Species-specific antimicrobials that could selectively kill pathogenic bacteria without targeting other species in the microbiome could limit these problems. The pathogen genome presents an excellent target for the development of such antimicrobials. In this study we report the design and evaluation of species-selective peptide nucleic acid (PNA) antibacterials. Selective growth inhibition of B. subtilis, E. coli, K. pnuemoniae and S. enterica serovar Typhimurium in axenic or mixed culture could be achieved with PNAs that exploit species differences in the translation initiation region of essential genes. An S. Typhimurium-specific PNA targeting ftsZ resulted in elongated cells that were not observed in E. coli, providing phenotypic evidence of the selectivity of PNA-based antimicrobials. Analysis of the genomes of E. coli and S. Typhimurium gave a conservative estimate of >150 PNA targets that could potentially discriminate between these two closely related species. This work provides a basis for the development of a new class of antimicrobial with a tuneable spectrum of activity.

Show MeSH
Related in: MedlinePlus