TY - JOUR

T1 - Uptake, Stability, and Activity of Antisense Anti-acpP PNA-Peptide Conjugates in Escherichia coli and the Role of SbmA

AU - Yavari, Niloofar

AU - Goltermann, Lise

AU - Nielsen, Peter E.

PY - 2021

Y1 - 2021

N2 - PNA oligomers conjugated to bacteria penetrating peptides (BPPs), such as (KFF)3K, targeting essential bacterial genes, such as acpP, can inhibit bacterial growth at one-digit micromolar concentrations. It has been found that the LPS of the outer membrane of Gram-negative bacteria is a barrier for cellular uptake of (KFF)3K-eg1-PNA and that the SbmA transporter protein is involved in the passage through the inner membrane. We now further elucidate the uptake mechanism of (KFF)3K-eg1-PNA by showing that the peptide part of (KFF)3K-eg1-PNA is unstable and is degraded by peptidases in the medium of a bacterial culture (t1/2 < 5 min) and inside the bacteria. Analysis of peptide-PNA conjugates present in the periplasmic space and the cytoplasm showed the presence of mainly PNA with only the FFK tripeptide and without a peptide, at a concentration 10-fold that added to the medium. Furthermore, the two main degradation products showed no antibacterial effect when added directly to a bacterial culture and the antibacterial effect decreased with peptide length, thereby demonstrating that an intact peptide is indeed crucial for uptake but not for intracellular antisense activity. Most surprisingly, it was found that although the corresponding series of the proteolytically stable D-form (kff)3k-eg1-PNAs exhibited an analogous reduction of activity with peptide length, the activity was dependent on the presence of SbmA for the shorter peptides (which is not the case with the full length peptide). Therefore, our results suggest that the BPP is necessary for crossing both the LPS/outer membrane as well as the inner membrane and that full length (KFF)3K may spontaneously pass the inner membrane. Thus, SbmA dependence of (KFF)3K-eg1-PNA is ascribed to peptide degradation in the bacterial medium and in periplasmic space. Finally, the results show that stability and metabolism (by bacterial proteases/peptidases) should be taken into consideration upon design and activity/uptake analysis of BPPs (and antimicrobial peptides).

AB - PNA oligomers conjugated to bacteria penetrating peptides (BPPs), such as (KFF)3K, targeting essential bacterial genes, such as acpP, can inhibit bacterial growth at one-digit micromolar concentrations. It has been found that the LPS of the outer membrane of Gram-negative bacteria is a barrier for cellular uptake of (KFF)3K-eg1-PNA and that the SbmA transporter protein is involved in the passage through the inner membrane. We now further elucidate the uptake mechanism of (KFF)3K-eg1-PNA by showing that the peptide part of (KFF)3K-eg1-PNA is unstable and is degraded by peptidases in the medium of a bacterial culture (t1/2 < 5 min) and inside the bacteria. Analysis of peptide-PNA conjugates present in the periplasmic space and the cytoplasm showed the presence of mainly PNA with only the FFK tripeptide and without a peptide, at a concentration 10-fold that added to the medium. Furthermore, the two main degradation products showed no antibacterial effect when added directly to a bacterial culture and the antibacterial effect decreased with peptide length, thereby demonstrating that an intact peptide is indeed crucial for uptake but not for intracellular antisense activity. Most surprisingly, it was found that although the corresponding series of the proteolytically stable D-form (kff)3k-eg1-PNAs exhibited an analogous reduction of activity with peptide length, the activity was dependent on the presence of SbmA for the shorter peptides (which is not the case with the full length peptide). Therefore, our results suggest that the BPP is necessary for crossing both the LPS/outer membrane as well as the inner membrane and that full length (KFF)3K may spontaneously pass the inner membrane. Thus, SbmA dependence of (KFF)3K-eg1-PNA is ascribed to peptide degradation in the bacterial medium and in periplasmic space. Finally, the results show that stability and metabolism (by bacterial proteases/peptidases) should be taken into consideration upon design and activity/uptake analysis of BPPs (and antimicrobial peptides).

U2 - 10.1021/acschembio.0c00822

DO - 10.1021/acschembio.0c00822

M3 - Journal article

C2 - 33684286

AN - SCOPUS:85103228765

VL - 16

SP - 471

EP - 479

JO - A C S Chemical Biology

JF - A C S Chemical Biology

SN - 1554-8929

IS - 3

ER -