Inhibition of Intracellular Growth of Salmonella enterica Serovar Typhimurium in Tissue Culture by Antisense Peptide-Phosphorodiamidate Morpholino Oligomer

Abstract

Two types of phosphorodiamidate morpholino oligomers (PMOs) were tested for inhibition of growth of Salmonella enterica serovar Typhimurium. Both PMOs have the same 11-base sequence that is antisense to the region near the start codon of acpP, which is essential for lipid biosynthesis and viability. To the 3 end of each is attached the membrane-penetrating peptide (RXR) 4 XB (R, X, and B indicate arginine, 6-aminohexanoic acid, and ␤-alanine, respectively). One peptide-PMO (AcpP PPMO) has no charge on the PMO moiety. The second PPMO has three cations (piperazine) attached to the phosphorodiamidate linkages (3؉Pip-AcpP PPMO). A scrambled-sequence PPMO (Scr PPMO) was synthesized for each type of PMO. The MICs of AcpP PPMO, 3؉Pip-AcpP PPMO, and either one of the Scr PPMOs were 1.25 M (7 g/ml), 0.156 M (0.94 g/ml), and >160 M (>900 g/ml), respectively. 3؉Pip-AcpP PPMO at 1.25 or 2.5 M significantly reduced the growth rates of pure cultures, whereas AcpP PPMO or either Scr PPMO had no effect. However, the viable cell count was significantly reduced at either concentration of 3؉Pip-AcpP PPMO or AcpP PPMO, but not with either Scr PPMO. In other experiments, macrophages were infected intracellularly with S. enterica and treated with 3 M 3؉Pip-AcpP PPMO. Intracellular bacteria were reduced >99% with 3؉Pip-AcpP PPMO, whereas intracellular bacteria increased 3 orders of magnitude in untreated or Scr PPMO-treated cultures. We conclude that either AcpP PPMO or 3؉Pip-AcpP PPMO inhibited growth of S. enterica in pure culture and that 3؉Pip-AcpP PPMO reduced intracellular viability of S. enterica in macrophages.

Bacterial Antisense RNAs: How Many Are There, and What Are They Doing?

“…They have been shown to inhibit growth of E. coli (28, 62, 68, 101), S. enterica (64), Staphylococcus aureus (67) and M. tuberculosis (32) when targeted to essential genes. Since antisense RNAs allow for the conditional repression of target genes, they also have been employed to study bacterial growth and metabolism (6, 27, 40, 76, 110) and to characterize known or putative virulence factors (38, 65).…”

Antisense Phosphorodiamidate Morpholino Oligomers Targeted to an Essential Gene InhibitBurkholderia cepaciaComplex

“…The conjugation of various antimicrobial peptides to the PMO (herein called PPMO) has led to dramatically increased cell permeability[16]. These molecules have been used successfully in other bacteria such as Escherichia coli and Salmonella [17, 18]. In the case of E. coli , the gene targeted was acpP (acyl carrier protein), which is essential for bacterial growth[17, 19].…”

Targeting Essential Genes in Salmonella enterica Serovar Typhimurium with Antisense Peptide Nucleic Acid

“…The ability of the PNAs to cross the cytoplasmic membrane was facilitated by the CPP via the linker. However, the relatively high molecular weights and possibly the polarity account for the decreased antibacterial activity that is observed when comparing activities in vitro and in cell culture (17,21). Conjugates needed to traverse two phospholipid membranes in addition to the cell membrane of the bacteria to reach the target mRNA and the introduction of these additional barriers constrained bioaccumulation of the PNA-peptide conjugates inside the bacterium, thereby reducing antibacterial activity compared to that in in vitro models.…”