Antisense phosphorodiamidate morpholino oligomers targeted to an essential gene inhibit Burkholderia cepacia complex

Abstract

Background: Members of the Burkholderia cepacia complex (Bcc) cause considerable morbidity and mortality in patients with chronic granulomatous disease and cystic fibrosis. Many Bcc strains are antibiotic resistant, which requires the exploration of novel antimicrobial approaches, including antisense technologies such as phosphorodiamidate morpholino oligomers (PMOs).

Methods: Peptide-conjugated PMOs (PPMOs) were developed to target acpP, which encodes an acyl carrier protein (AcpP) that is thought to be essential for growth. Their antimicrobial activities were tested against different strains of Bcc in vitro and in infection models.

Results: PPMOs targeting acpP were bactericidal against clinical isolates of Bcc (>4 log reduction), whereas a PPMO with a scrambled base sequence (scrambled PPMO) had no effect on growth. Human neutrophils were infected with Burkholderia multivorans and treated with AcpP PPMO. AcpP PPMO augmented killing, compared with neutrophils alone and compared with neutrophils alone plus scrambled PPMO. Mice with chronic granulomatous disease that were infected with B. multivorans were treated with AcpP PPMO, scrambled PPMO, or water at 0, 3, and 6 h after infection. Compared with water-treated control mice, the AcpP PPMO-treated mice showed an approximately 80% reduction in the risk of dying by day 30 of the experiment and relatively little pathology.

Conclusion: AcpP PPMO is active against Bcc infections in vitro and in vivo.

Figure 1

The AcpP PPMO is bactericidal in vitro against members of the Bcc. B. multivorans (CGD clinical isolate #1, Panel A); B. cepacia (CGD clinical isolate #3, Panel B); Panel C: B. cenocepacia (CGD clinical isolate #5, Panel C) were grown overnight alone, or in the presence of PPMOs. Scr PPMO = scrambled sequence control; AcpP PPMO #1 = acyl carrier protein PPMO; Peptide only = (RFF)₃RXB peptide without oligomer where X indicates 6-aminohexanoic acid and B indicates beta-alanine. Each bar represents the mean cfu/mL from 5 independent experiments. ** p<0.0001 compared to T0 Burkholderia only (paired t-test). The 24-hour counts for AcpP PPMO were 0 cfu/ml for all observations in panels A-C, with the limit of detection indicated as 10 cfu/ml.

Figure 2

Sequence alignment of the acpP gene in species of Bcc. There is high homology of the 5′ acpP gene sequence within the genus Burkholderia. Shown are the acpP sequences for various species of Bcc and other members of Burkholderia. The start ATG is in bold and underlined. AcpP PPMO #1 and AcpP PPMO #2 are two PPMOs designed against the acpP gene. Small letters in red represent a base that has a mismatch compared to the PPMO sequence. An asterisk represents strains where PPMOs were tested.

Figure 3

The AcpP PPMO #2 enhances PMN killing of B. multivorans (CGD clinical isolate #1). The symbols represent time points of cell harvest (2, 4 and 24 hours) and are displayed as the mean cfu/mL. Open circles = B. multivorans alone; Open squares = PMN + B. multivorans; Black circles = PMN + B. multivorans + Scr PPMO; Black squares = PMN + B. multivorans + AcpP PPMO #2. Each symbol represents the mean of 6 normal donors over 3 separate experiments (Panel A) and 3 CGD donors on 2 separate experiments (Panel B) and error bars represents the range.

Figure 4

AcpP PPMO increases survival of B. multivorans (CGD clinical isolate #1) infected mice. Mice were age- and sex-matched for all experiments. Mice were injected with 5 × 10⁶ cfu intraperitoneally (ip) followed by an immediate ip dose of 200μg (0.1 mL) of PPMO treatment (Black squares = AcpP PPMO #2 tx; Black circles = Scrambled PPMO tx) or Water alone (Black diamonds). **AcpP PPMO treatment had a reduction in 30-day mortality compared to water treatment with a hazards ratio (HR) of .21 (p<0.0001); and to scrambled PPMO treatment with a HR of .47 (p=0.037). *Scr PPMO treatment had a reduction in 30-day mortality compared to water treatment with a HR of .44 (p=0.023).

Figure 5

Histopathologic findings in mice infected with B. multivorans. Spleen and lymph nodes were fixed, sectioned and stained with hematoxylin and eosin. An AcpP PPMO-treated mouse that survived until day 30 has normal splenic architecture (Panel A; 2.5x magnification). The spleen of a water-treated mouse that survived until day 4 has expansion of red pulp with suppurative splenitis, which effaces normal hematopoietic elements (Panel B; 2.5x magnification). Panel C shows an aggregate of neutrophils at 40x. A section of the mesenteric lymph node from the same AcpP PPMO-treated mouse (Panel D) with normal architecture. The mesenteric lymph node from the water-treated mouse has multifocal suppurative lymphadenitis in the outer cortex of the node (Panel E; 2.5x magnification). Panel F shows a focus of suppurative inflammation from the affected lymph node at 40x.