Targeting the Bacterial Cytoskeleton of the Burkholderia cepacia Complex for Antimicrobial Development: A Cautionary Tale

Abstract

Burkholderia cepacia complex (BCC) bacteria are a group of opportunistic pathogens that cause severe lung infections in cystic fibrosis (CF). Treatment of BCC infections is difficult, due to the inherent and acquired multidrug resistance of BCC. There is a pressing need to find new bacterial targets for antimicrobials. Here, we demonstrate that the novel compound Q22, which is related to the bacterial cytoskeleton destabilising compound A22, can reduce the growth rate and inhibit growth of BCC bacteria. We further analysed the phenotypic effects of Q22 treatment on BCC virulence traits, to assess its feasibility as an antimicrobial. BCC bacteria were grown in the presence of Q22 with a broad phenotypic analysis, including resistance to H₂O₂-induced oxidative stress, changes in the inflammatory potential of cell surface components, and in-vivo drug toxicity studies. The influence of the Q22 treatment on inflammatory potential was measured by monitoring the cytokine responses of BCC whole cell lysates, purified lipopolysaccharide, and purified peptidoglycan extracted from bacterial cultures grown in the presence or absence of Q22 in differentiated THP-1 cells. BCC bacteria grown in the presence of Q22 displayed varying levels of resistance to H₂O₂-induced oxidative stress, with some strains showing increased resistance after treatment. There was strain-to-strain variation in the pro-inflammatory ability of bacterial lysates to elicit TNFα and IL-1β from human myeloid cells. Despite minimal toxicity previously shown in vitro with primary CF cell lines, in-vivo studies demonstrated Q22 toxicity in both zebrafish and mouse infection models. In summary, destabilisation of the bacterial cytoskeleton in BCC, using compounds such as Q22, led to increased virulence-related traits in vitro. These changes appear to vary depending on strain and BCC species. Future development of antimicrobials targeting the BCC bacterial cytoskeleton may be hampered if such effects translate into the in-vivo environment of the CF infection.

Keywords: Burkholderia; antimicrobial; cytoskeleton.

Figure 1

Growth and morphological changes induced by Q22 treatment. (A) Growth of B. cenocepacia J2315 in the absence or presence of increasing concentrations of Q22 (0, 20, 30, 40 μg/mL); (B) scanning electron microscope images of B. cenocepacia J2315, untreated and after treatment with 30 μg/mL Q22 at 6 h timepoint.

Figure 2

Survival of B. cepacia complex (BCC) strains exposed to H₂O₂ in vitro. Late-stationary-phase cells were treated with varying concentrations of H₂O₂, 0 mM (A), 10 mM (B), and 20 mM (C), as described under Section 4. Samples were plated in triplicate for colony counts, and percentage survival (expressed as % viability) was calculated relative to colony counts of untreated bacteria. * p < 0.05. Isolates included B. cenocepacia LMG16656 (J2315), B. cenocepacia LMG18829, B. multivorans LMG13010, and B. vietnamiensis LMG16232 (see Table 1).

Figure 3

Cytokine responses of THP-1 cells, stimulated with Q22-treated and untreated preparations. Cytokine responses at 6 h post-stimulation with whole cell lysates of BCC strains B. cenocepacia J2315, B. cenocepacia LMG18829, B. multivorans LMG13010, and B. vietnamiensis LMG16232. (A) TNFα; (B) IL-1β. TNFα responses at 6 h post stimulation with whole cell lysates, purified lipopolysaccharide (LPS) (C), or purified peptidoglycan (PG) (D) from BCC strain B. cenocepacia J2315. Comparison of profiles from cells stimulated with and without a CD14 antibody pre-incubation step (C). * p < 0.05.

Figure 4

Toxicity of Q22 in the zebrafish embryo model. Zebrafish embryos were exposed to increasing concentrations of Q22 (6.4 µg/mL; 64 µg/mL; 640 µg/mL) at 30 high power field and monitored for survival for up to 120 h post-incubation. Data expressed as percentage survival.