Exploring Roles of the Polysaccharide Capsule in Pathogenesis of Hypervirulent Acinetobacter baumannii Clinical Isolate Lac-4

Abstract

The frequently multidrug-resistant bacterial pathogen Acinetobacter baumannii is a leading cause of nosocomial infections, including ventilator-associated pneumonia, such that the World Health Organization and US Centers for Disease Control and Prevention have declared it a top priority candidate for novel drug development. Nearly all clinical A. baumannii strains express a thick surface polysaccharide capsule that protects against desiccation, host defenses, and disinfectants. In this study, we investigated the contribution of the polysaccharide capsule to virulence caused by the A. baumannii clinical isolate Ab Lac-4, which is rare in its ability to cause pneumonia and disseminated sepsis in healthy mice. We assessed the role of the capsule in wildtype Lac-4 (WT) by generating a premature stop codon in wza, which codes for the polysaccharide export protein. The wza# mutant was hypersensitive to killing by complement, whole blood, and healthy human neutrophils compared to WT and a revertant mutant (wza-Rev). Furthermore, the wza# mutant was highly attenuated in murine sepsis and unable to disseminate from the lungs during pneumonia. This study reinforces the capsule as a key contributor to Ab Lac-4 hypervirulence.

Keywords: Acinetobacter baumannii; antibiotic resistance; bacterial infections; complement; neutrophils; pneumonia; polysaccharide capsule; sepsis.

Figure 1

Gene deletion strategy: (A) Schematic depicting capsule locus (K Locus) for Acinetobacter baumannii Ab Lac-4. (B) Representative images showing the impact of capsule loss on buoyancy and growth on tryptic soy agar (TSA). Ab Lac-4 WT, wza#, and wza-Rev were grown to stationary phase and allowed to stand at RT for 3 h. (C) Turbidity (OD₆₀₀) of the liquid cultures; three independent experiments pooled. n.s. = not significant, **** p < 0.0001. (D) Growth curves from Ab Lac-4, wza#, and wza-Rev grown in tryptic soy broth (THB). Three independent experiments were pooled.

Figure 2

Loss of polysaccharide capsule significantly increases susceptibility to the innate immune system. Percent survival of Ab Lac-4, wza#, and wza-Rev in the presence of healthy human (A) whole blood, (B) heat-inactivated whole blood, (C) 10% active human serum, and (D) 10% heat-inactivated human serum for 1 or 3 h. (E) Percent survival of serum opsonized WT, wza#, and wza-Rev 3 h after incubation with healthy human neutrophils. The means of three independent experiments ± SEM are graphed. n.s. not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 3

The capsule is required to cause lethal sepsis and systemic dissemination from the lungs during pneumonia. (A) Survival data from C57BL/6 (B6) mice infected intraperitoneally (IP) with 1 × 10⁷ CFUs WT, wza#, or wza-Rev in PBS. n = 10, two independent experiments pooled. (B) CFUs from lungs, spleens, and blood collected 12 h post-infection with 1 × 10⁷ CFUs WT, wza#, or wza-Rev intratracheally (IT). Mice were humanely euthanized according to IACUC protocols; organs were collected, homogenized, serially diluted in sterile PBS, and plated for CFUs on TSA. Blood was collected using cardiac puncture. n = 10, two independent experiments were pooled. (C) Percent adherence to A549s relative to WT. WT, wza#, and wza-Rev were incubated with A549 cells for 30 min. Cells were washed 5× with PBS, trypsinized with 0.05% trypsin, serially diluted in sterile PBS, and plated for CFUs. Means ± SEM of three independent experiments. n.s. not significant, ** p < 0.01, *** p < 0.001, **** p < 0.0001.