Inactivation of Phospholipase D Diminishes Acinetobacter baumannii Pathogenesis

Growth characteristics of A. baumannii PFGE types in human serum. Graphed are the growth characteristics of representative strains of 12 distinct A. baumannii PFGE types following 0, 7.5, 15, 30, and 60 h of incubation in human serum, as determined by the OD₆₀₀ (starting inoculum, 1 × 10⁷ CFU). The black lines and symbols indicate strains that exhibited the ability to proliferate well in serum, and the light-gray lines and symbols indicate strains that exhibited diminished serum growth in comparison to all other isolates analyzed. All OD₆₀₀ values have been normalized to the optical density of serum alone, and standard deviations are shown (n = 6). (Inset) Growth curves of A. baumannii strain 98-37-09 (solid boxes) (PFGE type I; proliferates well in human serum) and ATCC 17978 (open boxes) (PFGE type E; reduced serum growth) grown in human serum; standard deviations are shown (n = 3).

Growth characteristics of A. baumannii mutant library members with increased serum susceptibility. Shown are the growth characteristics of the wild type (strain 98-37-09) and isogenic mutants in LB medium (solid bars) and human serum (hatched bars), as measured by the OD₆₀₀. All values have been plotted relative to wild-type growth (set at 1.0). Also shown are the growth curves of wild-type (solid boxes), ACJ2 (A1S_2989 mutant; solid diamonds), and ACJ2 harboring pACJ01 (wild-type copy of A1S_2989; open circles) in human serum (inset). Standard deviations are shown (n = 6).

Characterization of the ATCC 17978 A1S_2989 locus. (A) Predicted amino acid sequence of ATCC 17978 locus A1S_2989-encoded protein with putative PLD active sites underlined; the boldface residues correspond to conserved HXKX₄DX₆G(G/S) amino acids. The asterisk indicates a transposon insertion site within strain ACJ2. (B) Chromosomal organization of A. baumannii strain ATCC 17978 locus A1S_2989 with the strain ACJ2 transposon EZ-Tn5 <R6Kγori/KAN-2> insertion site shown. (C) Graph of the phospholipase D activity assay results for wild-type (strain 98-37-09) and ACJ2 (A1S_2989 mutant) cells harboring either vector (pWH1266) or a plasmid containing a wild-type copy of A1S_2989 (pACJ01). The results were normalized to equal amounts of total starting protein, as described in Materials and Methods; standard deviations are shown (n = 6).

Invasion properties of A. baumannii 98-37-09, ACJ2, and complementation strains. (A) Internalization characteristics of wild-type (98-37-09) and ACJ2 (A1S_2989 mutant) cells following incubation with human cervical cancer (HeLa) or human bronchial epithelial (BEAS-2B) cells. Plotted are the numbers of CFU ml of A. baumannii⁻¹ internalized following 4 h of incubation. (B) Internalization properties of wild-type and ACJ2 cells harboring plasmid vector (pWH1266) or an A1S_2989 complementation plasmid (pACJ01). The asterisks indicate statistically significant differences between wild-type and ACJ2 internalization as determined by Student's t test (A) or two-way analysis of variance (P < 0.05). Standard deviations are shown.

Pathogenic properties of A. baumannii strain 98-37-09 and ACJ2 cells in a murine-pneumonia model. (A) The averaged bacterial burdens of wild-type (98-37-09) and ACJ2 (A1S_2989 mutant) A. baumannii cells collected from the blood at 24 and 48 h after intranasal colonization (n ≥ 7); the horizontal line indicates the lower limit of detection. (B) The bacterial burdens of the wild type and ACJ2 obtained from lungs, hearts, and livers at 24 h (n ≥ 8) or 48 h (n ≥ 16) after intranasal colonization; the 48-h data points represent the results from two independent experiments; the horizontal lines indicate the lower limits of detection. The asterisks indicate statistically significant differences between the wild-type and ACJ2 bacterial burdens as determined by a two-tailed, unpaired Student t test (*, P < 0.05; **, P < 0.01). All data are shown as the log CFU ml blood or homogenized tissue⁻¹.