Fig 1.
Capsular gene loci for A. baumannii KL3 and KL22 and capsular carbohydrate composition and linkage of KL22, and KL3 capsule locus strains.
(A) Whole-genome sequencing of HUMC1 (a hypervirulent strain), 15827 (a hypovirulent strain) and ATCC 17978 (an avirulent strain) revealed distinct capsule loci organized into KL22 (HUMC1 and 15827) and KL3 (ATCC 17978) groups. KL22 differs from KL3 in that it contains an extra acetyltransferase gene pgt1, while HUMC1 (KL22) contains a transposon insertion sequence disruption in the coding region of the glycotransferase gtr6 (downward black arrow). We then disrupted the gtr6 gene in ATCC 17978 through the insertion of an antibiotic resistance cassette in its coding (upward black arrow), and then by replacing the entire gene with a defective copy from HUMC1. (B) (Top) Structural analysis of hypervirulent HUMC1 and the ATCC 17978 Δgtr6 mutant (KL3) revealed differential levels of acetylation at the A4 position marked in grey highlight (90% for pgt1+ HUMC1 and 50% for pgt1- ATCC 17978 Δgtr6). The two strains are isogenic at the capsule locus save for pgt1. (Bottom) Structural analysis of avirulent ATCC 17978 and hypovirulent 15827 (KL22) revealed the same pgt1-mediated difference in acetylation as well as an additional GlcNAc branch at position B (grey rectangle). Both KL22 and KL3 loci have a functioning gtr6 gene.
Table 1.
Strains by Locus Classification, Genotype, and Phagocytosis Phenotype.
All strains used in this study are described according to Kenyon classification capsule assembly locus type, genotype by gtr6 and pgt1, and relative phagocytic potential.:: = chromosomal gene insertion, / = plasmid insertion, * = generated mutant.
Fig 2.
Macrophage phagocytosis of ATCC 17978 Δgtr6, NIH1 Δgtr6, and HUMC1::gtr6, gentamycin protection assay, and representative micrographs.
(2A) RAW 264.7 cells were co-incubated with NIH1 (left) and ATCC 17978 (right) isogenic wild-type strains and Δgtr6 mutants. (2B) RAW 264.7 cells were co-incubated with ATCC 17978, the HUMC1::gtr6 mutant strain with repaired gtr6, or wild-type HUMC1. (2C) RAW 264.7 cells were co-incubated with ATCC 17978 wild type, Δgtr6, Δgtr6/pSC1a (the knockout mutant with a plasmid-borne functional copy) in the presence of complement-active serum, and Δgtr6/pSC1a in the presence of heat-inactivated serum. *p < 0.001. (2D) Gentamicin protection assay with RAW 264.7 cells and wild-type ATCC 17978 (black bars) or ATCC 17978 Δgtr6 (white bars). Cytochalasin D was added as an inhibitor of phagocytosis. Total bacteria plated for CFUs and expressed as a proportion of initial bacterial inoculum. * = significant vs. bacteria-only group, ⸸ = significant vs. bacteria + RAW 264.7 cell group. *,⸸ = p < 0.01 (2E) RAW 264.7 cells were incubated with ATCC 17978, HUMC1, ATCC 17978 Δgtr6, and HUMC1::gtr6. Stained with Wright-Giemsa stain, total magnification is 1000x. Results are from two repeat experiments with duplicate samples in each. White arrows denote adherent or internalized bacteria.
Fig 3.
Bacterial blood burden and in vivo lethality by gtr6 genotype.
(3A) Bacterial burden in the blood at 1-hour post-infection with 1.0 ×108 CFUs of ATCC 17978 WT and Δgtr6 (left) and NIH1 WT and Δgtr6 (right). *p < 0.001 (3B) C3HeB/Fe mice were infected intravenously with 2.4×108 CFUs of ATCC 17978 (black squares), 8.3 ×107 CFUs of ATCC 17978 Δgtr6 (white squares), 1.0 ×108 CFUs of NIH1 (black circles) and NIH1 Δgtr6 (white circles), 2.9 ×107 CFU of HUMC1 (black triangles), and 2.0 ×108 CFUs of HUMC1::gtr6 (white triangles). *p < 0.05, **p < 0.01. Wide bars denote median, error bars denote IQR. Experiments repeated once, n = 5 per group for in vivo.
Fig 4.
Quantification of capsule content, pre-incubation of phagocytes with purified bacterial capsule, and pre-incubation of phagocytes with soluble carbohydrates.
(4A) 2.0×108 CFU of ATCC 1778 and HUMC1 had total capsule carbohydrate capsule extracted in parallel and total carbohydrate content measured via phenol-sulfuric acid colorimetry. (4B) Incubation of macrophages and bacteria with purified capsule from gtr6+ (ATCC 17978, 15827) and gtr6- (HUMC1, ATCC 17978 Δgtr6) strains. Extract-free uptake was used as a control. *p < 0.0001 (4C) RAW 264.7 cells were pre-incubated with soluble mannan (0.5mg/mL), laminarin (0.5mg/mL), and dextran sulfate (0.1mg/mL) or an untreated control prior to co-incubation with ATCC 17978. *p < 0.0001. Two biological replicates for in vitro. Wide bars denote median, error bars denote IQR.
Fig 5.
Receptor blockade with siRNA knockdown, antibody neutralization of beta-glucan receptors prior to bacterial uptake, and phagocytosis of bacteria by peritoneal macrophages and neutrophils.
(5A) RAW 264.7 cells were pre-incubated with anti-Dectin-1, anti-CR3, anti-MR neutralizing monoclonal antibodies or an isotype control prior to co-incubation with ATCC 17978. *p < 0.0005, **p < 0.0001 (5B) Knockdown of Dectin-1 and/or CR3 in RAW 264.7 cells followed by incubation with ATCC 17978. *p < 0.0001 (5C) Primary peritoneally-elicited macrophages from C57BL/6 mice followed by phagocytosis assays with ATCC 17978. *p < 0.05, **p < 0.0001 (5D) Phagocytosis assays of ATCC 17978 with peritoneal neutrophils from wild-type mice with disruption of phagocytosis upon the addition of heat-inactivated serum (HI-S) or complement-active serum (CA-S). *p < 0.0001 (5E) Phagocytosis assays with RAW 264.7 macrophages with gtr6+ and capsule-free strains (ATCC 17978 WT, 15827, ATCC 17978 ΔitrA), and gtr6- strains (ATCC 17978 Δgtr6, HUMC1), with complement active (CA-S) or heat-inactivated (HI-S) serum. *p < 0.0001. Experiments repeated once with two biological replicates. Wide bars denote median, error bars denote IQR.
Fig 6.
Phagocytosis in the presence of a lectin domain inhibitor, phagocytosis by macrophages in serially diluted serum, and infection of complement-depleted mice.
(6A) Incubation of RAW 264.7 cells with ATCC 17978 in the presence of 100μg/mL GlcNAc (NAG), a CR3 lectin domain inhibitor. (6B) Serial two-fold dilutions of complement-active mouse serum in a RAW 264.7 cell phagocytosis assay with ATCC 17978. *p < 0.0001 (6C) Male C57BL/6 mice aged 10 weeks were infected intravenously with 2.0×108 CFUs of 15827, with or without administration of 15μg cobra venom factor (CVF) 48 h prior to infection. *p < 0.001. Experiments repeated once, n = 5 per group for in vivo and two technical replicates for in vitro.
Fig 7.
Flow cytometry of strains incubated with serum and anti-C3b antibodies.
(7A) Flow cytometry of bacteria following incubation in 10% complement active serum followed by anti-C3b antibodies. Strains denoted by known virulence (brackets) as well as gtr6 phenotype (upward arrows). p < 0.0001. (7B) Representative flow plot of initial forward and side scatter plot and sub-gating on single bacterial cells with FITC-A as the anti-C3b fluorophore. (7C) Representative histograms of anti-C3b fluorescent bacteria for HUMC1 (hypervirulent), 15827 (hypovirulent), ATCC 17978 ΔitrA (avirulent), and the isotype control. 20,000 events collected per condition for flow cytometry, gated for singlets via FSC/SSC, fluorescence gate set to exclude 99% of isotype control and copied across samples ran in parallel.