(B and C) Fluorescence microscopy of 5-min and 15-min serum-exposed Δ rfaH and WT cells (B) or 15-min serum exposed Δ lpp and WT cells (C) following incubation with APC-conjugated anti-C3b antibody. For the B5055 Δ rfaH mutant, data were collected only at the first two time points due to cell lysis detected by release of cytoplasmic fluorescent marker from labeled cells. Two-way repeated measures ANOVA with uncorrected Fisher’s least significant difference (LSD test revealed significance as follows: *, P ≤ 0.05 * *, P ≤ 0.01 ***, P ≤ 0.001 ****, P ≤ 0.0001.
Values were converted to AU, setting T 0 to 1. (A) Flow cytometry-based determination of C3b binding to ATCC 43816, B5055, NTUH-K2044, RH201207, and their respective mutants were measured after 15, 30, 60, 120, and 180 min of incubation in human pooled serum at 37☌ ( n = 3).
Overall significance for each strain was determined by one-way ANOVA, followed by Dunnett’s post hoc test to compare each WT or complemented strain to Δ lpp plus vector. This effect was not seen with the empty vector or with an Lpp construct lacking its C-terminal lysine (ΔK78). Induction of wild-type Lpp partially restored the hypermucoid phenotype of the NTUH-K2044 and B5055 Δ lpp mutants. The hypermucoidy assay for capsule was performed on stationary-phase, arabinose-induced cultures washed once in phosphate-buffered saline (PBS). pneumoniae Δ lpp mutants using an inducible vector ( n = 3). The RH201207 Δ rfaH mutant was compared to the WT by one-way ANOVA. Overall statistical significance for each strain was determined by one-way ANOVA the Δ rfaH mutant and complemented strains were compared to the WT by Dunnett’s post hoc test. (B) Comparison of capsule production in Δ rfaH mutant and complemented mutant strains ( n = 3). All three strains showed a significant reduction in cell-associated capsule, while total capsule was unchanged or increased (one-way ANOVA relative to WT *, P < 0.05 * *, P < 0.01 ** *, P < 0.001). Uronic acids were either quantified directly from culture or following a single wash and resuspension in LB (see Materials and Methods), and Δ lpp values were normalized to the WT from the same strain and condition. (A) Comparison of total and cell-attached capsule content of wild-type and Δ lpp mutants of K. Klebsiella Tn-Seq TraDIS capsule complement resistance functional genomics serum resistance transposon insertion sequencing.Įffects of Lpp and RfaH on capsule production and retention. pneumoniae adopts differing mechanisms and utilizes distinct gene sets to avoid complement attack. Our findings show that, even among this small selection of isolates, K. All rfaH and lpp mutants bound C3b and C5b-9 in large quantities. Binding experiments with the complement components C3b and C5b-9 showed that the underlying mechanism of evasion varied in the four strains: B5055 and NTUH-K2044 appeared to bypass recognition by complement entirely, while ATCC 43816 and RH201207 were able to resist killing despite being associated with substantial levels of C5b-9. The murein lipoprotein gene lpp promoted capsule retention through a mechanism dependent on its C-terminal lysine residue its deletion led to modest reductions in complement resistance. Deletion of the antiterminator rfaH, which controls the expression of capsule and O side chains, resulted in dramatic complement resistance reductions in all strains.
More than 90 genes contributed to resistance in one or more strains, but only three, rfaH, lpp, and arnD, were common to all four strains. pneumoniae strains (NTUH-K2044, B5055, ATCC 43816, and RH201207), and explored their recognition by key complement components. Here, we used functional genomic profiling to define the genetic basis of complement resistance in four diverse serum-resistant K. Identifying and characterizing the factors responsible for preventing activation of, and killing by, serum complement could inform new approaches to treatment of K. Resistance to killing by serum enhances the capacity of Klebsiella pneumoniae to cause infection, but it is an incompletely understood virulence trait. The serum complement system is a first line of defense against bacterial invaders.