Table 1.
Strains and plasmids used in this study.
Table 2.
Primers used in this study.
Figure 1.
Virulence of K. pneumoniae against D. discoideum can be modulated.
(A) The ability of Dictyostelium to grow on a bacterial lawn was assessed by depositing amoebae (from 10 to 10,000) on a lawn of bacteria grown on SM agar medium. A phagocytosis plaque was observed 5 days later when bacteria were permissive. Bacteria tested were: K. pneumoniae (Kp52145), cps mutant (52145-ΔwcaK2; ΔwcaK2), or control strain (K. aerogenes). (B) The ability of wild-type K. pneumoniae (Kp52145), cps mutant (52145-ΔwcaK2), or control strain (K. aerogenes) to resist predation by D. discoideum was tested on HL5-agar, pure or diluted. 1,000 amoebae were deposited on the bacterial lawns and plaques were recorded 5 days later. (C) The ability of Dictyostelium to grow on a bacterial lawn was assessed by depositing amoebae (from 10 to 10,000) on a lawn of bacteria grown on HL5–5% agar medium. A phagocytosis plaque was observed 5 days later when bacteria were permissive (K. aerogenes and 52145-ΔwcaK2; ΔwcaK2).
Figure 2.
Role of K. pneumoniae CPS on phagocytosis resistance.
(A) Dictyostelium cells were incubated with Klebsiella and the number of surviving bacteria (total or cell-associated) was determined at different times by killing the Dictyostelium and plating the bacteria on LB plates. Bars represent mean ± s.e.m (n = 4). *; P<0.05 (results are significantly different from the results for the wild-type strain [Kp52145]; two tailed t test). (B) Dictyostelium AX2/RFP cells were incubated in the presence of Klebsiella strains, K. pneumoniae (Kp52145), cps mutant (52145-ΔwcaK2; ΔwcaK2), containing plasmid pFPV25.1Cm for 30 min, and then fixed. Images are representative of three independent experiments. (C) Dictyostelium cells were incubated with Klebsiella and the number of intracellular bacteria was determined at different times by killing the Dictyostelium and plating the bacteria on LB plates. Each point represents the mean and standard deviation of twelve samples from four independent experiments. (D) Klebsiella phagocytois by MH-S mouse alveolar macrophages. Intracellular bacteria were determined by the gentamicin protection assay. Bars represent mean ± s.e.m (n = 4). *; P<0.05 (results are significantly different from the results for the wild-type strain [Kp52145]; two tailed t test). (E) Immunofluorescence confocal microscopy of MH-S mouse alveolar macrophages infected with Klebsiella strains Kp52145 or 52145-ΔwcaK2 (ΔwcaK2), containing plasmid pFPV25.1Cm. Actin cytoskeleton was stained with Phalloidin-RRX (red) and host cell nuclei were stained with Hoechst (blue). Images are representative of five independent experiments.
Figure 3.
Role of K. pneumoniae LPS polysaccharide section on phagocytosis resistance.
(A) K. pneumoniae 52145 (Kp52145) oligosaccharide structure based on a published study [35]. Lines denote the truncation level for the different core biosynthetic gene mutations. Residues J and K could be hydrogen (H) or GalA. (B) Dictyostelium cells were incubated with Klebsiella strains (wild type [Kp52145], LPS OPS mutants [52O21] and 52145-ΔwaaL [ΔwaaL], CPS mutant 52145-ΔwcaK2 [ΔwcaK2], or strain lacking the CPS and the OPS 52145-ΔwcaK2-ΔwaaL [ΔwcaK2-ΔwaaL]) and the number of surviving bacteria (total or cell-associated) was determined at different times by killing the Dictyostelium and plating the bacteria on LB plates. Bars represent mean ± s.e.m (n = 4). *; P<0.05 (results are significantly different from the results for the wild-type strain [Kp52145]; two tailed t test). ▵; P<0.05 (results are significantly different from the results for the cps mutant [52145-ΔwcaK2]; two tailed t test). (C) Dictyostelium cells were incubated with Klebsiella strains (wild type [Kp52145], LPS OPS mutant 52145-ΔwaaL [ΔwaaL], and LPS core mutants 52145-ΔwaaQ [ΔwaaQ], 52145-ΔwaaL-ΔwaaQ [ΔwaaL-ΔwaaQ], 52145-ΔwabG [ΔwabG] 52145-ΔwabM [ΔwabM], 52145-ΔwabH [ΔwabH], and 52145-ΔwabK [ΔwabK]) and the number of surviving bacteria was determined at different times by killing the Dictyostelium and plating the bacteria on LB plates. Bars represent mean ± s.e.m (n = 4). *; P<0.05 (results are significantly different from the results for the wild-type strain [Kp52145]; two tailed t test). ▵; P<0.05 (results are significantly different from the results for the waaL mutant [52145-ΔwaaL]; two tailed t test). (D) Dictyostelium cells were incubated with Klebsiella mutant lacking the CPS and the OPS 52145-ΔwcaK2-ΔwaaL (ΔwcaK2-ΔwaaL), or the OPS and the first, second or third sugar of the core (strains 52145-ΔwcaK2-ΔwabM [ΔwcaK2-ΔwabM], 52145-ΔwcaK2-ΔwabH [ΔwcaK2-ΔwabH], and 52145-ΔwcaK2-ΔwabK [ΔwcaK2-ΔwabK] respectively). The number of surviving bacteria was determined at different times by killing the Dictyostelium and plating the bacteria on LB plates. Bars represent mean ± s.e.m (n = 4). *; P<0.05 (results are significantly different from the results for 52145-ΔwcaK2-ΔwaaL; two tailed t test). (E) Phagocytosis of LPS polysaccharide mutants by MH-S mouse alveolar macrophages. Intracellular bacteria were determined by the gentamicin protection assay. Kp52145 (wild type); OPS mutants 52O21 and ΔwaaL (52145-ΔwaaL); LPS core mutants ΔwaaQ (52145-ΔwaaQ), ΔwaaL-ΔwaaQ (52145-ΔwaaL-ΔwaaQ), ΔwabG (52145-ΔwabG), ΔwabM (52145-ΔwabM), ΔwabH (52145-ΔwabH), and ΔwabK (52145-ΔwabK). Bars represent mean ± s.e.m (n = 4). *; P<0.05 (results are significantly different from the results for the wild-type strain [Kp52145]; two tailed t test). (F) MH-S cells engulfment of Klebsiella cps mutant, strain 52145-ΔwcaK2 (ΔwcaK2), or strains lacking the CPS and the OPS 52145-ΔwcaK2-ΔwaaL (ΔwcaK2-ΔwaaL), or the OPS and the first, second or third sugar of the core (strains 52145-ΔwcaK2-ΔwabM [ΔwcaK2-ΔwabM], 52145-ΔwcaK2-ΔwabH [ΔwcaK2-ΔwabH], and 52145-ΔwcaK2-ΔwabK [-ΔwcaK2-ΔwabK] respectively). *; P<0.05 (results are significantly different from the results for the cps mutant [52145-ΔwcaK2]; two tailed t test).
Figure 4.
Role of K. pneumoniae lipid A decorations on phagocytosis resistance.
(A) Dictyostelium cells were incubated with Klebsiella strains (wild type [Kp52145], pagP mutant (ΔpagP, 52145-ΔpagPGB), pmrF mutant (ΔpmrF, 52145-ΔpmrF), or pagP-pmrF double mutant (ΔpagP-ΔpmrF, 52145-ΔpagPGB-ΔpmrF). The number of surviving bacteria (total or cell-associated) was determined at different times by killing the Dictyostelium and plating the bacteria on LB plates. Bars represent mean ± s.e.m (n = 4). *; P<0.05 (results are significantly different from the results for the wild-type strain [Kp52145]; two tailed t test). ▵; P<0.05 (results are significantly different from the results for 52145-ΔpagPGB; two tailed t test). (B) Dictyostelium cells were incubated with Klebsiella lipid A mutants constructed in the background of the cps mutant (52145-ΔwcaK2 [ΔwcaK2]). The number of surviving bacteria was determined at different times by killing the Dictyostelium and plating the bacteria on LB plates. Bars represent mean ± s.e.m (n = 4). *; P<0.05 (results are significantly different from the results for the cps mutant [52145-ΔwcaK2]; two tailed t test). ▵; P<0.05 (results are significantly different from the results for 52145-ΔwcaK2-ΔpagPGB; two tailed t test). (C) Phagocytosis of lipid A mutants by MH-S cells. Wild type [Kp52145], pagP mutant (ΔpagP, 52145-ΔpagPGB), pmrF mutant (ΔpmrF, 52145-ΔpmrF), or double pagP-pmrF mutant (ΔpagP-ΔpmrF, 52145-ΔpagPGB-ΔpmrF). Bars represent mean ± s.e.m (n = 4) *; P<0.05 (results are significantly different from the results for the wild-type strain [Kp52145]; two tailed t test). ▵; P<0.05 (results are significantly different from the results for 52145-ΔpagPGB; two tailed t test). (D) Phagocytosis of lipid A mutants constructed in the background of the cps mutant (52145-ΔwcaK2 [ΔwcaK2]) by MH-S cells. Bars represent mean ± s.e.m (n = 4). *; P<0.05 (results are significantly different from the results for the cps mutant [52145-ΔwcaK2]; two tailed t test). ▵; P<0.05 (results are significantly different from the results for 52145-ΔwcaK2-ΔpagPGB; two tailed t test).
Figure 5.
Role of K. pneumoniae OMPs on phagocytosis resistance.
(A) Dictyostelium cells were incubated with Klebsiella strains (wild type [Kp52145], ompA mutant (ΔompA, 52OmpA2), ompK36 mutant (ΔompK36, 52OmpK36), and the corresponding complemented strains. The number of surviving bacteria (total or cell-associated) was determined at different times by killing the Dictyostelium and plating the bacteria on LB plates. Bars represent mean ± s.e.m (n = 4). *; P<0.05 (results are significantly different from the results for the wild-type strain [Kp52145]; two tailed t test). (B) Dictyostelium cells were incubated with Klebsiella OMPs mutants constructed in the background of the cps mutant (52145-ΔwcaK2, [ΔwcaK2]). The number of surviving bacteria (total or cell-associated) was determined at different times by killing the Dictyostelium and plating the bacteria on LB plates. Bars represent mean ± s.e.m (n = 4). *; P<0.05 (results are significantly different from the results for the cps mutant [52145-ΔwcaK2]; two tailed t test). (C) Phagocytosis of OMPs mutants by MH-S cells. Wild type [Kp52145], ompA mutant (ΔompA, 52OmpA2), ompK36 mutant (ΔompK36, 52OmpK36), and the corresponding complemented strains. Bars represent mean ± s.e.m (n = 4) *; P<0.05 (results are significantly different from the results for the wild-type strain [Kp52145]; two tailed t test). (D) Phagocytosis of OMPs mutants constructed in the background of the cps mutant (52145-ΔwcaK2) by MH-S cells. Bars represent mean ± s.e.m (n = 4). *; P<0.05 (results are significantly different from the results for the cps mutant [52145-ΔwcaK2]; two tailed t test).
Figure 6.
Virulence of K. pneumoniae ompK36 mutant.
Bacterial counts in mouse organs at 24 h post infection or 72 h post infection. Mice were infected intranasally with a bacterial mixture containing 5×104 bacteria of wild type (Kp52145, •) or ompK36 mutant (ΔompK36, ○) Results were reported as log CFU per gram of tissue (Log CFU/g). *, results are significantly different (P<0.05; two-tailed t test) from the results for Kp52145. (A) Trachea; (B) Lung; (C) Spleen, (D) Liver.