Fig. 6: K. pneumoniae promotes HCC via its surface protein PBP1B binding to and activating TLR4 in HCC cells.
a, Representative SEM (top) and TEM (bottom) images of Hep3B and Huh7 HCC cell lines after co-culture with K. pneumoniae (MOI = 10). After co-culture of HCC cells with K. pneumoniae, cell lysate was spread on BHI agar for bacterial culture. n = 3 independent experiments with similar results. b, Effect of pasteurized K. pneumoniae (MOI = 10) on HCC cell proliferation (top) (n = 3 biologically independent samples), colony formation (middle) (n = 3 biologically independent samples) and growth of HCC patient-derived organoid (bottom) (n = 20 biologically independent samples). c, Effect of pasteurized K. pneumoniae (MOI = 10) on PCNA expression in HCC cells. n = 3 (Ctrl), 6 (pasteurized KP). d, Screening of K. pneumoniae adhesins by biotin pull-down assay. e, Hep3B membrane protein was incubated with GST or GST–PBP1B together with GST magnetic beads for GST pull-down assay. Corresponding bands in GST–PBP1B and Hep3B cell membrane protein groups were subjected to mass spectrometry analysis. In d and e, n = 3 independent experiments with similar results. f, Binding affinity between PBP1B and TLR4 was detected using SPR. Kd, dissociation constant. g, Representative structure of PBP1B and TLR4 after molecular docking (left) and the residues involved in the interaction between PBP1B with TLR4 (right). The structure of TLR4 is coloured pink while the PBP1B is coloured yellow. The light green dash represents hydrogen bond. h, TLR4 from Hep3B and Huh7 cell lysates was pulled down by GST–PBP1B according to the GST pull-down assay. i, Immunoprecipitation of TLR4 from Hep3B or Huh7 cells lysates validated the binding between TLR4 and GST–PBP1B. In h and i, n = 3 independent experiments with similar results. j, Effect of PBP1B (0.05 μM) on HCC cell proliferation (top) (n = 3 biologically independent samples), colony formation (middle) (n = 3 biologically independent samples) and growth of HCC patient-derived organoid (bottom) (n = 20 biologically independent samples). n = 3 independent experiments with similar results. k, Effect of PBP1B (0.05 μM) on TLR4, MyD88, p-P65, P65 and PCNA protein expression in HCC cells, as determined by western blot. n = 3 biologically independent samples. l,m, TLR4i (30 μM) abolished the effect of PBP1B on HCC cell proliferation (l) and colony formation (m). n = 3 biologically independent samples. n, Effect of K. pneumoniae on TLR4 expression in liver tissues of both germ-free and SPF mice with or without DEN treatment by IHC staining. In b,c, j–m, data are presented as mean ± s.e.m. Each data point in bar plots represents one mouse. Cell proliferation was analysed using two-way ANOVA. In b,c,j,k,m, comparisons between two groups were analysed using Student’s t-test. Unless otherwise stated, statistical significance was calculated using one-way ANOVA. Adjustments were made for multiple comparisons.
