Fig. 6: citrOgen derived anti-KP T3F antibodies block biofilm formation.
A The strain CRT3F expresses the structural mrkABCDF genes under the control of the T3SS effector protein Map promoter. B mrkA mRNA was assessed by qRT-PCR from CRT3F under T3SS suppressing (LB) and inducing (DMEM) conditions. CRWT is also shown as a negative control. DMEM specifically induces mrkA transcription in CRT3F. 4 biological replicates, central tendency mean, error bars SEM. Two-sided paired T-test between LB and DMEM-grown CRT3F. C CRT3F and CRWT were grown in DMEM on glass coverslip slides. Attachment was assessed at 6 h and bacteria stained for MrkA. A dense lawn of MrkA-positive CRT3F is observed binding to the glass slide, indicating functional KP T3F expression. D At 8 dpi, faecal CRT3F+pespO levels are similar to CRWT. Central tendency: median. n = 15 mice per group, 3 biological repeats. Two-sided Mann–Whitney test, ns non-significant. GoF, gram of faeces. E At 30 dpi with CRWT or CRT3F+pespO sera was collected. An ELISA was conducted against heat-killed KPWT and an isogenic mutant lacking T3F expression (KP∆mrkA-F). CRWT infected mice had no detectable IgG responses to KPWT or KP∆mrkA-F. CRT3F +pespO infected mice had specific IgG responses against KPWT. n = 15 mice per group, 3 biological repeats. Central tendency: mean. Two-sided Mann–Whitney test. F Sera from CRWT and CRT3F+pespO-infected mice were used to stain KPWT. An α-MrkA pAb was used as a positive control. CRWT derived sera results in no detectable staining of KPWT. Sera from CRT3F+pespO-infected mice and α-MrkA pAb staining phenocopy each other. G CR preadsorbed sera from CRWT and CRT3F+pespO-infected mice were preincubated with KPWT or KP∆mrkA-F. No-antibody was used as an additional control. Following 6 h of growth sfGFP signal was assessed and presented as the KPWT normalised to KP∆mrkA-F binding. CRT3F+pespO-infected mice sera specifically inhibited binding compared to CRWT. 4 biological replicates, central tendency: mean, error bars: SEM. Repeated-measures One-way ANOVA corrected with Dunnett’s multiple comparisons test. H Biofilm formation from (G) was assessed by sfGFP fluorescence microscopy. A large reduction in adhered KPWT is observed when CRT3F pespO-infected cross adsorbed sera was incubated with KPWT compared to CRWT cross adsorbed sera. ns non-significant; *, p < 0.5; ****, p < 0.0001. Source data and exact p-values are provided in the Source Data file.
