Fig. 5: citrOgen derived anti-KP K2 antibodies can be used for serotyping.
A The cps in KPWT is c.22 kb long with 15 open reading frames. This was inserted into CRWT by substitution of the colanic acid biosynthesis locus generating the strain CRKPK2. B At 8 dpi faecal CRKPK2+pespO levels are similar to CRWT. Central tendency: median. n = 15 mice per group, 3 biological repeats. Two-sided unpaired T-test, ns non-significant. GoF, gram of faeces. C At 30 dpi blood was taken from mice that cleared infection with CRWT or CRKPK2+pespO. An ELISA was conducted against heat-killed KPWT (expressing KP K2) and an isogenic acapsular mutant (KP∆wcaJ). CRWT infected mice had no detectable IgG responses to KPWT or KP∆wcaJ. CRKPK2+pespO infected mice had IgG responses specifically against KPWT. n = 15 mice per group, 3 biological repeats. Central tendency: mean. Two-sided Mann–Whitney test. D Sera from CRWT and CRKPK2+pespO-infected mice were used to stain KPWT and image by immunofluorescence microscopy. An α-KP K2 pAb was used as a positive control. CRWT derived sera results in no detectable staining of KPWT, whereas sera from CRKPK2+pespO-infected mice and the α-KP K2 pAb phenocopy each other with punctate membrane-associated staining of KPWT. E Sera from CRKPK2+pespO-infected mice were used to serotype a collection of 100 diverse KP strains with serotypes assigned by genomic analysis. Each box represents one strain with the cps (KL) type described. The boxes are coloured according to the strength of binding by CRKPK2+pespO-infected mice sera, which was assessed by flow cytometry. The median fluoresence intensity (MFI) key is shown in the bottom right together with staining of KPWT and KP∆wcaJ controls. A box, centred on a KL2 isolate is shown. F The strains contained within the highlighted box in (E) were stained with the reference α-KP K2 pAb and imaged by immunofluorescence microscopy. The KL2 strain in the centre demonstrate punctate membrane staining in keeping with K2 capsule. The other strains (non-KL2) demonstrate no staining. Bacterial cell membranes were counterstained with FM-4-64 for imaging. Images representative of 2 biological repeats. ns non-significant; ****, p < 0.0001. Source data and exact p-values are provided in the Source Data file.
