Fig. 5: ArcZ and MlaA are crucial for Klebsiella virulence in mouse.

a Competition assay using a 1:1 mixture of WT and ΔarcZ mutant. Mice were inoculated with 10³ CFU of the mixed bacteria intranasally, and scarified 48 h pi for enumeration. Growth competition in LB serves as in vitro control. N = 10 biological replicates, lines indicate median value. **, p < 0.01; two-tailed Mann-Whitney U test. ArcZ overexpression significantly reduced Klebsiella pathogenesis in mice. Bacterial burden in lung (b), spleen (c), and liver (d) were enumerated 48 h pi. Samples below limit of detection (100 CFU) are drawn on X-axis. N = 5 biological replicates, lines indicate median value. *, p < 0.05; **, p < 0.01; two-tailed Mann-Whitney U test. One-way ANOVA test with post-hoc Tukey’s multiple comparisons: p < 0.05 (c). e Western blot verification of MlaA expression in the chromosomal deletion and complementation strains. 3xFLAG tag was introduced into the C-terminus of MlaA and used for detection by anti-FLAG antibody. GroEL serves as loading control. Experiment was performed three times. Uncropped blots in Source Data. f MlaA is required for HMV. The HMV level was determined with overnight cultures of indicated strains. Chromosomal complementation was constructed by inserting a copy of mlaA gene with its own promoter into putA locus using homologous recombination. The acapsular i-wza mutant served as negative control. N = 3 biological replicates, bars indicate mean ± SD. *, p < 0.05; **, p < 0.01; two-tailed Student’s t-test. g–i MlaA is required for Klebsiella pathogenesis. Mice were infected with 10³ CFU of indicated strains via intranasal instillation. Bacterial burdens were analyzed 48 h pi. N = 6 biological replicates, lines indicate median value. **, p < 0.01, two-tailed Mann-Whitney U test. One-way ANOVA test with post-hoc Tukey’s multiple comparisons: p < 0.001 (g, h). j Mutations introduced in the MlaA protein variants and their associated molecular functions. k HMV analysis for strains with chromosomal MlaA variants. The mlaA gene containing desired mutations was introduced back into the chromosome of ΔmlaA strain. N = 3 biological replicates, bars indicate mean ± SD. *, p < 0.05; **, p < 0.01; two-tailed Student’s t-test.