Fig. 3: The mutation of hmwC enhanced the stress response, biofilm formation, and the virulence of Y. pestis.

Analysis of the influence of hmwC deletion on bacterial proliferation and growth in LB medium (a), TMH medium (b), and LB medium with 2.5% NaCl (d). Y. pestis strains were grown at 26 °C and the absorbance at OD_620nm were continuously monitored every 2 h of for a total of 32 h. c Y. pestis strains were grown in 24-well plates. First measure the OD_620nm value of the bacterial solution. Then the biofilm was stained with 0.1% crystal violet solution and decolorized with dimethyl sulfoxide, and the OD_570nm value of the decolorized solution was measured. The amount of biofilm was expressed as OD_570nm/OD_620nm ratio. e Equal bacterial solutions of WT, ΔhmwC, and ΔhmwC/HmwC strain were incubated parallelly at 50 °C for 10, 20, and 40 min, then the viable count of bacteria was determined by plating the bacterial dilutions onto a Hottinger’s agar plate in triplicate. f Equal bacterial solutions of WT, ΔhmwC, and ΔhmwC/HmwC strain were placed parallelly at −20 °C or 4 °C for 24 h, then the viable count of bacteria was determined as described above. g Circular filter paper with 100 mM or 200 mM hydrogen peroxide solution was placed in the center of semi-solid petri dish, and the diameter of the inhibition circle was measured to reflect the hydrogen peroxide resistance. h WT, ΔhmwC, and ΔhmwC/HmwC strain cultures diluted parallelly with pH 3.5 20 mM glucose minimal medium were incubated at room temperature for 30 min and 60 min, then the viable count of bacteria was determined as described above. i RAW264.7 were infected with WT, ΔhmwC, and ΔhmwC/HmwC strain at a MOI of 10. At 0.5 hpi, gentamicin was added to kill the extracellular bacteria. At 0.5, 2, 4, 8, and 24 hpi., the viable count of intracellular bacteria was determined as described above. j Each 6–8-week-old female mice was subcutaneously challenged with 100 μl of the wild-type Y. pestis strain or ΔhmwC suspension (~100 CFU) (n = 10/group), and the survival of challenged mice was observed for 14 consecutive days. A log-rank test was used to calculate the statistical significance. k Livers, spleens and lungs of the challenged mice were harvested when the s.c. challenged Bal b/c mice were moribund, i.e. 4 dpi., and the collected tissues were homogenized in sterile PBS to measure live bacteria, as described above. One-way ANOVA was performed to analyze the significance of difference in bacterial loads in the different tissues. c n = 10 biological replicates in each group, (j, k) n = 10 mice in each group. a, b, d–i n = 3 biological replicates in each group. Data are presented as mean values ±SEM. p values were calculated by two-tailed Student’s t test. Note that some error bars are too small to be visualized.