Table 1.
Bacterial strains and plasmids used in this study.
Table 2.
Primers used for dnaK deletion and complementation.
Fig 1.
Construction of the ∆dnaK mutant and assessment of bacterial growth under aerobic conditions.
(A) Schematic representation of the in-frame deletion strategy used to generate the ∆dnaK mutant of Staphylococcus xylosus. Approximately 1-kb regions flanking the dnaK coding sequence were cloned into the temperature-sensitive shuttle vector pIMAY for allelic exchange. (B) Growth kinetics of wild-type, ∆dnaK, and ∆dnaK-C strains cultured in TSB at 37 °C with shaking. OD₆₀₀ measurements and CFU enumeration were performed at indicated time points. Data represent means ± SEM from three independent experiments. Statistical comparisons among groups were analyzed by one-way ANOVA followed by Tukey’s post hoc test. ns, not significant.
Fig 2.
Assessment of host cell adhesion, invasion, and intracellular survival.
(A) Quantification of bacterial adhesion to breast cancer cells at 2 h post-infection. (B) Quantification of bacterial invasion into breast cancer cells at 2 h post-infection. Salmonella enterica serovar Typhimurium was used as a positive control for adhesion and invasion, while E. coli DH5α served as a negative control. For both adhesion and invasion assays, Salmonella enterica serovar Typhimurium was used as a positive control, and Escherichia coli DH5α served as a negative control. (C) Quantification of total bacterial load per well at 24 h post-infection, determined by S. xylosus-specific qPCR. (D) Enumeration of intracellular CFUs of wild-type, ∆dnaK, and ∆dnaK-C strains at 24 h post-infection following gentamicin treatment and host-cell lysis. Data represent means ± SEM from three independent experiments performed in triplicate. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple-comparison test (p < 0.05; ns, not significant).
Fig 3.
DnaK is dispensable for resistance to hypoxic stress and antimicrobial peptide exposure.
(A) Growth of wild-type, ∆dnaK, and ∆dnaK-C strains under hypoxic conditions (1% O₂). OD₆₀₀ measurements and CFU enumeration were performed at indicated time points. (B) Survival of strains following treatment with LL-37 (10 µg/mL, 2 h). Viable CFUs were determined by plating. Data represent means ± SEM from three independent experiments. Differences among groups were analyzed by one-way ANOVA. p < 0.05; ns, not significant.
Fig 4.
Sensitivity of the dnaK-deficient strain to oxidative and acid stress.
(A) Survival of strains following exposure to 5 mM hydrogen peroxide for 1 h. (B) Survival after incubation in TSB adjusted to pH 5.5 for 2 h. Viability was quantified by CFU plating. Data are presented as means ± SEM from three biological replicates. Group comparisons were evaluated using one-way ANOVA with Tukey’s multiple-comparison test. p < 0.05.
Fig 5.
Microfluidic tumor-on-a-chip model for time-resolved shear-stress exposure of S. xylosus–infected tumor cells.
(A) Diagram of the breast cancer–on–a–chip platform used to simulate physiological laminar shear stress. (B) Time-course analysis of tumor-cell viability under laminar shear flow. MDA-MB-231 cells were uninfected or infected with wild-type, ΔdnaK, or ΔdnaK-C Staphylococcus xylosus. Cell viability was quantified at the indicated time points using a live/dead fluorescence assay and normalized to uninfected cells at 0 h. Data represent means ± SEM from three independent microfluidic experiments. (C) Quantification of tumor-cell viability at 4 h of shear-stress exposure. Each data point represents an independent microfluidic experiment. Statistical significance was determined by one-way ANOVA with Tukey’s post hoc test. p < 0.05; ns, not significant.