Effect of calcium on the interaction of Acinetobacter baumannii with human respiratory epithelial cells

Abstract

Background: Investigating the factors that influence Acinetobacter baumannii(Ab) adhesion/invasion of host cells is important to understand its pathogenicity. Metal cations have been shown to play an important role in regulating the biofilm formation and increasing the virulence of Ab; however, the effect of calcium on host-bacterial interaction has yet to be clarified. Here, the dynamic process of the interaction between Ab and human respiratory epithelial cells and the effect of calcium on host-bacterial interaction were explored using microscopic imaging, quantitative PCR and real time cellular analysis (RTCA).

Results: The concentration of calcium, multiplicity of infection and co-culture time were all demonstrated to have effects on host-bacterial interaction. A unique "double peak" phenomenon changed to a sharp "single peak" phenomenon during the process of Ab infection under the effect of calcium was observed in the time-dependent cell response profiles. Moreover, calcium can increase Ab adhesion/invasion of epithelial cells by regulating the expression of Ab-related genes (ompA, bfmRS, abaI).

Conclusions: Effective control of calcium concentrations can provide new approaches for the prevention and treatment of multi-drug resistant Ab.

Keywords: Acinetobacter baumannii; Calcium; host-bacterial interaction; Infection.

Fig. 1

Analysis of optimum MOI of Ab to human respiratory epithelial cells. Control: 0.45% NaCl. a Microscopy observations of the effect of Ab on epithelial cells (2 × 10⁵ cells per well) at different MOIs (co-cultured for 4 h). Scale bar = 30 μm. Red arrows: bubble-like (denucleated) dead cells with bacterial adhesion visible around (especially in the MOI 50 and MOI 100 experimental groups); Yellow arrows: the phenomenon of bacterial aggregation. b The qPCR results indicating Ab invasion of epithelial cells (including strong adhesion) at different MOIs (host cells and bacteria were co-cultured for 4 h and 1 × 10⁵ cells were isolated). The differences in the Ct values (MOI 100 group < MOI 50 group < MOI 10 group < MOI 1 group) among all the groups were statistically significant, as determined by the SNK test (P < 0.05). The differences between the MOI 1 and MOI 10 groups and the MOI 10 and MOI 50 groups were greater than between the MOI 50 and MOI 100 groups. The higher the MOI was, the greater the Ab invasion of epithelial cells. c Ab infection TCRPs of epithelial cells (20,000 cells per well) at different MOIs. t: point at which bacteria or NaCl were added (after background readings for the E-Plate were obtained and the plate was incubated at room temperature for 30 min). The “double peak” phenomenon emerged during Ab infection. The smaller the MOI was, the later and higher the peak CI was, the more significant the phenomenon was. Representative curves are an average of three replicate wells

Fig. 2

The effect of Ab on human respiratory epithelial cells in different incubation states. a Microscopy observation of the effect of Ab on epithelial cells (4 × 10⁵ cells per well) in different incubation states. Scale bar = 30 μm. Experimental group: addition of bacterial suspension; Control group: addition of 0.45% NaCl; Red arrows: bubble-like (denucleated) dead cells; Yellow arrows: the phenomenon of bacterial aggregation; Blue arrows: massive cell death. b Ab infection TCRPs of epithelial cells (20,000 cells per well) in different incubation states. I: addition of bacterial suspension (red arrow) at cell suspension (0 h); II: addition of bacterial suspension (green arrow) at 30–40% cell confluence (after 24 h); III: addition of bacterial suspension (blue arrow) at 80–90% cell confluence (after 48 h); IV: blank control (without intervention); t: treatment time point; t₁: cell suspension (0 h); t₂: 30–40% cell confluence (after 24 h); t₃: 80–90% cell confluence (after 48 h). Ab infection caused the CI to rise and fall. A unique “double peak” phenomenon emerged during Ab infection. The addition of 0.45% NaCl or sterile distilled water had no significant effect on the CI

Fig. 3

The effect of calcium on Ab proliferation (growth curves). Control I: calcium final concentration was 0 mmol/L (with EDTA treatment).Calcium can promote the proliferation of Ab

Fig. 4

Biofilm assay results. Control I: calcium final concentration was 0 mmol/L (with EDTA treatment). Calcium may promote Ab biofilm formation, and ompA has a positive effect on biofilm formation

Fig. 5

qPCR results of calcium effect on host-bacterial interaction. Ab and human respiratory epithelial cells were co-cultured in calcium-supplemented medium for 2 h, 4 h and 6 h, and 1 × 10⁵cells were isolated for qPCR detection. Control I: calcium final concentration was 0 mmol/L (with EDTA treatment). Calcium had positive effect on the interaction between Ab and epithelial cells. With the increase of calcium concentrations and prolonged co-culture times, the amount of Ab invasion into epithelial cells increased (the smaller the Ct value, the more bacterial invasion to cells)

Fig. 6

The effect of calcium on Ab infection TCRPs of human respiratory epithelial cells. A total of 20,000 cells per well were seeded into E-plates. t: treatment time point (48 h, 80–90% cell confluence). With increased calcium concentrations (bacteria-infected cells), the CI increased or decreased faster and reached a higher peak value. A sharp “single peak” phenomenon occurred during the infection

Fig. 7

Effect of calcium on the expression of Ab-related genes. The calcium supplementation final concentrations were as follows: Group a, 1.4 mmol/L; Group b, 2.4 mmol/L; Group c, 3.4 mmol/L; Group d, 4.4 mmol/L; Group control I: calcium final concentration was 0 mmol/L (with EDTA treatment); Abiotic environment: bacteria only were cultured in plates containing RPMI 1640 medium; Cellular environment: bacteria were cultured in epithelial cell covered plates (80–90% cell confluence) containing RPMI 1640 medium. The recA gene was used as an internal reference. The relative changes of Ab related gene expression between the experimental groups and control I group were calculated by the 2^-△△Ct method