Outer Membrane Protein F Is Involved in Biofilm Formation, Virulence and Antibiotic Resistance in Cronobacter sakazakii

Abstract

In some Gram-negative bacteria, ompF encodes outer membrane protein F (OmpF), which is a cation-selective porin and is responsible for the passive transport of small molecules across the outer membrane. However, there are few reports about the functions of this gene in Cronobacter sakazakii. To investigate the role of ompF in detail, an ompF disruption strain (ΔompF) and a complementation strain (cpompF) were successfully obtained. We find that OmpF can affect the ability of biofilm formation in C. sakazakii. In addition, the variations in biofilm composition of C. sakazakii were examined using Raman spectroscopy analyses caused by knocking out ompF, and the result indicated that the levels of certain biofilm components, including lipopolysaccharide (LPS), were significantly decreased in the mutant (ΔompF). Then, SDS-PAGE was used to further analyze the LPS content, and the result showed that the LPS levels were significantly reduced in the absence of ompF. Therefore, we conclude that OmpF affects biofilm formation in C. sakazakii by reducing the amount of LPS. Furthermore, the ΔompF mutant showed decreased (2.7-fold) adhesion to and invasion of HCT-8 cells. In an antibiotic susceptibility analysis, the ΔompF mutant showed significantly smaller inhibition zones than the WT, indicating that OmpF had a positive effect on the influx of antibiotics into the cells. In summary, ompF plays a positive regulatory role in the biofilm formation and adhesion/invasion, which is achieved by regulating the amount of LPS, but is a negative regulator of antibiotic resistance in C. sakazakii.

Keywords: Cronobacter sakazakii; LPS; adhesion/invasion; biofilm formation; ompF.

Figure 1

Construction and verification of the ompF deletion mutant. (a) Schematic representation of the ∆ompF construction via homologous recombination. (b) Verification of ompF homologous recombination events. Lane M, DL10000 marker; lanes 1 and 2, amplified with KF/KR, WT and ΔompF as templates, respectively; lanes 3 and 4, amplified with 413F/413R, ΔompF and WT as templates, respectively; lane 5, amplified with 413UF/413UR, ΔompF as template; lane 6, amplified with 413DF/413DR, ΔompF as template; lane 7, amplified with 413UF/413DR, ΔompF as template.

Figure 2

Growth and cell morphology of C. sakazakii in LB medium. (a) Growth of C. sakazakii in LB medium. Error bars represent the standard deviations from independent experiments performed in triplicate. (b) SEM to observe the cell morphology of WT, ΔompF and cpompF. Panels: WT, C. sakazakii ATCC BAA-894; ΔompF, mutant strain; cpompF, complementation strain.

Figure 3

Comparison of the biofilm formation ability of different C. sakazakii isolates. The asterisks indicate that the percent biofilm formation by the mutant was significantly different (p < 0.05) from that by the wild_type strain.

Figure 4

Comparison of the spectral features of C. sakazakii biofilms using Raman spectroscopy. (a) Principal component analysis of biofilm composition in C. sakazakii WT, ΔompF and cpompF strains. (b) Comparison of the spectral features of C. sakazakii biofilms using Raman spectroscopy. (A): complement strains, (B): mutant, (C): wild_type.

Figure 5

SDS-PAGE analysis of LPS extracted from C. sakazakii WT, ΔompF and cpompF strains.

Figure 6

Adhesion to or invasion of epithelial cells by different C. sakazakii isolates. The asterisks indicate that the percent invasion by the mutant was significantly different (p < 0.01) from that by the wild_type strain.

Figure 7

Antibiotic resistance of the C. sakazakii WT, ΔompF and cpompF strains. (a) 1, 2, 3 and the corresponding positions on the other plates represent gentamicin, ampicillin and penicillin, respectively; (b) 4, 5, 6 and the corresponding positions on the other plates represent tetracycline, ciprofloxacin and kanamycin, respectively.