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. 2023 Dec 21;89(12):e0107423.
doi: 10.1128/aem.01074-23. Epub 2023 Nov 30.

Anti-virulence and bactericidal activities of Stattic against Shigella sonnei

Mingfang Wang #  1 , Jia Zeng #  1 , Huihui Tan  1 , Quan Guo  1 , Xia Li  1 , Xiwen Ling  1 , Jinyue Zhang  2 , Shihao Song  1   2 , Yinyue Deng  1
Affiliations

Anti-virulence and bactericidal activities of Stattic against Shigella sonnei

Mingfang Wang et al. Appl Environ Microbiol. .

Abstract

Shigella sonnei is a major human enteric pathogen that causes bacillary dysentery. The increasing spread of drug-resistant S. sonnei strains has caused an emergent need for the development of new antimicrobial agents against this pathogenic bacterium. In this study, we demonstrate that Stattic employs two antibacterial mechanisms against S. sonnei. It exerted both anti-virulence activity and bactericidal activity against S. sonnei, suggesting that it shows advantages over traditional antibiotics. Moreover, Stattic showed excellent synergistic effects with kanamycin, ampicillin, chloramphenicol, and gentamicin against S. sonnei. Our findings suggest that Stattic has promising potential for development as a new antibiotic or as an adjuvant to antibiotics for infections caused by S. sonnei.

Keywords: GalU; Shigella sonnei; Stattic; antibiotic; virulence.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig 1
Fig 1
Effects of compounds on the virulence of S. sonnei. All compounds were dissolved in Dimethyl sulfoxide (DMSO). LDH release was used to assess cytotoxicity, and the amount of LDH released by S. sonnei infectious assays treated with the same volume of DMSO was used as a control and defined as 100%, aimed to normalize the LDH production of HeLa cells infected by S. sonnei in the presence of compounds. The data are presented as the mean ± SD (n = 3, independent measurements). One-way ANOVA was used to determine the significance of the results (*P < 0.05; **P < 0.01; ***P < 0.001; ns = no significance).
Fig 2
Fig 2
Stattic binds to GalU. (a) Genomic organization of the galU region in S. sonnei Ss046. (b) Analysis of the domain structure of GalU. (c) SDS‒PAGE analysis of the GalU protein. (d) MST analysis of the binding of Stattic to GalU.
Fig 3
Fig 3
The effect of Stattic on the enzyme activity of GalU. (a) Catalytic reaction of GalU. (b) The change in GalU activity in the presence of different concentrations of Stattic. (c) The structure model of the binding of Stattic to GalU. Cyan represents the solid surface of GalU protein, yellow represents the stick model of Stattic, and magenta represents the amino acid residues that may interact with Stattic. (d) MST analysis of the binding of Stattic to GalU mutants. The data are presented as the mean ± SD (n = 3, independent measurements). One-way ANOVA was used to determine the significance of the results (*P < 0.05; **P < 0.01; ***P < 0.001; ns = no significance). ND, not detected.
Fig 4
Fig 4
Effects of Stattic on the virulence-related phenotypes of S. sonnei. The virulence-related phenotypes of (a) biofilm formation (n = 5, independent measurements) and (b) EPS production (n = 5, independent measurements) in S. sonnei in the presence of different concentrations of Stattic were examined. (c) The cell cytotoxicity of S. sonnei in the presence of different concentrations of Stattic was evaluated by LDH assay (n = 3, independent measurements). The effects of Stattic on biofilm formation (d), EPS production (e), and cell cytotoxicity (f) of S. sonnei wild-type, galU mutant, galU complemented, and galU mutant complemented with galUQ109A were also examined (n = 3, independent measurements). S. sonnei was treated with different concentrations of Stattic and incubated statically at 37°C. The data are presented as the mean ± SD. Error bars indicate the SDs. One-way ANOVA was used to determine the significance of the results (*P < 0.05; **P < 0.01; ***P < 0.001; ns = no significance).
Fig 5
Fig 5
The synergistic effect of Stattic with different antibiotics on the cell cytotoxicity of S. sonnei. The following antibiotic concentrations were used in this experiment: Kan, kanamycin, 6.25 µg mL−1; Amp, ampicillin, 1.56 µg mL−1; Chl, chloramphenicol, 1.56 µg mL−1; Gen, gentamicin, 1.56 µg mL−1; and Spec, spectinomycin, 12.5 µg mL−1. The concentration of Stattic was 10 µM. The data are presented as the mean ± SD (n = 3, independent measurements). One-way ANOVA was used to determine the significance of the results (*P < 0.05; **P < 0.01; ***P < 0.001; ns = no significance).
Fig 6
Fig 6
Effects of Stattic on the virulence-related phenotypes of E. coli and K. pneumoniae. The virulence-related phenotypes of biofilm formation (n = 3, independent measurements) (a), EPS production (n = 3, independent measurements) (b), and cell cytotoxicity (c) of E. coli, and those phenotypes (d–f) of K. pneumoniae, are presented with the addition of different concentrations of Stattic. The cell cytotoxicity of E. coli and K. pneumoniae in the presence of different concentrations of Stattic were evaluated by LDH assay (n = 3, independent measurements). The data are presented as the mean ± SD. Error bars indicate the SDs. One-way ANOVA was used to determine the significance of the results (*P < 0.05; **P < 0.01; ***P < 0.001; ns = no significance).
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