Inhibition of Fusarium solani Infection in Murine Keratocytes by Lactobacillus salivarius ssp. salivarius JCM1231 Culture Filtrate In Vitro

ABSTRACT

Purpose: To explore the inhibitory activity of Lactobacillus salivarius ssp. salivarius JCM1231 (L. salivarius JCM1231) culture filtrate against Fusarium solani (F. solani) and its effects on murine keratocytes (MKs) infected with F. solani.

Methods: L. salivarius JCM1231 was cultured in an anaerobic incubator for 24 h, and the L. salivarius culture filtrate (LSCF) was prepared .The antifungal activity of L. salivarius JCM1231 against F. solani was determined with a plate overlay assay, agar diffusion assay, and conidial germination inhibition test. The effects of temperature, pH, and proteolytic enzymes on the antifungal activity of LSCF were detected with microtiter plate-well assay and conidial germination inhibition assay. Furthermore, the effects of LSCF on MKs infected with F. solani were detected. Cell activity and apoptosis were measured using methylthiazoletetrazolium assays and flow cytometry analysis, respectively. The levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) cytokines were measured using real-time polymerase chain reactions and enzyme-linked immunosorbent assays (ELISA), and mycotoxin production was detected with high-performance liquid chromatography tandem mass spectrometry.

Results: Conidial germination and mycelia growth of F. solani were significantly inhibited by LSCF. The antifungal substances produced by L. salivarius JCM1231 were heat unstable, proteinaceous, and sensitive to proteolytic enzymes and were active within a narrow acidic pH range between 2.0 and 4.0. In the presence of 15 µg/ml of LSCF, cell activity was significantly increased, and cell apoptosis, the level of IL-6 and TNF-α expressions, and mycotoxin (zearalenone and fumonisin B1) productions were decreased significantly in MKs infected with F. solani.

Conclusion: L. salivarius JCM1231 culture filtrate can effectively inhibit F. solani growth and protect MKs against F. solani infection.

KEYWORDS:

• F. solani
• fungal keratitis
• high-performance liquid chromatography
• L. salivarius JCM1231
• mycotoxin

Acknowledgments

We thank Ye Wang, Qingjun Zhou, and Zhiyuan Li (Shandong Eye Institute, Qingdao, Shandong, China) for laboratory technical support.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

Funding

This work was supported in part by the National Natural Science Foundation of China, Beijing, China (grant no.: 81270976), and Fujian Province Medical Project for Middle-aged and Young Talents (BC), Fuzhou, China (grant no.: 2015-ZQN-ZD-20).

Additional information

Funding

This work was supported in part by the National Natural Science Foundation of China, Beijing, China (grant no.: 81270976), and Fujian Province Medical Project for Middle-aged and Young Talents (BC), Fuzhou, China (grant no.: 2015-ZQN-ZD-20).