Function analysis of LIPI-4 in Listeria monocytogenes reveals a key role of flagella formation in the regulation of virulence

Abstract

Listeria pathogenicity island 4 (LIPI-4), a newly discovered virulence gene cluster, plays a critical role in the development of central nervous system infections and maternal-fetal infections caused by Listeria monocytogenes. This study aimed to clarify the pathogenic role of LIPI-4 in L. monocytogenes. Our findings revealed that LIPI-4 is involved in the invasion and intracellular proliferation of L. monocytogenes in Caco-2, hCMEC/D3, HTR-8/Svneo, and RAW264.7 cells and facilitates its intercellular migration. Further investigations demonstrated that the deletion of LIPI-4 significantly reduces bacterial motility and impairs flagellum formation. Transcriptomic analysis further indicated that LIPI-4 regulates flagellum formation by modulating microbial metabolic processes and carbon metabolism. Our research demonstrates that LIPI-4 promotes flagellum formation through metabolic regulation, thereby enhancing L. monocytogenes colonization and cellular invasion. This discovery provides novel insights into the pathogenic mechanisms of L. monocytogenes.

Keywords: LIPI-4; Listeria monocytogenes; Virulence; flagella; transcriptomics.

Figure 1.

The growth curves of L. monocytogenes at 37°C. A: BHI; B: basic broth culture medium with lactose as sole carbon source; C: basic broth culture medium with cellobiose as sole carbon source.

Figure 2.

Comparative analysis of adhesion and invasion capacities between LM928 and ΔLIPI-4 strains. The two strains were used to infect Caco-2, hCMEC/D3, HTR-8/Svneo, and RAW264.7 cells at different multiplicities of infection (MOI). After 1 h of infection, the adhesion rate was determined by counting the number of cell-associated bacteria (A – D). The invasion rate was calculated after 1 h of incubation with 100 μg/mL gentamicin-containing medium (E – H). Values represent the mean ± SEM (n = 3), ns: no significance, *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3.

Intracellular proliferation of L. monocytogenes. LM928, ΔLIPI-4, and CΔLIPI-4 strains were used to infect Caco-2, hCMEC/D3, HTR-8/Svneo, and RAW264.7 cells. After 1 h of infection, the adhesion rate was determined by counting the number of cell-associated bacteria. The invasion rate was calculated by incubating the cells with 100 μg/mL gentamicin-containing medium for 1 h (recorded as 0 h). The number of intracellular bacteria was determined by adding 10 μg/mL gentamicin-containing medium, and samples were collected at 4, 8, and 12 h, respectively (A – D). Values represent the mean ± SEM (n = 3). ns: no significance; *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4.

Comparison of intercellular migration abilities in L929 cells infected with L. monocytogenes LM928, LIPI-4 deletion, and complemented strains. A: plaque formation in L929 cells after infection with L. monocytogenes. B: plaque numbers. Values represent the mean ± SEM (n = 3). ns: no significance, ***p < 0.001.

Figure 5.

Flagellum synthesis is absent in L. monocytogenes after LIPI-4 deletion. (A) Swarming ability of LM928, ΔLIPI-4, and CΔLIPI-4 strains at 28°C, 37°C, and 42°C. The motility zone diameters of each strain were measured and statistically analyzed. (B) Puncture tests of LM928, ΔLIPI-4, and CΔLIPI-4 strains in a semisolid medium were conducted to determine motility at 28°C, 37°C, and 42°C. (C) Flagella production in LM928 and ΔLIPI-4 strains at 28°C as observed by transmission electron microscopy (TEM). (D) Percentage of bacteria with at least one visible flagellum in 15 fields of view in TEM observations of LM928 and ΔLIPI-4 strains. (E) Changes in the expression levels of the flaA gene in LM928 strains in the absence of LIPI-4. Values represent the mean ± SEM, ns: no significance, *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 6.

LM928 and ΔLIPI-4 transcriptome analysis at 28°C and 37°C. Volcano map of DEGs for ΔLIPI-4 vs LM928 at 28°C (A) and 37°C (B); KEGG pathways analysis at 28°C (C) and 37°C (D); GO enrichment analysis at 28°C (E) and 37°C (F). The size and color of the dot represented the number of DEGs and the range of padj value (adjusted p value).

Figure 7.

Verification of transcriptome significant difference gene by using RT-PCR method.