Campylobacter fetus is Internalized by Bovine Endometrial Epithelial Cells

Abstract

Campylobacter fetus is an important venereal pathogen of cattle that causes infertility and abortions. It is transmitted during mating, and it travels from the vagina to the uterus; therefore, an important cell type that interacts with C. fetus are endometrial epithelial cells. Several virulence factors have been identified in the genome of C. fetus, such as adhesins, secretion systems, and antiphagocytic layers, but their expression is unknown. The ability of C. fetus to invade human epithelial cells has been demonstrated, but the ability of this microorganism to infect bovine endometrial epithelial cells has not been demonstrated. Bovine endometrial epithelial cells were isolated and challenged with C. fetus. The presence of C. fetus inside the endometrial epithelial cells was confirmed by the confocal immunofluorescence. C. fetus was not internalized when actin polymerization was disturbed, suggesting cytoskeleton participation in an internalization mechanism. To evaluate the intracellular survival of C. fetus, a gentamicin protection assay was performed. Although C. fetus was able to invade epithelial cells, the results showed that it did not have the capacity to survive in the intracellular environment. This study reports for the first time, the ability of C. fetus to invade bovine endometrial epithelial cells, and actin participation in this phenomenon.

Campylobacter fetus is an important venereal pathogen of cattle that causes infertility and abortions. It is transmitted during mating, and it travels from the vagina to the uterus; therefore, an important cell type that interacts with C. fetus are endometrial epithelial cells. Several virulence factors have been identified in the genome of C. fetus, such as adhesins, secretion systems, and antiphagocytic layers, but their expression is unknown. The ability of C. fetus to invade human epithelial cells has been demonstrated, but the ability of this microorganism to infect bovine endometrial epithelial cells has not been demonstrated. Bovine endometrial epithelial cells were isolated and challenged with C. fetus. The presence of C. fetus inside the endometrial epithelial cells was confirmed by the confocal immunofluorescence. C. fetus was not internalized when actin polymerization was disturbed, suggesting cytoskeleton participation in an internalization mechanism. To evaluate the intracellular survival of C. fetus, a gentamicin protection assay was performed. Although C. fetus was able to invade epithelial cells, the results showed that it did not have the capacity to survive in the intracellular environment. This study reports for the first time, the ability of C. fetus to invade bovine endometrial epithelial cells, and actin participation in this phenomenon.

Fig. 1.

Epithelial cells from bovine endometrium maintained in DMEM supplemented with 10% fetal bovine serum. A) Normal appearance of the epithelial-like cells (20×). B) Immunofluorescence. Cytokeratin 18 of epithelial cells of bovine endometrium stained with Alexa 488 (green) (40×). C) The results of RT-PCR for keratin 8 on 2% agarose gel after staining with ethidium bromide. In left lane: DNA ladder (Thermo Fisher Scientific); right lane: the amplicon of keratin 8 (215 pb).

Fig. 2.

The adherence assay. Epithelial cells from bovine endometrium were challenged with C. fetus (A) or E. coli (B) for 1 h, fixed with methanol and stained with Giemsa (100×). The arrows show the adhered bacteria.

Fig. 3.

The intracellular survival assay. Epithelial cells of endometrium of bovine were infected with C. fetus ATCC 27374 (a MOI of 1000:1). The intracellular bacteria were recovered and plated on the Campylobacter selective agar supplemented with 5% of blood. Average log CFU are shown at 0, 2, 4, 10 and 24 h p.i.

Fig. 4.

Confocal differential fluorescent staining of internal C. fetus ATCC 27374 on the infected epithelial cells of bovine endometrium. Epithelial cells were grown on coverslips and infected with C. fetus ATCC 27374 at a MOI of 1000:1. Cytoskeleton was stained with phalloidin-FITC (green), and the bacteria with Alexa 594 (red) 2 h p.i. White arrows show intracellular C. fetus (70×).

Fig. 5.

The cytoskeleton inhibition assay. Epithelial cells of endometrium of bovine were treated with cytocalasin D or nocodazole before and during infection. The cells were infected with C. fetus ATCC 27374 (a MOI of 1000:1). The intracellular bacteria were recovered and plated on the Campylobacter selective agar supplemented with 5% of blood. Average log CFU are shown at 0 and 2 p.i. T-test was performed. All treatments were compared to the not-treatment control, *(p < 0.001).