Flavoridin inhibitsYersinia enterocoliticauptake into fibronectin-adherent HeLa cells

Abstract

In this study, three structurally distinct disintegrins (flavoridin, echistatin, kistrin) were used as molecular probes to further characterize the molecular mechanisms underlying Yersinia enterocolitica infection of host cells. The activity of the three disintegrins on Y. enterocolitica uptake into fibronectin-adherent HeLa cells was evaluated at disintegrin doses which were non-cytotoxic and unable to induce cell detachment. Flavoridin resulted to be the most effective in inhibiting bacterial entry into host cells; echistatin was almost 50% less effective than flavoridin, whereas kistrin was definitely inactive. Our results suggest that alpha(5)beta(1) integrin receptor, which binds flavoridin with higher affinity than the other two disintegrins, plays a major role in Y. enterocolitica uptake into HeLa cells. Furthermore, flavoridin binding to this integrin prevented the disruption of the functional complex FAK-Cas, which occurs in the Y. enterocolitica uptake process.

Integrin receptors play a role in the internalin B-dependent entry of Listeria monocytogenes into host cells

“…Before evaluating the ability of kistrin, echistatin and flavoridin to inhibit L. monocytogenes uptake into HeLa cells, we determined their cytotoxicity by exposing the cells to increasing concentrations of each disintegrin (0-100 μg/ml) for different time intervals (10 min to 24 h). The viability of the cells, as measured uding the trypan blue exclusion method [21], was higher than 90% after 24 h of cell exposure to 100 μg/ml of each disintegrin (data not shown). However, for the uptake inhibition studies, echistatin, flavoridin and kistrin were used at the low doses of 0.05-1 μg/ml.…”

“…Due to structural motifs other than the RGD sequence present in their molecules, disintegrins show a high selectivity in their binding to different integrin receptors, and are thus a useful tool for studying integrin-mediated cell functions and bacterial pathogenesis [21]. Kistrin binds α v β 3 with high affinity, and it is recognized by α 5 β 1 , albeit with a lower affinity than flavoridin and echistatin [20].…”

“…Then, the cells were washed three times with DMEM, and lysed with Triton X-100 (0.3% final concentration). The number of bacteria internalized by the cells was counted on agar plates using the colony-forming unit (CFU) method [21]. The percentage of bacterial uptake into the cells was determined by calculating the number of bacteria internalized into infected cells with respect to the total number of bacteria used for the infection.…”

Salmonella engineered to express CD20-targeting antibodies and a drug-converting enzyme can eradicate human lymphomas

“…Salmonella localization was then examined via immunofluorescence under conditions allowing for the discrimination of intracellular and extracellular bacteria, based on differentially staining before and after permeabilization. 22 As shown in Figure 2A-B, 2G9-Salmonella bound and invaded the CD201 cells at least as efficiently as unmodified AT-Salmonella, but significantly more than GSTb1-Salmonella (displaying the irrelevant control VHH). 2G9-Salmonella bound cells 5.6-fold (MCA203) (P 5 .006) and 7.9-fold better (CT26) (P 5 3 3 10…”

“…Slides were then subjected to immunofluorescence staining to discriminate extracellular (doubly positive for Cy5-and Cy3-secondary antibodies) and intracellular bacteria (Cy5-singly-positive) 22 using an anti-Salmonella antibody (Virostat). Cytoplasm was visualized via phalloidin-fluorescein isothiocyanate (FITC) and nuclei by 49,6 diamidino-2-phenylindole.…”