Effect on human cells of environmental Vibrio parahaemolyticus strains carrying type III secretion system 2

Abstract

Vibrio parahaemolyticus is an inhabitant of estuarine and marine environments that causes seafood-borne gastroenteritis worldwide. Recently, a type 3 secretion system (T3SS2) able to secrete and translocate virulence factors into the eukaryotic cell has been identified in a pathogenicity island (VP-PAI) located on the smaller chromosome. These virulence-related genes have previously been detected only in clinical strains. Classical virulence genes for this species (tdh, trh) are rarely detected in environmental strains, which are usually considered to lack virulence potential. However, during screening of a collection of environmental V. parahaemolyticus isolates obtained in the North Adriatic Sea in Italy, a number of marine strains carrying virulence-related genes, including genes involved in the T3SS2, were detected. In this study, we investigated the pathogenic potential of these marine V. parahaemolyticus strains by studying their adherence ability, their cytotoxicity, their effect on zonula occludin protein 1 (ZO-1) of the tight junctions, and their effect on transepithelial resistance (TER) in infected Caco-2 cells. By performing a reverse transcription-PCR, we also tested the expression of the T3SS2 genes vopT and vopB2, encoding an effector and a translocon protein, respectively. Our results indicate that, similarly to clinical strains, marine V. parahaemolyticus strains carrying vopT and vopB2 and that other genes included in the VP-PAI are capable of adhering to human cells and of causing cytoskeletal disruption and loss of membrane integrity in infected cells. On the basis of data presented here, environmental V. parahaemolyticus strains should be included in coastal water surveillance plans, as they may represent a risk for human health.

FIG. 1.

Amplification products obtained by reverse transcription of gyrB (housekeeping gene) (A), vopT (encoding effector T3SS2) (B), and vopB2 (encoding part of the translocon T3SS2) (C). Each panel contains a molecular marker in the left lane with six sample lanes as follows: lanes 1 and 2, VPeVEpan; lanes 3 and 4, VPe23; lanes 5 and 6, AN2416 (reference KP-positive strain, given by R. Y. C. Kong). Lanes 1, 3, and 5 are RT⁺; lanes 2, 4, and 6 are RT⁻.

FIG. 2.

Adherence test micrographs showing HeLa cells infected with representative environmental V. parahaemolyticus strains. (A) KP-positive clinical strain (QM 97097); (B) VPeVEpan (tdh⁺ T3SS2⁺); (C) strain not carrying virulence-related genes (VPe93); (D) strain carrying only T3SS2 genes (VPe23); (E) trh-positive environmental V. parahaemolyticus strain (VPe10).

FIG. 3.

Percent cytotoxic activity, measured as LDH concentrations released from Caco-2 cells infected with environmental V. parahaemolyticus strains. From left to right: strain VPe93 without virulence genes (tdh and T3SS2 negative); strain VPe23 (tdh negative, T3SS2 positive) carrying only the genes belonging to the secretion system; the pandemic strain VPeVEpan (tdh and T3SS2 positive) carrying the secretion system genes and the tdh gene, and, in black, the KP-positive clinical strain (QM 97097) as a reference. The results represent the means of six independent determinations, with bars showing one standard deviation. The differences in cytotoxicity values between strain VPe23, strain VPe93, and the clinical strain QM 97097 are statistically significant (Student's t test, P < 0.01).

FIG. 4.

Transepithelial resistance values of Caco-2 cell monolayers infected with V. parahaemolyticus environmental strains. Bacterial strains used in this experiment were the pandemic strain VPepan, carrying the tdh and T3SS2 genes, the strain VPe23, carrying only the T3SS2 genes, and the strain VPe93, as a representative of strains without virulence genes. A reference strain (QM97097) was also included. The same experiment conducted with strains VPepan2, VPe28, and VPe122 gave similar results without statistically significant differences (Student's t test) compared with the one presented in the figure. Three separate experiments were conducted in duplicate, and results were averaged.

FIG. 5.

Immunofluorescence micrographs of Caco-2 cells double labeled with Alexa488 phalloidin for filamentous actin (green) and Alexa568 for rabbit antibodies against ZO-1 (red). (A) Uninfected cells; (B) cells infected with a strain not carrying virulence-related genes (VPe93); (C) cells infected with a strain carrying T3SS2 genes (VPe23); (D) cells infected with KP-positive reference strain QM 97097; (E and F) cells infected with the VPeVEpan strain (T3SS2 and tdh positive). Photographs are representative of the results obtained in two separate experiments.