Type III secretion system 1 of Vibrio parahaemolyticus induces oncosis in both epithelial and monocytic cell lines

Abstract

The Vibrio parahaemolyticus type III secretion system 1 (T3SS1) induces cytotoxicity in mammalian epithelial cells. We characterized the cell death phenotype in both epithelial (HeLa) and monocytic (U937) cell lines following infection with V. parahaemolyticus. Using a combination of the wild-type strain and gene knockouts, we confirmed that V. parahaemolyticus strain NY-4 was able to induce cell death in both cell lines via a T3SS1-dependent mechanism. Bacterial contact, but not internalization, was required for T3SS1-induced cytotoxicity. The mechanism of cell death involves formation of a pore structure on the surface of infected HeLa and U937 cells, as demonstrated by cellular swelling, uptake of cell membrane-impermeable dye and protection of cytotoxicity by osmoprotectant (PEG3350). Western blot analysis showed that poly ADP ribose polymerase (PARP) was not cleaved and remained in its full-length active form. This result was evident for seven different V. parahaemolyticus strains. V. parahaemolyticus-induced cytotoxicity was not inhibited by addition of the pan-caspase inhibitor carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD-FMK) or the caspase-1 inhibitor N-acetyl-tyrosyl-valylalanyl-aspartyl-aldehyde (Ac-YVAD-CHO); thus, caspases were not involved in T3SS1-induced cytotoxicity. DNA fragmentation was not evident following infection and autophagic vacuoles were not observed after monodansylcadaverine staining. We conclude that T3SS1 of V. parahaemolyticus strain NY-4 induces a host cell death primarily via oncosis rather than apoptosis, pyroptosis or autophagy. INTRODUCTIONVibrio parahaemolyticus is a Gram-negative food-borne pathogen that is commonly associated with consumption of raw or undercooked seafood (Joseph et al., 1982). Symptoms of infection include diarrhoea, nausea, vomiting, headache, fever and chills. In addition to typical gastrointestinal infections, approximately 5 % of V. parahaemolyticus infections advance to septicaemia (Hlady & Klontz, 1996) and these infections may be fatal, especially in immunocompromised patients or those with a preexisting medical condition such as liver disease or diabetes (Yeung & Boor, 2004).Thermostable direct haemolysin (TDH) is considered the primary virulence factor in V. parahaemolyticus. In addition to its ability to form pores in red blood cells, TDH causes increased short circuit current, increased Cl 2 secretion in human colonic epithelial cells, and enhanced Ca 2+ entry from the extracellular medium (Takahashi et al., 2001). An early study of a tdh deletion mutant demonstrated significantly reduced ability to cause fluid accumulation in ileal loops of a rabbit model (Nishibuchi et al., 1992). In contrast, Park et al. (2004) showed that a tdh deletion mutant has the ability to cause fluid accumulation in the ligated intestine of rabbits. Furthermore, both TDH-positive and -negative strains of V. parahaemolyticus can disrupt the epithelial tight junction, a precursor to dissemination of bacteria into the circulation (Lynch et a...

The type III secretion system of Vibrio alginolyticus induces rapid apoptosis, cell rounding and osmotic lysis of fish cells

“…Interestingly, V. alginolyticus induces apoptosis during the process of T3SS-mediated cell death, whereas V. parahaemolyticus does not. In contrast to our findings, the T3SS1-mediated cell death of V. parahaemolyticus preserves the integrity of the host-cell nuclear membrane and does not activate caspase molecules (Burdette et al 2008;Zhou et al, 2009). This difference may be due to the inherent differences in the T3SS effectors of V. alginolyticus and V. parahaemolyticus, as there were some minor differences between the hypothetical regions encoding them ( Supplementary Fig.…”

“…It has been suggested that many bacteria use their T3SS to create pores in host cell membranes directly, thereby causing osmotic lysis (Dacheux et al, 2001;Fink & Cookson, 2006;Neyt & Cornelis, 1999;Nonaka et al, 2003;Zhou et al, 2009). Based on the above observations, we hypothesized that LDH release caused by V. alginolyticus is a result of osmotic lysis.…”

“…Recently, Burdette et al (2008) reported that V. parahaemolyticus T3SS1-mediated cytotoxicity is caspaseindependent and involves the induction of autophagy, followed by cell rounding and lysis. More recent studies also suggested that the bacterium kills host cells primarily via oncosis rather than apoptosis, pyroptosis or autophagy (Zhou et al, 2009). However, few reports are available regarding cell death caused by the T3SSs of other Vibrio species.…”

“…Osmoprotection experiments were carried out as described previously (Fink & Cookson, 2006;Zhou et al, 2009). Briefly, EPC cells were infected with the ZJO strain or treated with 10 mM H 2 O 2 in the presence of 30 mM solutions of sucrose (Amresco), PEG600, PEG1000, PEG1450 or PEG 3350 (Sigma) made up in serum-free TC199 medium and filter-sterilized prior to use.…”

Virulence gene expression of Vibrio parahaemolyticus in the viable but nonculturable state

“…Similarly, Coutard et al (2007b) found that tdh2 was expressed but not upregulated. On the contrary, Coutard et al (2005) and T3SS2 associated genes (translocator genes and their chaperones, regulators, structural, and effector proteins) showed substantial increases over the course of infection in HeLa cells Nydam et al, 2014;Zhou et al, 2009). …”