Vesicular stabilization and activity augmentation of enterohaemorrhagic Escherichia coli haemolysin

Haemolysin from enterohaemorrhagic Escherichia coli (EHEC-Hly), a putative EHEC virulence factor, belongs to the RTX (repeat-in-toxin) family whose members rapidly inactivate themselves by self-aggregation. By investigating the status of EHEC-Hly secreted extracellularly, we found the toxin both in a free, soluble form and associated, with high tendency and independently of its acylation status, to outer membrane vesicles (OMVs) extruded by EHEC. We compared the interaction of both toxin forms with erythrocytes using scanning electron microscopy and binding assays. The OMV-associated toxin was substantially (80 times) more stable under physiological conditions than the free EHEC-Hly as demonstrated by prolonged haemolytic activity (half-life time 20 h versus 15 min). The haemolysis was preceded by calcium-dependent binding of OMVs carrying EHEC-Hly to erythrocytes; this binding was mediated by EHEC-Hly. We demonstrate that EHEC-Hly is a biologically active cargo in OMVs with dual roles: a cell-binding protein and a haemolysin. These paired functions produce a biologically potent form of the OMV-associated RTX toxin and augment its potential towards target cells. Our findings provide a general concept for stabilization of RTX toxins and open new insights into the biology of these important virulence factors.     

Hemolysin from Shiga toxin-negative Escherichia coli O26 strains injures microvascular endothelium

We identified Shiga toxin gene (stx)-negative Escherichia coli O26:H11 and O26:NM (nonmotile) strains as the only pathogens in the stools of five patients with hemolytic-uremic syndrome (HUS). Because the absence of stx in E. coli associated with HUS is unusual, we examined the strains for potential virulence factors and interactions with microvascular endothelial cells which are the major targets affected during HUS. All five isolates possessed the enterohemorrhagic E. coli (EHEC)-hlyA gene encoding EHEC hemolysin (EHEC-Hly), expressed the enterohemolytic phenotype, and were cytotoxic, in dose- and time-dependent manners, to human brain microvascular endothelial cells (HBMECs). Significantly reduced cytotoxicity in an EHEC-Hly-negative spontaneous derivative of one of these strains, and a dose- and time-dependent cytotoxicity of recombinant E. coli O26 EHEC-Hly to HBMECs, suggest that the endothelial cytotoxicity of these strains was mediated by EHEC-Hly. The toxicity of EHEC-Hly to microvascular endothelial cells plausibly contributes to the virulence of the stx-negative E. coli O26 strains and to the pathogenesis of HUS.     

Enterohaemorrhagic Escherichia coli haemolysin is cleaved and inactivated by serine protease EspPα

The haemolysin from enterohaemorrhagic Escherichia coli (EHEC-Hly) and the serine protease EspPα are putative virulence factors of EHEC. We investigated the interplay between these secreted factors and demonstrate that EspPα cleaves the 107 kDa large EHEC-Hly. Degradation was observed when purified EspPα was added to a growing culture of an EHEC-Hly-expressing strain, with isolated proteins and with coexpressing strains, and was independent of the EHEC serotype. EHEC-Hly breakdown occurred as a multistage process with the formation of characteristic fragments with relative molecular masses of ~82 kDa and/or ~84 kDa and ~34 kDa. The initial cleavage occurred in the N-terminal hydrophobic domain of EHEC-Hly between Leu(235) and Ser(236) and abolished its haemolytic activity. In a cellular infection system, the cytolytic potential of EHEC-Hly-secreting recombinant strains was abolished when EspPα was coexpressed. EHEC in contact with human intestinal epithelial cells simultaneously upregulated their EHEC-Hly and EspP indicating that both molecules might interact under physiological conditions. We propose the concept of bacterial effector molecule interference (BEMI), reflecting the concerted interplay of virulence factors. Interference between effector molecules might be an additional way to regulate virulence functions and increases the complexity of monomolecular phenotypes.     

Outer Membrane Vesicles Mediate Transport of Biologically Active Vibrio cholerae Cytolysin (VCC) from V. cholerae Strains

Background

Outer membrane vesicles (OMVs) released from Gram-negative bacteria can serve as vehicles for the translocation of virulence factors. Vibrio cholerae produce OMVs but their putative role in translocation of effectors involved in pathogenesis has not been well elucidated. The V. cholerae cytolysin (VCC), is a pore-forming toxin that lyses target eukaryotic cells by forming transmembrane oligomeric β-barrel channels. It is considered a potent toxin that contributes to V. cholerae pathogenesis. The mechanisms involved in the secretion and delivery of the VCC have not been extensively studied.

Methodology/Principal Findings

OMVs from V. cholerae strains were isolated and purified using a differential centrifugation procedure and Optiprep centrifugation. The ultrastructure and the contents of OMVs were examined under the electron microscope and by immunoblot analyses respectively. We demonstrated that VCC from V. cholerae strain V:5/04 was secreted in association with OMVs and the release of VCC via OMVs is a common feature among V. cholerae strains. The biological activity of OMV-associated VCC was investigated using contact hemolytic assay and epithelial cell cytotoxicity test. It showed toxic activity on both red blood cells and epithelial cells. Our results indicate that the OMVs architecture might play a role in stability of VCC and thereby can enhance its biological activities in comparison with the free secreted VCC. Furthermore, we tested the role of OMV-associated VCC in host cell autophagy signalling using confocal microscopy and immunoblot analysis. We observed that OMV-associated VCC triggered an autophagy response in the target cell and our findings demonstrated for the first time that autophagy may operate as a cellular defence mechanism against an OMV-associated bacterial virulence factor.

Conclusion/Significance

Biological assays of OMVs from the V. cholerae strain V:5/04 demonstrated that OMV-associated VCC is indeed biologically active and induces toxicity on mammalian cells and furthermore can induce autophagy.     

Serine Protease EspP from Enterohemorrhagic Escherichia Coli Is Sufficient to Induce Shiga Toxin Macropinocytosis in Intestinal Epithelium

Life-threatening intestinal and systemic effects of the Shiga toxins produced by enterohemorrhagic Escherichia coli (EHEC) require toxin uptake and transcytosis across intestinal epithelial cells. We have recently demonstrated that EHEC infection of intestinal epithelial cells stimulates toxin macropinocytosis, an actin-dependent endocytic pathway. Host actin rearrangement necessary for EHEC attachment to enterocytes is mediated by the type 3 secretion system which functions as a molecular syringe to translocate bacterial effector proteins directly into host cells. Actin-dependent EHEC attachment also requires the outer membrane protein intimin, a major EHEC adhesin. Here, we investigate the role of type 3 secretion in actin turnover occurring during toxin macropinocytosis. Toxin macropinocytosis is independent of EHEC type 3 secretion and intimin attachment. EHEC soluble factors are sufficient to stimulate macropinocytosis and deliver toxin into enterocytes in vitro and in vivo; intact bacteria are not required. Intimin-negative enteroaggregative Escherichia coli (EAEC) O104:H4 robustly stimulate Shiga toxin macropinocytosis into intestinal epithelial cells. The apical macropinosomes formed in intestinal epithelial cells move through the cells and release their cargo at these cells’ basolateral sides. Further analysis of EHEC secreted proteins shows that a serine protease EspP alone is able to stimulate host actin remodeling and toxin macropinocytosis. The observation that soluble factors, possibly serine proteases including EspP, from each of two genetically distinct toxin-producing strains, can stimulate Shiga toxin macropinocytosis and transcellular transcytosis alters current ideas concerning mechanisms whereby Shiga toxin interacts with human enterocytes. Mechanisms important for this macropinocytic pathway could suggest new potential therapeutic targets for Shiga toxin-induced disease.     

Mitochondria hyperpolarization is an early event in oxidized low-density lipoprotein-induced apoptosis in Caco-2 intestinal cells

We investigated the mechanisms underlying the pro-apoptotic activity exerted by oxidized low-density lipoproteins (oxLDL) in Caco-2 intestinal cells, a cell line which retains many morphological and enzymatic features typical of normal human enterocytes. We found that: (i) oxLDL induced mitochondrial-mediated apoptosis by provoking first an increase in mitochondrial membrane potential, followed, later, by the typical apoptosis-associated depolarization (type II apoptosis); accordingly, (ii) caspase-9 inhibition significantly hindered apoptosis while caspase-8 inhibition did not; and finally (iii) dietary phenolic antioxidizing compounds exerted a significant protective antiapoptotic activity. These results point to mitochondrial hyperpolarization as 'sensitizing feature' in apoptotic proneness of Caco-2 intestinal cells to oxLDL exposure.     

Vibrio vulnificus RTX toxin plays an important role in the apoptotic death of human intestinal epithelial cells exposed to Vibrio vulnificus

During Vibrio vulnificus infection, V. vulnificus reaches the intestine and then invades the bloodstream by crossing the intestinal mucosal barrier of the host, which results in systemic septicemia. Previously, we reported that the RtxA toxin secreted through the RtxE transporter contributes to the cytotoxicity of V. vulnificus against intestinal epithelial cells. Here, we used gene mutants of rtxE and rtxA to determine the role that V. vulnificus RtxA toxin plays in the apoptotic death of human intestinal epithelial cells. The levels of DNA fragmentation were lower in human epithelial cells infected with an rtxE mutant of V. vulnificus than in those that were infected with the wild type. In addition, the rtxE mutant was found to induce lower levels of TUNEL positive cells and cell cycle arrest at the subG(1) than the wild type V. vulnificus. Furthermore, the decreased levels of DNA fragmentation, TUNEL positive cells and subG(1) arrest by the rtxE gene mutation were restored by the complementation of an rtxE gene into the rtxE mutant V. vulnificus. Finally, the rtxA mutant induced significantly lower levels of apoptotic cell death than the wild type. The levels of the PARP, cytochrome c, caspase-3, and mitochondrial membrane depolarization were lower in human epithelial cells infected with the rtxE and rtxA mutants, compared with the wild type and rtxE gene-complemented strains of V. vulnificus. Taken together, these results indicate that V. vulnificus RtxA toxin induces the apoptotic death through a mitochondria-dependent pathway in human intestinal epithelial cells exposed to V. vulnificus.     

LPA protects intestinal epithelial cells from apoptosis by inhibiting the mitochondrial pathway

We previously showed (Gastroenterology 123: 206-216, 2002) that lysophosphatidic acid (LPA) protects and rescues rat intestinal epithelial cells (IEC-6) from apoptosis. Here, we provide evidence for the LPA-elicited inhibition of the mitochondrial apoptotic pathway leading to attenuation of caspase-3 activation. Pretreatment of IEC-6 cells with LPA inhibited campothecin-induced caspase-9 and caspase-3 activation and DNA fragmentation. A caspase-9 inhibitor peptide mimicked the LPA-elicited antiapoptotic activity. LPA elicited ERK1/ERK2 and PKB/Akt phosphorylation. The LPA-elicited antiapoptotic activity and inhibition of caspase-9 activity were abrogated by pertussis toxin, PD 98059, wortmannin, and LY 294002. LPA reduced cytochrome c release from mitochondria and prevented activation of caspase-9. LPA prevented translocation of Bax from cytosol to mitochondria and increased the expression of the antiapoptotic Bcl-2 mRNA and protein. LPA had no effect on Bcl-xl, Bad, and Bak mRNA or protein expression. These data indicate that LPA protects IEC-6 cells from camptothecin-induced apoptosis through G(i)-coupled inhibition of caspase-3 activation mediated by the attenuation of caspase-9 activation due to diminished cytochrome c release, involving upregulation of Bcl-2 protein expression and prevention of Bax translocation.     

大肠杆菌yijP侵袭蛋白诱导人脑微血管内皮细胞凋亡

为研究大肠杆菌的脑微血管内皮细胞侵袭基因yijP的功能,将yijP基因（1．04kb）克隆到pQE30表达载体,构建表达产物为N末端带有6个组氨酸（His）序列的yijP汇合蛋白,以M15（pREP4）为受体菌,大量表达（His）6-yijP汇合蛋白,利用Ni—NTA亲和层析纯化汇合蛋白,将经透析法复性的一定浓度的（His）6-yijP蛋白加入到体外培养的人脑微血管内皮细胞中,结果显示yijP蛋白对人脑微血管内皮细胞有较强的细胞毒作用：在相差显微镜下可观察到细胞皱缩、胞膜呈泡状膨出,随着时间延长细胞逐渐脱落;荧光显微镜下可见细胞核呈现为致密团块状或圆形浓染颗粒状,呈凋亡样改变：DNA琼脂糖凝胶电泳可见DNA阶梯状条带;流式细胞仪显示在正常二倍体峰之前出现一个亚二倍体峰;Western印迹可检测到caspase-3的活性片段。这些现象均出现在yijP蛋白作用于人脑微血管内皮细胞的16h之后,提示在大肠杆菌侵袭人脑微血管内皮细胞过程中,yijP蛋白可能起到诱导脑微血管内皮细胞迟发性凋亡的毒素作用。     

Herpes simplex virus blocks apoptosis by precluding mitochondrial cytochrome c release independent of caspase activation in infected human epithelial cells

Expression of HSV-1 genes leads to the induction of apoptosis in human epithelial HEp-2 cells but the subsequent synthesis of infected cell protein prevents the process from killing the cells. Thus, viruses unable to produce appropriate prevention factors are apoptotic. We now report that the addition of either a pancaspase inhibitor or caspase-9-specific inhibitor prevented cells infected with an apoptotic HSV-1 virus from undergoing cell death. This result indicated that HSV-1-dependent apoptosis proceeds through the mitochondrial apoptotic pathway. However, the pancaspase inhibitor did not prevent the release of cytochrome c from mitochondria, implying that caspase activation is not required for this induction of cytochrome c release by HSV-1. The release of cytochrome c was first detected at 9 hpi while caspase-9, caspase-3 and PARP processing were detected at 12 hpi. Finally, Bax accumulated at mitochondria during apoptotic, but not wild type HSV-1 infection. Together, these findings indicate that HSV-1 blocks apoptosis by precluding mitochondrial cytochrome c release in a caspase-independent manner and suggest Bax as a target in infected human epithelial cells.     

Biogenesis of outer membrane vesicles in Serratia marcescens is thermoregulated and can be induced by activation of the Rcs phosphorelay system

Outer membrane vesicles (OMVs) have been identified in a wide range of bacteria, yet little is known of their biogenesis. It has been proposed that OMVs can act as long-range toxin delivery vectors and as a novel stress response. We have found that the formation of OMVs in the gram-negative opportunistic pathogen Serratia marcescens is thermoregulated, with a significant amount of OMVs produced at 22 or 30°C and negligible quantities formed at 37°C under laboratory conditions. Inactivation of the synthesis of the enterobacterial common antigen (ECA) resulted in a hypervesiculation phenotype, supporting the hypothesis that OMVs are produced in response to stress. We demonstrate that the phenotype can be reversed to wild-type (WT) levels upon the loss of the Rcs phosphorelay response regulator RcsB, but not RcsA, suggesting a role for the Rcs phosphorelay in the production of OMVs. MS fingerprinting of the OMVs provided evidence of cargo selection within wild-type cells, suggesting a possible role for Serratia OMVs in toxin delivery. In addition, OMV-associated cargo proved toxic upon injection into the haemocoel of Galleria mellonella larvae. These experiments demonstrate that OMVs are the result of a regulated process in Serratia and suggest that OMVs could play a role in virulence.     

IL-10 Prevents apoptosis of brain endothelium during bacteremia

IL-10-deficient mice infected with the relapsing fever bacterium Borrelia turicatae rapidly succumb to a brain hemorrhage if they are unable to clear peak bacteremia. In this study, we investigated the protective role of IL-10 during relapsing-remitting bacteremia and explored the molecular events involved in the protection of brain endothelium by IL-10. Brain endothelial injury was measured with cytotoxicity and diverse apoptotic assays, whereas the signaling pathway analysis was done by quantitative PCR array. The results showed that severe endothelial cell injury leading to hemorrhage in the brain and other organs occurred in IL-10-deficient mice during relapsing-remitting infection. Human brain microvascular endothelial cells (HBMEC) produced abundant proinflammatory mediators upon exposure to whole bacteria or purified bacterial lipoprotein but did not produce any detectable IL-10. Whole bacteria and purified outer membrane lipoprotein rapidly killed HBMEC by apoptosis in a time- and concentration-dependent manner. Exogenous IL-10 protected HBMEC from apoptosis. HBMEC apoptosis during exposure to a low number of bacteria was associated with downregulation of TNF and TNFAIP3 and upregulation of BAX. In contrast, HBMEC apoptosis during exposure to high concentrations of purified outer membrane lipoprotein was associated with marked upregulation of FAS, FAS ligand, and the adaptor molecules RIPK1 and CFLAR. Exogenous IL-10 reversed all the apoptotic signaling changes induced by whole bacteria or its purified lipoprotein. The results indicate that prominent brain endothelial cell apoptosis occurs during relapsing-remitting bacteremia in the absence of IL-10 and point to a prominent role for bacterial lipoprotein-mediated activation of FAS and caspase-3 in this process.     

Enteropathogenic Escherichia coli EspF is targeted to mitochondria and is required to initiate the mitochondrial death pathway

Enteropathogenic Escherichia coli (EPEC) is a causative agent of infant diarrhoea in developing countries. The EspF protein is the product of the espF gene found on the locus of enterocyte effacement, the key pathogenicity island carried by EPEC and enterohemorrhagic E. coli. EspF is injected from adherent EPEC into host cells via a type III secretion system and was previously shown to induce apoptotic cell death and to be required for disruption of host intestinal barrier function. In this work, we show by immunofluorescence and fractionation studies that EspF is targeted to host mitochondria. The N-terminal region of EspF serves as a mitochondrial import signal and, when expressed within cells, can target hybrid green fluorescent protein to mitochondria. Assessment of mitochondrial membrane potential in infected epithelial cells indicated that EspF plays a role in the mitochondrial membrane permeabilization induced by EPEC infection. Furthermore, EspF was associated with the release of cytochrome c from mitochondria into the cytoplasm and with caspase-9 and caspase-3 cleavage. These findings indicate a role for EspF in initiating the mitochondrial death pathway.     

Effect of Porphyromonas gingivalis outer membrane vesicles on gingipain-mediated detachment of cultured oral epithelial cells and immune responses

Porphyromonas gingivalis is a major etiological agent of periodontal diseases and the outer membrane vesicles (OMVs) contain virulence factors such as LPS and gingipains. In this study, we investigated the potential role of the OMVs in host immune response and tissue destruction during P. gingivalis infection. Firstly, we found that sera from periodontitis patients had significantly stronger reactivity against an OMV-producing wild type strain than the isogenic OMV-depleted strain. OMVs were found to be highly antigenic, as absorption of patient sera with OMVs greatly reduced reactivity with whole cells of P. gingivalis. LC-MS/MS analysis of OMVs revealed multiple forms of gingipains and several gingipain-related proteins. Western blots of OMVs using patient sera revealed a conserved immunoreactive antigen profile resembling the profile of OMV antigens that were recognized by gingipain antiserum, suggesting a potential role of OMV-associated gingipains in triggering antibody-mediated immune responses to P. gingivalis infection. When OMVs were added to a monolayer of an oral squamous epithelial cell line, OMVs caused cell detachment, which was inhibited by preincubating OMVs with anti-gingipain antiserum. These data suggest that gingipain-laden OMVs may contribute to tissue destruction in periodontal diseases by serving as a vehicle for the antigens and active proteases.     

Bringing down the host: enteropathogenic and enterohaemorrhagic Escherichia coli effector‐mediated subversion of host innate immune pathways

Enteric bacterial pathogens commonly use a type III secretion system (T3SS) to successfully infect intestinal epithelial cells and survive and proliferate in the host. Enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC; EHEC) colonize the human intestinal mucosa, form characteristic histological lesions on the infected epithelium and require the T3SS for full virulence. T3SS effectors injected into host cells subvert cellular pathways to execute a variety of functions within infected host cells. The EPEC and EHEC effectors that subvert innate immune pathways – specifically those involved in phagocytosis, host cell survival, apoptotic cell death and inflammatory signalling – are all required to cause disease. These processes are reviewed within, with a focus on recent work that has provided insights into the functions and host cell targets of these effectors.     

The N-Terminal Domain of EspF Induces Host Cell Apoptosis after Infection with Enterohaemorrhagic Escherichia coli O157:H7

Enterohemorrhagic Escherichia coli (EHEC) employs a type III secretion system (TTSS) to export the translocator and effector proteins required for mucosal colonization. As an important bacterial effector protein in locus of enterocyte effacement four, the EspF protein causes F-actin filament aggregations to form attaching and effacing (A/E) lesions, and induces the destruction of brush-border microvilli and cytoskeletal rearrangements to form pedestals. However, the molecular pathogenesis of A/E lesions due to EHEC O157:H7 infection is unclear. In this study, we constructed an espF-deficient mutant (ΔespF) with a 162-bp deletion in the N-terminal domain by using overlap extension PCR. The results showed that EHEC EspF translocated into intestinal epithelial cells, targeted mitochondria and induced apoptosis. The ΔespF mutant, compared to EHEC prototype Guangzhou strain, had lower cell attachment and effacement abilities, lower caspase-9/3 and lactate dehydrogenase levels, lower bacterial adhesion, weaker mitochondria apoptosis, and a higher mouse survival rate. Our results demonstrate the probable function of the EspF N-terminal domain, which targets mitochondria and binds mitochondria heat shock protein 70 to induce cell apoptosis via A/E lesions. These findings may be invaluable in clarifying the molecular pathogenesis of EspF of EHEC O157:H7.     

Latrunculin B facilitates Shiga toxin 1 transcellular transcytosis across T84 intestinal epithelial cells

Shiga toxins (Stx), released into the intestinal lumen by enterohemorrhagic Escherichia coli (EHEC), are major virulence factors responsible for gastrointestinal and systemic illnesses. These pathologies are believed to be due to the action of the toxins on endothelial cells, which express the Stx receptor, the glycosphingolipid Gb3. To reach the endothelial cells, Stx must translocate across the intestinal epithelial monolayer. This process is poorly understood. We investigated Stx1 movement across the intestinal epithelial T84 cell model and the role of actin turnover in this transcytosis. We showed that changes in the actin cytoskeleton due to latrunculin B, but not cytochalasin D or jasplakinolide, significantly facilitate toxin transcytosis across T84 monolayers. This trafficking is transcellular and completely inhibited by tannic acid, a cell impermeable plasma membrane fixative. This indicates that actin turnover could play an important role in Stx1 transcellular transcytosis across intestinal epithelium in vitro. Since EHEC attachment to epithelial cells causes an actin rearrangement, this finding may be highly relevant to Stx-induced disease.     

The early phase of programmed cell death in Caco-2 intestinal cells exposed to PTH

The regulation of apoptosis is critical for ensuring the homeostasis of an organism. As such, the cell has derived various mechanisms to precisely control the balance between survival and apoptotic signaling. Parathyroid hormone (PTH) function as a major mediator of bone remodeling and as an essential regulator of calcium homeostasis. Depending on the cell type involved, PTH also inhibits or promotes the apoptosis. In a previous work we found that PTH promotes the apoptosis of human Caco-2 intestinal cells. In the current study, we demonstrate, for the first time, that stimulation of Caco-2 cells with PTH (10(-8) M) results in the dephosphorylation and translocation of pro-apoptotic protein Bad from the cytosol to mitochondria and release of cytochrome c and Smac/Diablo. The hormone also triggers mitochondria cellular distribution to the perinuclear region, morphological features consistent with apoptosis. PTH increases the enzymatic activity of caspase-3 (48 h) that is also evidenced from the appearance of its cleaved fragments in western blot experiments. Moreover, active caspase-3 is present in nucleus after PTH treatment. In addition, a caspase-3 substrate, poly (ADP-ribose) polymerase (PARP), is degraded by 48 h of PTH treatment. Taken together, our results suggest that, in Caco-2 cells, the induction of apoptosis in response to PTH is mediated by translocation of mitochondria to the perinuclear region, dephosphorylation of Akt, dephosphorylation of Bad and its movement to the mitochondria and subsequent release of cytochrome c and Smac/Diablo which result in activation of downstream caspase-3.     

Free-soluble and outer membrane vesicle-associated VacA from Helicobacter pylori: Two forms of release, a different activity

Helicobacter pylori releases VacA both as free-soluble and as outer membrane vesicle (OMV)-associated toxin. In this study, we investigated the amount of VacA released in each of the two forms and the role of each form in VacA-induced cell vacuolation in vitro. We found that: (1) free-soluble toxin accounted for about 75% of released VacA, while the remaining 25% was OMV-associated; (2) although OMV-associated VacA caused a statistically significant vacuolation, virtually all the vacuolating activity of a H. pylori broth culture filtrate was due to free-soluble VacA. While it is widely accepted that OMVs may represent an important vehicle for delivering virulence factors to the gastric mucosa, our results suggest that OMV-associated VacA could play a pathobiological role different from that of free-soluble toxin. This conclusion fits with mounting evidence that VacA exerts a large pattern of pathobiological effects among which cell vacuolation might not be the main one.