Jpn. J. Infect. Dis., 55, 1‑5, 2002

Review

Helicobacter pylori Vacuolating Cytotoxin, VacA
Toshiya Hirayama* , Akihiro Wada,Kinnosuke Yahiro,
MiyukiKimura and Takahiro Kimura
Department ofBacteriology, Institute of Tropical Medicine, Nagasaki University
Sakamoto 1112‑4, Nagasaki 852‑8523, Japan(Received October 3 1 , 2001. Accepted January 23, 2002)

CONTENTS:
1. Introduction

2. Stmcture and biological activity

3. Receptor
4. Vacuolation and cell death

5. Conclusion

SUMMARY: Helicobacter pylori is the leading bacterial cause of food‑bone illness worldwide and plays amaJOr role in the development of chronic gastritis, peptlC ulcer, and gastric cancer･ Strains isolated from patientscontain the cagA gene (cytotoxin‑associated gene A) and produce the vacuolating cytotoxin, VacA･ Recent
molecular and cellular studies ofVacA action have begun to uⅣavel its structure and the details of the mecha‑

nism of gastric Injury Caused by H･ pylori infection･a potent urease to neutralize gastric acidity by producing

1. Introductionammonia from urea present in mu.cosal secretions (4,5)･

Helicobacter pylori is a motile, spiral‑shaped, andH. pylori is believed to be a major Cause Of chronic active

microaerophilic Gram‑negative bacterium, adapted to colonize

gastritis, peptlC ulcer, and atrophic gastritis; it is associatedand suⅣive in the stomach mucosa; it is believed to colonize

with anincreasedrisk of gastric adenocarcinoma and mucosa‑

more than 50% of the world population (1‑3). Although H･

associated lymphoid tissue (MALT) 1ymphoma･ As shown inpylorl generally survives within the gastric mucus layer, lt

Fig. 1 , H. pylori infection is responsible for human diseases,

can attach to host cells. H. pylori canuse at least five different
adhesins for successful attachment to gastric epithelial cells.

whenuntreated. Persistent H. pylori infection results in the

Adaptation requlreS expression of several adhesins and or

induction of an innammatory response and epithelial celland is acquired mostly ln Childhood and persists chronicallyI nfection

Acute Gastritis

Chronic Gastritis
Atrophy
暮ntestinalMetaplasia

Dysplasia

Gastric Carcinoma
Fig. 1. H. pylori is responsible for human disease. H.pylori infection induces diseases listed in the boxes years or decades
later as shown by time scale on the right‑hand side (6)･

*Corresponding author: Tel: +81‑95‑849‑783 1, Fax: +81‑951849‑
7805

damage, which is mamifested histologically as chronic superfi‑

pylori‑infected humans (151 I 7).

cial gastritis. Although most infections remain asymptomatic,
about I 0% ofH･ pylori‑infected people become ill, sufferingfrom peptlC ulcer, gastric adenocarcinoma, and/or MALT

2. Structure and biological activity

Intracellular vacuolization has been obseⅣed by electron

In 1988, Leunk et al. hst reported the presence ofa cytotoxin
with vacuolating activityin culturefiltrates ofH. pylori (1 8).

microscopy of gastric epithelial cells from patients with

In 1 992, Cover and Blaser reported the purification ofVacA

chronic gastritiSinfected with H. pylon'(7,8). H. pylori producesfrom a culture supematant (1 9) and its sequencing revealed alymphoma, years or decades later (6).

and secretes a protein toxin,血e vacuolating cytotoxin, ⅥlCA,

1 39‑kDa protein (20). As shown in Fig. 2, the 87‑kDa ma山rewhich induces cytoplasmic vacuolation in some eukaryotic

toxin is generated by proteolytic cleavage or a 140‑kDa

cells, leading eventually to cell destruction. So far, VacA is

precursor at the bacterial outer membrane (2 1).

the only protein toxin known to be produced by H. pylori.
Before purification and characterization or VacA, an

under denaturatlng COnditions, whereas the native toxin was

immunodominant proteinencoded bythe cagA gene (cytotoxin‑

an oligomeric complex of about 1 ,000 kDa, suggestlng Self‑

Purified VacA behaved as a molecule of about 90 kDa

associated gene A), which is located at the border ofa path0‑

association. Two different VacA genotypes, ml and m2, havegenicityisland ofH. pylori, was mistaken for VacA.

been found in clinical isolates ofH. pylori (22,23). The m2

Although the function of CagA, which is translocated into

genotype is distinguished from the ml genotype VacA by thethe host cell leading to tyrosine‑phosphorylation (9‑1 I), ispresence of a unlque middle‑reglOn locus. The ml and m2

still unknown,type I strains, which produce both VacA andproteins differ in cell specificity, suggestingthatthe two VacA

CagA, are considered more pathogenic and are associated withmolecules have different receptor‑binding characteristics (24).

VacA of the ml genotype is an oligomeric toxin, composed

the more severe diseases (i.e., ulceration, adenocarcinoma)
than the type II strains lacking ⅥcA activity and the path0‑

6

genicity island (12).

proteolytically cleaved into two fragments of about 37 andAlthough a clear functional association between VacA andor 7

subunits. The monomer or VacA is sometimes

5 8 kDa a洗er secretion. The amino‑temina1 3 7‑kDafragment

the clinical outcome or any type of gastroduodenal diseaseis the enzymatically active "A portion" and the carboxy‑

carmot be drawn yet, VacA has been proven epidemiologicallytermina1 5 8‑kDa fiagment is the receptor‑binding HB portionM

to be a virulence factor associated with peptic ulcer (1 3, 14).

like o血er bacterial "AB toxin"S (25‑27). Within several hours

h addition, oral administration ofVacA causes gastric mucosal
damage in mice, suggestlng that血e action orthis toxin mayof the addition of VacA to human gastric adenocarcinoma,
AZ‑521 cells, large intracellular vacuoles with an acidic

contribute to epithelial cell injury Or PePtlC ulceration in H.

intravacuolar pH, become visible, as shown in Fig. 3. In

Signal($1/$2)

peptidemid･region
(

rrll

/m2)

Fig･ 2･ StructuralorganiZation ofVacA produced by H･ pylori (ATCC49503 strain)･ VacA ofstrainATCC49503 Consists of 1 287‑amino acids, and undergoes
cleavage of a 33‑amlnO acid amino‑terminal slgnal sequence and a 400‑amino acid carboxy‑teminal peptides sequence to yield a mature secreted toxin(821 amino acids)･ Two majortypes of signal sequences and mid‑regions were detected, designated as sl and s2, and ml and m2, respectively. The mld‑
region spans approximately 300 amino acids, and ml and m2 sequences have about 55% amillO acid identityin this region.

Fig. 3. MorphologlCal change of human gastric adenocarcinoma cell tine AZ‑521 cells caused by VacAuslng Phase‑contrast
mlCrOSCOPe. All photographs were taken at a magnification of x 400. (a) Control, (b) AZl52l cells treated with120 nM
VacAfbr24h.

2

general, to quantifythe vacuolating activityofVacA, the uptake
orneutral red into the vact101es in VacA‑treated cells wasdetemined by subtractlng the absorbance of cells incubatedwithout toxin from that of toxin‑treated cells.

Incubation of VacA at pH 2 increased its vacuolatlng

Nontransfo￠ted cells

activity, and its resistance to acid and pepsin degradation. It
was notable that acid‑activated VacA contributed to thedevelopment of duodenal lesions, independent of HI Pylori(28). In addition, VacA was activated not only under acidic
but also alkaline conditions resulting ln its enhanced binding
to target cells (29). Although the detailed mechanism is the

Don i nant･negative
dynamjnsubject of much speculation, taken together with the recent

report on the tertlary Structure OfVacA, the acid‑ and alkali‑

activation of VacA might reflect a molecular change form
hexamer to heptamer, resultinginthe exposure of some portionof the protein surface needed for receptor binding (29).

W ild･type dynaminFig. 4. Numbers ofVacA‑vacuolatlng Cells in HeLa cells transiently

transfected with dominant‑negative dynamln‑1 orwild‑type, and

3. Receptor

nontransfected HeLa cells. Results are expressed aspercent of vacuolated
cells (38).

VacA binding to speciflC high‑affinitycell surface receptors
was shown by uslng indirect immunofluorescence and now

cytometry; high‑afrlnitytoxin binding was necess.ary for cell

apoptosis (41). VacA might be at least one factor causing cell

intoxication (30,3 1). A 250‑kDa receptor proteln tyrOSine

death by apoptosis observed in H. pylori infection (42).

phosphatase (RPTP) a served as a receptor for VacA on AZ‑

521 cells; a second protein, pl40, was also commonly detected

5. Conclusion

in VacA‑susceptible cells (29,32,33). The overexpression of
RPTPβ conferred VacA sensitivity on BHKl2 1 cells transfected

H. pylori produces and secretes a potent cytotoxin, VacA,

with the RPTPB CDNA, consistent with RPTP B acting as awhich induces cytoplasmic vacuolation and cell destmction,

receptor fb∫ VacA (33). Increased binding of acid‑ or alkali‑

activated VacA to RPTPB may alter its activityand pQssibly

leading finally to gastric Injury. Our recent report suggeststhat VacA interacts with RPTPB ‑associated signal transduc‑accelarate or inhibit the dephosphorylation of tyroslne Ontion pathways, which are essential fわr toxicity. Althoughcytosolic proteins. Further study of the function of RPTPB
in target cells may well provide further information concemlng

Ogura et al. reported that Mongolian gerbils challenged by
VacA mutant strain induced severe 'gastritis (43), further

the mechanism ofVacA toxicity on host cells.

understanding of the pathological responses of wild type and4. Vacuolation and cell death
The membranes of VacA‑induced vacuoles are intermediate

RPTPB‑deficientanimal models may well provide valuable
infbmation regarding the mechanism of VacA toxicity, thatis, whether RPTPβ is essential f♭r the induction orgastriculcers by ⅥcA.

compartments between late endosomes and lysosomes; thevacuoles contain both a late endosomal marker,the small GTP‑ACKNOWLEDGMENTS

binding protein Rab7, and a lysosomal marker, the membraneproteinLgp 1 10 (34). Accordingly, VacAmight disrupt normal

We thank Dr. Joel Moss and Dr. Martha Vaughan, NHLBI,

membrane trafrlCking at or near the level of late endosomes.

National Institutes of Health, USA, for their critical review

The microlnjeCtion of VacA or the transfection ofplasmids
containingthe VacA gene into HeLa cells resulted inthe forma‑of this manuscript.

tion or intracellular vacuoles (35), providing evidence that

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