Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02233328 1998-03-27
W O 97/12910 PCT/GB96/02404
HELICOBACTER PYLORI PROTEIN
..
The present invention relates to a novel antigen derived
from the coccoid form of H. pylori, its general use in
medicine, its use in the preparation of vaccines, as well
as its use in the detection of the coccoid form and the
diagnosis of H. pylori infection, as well as
determination of the disease prognosis of a subject.
~. py7ori is a Gram negative bact~ria that ha~ been
strongly implicated in chronic active gastritis and
peptic ulcer disease (Marshall et al, Medical ~ournal of
Australia, 142 :439-444 (1985); Buck, G.E., Journal of
clinical Microbiology, 3:1-12 (1990)). In in vitro
culture, H. pylori exists in two distinct morphological
forms, the culturable spiral form and the non-culturable
coccoid form (Marshall et al, Microbios letters, 25: 83-88
(1984); Kung, J.S.L., and HO, B., Workshop on
Gastroduodenal Pathology and Campylobacter pylori
(abstract P9), edited by F. Megraud and H. Lamouliatte,
Bordeaux, France (1988)). The spiral form of the
bacterium does not survive beyond about 2 hrs when
exposed to air. Under unfavourable conditions, the spiral
form undergoes differentiation into the coccoid form
(Vijayakumari and Ho, Acta Gastro-enterologica Belgica,
56:101 (1993)).
To date, there has been only a single report of the
successful in vitro transformation of coccoids to
spirals, which experiment has proved unrepeatable (Mai et
al, Gastroduodenal Pathology and Campylobacter pylori,
pp28-33, edited by F. Megraud and H. Lamouliatte,
Elsevier Science Publishers (1989). Although the
formation o~ coccoids in vi tro could be induced by
CA 02233328 l998-03-27
W O 97/12910 PCT/GB96/02404
antibiotics or by deprivation of nutrients (Nilius et al,
Zbl. Bakt. 280: 259-272 (1993), studies of the coccoid
form have been hampered due to the lack of inrormation
regarding this form, and its role in the life cycle of H.
pylori, as well as the lack of any method for obtaining
a synchronous culture of this form. Up to now the coccoid
form of H.pylori has been regarded effectively as a
"dead", non-viable form whose role in the life-cycle of
the bacterium is unclear.
The results discussed herein indicate that the coccoid
form can indeed exist in a viable form and does have a
role in the life cycle of H. pylori, and that this role
has implications in the diagnosis, prognosis and
treatment of H. pylori infections.
At present, various antigens of the H. pylori spiral form
have been identified (see, for example, WO 93/22682) and
are used, for example, in diagnostic kits for the
detection of H. pylori, e.g. the ~TT.~T-~ test marketed by
CORTECS LTD. However, the results described herein
indicate that it is essential to be able to detect _he
coccoid form o~ the bacterium to ensure accurate and
complete (i.e. coccoid only infection) diagnosis. At
present, no specific antigens for the coccoid form have
been identified.
Thus, ln a first aspect, the present invention provides
an antigenic protein having a molecular weight of 60 kDa,
as determined by native PAGE, obtainable from the coccoid
form of H. pylori. In particular, the protein can
furhter be characterised in that it has the following N-
terminal amino acid sequence:
D-T-H-K-S-E-I-A-H-R-F-N-D-L-G.
_
CA 02233328 1998-03-27
W O 97/12910 PCT/GB96/02404
Given the antigenicity of this protein, and its unique
presence in the coccoid form, it is useful as a means of
detecting the presence of the coccoid form of H. pylori.
r
Therefore, in a second aspect, the invention provides the
use o~ the antigen of the invention in the detection of
antibodies against H. pylori, and specifically detecting
the coccoid form In addition, the novel antigen o~ the
invention can be used in combination with other antigens,
particLIlarly thQse obtainable frcm the spiral form of
H.pylori to provide more sensitive methods of detecting
H . pyl ori .
In addition, the coccoid antigen of the invention can be
used to raise antibodies, which antibodies can then be
used to detect the antigen, including the antigen when
present as part of the intact coccoid. For example,
therefore, the antibodies could be labelled and used as
a means of detecting coccoids in tissue samples and the
like. Methods of raising antibodies using the antigen
are well known to the skilled man, as are means of
labelling such antibodies for use in such methods. Thus,
a third aspect the present invention provides the use of
the coccoid antigen of the invention in the preparation
of antibodies, e..g polyclonal antibodies. In a further
aspect, the invention provides the use of such antibodies
in the detection of the coccoid form of H. pylori which
comprises antibodies raised against the coccoid antigen
of the invention.
The antigen of the invention also finds use as part of an
antigen composition, which may contain antigens against
~oth the spiral and coccoid forms of H. pylori. In a
sixth aspect, thererore, the invention provides a
CA 02233328 1998-03-27
W O 97/12910 PCT/GB96/02404
. .
composition comprising the antigenic protein of the
invention, optionally together with one or more other H.
pyl ori antigens, these one or more other antigens being
obtainable from either the spiral or coccoid form of H.
pyl ori .
In a seventh aspect, the invention provides a method of
detecting the coccoid form of H. pylori, e.g. by
detecting antibodies, which includes the step of
contacting the antigen of the in~en~ion, or the antigen
composition o~ the in~ention, with a sample. Usually the
sample will be a biological sample, eg a blood sample, a
urine sample or a saliva sample. The antigen or antigen
composition can be brought into direct contact with the
biological sample. Alternatively, the biological sample
can first be treated to render it more suitable, eg by
filtration, pH adjustment etc. Examples of suitable
methods are those described in UK patent application no.
9422991.1.
In an eighth aspect the present invention provides a
method ~or the diagnosis of H.pylori infection which
includes the step of contacting the antigenic protein of
the invention with a biological sample obtained from a
subject. The antigen can be provided in the form of an
antigenic composition as described herein. In general,
the diagnostic method of the invention will also include
rhe step of detection o~ other an~igens obtainable ~rom
either the spiral or coccoid form of H. pyl ori . In this
way a more sensitive method of diagnosis for H. pyl ori
infection is provided. Suitably, the diagnostic method of
the invention is carried out on a sample of blood, a
sampie of urine or a sample o~ saliva.
-
CA 02233328 1998-03-27
PCT/GB96/02404
W O 97/12910
Suitably, the diagnostic method of the invention will be
be carried using a test device or test kit, e.g. that
used in the '~ T~T-~ test. In a further aspect, therefore,
the present invention provides a kit for use in the
diagnosis o~ H. pylori infection which comprises the
antigenic protein o~ the invention. Generally, the kit of
the invention will also include one or more other
antigens obtainable from either the spiral or coccoid
form of H.pylori.
The identification of the unique antigen of the invention
also opens up the possibility of providing a vaccine
against H. pyl ori which will be active against both the
spiral and coccoid forms of the bacterium. In a tenth
aspect, therefore, ~he present invention provides a
vaccine for the prophylaxis or treatment of H.pylori
infections which comprises the antigen of the invention
together with one or more adjuvants and/or carriers. In
a prererred embodiment of this aspect the vaccine
includes one or more antigens derived from the spiral
form of H. pyl ori . Suitably, these additional antigens
will include at least one which is unique to the spiral
form.
In an alternative embodiment, the vaccine can comprise
the coccoid form (either killed or "live'l) of H. pylori
itself since the cells could be taken up in the GI tract
and then induce an immune response. Furthermore, forms
o~ H. pyl ori which exist between the true coccoid and
true spiral forms could be used on the basis that they
are expressing the novel antigen. In other words, the
coccoid, or intermediate, form(s) of H. pylori are used
~ as a vehicle for delivery of the novel antigen to achieve
an immune response.
CA 02233328 1998-03-27
W O 97/12910 PCT/GB96/02404
. .
In addition, it has been determined that the novel
antigen o~ the invention can be used to detect IgM
antibodies produced in children in response to H. pyl ori
infection. In an eleventh aspect, therefore, the present
invention provides a method of detecting IgM antibodies
against the coccoid form of H. pyl orl in children, which
comprises the step of bringing the antigen of the
invention into contact with a biological sample obtained
from a child. Suitably, the biological sample will be a
blood sample, a urine sample or a saliva sample.
As described herein, there has now been devised a method
for culturing the coccoid form of H. pyl ori so that it is
viable. Thus, in a final_aspect, the present invention
provides a method of culturing the coccoid form of H.
pyl ori which comprises the step oE regularly adding
carbon dioxide to a culture medium comprising the spiral
form of H. pyl ori such that conversion to the coccoid
form occurs and wherein the coccoid form obtained is
viable. Suitably, CO2 is added at least twice a day and
the culture is allowed to run for nine weeks to ensure
conversion.
The invention will now be described wi.th reference to the
following examples, which should not be construed as
limiting the invention. The examples refer to the figures
in which:
FIGURE 1: shows a typical growth cur~e ~or
3 0 H. pyl ori grown in a chemostat with concurrent
pH and urease measurements;
FIGURE 2: shows wet preparations of (a)
spirals and (b) coccoids seen under phase
,
CA 02233328 1998-03-27
W O 97/12910 PCT/GB96/02404
.
contrast microscope, magnification xlO00.
Spiral cells were uniformly dense while
coccoids were of two types: (A) compact with
dense cytoplasm and (B) those with loose
cy~oplasm like "ghost" cells;
FIGU~E 3: shows a transmission electron
micrograph of a coccoid, magnification x
80,000;
FIGURE 4: shows a transmission electron
micrograph of a coccoid with flagella,
magnification x 80,000;
FIGURE 5: shows a silver stained SDS-PAGE
protein profile. Lanes: 1 high molecular weight
marker (Sigma): 2 low molecular weight marker
(Sigma): 3 spiral NCTC 11637: 4 coccoid NCTC
11637: 5 spiral V2: 6 coccoid V2;
FIGURE 6: shows modified periodic acid Schiff
stained smears of (a) spirals and (b) coccoids,
magnification x 1000;
FIGURE 7: shows modified gram staining of
coccoids, wherein in (a) they have been treated
with the salivary enzyme ~ amylase and in (b)
they have not;
FIGURE 8: shows DNA of H.pylori. Lanes: 1
HindIII cut A DNA: 2 spiral NCTC 11637: 3
coccoid NCTC 11637: 4 spiral V2: 5 coccoid V.;
FIGURE 9: shows the results of modified
CA 02233328 l998-03-27
W O 97/12910 PCT/GB96/02404
Albert's stain, magni:~ication x 1000;
FIGURE 10: shows detection o:E urease enzyme
acti~ity on PAGE. Lanes: 1 high molecular
weight marker (Pharmacia): 2 spiral NCTC 11637:
3 coccoid NCTC 11637: 4 spiral Vz: 5 coccoid V2 i
FIGTJRE 11: shows silver stained native PAGE
protein profile. Lanes: 1 high molecular weight
marker (Pharmacia): 2 spiral NCTC 11637: 3
coccoid NCTC 11637: 4 spiral V2: 5 coccoid Vai
FIGURE 12: shows a western immunoblot under
non-denaturing conditions with the coccoid
antigen. Lanes: 1 molecular weight marker
(Pharmacia): A preimmune anti-spiral serum: B
anti-spiral serum: C preimmune anti-coccoid
serum: D anti-coccoid serum;
FIGlJRE 13: shows indirect fluorescent
antibody test of coccoids, magnification xlOOO,
wherein it can be seen ~chat, like the spirals,
the coccoids fluoresce under ultra violet
light, indicating tha~ their surface antigens
are similar.
Ea~MPLE 1
(a) Bacterial train and preparation of the
d~ferentiated forms of N. pylo~i .
A local H. pylori strain V isolated ~rom a pa~ient with
non-ulcer dyspepsia was used. This strain was initially
grown on chocolate blood agar (CBA) to check for purity.
CA 02233328 1998-03-27
W O 97/12910 PCT/GB96/02404
-
The plate culture was then used as inoculum for a 250ml
Schott flat-bottomed round bottle containing 3Oml BHIH
(brain neart in~usion supplemented with 10~ horse serum
and 0.4~ yeast extract), and incubated at 37~C for 72h.
This ln turn serves as the inoculum for chemostat or
batch cultures.
A 1.5L fermenter containing 540ml BHIH was set up as
described in Ho and Vijayakumari (Microbios, 76:59-66
(1993). The medium was inoculated with 2x30ml of 3 day
old H.pylori culture, giving a ratio of 1:10
(inoculum:medium). Hence, carbon dioxide was supplied
twice daily, for five seconds each time, and dissolved
oxygen was maintained by a feedback control of the
impeller speed of around 35-40 rpm. Samples were
withdrawn at time intervals and checked for urease
activity, pH, viability, and microscopic ~X~m; n~tion for
morphological changes.
The culture was maintained under these conditions for up
to 3 months during which daily monitoring of the cells
was continued. When homogenous/synchronous culture was
oDserved, the cells were harvested by centrifugation at
lO,OOOg for 40min. and washed once. The pellet was then
used to prepare coccoid antigen using the modified
glycine method (Ho, B., and Jiang, B., European Journal
of Gastroenterology and Hepatology, 7:121-124 (1995).
Alternatively, a lL Schott round-bottomed bottle or lL
Erlenmeyer flask with a side-arm and fitted with a tight
-itting rubber bung, containing 270ml BHIH was used. A
7mm diameter hole was bored so as to accomodate the
--~ting of a disposable filter unit containing a 0.22~m
filter having a diame~er of 50mm (e.g. Gelman). Each
CA 02233328 1998-03-27
W O 97/12910 PCT/GB96/02404
270ml of BHIH was inoculated with 3Oml of 3 day old
H.pylori culture. Carbon dioxide was supplied twice daily
via the 0.22~m filter.
The cul~ure was incubated in a 37~C shaker incubator (New
Brunswick) maintained at 90rpm for up to nine weeks and
the cells were subsequently harvested by centrifugation
at lO,OOOg for 40min. The cell pellet was washed once and
antigen prepared as described above.
A 48 hr old culture on chocolate blood agar was used to
provide ~'synchronous" spirals.
( i i ) Ser7~m specimens .
Sera from 50 patients with gastroduodenal disease and 50
blood donors were kindly provided by A/Prof K M Fock of
Toa Payoh Hospital and Dr D Kuperan of National
University Hospital, Singapore, respectively.
(iii) ~r T-~ and Western ; mm-7n~ h 7 ott~.ng.
Indirecr ELISA and Western immunoblotting were performed
according to the method of Khia and Ho, Biomed. Letts.
50: 71-78 (1994).
RESULTS
Figure 1 shows viability, pH and urease specific acti~ity
for a typical culture. From this it can be seen that at
9 weeks the culture had become a coccoid culture. This
was confirmed by microscopic ~ mi n~tion failure of
spiral ~or growth on CBA. The time taken for the spiral
form to differentiate into the coccoid form is dependent
on the cons~ant supply of Carbon Dioxide.
CA 02233328 1998-03-27
W O 97/12910 PCTtGB96/02404
It is also clear that there are two forms of coccoid. One
has a aense cytoplasm while the other has a "ghost"-like
appearance. This latter form is considered to be non-
viable. In contrast to other reports (Nilius et al,
5 infra), the chemostat culture showed mostly dense
coccoids. These coccoids were also harvested and
suspended in BHIH supplemented with 20~ glycerol. The
suspended coccoids were then stored at -80~C until
needed.
ELISA.
By ELISA, 37 out of 50 patients (74%) were sero-positive
for H. pylori IgG antibodies to both the coccoid and
spiral antigens (Table 1). Of the 50 blood donors, 16
(32~) and 14 (28~) were found to be sero-reactive to IgG
antibodies to coccoid and spiral antigens, respectively
(Table l). Using spiral antigen as the standard for
comparison, the sensitivity and specificity of IgG ELISA
to coccoid antigen were 98 and 94~, respectively.
TABLE l. Serum IgG antibodies to H. pylori coccoid and
spiral antigens by ELISA.
Patlents Blood donors
(n=50) (n=50)
Spiral Positive Negative Positive Negative
Coccoid
Positive 36 1 14 2
Negative 1 12 - 34
Sensitivity: 98
Specif~city: 94
CA 02233328 1998-03-27
W O 97/12910 PCT/GB96/02404
.,
12
These results show that in the sample used a 4~ higher
detectlon rate was achieved when using both the coccoid
specific antigen as well as spiral antigen(s). Clearly,
therefore, better rates of diagnosis, and thus treatment
will result from the use of the coccoid specific antigen.
Western blottin~.
By Wes~ern blotting, serum IgG was shown to recognise
similar major protein bands in both the coccoid and
spiral antigens. The conser~ed protein bands in both
antigens detected were 128, 116, 110, 95, 91, 66, 60, 54,
50 and 33 kDa.
EXAMP~E 2
Bac teri al s trains .
Two strains of H. pylori were used, the local H. pylori
strain V2 referred to in example l above isolated from a
patient with non-ulcer dyspepsia and the standard strain
NCTC 11637.
Pre~aration of cult~res.
Coccoid and spiral sultures were prepared as described in
example 1. For coccoids, a batch culture was grown as
described earlier (Ho and Vijayakumari, infra) . A small
aliquot was aseptically removed at time intervals to
assess culturability on chocolate blood agar, and the
viable count enumerated using the Miles and Misra
technique (Miles and Misra, ~ournal of Hygiene, 38:732-
738 (1938). The percentage of coccoids was estimated by
counting in ~riplica~e the number of spirals to coccoids
using a Neu~auer bacterial cell counting chamber under a
phase con~rast microscope. Urease specific activity was
measured using the phenol spectrophotometric method of
CA 02233328 1998-03-27
W 097/12910 PCT/GB96/02404
Hamilton-Miller and Gergan, infra, while pH o~ the
culture medium was monitored.
Lig~ t and el ec tron mi croscopi c ~Y~min~tion of cul tu~es .
Cultures were observed for motility and morphology using
phase contrast microscopy. Air dried smears of cultures
were stained using the modified Gram stain in which
counter staining with dilute car~ol fuschin was carried
out for 10 to 30 minutes instead of the usual 1 minute.
In the Periodic acid Schiff staining for polysaccharide,
the method of Hotchkiss (Archives in biochemistry,
16:131-141 (1948)) was modified with a longer st~;n;ng
time of 90 minutes compared with the normal 15-45 minutes
in ~uschin sulphite and a prolonged counter st~-ning for
45 minutes with malachite green.
Laybourn's modification of Albert's stain for volutin
granules (Cruickshank, Medical Microbiology:A guide to
laboratory diagnosis and control of infection, pp656-657
(1968) ) was further modified for the coccoids with a
longer staining time of 45 mins instead of the usual 3-5
mins used for the spirals.
Cells for transmission electron microscopy were fixed in
4~ glutaraldehyde, dried on copper grids and negatively
stained with 0.5% phosphotungstic acid (pH 6.8). Gridg
were viewed using Philips JOEL-JEM-1200EX transmigsion
electron microscope.
Biochemical assay~.
The presence of 19 different enzymatic reactions and the
biotype were determined using the commercial strip API
ZYM kit 2S20 (Kung et al, Journal of Medical
CA 02233328 1998-03-27
W O 97/12910 PCT/GB96/02~04
, .
14
Microbiology, 29:203-206 (1989)). Urease specific
activity was determined quantitatively using the phenol
spectrophotometric method of Hamilton-Miller and Gergan
(Investigative Urology, 15:327-328 (1979)) while protein
S content was assayed by the modified Lowry assay
(Schacterle & Pollack, Analytical Biochemistry, 51: 654-
655 (1973)). ATP was quantitated using the
bioluminescence assay kit (Bio-Orbit, Finland) and the
polysaccharide content measured by the L-cysteine
sulphuric acid assay as described by Chaplin and Kennedy
(Carbohydrate Analysis:a practical approach, ppl-2.
Edited by M.F. Chaplin and J.F. Kennedy, Oxford:IRL press
(1986)).
DNA extraction and microassay.
The DNA of both forms were extracted according to the
procedure of Clayton et al (Infection and Tmm~n7ty,
57:623-529 (1988)) and electrophoresed on a 1~ agarose
gel. Total DNA content per cell was assayed according to
the method by Kapuscinski and Skoczylas (Analytical
Biochemistry, 83:252-257 (1977)).
Protein profile and Western imm~oh70tting~
Protein profiles were elucidated by polyacrylamide gel
electrophoresis (PAGE) according to the method of Laemmli
(Nature, 227: 680-685 (1970)). In the non-denaturing
native PAGE, 30 ~g total procein of whole cell
preparations were electrophoresed on a 6~ separating gel
and 5~ stacking gel. In the sodium dodecyl sulphate
(SDS) denaturing PAGE, the same amount of protein was
electrophoresed on a 10~ separating gel and 5~ stacking
gel. Relative molecular weight was determined with
-eference proteins run under the respective
electrophore~ic conditions. Both types o~ gels were
CA 02233328 1998-03-27
W O 97/12910 PCT/GB96/02404
. .
visualised by silver staining according to the procedure
of Sambrock et al (Molecular Cloning:A Laboratory MAnl~7,
2nd edn. Cold Spring Harbour, New York:Cold Spring
Harbour Laboratory (1989)). In addition, the protein
bands oi~ both the native and SDS PAGE were electro-
transblotted onto Immobilon P (Millipore) membrane using
a modi:Eication of the method of Towbin et al (PNAS USA
76: 4350-4354 ~1979)).
Antibodies raised in rabbits against either the spiral or
the coccoid were used as probes to identify the specific
and immunogenic proteins in both forms.
~aemagg7utination and h~m~gglutination inh;h;tion assay.
A slight modification of the microtitration plate assay
of Morgan et al (~ournal of Clinical Microbiology 29:
395-397 (1991)) was carried out with 20 ~l of 2~ v/v red
blood cells (human or rabbit) which were added to 25 ~11
o~ bacterial culture containing a range of l07-10l2
cells/ml in individual microtitration wells. Each
mixture was incubated in quadruplicates at 4~C overnight
before the haemagglutination patterns were read. The
haemagglutination inhibition assay was performed with
bacteria pretreated with 1 mg ml-l protease (pronase E,
Sigma) at 37~C for 60 minutes or heated at 60~C for 10
minutes. Similarly, the red blood cells were pretreated
with 4.0 ~g ml~1 Neuraminidase (Sigma) or 1 mg ml~1
protease at 37~C for 60 minutes before haemagglutination
assay.
Indirect fluorescent antibody test.
Smears of coccoids and spirals were dried on glass slides
(HTC, Wellcome) before being treated with human sera
positive for IgG antibodies against H. pylori. After 30
CA 02233328 1998-03-27
W O 97tl2910 PCT/G B96/02404
~ .
16
minutes' incubation, the smears were washed three times
for ten minutes each with phosphate buffered saline (PBS
- pH 7.6). Smears were then treated with 1:40 dilution o~
goat anti-human IgG conjugated FITC (Wellcome) for
another 30 minutes. Smears were washed four times for
fifteen minutes each with PBS, mounted with buffered
glycerol and viewed under ultra violet light using the
Reichert-Jung fluorescence microscope.
RESULTS AN~ DTSC~SS~C~
nSynchro~ous " cul ture of coccoids .
A "synchronous" culture of coccoids has been
successfully prepared in a chemostat environment. Figure
1 shows a typical growth curve for H. pylori in a
chemostat. Growth in the first two weeks was similar to
that described by Ho and Vijayakumari (infra). The late
stationary phase showed a gradual decrease in viable
counts to 105 CFU/ml within the next two weeks.
Subsequently, the declining death phase continued
linearly for the following five week.s. Throughout the
approximateiy nine week culturing peri.od, the percentage
of coccoids was shown to be inversely proportional to
spirals.
The pH of culture medium decreased from neutral to 6.58
in the first 3 days and remained at 6.53 +/- 0.13 in the
stationary phase ~or the nex~ four weeks. It then
increased to a maximum of 6.98 on the 7th week, after
which the pH was stable at 6.84 +/- 0.02 in the following
two weeks. Catrenich & Makin (Sc~n~in~vian ~ournal of
Gastroenterology, 6 (suppl. 181): 58-64 (1991)) reported
a similar inversely proportional relationship between pH
anG viable counts and postulated that the loss of
CA 02233328 1998-03-27
W O 97/12910 PCT/GB96/02404
-
17
viability and conversion to coccoids was due to basic pH
endogenously produced by deAmin~se activity. However, in
our study, as the pH of the culture medium rem~'n~d
within the tolerable range for the growth o~ H. pylori
(pH 6.5-7.5) as reported by Marshall et al (infra) and
Dick (Ann. Rev. Microbiology, 44: 249-269 (1990)), the
conversion o~ spirals to coccoids was most probably due
to nutrient depletion or metabolite inhibition rather
than pH changes.
The average urease specific activity (USA) as 4 . 90 +/-
4.42 U mg~1 protein in the exponential phase and increased
to 11.58 +/- 10.03 U mg~1 protein in the stationary phase.
During this period, a trimodal peak was observed at the
18th, 156th and a highest peak at the 333rd hour
respectively. The USA then decreased linearly with the
declining death phase. At the end of the culture period,
USA was 0.18+/- 0.03 U mg~1 protein. This indicates that
urease activity is valuable for the actively reproducing
spirals and decreases with the formation of coccoids.
Marshall et al (Gastroenterology, 99:697-702 (1990))
reported that the primary function of urease activity of
H. pylori in vi tro is to protect the bacterium against
acidity. As such, the increase in USA in the stationary
phase may be an adaptive response of the spirals to the
increasing acid pH due to metabolism. Concomitantly, as
the pH increases in the declining phase, the USA is
decreased.
3 0 Microscopy.
Under phase contrast microscopy, the spirals appeared as
curved or S-shaped rods with uniform contrast in
cytoplasmic density (fig 2a). The coccoids on the other
hand were circular and consisted of two types: one type
CA 02233328 1998-03-27
W O 97/12910 PCT/GB96/02404
18
was shown to be compact with dense cytoplasm, while the
other type was with loose cytoplasm and had the
appearance of "ghost" cells (Fig 2b). It was not
possible to separate the two types of coccoids using
sucrose density gradient centrifugatlon. In transmission
electron microscopy, our observations were similar to
earlier reports by Marshall et al (1984 infra) where the
spirals were shown to be curved rods with cell ~-~en~ions
of 0.3-0.5 ~ x 1.0-3.0 ~ and possessing tufts of 1-6
flagella ending in bulbous tips. In contrast, the
coccoids were circular with diameters ranging from 200-
300 nm and had intact cell membranes (Fig 3).
Unlike the spirals, which had characteristic darting
motility, the coccoids were non-motile when observed
under the phase contrast microscope. Transmission
electron microscopy, on the other hand, showed the
presence of flagella in some coccoids (Fig 4). This
could either mean that the flagella is a remnant of the
spirals (Marshall et al, 1984 infra) or that the coccoids
actually possess ~lagella but they are inactive due to
the dormant s~ate or the lack of energy to drive it.
Suerbaum et al ~ Journal of Bacteriology, 175:3278-3288
(1993) having cloned the flagellin genes, observed that
mutation in the major flagellin gene resulted in non-
motile and a flagellated H. pylori while mutation in the
minor flagellin gene produced normally flagellated and
motile bacterium. In this study, the denaturing SDS PAGE
protein profile showed that the 58 kDa minor flagellin
protein is present in equal intensities in the spirals
and coccoids, but the major 52 kDa flageilin protein is
reduced in intensity in the coccoids as compared to the
spirals (Fig 5).
-
CA 02233328 1998-03-27
W O 97/12910 PCT/GB96/02404
-
19
Since the minor flagellin is located proximal to the
flagella hook, a structure re~uired for flagella
attachment (Kostrzynska et al, Journal o~ Bacteriology,
173:937-946 (l991)), the findings could explain the
intact flagella on the coccoids but absence of motility.
On modified Gram staining, the spirals appeared Gram
negative with counter staining for 10 minutes while the
coccoids remained weakly Gram negative even after counter
staining for 30 minutes. In the modified Periodic Acid
Schiff staining, spirals stained green (Fig 6a) but the
coccoids were bright red, indicating the presence of
polysaccharides on their cell wall (Fig 6b). This
reaction was sustained by the fuzzy halo of approximately
50-60 nm radius around the coccoids as observed on
transmission electron microscopy (Fig 3). This
polysaccharide layer which contributes > 50~ of the
coccoid cell component could account for the poor
staining of the coccoids in the modified Gram st~in;ng
reaction. Consistently, treatment of the coccoids with
salivary enzyme ~ amylase before staining to digest the
polysaccharide content of the coccoids greatly improved
the Gram reaction (Fig 7). Moreover, the polysaccharide
content of the coccoids was 10 x more than the spirals
(Table 2~. It has been shown that the presence of a
polysaccharide coat could help the survival of bacteria
under adverse environmental conditions. The hygroscopic
layer could act as a cellular buffer controlling gaseous
~ch~nge and preventing excessive absorption or loss of
fluid that could lead to cell lysis and death (Wilkinson,
Bacteriological Review, 22:46-73 (1958)).
CA 02233328 1998-03-27
W O 97/12910 PCT/GB96/0240
TA~E 2: Constltuents of Spirals and Coccoids
Constituent NCTC 11637 V2
(~g/cell)
SPIRAL COCCOID SPIRAL COCCOID
PROTEIN 7.84x 3.92x 7.86x 4.62x
10-3 10-3 1~-9 10-g
DNA 6.21x 3.12x 7.25x 3.79x
lo-lQ 10-1~ lo-lC 10-~
POLY- 6.38x 5.01x 2.95x 2.15x
SACCHARIDE lo-ll 10-lo -ll lo-lo
ATP* 1.02x 1.27x 6.19x 8.40x
10-~6 1o-18 1o-16 1o-18
*Units in ~mol/cell
Thus, the coccoids could possibly survive outside the
human body with protection offered by the thick
polysaccharide layer from atmospheric oxygen tension as
well as the unfavourable environment. Similar
observations were rendered for Campylobacter jejuni by
Rollins and Colwell (Applied and En~ironmental
Microbiology, 52:531-538 (1985)) where an increase in
viscosity of the culture suspension was noted as it
underwent transition from the spirals to coccoids. They
suggested that the production of an extracelluar viscous
polysaccharide as an adaption to ensure extended survival
for C . j ej uni .
In H. pylori cultures, this increase in viscosity was not
observed bu~ the granular spiral culture tended to
flocculate. rJnder phase contras~ microscopy, the
CA 02233328 1998-03-27
W O 97/12910 PCT/GB96/02404
.
21
"synchronous" coccoids were seen in large clumps. The
polysaccharide iayer could function to hold the coccoids
together resulting in the floccules.
DNA content.
Intact chromosomal DNA was extractable ~rom coccoids
indicating that these ~orms are probably viable (Fig 8).
total DNA content of spirals was approximately 5.22 x 10-7
. ng/cell, while that oi~ coccoids was 3.13 x 10-7 ng/cell
(Table 2). The decreased DNA in the coccoids as compared
to the spirals could be accounted for by the population
of coccoids having loose and leaky cytoplasm as observed
under phase contrast microscopy (Fig 2b). On the other
hand, possession of only half the total amount o~ DNA in
lS the coccoids as compared to the spirals, could also
indicate the dormancy or survival strategy of this
differentiated ~orm.
Novitsky and Morita (Applied and Environmental
Microbiology, 33: 635-641 (1977) ) reported a 48~6
reduction in DNA content of the marine vibria ANT 300
under starvation survival. They suggested that this
could be a strategy for conservation of energy resulting
in the degradation of extraneous or partially replicated
DNA.
Biochemica7 characteristics.
The spirals were oxidase and catalase positive but the
coccoids did not show any visible reaction in these
qualitative tests (Table 3). In the API ZYM test, both
the spirals and the coccoids showed similar enzyme
profiles belonging to Biotype II as described by Kung et
al (~ournal o~ Medical Micro~iology, 29:203-206 (1989)).
Interestingly, equal amounts o~ acid and alkaline
CA 02233328 l998-03-27
W O 97/12910 PCT/GB96/02404
.
22
phosphatase activities were observed in the coccoids and
spirals (Table 3). The presence of these enzymes in the
dormant coccoids could function in the transport of
inorganic phosphates and generation of energy source in
the form of ATP. The coccoids contained ATP but 100 x
less than in the spirals (Table 2). This signifies that
the coccoids are a viable but dormant form. Similarly,
a 99% reduction in endogenous respiration was exhibited
by the marine vibrio ANT 300 as part of its survival
strategy under long term nutrient star~ation (Novitsky &
Morita, Applied and Environmental Mi~:~robiology, 32: 617-
622 (1976)).
CA 02233328 1998-03-27
W O 97/12910 PCTIGB96/02404
TABLE 3: Enzyme profile of spirals and coccoids
ENZYME SPIRALS COCCOIDS
Oxidase +
Catalase +
Urease + W
3.61U/mg protein 0.18U/mg protein
Acid + (240nM) + (240nM)
phosphatase
Alkaline + (240nM) + (240nM)
phosphatase
Napthol AS- + (20nM~ + (20nM)
Bl phospho-
hydrolase
Leucine + (240nM) + (5nM)
arylamidase
+ = positive
- = negative
W = weak positive
In addition, recent work has elucidated the formation of
intracytoplasmic polyphosphates in spirals of H. pylori
under adverse conditions (Bode et al, ~ournal of General
Microbiology, 139:3029-3033 (1993); Caselli et al, Gut,
34:1507-1509 (1993)). These structures represent a
reservoir o~ stored energy and phosphorus and may be
regarded as an alternative energy source when ATP is in
short supply. Volutin granules (polyphosphates) were
observed in the coccoids by Albert's stain (Fig 9) and
CA 02233328 1998-03-27
W O 97/12910 PCT/G B96/02404
24
could constitute a survival strategy. Furthermore, the
presence of the phosphohydrolase enzyme could play a role
in the metabolism of these polyphosphates.
The mean urease specific activity of coccoids was twenty
times less than that of the spirals having an activity o~
0.18 +/- 0.03 U mg~l protein as compared to 3.61 +/- 0.52
U mg~l protein in the spirals. The low urease actvity in
the coccoids could either be due to the preformed enzymes
left in the coccoids or that the dormant coccoids do not
require as much urease enzyme activity as the actively
reproducing spirals. This could also be the reason why
the rapid urease test on coccoids was negative even after
48 hours and that the urease activity band could not be
detected by the sensitive staining on PAGE by Shaik's
method (Shaik et al, Analytical Biochemistry, 103:140-143
(1980)) as shown in Fig 10. Nevertheless, the urease
subunits A and B of 29 and 66 kD respectively seem
conserved in the cocoids as shown on the denaturing SDS
PAGE (Fig 5). It is interesting to note that equal band
intensities of these two urease subunits were observed in
the spirals and coccoids in spite of the 20 times lower
urease activity in the coccoids.
The dominant presence of only the urease A and B subunits
in the coccoids could explain the decreased urease
activity as urease subunits C and D which are also
essential ~or urease activity in H. pylori (Labigne et
al, ~ournal of Bacteriology, 173:1920 1931 (1991)) were
absent.
Protein profile.
It was also obser~ed as shown in the SDS-PAGE (Fig 5)
that there was a decrease in the number and intensity of
CA 02233328 1998-03-27
W O 97/12910 PCT/GB96/02404
2 5
various protein bands o~ molecular weight ~ 14 ~ 200 kD
in the coccoids indicating the dormant, inactive state of
this form. This is further strengthened by the fact that
the total protein content per cell in the coccoids was
only half that found in the spirals (Table 2). ~eeve et
al (Journal of Bacteriology, 160:1041-1046 (1984))
demonstrated that protein degradation was nece~sary for
starvation survival of Escherichia coli and .~7m~e71a
typhim~rium as part of energy conservation. On the other
hand, conservation Gf the possible urease subullits A and
B as well as the presence of flagellar proteins could
mean that the urease enzyme and flagella are necessary
~or the conversion of the coccoids to spirals and
subsequent survival of the spirals in the gastric
environment.
In the native PAGE, the presence of three novel proteins
of 955, 871 and 661 kDa proteins in trace amount and a
distinct band of 60 kDa were observed (Fig 11).
Appearance of the novel protein bands would appear to
indicate that the coccoids have a purpose and do not
simply represent a degenerative form of H. pylori. In
addition, western im~llnohlotting identified the 60 kDa
protein to be specific and immunogenic for the coccoids
(Fig 12).
F~ ~gglutination and haemagglutination inh;hi tion assay.
Majority of strains of H. pylori are known to agglutinate
various red blood cells including human and rabbit
(Morgan et al, 1991 infra; Taylor et al, ~ournal of
Medical Microbiology, 37 :299-303 (1992)). In this study,
both the spirals and coccoids agglutinated human red
blood cells equally well at bacterial concentrations of
~ 10a cells. With the rabbit red blood cells, the spirals
CA 02233328 1998-03-27
W O 97/12910 PCT/GB96/02404
.
26
agglutinated at a minimllm of lOa cells but the coccoids
required 109 cells to produce visible haemagglutination.
Haemagglutination was inhibited by heat and protease
treatment of the red blood cells. This indicated that
the haemagglutinin in the coccoid, similar to that
observed by Huang et al (FEMS Microbiology Letters, 56:
109-112 (1988)) in the spirals, is a protein while the
receptor is not a protein but sialic acid. Hence, the
haemagglutinating property of the spirals is retained in
the coccoid as was also observed by Wadstrom et al
(European Journal of Gastroenterology and Hepatology,
5 (suppl.2):S12-S15 (1993~).
Indirect fluorescent anti~ody test.
The coccoids were shown to fluoresce under ultra violet
light (Fig 13) indicating that their surface proteins are
intact and similar to spirals. This property could be
utilised to detect the speci~ic presence and survival of
H. pylori in the environment and may help to elucidate
the route of transmission. Similar properties have been
employed to study the survival of viable but non-
culturable forms of .~7mnnella enteritides (Roszak et al,
C~n~ian Journal of Microbiology 30: 334-338 (1983)) and
Vibrio cholerae as well as E. coli (Xu et al, Microbiol.
Ecology 8: 313-323 (1982)) in the environment.
From the results it would appear that the coccoid form
can exist in a viable form, contrary to what was believed
previously. It has intact DNA, ATP enzyme activities,
presence of novel and conserved protei.n and the presence
of a thick polysaccharide coat to protect it under
adverse conditions in the environment.
The fastidious nature of the spirals in both nutritional
,
CA 02233328 l998-03-27
W O 97/12910 PCT/GB96/02404
27
and physiological requirements inhibits their survival in
the environment (Marshall et al, 1984 infra; Dick, 1990
infra) . The microaerophilic spirals are sensitive to
oxygen and have been reported to become non-viable if
exposed to air for more than 2 hours (Soltesz et al,
~ournal of Clinical Microbiology, 30:1453-1456 (1992)).
Thus, the coccoids, as an alternative morphological
state, might be the link in the cell cycle and possibly
in the mode of transmission of H. pylori. S;l~ r viable
1~ but non-culturable dif~erentiated forms have been
observed in the cell cycle of other microorgAniRm~ like
Myxococcus xanthus (White et al, ~ournal of Bacteriology,
95:2186-2197 (1968)), marine vibrio ANT 300 (Novitsky &
Morita, 1977 infra) and Art~robacter crystallopoietes
(Boylen ~ Ensign, Journal of Bacteriology, 103: 569-577
(1970)) and shown to play an integral role in their
survival strategy under adverse conditions.
Many reports have cited the detection of H. pylori in
water by non-culturable methods like the 3H thymidine
uptake studies (~h~h~m~t et al, Klin.Wochenschr., 67:62-
62 (1989)) and detection by polymerase chain reaction in
faeces (Mapstone et al, Lancet, 341:447 (1993)) and in
the water supply in Peru (Westblom et al, Acta
Enterolgica Belgica, 56(suppl):47 (1993)). H. pylori
prevalence studies by 3C urea breath test has also been
associated with consumption of river water (Klein et al,
Lancet, 337:1503-1508 (1991)). These indirect detection
procedures do not exclude the possibility of detecting
coccoids in the environment as this study shows the
presence of DNA in the coccoids. Further, the isolation
of H. pylori from human faeces immediately after
excretion does not exclude the possibility that the
spirals would not survive in the normal atmosphere for
CA 02233328 1998-03-27
W O 97/12910 PCT/GB96/02404
greater than ~ hours (Soltesz et al, 1992 infra) owing to
their microaerophilic nature and oxygen toxicity (Krieg
& Hoffman, Ann.Rev.Microbiol., 40:107-130 (1989)). These
earlier reports only serve to establish the fact that
there exists more than a single form of H. pylori.
