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Previous Article | Next Article 
Infection and Immunity, November 1998, p. 5555-5560, Vol. 66, No. 11
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Diversity in Protein Synthesis and Viability of
Helicobacter pylori Coccoid Forms in Response to
Various Stimuli
Hiromoto
Mizoguchi,1,*
Toshio
Fujioka,1
Kenji
Kishi,1
Akira
Nishizono,2
Reiji
Kodama,1 and
Masaru
Nasu1
Second Department of Internal
Medicine1 and
Department of Infectious
Disease Control,2 Oita Medical University,
Hasama-machi, Oita 879-5593, Japan
Received 29 April 1998/Returned for modification 17 June
1998/Accepted 13 August 1998
 |
ABSTRACT |
The viability of the coccoid forms of Helicobacter
pylori was evaluated by assessing protein synthesis. Metabolic
labeling studies showed the synthesis of proteins and the specific
protein profiles of H. pylori coccoids produced under
various conditions. Harsh conditions such as aerobiosis and starvation
(lack of horse serum) in the culture did not affect the synthesis of
proteins in the coccoids. Lowering of the pH to that of gastric
secretions induced expression of several proteins in the coccoids.
However, the coccoids produced under prolonged microaerobic conditions exhibited a profile of acid stress-induced protein expression different
from that induced by aerobiosis or starvation. Our data suggest that
coccoid H. pylori exhibits diversity in viability following
exposure to different stresses and that the response to acid stress of
coccoid H. pylori could be involved in infection of the
host stomach.
| |
TEXT |
Morphological conversion from spiral
to coccoid forms has been described for Helicobacter pylori
cultured under several suboptimal conditions. These conditions include
aerobiosis (4, 6), alkaline pH (4, 6, 15), high
temperature (22), extended incubation (6), or
treatment with a proton pump inhibitor (6) or antibiotics
(3). This coccal form conversion phenomenon, which has been
thought to result in a viable but nonculturable form of the bacterium
(3), is not exclusive to H. pylori, as it is
common for other enteric pathogens (16, 20, 21). Several investigators have suggested that the coccoidal form of H. pylori represents a degenerative form with no infectious
capability (7, 9, 17). Others have reported that the coccoid
form retains a weak metabolic activity (3, 23), important
structural components (2, 13), and pathogenicity
(26). Successful in vitro culture of coccoid forms, however,
has not as yet been established. Therefore, whether the coccoid form is
in the process of degeneration or in the dormant stage prior to
subsequent bacterial transmission remains an open question. Recently,
successful infection with coccoid forms of H. pylori or
Campylobacter jejuni in animal models has been reported
(5, 16). These findings have highlighted the possible role
of the coccoid forms in transmission of infection and morphological
conversion of coccoids to the spiral form. The acidity of gastric
secretions is considered to be harmful to most organisms in the gastric
environment. A recent study, however, suggested that acidity is
essential for the establishment of colonization of the stomach by
H. pylori, as brief exposure of H. pylori to a
low pH increases the expression of heat shock proteins (hsp's), which enhance the attachment of the organism to the gastric epithelium (14).
The present studies were performed to investigate whether coccoid forms
of H. pylori had the capacity to synthesize proteins and, if
so, whether coccoids produced by various procedures exhibited diversity
in protein synthesis levels or viability. We also examined the
potential morphological conversion of the coccoid to the spiral form
following acid exposure. Our results demonstrated protein synthesis and
induction of specific proteins by acid shock in the coccoid form. Under
unfavorable conditions, this metabolism appears to be more stable in
the coccoid form than in the spiral form.
Morphological change and culturability of coccoid forms.
H.
pylori ATCC 43504 was used in our studies. The frozen bacterial
suspension was thawed from storage at 
80°C and smeared onto 7%
horse blood-agar plates containing Mueller-Hinton agar (Becton
Dickinson, Cockeysville, Md.). Cultures were incubated under
microaerobic conditions in anaerobic jars (Campypak System; BBL
Microbiology Systems) with high humidity at 37°C for 3 days. Subsequently, a portion of the culture was harvested and resuspended in
8 ml of liquid brucella broth (Difco, Detroit, Mich.) supplemented with
10% (vol/vol) horse serum (Gibco, Grand Island, N.Y.) and incubated in
a microaerobic environment at 37°C for 2 days on a rotary shaker.
To induce coccoid forms via various environmental conditions and
subsequently compare their biological features, we employed different
culture conditions, such as aerobiosis, prolonged culture, and nutrient
starvation. Aerobiosis was performed as follows: when bacteria became
subconfluent in liquid microaerobic culture, the culture was
transferred into an aerobic atmosphere and incubated for a further 3 or
7 days with agitation (200 rpm). Prolonged H. pylori culture
was performed in liquid medium for up to 20 days at 37°C, without
supplementation with fresh medium, under microaerobic conditions, and
with the Campypak being changed every 3 days. For nutrient starvation
conditions, bacteria in exponential growth were washed three times with
phosphate-buffered saline (PBS) (pH 7.4) and then incubated in PBS at
4°C for 3 months without agitation. The culturability of coccoid
populations was determined by plating 100 µl of a 1- or 10-fold
dilution of the broth culture (adjusted to a 5 McFarland concentration)
in PBS onto blood agar plates. Plates were incubated at 37°C under
microaerobic conditions for 3 or 7 days, respectively. Coccoid
populations produced under all of the conditions assayed in this study
lacked colony-forming ability. No spiral forms were detected by light
microscopy in the randomly chosen fields of coccoid populations induced
by any condition used. In contrast, the 3-day microaerobic culture
containing >95% spiral forms exhibited 108 to
109 CFU/ml.
In an attempt to clarify whether different suboptimal culture
conditions induced morphologically different coccoid forms, we compared
coccoid forms induced by aerobic culture and prolonged culture with
respect to ultrastructural morphology. The morphology of H. pylori incubated under various conditions was determined by
scanning electron microscopy. For this purpose, samples of each culture
were placed on a 0.4-µm-pore-size polycarbonate filter (Isopore
Track-Etched Membrane Filter; Millipore, Bedford, Mass.) and fixed in
2% glutaraldehyde in PBS for 1 h. The samples were then
dehydrated for 10 min in serial concentrations of ethyl alcohol (50, 70, 80, 90, 95, and 100%) and t-butyl alcohol and subjected to critical-point drying and gold-palladium coating. The samples were
examined by scanning electron microscopy (S 800 model electron microscope; Hitachi Co., Tokyo, Japan).
Figure 1A shows
coexistence of spiral and coccoid forms after a 5-day microaerobic
culture. After a 3-day aerobic culture (Fig. 1B), almost all organisms
were spherical and no spiral forms were observed; a few U-shaped and
doughnut-shaped forms (the latter are thought to be an intermediate
state between U-shaped and completely spherical coccoid forms)
and degraded forms were also present. After a 7-day aerobic culture
(Fig. 1C), most organisms appeared degraded although a small subset
maintained spherical forms. Prolonged culture for 20 days generated
coccoid forms exclusively (Fig. 1D). Coccoids from prolonged culture
exhibited a rougher surface than that of coccoids from a 3-day
aerobiosis; the latter resembled coccoids from a 3-day microaerobic
culture. Furthermore, most of the coccoids from prolonged culture
appeared to fuse and aggregate, probably due to cell wall degeneration.
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FIG. 1.
Morphology of coccoid forms of strain ATCC 43504 produced under various conditions, as observed by scanning electron
microscopy. (A) Coexistence of spiral (S) and coccoid forms in 5-day
microaerobic culture; (B) complete spherical coccoid (C), U-shaped (U),
doughnut-shaped (O), and degraded (E) forms produced by 3-day
aerobiosis; (C) coccoid forms and degraded forms produced by 7-day
aerobiosis; (D) coccoid forms produced by 20-day microaerobic
culture.
|
|
Protein synthesis in coccoid forms.
To determine whether the
coccoid form was viable, trans-35S metabolic
labeling of H. pylori with methionine and cysteine was performed, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Spiral or coccoid H. pylori
cells (109 bacteria) were suspended in 300 µl of serum-
and methionine-free RPMI 1640 medium and then plated in 24-well plates.
The bacteria were metabolically labeled in the medium with 300 µCi of trans-35S per ml at 37°C for 1 or 5 h under one of several conditions: in a microaerobic or
aerobic environment, with or without 2% horse serum, or in medium at
pH 2.0, 3.5, 5.0, or 7.4. 35S-labeled bacteria were washed
three times with PBS and then lysed in 100 µl of Laemmli sample
buffer containing 5% (vol/vol) 
-mercaptoethanol, 2% (wt/vol) SDS,
10% (vol/vol) glycerol, and 125 mM Tris-HCl (pH 6.8). Samples were
heated at 100°C for 5 min, and 50 µl of each sample was subjected
to SDS-PAGE on a 9% acrylamide slab gel (27). The gels were
dried and processed for fluorography using Kodak XAR film.
As shown in Fig. 2, H. pylori
coccoids subjected to a 3-day aerobic culture synthesized proteins.
This synthetic ability was maintained in coccoids subjected to a 7-day
aerobic culture, but the level of synthesized proteins in coccoids from
the 7-day aerobic culture was much reduced, apparently due to the
degeneration of coccoid forms (Fig. 1C). Coccoid forms produced <1%
of the amount of proteins synthesized by spiral forms. Furthermore, the
protein profiles of coccoid and spiral forms were apparently different. Restoring the 3-day aerobically cultured coccoid forms to adequate culture conditions for 3 days did not lead to morphological conversion of coccoids or restoration of the pattern of synthesized proteins.
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FIG. 2.
Protein synthesis in coccoid forms of H. pylori. Coccoid forms were produced by 3-day (d3) and 7-day (d7)
aerobic cultures. Each form was 35S labeled for 5 h
under microaerobic conditions. Samples of spiral forms were diluted to
1-, 10-, and 100-fold and subjected to SDS-PAGE. Samples of coccoid
forms were undiluted.
|
|
The demonstration of DNA synthesis (by incorporation of
5-bromodeoxyuridine) (3) and preservation of intact cellular
structures and certain enzymes (2, 13) in coccoid forms have
confirmed that coccoid forms are viable but in a nonculturable state.
This viability appears to be stable under optimal conditions since DNA
synthesis was observed in coccoids after 3 months of storage in
physiological saline at 4°C (3). Our data from the
metabolic labeling study support this finding (see Fig. 5B).
Stability of protein synthesis in coccoid forms.
H.
pylori requires a microaerobic atmosphere and serum for in vitro
propagation. We investigated the effect of these factors on metabolism
in spiral and coccoid forms of H. pylori. Figure 3 demonstrates that no difference in the
pattern or intensity of labeled proteins in coccoids from microaerobic
and aerobic cultures was detected by SDS-PAGE. However, the amount of
protein produced from spiral forms markedly decreased under aerobic
conditions. On the other hand, the addition of horse serum for up to
5 h during labeling did not alter the profile or amount of
synthesized protein in either spiral or coccoid forms. The long-term
survival of this spherical shape, even under harsh conditions outside
the host stomach, is indicated by the lack of inhibition of protein
synthesis in coccoids under aerobic conditions (Fig. 3) and
conservation of the ability to synthesize proteins under starvation
conditions for at least 3 months (see Fig. 5B). However, coccoids
readily lose viability under high-temperature (37°C) storage
conditions while at low temperatures (<15°C) coccoids can maintain
viability for at least 3 months (11, 22). It is thus likely
that storage temperature affects the viability of coccoids.
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FIG. 3.
Effect of horse serum or aerobic conditions on protein
synthesis in H. pylori. Each form was 35S
labeled for 5 h in the presence (+) or absence () of 2% horse
serum (HS) under microaerobic conditions (lanes M) or aerobic
conditions (lanes A).
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|
Acid shock induces the expression of proteins in coccoid
forms.
The acid nature of gastric secretions is a critical barrier
in preventing infections in the stomach. To examine the response of
H. pylori to acid stress under the condition without urea, we exposed spiral forms and coccoids from 3-day aerobic culture to
low-pH-adjusted media during metabolic labeling. The exposure of spiral
forms to a pH of 5.0 enhanced the production of proteins with molecular
masses of 68, 40, and 30 kDa. In contrast, protein synthesis was
diminished when pH was lowered further to 3.5 or 2.0. In the coccoid
forms, lowering the pH to 3.5 induced expression of 90- and 68-kDa
proteins and exposure to pH 2.0 induced a 35-kDa protein (Fig.
4).
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FIG. 4.
Induction of proteins in coccoid forms of H. pylori by acid shock. Spiral and coccoid forms produced by a 3-day
aerobic culture were 35S labeled for 1 h during
exposure to acid at the indicated pH in serum- and methionine-free RPMI
1640 medium under microaerobic conditions. Proteins induced by exposure
of coccoids to pH 3.5 and 2.0 are indicated by arrowheads.
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|
In another series of experiments, we examined the stability of coccoids
from a 3-day aerobic culture under adverse conditions by storing the
coccoids for 20 days at 4°C in distilled water. Exposure of this
population of coccoids to pH 2.0 induced the same profile of proteins
as that observed in coccoids from a 3-day aerobic culture. However, the
viability of 3-day aerobically cultured coccoids was completely lost
after incubation in distilled water at room temperature (data not
shown).
It is conceivable that surface urease activity enables H. pylori to survive in the gastric environment, as a
urease-deficient mutant of H. pylori lacks the ability to
colonize the nude mouse stomach (18, 25). Since coccoid
forms of H. pylori have no urease activity (13,
19), their survival in the stomach in such a low-pH environment
is unlikely. However, our data show that acid shock, even for a period
of 1 h, did not inhibit the production of proteins but,
conversely, induced the synthesis of proteins (Fig. 4 and
5), which might be hsp's
(14). Members of the hsp family function as molecular
chaperones and protect intracellular proteins from denaturation
(8, 12) but are also likely to be relevant to cell cycle
progression (24) and rearrangement of cytoskeletal proteins
(10). The preservation of polyphosphate granules in coccoid
forms, probably as energy, supports the possibility of subsequent
transformation of coccoids into spiral forms (3). These
findings suggest that gastric acidity is an essential factor for
initiation of morphological conversion and regrowth of coccoid H. pylori. However, acid shock alone was insufficient for restoration
of either morphology or growth of coccoid forms in vitro. If acid
stress is an initiator for conversion to the spiral form, additional
stimuli and/or appropriate circumstances might be necessary for such
conversion.
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FIG. 5.
Comparison of profiles of proteins induced by acid
exposure of coccoid forms produced by a 20-day microaerobic culture (A)
and a 3-month incubation in PBS at 4°C (B). Coccoids were
35S labeled for 1 h in methionine-free RPMI 1640 medium under microaerobic conditions at pH 7.4 or 2.0.
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|
Differential profiles and amount of proteins synthesized by coccoid
forms induced under various conditions.
Many conditions are known
to induce the coccoid form; however, little is known about variations
in biological features among coccoids produced by different procedures.
To compare the characteristics of metabolism in coccoid forms produced
by various procedures, we employed three different culture conditions:
aerobiosis, prolonged culture, and long-term starvation at low
temperatures. All coccoid populations produced under these conditions
exhibited protein-synthetic ability and responsiveness to acid shock
(by expression of stress proteins) despite the absence of
colony-forming ability.
In coccoids produced by long-term starvation, the pattern of both
constitutively synthesized and acid-induced proteins (35-, 68-, and
90-kDa proteins) was similar to that in coccoids from the 3-day aerobic
culture. However, the amount of proteins synthesized (after starvation)
was much greater than in the 3-day aerobic culture or prolonged culture
(Fig. 4 and 5). In coccoids obtained from prolonged culture, only one
protein of 55 kDa was induced by exposure to pH 2 (Fig. 5A). Further
incubation of coccoids from 20 to 40 days was accompanied by a loss of
responsiveness to acid shock, although small amounts of proteins were
still produced.
Bode et al. (3) reported that the diameter of the
amoxicillin-induced coccoid is markedly smaller than that of coccoids induced by bismuth agents or erythromycin. Sequential alterations of
the activity of several enzymes in the coccoid form during extended
culture have been described recently by Hua and Ho (13). These investigators showed that the activity of some enzymes in coccoids is fully conserved for up to 35 days in culture, with levels
as high as those observed in the spiral form, while others gradually
decrease after 21 days in culture, without alteration at the DNA level.
Our results showed differences in the profiles of acid-induced proteins
between coccoid forms produced by aerobiosis and prolonged culture
(Fig. 4 and 5). This difference in biological response may be partly
related to slight differences in shape between coccoids from prolonged
culture with rough surfaces and those from aerobic culture with smooth
surfaces (Fig. 1). This minimal change in morphology in coccoids in
prolonged culture may mark the onset of degeneration. Thus, it is
likely that coccoids exhibit variation in viability. Recently,
successful colonization by a nonculturable population of mostly coccoid
H. pylori cells in a mouse model was reported (1,
5); however, Eaton and coworkers (9) could not achieve
this in a gnotobiotic piglet model. These contradictory results may
reflect the differential viability of coccoids, although strain
diversity, contamination with spiral forms, and differential host
species specificities should be considered.
Based on the present results, we speculate that the protein synthesis
response of coccoid H. pylori to acid stress plays an important role in triggering conversion from the coccoid to the spiral
form and also that diversity in the viability of coccoids correlates
with their ability to cause infection. Together with future in vivo
studies using animal models, our in vitro approach evaluating the
viability of coccoids may help distinguish "reversible" coccoids,
capable of transition to the spiral form, from "irreversible" coccoids, which progress to cell death.
| |
ACKNOWLEDGMENTS |
We thank K. Arita and M. Kimoto for technical assistance. We also
thank F. G. Issa, Department of Medicine, University of Sydney,
Sydney, Australia, for careful reading and editing of the manuscript.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Second
Department of Internal Medicine, Oita Medical University, Hasama-machi,
Oita 879-5593, Japan. Phone: 81-97-549-4411, ext. 5804. Fax:
81-97-549-4245.
Editor:
E. I. Tuomanen
| |
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Infection and Immunity, November 1998, p. 5555-5560, Vol. 66, No. 11
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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