Diversity in Protein Synthesis and Viability of Helicobacter pylori Coccoid Forms in Response to Various Stimuli

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.

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.

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) β-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.

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).

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.

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.

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).

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).

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.

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.

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.