Clinical significance of the coccoid form and comparison of three methods for generating the coccoid H. pylori

H. pylori, a gram-negative and microaerophilic bacterium, plays an important role in gastritis, peptic ulcer disease, and gastric cancer [2]. It is known as a spiral-shaped bacterium with flagella that moves in the thick gastric mucus, but changes to a coccoid form when exposed to certain situations or stresses [6]. This morphological change is considered to be one of the survival mechanisms of H. pylori under adverse environmental conditions, including aerobic conditions, alkaline pH conditions, high temperature, nutrient depletion, antibiotic treatment, proton pump inhibitor treatment, and co-culture with other bacteria [5, 14, 24,25,26]. These coccoid H. pylori cells are usually in the VBNC state. The important point of this state is that they have minimal metabolic activity but express virulence factors such as urease, HpaA, CagA, CagE, VacA, and BabA [27, 28]. These factors affect the development of chronic inflammation and diseases such as gastric cancer [29].

In this study, we induced morphological changes in H. pylori to coccoid forms using three clinically important methods: starvation, amoxicillin antibiotic treatment, and S. mitis supernatant treatment. We experimentally confirmed the VBNC state by comparing colony formation on culture plates and fluorescence staining with SYTO9/PI reagents (LIVE/DEAD bacterial viability staining kit).

In our preliminary study comparing the morphology of H. pylori by Gram stain, DAPI stain, Hoechst, and SYTO9 staining, the most distinct morphology was observed with SYTO9 staining, and SYTO9/PI staining could simultaneously confirm viability; therefore, we used this method to monitor coccoid changes. We observed that spiral-shaped H. pylori changed to coccoid shapes and various shapes, such as the U, donut, and short rod forms, which are considered intermediate forms in the process of becoming coccoid forms. These shape changes are thought to be an adaptive mechanism of H. pylori to unfavourable conditions and are related to peptidoglycan and cytoskeleton rearrangement [11]. We also observed that some cells changed to filament shapes, which later disappeared, probably due to their septation into shorter cells. In this study, we counted and assessed only the spiral and coccoid forms (including the U and donut forms) because the significance of the rod and filamentous forms is not clear. The proportion of H. pylori that changed to a coccoid shape over time was greater when only living cells were considered. In the amoxicillin-treated group, some spiral H. pylori cells stained with SYTO9 but not with PI were observed on the third day, but they did not form colonies on chocolate agar plates, suggesting that these cells might have intact cell membranes but lost the ability to proliferate (Figs. 4 and 5B). As a survival strategy, H. pylori cells undergo coccoid transformation, which enables them to persist for longer periods of time with reduced metabolic activity [30].

The first method we applied to generate the coccoid form was starvation by long broth culture. Starvation can be an unfavourable condition that H. pylori encounters under various environmental conditions. For starvation induction, we cultured H. pylori in two broths: one in BHI broth and the other in BHI broth with 10% FBS. Through repeated experiments, we observed minimal differences in the rate of coccoid formation between the two groups (Supplementary Fig. 1). This finding indicates that H. pylori transform into the coccoid form in prolonged broth culture as nutrients are depleted, regardless of the presence of serum. Thus, broth culture itself is an unfavourable condition for bacterial growth. Consequently, we established a baseline condition of starvation method in BHI broth and compared three groups under the same conditions.

We observed the coccoid transformation of H. pylori by treating the bacteria with amoxicillin, a commonly used antibiotic used for H. pylori eradication therapy, at a half the MIC. The clinical significance of the coccoid conversion of H. pylori by antibiotic treatment is that it may result in the failure of eradication therapy. In this study, we observed rapid coccoid transformation in a culture containing amoxicillin, the most potent coccoid form changer among antibiotics [17], and confirmed that the cells were in the VBNC state for more than 14 days. When H. pylori is exposed to amoxicillin, it can rapidly transform into a coccoid form that is tolerant to the antibiotic, potentially leading to treatment failure. Therefore, the impact of the coccoid form in H. pylori eradication should not be ignored.

The third method that we used to generate the coccoid form was supplementing the culture broth of H. pylori with the supernatant of S. mitis. We compared the culture conditions, we found that S. mitis grew much better under microaerophilic condition, which is more similar to the conditions under which S. mitis grows in the human body. It has been confirmed in gerbil and mouse studies that long-term infection by H. pylori changes the composition and diversity of the gastric microbiota due to increased gastric pH, epithelial cell destruction, and metabolic products from infection [19, 31, 32]. When the gastric microbiota is altered by H. pylori infection, S. mitis, which is part of the human oral microbiota, is significantly increased in the gastric mucosa of patients with atrophic gastritis or gastric cancer [18]. Khosravi et al. showed the coccoid transformation of H. pylori by S. mitis using an in vitro co-culturing system as well as by supplementing the culture supernatant of S. mitis [24]. These authors suggested that fast-growing S. mitis contributed to nutrient depletion and induced a morphological change in H. pylori from a spiral to a coccoid form when they were co-cultured. The authors asserted that S. mitis produced and released diffusible factor(s) that induce coccoid conversion of H. pylori cells. However, in our study, H. pylori treated with the S. mitis supernatant was culturable for the most extended period among the three methods, showing different results from those of Khosravi et al. Moreover, in this experiment, the group that added S. mitis supernatant, even though it was nutritionally deficient, remained culturable and maintained the spiral form longer. In our experiments, the number of colonies in the S. mitis supernatant-treated H. pylori group initially decreased but increase again after approximately 4 days. This suggests that the culturability of H. pylori initially declined due to nutrient deficiency but was later restored, possibly due to substances present in the S. mitis supernatant. Based on our results, we speculated that there may be some substances in the supernatant of S. mitis that allow H. pylori to maintain culturability for a long time. These findings imply that S. mitis may prolong the survival of H. pylori when they coexist in the stomach, affecting the pathogenicity of H. pylori. However, more research is necessary to understand the interaction of S. mitis with H. pylori and its subsequent effects on the gastric diseases.

As confirmed in this study, the coccoid H. pylori can survive for a long time under unfavourable conditions. The extended survival of the coccoid form in unfavourable gastric environments, potentially due to antibiotic tolerance, may contribute to eradication failure [28]. Furthermore, these coccoid H. pylori can affect the occurrence and progression of gastric cancer [7, 9]. This suggests that it would be necessary to eradicate both the spiral and coccoid forms to successfully eradicate and prevent gastrointestinal diseases.

Proteomic analysis of the coccoid H. pylori

We performed proteomics analysis of the spiral and coccoid forms of H. pylori generated by three methods. Through this analysis, we were able to identify proteins that are detected only in spiral form, proteins that are detected only in coccoid form, proteins that are detected in both forms, and proteins that are commonly detected in all three coccoid forms of H. pylori. (Fig. 6; Table 1). Although the 60-kDa chaperonin (GroEL) was detected in the spiral form, it was more abundant in the coccoid form in our study. GroEL belongs to the chaperonin family and is required for proper folding of proteins in many bacteria [33]. Protein folding, which induces protein denaturation, is important for bacterial survival under stressful conditions. In addition to its chaperone activity, GroEL is reported to bind to iron [34]. The acquisition of iron is essential for nearly all organisms and is involved in the efficiency of several metabolic processes, such as respiration and oxygen transport [35]. The abundance of these proteins in the coccoid H. pylori may be advantageous for the nutritional supply and stabilisation of cellular proteins. Furthermore, GroEL is a heat shock protein (HSP60) that can induce the expression and secretion of tumor necrosis factor-α (TNF-α) in host cells, contributing to disease development by producing proinflammatory cytokines [36, 37]. Tanaka et al. reported that the serum antibody levels against H. pylori HSP60 were greater in patients with gastritis and gastric cancer, suggesting that HSP60 may be associated with gastric carcinogenesis [37]. HSP60, which is expressed in the coccoid form, may continuously affect the occurrence of gastric cancer even after H. pylori becomes dormant, but this needs to be further studied in the future. Elongation factor (EF) Tu is the protein with the highest scores in the spiral and coccoid forms of H. pylori. It is related to transcription and translation, contributing to peptide chain elongation during protein translation. Increased EF Tu expression levels in the coccoid form of H. pylori demonstrate continued protein translation [38].

Our study revealed that the CTP synthase (CtpS) score is more than twice as high in the spiral form of H. pylori than in its coccoid form (Supplementary Table 5). This finding is consistent with the known role of CtpS in other bacteria, such as Caulobacter and E. coli, where CtpS is involved in cell shape regulation and enzymatic activity. In Caulobacter, CtpS forms filaments that regulate cell curvature by interacting with crescentin, a cytoskeletal element. This interaction is crucial for maintaining the characteristic curved shape of Caulobacter. The higher expression of CtpS in the spiral form of H. pylori suggests that CtpS may play a role in maintaining the spiral shape, which is critical for its motility and colonisation in the gastric mucosa [39].

The ATP-dependent Clp protease proteolytic subunit (ClpP) and AAA family ATPases were expressed only in the spiral form of H. pylori, but not in the coccoid form. The Clp proteases consist of a self-compartmentalized peptidase and ATPases associated with diverse cellular activities (AAA family ATPases). These components collaborate to carry out regulated protein degradation in a variety of physiological processes, including homeostatic protein quality control, responses to environmental stress, and virulence in pathogenic bacteria [40]. The active state likely involves high metabolic activity, where the demand for protein synthesis and degradation is elevated. The presence of ATP facilitates the binding and activation of AAA + family, enabling efficient protein unfolding, translocation, and degradation. This process ensures that misfolded or damaged proteins are promptly removed, maintaining cellular homeostasis and promoting bacterial growth and virulence. In our study, in the coccoid form of H. pylori, these proteolytic components were not expressed. As reported, dormant Mycobacterium carries out less Clp-mediated protein degradation, with ClpP1 and ClpP2 existing predominantly in inactive conformations [40]. Similarly, the lack of expression of these proteins in the coccoid form of Helicobacter pylori would be a strategic adaptation to conserve energy during periods of low metabolic activity. The inactivity of ClpP in the absence of sufficient ATP and AAA + family binding prevents unnecessary degradation of nascent polypeptides or transiently disordered proteins [40]. By minimizing energy expenditure on protein degradation during dormancy, H. pylori might enhance its long-term viability and persistence.

There were proteins that were commonly expressed in the coccoid forms induced by the three methods, the highest scoring of which was type I glutamate-ammonia ligase (Table 1). Type I glutamate-ammonia ligase, also known as glutamin synthetase, plays an essential role in nitrogen assimilation [41]. This enzyme catalyses the ATP-dependent conversion of glutamate and ammonia into glutamine. And in some bacteria, it is linked to responses to environmental stresses, such as nutrient depletion [41]. Thioredoxin reductase was commonly detected in three methods inducing coccoid forms, with the highest score observed in the S. mitis supernatant-treated group (Table 1). This protein increases tolerance against oxidative stress, suggesting that the coccoid H. pylori may survive under unfavourable conditions such as oxidative stress [42]. There were differences in protein expression in the coccoid forms induced by each method, suggesting that the predominant mechanisms of coccoid transformation in response to external stress may differ depending on the situation. Additionally, the proteins commonly observed in the spiral form and across all three groups exhibited varying scores for each method, indicating that the key proteins utilized in each group might vary. In this study, some proteins that are highly expressed in the coccoid H. pylori were shown to play a role in securing nutrition in extreme situations or resisting various stresses, thus promoting survival under unfavourable conditions. Further studies on the proteins expressed in the coccoid form of H. pylori are necessary to understand the significance of these proteins.

In this study, we set up BHI broth culture without serum as a method to induce the coccoid form by starvation. Even with the inclusion of serum, prolonged broth culture itself acts as a stress factor for the survival of H. pylori. In our experiments, adding serum to BHI broth did not significantly impact the transformation to the coccoid form (Supplementary Fig. 1). To ensure consistent growth conditions across the three groups, serum was not added to any of the samples. This could be a limitation of the experiment, as the absence of serum might have influenced the starvation effects on the amoxicillin and S. mitis groups. However, we reliably confirmed that even under nutrient-deficient conditions, the addition of S. mitis supernatant significantly prolonged the culturability and maintenance of the spiral form of H. pylori. Another limitation of this study is that the H. pylori strain we tested was a standard strain, so different clinical strains may yield different results. Third, the proteins expressed when H. pylori changes to the coccoid form in the stomach may differ from those expressed in vitro. Therefore, it may not establish a relationship between the expression of these proteins and the development of gastritis or gastric cancer.