Comparison of three methods for generating the coccoid form of Helicobacter pylori and proteomic analysis

Abstract

Background: Helicobacter pylori changes from spiral to coccoid depending on the host state, environmental factors, and surrounding microbial communities. The coccoid form of H. pylori still maintains its complete cellular structure, retains virulence genes, and thus plays a role in pathogenicity. To understand the coccoid form, it is crucial to establish the in vitro generation of the coccoid H. pylori. Although some conditions have been studied for the generation of the coccoid form, few studies have compared these conditions for coccoid generation. Here, we generated coccoid forms via three methods and compared the differences in morphology, viability, culturability, and protein expression.

Results: The coccoid H. pylori was generated in vitro via three methods: a starvation method, a method using amoxicillin, and a method using the culture supernatant of Streptococcus mitis. The morphology and viability of the cells were examined by fluorescence microscopy after staining with SYTO9 and propidium iodide. The culturability of H. pylori was examined by counting colony-forming units on chocolate agar plates. In the starvation group, no colonies formed after 7 days, but viable coccoids were continuously observed. In the amoxicillin-treated group, the culturability decreased rapidly after 12 h, and showed a viable but non culturable (VBNC) state after the third day. Most cells treated with S. mitis supernatant changed to coccoid forms after 7 days, but colonies were continuously formed, probably due to living spiral forms. We performed proteomics to analyse the differences in protein profiles between the spiral and coccoid forms and protein profiles among the coccoid forms generated by the three methods.

Conclusion: Amoxicillin treatment changed H. pylori to VBNC cells faster than starvation. Treatment with the S. mitis supernatant prolonged the culturability of H. pylori, suggesting that the S. mitis supernatant may contain substances that support spiral form maintenance. Proteomic analysis revealed that the expression of proteins differed between the spiral form and coccoid form of H. pylori, and this variation was observed among the coccoid forms produced via three different methods. The proteins in the coccoid forms produced by the three methods differed from each other, but common proteins were also observed among them.

Keywords: Helicobacter pylori; Streptococcus mitis; Amoxicillin; Coccoid form; Proteomics; Starvation.

Fig. 1

Study design of coccoid generation by three methods and the experimental timeline. (A) Coccoid forms of H. pylori were generated in vitro by three methods: starvation, amoxicillin treatment, and S. mitis supernatant treatment. (B) The culturability, morphology, and viability of H. pylori cells under the three conditions were examined at the designated time points

Fig. 2

SDS-PAGE analysis of whole cell proteins from Helicobacter pylori cell lysates. Protein patterns of spiral form Helicobacter pylori and coccoid form Helicobacter pylori generated by various methods (starvation, S. mitis supernatant treatment, and amoxicillin treatment) were examined on 14% SDS-PAGE. SDS-PAGE, Sodium Dodecyl Sulfate-PolyAcrylamide Gel Electrophoresis; MIC, minimum inhibitory concentration

Fig. 3

Morphological changes in H. pylori from spiral to coccoid forms. H. pylori cells were collected from chocolate agar plates and suspended in BHI broth for coccoid generation via three methods. (A) The SYTO9 fluorescent image of the spiral form of H. pylori was observed by immediately staining the sample obtained from the colony. (B) H. pylori cells from samples subjected to three methods (starvation, amoxicillin, and S. mitis supernatant) were stained with SYTO9 to observe morphological changes. The scale bars indicate 10 μm. (C) Percentages of coccoid form were calculated at 12 h, 3 days, and 7 days of culture for the three methods. Means and standard deviations (SDs) (n = 3) (n = 3: technical replicates) are shown as columns and error bars, respectively

Fig. 4

Culturability of H. pylori cells from the three methods. (A) At the designated time points, H. pylori cells were plated on chocolate agar plates and cultured for 2 days. In the S. mitis supernatant-treated H. pylori group, colonies were observed to continue growing at 14 days, so culturability was also checked on days 21, 24 and 27 (data not shown for days 27). Images of the culture plates were obtained using a stereoscopic microscope. Colonies were observed as white due to the reflection of the light. The scale bars indicate 500 μm. (B) CFUs of H. pylori were counted to quantitatively analyse culturability. The means and SDs (n = 3) (n = 3: technical replicates) are shown as dots and error bars, respectively

Fig. 5

Viability of H. pylori cells. (A) At the designated time points, cells from the three methods were stained with SYTO9/PI (LIVE/DEAD) staining reagents. SYTO9 stained both live and dead cells with green fluorescence. PI stained only dead cells with red fluorescence. In merged images, dead cells are shown as an orange fluorescent color. The scale bars indicate 10 μm. (B) Percentages of coccoid and spiral forms, along with the proportions of live and dead cells, were counted at 3, 7, and 14 days of culture for the three methods

Fig. 6

Summary of proteomics results. (A-C) Venn diagrams showing unique and common proteins between the spiral and coccoid forms generated by starvation (A), amoxicillin treatment (B), and S. mitis supernatant treatment (C). (D) Venn diagram showing the unique and common proteins among the coccoid-specific proteins identified by the three methods