Cholesterol sulfate signaling facilitates persistent colonization of mucoid-coccoid H. pylori in gastric environment
Elham Godini, Farideh Siavoshi
TL;DR
The study shows how cholesterol sulfate helps Helicobacter pylori bacteria survive in the stomach by transforming into a more resilient form.
Contribution
The novel finding is that cholesterol sulfate acts as a signaling molecule triggering transformation and stress resistance in H. pylori.
Findings
Mucoid-coccoid H. pylori formed in cultures with cholesterol and showed increased resistance to stressors.
Cholesterol sulfate likely activates signaling systems leading to transformation into m-coccoids.
Adding atorvastatin after 54 hours resulted in m-coccoid formation, suggesting a time-dependent transformation.
Abstract
Cholesterol incorporation into membrane protects H. pylori against stresses. Culture of spiral H. pylori in brucella broth (BBR) containing 250 µM cholesterol produced mucoid colonies with coccoid bacteria (m-coccoids). Since H. pylori do not metabolize cholesterol as a carbon source, we examined the possible role of cholesterol or its water-soluble impurity cholesterol sulfate as signaling molecules. Cultures of spiral H. pylori in BBR containing 50–250 µM cholesterol were examined for bacterial growth and morphology. After 72 h microaerobic incubation, a 50-µL volume of each culture was surface-inoculated on brucella blood agar (BBA). After 72 h, cultures of H. pylori in 150 and 250 µM cholesterol produced m-coccoids on BBA. Compared with spiral H. pylori, m-coccoids exhibited enhanced motility, flagellation, biofilm formation, expression of signaling genes, hom B, lep A and lux S and…
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Taxonomy
TopicsBrucella: diagnosis, epidemiology, treatment · Escherichia coli research studies · Probiotics and Fermented Foods
Introduction
It is well known that H. pylori have evolved to establish in human stomach to benefit from its unique environment for persistent colonization, being able to survive and replicate despite the immune responses (Wunder et al. 2006) and dynamic processes of mucus renewal and epithelial cell turnover (Raff 1998). There are many unknown details relating to the rationale of H. pylori colonization in human stomach as a symbiont or a pathogen. One suggested reason for H. pylori settlement in human stomach is bacterial access to cholesterol abundantly found in gastric environment (Hirai et al. 1995; Iwamori et al. 2005). Cholesterol is present in all animal tissues and much of it occurs in plasma membrane as un-esterified form. Animal cells not only synthesize their own cholesterol endogenously but also obtain it from plasma lipoproteins by receptor-mediated endocytosis (Brown and Goldstein 1986). Cholesterol sulfate (CS) that is relatively stable at acidic conditions is the major acidic lipid present in epithelial tissues of mammalian digestive tract (Cui and Iwamori 1997). The uniquely higher content of CS in antral and fundal epithelia of human digestive tract, compared with other visceral organs, shows their critical role in regulating homeostasis of gastric mucosa (Iwamori et al. 2005).
Cholesterol is not an essential nutrient for H. pylori (Testerman et al. 2001) but gastric epithelial cell membrane (Wunder et al. 2006) as well as gastric mucus and consumed food (McGee et al. 2011) are sources that may provide cholesterol for H. pylori. Reports show that H. pylori specifically incorporate cholesterol into their cell membrane to strengthen it (Trampenau and Müller 2003). H. pylori also α-glucosylate the cholesterol and incorporate it into their cell membrane (Haque et al. 1996; Hirai et al. 1995) to evade phagocytosis and disrupt T-cell activation (Wunder et al. 2006). It appears that glucosylated cholesterol plays a critical role in H. pylori resistance to antibiotics (McGee et al. 2011). Furthermore, steroids such as sex hormones, estrogens or androgens, that are abundant in human stomach (Kominea et al. 2004) could be used by H. pylori as components of membrane lipids to strengthen their membrane against acidic pH or antibiotics (Shimomura et al. 2009). Cholesterol has also been implicated in the biosynthesis of lipopolysaccharide and presentation of Lewis antigens at the surface of H. pylori cell, both playing role in pathogen-host interactions. It seems that cholesterol supports both increased display of Lewis antigens and the modification of LPS core/lipid A structure (Hildebrandt and McGee 2009). Dependence of H. pylori to cholesterol and cholesterol derivatives in the gastric environment may refer to many more cholesterol-dependent biological activities of H. pylori that remain to be elucidated (Hildebrandt and McGee 2009; McGee et al. 2011). In our previous study, a mucoid H. pylori strain that was resistant to metronidazole showed considerable susceptibility to the combination of metronidazole and simvastatin (Atif et al. 2024). While the mechanisms of action of statins as cholesterol-lowering, anti-inflammatory, immunomodulatory and anticancer drugs are well studied, little is known about their target (s) in bacterial cell (Hennessy et al. 2016).
Considering that human intestine is a cholesterol- rich environment (Kruit et al. 2006) where several enteric bacteria such as Escherichia coli, have evolved to colonize it to benefit from cholesterol for their biological activities (Reis and Horn 2010) and knowing that H. pylori lack the machinery to synthesize cholesterol (Hirai et al. 1995) or utilize it as a carbon source (Albertson et al. 1998; Testerman et al. 2001), we were wondered why are H. pylori so dependent on the cholesterol exclusively found in human gastric environment and not in the intestine? Furthermore, how does this gastric cholesterol affect H. pylori vital activities? H. pylori have been recognized as members of Epsilonproteobacteria, a versatile group of chemolithoautotrophic thermophiles and mesophiles (Campbell et al. 2006; Longnecker and Reysenbach 2001) that live in environments with harsh physicochemical conditions such as deep-sea hydrothermal vents, rumen of cows and human stomach (Globisch et al. 2012). They are also symbionts of invertebrates and commensals or pathogens of mammals including humans (Ruehland and Dubilier 2010). The ability of Epsilonproteobacteria to colonize their host is related to characteristics typical of marine bacteria, such as surface attachment followed by rapid growth and replication, morphological changes and biofilm formation (Campbell et al. 2006; Dang and Lovell 2016). Considering that natural aquatic environments are generally rich in sterols (Carvalhal et al. 2018), it seems reasonable to suggest that dependence of H. pylori to cholesterol could have initiated long time ago when they, as Epsilonproteobacteria, evolved to live in such habitats.
It has been demonstrated that H. pylori exist in three forms; viable and culturable spiral bacteria, viable but non-culturable coccoids and non-viable degenerative forms (Hirukawa et al. 2018; Percival and Suleman 2014). Results of different studies propose different definitions of coccoid H. pylori. It has been indicated that spiral H. pylori are able to turn into coccoids to survive in stressful temperature, pH and oxygen tension as well as exposure to antibiotics (Citterio et al. 2004; Li et al. 2014). Furthermore, coccoid H. pylori can enter viable but non-culturable state in response to stressful conditions but revert to metabolically active and culturable spiral forms under favorable conditions (Kusters et al. 1997). In fact, in vitro and in vivo experiments proved that coccoid forms of H. pylori may survive in alkaline pH, aerobiosis, high temperatures and prolonged incubation in water and when exposed to proton pump inhibitors or antibiotics (Berry et al. 1995; Nilsson et al. 2002; Park et al. 2004; Tominaga et al. 1999). It has been demonstrated that spiral H. pylori in broth culture turned into coccoid after sixteen days microaerobic incubation but were not culturable and did not colonize gnotobiotic piglets. This coccoid form was considered as degenerated and non-viable morphologic phase of H. pylori (Eaton et al. 1995). It has been revealed that coccoid H. pylori produced after 3-months culture showed ability to multiply as coccoids without reverting to spiral form. These viable coccoids retained their infectivity in laboratory animals (Loke et al. 2016). From the pathological point of view, the coccoid H. pylori formed after failure in antibiotic therapy and under anaerobic conditions (Yamaguchi et al. 1999) or upon aging (Azevedo et al. 2007) were speculated as dormant or stressed forms of H. pylori. Altogether, it is not clear whether all studies report the same coccoid phenomenon and these coccoid H. pylori are viable and infectious. Finally, it is not known how transformation of spiral H. pylori to coccoid or mucoid colonies with coccoid bacteria (m-coccoids) occurs. In this study, cultures of spiral H. pylori in brucella broth (BBR) containing 150 and 250 µM cholesterol were transformed into m-coccoid H. pylori. It was speculated that this transformation could be based on signal transduction initiated by cholesterol or its water-soluble derivative, CS.
It has been revealed that several sensing, signaling and responding systems exist in bacteria that by sensing the extracellular signals mediate regulation of biological activities of bacteria such as production of flagella, motility, surface attachment and biofilm formation (Kalivoda et al. 2013). These regulating systems include quorum sensing (QS), two-component signal transduction system (TCS), chemotaxis, posttranscriptional regulation by small RNAs and centralized regulation by second Messengers (cyclic GMP and cyclic di-GMP) (Dang and Lovell 2016). Interestingly, there are bacterial species that possess several numbers of each of these signaling systems like TCS that operate simultaneously for adaptive response to various environmental conditions (Kimes et al. 2012). Furthermore, several different signaling systems may operate as a network to confer fitness advantages for bacterial adaptation to the continuously changing environments (Mo et al. 2022). It has been proposed that in bacterial world, evolution of signaling networks is driven by ecological adaptation (Galperin 2010).
In this study, spiral H. pylori were cultured in BBR supplemented with 50–250 µM cholesterol. After 72 h, cultures of spiral H. pylori in 50 and 100 µM cholesterol remained spiral and produced typical colonies on brucella blood agar (BBA) and those in 150 and 250 µM cholesterol produced m-coccoids. Because spiral to m-coccoid transformation only occurred after 72 h, we repeated the cultivation of spiral H. pylori in BBR containing 250 µM cholesterol and followed the changes in bacterial morphology and growth on BBA every 6 h and up to 72 h. Expression of signaling genes homB, lep A and lux S was examined during spiral to m-coccoid transformation. Furthermore, the effect of cholesterol on biofilm formation, motility and flagellation as well as resistance to stressful temperature, pH, NaCl, aerobic atmosphere, antibiotics and atorvastatin (ATV) was examined. Finally, ATV was added to spiral H. pylori cultures in BBR containing 250 µM cholesterol at 6-hr intervals and the changes in bacterial growth were assessed. We used published reports to find out if there is any relation between cholesterol and signal transduction in bacteria and how this signaling could be inhibited by ATV, the inhibitor of cholesterol synthesis in animals.
Materials and methods
H. pylori isolate
H. pylori strain used in this study was isolated from gastric biopsy of a patient with gastritis. This isolate was characterized by amplification of 16 S rRNA gene and sequencing, as reported in our previous study (Kadkhodaei et al. 2024).
Preparation of Brucella broth (BBR) and Brucella blood agar (BBA) media
BBR was prepared by dissolving 28.1 g Brucella Broth powder (Pronadisa, CONDA, Spain) in 1 L of distilled water and autoclaving at 121 °C for 15 min. BBA was prepared by dissolving 43 g Brucella Agar powder (Pronadisa, CONDA, Spain) in 1 L of distilled water and autoclaving at 121 °C for 15 min. After cooling to 45–50 °C, 70 mL of defibrinated sheep blood was added to one liter of cooled brucella agar, mixed thoroughly and dispensed into sterile Petri dishes.
Selection of stressful conditions
In this study, we postulated that cholesterol may play critical roles in H. pylori transformation from spiral to m-coccoid as well as bacterial resistance to different stresses. We examined spiral and m-coccoid H. pylori response to generally- used stresses such as high temperatures, acidic pH, high salinity, antibiotics and aerobiosis (Citterio et al. 2004; Li et al. 2014).
Selection of cholesterol concentrations and preparation of cholesterol solutions
We used information reported in previous studies for selecting cholesterol concentrations of 50 µM (Hosoda et al. 2009) to 250 µM (Qaria et al. 2018; Wunder et al. 2006). The origin of purchased cholesterol (AppliChem, Germany) used in this study was sheep wool grease with 95% purity. Stock solution of cholesterol (5 mM) was prepared in ethanol and added to BBRs to reach the final concentrations of 50–250 µM.
Impact of different concentrations of cholesterol (50–250 µM) on the growth and morphology of spiral H. pylori
Tubes containing 3 mL of BBR supplemented with 50, 100, 150 and 250 µM cholesterol were inoculated with fresh (72 h) culture of spiral H. pylori on BBA and their turbidity was adjusted to MacFarland standard No 2 (6 × 10^8^ CFU/mL). BBR tubes with 10% horse serum (Baharafshan, Iran) or without cholesterol were used as controls. Tubes were incubated at 37 °C in CO_2_ (10% CO_2_, 5% O_2_, 85% N_2_) shaker incubator (x 150 rpm). Optical density (OD) of BBR cultures was measured at 600 nm after 72 h, using spectrophotometer (pg instruments, T70, United kingdom). Furthermore, 50-µl volumes of each 72-hr BBR culture were used for Gram staining, staining with LIVE/DEAD BacLight Bacterial Viability Kit L-7012 according to manufacturer’s instruction (Invitrogen, Eugene, Oregon, USA) and also for surface- inoculation on BBA. Microscopic observations were performed using light (zeiss, Germany (and fluorescence (BX51; Olympus, Tokyo, Japan) microscopes. After 72 h microaerobic incubation at 37 °C, plates were examined for bacterial growth and Gram stain was used for observing bacterial morphology. It is noteworthy that fresh culture of H. pylori on BBA was also directly surface-inoculated on BBA supplemented with 50–250 µM cholesterol and examined for growth and morphology after 72 h microaerobic incubation at 37 °C. Since at 150 and 250 µM cholesterol, spiral H. pylori turned into m-coccoids, the similar identity of both bacteria was confirmed by biochemical and molecular methods presented in the Supplementary Information.
Using light and transmission electron microscopy methods to determine the time point when spiral H. pylori culture in 250 µM cholesterol transform into m-coccoids with enhanced motility and flagellation
Fresh culture of spiral H. pylori was inoculated into a flask containing 250 mL BBR supplemented with 250 µM cholesterol and the turbidity was adjusted to MacFarland standard No 2 (6 × 10^8^ CFU/mL). The flask was incubated at 37 °C in CO_2_ shaker incubator. Bacterial growth and morphology were examined every 6 h and up to 72 h, by using 50-µL volumes of bacterial culture for Gram-staining and surface-inoculation on BBA. Plates were examined for bacterial growth after 72 h microaerobic incubation at 37 °C. Fresh cultures of spiral and m-coccoid H. pylori on BBA were used for preparing wet mounts and observing bacterial motility by the light microscope. These cultures were also used for flagella staining according to M. West (West et al. 1977). Furthermore, samples from 24, 48 and 72 h cultures of spiral H. pylori in BBR supplemented with 250 µM cholesterol were prepared for transmission electron microscopy (Philips, EM208S) as previously described (Heydari et al. 2021).
Detection of the expression of hom B, lep A and lux S genes during transformation of spiral H. pylori into m-coccoids in the presence of 250 µM cholesterol
Real-time polymerase chain reaction (PCR) was used for measuring the expression of three signaling genes hom B,* lep A* and lux S during transformation of spiral H. pylori into m-coccoids; hom B an outer membrane protein involved in surface adherence and biofilm formation (Servetas et al. 2018), lep A a membrane GTPase that is probably related to membrane-signaling pathways (Bijlsma et al. 2000) and lux S involved in the production of autoinducer-2 (AI-2) and microbial communication as well as in biofilm formation and stress resistance (Liu et al. 2018). A fresh suspension of spiral H. pylori was prepared in BBR with the turbidity mentioned earlier. One-mL volume of bacterial suspension was inoculated into two flasks containing 30 mL of BBR, one supplemented with 250 µM cholesterol and one without, as a control. After 24, 48 and 72 h microaerobic incubation at 37 °C in CO_2_ shaker incubator, bacterial cultures were centrifuged at 6000 rpm for 10 min. Pellets were washed with phosphate-buffered saline (PBS) and used for RNA extraction with a commercial kit (viragene, Iran). Oligo(dT)-primed cDNA synthesis was carried out using the AddScript cDNA Synthesis Kit (add bio) according to manufacturer’s instruction. Real- time PCR was performed as previously described (Zhu et al. 2009) with primers (Cellini et al. 2005; Kadkhodaei et al. 2020; Servetas et al. 2018) provided in the Supplementary Information. The H. pylori-specific 16 S rRNA gene was used as the internal reference gene for normalization. Transcription levels of the target genes were normalized against 16 S rRNA expression. All the reactions were performed in triplicate and no-template controls (NTCs) were included in each run. Real-time assays were conducted using MyGo Pro ESR real-time PCR system. Quantification cycle (Cq) values were determined using MyGo Pro ESR software (version 3.6.4). Relative gene expression levels were calculated using the 2^⁻ΔΔCt^ method. Primer efficiency was set to 2 (100%) for all the primer pairs.
Statistical analysis
For data analysis (Fold-change values (2^⁻ΔΔCt^) analysis), non-parametric statistical tests were used. The Friedman test was applied to compare the mean gene expression levels among three related groups (24 h, 48 h and 72 h). A statistically significant difference in the expression of lepA, homB and luxS genes was observed (p-value < 0.05). The level of statistical significance was set at 0.05. All analyses were performed using SPSS version 27.
Biofilm formation by spiral H. pylori on BBA immersed in BBR supplemented with 250 µM cholesterol
Three tubes containing BBR, 250 µM cholesterol and 1cm^3^ piece of BBA were inoculated with fresh culture of spiral H. pylori and incubated microaerobically at 37 °C. BBA pieces were taken out after 24, 48 and 72 h and washed in physiological saline by 5 min centrifugation (x 1500 rpm) to eliminate non-adsorbed bacteria. BBA cubes were prepared for scanning electron microscopy (Hitachi S4160 FESEM, Tokyo, Japan) according to Paweł Krzyz˙ek 2022 (Krzyżek et al. 2022). Electron micrographs were taken to show the change in bacterial morphology and biofilm formation by time.
Impact of cholesterol on the viability of spiral and m-coccoid H. pylori when exposed to stressful conditions; high temperatures (45–95 °C), extremes of pH (2–8), high concentrations (5–15%) of NaCl, aerobic conditions, antibiotics and ATV (30, 50, 100 and 250 µg/mL)
Suspensions of spiral and m-coccoid H. pylori were prepared in tubes containing 3 mL BBR, with or without 250 µM cholesterol, and their turbidity was adjusted as mentioned earlier. These tubes were used to examine the viability of spiral and m-coccoid H. pylori when exposed to different stresses. Bacterial suspensions in BBR were incubated in water bath with different temperatures (37, 45, 65, 75 and 95 °C) for 15 and 30 min and 1–4 h. BBRs with their pH adjusted to 2, 4, 6, 7 and 8 were inoculated with bacterial suspensions and examined after 15 and 30 min and 1–4 h. BBRs with 5, 10 and 15% NaCl were inoculated with bacterial suspensions and examined after 15 and 30 min and 1–4 h. All the treated cultures were tested for viability after 15 and 30 min and 1–4 h. For testing bacterial viability, a 50- µL volume of treated spiral or m-coccoid H. pylori cultures was surface inoculated on BBA and examined for growth and morphology after microaerobic incubation at 37 °C. Furthermore, 50-µL volumes of spiral and m-coccoid H. pylori suspensions were surface- inoculated on four BBA plates; two plates of spiral and m-coccoid were incubated at 37 °C in CO_2_ incubator and the other two at 37 °C in regular incubator with normal aerobic conditions.
For testing antibiotic susceptibility of bacteria, fresh cultures of spiral and m-coccoid H. pylori were used for preparing bacterial suspensions with the turbidity of MacFarland unit No 2 (6 × 10^8^ CFU/mL). A 50-µL volume of each bacterial suspension was surface-inoculated on BBA, using a sterile glass rod. Sterile blank paper discs were superimposed on the surface of plates and impregnated with 10 µL of concentrated solution of each antibiotic, in dimethyl sulfoxide, to reach their minimum inhibitory concentrations. Tested antibiotics (µg/mL) included metronidazole (8), clarithromycin (2), Amoxicillin (1), tetracycline (0.5), furazolidone (0.5), ciprofloxacin (1), levofloxacin (1) and rifampin (4). BBA plates were incubated in CO_2_ incubator at 37 °C for up to 7 days. Inhibition zone diameters were measured to evaluate bacterial susceptibility (Mansour-Ghanaei et al. 2022).Tubes containing BBRs supplemented with different concentrations of ATV (30, 50, 100 and 250 µg/mL), with or without 250 µM cholesterol, were inoculated with suspensions (6 × 10^8^ CFU/mL) of spiral and m-coccoid H. pylori. Bacterial cultures were incubated in CO_2_ shaker incubator at 37 °C for 24, 48 and 72 h and their viability was examined by surface inoculation on BBA and microaerobic incubation at 37 °C.
Impact of ATV on transformation of spiral H. pylori to m-coccoids in the presence of 250 µM cholesterol
ATV was added every 6 h to cultures of spiral H. pylori in BBR containing 250 µM cholesterol up to 72 h. Thirteen tubes containing BBR and 250 µM cholesterol were inoculated with spiral H. pylori and their turbidity was adjusted as mentioned earlier. Tubes were incubated at 37 °C in CO_2_ shaker incubator (x 150 rpm) and 100 µg/mL ATV was added to tubes after 0 to 72 (0, 6,12,18,24,30,36,42,48, 54, 60, 66,72) hr. Volumes of 50 µL were taken from every 13 tubes at 6-hr intervals and used for Gram staining and surface- inoculation on BBA. Plates were examined for bacterial growth and morphology after microaerobic incubation at 37 °C.
Results
Impact of different concentrations of cholesterol (50–250 µM) on the growth and morphology of spiral H. pylori
Microscopic examination of Gram-stained preparations of 72-hr cultures of spiral H. pylori in BBR supplemented with 50 and 100 µM cholesterol showed similar results; spiral bacteria some as single and dispersed but mostly as aggregates. When these cultures were surface-inoculated on BBA, H. pylori produced typical pinpoint colonies (Fig. 1A1). Gram- and Live/Dead- stained preparations showed Gram-negative spirals (Fig. 1A2) and live green bacteria (Fig. 1A3), respectively. Similar results were observed in BBR with or without 10% serum. OD measurements after 72 h showed that compared with control tube (OD = 0.215), spiral H. pylori showed negligible growth in 50 µM (OD = 0.377) and 100 µM (OD = 0.394) cholesterol. It is noteworthy that when fresh culture of spiral H. pylori on BBA was directly surface-inoculated on BBA supplemented with 50 or 100 µM cholesterol, H. pylori retained spiral morphology and produced typical colonies. Microscopic examination of Gram-stained preparations of 72-hr cultures of spiral H. pylori in BBR supplemented with 150 and 250 µM cholesterol showed similar results; all the bacteria were seen as coccoids. When 72-hr cultures were surface-inoculated on BBA, H. pylori formed mucoid colonies (Fig. 1B1) of coccoid bacteria (Fig. 1B2) that stained green with Bacterial Viability Kit (Fig. 1B3). OD measurements after 72 h showed that compared with control tubes with spiral H. pylori (OD = 0.271), m-coccoid H. pylori showed about three-time increase in 150 µM (OD = 0.725) and 250 µM (OD = 0.789) cholesterol. It is noteworthy that when fresh culture of spiral H. pylori on BBA was directly surface-inoculated on BBA supplemented with 150 or 250 µM cholesterol, H. pylori remained spiral and produced typical colonies.
Fig. 1. Impact of 50–250 µM cholesterol on the growth and morphology of spiral H. pylori. At 50 and100 µM cholesterol, spiral H. pylori produced typical glistening pinpoint colonies (A1) with Gram-negative spirals (A2) that stained green with Live/Dead stain (A3). At 150 and 250 µM cholesterol, spiral H. pylori produced mucoid colonies (B1) with Gram-negative coccoids (B2) that stained green with Live/Dead stain (B3). Original magnifications of the light and fluorescence microscopes were x 1250 and x 1000, respectively
Using light and transmission electron microscopy methods to determine the time point when spiral H. pylori culture in 250 µM cholesterol transform into m-coccoids with enhanced motility and flagellation
Gram stained preparations from BBR cultures showed that after 6–24 h, H. pylori appeared as dispersed spirals (Fig. 2A1) that produced typical colonies on BBA (Fig. 2A2) with Gram-negative spirals (Fig. 2A3). Flagella staining showed only few spirals with polar flagella (Fig. 2A4). After 30–48 h, H. pylori formed aggregates (Fig. 2B1) of spirals that produced typical colonies on BBA (Fig. 2B2) with Gram-negative spirals (Fig. 2B3). Flagella staining showed only few spirals with polar flagella (Fig. 2B4). However, after 54–60 h, spirals were transformed into coccoids that were interconnected with delicate lattices (Fig. 2C1) and produced mucoid colonies on BBA (Fig. 2C2) with Gram-negative coccoids (Fig. 2C3). Flagella staining showed coccoids with enhanced flagellation (Fig. 2C4). After 72 h, all the bacterial cells appeared as coccoids (Fig. 2D1) that produced mucoid colonies (Fig. 2D2) with Gram-negative coccoids (Fig. 2D3). Flagella staining showed coccoids with enhanced flagellation (Fig. 2D4). Videos taken from wet mounts showed coccoids with enhance motility and spirals with slow motility (Supplementary videos). Transmission electron micrographs showed intermediate steps in H. pylori transformation from spiral to coccoid forms. After 24 h of incubation, H. pylori showed spiral morphology that changed to intermediate morphology after 48 h and finally turned into coccoid after 72 h (Fig. 3).
Fig. 2. Transformation of spiral H. pylori into m-coccoids within 72 h culture in BBR supplemented with 250 µM cholesterol. Microscopic examination after 6–24 h showed dispersed spirals (A1) that produced typical H. pylori colonies on BBA (A2) with Gram-negative spirals (A3) and few with polar flagellation (A4). After 30–48 h, spirals formed aggregates (B1) and produced typical H. pylori colonies (B2) with Gram-negative spirals (B3) and few showing polar flagellation (B4). After 54–60 h, spirals were transformed into coccoids that were interconnected with delicate lattices (C1) and produced mucoid colonies (C2) with Gram-negative coccoid bacteria (C3) and enhanced flagellation (C4). After 72 h, transformed H. pylori appeared as dispersed coccoids (D1) that produced mucoid colonies (D2) with Gram-negative coccoids (D3) and enhanced flagellation (D4). Many detached flagella are seen in the background of C4 and D4. Original magnification x 1250
Fig. 3. Transmission electron micrographs showed transformation of spiral H. pylori into coccoids when cultured in BBR supplemented with 250 µM cholesterol for 72 h. H. pylori showed spiral morphology after 24 h (A1-A5), transition from spiral into coccoid morphology after 48 h (B1-B5) and total coccoid morphology after 72 h (C1-C5). Bars: 200 nm–1 μm
Detection of the expression of hom B, lep A and lux S genes during transformation of spiral H. pylori to m-coccoid in the presence of 250 µM cholesterol
Results of Real- time PCR showed that compared with control, there was a modest increase in the expression of three signaling genes after 24, 48 and 72 h; hom B (1.19, 1.29 and 1.59), lep A (1.08, 1.24 and 1.26) and lux S (0.78, 0.89 and 1.18) (Fig. 4). Statistical analysis showed that this increase was statistically significant (p-value < 0.05). It has been demonstrated that even modest changes in key regulatory gene expression can produce large phenotypic and pathway-level consequences. Supporting this suggestion, Laurent et al. showed that many transcripts with fold changes below the typical 2–3 fold threshold are biologically meaningful and low fold-change RNAs are often enriched for functionally relevant roles (Laurent et al. 2013).
Fig. 4. Fold-change expression of lepA, homB, and lux S genes in H. pylori cultured in the presence of 250 µM cholesterol after 24–72 h. Results of Real- time PCR showed increase in the expression of three signaling genes after 24, 48 and 72 h; hom B (1.19, 1.29 and 1.59), lep A (1.08, 1.24 and 1.26) and lux S (0.78, 0.89 and 1.18)
Biofilm formation by spiral H. pylori on BBA immersed in BBR supplemented with 250 µM cholesterol
Scanning electron micrographs taken from H. pylori culture in BBR with 250 µM cholesterol and a piece of BBA, showed change of H. pylori morphology from spiral into coccoid upon adsorption to BBA, burst of bacterial cell division and progress in biofilm formation during 24–72 h (Fig. 5A, B and C).
Fig. 5. Scanning electron micrographs showed transformation of spiral H. pylori into coccoids and biofilm formation in BBR supplemented with 250 µM. Spiral H. pylori attached to the surface of BBA, turned into coccoid and formed biofilm. Compared with 24 h culture with no biofilm (A), growth of biofilm is observed after 48 (B) and 72 h (C). Bars: 20–100 μm
Impact of cholesterol on the viability of spiral and m-coccoid H. pylori when exposed to stressful conditions; high temperatures (45–95 °C), extremes of pH (2–8), high concentrations (5–15%) of NaCl, aerobic conditions, antibiotics and ATV (30–250 µg/mL)
The impact of stressful temperature, pH, NaCl and ATV on H. pylori suspensions in BBR with or without 250 µM cholesterol and after different exposure times are summarized in Tables 1 and 2. It is noteworthy that microscopic observations on Gram-stained preparations showed that spiral or coccoid morphology of H. pylori remained unchanged after exposure to stressful conditions. Compared with spiral H. pylori that did not withstand high temperatures except 45 °C for 15 min in the presence of cholesterol, m-coccoid H. pylori could grow after 4 h exposure to 45 °C with or without cholesterol and up to 30 min to 65 °C with cholesterol. Spiral H. pylori remained viable after exposure to pH 6–8 for 4 h. However, at pH 4 they remained viable after 2 h with cholesterol and 1 h without it. m-coccoid H. pylori were able to withstand pH of 6–8 as well as acidic pH (2–4) for 4 h even without cholesterol. Spiral H. pylori did not withstand high concentrations of NaCl even in the presence of cholesterol, except 5% NaCl with cholesterol for 15 min. However, m-coccoid H. pylori could only grow after exposure to 5% and 10% NaCl with cholesterol up to 4 h (Table 1). Spiral H. pylori showed confluent growth under microaerobic conditions but no growth when exposed to atmospheric oxygen. However, m-coccoid H. pylori showed confluent growth under microaerobic as well as aerobic incubations. Spiral H. pylori showed susceptibility to all antibiotics, except tetracycline, with the inhibition zone diameters between 35 and 41 mm. However, m-coccoid H. pylori were resistant to all the antibiotics except rifampin with inhibition zone diameter of 40 mm. m-coccoid H. pylori showed confluent growth after 24–72 h exposure to all concentrations of ATV with or without cholesterol and retained its mucoid and coccoid characteristics. Spiral H. pylori showed growth after 24 h exposure to 30, 50 and 100 µg/mL ATV with or without cholesterol. However, they grew after 48–72 h exposure to 30, 50 and 100 µg/mL ATV only in the presence of cholesterol. Furthermore, spiral H. pylori showed growth only after 24 h (and not 48 and 72 h) exposure to 250 µg/mL ATV and in the presence of cholesterol (Table 2).
Table 1. Viability of spiral (S) and m-coccoid (M) H. pylori when exposed to stressful temperature, pH and NaCl for 15 min–4 h with (C) or without (N) cholesterol. Growth (+), no growth (-)Exposure timeTemperature (^⸰^C)pHNaCl (%)456524510SMSMSMSMSMSM N C N C N C N C N C N C N C N C N C N C N C N C15 min– +
+
+ ––– + – +
+
+ ++ +
+ – + – + ––– + 30 min–– +
+ ––– + –– +
+ ++ +
+ ––– + ––– + 1 h–– +
+ –––––– +
+ ++ +
+ ––– + ––– + 2 h–– +
+ –––––– +
+ – +
+
+ ––– + ––– + 3 h–– +
+ –––––– +
+ –– +
+ ––– + ––– + 4 h–– +
+ –––––– +
+ –– +
+ ––– + ––– +
Table 2. Viability of spiral (S) and m-coccoid (M) H. pylori when exposed to Atorvastatin for 24–72 h with (C) or without (N) cholesterol. Growth (+), no growth (-)Exposure timeAtorvastatin (µg/ml)3050100250SMSMSMSM N C N C N C N C N C N C N C N C24 h++++++++++++–+++48 h–+++–+++–+++––++72 h–+++–+++–+++––++
Impact of ATV on transformation of spiral H. pylori into m-coccoid in the presence of 250 µM cholesterol
Cultures of spiral H. pylori with ATV added at 0–48 h of incubation produced typical colonies of spiral H. pylori on BBA. However, cultures with ATV added after 54–72 h produced mucoid colonies of coccoid H. pylori. Microscopic examination of Gram-stained smears from ATV- treated BBR cultures showed that ATV added before 54 h could inhibit transformation of spiral H. pylori into m-coccoids but not when added after 54 h (Fig. 6).
Fig. 6. Inhibition of spiral H. pylori transformation into coccoids by ATV. Gram staining showed that spiral H. pylori grown in BBR containing 250 µM cholesterol with 100 µg/mL ATV added before 54 h formed dispersed aggregates of spirals within 6–24 h (A1) that turned into more compact spirals after 30–48 h (A2) and 54–72 h (A3). Gram staining of these 6–72 h H.pylori cultures on BBA showed Gram-negative spirals (A4). Spiral H. pylori grown in BBR containing 250 µM cholesterol with 100 µg/mL ATV added after 54 h formed dispersed aggregates of spirals within 6–24 h (B1) that turned into more compact spirals after 30–48 h (B2) and lattices of dispersed coccoids after 54–72 h (B3). Gram staining of these 54–72 h H.pylori cultures on BBA showed Gram-negative coccoids (B4). Original magnification x 1250
Discussion
Reports show that several bacteria, including members of the enteric group incorporate cholesterol into their membrane directly from the host for a range of important biological activities. They use cholesterol for signaling (Carabeo et al. 2004), cell membrane integrity and trafficking as well as internalization and maintenance of the intracellular niche (Lafont et al. 2002). In the other side, incorporation of cholesterol into host’s cell membranes can protect intracellular bacteria by inhibiting phagolysosomal fusion and deleterious acidification (Catron et al. 2002). Furthermore, cholesterol-rich microdomains are involved in bacterial attachment to the cell surface and delivery of effector proteins (Carabeo et al. 2004; Schraw et al. 2002). Bacteria also use cholesterol as a nutrient source and for modifying their cell membrane to resist stressful conditions and antibiotics (Toledo and Benach 2016). Considering that H. pylori are not capable of synthesizing cholesterol or consuming it as a carbon source, results of this study propose that H. pylori are dependent on cholesterol for its signals that facilitate their persistent colonization in human stomach. It is well known that cholesterol as the precursor of all steroid hormones can act as a signaling molecule (Cortes et al. 2014). Furthermore, CS as a normal constituent of human tissues (Strott and Higashi 2003), including gastric fluid and epithelium (Iwamori et al. 2005) can function as a signaling molecule (Denning et al. 1995; Strott and Higashi 2003). CS is a cholesterol derivative in which 3β-hydroxyl is substituted by sulfate group (Bukiya and Dopico 2017). This sulfonation process converts the hydrophobic water-insoluble cholesterol to its hydrophilic water-soluble derivative. It has been demonstrated that the rate of inter-membrane exchange of CS is about an order of magnitude faster than cholesterol (Rodrigueza et al. 1995). Interestingly, sulfated steroids constitute the largest subset of sulfated secondary metabolites in marine environments (Carvalhal et al. 2018) and marine organisms (Kornprobst et al. 1998), playing critical role in signal transduction due to their water solubility (Elyakov and Stonik 2003).
Results of this study showed that when spiral H. pylori were grown in liquid culture medium (BBR) containing 250 µM cholesterol, they first (within 24–48 h) formed aggregated spirals but transformed into coccoids after longer incubation (after 54–72 h). When these spirals and coccoids were surface inoculated on BBA, they produced pinpoint and mucoid colonies, respectively. Observations with the light microscope revealed that these transformations could have started with attachment and aggregation of spirals that proceeded with a burst of bacterial division and production of exopolysaccharide, resulting in the formation of coccoids that produced mucoid colonies. Micrographs taken by transmission electron microscope showed changes from spiral to coccoid morphology by time and those taken by scanning electron microscope showed attachment of coccoids to the surface of blood agar cubes and burst of cell division. Compared with spiral H. pylori, m-coccoids appeared more flagellated with enhanced motility. Finally, compared with spiral H. pylori, m-coccoids showed resistance to high temperature (65° C), acidic pH (2–4), high NaCl concentration (10%) and grew under aerobic conditions. While spiral H. pylori were susceptible to all the tested antibiotics except tetracycline, m-coccoids exhibited resistance to all the antibiotics except rifampin. It has been indicated that bacterial susceptibility to rifampin could be due to its lipid solubility (Levison and Levison 2009). ATV-treated spiral H. pylori did not show growth on BBA however ATV-treated m-coccoids remained viable and showed confluent growth.
In our previous studies, we have reported isolation of three mucoid H. pylori strains from three gastric biopsies of dyspeptic patients; two with short rod morphology (Siavoshi et al. 2012) and one with coccoid morphology (m-coccoids). The three mucoid isolates showed resistance to all the antibiotics currently used for H. pylori eradication. It was revealed that m-coccoid H. pylori besides accumulating high amounts of cholesterol, their polyunsaturated fatty acids content (82%) was higher than control spiral H. pylori (36%) (Kadkhodaei et al. 2020). Unsaturated fatty acids in bacteria have been implicated in antioxidant activity (De Carvalho and Caramujo 2018) and antibiotic efflux (Nishida et al. 2010). In the one side, it has been revealed that accumulation of cholesterol and production of exopolysaccharide in H. pylori could lead to decrease in antibiotic uptake and emergence of resistance (Trainor et al. 2011). In the other side, a mucoid H. pylori that showed resistance to metronidazole was turned into susceptible when metronidazole (8 µg/mL) was combined with simvastatin (100 µg/mL), indicating that synergistic effect of both drugs led to disruption of permeability barrier and effective antibacterial activity of metronidazole. It is noteworthy that our microscopic examinations of Gram-stained gastric biopsies revealed that some patients carry only spiral H. pylori or only coccoids and some have a mixture of both (Kadkhodaei et al. 2020). Despite several studies on transformation of spiral H. pylori to coccoid morphology or m-coccoids, the reasons and mechanisms of these morphological changes are not known.
Results of this study suggested that cholesterol or CS may have acted as signaling molecules when bacterial cells reached certain density at late exponential phase of growth curve (after 48–54 h), presenting enough interacting surface receptor molecules to sense the exogenous cholesterol/CS signals and initiate several cascades of altered biological activities. We first considered QS mechanism that was dependent on the increased bacterial density and release of homB, lep A and lux S gene products for controlling expression of genes such as those related to biofilm formation (Keller and Surette 2006), flagellation and host colonization (Rader et al. 2007). It has been demonstrated that lux S gene is part of Epsilonproteobacteria genome and represents an evolutionary link between vent Epsilonproteobacteria and human pathogens (Pérez-Rodríguez et al. 2015). Although, results of Real-time PCR showed a significant increase in the expression of QS genes in m-coccoid H. pylori cultured in the presence of 250 µM cholesterol, we looked in the literature for the likelihood of existence of other signaling systems in H. pylori.
It is noteworthy that while spiral to m-coccoid transformation happened in BBR cultures supplemented with 150 and 250 µM, it did not happen when fresh spiral H. pylori grown on BBA was directly cultured on solid BBA containing 150 or 250 µM cholesterol. These results showed that the reason for spiral to m-coccoid transformation could be related to initiation of signaling pathway(s) that was only possible in liquid conditions. It has been indicated that signaling molecules need to be water-soluble to bind to specific receptors on the cell surface, transducing the extracellular signals into the cell by activating intracellular signaling pathways. Furthermore, cell surface receptors are often associated with G-proteins or have serine/ threonine/ tyrosine kinase activities (Stancu and Sima 2001). Since stock solution of cholesterol in ethanol was diluted with BBR, dissolved in water, to reach the final concentrations of 50–250 µM, we looked for the properties of cholesterol purchased for our study. The purchased cholesterol was prepared from sheep wool grease with the purity of 95%. We found that CS is among the minor contents of natural sheep wool (Coderch et al. 2023). It was hypothesized that impurity of cholesterol could be composed of the water-soluble moieties, including CS that acted as a signaling molecule (Denning et al. 1995; Strott and Higashi 2003). This water-soluble CS could activate one or several types of receptors on the surface of H. pylori cell, probably related to G-proteins or protein kinases (PKs) group (Stancu and Sima 2001). Our search in the literature revealed that CS selectively regulates the activity of protein kinase C (PKC) isoforms (Bukiya and Dopico 2017). Furthermore, protein-serine kinase activity has been detected in H. pylori (Grangeasse et al. 1999).
Many bacteria have evolved to either exist as free-living or interact with their host as a pathogen or a symbiont (Rose et al. 2012). For adapting to their niche, bacteria need to sense the signal, transduce it and respond. Protein kinases (PKs) that regulate specific signal transduction pathways are widespread in all the three domains of life, Eukarya, Archaea and Bacteria (Kennelly 2002). In humans, PKs play major roles in cellular homeostasis and dysregulated kinase activity has been linked to a variety of pathological conditions such as cancer (Lapenna and Giordano 2009) and cardiovascular diseases (Amin et al. 2019). Accordingly, PKs are the second most therapeutically targeted group of proteins after G-protein-coupled receptors (Silnitsky et al. 2023). Five classes of PKs have been identified in bacteria, among them tyrosine kinases (Tyr kinases) and Hanks-type Serine/Threonine kinases (STKs) (commonly known as eukaryotic-like STKs) can phosphorylate various proteins and regulate bacterial physiology (Mijakovic et al. 2016). It has been demonstrated that ancestral PKs existed prior to divergence of Eukaryotes, Bacteria and Archaea (Leonard et al. 1998). Furthermore, Tyr kinases (Whitmore and Lamont 2012) and STKs (Ortiz-Lombardıa et al. 2003) that exhibit structural and functional properties similar to those of their eukaryotic counterparts regulate many biological activities in bacteria, such as growth and cell division, biofilm formation, stress response, as well as pathogenic or symbiotic interactions with eukaryotic hosts. Interestingly, CS that is ubiquitous in biological fluids (Gurpide et al. 1966) has been extensively recognized as one of the most important sulfonated steroids that selectively regulates the activity of PKC isoforms (Bukiya and Dopico 2017).
Results of this study showed that when ATV was added to BBR culture of spiral H. pylori before 48 h, it inhibited the initiation of signaling cascades and transformation of spiral H. pylori into m-coccoids with altered biological activities. However, when ATV was added after 54–72 h, no inhibition happened and the cascades of signaling and transformation of spiral H. pylori to m-coccoids continued. These results suggested that the cascades of signaling that initiated by CS were sensitive to ATV if added before a critical time point of bacterial growth curve. In this regard, we looked in the literature for the role of statin as the inhibitor of signal transduction. We found that statins in addition to lowering cholesterol by inhibiting mevalonate synthesis, inhibit the isoprenylation of heterotrimeric G proteins and small GTP-binding proteins belonging to the family of signaling molecules Ras, Rho, Rap, and Rab GTPases responsible for the control of trafficking of a number of membrane proteins (Van Aelst and D’Souza-Schorey 1997). Statins by inhibiting these signaling molecules exhibit pleiotropic effects that may help curing different diseases. Statins can also prevent Rab-GTPase proteins that mediate PKC-dependent signaling (Köhnke et al. 2013), suggesting that patients whose diseases are associated with chronic PKC activation may also benefit from statins (Ronzier et al. 2019). The mechanism of antibacterial action of statins on bacteria is not well understood (Hennessy et al. 2016). However, several studies have reported the pleiotropic effects of statins on the growth and virulence of bacterial pathogens such as those involved in sepsis and pneumonia (Chopra et al. 2012; Hennessy et al. 2016). It has been demonstrated that consumption of statins with antibiotics increases the efficacy of treatment of bacterial infections by inhibiting production of virulence factors such as toxin or biofilm (Hennessy et al. 2016). Furthermore, the antibacterial effect of rifampin against M. tuberculosis and M. leprae infection was enhanced when used combined with statins (Lobato et al. 2014). Statins also inhibited the invasion of E. coli into bladder epithelial cells (Martinez and Hultgren 2002) that could be due to inhibition of Rho GTPase signaling (Goldstein and Brown 1990).
Results of this study suggest that CS signaling that was sensed by H. pylori surface receptors resulted in spiral transformation into m-coccoids with altered biological activities, including enhanced resistance to stressful conditions. Our results propose that there should be more than one signaling system for initiating expression of these activities. One candidate signaling system could be QS that is dependent on the increased bacterial density to release effective concentrations of autoinducer-2 (AI-2) (Rader et al. 2007). Furthermore, additional signaling systems could exist in H. pylori such as G-proteins and PKCs that were initiated with CS. In this study, these cascades of signaling were inhibited by ATV at early steps (0–48 h) of growth but not at later steps (54–72 h). It has been indicated that CS is directly involved in activation of PKC isoform eta (PKC-eta) which is expressed predominantly in epithelial tissues including stomach (Kuroki et al. 2000), playing roles in cell proliferation, differentiation and death (Zurgil et al. 2014). However, PKC-eta is resistant to translocation and down regulation when stimulated by cancer promoter phorbol esters or CS (Murakami et al. 1996).
Conclusions
For successful surface-associated lifestyle, bacteria certainly need to sense and respond to surface-related signals (Janczarek et al. 2018). It has been suggested that sensing signals from the host can exert selective pressure on bacteria that transiently or chronically colonize humans to cause disease or establish commensalism (Yang et al. 2016). When searching for the properties of H. pylori as members of Epsilonproteobacteri, it seems logical to go back millions of years when H. pylori as a marine bacterium evolved to adapt to their aquatic niche by acquiring abilities for surface attachment, rapid replication, flagellation and motility, biofilm formation and stress resistance. In aquatic environments, H. pylori may have become dependent on water-soluble CS for initiating their signaling mechanisms for adapting to harsh conditions of marine water or human stomach. Results of this study suggest that CS initiated transformation of H. pylori to m-coccoids with altered biological properties. This transformation not only involved QS but also statin-sensitive signaling systems such as PKC group and G-proteins. It would be interesting to study the effect of statins on this transformation in vivo. It is concluded that access to CS that initiates bacterial adaptive responses to human stomach could be regarded as an evolutionary privilege that facilitates persistent colonization of H. pylori in gastric environment, despite immune responses and turnover of gastric epithelium. CS availability in gastric environment may determine the fate of H. pylori colonization in human stomach as spiral or m-coccoid colonizers, the consequences of their colonization as symbionts or pathogens and therapeutic strategies for bacterial eradication.
Supplementary Information
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