Biofilm and Quorum Sensing in Helicobacter pylori

Abstract

Helicobacter pylori (H. pylori) is a gram-negative bacterium living in the human gastrointestinal tract considered as the most common cause of gastritis. H. pylori was listed as the main risk factor for gastric cancer. Triple therapy consisting of a proton pump inhibitor and combinations of antibiotics is the main treatment used. However, this line of therapy has proven less effective mainly due to biofilm formation. Bacteria can regulate and synchronize the expression of multiple genes involved in virulence, toxin production, motility, chemotaxis, and biofilm formation by quorum sensing (QS), thus contributing to antimicrobial resistance. Henceforth, the inhibition of QS called quorum quenching (QQ) is a promising target and alternative to fight H. pylori resistance to antimicrobials. Many phytochemicals as well as synthetic compounds acting as quorum quenchers in H. pylori were described in vitro and in vivo. Otherwise, many other compounds known as quorum quenchers in other species and inhibitors of biofilm formation in H. pylori could act as quorum quenchers in H. pylori. Here, we summarize and discuss the latest findings on H. pylori’s biofilm formation, QS sensing, and QQ mechanisms.

1. Introduction

Helicobacter pylori (H. pylori) is a microaerophilic, spiral-shaped, gram-negative bacterium that belongs to Epsilonproteobacteria [1]. H. pylori establishes about 50% life-long infections. While it is asymptomatic in 85% of cases, individuals with chronic gastritis linked to H. pylori have a 10–20% chance to develop peptic ulcers and 1% chance to develop gastric carcinoma [2]. Barry Marshall and Robin Warren were the first to successfully isolate and culture H. pylori from the human stomach in 1983 [3]. The pair later conducted self-ingestion experiments that confirmed H. pylori’s colonization of the human stomach, thereby inducing inflammation of the gastric mucosa. Marshall first reported the development of persistent gastritis after ingestion, which was treated with doxycycline and bismuth subsalicylate [4]. These findings promoted more research, which ended up showing that high amount of H. pylori in the stomach promotes multiple gastrointestinal troubles, including chronic gastritis, peptic ulcer disease, gastric mucosa-associated lymphoid tissue (MALT) lymphoma, and gastric cancer [3].

In the early 1980s, Robin Warren and Barry Marshall showed for the first time that a bacterium named H. pylori could be associated with cancer development. In 2005, the Nobel Prize in Physiology or Medicine was awarded to R. Warren and B. Marshall for the “discovery of the bacterium H. pylori and its role in gastritis and peptic ulcer disease.”

Furthermore, the International Agency for Research on Cancer classified H. pylori in group 1 of carcinogens [5]. It has been shown that H. pylori infection may as well be correlated with insulin resistance, the increase of total and low-density lipoprotein cholesterol, and the decrease of high-density lipoprotein [6]. Due to differences in socioeconomic and hygienic conditions, H. pylori prevalence varies between and within countries. In general, it is estimated to range from 85–95% in developing countries and between 30% and 50% in developed countries [7]. The prevalence of the infection cannot be summarized in a single figure due to unreliable diagnostic methods in some regions, poor representation of some countries, and differences in data quality [8].

Currently, the first line therapy used to treat H. pylori infection is a combination of proton pump inhibitors (PPIs) with amoxicillin or metronidazole and clarithromycin. This triple therapy fails in about 20–30% of cases, requiring the use of a quadruple therapy consisting of a PPI, bismuth, tetracycline, and metronidazole [9, 10]. Nevertheless, an alarming increase in multidrug-resistant strains of H. pylori to ampicillin, penicillin, co-amoxiclav, amoxicillin, clarithromycin, metronidazole, tetracycline, doxycycline, erythromycin, and doxycycline has been reported [11, 12, 13]. This is ascribed to antibiotic abuse, therapeutic failures, and phenotypical mechanisms promoting resistance and/or tolerance to antimicrobials, notably, biofilm formation [14, 15]. Biofilm formation is a process in which organisms firmly adhere to abiotic, and/or biotic surfaces then grow together to form a complex community that often forms a special structure through four stages: (i) reversible bacterial adhesion; (ii) irreversible adhesion; (iii) formation and maturation of matrix; and (iv) dispersal of cells [16]. Biofilms mainly consist of extracellular polymeric substances composed of polysaccharides, proteins, nucleic acids, and lipids forming a protective barrier against adverse conditions and decreasing the penetration of antibiotics [17]. In H. pylori, flagella play a major role in biofilm formation in the gastrointestinal tract [18].

Most bacteria use quorum sensing (QS) as a communication system, relying on the secretion and perception of small molecules called auto-inducers (AIs) [19, 20]. The QS system can activate and/or regulate gene expression of many phenotypes that can be problematic for humans, i.e., biofilm formation, so that bacteria as a group can jointly cope with changes in the surrounding environment, resulting in adverse consequences such as drug resistance and virulence [21, 22]. A new tactic for outsmarting bacteria called quorum quenching (QQ) is currently explored to reduce their virulence without interfering with their growth, causing less Darwinian selection pressure for bacterial resistance [23]. This paradigm shift has become a promising antibacterial strategy, which not only prevents the development of antimicrobial resistance but also the disturbance of human gastrointestinal microflora, as well as the prevention of adverse side effects commonly associated with the available treatment [24]. Since the main steps of QS are the production and detection of signal molecules, QQ can interfere with this system in different ways, either intracellularly or extracellularly by application of inhibitors of AI biosynthesis and perception [25], application of AI antagonists (mimicking AIs), chemical inactivation of AI, sequestering antibodies [26] or macromolecules such as cyclodextrins [27], and degrading enzymes [28]. This strategy showed promising effect in vitro and in vivo, as well as synergistic effects with antibiotics by increasing bacterial susceptibility to antibiotics [29].

Here, we summarize the biofilm formation regulated by the QS system involved in the antimicrobial resistance in H. pylori. Meanwhile, we also provide the latest development of QS inhibitors (QSIs) or QQ enzymes (QQEs) as a potential strategy for the design of new antimicrobial agents to manage H. pylori infections.

2. Biofilm formation in H. pylori

Biofilms have been recognized as a microbial sessile community, irreversibly attached to either animate and inanimate objects [30]. Biofilms are contained in a self-produced extracellular polysaccharide (EPS) layer. This matrix is commonly rich in proteins including enzymes, polysaccharides (1–2%), nucleic acids (<1%), and water (up to 97%) [31]. Temperature, pH, osmolarity, UV radiation, desiccation, oxygen tension, and nutrient availability are all environmental stressors that directly affect the phenotype of biofilms [16, 32]. In vitro analyses have further confirmed that H. pylori biofilms reduce drug permeability and decrease the susceptibility to antibiotics. In fact, cells in the bacterial biofilm are 10–100 times more resistant toward antimicrobial agents than cells in a planktonic state [33, 34]. H. pylori colonizing the stomach has developed three patterns of drug resistance, including single drug resistance (SDR), heteroresistance (HR), and multidrug resistance (MDR), which probably overlap and are linked in their molecular mechanisms and their clinical implications [35, 36, 37, 38, 39, 40, 41, 42].

In the human stomach, H. pylori biofilms are found on the surface of gastric mucosa. Once introduced into the stomach, H. pylori appears in a spiral form, which is very mobile and associated with the colonization of new niches [43, 44, 45, 46]. Subsequently, it comes into contact with the mucin layer that covers the epithelial cells, resulting in tension-dependent adhesion between the mucin and H. pylori [47]. After an efficient adhesion and multiplication, a morphological transformation occurs, which is accompanied by the creation of multiple shapes (spiral, rod, curved, coccoid, and filamentous forms) to establish a biofilm [48]. However, in the case of prolonged colonization, all biofilm cells eventually transform into a coccoid form involved in survival and greater tolerance to adverse environmental factors [49, 50]. Biofilm formation in H. pylori involves many factors shown in Table 1.

Factors	References
Flagella and pili	[18]
Outer membrane vesicles (OMV)	[43]
Extracellular DNA (e-ADN)	[43]
Adhesin (outer membrane proteins namely Hop & Hom)	[51]
Lipopolysaccharides (LPS)	[52]
Flagellar proteins	[52]
Efflux pumps	[53]
Enzymes regulating pH (urease and arginase)	[54]
luxS gene	[54]
Chemoreceptors	[54]
Toxin-antitoxin system proteins	[55]
H. pylori neutrophil-activating protein (HP-NAP)	[55, 56]
Mannose-related proteoglycans (proteomannans)	[57]

Table 1.

Factors involved in the formation of biofilms in Helicobacter pylori.

3. Biofilm formation and QS in H. pylori

The discovery of QS in Vibrio fischeri and Vibrio harveyi, two species that achieve bioluminescence using QS signaling molecules, sparked research into this complex signaling system [58]. The regulation of gene expression under QS control was investigated in multiple gram-negative bacteria species, including H. pylori [52, 59, 60]. For H. pylori, QS is involved in motility, biofilm development, and antibiotic resistance [32, 47, 59, 61]. Once biofilm formation is elicited from planktonic cells, the aggregated cells surrounded with extracellular polymeric substances (EPS) modify their phenotype, exchange genetic material, produce AI, and provide physical protection [33]. Owing to the formation of biofilms, H. pylori infections became typically persistent and rarely resolved by traditional antimicrobial therapies [34].

Overall, the QS system includes the following steps: (i) AI production; (ii) excretion of AI to the surrounding environment; (iii) sensing and binding of the AI to receptors at high cell density; (iv) retrieval of the receptor-signal complex from the cell and its binding to the promoter region; and (v) activation of genes expression [62, 63]. There are four different signals involved in QS. The most common are N-acyl homoserine lactones (AHLs), also known as autoinducer-1 (AI-1), which are fatty acid derivatives produced and used by gram-negative bacteria [64], while gram-positive bacteria use peptides or modified peptides. Furanosyl borate diesters or autoinducer-2 (AI-2) are derived from the recycling of S-adenosyl-homocysteine and used by both gram-positive and gram-negative bacteria [64]. There is also the autoinducer-3 (AI-3), which allows the cross-talking with mammalian epinephrine host cell signaling systems [65].

H. pylori, when located in the gastric mucosa, responds to several specific chemical signals. The chemotactic response is mediated by chemoreceptors called chemotaxis proteins [59]. H. pylori genome encodes four chemoreceptors: TlpA (effector; arginine, bicarbonate), TlpB (effector; AI2, urea, hydroxyurea, formamide acetamide.), TlpC (effector; unknown), and TlpD (effector; hydrogen peroxide) [66]. The H. pylori QS network involves the chemoreceptor TlpB responding to the AI-2 signaling molecule, a class of furanosyl borate diesters synthesized by the LuxS protein [59, 66] (Figure 1). The 4,5-dihydroxy-2,3-pentanedione (DPD), which is the precursor of AI-2 in H. pylori, is produced by LuxS protein [67]. First, LuxS produces the homocysteine through the cleavage of S-ribosylhomocysteine (SRH), which is a part of the S-adenosylmethionine (SAM) pathway. The process involves two main enzymes, i.e., 5′-methylthioadenosine/adenosylhomocysteine nucleosidase (MTAN) and metalloenzyme [68]. The DPD generated is rearranged into an assortment of chemically related molecules known as AI-2 through a process of dehydration and cyclization [69]. Usually, there are two types of chemoreceptor binding to their AIs, either through direct binding with AI or through interactions with AI binding proteins that transduce signals to the chemoreceptor [70]. In H. pylori, TlpB does not bind to AI-2 in vitro with high affinity and requires two periplasmic binding proteins, AibA and AibB, which bind to AI-2 independently. AibA and AibB are conserved at greater than 95% identity at the amino acid sequence level in all species of H. pylori [32]. The structures of AibA and AibB are not yet elucidated. However, protein sequence homology identifies AibA as homologous to dipeptide binding proteins (39% identity to E. coli dipeptide binding protein (PDB ID: 1DPP) and AibB as homologous to proteins of E. coli molybdate binding (36% identity to the periplasmic molybdate binding protein of Azotobacter vinelandii (PDB ID: 1ATG) [32].

Figure 1.

QS in Helicobacter pylori: LuxS produces AI-2 from the methyl cycle. At high cell density, high concentration of AI-2 in the environment bind to TlpB to active chemotaxis. The binding to the periplasmic proteins AibA and AibB active chemorepulsion. Moreover, AI-2 signals upstream of FlhA manages the branching pathways of gene expression under control of FlgS, FlgM, and σ28 proteins.

The QS system regulates several mechanisms to assure H. pylori colonization in the harsh conditions of the stomach. These include flagellar motility, chemotaxis, and the cag pathogenicity island (Cag PAI) expression, which are all involved in biofilm formation [18, 32, 60]. This indicates that the QS system regulates the various stages of biofilm development from the initial adhesion to the final detachment of the cells [46, 69]. The deletion of luxS gene altered the expression of flagellar genes, i.e., flaA, flaE, flhA, and fliI [69]. Otherwise, the addition of AI-2 or DPD restored the altered phenotype and transcription of these genes. This evidenced that AI-2 is involved in flagellar morphology in H. pylori as it influences the first steps of the flagellar gene expression (Figure 1) [69]. The presence of flagella provides motility that enhances the recruitment of planktonic cells to the biofilm, a crucial step in biofilm formation [18].

CagA protein, encoded by cag PAI, has been identified to be induced in H. pylori biofilms [54]. A significant decrease in biofilm biomass was observed following mutations in cagA and cag PAI, confirming its important role in biofilm formation [52]. The QS system regulates the cag PAI through its repression by AI-2, which, in turn, attenuates inflammatory response [60]. The type IV secretion system (T4SS), also encoded by cag PAI, is essential in direct cell–cell contact [71]. It is believed that this direct cell–cell contact can also control the biofilm behavior in H. pylori [33]. While cag PAI is involved in bacteria-host interaction, it could also be involved in H. pylori bacteria-bacteria interaction, as well as biofilm formation. Besides, bacterial outer membrane proteins (OMPs) are crucial for ion transport, osmotic stability, bacterial virulence, and adherence. Adhesion to gastric cell mediated by Omp18, a peptidoglycan-associated lipoprotein precursor, was reported in H. pylori [72]. After adhesion, the cell envelope gene (lpxD) is upregulated [73]. H. pylori urease enzyme (ureA) is important for pH regulation; it prevents the acidification of the biofilm, increasing its stability [74, 75]. Thus, omp18, lpxD, and ureA genes could be directly involved in H. pylori biofilm formation [76].

4. QQ in H. pylori

In H. pylori, AI-2 has been involved in the regulation of motility, type IV secretion, and, most importantly, biofilm formation [32]. The QS plays a critical role in multidrug resistance of H. pylori by upregulating both biofilm-associated matrix and efflux pump genes to improve bacterial resistance [77]. Cells in the bacterial biofilm are 100–1000 times more resistant toward antimicrobials than cells in a planktonic state [34]. The inhibition of QS results in a decrease in biofilm formation, making bacteria more susceptible [78].

Since the main component of QS is the production and detection of signal molecules, QQ can interfere with this system in different ways, either intracellularly or extracellularly. It includes: (i) the inhibition of signal synthesis; (ii) the inhibition of signal transmission; (iii) the enzymatic degradation of AI; and (iv) the inhibition of signal detection [25, 28] (Figure 2). These strategies showed promising effect in vitro and in vivo, as well as synergistic effects with traditional antibacterial treatments by increasing bacterial susceptibility to antibiotics [79].

Figure 2.

Different ways to inhibit QS in Helicobacter pylori.

To date, few H. pylori QSIs were described, whether synthetic or produced by living organisms, such as plants, animals, and bacteria [80, 81, 82]. Flavonoids, i.e., naringenin, quercetin, myricetin, baicalein, catechin, flavone, and turmeric, exhibited promising antibiofilm and antiadhesive properties against H. pylori [83, 84, 85, 86, 87, 88] (Table 2). Notably, a study conducted to assess the effect of Acorus calamus on H. pylori cultures demonstrated strong antibiofilm and antiadhesive properties [89]. Molecular interaction studies were later performed by the same group of researchers through molecular docking of β-sitosterol, a phytobioactive component of A. calamus, toward QS proteins ToxB, DnaA, PhnB, and Sip. Exceptionally high binding affinity and molecular interaction were exhibited, linking the antibiofilm properties of A. calamus to the inhibition of QS proteins by β-sitosterol [89]. The most direct and effective way to inhibit the QS system is the enzymatic degradation of the QS molecules, which stops signal transduction [93]. In gram-negative bacteria, two types of hydrolases were described, namely, AHL-lactonase and AHL-acylase. Today, few studies investigated the enzymatic lysis of QS signals in H. pylori. By degrading AHL produced by H. pylori, N-acylhomoserine lactonase produced by Bacillus licheniformis inhibited the biofilm formation and attenuate virulence [90].

Quencher	Effect on H. pylori	Test	Mechanism of QQ	Reference
β-sitosterol (Acorus calamus)	Antibiofilm, Antibacterial	in silico & in vitro	AI-2 antagonist	[89]
N-acylhomoserine lactonase (Bacillus licheniformis)	Antibiofilm & antibacterial	in vitro	Degradation of AHL (Ais)	[90]
Methylthio-DADMe-immucillin-A	MTAN inhibitor	in silico	Binding to the MTAN target	[91]
Parachlorophenylthio-DADMe-immucillin-A	MTAN inhibitor	in silico	Binding to the MTAN target	[91]
-SH Furanosyl Borate Diester	Antibiofilm, Antibacterial^2	in silico	AI-2 antagonist	[92]

Table 2.

QSIs and QQEs in Helicobacter pylori.

Another effective way to inhibit QS is the blockage of signaling cascade through the inactivation of downstream response regulators. The precursor SRH of AI-2 results from the action of MTAN on SAH. The inhibition of MTAN induces an accumulation of 5-methylthioadenosine (MTA) and SAH, which, in turn, inhibits AI-2 production [91, 94]. In silico testing of DADMe-ImmA derivatives further confirmed this as a viable QQ technique, since it displayed MTAN inhibition by tight binding to the receptor [95]. More in silico studies investigated the possibility of designing furanosyl borate diester derivatives from its pharmacophore modeling by substituting the –OH groups of AI-2 and DPD by -SH making it a potent competitive inhibitor to AI-2 [92].

Based on previous studies, various phytochemicals from medicinal plants with known antibiofilm activity could act via inhibition of QS in H. pylori (Table 3). Baicalin from medicinal plants exhibited, in vivo, bactericidal and antiadhesive activities as well as limited urease production and reduced vacA gene expression, leading to virulence reduction. Baicalin limited the bacterial adhesion and colonization and enhanced bacterial sensitivity via suppression of urease and blockage of the sulfhydryl group. This makes Baicalin a potential quorum quencher in H. pylori [83, 88]. Quercetin from Vitis rotundifolia inhibited the growth of H. pylori [84], while in P. aeruginosa, quercetin inhibited AHL production suggesting its action through QQ against H. pylori. In parallel, catechin was described as a quorum quencher in P. fluorescens suggesting its potential inhibition of QS in H. pylori. Catechin from Chamomilla recutita inhibited the growth of H. pylori and urease production in H. pylori (which increases bacterial sensitivity) as well as caused membrane disruption [86]. Naringenin produced by Hibiscus rosa sinensis showed a potent bactericidal effect to MDR bacteria and also the inhibition of growth and biofilm formation in H. pylori [96]. Moreover, naringenin exhibited a potent competition with AHL for binding in P. aeruginosa. Taken together, it seems that naringenin inhibits biofilm formation in H. pylori by acting as quorum quencher. Turmeric (Curcuma longa) exhibited a good antibiofilm effect toward H. pylori [97, 101]. Besides, turmeric decreased AHL production in Aeromonas sobria and limited interaction with LuxI-type synthases and downregulated LuxI-type and LuxR-type genes in various bacterial species. This makes turmeric a potential quencher toward H. pylori. Vaccinium oxycoccus produces proanthocyanidins with antibiofilm and bacteriostatic activities against H. pylori [98]. Proanthocyanidins also limited the siallylactose-specific (S-fimbriae) adhesion of H. pylori to human mucus, erythrocytes, and gastric epithelial cells. In P. aeruginosa, proanthocyanidins was shown to inhibit AI production and to limit the activation of QS transcriptional regulators. Taken together, proanthocyanidins could be considered as a potent quorum quencher in H. pylori.

Molecule	Effect on H. pylori	Test	Possible mechanism	Reference
Baicalin	Antibiofilm; Adhesion inhibition; Bactericidal; Virulence reduction; Urease inhibition	in vivo	Reduction of binding and colonization; Suppression urease and blockade of sulfhydryl group.	[83, 88]
Quercetin; (V. rotundifolia)	Antibiofilm; Growth inhibition	in vitro	QSI in P. aeruginosa	[84]
Catechin; (Chamomilla recutita)	Antibiofilm; Growth inhibition Urease inhibition Membrane disruption	in vivo	QSI in P. fluorescens	[86]
Naringenin; (H. rosa sinensis)	Antibiofilm Bactericidal	in vitro	QSI in P. aeruginosa	[96]
Turmeric; (C. longa)	Antibiofilm Antiadhesive Immunostimulant (igG toward H. pylori)	in vitro	Inhibition of AHL production in A. sobria; Interaction with LuxI; Down-regulation of LuxI-type & LuxR	[97, 98]
Proantho-cyanidins; (Vaccinium oxycoccus)	Antibiofilm, Bacteriostatic,; Inhibits siallylactose-specific (S-fimbriae)	in vitro & in vivo	Inhibition of AHL production; Anti-QS regulators in P. aeruginosa	[98]
Emodin; (A. vera)	Antibiofilm; Antiadhesion; Affects n-acetyl transferase	in vitro	Inhibition of the HefA gene	[99]
Niclosamide	Antibiofilm; Bacteriostatic, Decreasing the secretion of IL-8,; Disruption of H. pylori proton motive force.	in vitro &; in vivo	QSI in P. aeruginosa; Affects transcription of QS genes in P. aeruginosa	[100]

Table 3.

Inhibitors of biofilm formation potentially via inhibition of QS in Helicobacter pylori.

5. Conclusion

Despite the advancements in the medical field, the treatment of H. pylori infections has lost its efficacy. H. pylori QS-mediated behavior is the main contributor to bacterial survival and pathogenicity. The significance of bacterial communication in the expression of pathogenic factors makes QS a great target to treat H. pylori infection or increase antibiotic efficacy by synergy. In the past two decades, researchers have discovered plenty of QSI agents that can prevent biofilm formation and decrease virulence. The development of new QSI/QQE that can be combined with antibiotics has been a hot topic in the antibacterial research field. More studies are required to demonstrate their mechanisms of action and the optimal doses of the QS inhibitory compounds that are safe and effective.

Conflict of interest

The authors declare no conflict of interest.