Helicobacter pylori is a gram-negative, flagellated, microaerophilic bacterium that selectively colonizes the gastric mucosa. H. pylori is one of the most common infectious agents worldwide, and approximately 50% of the world's population is estimated to be infected (Marshall and Warren, 1983, 1984). Although the detailed transmission route of H. pylori remains uncertain, an oral-oral or fecal-oral route during childhood is thought to be the most plausible method of human-to-human transmission (Goh et al., 2011). Once established, H. pylori has no significant bacterial competitors (Peek and Blaser, 2002). The prevalence of H. pylori infection varies widely by geographic area, age, race, and socioeconomic status (SES), and developing appear to have higher infection rates than developed countries (Brown, 2000). Indeed, the prevalence exhibits country-to-country variation, with values as low as 15.4% in Australia to values as high as 90% in developing countries such as Iran (Moujaber et al., 2008; Hosseini et al., 2012; Siao and Somsouk, 2014). H. pylori is unique in that the bacterium can persist for decades in the harsh stomach environment, where it damages the gastric mucosa and alters the pattern of gastric hormone release, thereby affecting gastric physiology (Wang et al., 2014). The slow development of cancer known as Correa's cascade (Correa, 1992) includes a series of intermediate stages (precancerous lesions) before malignancy per se occurs. These precancerous lesions occur in the following order: gastritis, atrophy, intestinal metaplasia (IM), and eventually dysplasia. H. pylori represents the most significant risk factor for gastric malignant tumors (Wang et al., 2014). Gastric cancer (GC) is an insidious disease, with symptoms that often manifest at an advanced stage, a time when the few remaining therapeutic options have low efficiency (Boreiri et al., 2013). Approximately 10% of infected individuals develop severe gastric lesions, such as those in peptic ulcer disease; 1–3% progress to GC, with a low 5-year survival rate (Cirak et al., 2007), and 0–1% develop mucosa-associated lymphoid tissue (MALT) lymphoma (Noto and Peek, 2012; Parreira et al., 2013; Wang et al., 2014). Compared with uninfected individuals, individuals infected with H. pylori are estimated to have a 2–8-fold increased risk of developing GC (Huang et al., 1998; Eslick et al., 1999; Helicobacter and Cancer Collaborative Group, 2001; Kamangar et al., 2006; Wang et al., 2007), and the International Agency for Research on Cancer (IARC) has classified H. pylori as a class I carcinogen (IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, 1994). However, the inability of the immune system to clear H. pylori infection is not well described. Furthermore, the mechanisms controlling the induction and maintenance of H. pylori-induced chronic inflammation are only partly understood.

In the past, H. pylori was considered to be a non-invasive bacterium that generally only adhered to gastric mucosa epithelial cells or survived in the gastric lumen. In contrast, recent studies have shown that H. pylori is invasive, and it is now regarded as a special intracellular pathogen (Petersen and Krogfelt, 2003; Dubois and Borén, 2007). H. pylori microcolonies form on the surface of the cell membrane, and this area then becomes the microenvironment for bacterial reproduction (Tan et al., 2009). H. pylori can invade cells and replicate to reproduce and complete an entire biological cycle by cell division (Chu et al., 2010). Nonetheless, the mechanism of invasion remains unclear, and studies to date have mainly concentrated on receptor-mediated endocytosis and a tyrosine kinase-dependent process (Evans et al., 1992; Birkness et al., 1996; Su et al., 1999; Kwok et al., 2002). A phagosome forms after H. pylori invades gastric epithelial cells; the bacterium then exits cells to colonize again while conditions are suitable and repeatedly infects cells. These findings suggest that invasion might play an important role in disease induction, immune escape, and chronic infection (Kwok et al., 2002; Dubois and Borén, 2007; Jang et al., 2013). In this “cellular internalization” process, a bacterium specifically binds to a host cell receptor and enters the cytoplasm via phagocytic vacuoles formed through cell membrane invagination. Therefore, to provide insight into the pathogenic mechanism of H. pylori, this article will review relevant studies and describe evidence for the adhesion and invasion of gastric mucosa epithelial cells by this bacterium.

Within the gastric mucus layer, bacteria can be found relatively close to the gastric lumen or deep within the gastric glands, and these microbes can either swim freely (Hazell et al., 1986; Schreiber et al., 2004; Celli et al., 2009) or attach to gastric epithelial cells (Hessey et al., 1990). To achieve successful engraftment, the most important step in H. pylori infection, the bacterial cells must survive under various unfavorable conditions, such as exposure to pepsin and an extremely low pH. Engraftment occurs primarily when adhesion molecules or other molecules on the surface of H. pylori bind to mucins, allowing the bacterial cells to colonize the gastric mucosa epithelium. This event triggers expression of several bacterial genes, including some that encode virulence factors and protect the pathogen from clearance mechanisms, such as liquid flow, peristaltic movement or shedding of the mucous layer (Kim et al., 2004). This process includes at least two steps: (1) H. pylori quickly moves through the mucus layer to the surface of the gastric mucosa, which has a relatively neutral pH, under the aegis of the pH buffer mechanism; and (2) H. pylori firmly adheres to gastric mucosa epithelial cells via the outer membrane protein (OMP).

Mucins, which are high molecular weight, heavily glycosylated glycoproteins secreted by epithelial cells, play a key role in the adhesion process. Mucins are located on the surface of the stomach cavity and are the main components of the gastric mucosa epithelial mucus layer. At this location, mucins form a barrier in the mucosal defense system that protects gastric epithelial cells against chemical, enzymatic, microbial, and mechanical damage. Mucin-1 (MUC-1), Mucin-5AC (MUC-5AC), and Mucin-6 (MUC-6) are expressed in the gastric mucosa of normal adults, and the genes encoding these mucins are located on different chromosomes. The main structures formed by mucins are protein scaffolds based on the core peptide, the typical structure of which is a variable number of tandem repeats (VNTRs) rich in serine, threonine and proline, which are potential glycosylation sites. One study has to date demonstrated that MUC-5AC and MUC-6 are separated in the gastric mucus gel, resulting in a layered linear array (Ho et al., 2004). The former is mainly found on the surface and bottom of the mucus gel, and the latter is present among the various layers. This natural stratification of mucins increases the viscosity of gastric mucosa epithelial mucus gel and provides an independent system to completely protect gastric mucosa epithelial cells. Therefore, H. pylori must pass through mucins to successfully adhere to to host cells.

Both urease and flagella-mediated, chemosensory-directed motility are essential and relevant to the process of colonization (Eaton et al., 1991, 1996; Nakamura et al., 1998; Rolig et al., 2012). H. pylori secretes a large amount of urease, and the surface-bound enzyme catalyzes hydrolysis of urea to generate ammonia and bicarbonate, which are then released into the cytosol and periplasm, forming a neutral environment around the bacterial cells (Weeks et al., 2000). This process decreases the viscosity and elastic modulus of the mucus layer, which changes from a gel to a viscous solution with increasing pH, thereby facilitating the passage of the bacteria through the mucus. Ammonia protects the metabolic activity of H. pylori, which remains at 50–60% of its normal activity level in the highly acidic environment of pH 2.5 (Celli et al., 2009; Follmer, 2010). Microscopy studies of the motility of H. pylori in the gastric mucosa at acidic and neutral pH values in the absence of urea showed that the bacteria swim freely at high pH but are highly constrained at low pH (Celli et al., 2009). From a hydrodynamics viewpoint, the shape of the H. pylori cell can also affect its swimming speed. Previous studies have suggested that H. pylori possesses numerous long flagella that may allow the cell to swim through the sticky viscoelastic mucus gel in a manner similar to how a screw passes through a cork (Berg and Turner, 1979; Karim et al., 1998). Swimming speeds also decrease with increasing viscosity of the polymer solution (Worku et al., 1999). The results of Martínez et al. (2016) are consistent with the above observations. On this basis, these authors provide an in-depth, quantitative analysis of H. pylori's natural variation in helical cell and flagellum morphology, indicating that both cell shape and flagellum number independently affect swimming speed in viscous environments, with flagellum number contributing to a greater degree. In addition, as for other commensal microbes that colonize a host over long periods of time, H. pylori must sense and integrate many signals emanating from the gastric epithelium that attract the microbes to the cell surface for colonization and persistence. This ability to move in response to chemical cues, i.e., chemotaxis, is determined by the core signaling complex proteins CheW, CheA, and CheY (Beier et al., 1997; Foynes et al., 2000; Pittman et al., 2001). H. pylori also has three membrane-bound chemoreceptors, TlpA, TlpB, and TlpC, and one cytoplasmic chemoreceptor, TlpD (Lertsethtakarn et al., 2011). Rolig et al. (2012) found that different regions of the stomach contain unique chemotactic signals. For example, in the corpus, H. pylori utilizes chemotaxis for initial localization but not for subsequent growth. In contrast, in the antrum and the corpus-antrum transition zone, chemotaxis does not help initial colonization but does promote subsequent proliferation. The main chemoreceptor that allows H. pylori to thrive in the antrum is TlpD, with the other chemoreceptors playing minor roles. Thus, chemotaxis may be necessary to locate the antrum or to maintain colonization at this site. Indeed, chemotaxis of H. pylori toward the urea present on the epithelial cell surface may also be crucial for survival in the stomach (Nakamura et al., 1998). It was recently demonstrated that H. pylori swims toward injured epithelia, suggesting that the bacterial cells are attracted to host-derived molecules (Aihara et al., 2014). The main bacterial chemoreceptor responsible for this chemoattraction is TlpB, and urea has been identified as the host metabolite that attracts H. pylori (Huang et al., 2015). In addition, Huang et al. (2015) revealed a function for H. pylori urease in facilitating sensitive detection of urea at concentrations as low as 50 nanomolar. Therefore, H. pylori has evolved a sensitive urea chemodetection and destruction system involving a high-affinity chemoreceptor-ligand interaction that functions at the nanomolar concentrations created locally by urease, allowing the bacterium to dynamically and locally modify the host environment to locate the epithelium. Overall, we believe that the ability of H. pylori to bore through the mucus gel might be achieved in two ways: flagella and chemotaxis provide some contribution, but changes in the rheological properties of the environment are also important.

Adhesins, which attach to the surface of the bacterium, play a vital role because they can recognize the structures of glycans expressed in the gastric mucosa, and H. pylori must swim through the mucus gel (Ilver et al., 1998; Mahdavi et al., 2002). Using adhesins, H. pylori can identify peptidoglycans on the surface of gastric epithelial cells, molecules that are mainly found on mucins in the gastric mucus gel. Furthermore, any H. pylori cell that does not adhere to an epithelial cell would be quickly removed from the epithelial cell surface and the mucus gel. At present, it is generally believed that OMPs are the main adhesins of H. pylori. Among these adhesins, blood group antigen-binding adhesion (BabA) can identify the difucosylated ABO/Lewis b (LeB) antigen present on red blood cells and gastrointestinal mucosa epithelial cells. In normal gastric tissue, LeB-binding strains always bind to MUC-5AC- and LeB-positive epithelial cells (Van de Bovenkamp et al., 2003). Moreover, the BabA-Leb interaction is important not only for H. pylori to adhere to the stomach surface but also to anchor the bacterial secretion system to the host cell surface for effective injection of bacterial factors into the host cell cytosol, which is the cause of clinical outcomes. In other words, once BabA binds to LeB, Type IV secretion system (TFSS)-dependent host cell signaling is triggered to induce the transcription of genes that enhance inflammation, intestinal metaplasia development, and associated precancerous transformation (Ishijima et al., 2011). Another adhesin is sialic acid-binding adhesion (SabA), which can bind to the antigens Lewis X (sLex) and Lewis a (sLea). Lewis antigens are common in infected and inflamed gastric mucosa (Lindén et al., 2008). SabA expression can rapidly respond to changing conditions in the stomach or in different regions of the stomach that permit H. pylori to adapt to varying microenvironments or host immune responses to ensure long-term colonization and infection (Goodwin et al., 2008). Accordingly, SabA-mediated adherence is positively correlated with the sLex concentration in vitro (Lindén et al., 2004). During persistent infection and chronic inflammation, H. pylori triggers an alteration in the glycosylation patterns in the gastric mucosa, including upregulation of inflammation-associated sLex antigens, and H. pylori is likely to adhere to the gastric mucosa with SabA. In addition, SabA interacting with the sLex antigen can enhance H. pylori colonization in patients with weak or no Leb expression (Yamaoka, 2008). SabA production is indeed reported to be associated with severe intestinal metaplasia, gastric atrophy, and the development of gastric cancer (Yamaoka et al., 2006). However, de Klerk et al. (2016) also found certain Lactobacillus strains could inhibit SabA expression at the transcriptional level by releasing an effector molecule into the medium, further affecting H. pylori binding capacity and thereby reducing its adherence. In particular, Lactobacillus, a well-known member of the normal microbiota in the human gastrointestinal tract, has always been studied in relation to H. pylori but largely as a possible supplement for antibiotic treatment (Patel et al., 2014), and interference with H. pylori colonization may contribute to the molecular mechanism for this association (de Klerk et al., 2016). AlpA and AlpB as OMPs also play a role in adherence to host cells and tissues (Odenbreit et al., 1999, 2002a). The alpAB locus, which is ubiquitously present in H. pylori strains, is expressed during infections (Rokbi et al., 2001; Odenbreit et al., 2009), and alpAB mutants are defective in adherence to human gastric tissue sections (Odenbreit et al., 1999). One study demonstrated that laminin is the host receptor of both AlpA and AlpB, and H. pylori deficient in these factors causes more severe inflammation than the isogenic wild-type strain in gerbils (Senkovich et al., 2011). The reason may be that the alpAB locus influences host cell signaling and cytokine production (Odenbreit et al., 2002a,b; Loke et al., 2007; Lu et al., 2007). H. pylori CagL protein is a specialized adhesin that is targeted to the pilus surface, where it binds to and activates integrin alpha5beta1 receptor on gastric epithelial cells through an arginine-glycine-aspartate motif. This interaction can mediate receptor-dependent delivery of CagA into gastric epithelial cells and may assist in certain intracellular signaling events.(Kwok et al., 2007; Barden et al., 2013). Current evidence suggests that MUC-1 in fact inhibits H. pylori colonization in vitro and in vivo (McGuckin et al., 2007; Lindén et al., 2009). Lindén et al. (2009) showed that MUC1 could inhibit H. pylori binding to epithelial cells, a process that occurs through both BabA and SabA. When the pathogen fails to bind to MUC1, the mucin sterically inhibits adhesion to other potential cell surface ligands. However, when the pathogen does bind to MUC1, the extracellular domain of the mucin is released from the epithelial surface and acts as a releasable decoy to prevent prolonged adherence. H. pylori also can modulate host cell glycosylation patterns to enhance adhesion. A highly pathogenic strain was able to alter expression of β3GlcNAcT5 (β3GnT5), a GlcNAc transferase essential for the biosynthesis of Lewis antigens, to increase sLex expression and H. pylori adhesion in human gastric carcinoma cell lines (Marcos et al., 2008). Furthermore, although the urease and alkyl hydroperoxide reductase (AhpC) located on the surface of H. pylori are not OMPs, these enzymes still have good affinity for stomach mucins because they are components of the membrane (Nilsson et al., 2000). Loke et al. (2007) confirmed that both polysaccharides and mucin could bind to AhpC and UreA, so polysaccharides may function as a potential anti-adhesive agent against H. pylori colonization of gastric mucin by competing for mucin-binding sites. This result indirectly indicated the probable role of these two H. pylori proteins in colonization. Other adhesins, such as HopZ and OipA, are also involved in the adhesion process, though the specific ligands need to be further clarified.

In the gastric mucosa, the basement membrane is separated from the gastric lumen by only a single cell layer, and H. pylori can swim freely in the mucus layer in close contact with the epithelial cells, preferentially at the apical side of intercellular contacts (Hazell et al., 1986; Necchi et al., 2007; Tan et al., 2009). Any damage to the epithelial cell layer will expose extracellular matrix proteins, and disruptions of tight junctions by H. pylori will also allow the bacterial cells access to the basement membrane (Necchi et al., 2007). H. pylori targeting of adherens junctions may be beneficial for colonization and persistence at the host epithelial surface, and it may also cause abnormal receptor activation and stimulation of signaling pathways involved in inflammation, proliferation, migration, and invasion, which will have detrimental consequences and result in disease development (Costa et al., 2013).

H. pylori is the main pathogenic bacteria of chronic active gastritis, peptic ulcers, gastric mucosa-associated lymphoid tissue lymphoma, and gastric cancer. This bacterium seeks refuge inside cells when the external environment is not suitable, and it can also complete an entire biological cycle, including proliferation and apoptosis, within gastric epithelial cells. Furthermore, once the external conditions become suitable, undegraded H. pylori cells leave the host cell and can be released to infect other cells and cause repeated infection. The above findings suggest that invasion plays an important role in the induction of disease, immune escape and chronic infection. Moreover, these results provide a new direction for research on the pathogenic mechanism of H. pylori-associated gastric diseases. The latest consensus recommendations for treating H. pylori infection are non-bismuth quadruple therapy and traditional bismuth quadruple therapy as first-line strategies (Fallone et al., 2016). To date, studies on the reasons for eradication failure have concentrated on antibiotic resistance. However, H. pylori internalization provides a new research focus. The antibiotics in the recommended regimens include both cell membrane-penetrating antibiotics, such as clarithromycin or metronidazole, and antibiotics that do not penetrate the cell membrane, such as amoxicillin. Increasing the concentration and treatment time of cell membrane-penetrating antibiotics that effectively penetrate epithelial cells to kill intracellular H. pylori might help in achieving complete eradication.

YH, QW, DC, WX, and NL contributed equally to this review.

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