Prevalence of Various Helicobacter Pylori Virulence Factors in Patients

Introduction

Helicobacter pylori

Helicobacter pylori, which was earlier classified as Campylobacter pylori, is a gram negative, curve-shaped bacterium (Goodwin and Armstrong, 1990) (Figure 1). It is a capnophilic organism, i.e., it requires high concentrations of CO₂ (typically 5-10%) in the environment to grow. It is also an oxygen-sensitive microaerophilic organism (Cover, 2012). H. pylori was first isolated by Marshall and Warren in 1984 from patients with ulcers (duodenal or gastric) or chronic gastritis (Marshall and Warren, 1984). The bacterium has 4-6 polar sheathed flagella which helps in motility (Goodwin and Armstrong, 1990). An interesting biochemical feature of H. pylori is its ability to secrete urease. Urease is an enzyme which catalyses the production of ammonia and carbonic acid from urea. Production of this enzyme helps the bacteria to survive in the harsh acidic conditions of the stomach where it is usually found (Mobley, 1996). Rapid urease tests (RUTs) and urease breath tests (UBTs) have been developed for easy diagnosis of H. pylori infections (Graham and Miftahussurur, 2018).

Figure 1: Helicobacter pylori. Gram-negative, curved-shaped bacteria with 4-6 polar flagella (McColl, 2010).

H. pylori infections

Since H. pylori is found on the surface of the gastric epithelium, it causes disorders of the upper gastrointestinal tract. Nearly 10 years after this organism was first isolated, the International Agency for Research on Cancer (IARC) classified H. pylori as a group I carcinogen and the primary cause for the development of gastric cancer (1994). There are 3 main disorders of the upper gastrointestinal tract in which H. pylori is a cofactor – ulcers (duodenal and gastric), gastric adenocarcinoma and gastric mucosa-associated lymphoid tissue (MALT) lymphoma. Ulcers occur in 1-10% of infected individuals, around 3% people develop gastric cancer and <0.01% people develop gastric MALT lymphoma; thus, majority of the people infected with H. pylori remain asymptomatic (McColl, 2010). This organism has also been found to be associated with a few non-gastrointestinal tract disorders such as iron deficiency anaemia, cardiovascular diseases and immune thrombocytopenia (Crowe, 2019).

According to a global systemic review conducted in 2015, almost 4.4 billion people in the world were infected with H. pylori. The prevalence of infection was higher in developing nations with 79% people positive for H. pylori in Africa. Developed nations on the other hand had lower prevalence, the lowest being in North America (37.1%). Socioeconomic status and urbanisation are among the various reasons for this huge difference in the prevalence of H. pylori between countries. Another major problem observed in developing countries is the recurrence of infection. There is also a difference in prevalence among different age groups and it has been observed that there is an increase in prevalence with older age (Hooi et al., 2017).

Gastric and duodenal ulcers

H. pylori has been strongly linked to the development of both gastric and duodenal ulcers. Studies which were conducted in the first 10 years after H. pylori was first isolated reported that approximately 90% of all ulcer patients were positive for H. pylori (Kuipers et al., 1995). Peptic ulcers (also called gastric or duodenal ulcers) are mucosal defects, caused due to an infection with H. pylori, with penetrations through the muscularis mucosa. Infection with H. pylori causes inflammation of the gastric mucosa and induces higher secretion of gastrin and gastric acid. Excess acid causes damage to the duodenal mucosa and leads to ulceration. Gastric ulcers are most commonly observed on the lesser curved wall of the stomach while duodenal ulcers occur in areas which are exposed to higher concentrations of gastric acid (duodenal bulb) (McColl, 2010). Gastric ulcers usually occur in individuals over the age of 40 but duodenal ulcers can occur between the age of 20-50. Both host and bacterial factors influence the development of ulcers in the presence of H. pylori. Gastroduodenal ulcers can also be caused by non-steroidal anti-inflammatory drugs (NSAIDs) and are called H. pylori-negative ulcers. Treatment for ulcers usually involves eradication of infection (Kusters et al., 2006).

Gastric cancer

Gastric cancer is the fifth most common form of cancer in the world and is the third leading cause of death by cancers (Jemal et al., 2011). Studies have been conducted which show that pre-malignant lesions can be treated by eradication of H. pylori infections (Wroblewski et al., 2010).

Both genetic and environmental factors play a role in the pathogenesis of gastric cancer and the clinical outcome of H. pylori infections. The risk of developing gastric cancer increases by 2-3 folds in individuals with proinflammatory polymorphisms of certain genes such as interleukin 1-β and IL1 receptor antagonist genes. (El-Omar et al., 2001). Polymorphisms in genes that regulate the TNFα and IL16 are also associated with higher risk of gastric cancer (Gao et al., 2009). Development of gastric cancer is also influenced by environmental factors, diet being an important one. Diets involving higher amounts of salted, smoked or pickled foods, processed carbohydrates (sugars and refined grains) and meat and dried fish contribute to the increased risk of gastric cancer (Chen et al., 2002). Another risk factor which increases the chances of gastric cancer development is smoking. A strong increased risk of developing gastric cancer was observed in patients who smoked and were infected with cagA positive strains of H. pylori (Malfertheiner et al., 2010).

Gastric mucosa-associated lymphoid tissue (MALT) lymphoma

Gastric lymphomas from T cells are rare and almost all lymphomas of the stomach arise from B cells. Approximately 50% cases of gastric lymphoma are of the mucosa-associated lymphoid tissue. One of the main causes of gastric MALT lymphoma is H. pylori related gastritis (Zullo et al., 2014). Countries which have higher prevalence of H. pylori also show higher incidences of gastric cancer and lymphomas. Thus, the incidences of gastric cancer are higher in developing countries than developed countries. Gastric lymphoma can be effectively treated by eradicating the H. pylori infection. Patients treated by this method show long term remissions and hence this could be a potential cure for gastric lymphomas (Fischbach et al., 2004).

Virulence factors

The pathogenicity of the bacteria depends on its virulence factors. These play a major role in determining the clinical outcomes and possibly the treatment outcomes of an infection. H. pylori has various virulence factors which can be divided into 3 broad categories. The first category contains those genes which are only present in some strains. These are called strain specific genes and the best studied example of this category is cagPAI (cytotoxin-associated gene Pathogenicity Island). The next group of genes are those which help the bacteria to adapt under different environmental conditions (phase variable genes). These include the 6 genes which encode for proteins of the outer membrane of the bacteria. The status of these genes changes throughout growth and undergo phase variation. The final category of genes is those whose genotype depends on the strain (variable structures) and an example is the vacA gene (Roesler et al., 2014).

Vacuolating cytotoxin gene (vacA)

The vacA gene is present in all strains of H. pylori and it encodes for a cytotoxin VacA which is delivered in an active form to the host cells. However, the vacuolating activity differs between different strains of H. pylori. This is due to the allelic diversity seen in the 3 main regions of the gene – signal, middle and the recently discovered intermediate region (Camilo et al., 2017) (Figure 2). The N-terminus is encoded by the S region while the C-terminus is encoded by the M region. The intermediate region lies between the signal and middle region. Different combinations of the 2 major alleles of each region exist and this contributes to the degree of virulence of that strain. The vacAs1/m1 strains are capable of inducing vacuolation and considered the most virulent. They are found to be associated with peptic ulcers and gastric cancer. On the other hand, vacAs2/m2 strains show no vacuolating activity and are not virulent. The vacAs1/m2 strains may or may not have the ability to cause vacuolation depending on the allele of I region. The intermediate region plays an important role in the vacuolating activity of a strain as vacAs1/m2/i1 strains can induce vacuolation but s1/m2/i2 strains cannot (Roesler et al., 2014).

Figure 2: vacA gene structure. Signal region encodes the N-terminus while the middle region encodes the C-terminus of the protein. Intermediate region lies between the signal and middle region (Palframan et al., 2012).

VacA protein is a secreted cytotoxin. The polypeptide is cut at both ends before being delivered in an active form to the host cell. Once inside the host cell, it can induce multiple cellular activities leading to the vacuolation of the epithelial cell. The formation of vacuoles occurs because of disruption of the endosomal compartments. It can also cause the gastric epithelial barrier function to disrupt. VacA cytotoxin also targets the mitochondria and decreases its membrane potential leading to release of cytochrome c and ultimately inducing apoptosis. VacA binds to receptor-type protein tyrosine phosphatase which is present on the gastric epithelial cells. The receptor is responsible for regulating cell proliferation and differentiation which could play a role in the development of gastric and duodenal ulcers. While vacA and cagPAI genes are not located in close proximity on the chromosome of H. pylori, there have been reports that both genes can downregulate the effects of the other gene (Wroblewski et al., 2010).

Cag pathogenicity island (CagPAI)

There are 2 broad categories in which all strains of H. pylori can be divided, depending on whether they express the antigens vacuolating cytotoxin and cagA. In mouse models, both strain types show the ability to colonise, but only type I strains cause gastric disorders similar to the ones observed in humans. Thus, only type I strains are found in patients with duodenitis, duodenal ulcers and gastric tumours. Upon genotyping both strain types, it was observed that cagA gene is only present in those strains that are associated with severe forms of gastroduodenal disease, i.e., type I strain, while both strains have vacA gene. Type I strains secrete an active toxin via the type IV secretion system which makes it virulent (Censini et al., 1996). CagPAI (cytotoxin associated gene Pathogenicity Island), is one of the virulence factors of H. pylori which has been extensively studied. 31 genes are encoded by this ~40kb pathogenicity island. These genes encode for the bacterial type IV secretion system (Roesler et al., 2014). CagA, which is an oncoprotein, is delivered into the host gastric epithelial cell via this secretion system. The oncoprotein enters the host cytosol and interacts with ZO-1 and E-cadherin (components of the apical junctional complex) which are present on the cell membrane of host cells. A receptor tyrosine kinase (RTK) signalling cascade is activated when c-Src/Lyn and Abl kinases phosphorylate the tyrosine present in the EPIYA motif of CagA proteins. Loss of cell polarity is induced by this signalling cascade which also increases the invasiveness of the cell. These are both properties of an epithelial to mesenchymal transition (EMT) phenotype. The C-terminal and N-terminal domains of the CagA protein have distinct features but are equally important for the complete functioning of the protein (Jones et al., 2010). Strains which are positive for cagA gene are more pathogenic and induce the secretion of interleukin 1β and hence are found to be closely linked with atrophic gastritis and gastric cancer (Roesler et al., 2014). Another less studied gene present on the cagPAI is the cagE gene which encodes the CagE protein. This protein has shown to increase the production of IL-8, a neutrophil chemoattractant, from host epithelial cells. This could mediate the initial immune response of the host to a H. pylori infection. In a study conducted by Day et al (2000), in the Hospital for Sick Children, Toronto, they observed that 86% of the children with H. pylori infection had CagA positive strains. They also demonstrated that if the infecting H. pylori strain was CagE positive, children were likely to develop peptic ulcer disease (Day et al., 2000).

Outer membrane proteins (OMPs)

Outer membrane proteins are found only in the outer membranes of gram-negative bacteria. These proteins play an important role in the initial colonisation after pathogenic bacteria infects a human host. In H. pylori, OMPs help the organism to attach to the gastric epithelial cells and colonise the stomach (Matsuo et al., 2017). There are more than 30 OMP genes present in the genome of H. pylori which include babA, sabA and oipA (Roesler et al., 2014). The OMPs interact with cell receptors present on the gastric epithelial cells and prevent the bacteria from getting displaced from the stomach by peristaltic movements (Kao et al., 2016).

BabA: The most extensively studied H. pylori OMP is BabA which binds to ABO/Lewis b (Le^b) blood group antigens in the gastric mucosa (Kabamba et al., 2018). The H. pylori strains found in Europe are called “generalist strains” because they can bind to blood group A, B and O. However, in the south American native population where only blood group O is present, the H. pylori strains are called “specialist strains” (Ansari and Yamaoka, 2017). These strains can only bind to blood group O. BabA helps in the translocation of cagA oncoprotein via the type IV secretion system and it also causes inflammation in the stomach (ref).

OipA: Outer inflammatory protein plays a role in the adhesion of bacteria to the gastric epithelial cells. Other functions of OipA are unclear and many studies have been conducted to determine its role in the virulence of H. pylori. Strains with mutated oipA gene show a decrease in the production of IL-8 in the gastric epithelial cell lines. Thus, OipA functions as an inducer of pro-inflammatory response. In a study conducted by Yamaoka et al (2002), they showed that a functional OipA results in a higher density of H. pylori in the stomach and higher inflammation. The status of this gene can also predict whether an infection will lead duodenal ulcers (from gastritis) (Yamaoka et al., 2002). In another study conducted by Tabassam et al (2007), they discovered a novel function of OipA and showed that this protein may be involved in site-specific activation of FAK. The interaction between OipA and cell receptors causes downstream signalling and activates FAK (Tabassam et al., 2008)

Treatment of H. pylori infections

The standard regimen for the treatment of H. pylori infections includes both antimicrobials and antisecretory agents. The antisecretory agents are essential to increase the pH of the gastric environment and create optimal conditions for the antimicrobial agents to exert bactericidal effects (Yang et al., 2014). The antimicrobial agents generally used are clarithromycin, levofloxacin, amoxicillin and metronidazole and their efficacy depends on their plasma concentration. The most commonly used antisecretory agent is a proton pump inhibitor (PPI). PPIs are also the most effective in increasing the gastric pH. Their mechanism of action is inhibition of the gastric acid pump which leads to lower or no secretion of hydrochloric acid. H₂-receptor antagonists are also an alternate option but are not as effective as PPIs (Yang et al., 2014).

The recommended first line treatment for an H. pylori infection is a standard triple therapy consisting of 2 antibiotics (usually clarithromycin and amoxicillin) and 1 PPI for 7-14 days (Kamboj et al., 2017). A successful treatment aims to achieve an eradication rate of 80%, however this rate has dropped below 80% in many countries including Ireland (Brennan et al., 2018). A randomised-control study conducted in Ireland showed that the eradication efficacy of a 7 day standard triple therapy for H. pylori infected patients was 56.8%, which is significantly lower than the desired rate of 80% (Haider et al., 2015).

Antibiotic resistance

One of the main reasons for failure of first line treatment of H. pylori is antibiotic resistance. Antibiotic resistance can either be primary or secondary. Primary resistance occurs in patients with no history of previous H. pylori treatment. When patients develop resistance during the course of treatment it is called secondary resistance. Studies have shown that previous use of antibiotics is linked to primary antibiotic resistance (Smith et al., 2019). World Health Organisation (WHO) has declared research of clarithromycin-resistant H. pylori infection as high priority (Tacconelli et al., 2018).  In a meta-analysis study conducted in all WHO regions, it was observed that the resistance rates of H. pylori to antibiotics are increasing rapidly. In all WHO regions, resistant rates to clarithromycin and metronidazole are more than 15% (Savoldi et al., 2018).  Association between previous use of macrolides and clarithromycin resistance has been observed (Megraud et al., 2013). In a study conducted by Taneike et al, they studied the relationship between cagA genotype and metronidazole resistance in Irish H. pylori strains. They observed that patients who were negative for cagA showed higher rates of metronidazole resistance compared to patients who were cagA positive. Thus, they concluded that one of the reasons for the development of metronidazole resistance could be absence of cagA (Taneike et al., 2009).

Study aims

The aims of this study were to determine the prevalence of various H. pylori virulence factors among patients attending Tallaght University Hospital and to determine whether there is an association between virulence factor genotype and antibiotic resistance. These aims were addressed through the specific objectives below:

• Perform genotyping for the virulence factors genes vacA, cagE, oipA and babA1 using DNA isolated from stomach biopsy of H. pylori-infected patients
• Evaluate the relationship between virulence factor genotype and resistance to clarithromycin and levofloxacin.

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