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ABSTRACT

The human peptide LL-37 is a cationic peptide with antimicrobial activity against both Gram-positive and Gram-negative microorganisms. It has been shown to protect against gastritis caused by Helicobacter pylori infection. Most of the current synthetic expression systems for LL-37 depend on the construction of soluble fusion partners to avoid cytotoxic effects of the antimicrobial peptide in the E. coli host strain. However, the fusion systems require additional cleavage steps using enzymatic or chemical methods, which makes them impossible to express an active LL-37 peptide in vivo. In order to create a resistant host that can export LL-37 to the media, we intend to overexpress the E. coli marRAB operon, which activates the AcrAB-TolC efflux pump, a mechanism that has been related with resistance to this and similar antimicrobial peptides by expulsion. Our aim is to create a system in which E. coli expels LL-37 only when H. pylori and other pathogenic bacteria are present. In order to do this, we are using the LsrA promoter, which allows transcription in presence of AI-2, a molecule produced by bacteria to communicate via quorum-sensing.

THE PROBLEM

Helicobacter pylori colonizes the stomach of 50% of the world's human population throughout their lifetimes. Colonization with this organism is the main risk factor for peptic ulceration as well as for gastric adenocarcinoma and gastric MALT (mucosa-associated lymphoid tissue) lymphoma. Treatment for H. pylori has revolutionized the management of peptic ulcer disease, providing a permanent cure in most cases. Such treatment also represents first-line therapy for patients with low-grade gastric MALT lymphoma. Treatment of H. pylori is of no benefit in the treatment of gastric adenocarcinoma, but prevention of H. pylori colonization could potentially prevent gastric malignancy and peptic ulceration [1,2].

Etiologic Agent

H. pylori is a gram-negative bacillus that has naturally colonized humans for at least 50,000 years—and probably throughout human evolution. It lives in gastric mucus, with a small proportion of the bacteria adherent to the mucosa and possibly a very small number of the organisms entering cells or penetrating the mucosa; its distribution is never systemic. Its spiral shape and flagella render H. pylori motile in the mucus environment. The organism has several acid-resistance mechanisms, most notably a highly expressed urease that catalyzes urea hydrolysis to produce buffering ammonia. H. pylori is microaerophilic (requiring low levels of oxygen), is slow-growing, and requires complex growth media in vitro. Publication of several complete genomic sequences of H. pylori since 1997 has led to significant advances in the understanding of the organism's biology [3].
A very small proportion of gastric Helicobacter infections are due to species other than H. pylori, possibly acquired as zoonoses. Whether these non-pylori gastric helicobacters cause disease remains controversial. In immunocompromised hosts, several nongastric (intestinal) Helicobacter species can cause disease with clinical features resembling those of Campylobacter infections.

Epidemiology

The prevalence of H. pylori among adults is 30% in the United States and other developed countries as opposed to >80% in most developing countries. In the United States, prevalence varies with age: 50% of 60-year-old persons, 20% of 30-year-old persons, and <10% of children are colonized.H. pylori is usually acquired in childhood. The age association is due mostly to a birth-cohort effect whereby current 60-year-olds were more commonly colonized as children than are current children. Spontaneous acquisition or loss of H. pylori in adulthood is uncommon. Other strong risk factors for H. pylori colonization are markers of crowding and maternal colonization. The low incidence among children in developed countries at present is due, at least in part, to decreased maternal colonization and increased use of antibiotics.
Humans are the only important reservoir of H. pylori. Children may acquire the organism from their parents (more often from the mother) or from other children. Whether transmission takes place more often by the fecal-oral or the oral-oral route is unknown, but H. pylori is easily cultured from vomitus and gastroesophageal refluxate and is less easily cultured from stool.
In Mexico, seroprevalence studies were positive in 20% of children under 1 year, 50% at 10 years and 80% in those over 25 years [11, 12].

Pathology and Pathogenesis

H. pylori colonization induces a tissue response in the stomach, chronic superficial gastritis, which includes infiltration of the mucosa by both mononuclear and polymorphonuclear cells. (The term gastritis should be used specifically to describe histologic features; it has also been used to describe endoscopic appearances and even symptoms, which do not correlate with microscopic findings or even with the presence of H. pylori.) Although H. pylori is capable of numerous adaptations that prevent excessive stimulation of the immune system, colonization is accompanied by a considerable persistent immune response, including the production of both local and systemic antibodies as well as cell-mediated responses. However, these responses are ineffective in clearing the bacterium. This inefficient clearing appears to be due in part to H. pylori's downregulation of the immune system, which fosters its own persistence.
The pattern of gastric inflammation is associated with disease risk: antral-predominant gastritis is most closely linked with duodenal ulceration, whereas pangastritis is linked with gastric ulceration and adenocarcinoma. This difference probably explains why patients with duodenal ulceration are not at high risk of developing gastric adenocarcinoma later in life, despite being colonized by H. pylori.
How gastric colonization causes duodenal ulceration is now becoming clearer. H. pylori–induced inflammation diminishes the number of somatostatin-producing D cells. Since somatostatin inhibits gastrin release, gastrin levels are higher than in H. pylori–negative persons, and these higher levels lead to increased meal-stimulated acid secretion in the gastric corpus, which is only mildly inflamed in antral-predominant gastritis. How this increases duodenal ulcer risk remains controversial, but the increased acid secretion may contribute to the formation of the potentially protective gastric metaplasia found in the duodenum of duodenal ulcer patients. Gastric metaplasia in the duodenum may become colonized by H. pylori and subsequently inflamed and ulcerated.
The pathogenesis of gastric ulceration and that of gastric adenocarcinoma are less well understood, although both conditions arise in association with pan- or corpus-predominant gastritis. The hormonal changes described above still occur, but the inflammation in the gastric corpus means that it produces less acid (hypochlorhydria) despite hypergastrinemia. Gastric ulcers usually occur at the junction of antral and corpus-type mucosa, and this region is particularly inflamed. Gastric cancer probably stems from progressive DNA damage and the survival of abnormal epithelial cell clones. The DNA damage is thought to be due principally to reactive oxygen and nitrogen species arising from inflammatory cells and perhaps in relation to other bacteria that survive in a hypochlorhydric stomach. Longitudinal analyses of gastric biopsy specimens taken years apart from the same patient show that the common intestinal type of gastric adenocarcinoma follows stepwise changes from simple gastritis to gastric atrophy, intestinal metaplasia, and dysplasia. A second, diffuse type of gastric adenocarcinoma may arise directly from chronic gastritis alone.

Pathology and Pathogenesis

H. pylori colonization induces a tissue response in the stomach, chronic superficial gastritis, which includes infiltration of the mucosa by both mononuclear and polymorphonuclear cells. (The term gastritis should be used specifically to describe histologic features; it has also been used to describe endoscopic appearances and even symptoms, which do not correlate with microscopic findings or even with the presence of H. pylori.) Although H. pylori is capable of numerous adaptations that prevent excessive stimulation of the immune system, colonization is accompanied by a considerable persistent immune response, including the production of both local and systemic antibodies as well as cell-mediated responses. However, these responses are ineffective in clearing the bacterium. This inefficient clearing appears to be due in part to H. pylori's downregulation of the immune system, which fosters its own persistence.
The pattern of gastric inflammation is associated with disease risk: antral-predominant gastritis is most closely linked with duodenal ulceration, whereas pangastritis is linked with gastric ulceration and adenocarcinoma. This difference probably explains why patients with duodenal ulceration are not at high risk of developing gastric adenocarcinoma later in life, despite being colonized by H. pylori.
How gastric colonization causes duodenal ulceration is now becoming clearer. H. pylori–induced inflammation diminishes the number of somatostatin-producing D cells. Since somatostatin inhibits gastrin release, gastrin levels are higher than in H. pylori–negative persons, and these higher levels lead to increased meal-stimulated acid secretion in the gastric corpus, which is only mildly inflamed in antral-predominant gastritis. How this increases duodenal ulcer risk remains controversial, but the increased acid secretion may contribute to the formation of the potentially protective gastric metaplasia found in the duodenum of duodenal ulcer patients. Gastric metaplasia in the duodenum may become colonized by H. pylori and subsequently inflamed and ulcerated.
The pathogenesis of gastric ulceration and that of gastric adenocarcinoma are less well understood, although both conditions arise in association with pan- or corpus-predominant gastritis. The hormonal changes described above still occur, but the inflammation in the gastric corpus means that it produces less acid (hypochlorhydria) despite hypergastrinemia. Gastric ulcers usually occur at the junction of antral and corpus-type mucosa, and this region is particularly inflamed. Gastric cancer probably stems from progressive DNA damage and the survival of abnormal epithelial cell clones. The DNA damage is thought to be due principally to reactive oxygen and nitrogen species arising from inflammatory cells and perhaps in relation to other bacteria that survive in a hypochlorhydric stomach. Longitudinal analyses of gastric biopsy specimens taken years apart from the same patient show that the common intestinal type of gastric adenocarcinoma follows stepwise changes from simple gastritis to gastric atrophy, intestinal metaplasia, and dysplasia. A second, diffuse type of gastric adenocarcinoma may arise directly from chronic gastritis alone.

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