Chronic Salmonella Typhi Infection and Gallbladder Cancer
Introduction
Chronic S. Typhi (S. enterica enterica serovar Typhi) infection is correlated with gallbladder cancer. This rod shaped, flagellated, aerobic, Gram-negative bacterium is a pathogenic serovar of the S. enterica enterica subspecies. It is restricted to humans and reported to cause 21 million acute cases of typhoid fever annually, with 200,000 fatalities. It invades the mucosal surface of the intestine but spreads to deeper tissues such as liver, spleen, and bone marrow following phagocytosis by macrophages.1 Bacteria can also spread to the gallbladder via ducts from the liver.2 They are transmitted between individuals mainly through feces.
A small percentage of the individuals who suffer an acute infection—about 3-5%—become asymptomatic carriers whose infections persist for many years following the acute illness. S. Typhi achieves this persistent carrier state by creating biofilms on the cholesterol-based gallstones residing within the gallbladder. Unsurprisingly, over 90% of chronic carriers have gallstones.2
This chronic typhoid carrier state can lead to chronic inflammation of the gallbladder, in which the bacteria metabolize primary bile acids to produce potentially carcinogenic toxins and metabolites. One such carcinogen is bacterial β-glucuronidase, which, like many of the other secondary bile acids that are produced from bacterial enzyme processing and concentration in the gallbladder, is mutagenic.4 The result is carcinoma of the gallbladder epithelium.3 One defining feature of the gallbladder is its efficacy in concentrating not only bile salts but also toxins—an effect that amplifies their mutagenic effects so that carcinoma develops here instead of in other organs in chronic S. Typhi infection.
Epidemiologically, the chronic typhoid carrier state has been demonstrated to be the single most important risk factor for development of gallbladder cancer—which is generally very rare—in patients with cholesterol-based gallstones.3
Role of cholesterol gallstones and biofilms in chronic S. Typhi carriage
S. Typhi colonizes the gallbladder and persists in an asymptomatic carrier state.5 This carriage is facilitated by formation of biofilms on cholesterol gallstones.6 Crawford et al (2010) induced gallstones in mice then infected them and controls with S. Typhi. They found that significant amounts of S. Typhi could be recovered from the gallstones of mice with gallstones. These were induced by feeding with a lithogenic diet, which was supplemented with 1% cholesterol and 0.5% cholic acid. Bacteria could furthermore be recovered in stool samples of the mice with cholesterol gallstones—but not the control mice—for over a year. Significantly, it also took much longer for the mice with gallstones to recover from acute S. Typhi infection. Consistent results were obtained in vitro, affirming the specific role of cholesterol in this process.6
The S. Typhi O-antigen capsule is crucial to the specific binding affinity between the bacteria and cholesterol as well as the biofilm formation process. The genes involved in the process of creating this capsule are upregulated when S. Typhi is grown in a concentrated bile environment. The O-antigen capsule plays a structural role in the EPS matrix of the biofilm and has accordingly been identified as is a potential therapeutic target.7 It is crucial to successful carriage, aids the bacteria in adapting to its host, and has been characterized in multiple studies.8
Mechanisms of S. Typhi carcinogenicity in the gallbladder: how chronic carriage can lead to cancer
Bile as a mutagen of S. Typhi
Bile salts are able to damage DNA, which has a mutagenic effect on S. Typhi bacteria that allows them to thrive in the gallbladder. 9 One proposed mechanism through S. Typhi is mutated relates to oxidative damage related to the conjugation that characterizes bile salt chemistry. A main gallbladder function is to concentrate bile. This provides S. Typhi with an increased concentration of possible substrate mutations(15%???). Thus, this factor could select for S. Typhi survival within the gallbladder niche environment through genome rearrangements and subsequent polymorphisms that select for changes resulting in increased fitness of S. Typhi in chronic carriers.9 Analysis of the frequency of S. Typhi genome rearrangements post exposure to bile salts shows that these mutations tend to occur in several common locations and have particular manifestations. Bile, particularly in very high concentrations, can act as a mutagen in vivo for S. Typhi for a variety of particular gene targets including DNA adenine methylase, which works to decrease bile sensitivity and reduce rate of mutation in normal bacteria. This can lead to further mutations. 10 Although bile is not generally characterized as a strong mutagen, the high concentration and long exposure time are demonstrated to contribute to mutagenic potential and lead to cancerous changes of the gallbladder epithelium.10 Furthermore, by metabolizing these salts and also the cholesterol of the gallstones, S. Typhi bacteria themselves are capable of producing compounds that are mutagenic to the gallbladder epithelium.9
Chemical mechanisms of S. Typhi mutagenic action
Bile salt metabolism produces carcinogenic compounds in long-term S. Typhi carriers. Chemical mechanisms have been proposed. For example, the action of the β-glucuronidase enzyme, which leads to deconjugation of conjugated toxins and bile acids in this niche under high concentrations, could render them carcinogenic to the host.4 Another mechanism that leads to mutagenicity in the host cells depends on β-glucuronidase activity.12 β-glucuronidase metabolizes bile salt substrates—a process that leads to the production of a high-energy, active intermediates due to their aromaticity and conjugation. This intermediate, in turn, binds to DNA and has mutagenic potential in S. Typhi itself.13
Another proposed mechanism of mutagenicity relates to interactions with the cholesterol that
form the structural basis of the gallstones. Bacteria that colonize the gut can not only alter bile salts to a secondary bile form but also convert the cholesterol itself into carcinogenic compounds, including cholesterol 5alpha,6alpha-epoxide.11 Additional studies demonstrated that S. Typhi bacteria are capable of metabolizing cholesterol with multiple pathways to form mutagenic cholic acid derivative forms in the presence of bile and cholesterol.12
Other mechanisms including the S. Typhi genotoxin Cytolethal Distending Toxin B (CDT) may also play a role in this mutagenic and pathogenic process. This toxin is encoded by S. Typhi but is able to create DNA lesions in target cells—including, potentially, cells of human hosts.4,14
Epidemiological links between chronic S. Typhi carriage and gallbladder cancer
From biochemistry to epidemiology
The relationship between chronic S. Typhi carriage and gallbladder cancer has been researched and characterized in studies conducted in sites worldwide, including sites such as India, Scotland, the United States, and Mexico. These data include the link among cholesterol-based gallstones, S. Typhi carriage, and biofilm presence. One major case-control study performed in India—an endemic typhoid region—found that gallbladder cancer patients had significantly higher incidences of S. Typhi than controls and cholelithiasis patients did, at 29.4%. Furthermore, the risk of developing gallbladder carcinomas in typhoid carriers was 8.47 times higher than it was in non-carriers.15 Furthermore, as found in a study conducted in Mexico, typhoid carriage and biofilms were identified in 4.9% of surgically removed gallstones, but neither was present without the other—thus corroborating the idea that the mechanism of carriage is biofilm formation.6 The specificity of the niche gallbladder environment and the long time frame are key aspects of S. Typhi mutagenicity. A study conducted in Scotland thirty years following the 1964 typhoid outbreak found 16% of acutely infected individuals to be chronic carriers. Furthermore, these individuals were 167 times more likely to develop gallbladder cancer than were the patients who had suffered acute infections but not chronic carriage. Although risk of other cancers of digestive system organs was also elevated, this elevation was one to two orders of magnitude less intense by 1-2 orders on a logarithmic scale, thus stressing the specificity of gallbladder conditions.16 An earlier study conducted with diverse American populations corroborates this specificity, suggesting that variations of bile salts acting as a carcinogen within the gallbladder, bile duct, and small bowel. The most marked finding, however, was that individuals identified as chronic typhoid carriers died of hepatobiliary cancer six times more often—a significant difference—than the control subjects did. 17
Specificity of association cancer of the gallbladder as opposed to other pathologies
Bacterial degradation of primary bile acids in the gallbladder has been described be a factor in S. Typhi carcinogenesis in gallbladder carcinoma patients. One study found that, when patients with gallbladder carcinoma were compared to patients presenting only with gallstones, S. Typhi bacteria were identified in the bile of 40% of the gallbladder carcinoma patients and 30% of the cholelithiasis patients. However, cancer patients but not cholelithiasis patients had significantly elevated secondary bile acid levels—specifically, lithocholate and deoxycholate, which have both been linked to carcinogenicity in humans. Although gallbladder carcinoma patients consistently had higher secondary bile levels than cholelithiasis patients did regardless of S. Typhi carriage state, the cancer patients with bacteria in the bile had significantly higher secondary bile acid levels than the non-carrier cancer patients did.18
Conclusion
Long-term S. Typhi carriers tend to be asymptomatic—though highly contagious—and have a significantly elevated risk of developing gallbladder carcinoma. S. Typhi bacteria survive in the gallbladder niche by forming biofilms on cholesterol gallstones. The mutagenic effects of bile salts on S. Typhi further facilitate its survival, which is initially favored by structural components such as the O-antigen capsule, over long periods of time. Furthermore, S. Typhi itself has a mutagenic effect on the host through metabolism of bile salts into carcinogenic secondary bile compounds and other genotoxic effects. This connection has been characterized pathologically and epidemiologically by studies performed on a global scale, in which the chronic S. Typhi carrier state constitutes a key risk factor for gallbladder carcinoma.
References
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Edited by Hannah Moore, a student of Nora Sullivan in BIOL187S (Microbial Life) in The Keck Science Department of the Claremont Colleges Spring 2013.
