Bacterial Responses To The Host In Vitro Approaches

Due to the complex nature of host-specific and chemical stress conditions that are met by bacteria in the GI tract many studies describe the in vitro response of intestinal bacteria to a simplified model that mimics (a component of) the stress encountered in the host's GI tract.

Historically, these studies have been performed in pathogens, including studies describing the response towards acid stress in enteropathogenic bacteria such as Salmonella and Escherichia coli, which revealed that RpoS, Fur, PhoP, and OmpR are important pH-response regulators (10). More recent studies describe food-grade bacteria and their tolerance to acid stress. These studies have focused mainly on physiological aspects such as determination of levels of acid-tolerance (11,12). Changes in protein synthesis during acid adaptation have been studied in Propionibacterium freudenreichii using 2D-gel electrophoresis, indicating an important role in the early acid tolerance response for a biotin carboxyl carrier protein and enzymes involved in DNA synthesis and repair, as well as a role in the late response for the universal chaperones GroEL and GroES (13).

Several studies describe the defense mechanisms of Gram-negative enteric bacteria towards bile acids, which include the synthesis of porins, transport proteins, efflux pumps and lipopolysaccharides (14). In addition, a few genome-wide approaches aiming at the identification of proteins important for bile salt resistance in Gram-positive bacteria have been described. In Propionibacterium freudenreichii, Listeria monocytogenes and Enterococcus faecalis differential proteome analysis using 2D-gel electrophoresis led to the identification of several proteins that were expressed at a higher level in the presence of bile salts relative to control conditions lacking bile salts (15-17). In Propionibacterium freudenreichii these bile-induced proteins were further analyzed by N-terminal sequencing and peptide mass fingerprinting, leading to the identification of 11 proteins important in bile stress response. The induced proteins include general stress proteins such as ClpB and the chaperons DnaK and Hsp20 (16). Analogously, a subset of the proteins identified in E. faecalis appeared to be inducible by multiple sublethal stresses, including heat, ethanol, and alkaline pH (18). The fact that these general stress proteins are induced by bile is in agreement with the cross protection against bile after thermal or detergent pre-treatment that has been observed in several bacteria, including Enterococcus faecalis, Listeria monocytogenes and Bifidobacterium adolescentis (15,19,20). Moreover, in Escherichia coli an rpoS mutant failed to develop starvation-mediated cross protection after in vitro mimicking of osmotic, oxidative, and heat stresses (21). Two other bile-induced proteins in Propionibacterium freudenreichii are the superoxide dismutase and cysteine synthase, which could be involved in the protection against the oxidative stress imposed on Propionibacterium freudenreichii by bile. In addition, other studies describe the oxidative stress response of GI-tract organisms, including Campylobacter coli,

Escherichia coli and several Shigella species (21-23). A deletion mutant in the gene encoding superoxide dismutase in Campylobacter coli displayed poor survival and colonization during infection of an animal model (23). Moreover, proteins involved in signal sensing and transduction, and an alternative sigma factor appeared to be bile-inducible (16). Next to these proteomic approaches, random gene disruption strategies have been applied to Listeria monocytogenes and Enterococcus faecalis, resulting in strains that are more susceptible to bile salts than the wild-type strains. Subsequent genetic analysis of the mutants revealed that the disrupted genes encode diverse functions, including an efflux pump homologue (19) and genes involved in oxidative stress response, and cell wall and fatty acid biosynthesis (24). In Lactobacillus plantarum a genetic screen resulted in the identification of 31 genes of which the expression appeared to be induced by bile. In analogy with the random gene disruption strategies applied in other species, this genetic screen in L. plantarum led to the observation that efflux pumps and changes in the architecture in the cell envelope are important for bile resistance of these bacteria (25). Moreover, these findings are in agreement with several physiological studies in Gl-tract bacteria such as L. plantarum, Propionibacterium freudenreichii and L. reuteri that demonstrated that bile salts induce severe changes in the morphology of the cell membrane and/or cell wall of these organisms (Fig. 1) (16,25,26).

Overall, the aforementioned in vitro experiments have provided insight in the response of specific bacteria towards components of the complex mixture of stress conditions that is met by these bacteria during residence in or transit through the GI tract of their hosts. Although these approaches have helped to unravel the response of specific micro-organisms towards certain GI-tract conditions, they will not suffice to describe their behavior in the GI tract. The full response repertoire will only be triggered in vivo, where all physicochemical conditions are combined with specific host-microbe and microbe-microbe interactions. Therefore, more sophisticated approaches have aimed at the development of tools that allow the in vivo identification of genes that are important in the GI tract.

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