Bacterial adaptation to survive and colonize in the gastrointestinal tract

The intestinal tract of mammals is the most densely populated ecosystem on earth, comprising 10¹⁴ microbes (Ley et al. 2006), belonging to an estimated 500-1000 different species (Xu et al. 2007), with the Bacteroidetes and Firmicutes being the dominant phyla (Eckburg et al. 2005).

The entire gastrointestinal tract is covered by mucosal surfaces that are composed of water and glycoproteins called mucins. This layer is important as a lubricant, as a selective barrier allowing passage of nutrients, and as a defence system that protects the underlying epithelial cells from mechanical damage or entrance of harmful substances and pathogens.

This layer interacts directly with bacteria in the lumen, and represents the first line of defence against bacterial penetration (Derrien et al. 2010). The human intestinal microbiota play many beneficial roles to the host including enhancement of digestive efficiency, promotion of proper immune responses, and limiting pathogen colonization. In return, the mucin provides attachment sites to the bacteria and offers an important carbon source. Importantly, the success of this symbiotic relationship between host and microbe depends on restriction of bacterial penetration of host tissues (Duerkop et al. 2009). The ability to degrade mucin and to use released carbohydrates and amino acids as nutrients has been shown for a number of bacterial species including Lactobacillus rhamnosus GG (Sanchez et al. 2010),

Bifidobacterium species (Ruas-Madiedo et al. 2008; Ruiz et al. 2011), and Akkermansia muciniphila (Derrien et al. 2004). While this ability may be beneficial in healthy symbiontic relationships it also presents a potential problem, since disturbance of the structure and function of the mucus layer can be deleterious for the host and is characteristic for the pathology of many diseases.

While it may seem that mucin affects the microbial ecosystem in the intestinal tract beneficially, other factors may be detrimental for survival. Both commensal and pathogenic bacteria must resist the deleterious actions of a number of potential stress factors present in the intestinal tract in order to survive. These stress factors include low pH, low oxygen levels, nutrient limitations, elevated osmolarity and the presence of bile, a powerful surfactant. The

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ability to quickly sense and respond to these stress factors with appropriate alterations in gene expression and protein activity are crucial for survival (van de Guchte et al. 2002).

LAB have evolved a range of cellular defence mechanisms which allow them to withstand harsh conditions and sudden environmental changes. This includes chaperones to aid protein folding, catalases and superoxide dismutases to combat reactive oxygen species, proton pumps, decarboxylases and transporters to increase intracellular pH following acid exposure, and transport systems to maintain cellular osmolarity (Corcoran et al. 2008).

E. faecalis are robust bacteria that resist many kinds of stress factors, including heat, acid, hydrogen peroxide (H2O2), hyperosmolarity, NaOCl, UV irradiation and bile (Giard et al. 2001). Studies have shown that part of the bacterial stress response is of a general nature, involving general factors such as chaperones, that create simultaneous resistance towards several stress factors (van de Guchte et al. 2002). For inhabitants of the intestinal tract, like enterococci, several stress factors like reactive oxygen species, bile salts, osmolarity and acid are quite severe. One study showed that prolonged exposure to stress factors like bile salts, acid and heat, induced tolerance toward bile salts and acid that was maintained for a longer period compared to the tolerance toward heat, which reflects the conditions in their natural environment (Flahaut et al. 1996).

1.4.2.1 The acid stress response

Intestinal bacteria have to survive the transit from the oral cavity to the intestinal tract, a journey characterized by acidic conditions. In addition, acidic end products as a result of fermentation by LAB accumulate and may locally create unfavourable conditions for many bacteria (van de Guchte et al. 2002). Acid exposure causes intracellular accumulation of protons, which reduces the intracellular pH and affects the transmembrane ∆pH. This alters the PMF, which is required for transport across the membrane (Corcoran et al. 2008). Acid stress can also cause structural damage to the cell membrane, to DNA and to proteins (van de Guchte et al. 2002). A number of proteins have been identified by two-dimentional- (2D) gel electrophoresis as contributing to the acid tolerance of LAB, including chaperones (e.g.

GroEL, GroES, DnaK, ClpE and GrpE), proteins involved in handling oxidative stress (e.g.

superoxide dismutase), and heat shock proteins (Frees et al. 2003). Additional mechanisms potentially associated with acid tolerance relate to DNA repair, changes in the fatty acid composition of the cells (Fozo et al. 2004), alkalization of the external environment,

expression of transcriptional regulators, and alteration of metabolism and responses (Cotter et al. 2003). One important system that contributes to acid tolerance is the F0F1-ATPase. The

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catalytic portion F1 consists of the α, β, γ, δ and ε subunits for ATP hydrolysis, whereas the integral membrane portion F0 includes the a, b and c subunits which function as a channel for proton translocation (Sebald et al. 1982). While this F0F1-ATPase normally is used to convert the PMF to ATP, it may also generate a PMF via proton expulsion at the expense of ATP.

This system is crucial for maintaining pH homeostasis at low pH in LAB (Corcoran et al.

2008). For E. faecalis, it has been shown that the acid stress response is a rather specific response, since no cross-protection has been observed between this stress factor and others (Flahaut et al. 1996; Rince et al. 2003).

1.4.2.2 The bile stress response

Each day the liver secretes one litre of bile into the gastrointestinal tract. Bile affects

phospholipids and proteins of cell membranes and disrupts cellular homeostasis. Furthermore, bile induces secondary structure formation in RNA, induces DNA damage, activates enzymes involved in DNA repair, and alters the conformation of proteins (Begley et al. 2005).

Mechanisms involved in the bile stress response include changes in the fatty acid composition of the cell, expression of bile salt hydrolases that deconjugate bile acids, as well as expression of chaperones and general stress proteins (Corcoran et al. 2008). Studies on E. faecalis have shown that adaptation to bile salts leads to cross-protection towards heat challenge and, to some extent vice versa. This is due to the fact that both types of stress induce production of heat shock proteins. On the basis of this observation it has been claimed that the bile salt and the heat shock responses are closely related in E. faecalis (Flahaut et al. 1996; Rince et al.

2003).

1.4.2.3 The osmotic stress response

Intestinal bacteria are surrounded by nutrient solutions of various osmolarities. Exposure to osmosis results in a decrease in their cytoplasmic water activities which leads to changes in volume and pressure of the cell. Generally, bacteria respond to osmotic stress by increasing the concentration of osmolytes (Csonka et al. 1991). E. faecalis responds to osmotic shock provided by NaCl by an increase in intracellular potassium ion and glycine concentrations (Kunin et al. 1991).

1.4.2.4 The oxidative stress response

Facultatively anaerobic bacteria such as the enterococci do not need oxygen for growth, and the presence of oxygen may be toxic. This toxic effect is attributed to reactive

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oxygen species like hydrogen peroxide (H2O2) and superoxide (O2-) that attack proteins, lipids and nucleic acids (van de Guchte et al. 2002). Interestingly, phagocytic cells in the immune systems of mammals use a mechanism resulting in generation of oxidative stress in their attempts to kill pathogenic bacteria during infections (Klebanoff 1980; Hassett et al. 1989).

For successful infection the bacteria have to respond to this oxidative stress. The genome sequence of E. faecalis V583 reveals several antioxidant defence systems. Using knockouts it has been shown that manganese-containing superoxide dismutase (MnSOD) is induced by oxygen and that this affects the survival of the bacterium inside macrophages due to a better capability to handle reactive oxygen species (Verneuil et al. 2006). Furthermore, despite the fact that E. faecalis is generally considered a catalase negative bacterium, a gene coding for a catalase is present in the genome of E. faecalis V583 and catalase production has indeed been detected when E. faecalis V583 was cultured in the presence of heme (Frankenberg et al.

2002). E. faecalis V583 also seems to have three peroxidases which all are important for the defence against H2O2: a NADH peroxidase which reduces H2O2 to water, an alkyl

hydroperoxide reductase, and a protein (EF2932), which encodes a thiol peroxidase that is part of the regulon controlled by the hydrogen peroxide regulator HypR (La Carbona et al.

2007).