Piglets around weaning are known to be exceedingly susceptible for gut infections, since they undergo environmental and psychological stress as well as nutritional changes. The postweaning diarrhea, starting around 3–10 days after the piglets are weaned, has been a focus of research due to the high economic losses it entails in pig production (Fairbrother, Nadeau, & Gyles, 2005; Hampson, Woodward, & Connaughton, 1993; Tsiloyiannis, Kyriakis, Vlemmas, & Sarris, 2001).
These losses are not only caused by severe diarrhea and the resulting higher mortality but also by decreased growth performance and reduced weight gain. A characteristic pathomechanism of enteropathogenic Escherichia coli (EPEC) to cause diarrhea in humans and animals is the ability to provoke attaching and effacing lesions (AE lesions) (Bruant et al., 2009; DebRoy & Maddox, 2001; Girard, Batisson, Frankel, Harel, & Fairbrother, 2005; Kim, Kim, Hur, & Lee, 2010; Nataro & Kaper, 1998). AE lesions are histopathological alterations in epithelial cells of the intestine. They are characterized by the effacement of microvilli through rearrangement of the epithelial cell cytoskeleton (Kaper, McDaniel, Jarvis, & Gomez-Duarte, 1997), leading to a pedestal-forming actin accumulation directly beneath the adherent bacteria (Kalman et al., 1999; Kaper
et al., 1997; Kaper, Nataro, & Mobley, 2004). The concurrent destruction of the enteric brush border results in enteric malfunction and diarrhea.
Since infections with EPEC usually occur orally, EPEC, as well as many other bacteria, have evolved a system to cope with low pH conditions using the enzyme glutamic Cell Cycle Compound Library
decarboxylase (Gad), which converts glutamic acid into γ-aminobutyric acid (GABA). The subsequent release of GABA via a GABA-glutamate antiporter represents the factual extrusion of protons and stabilizes the bacteria\'s inner pH milieu (Foster, 2004; Hersh, Farooq, Barstad, Blankenhorn, & Slonczewski, 1996; Richard & Foster, 2003). In a previous study, we have shown that luminal GABA has significant local effects in the small intestine, namely a selective upregulation of mucin 1 (MUC1) (Braun, Sponder, Pieper, Aschenbach, & Deiner, 2015). The MUC1 protein is the best characterized transmembrane mucin and is an important player of intestinal defense. This implies that GABA ingested with vegetable food or produced by bacteria can have a direct stimulating effect on a key component of the mucosal barrier function.
In E. coli two functionally undistinguishable isoforms of Gad are known: GadA and GadB (De Biase, Tramonti, John, & Bossa, 1996; Smith, Kassam, Singh, & Elliott, 1992). Gene expression of gadA and gadB is regulated by the transcription factor gadX, located downstream of gadA. In case of acidic environmental conditions, gadX expression is increased, thereby leading to an upregulation of GadA/B production (Tramonti, Visca, De Canio, Falconi, & De Biase, 2002). As the pH optimum of bacterial Gad activity is discussed to be between 3.8 and 5 (De Biase, Tramonti, Bossa, & Visca, 1999; Shukuya & Schwert, 1960), it can be speculated that the more acidic the environment (e.g. stomach, cecum), the more GABA is produced, thereby solidifying the mucosal barrier. However, growth circumstances have to be considered because the presence of the gadA/B gene transcripts did not necessarily correlate with Gad enzyme activity, especially when cells
were grown under acidic conditions (Castanie-Cornet, Penfound, Smith, Elliott, & Foster, 1999).
In addition to its upregulating effect on the Gad system, GadX has also been shown to downregulate the expression of genes of the locus of enterocyte effacement (LEE) (Shin et al., 2001). It is widely accepted that LEE is largely responsible for the pathogenicity of EPEC as it includes almost all virulence genes necessary for the formation of AE lesions (An et al., 2000; Elliott et al., 1998; McDaniel & Kaper, 1997; McDaniel, Jarvis, Donnenberg, & Kaper, 1995; Shaw, Cleary, Murphy, Frankel, & Knutton, 2005). LEE comprises EscU, a component of the type III secretion system (T3SS), a needle-forming multiprotein complex, which spans through the i