Proteomic View of Interactions of Shiga Toxin-Producing Escherichia coli with the Intestinal Environment in Gnotobiotic Piglets

Abstract

Background: Shiga toxin (Stx)-producing Escherichia coli cause severe intestinal infections involving colonization of epithelial Peyer's patches and formation of attachment/effacement (A/E) lesions. These lesions trigger leukocyte infiltration followed by inflammation and intestinal hemorrhage. Systems biology, which explores the crosstalk of Stx-producing Escherichia coli with the in vivo host environment, may elucidate novel molecular pathogenesis aspects.

Methodology/principal findings: Enterohemorrhagic E. coli strain 86-24 produces Shiga toxin-2 and belongs to the serotype O157:H7. Bacterial cells were scrapped from stationary phase cultures (the in vitro condition) and used to infect gnotobiotic piglets via intestinal lavage. Bacterial cells isolated from the piglets' guts constituted the in vivo condition. Cell lysates were subjected to quantitative 2D gel and shotgun proteomic analyses, revealing metabolic shifts towards anaerobic energy generation, changes in carbon utilization, phosphate and ammonia starvation, and high activity of a glutamate decarboxylase acid resistance system in vivo. Increased abundance of pyridine nucleotide transhydrogenase (PntA and PntB) suggested in vivo shortage of intracellular NADPH. Abundance changes of proteins implicated in lipopolysaccharide biosynthesis (LpxC, ArnA, the predicted acyltransferase L7029) and outer membrane (OM) assembly (LptD, MlaA, MlaC) suggested bacterial cell surface modulation in response to activated host defenses. Indeed, there was evidence for interactions of innate immunity-associated proteins secreted into the intestines (GP340, REG3-γ, resistin, lithostathine, and trefoil factor 3) with the bacterial cell envelope.

Significance: Proteomic analysis afforded insights into system-wide adaptations of strain 86-24 to a hostile intestinal milieu, including responses to limited nutrients and cofactor supplies, intracellular acidification, and reactive nitrogen and oxygen species-mediated stress. Protein and lipopolysaccharide compositions of the OM were altered. Enhanced expression of type III secretion system effectors correlated with a metabolic shift back to a more aerobic milieu in vivo. Apparent pathogen pattern recognition molecules from piglet intestinal secretions adhered strongly to the bacterial cell surface.

Figure 1. Global adaptation of EHEC cells to the intestinal milieu.

Fifteen biological role categories, as defined in Dataset S1, are displayed in the graph. The bar length represents the sum of APEX_i quantities of all proteins with a statistically significant abundance change (in vitro versus in vivo) assigned to a given biological role category. Blue bars represent the in vitro (cell culture) growth, red bars the in vivo (intestinal) environments.

Figure 2. Metabolic pathways active in intestinal EHEC cells.

From the left to the right: import and metabolism of glycerol derivatives and dicarboxylic acids; import of mono- and disaccharides and their entry into the glycolytic pathway (GP); metabolism of ascorbic acid; mixed acid fermentation utilizing GP products; high affinity phosphate and ammonia import and metabolism; NADPH generation and menaquinone/menaquinol cycle; glutamate decarboxylase acid resistance system (Gad); oxygen-dependent and -independent electron transport chains; *periplasmic electron transporters. Symbols/color codes: substrates and products of pathways are denoted in black script, proteins in red script (if increased in abundance in vivo), in blue script (if there was no abundance change but high expression in vitro and in vivo), in green script (if decreased in abundance in vivo); full arrows indicate a catalytic step, dotted arrows a transport step. In the top right corner, seven global transcriptional regulators are depicted; their color-coded symbols are included in the graphic to illustrate where they influence gene expression. The area between the green line (outer membrane) and inner line (inner membrane) represents the periplasmic space.

Figure 3. Changes in the EHEC cell envelope during effacement of the host environment and molecular responses initiated by the piglet host.

At the top of the schematic, generally from left to right: Sus scrofa proteins identified as adhesion factors to the bacterial surface; proteins implicated in capsular poly-N-acetylglucosamine (PNAG) and lipopolysaccharide (LPS) biosynthesis; proteins implicated in β-barrel OM protein synthesis (YaeT and NlpB), lipoprotein export (LolA, LolB and LolD), OM asymmetry (Imp, MlaA and MlaC); T2SS and T3SS effectors and some of the established interactions with host cells and the extracellular matrix (StcE, EspA/B, Eae/Tir and OmpA); putative peptidoglycan-modulating proteins (YdhA and YkfE). Below from left to right: various extracytoplasmic stress responses; regulators of cell envelope stress and the connection to acetyl phosphate (Ac-P) signaling; tryptophan/indole biosynthesis; ROS and NOS mediated stress responses by peroxidases, catalases, dismutases and NO-detoxifying enzymes.