External factors influence intrinsic differences in Stx2e production by Porcine Shiga Toxin-producing Escherichia coli strains

Abstract

Porcine Shiga toxin-producing Escherichia coli (STEC) strains pose significant challenges to the pig industry. The toxins produced by these strains, particularly Shiga toxin subtype 2e (Stx2e), are associated with a range of clinical symptoms such as diarrhoea and oedema disease, which in severe cases result in death. Understanding the factors that influence the production and secretion of Stx2e is crucial to elucidate porcine STEC pathogenesis and to develop effective therapeutic strategies. Therefore, this study aimed to characterize the variability in Stx2e production among different porcine STEC strains and assess the effect of several external factors, including bile acids and antibiotics. Our results highlighted a substantial variation in extracellular Stx2e levels by porcine STEC strains. In addition, bile acids, especially the bile acid deoxycholate, exerted strain-specific effects on these extracellular Stx2e levels. Antibiotics also affected extracellular Stx2e levels with ciprofloxacin and enrofloxacin inducing a substantial increase in toxin production in certain strains. Genome analysis revealed that these strains encode a holin gene downstream of the Stx2e operon. Deleting this holin gene abolished the antibiotic-induced increase in extracellular Stx2e levels, while introducing holin expression in unresponsive strains increased the presence of Stx2e in the extracellular environment. These findings unravel a role for phage holins in Stx2e secretion and highlight the intricate interplay between genetic and environmental factors in regulating Stx2e production in porcine STEC strains. Together, our results offer insights into STEC pathogenesis.

Fig 1. Inter-strain differences in Stx2e production levels – STEC strains were grown overnight in LB and the secreted and total (secreted and intracellular) Stx2e levels in (A) the bacterial supernatants or (B) after cell lysis (0.1 mM polymyxin B) were determined by ELISA, respectively.

The toxin concentration (ng/mL) is plotted for 58 strains (57 STEC strains and one negative control strain, strain 1705). Each symbol represents an independent experiment (n = 3), and bars denote the geometric mean.

Fig 2. Inter-strain difference of genetically distinct STEC strains in extracellular Stx2e levels and their Stx2e operon landscape – (A) Maximum-Likelihood (ML) phylogenetic tree of all included STEC strains (n = 52), including strain 1705 as negative control and outgroup (red).

Bootstraps <95 are indicated next to branches. Strains that responded to fluoroquinolones are indicated with a green circle. Underlined strains were used in an in-depth characterization of the impact of external factors (see above) and strains highlighted in green were used in holin deletion experiments. (B) Bar chart of extracellular Stx2e levels (ng/mL) linked to their phylogenetic relatedness. Bars represent the mean, errors bars represent the sd (n = 3). (C) Graphical representation (clinker plot) of the 10,000 bp flanking sequences at both sides of the stx2e operon highlighting immediate up- and downstream annotated coding sequences (CDS). For each differing genomic landscape (i.e., different up- and/or downstream CDS), a clustering/group number was given, ranging from 1-16. MTase = Methyltransferase. Stx2eB (E46*) represent a mutation in the stx2eB gene resulting in the introduction of a stop codon. (D) Positional amino acid (AA) conservation of polymorphic sites within the Stx2eA protein. All other positions showed 100% AA conservations.

Fig 3. Influence of bile acids on extracellular Stx2e levels – STEC strains were grown overnight in LB medium with either 0.3% bovine bile (A), 0.3% porcine bile (adult) (B), 0.3% bile obtained from recently weaned piglets (C), or 3 separate bile acids each at 0.15% (D; cholate (♦), deoxycholate(▼), glycocholate(•)).

The Stx2e levels in the bacterial culture supernatants were determined by ELISA. Strains that showed a response were divided into two groups: decreased (A,B,C1) or increased (A,B,C2) extracellular Stx2e levels. The three strains used for testing the separate bile salts were strains that showed a pronounced decrease (1, red) or increase (2, green) under the influence of bile acid mixtures. (A-C) Each symbol represents the mean of three independent results (n = 3); D) each symbol represents an independent experiment (n = 3), bars denote the geometric mean. Either the concentration (A-C) or the fold change to the control in mean toxin secretion (D) was plotted for each strain. *, p < 0.05; ***, p < 0.001 vs control.

Fig 4. Influence of nor- and epinephrine on growth and extracellular Stx2e levels – All STEC strains were grown overnight in LB medium with 50 μM norepinephrine (NOR, •) or epinephrine (EN, ▲).

The optical density at 600nm (OD₆₀₀) was used as a measure of bacterial growth (A), and the presence of Stx2e in bacterial culture supernatants was determined by ELISA and presented as a fold change to the control (B). Each symbol represents the mean from three independent experiments, bars denote the geometric mean of all strains.

Fig 5. Influence of antibiotics on STEC growth and extracellular Stx2e levels – STEC strains were grown overnight in LB medium with amoxicillin (Amox), tetracycline (Tetra), erythromycin (Ery), ciprofloxacin (Cipro), and enrofloxacin (Enro) at ¼ the MIC.

(A) The difference in optical density at 600nm (OD₆₀₀) is compared to the control culture. Each symbol represents the geometric mean of three independent experiments, bars denote the geometric mean of all strains. (B) The Stx2e levels in bacterial culture supernatants (1 mL from standardized bacterial cultures at OD₆₀₀ = 1.0) was determined by ELISA and the fold change compared to the no-antibiotic control culture is plotted. Each symbol represents the geometric mean of three independent experiments. ***, p < 0.001 vs control. (C) Stx2e levels in perfusion outflow of the SISP experiment. STEC were pre-treated with perfusion fluid (STEC), bile acid mixture (STEC + Mix), deoxycholate (STEC + Deoxy) or enrofloxacin (STEC + Enro). Significant differences in Stx2e concentration were observed between the test conditions with enrofloxacin compared to those without. Each symbol represents one animal and represents the mean of two technical replicates. Bars denote the geometric mean of 6 animals. ***, p < 0.001 to control.

Fig 6. Influence of enrofloxacin on extracellular Stx2e levels by wild type and Δholin/ +holin mutant STEC strains – (A) In all strains where the holin gene was present, one of these genetic configurations were observed; Stx2e operon followed by I) holin in the same orientation, II) holin in reverse orientation, or III) holin in either orientation but preceded by additional (hypothetical) proteins. All 17 inducible STEC strains in this study belong to configuration (Conf.) I.

The unresponsive strains belong to configuration II or III. (B) Two STEC strains that responded to fluoroquinolones (4056 and 1717) and their Δholin mutants (B1), as well as two STEC strains that did not respond to fluoroquinolones (4055 and 4063) but were transformed with an inducible plasmid containing the holin gene (B2), were grown overnight in LB medium with either no antibiotic (•), enrofloxacin at 1/4^th of the MIC (▼), 1 mM IPTG (◼) or enrofloxacin at 1/4^th of the MIC + 1 mM IPTG (♦). The presence of Stx2e in the bacterial culture supernatants was determined by ELISA. The mean concentration of secreted toxin was plotted for each strain. Each symbol represents one independent experiment. **, p < 0.01***, p < 0.001. (C) The proposed 3D model of the holin protein using Alphafold2.