Distribution and Molecular Analysis of Subtilase Cytotoxin Gene (subAB) Variants in Shiga Toxin-Producing Escherichia Coli (STEC) Isolated From Different Sources in Iran

Background Subtilase is a potent cytotoxin that was first described in O113:H21 strain in Australia as a plasmid- encoded cytotoxin (subAB1). Subsequently, chromosomal variants including subAB2-1, subAB2-2, and subAB2-3 were described. Results In the present study a collection of 101 archived STEC strains isolated from various sources in Iran (2009–2016) were analyzed for the detection of different genes encoding the subtilase variants, plasmidic and chromosomal virulence genes, together with the phylogroup and serogroups. Overall, 57 isolates (56.4%) carried at least one variant of subAB. Most strains from small ruminants including 93% of sheep and 96% of caprine isolates carried at least one chromosomally encoded variant (subAB-2-1 and/or subAb2-2). In contrast, 12 cattle isolates (24%) only harbored the plasmid encoded variant (subAB1). STEC strains from other sources including deer, pony and humans were positive for subAB-2-1 and/or subAb2-2. Concerning the virulence markers, some strains showed an association with hosts the bacteria were isolated from. In particular, tia was associated with sheep, goats and pony isolates and astA gene was present in deer, pony and goats and terD was only found in deer and pony isolates. Only cattle STEC carried espP and epeA, the important markers of pO113 plasmid. Some genes were widespread among strain of various sources like ehly, iha and lpfO113 and some genes were not detected such as efa1, toxB and katP. Most strains belonged to phylogenetic group B1 (89.47%), but five strains from cattle, deer, pony and a goat were assigned to A phylogroup. Most cattle strains belonged to O113, while O5 was just detected in ovine isolates, and O128 and O113 were present in caprine strains. Conclusions the present study reveals the presence of potentially pathogenic genotypes among LEE-negative isolates and some host specificity related to subtilase variants and other virulence markers that may aid in source tracking of STEC during outbreak investigations.

Combined Action of Shiga Toxin Type 2 and Subtilase Cytotoxin in the Pathogenesis of Hemolytic Uremic Syndrome

Shiga toxin-producing E. coli (STEC) produces Stx1 and/or Stx2, and Subtilase cytotoxin (SubAB). Since these toxins may be present simultaneously during STEC infections, the purpose of this work was to study the co-action of Stx2 and SubAB. Stx2 + SubAB was assayed in vitro on monocultures and cocultures of human glomerular endothelial cells (HGEC) with a human proximal tubular epithelial cell line (HK-2) and in vivo in mice after weaning. The effects in vitro of both toxins, co-incubated and individually, were similar, showing that Stx2 and SubAB contribute similarly to renal cell damage. However, in vivo, co-injection of toxins lethal doses reduced the survival time of mice by 24 h and mice also suffered a strong decrease in the body weight associated with a lowered food intake. Co-injected mice also exhibited more severe histological renal alterations and a worsening in renal function that was not as evident in mice treated with each toxin separately. Furthermore, co-treatment induced numerous erythrocyte morphological alterations and an increase of free hemoglobin. This work shows, for the first time, the in vivo effects of Stx2 and SubAB acting together and provides valuable information about their contribution to the damage caused in STEC infections.

Distribution and molecular analysis of Subtilase cytotoxin gene (subAB) variants in Shiga toxin-producing Escherichia coli (STEC) isolated from different sources in Iran

Subtilase is a potent cytotoxin that was first described in O113:H21 strain in Australia as a plasmid- encoded cytotoxin (subAB1). Subsequently, chromosomal variants including subAB2-1, subAB2-2, and subAB2-3 were described. In the present study a collection of 101 STECs isolated from various sources in Iran (2009-2016) were analyzed for the detection of different genes encoding the subtilase variants, plasmidic and chromosomal virulence genes, together with the phylogroup and serogroups. Overall, 57 isolates (56.4%) carried at least one variant of subAB. Most strains from small ruminants including 93% of sheep and 96% of caprine isolates carried at least one chromosomally encoded variant (subAB-2). In contrast, 12 cattle isolates (24%) only harbored the plasmid encoded variant (subAB1). STEC strains from other sources including deer, pony and humans were positive for subAB-2-1 and/or subAb2-2. Concerning the virulence markers, some strains showed an association with hosts the bacteria were isolated from. In particular, tia was associated with sheep, goats and pony isolates and astA gene was present in deer, pony and goats and terD was only found in deer and pony isolates. Only cattle STEC carried espP and epeA, the important markers of pO113 plasmid. Some genes were widespread among strain of various sources like ehly, iha and lpfO113 and some genes were not detected such as efa1, toxB and katP. Most strains belonged to phylogenetic group B1 (89.47%), but five strains from cattle, deer, pony and a goat were assigned to A phylogroup. Most cattle strains belonged to O113, while O5 was just detected in ovine isolates, and O128 and O113 were present in caprine strains. In conclusion, the present study reveals the presence of potentially pathogenic genotypes among LEE-negative isolates and some host specificity related to subtilase variants and other virulence markers that may aid in source tracking of STEC during outbreaks.

Subtilase cytotoxin induces a novel form of Lipocalin 2, which promotes Shiga-toxigenic Escherichia coli survival

Shiga-toxigenic Escherichia coli (STEC) infection causes severe bloody diarrhea, renal failure, and hemolytic uremic syndrome. Recent studies showed global increases in Locus for Enterocyte Effacement (LEE)-negative STEC infection. Some LEE-negative STEC produce Subtilase cytotoxin (SubAB), which cleaves endoplasmic reticulum (ER) chaperone protein BiP, inducing ER stress and apoptotic cell death. In this study, we report that SubAB induces expression of a novel form of Lipocalin-2 (LCN2), and describe its biological activity and effects on apoptotic cell death. SubAB induced expression of a novel LCN2, which was regulated by PRKR-like endoplasmic reticulum kinase via the C/EBP homologous protein pathway. SubAB-induced novel-sized LCN2 was not secreted into the culture supernatant. Increased intracellular iron level by addition of holo-transferrin or FeCl3 suppressed SubAB-induced PARP cleavage. Normal-sized FLAG-tagged LCN2 suppressed STEC growth, but this effect was not seen in the presence of SubAB- or tunicamycin-induced unglycosylated FLAG-tagged LCN2. Our study demonstrates that SubAB-induced novel-sized LCN2 does not have anti-STEC activity, suggesting that SubAB plays a crucial role in the survival of LEE-negative STEC as well as inducing apoptosis of the host cells.

Developmentally regulated PERK activity renders dendritic cells insensitive to subtilase cytotoxin-induced integrated stress response

In stressed cells, phosphorylation of eukaryotic initiation factor 2α (eIF2α) controls transcriptome-wide changes in mRNA translation and gene expression known as the integrated stress response (ISR). We show here that dendritic cells (DCs) display unusually high eIF2α phosphorylation, which is mostly caused by a developmentally regulated activation of the ER kinase PERK (EIF2AK3). Despite high p-eIF2α levels, differentiated DCs display active protein synthesis and no signs of a chronic ISR. eIF2α phosphorylation does not majorly impact DC differentiation nor cytokines production. It is however important to adapt protein homeostasis to the variations imposed on DCs by the immune or physiological contexts. This biochemical specificity prevents translation arrest and expression of the transcription factor ATF4 during ER-stress induction by subtilase cytotoxin or upon DC stimulation with bacterial lipopolysaccharides. This is also exemplified by the influence of the actin cytoskeleton dynamics on eIF2α phosphorylation and the migratory deficit observed in PERK-deficient DCs.

Variants of Escherichia coli Subtilase Cytotoxin Subunits Show Differences in Complex Formation In Vitro

The subtilase cytotoxin (SubAB) of Shiga toxin-producing Escherichia coli (STEC) is a member of the AB5 toxin family. In the current study, we analyzed the formation of active homo- and hetero-complexes of SubAB variants in vitro to characterize the mode of assembly of the subunits. Recombinant SubA1-His, SubB1-His, SubA2-2-His, and SubB2-2-His subunits, and His-tag-free SubA2-2 were separately expressed, purified, and biochemically characterized by circular dichroism (CD) spectroscopy, size-exclusion chromatography (SEC), and analytical ultracentrifugation (aUC). To confirm their biological activity, cytotoxicity assays were performed with HeLa cells. The formation of AB5 complexes was investigated with aUC and isothermal titration calorimetry (ITC). Binding of SubAB2-2-His to HeLa cells was characterized with flow cytometry (FACS). Cytotoxicity experiments revealed that the analyzed recombinant subtilase subunits were biochemically functional and capable of intoxicating HeLa cells. Inhibition of cytotoxicity by Brefeldin A demonstrated that the cleavage is specific. All His-tagged subunits, as well as the non-tagged SubA2-2 subunit, showed the expected secondary structural compositions and oligomerization. Whereas SubAB1-His complexes could be reconstituted in solution, and revealed a Kd value of 3.9 ± 0.8 μmol/L in the lower micromolar range, only transient interactions were observed for the subunits of SubAB2-2-His in solution, which did not result in any binding constant when analyzed with ITC. Additional studies on the binding characteristics of SubAB2-2-His on HeLa cells revealed that the formation of transient complexes improved binding to the target cells. Conclusively, we hypothesize that SubAB variants exhibit different characteristics in their binding behavior to their target cells.

Crosstalk between Human Microvascular Endothelial Cells and Tubular Epithelial Cells Modulates Pro-Inflammatory Responses Induced by Shiga Toxin Type 2 and Subtilase Cytotoxin

Hemolytic uremic syndrome (HUS) is a consequence of Shiga toxin (Stx)-producing Escherichia coli (STEC) infection and is the most frequent cause of acute renal failure (ARF) in children. Subtilase cytotoxin (SubAB) has also been associated with HUS pathogenesis. We previously reported that Stx2 and SubAB cause different effects on co-cultures of human renal microvascular endothelial cells (HGEC) and human proximal tubular epithelial cells (HK-2) relative to HGEC and HK-2 monocultures. In this work we have analyzed the secretion of pro-inflammatory cytokines by co-cultures compared to monocultures exposed or not to Stx2, SubAB, and Stx2+SubAB. Under basal conditions, IL-6, IL-8 and TNF-α secretion was different between monocultures and co-cultures. After toxin treatments, high concentrations of Stx2 and SubAB decreased cytokine secretion by HGEC monocultures, but in contrast, low toxin concentrations increased their release. Toxins did not modulate the cytokine secretion by HK-2 monocultures, but increased their release in the HK-2 co-culture compartment. In addition, HK-2 monocultures were stimulated to release IL-8 after incubation with HGEC conditioned media. Finally, Stx2 and SubAB were detected in HGEC and HK-2 cells from the co-cultures. This work describes, for the first time, the inflammatory responses induced by Stx2 and SubAB, in a crosstalk model of renal endothelial and epithelial cells.