Effects of bacterial toxins on calcium homeostasis in renal inflammation

Calcium (Ca2+) is a highly versatile ion used for intra- and inter-cellular signalling in eukaryotic cells, and numerous cellular events are regulated by alteration of the intracellular Ca2+ concentration. We hypothesized that bacterial virulence factors may alter the Ca 2+ homeostasis in epithelial cells such that the innate immune response is affected. This thesis focuses on the effects of the two E.coli pore-forming toxins alpha-hemolysin (HlyA) and Cytolysin A (ClyA) on Ca2+ homeostasis and innate immune responses of renal epithelial cells.

By loading primary preparations of rat proximal tubule (PT) cells with a Ca2+-sensitive dye, we monitored the intracellular Ca 2+ concentration in single cells after toxin exposure. HlyA exerted a dose-dependent effect where high concentrations caused elevated levels of Ca 2+ followed by cell lysis, whereas sub-lytic concentrations induced intracellular Ca2+ oscillations. Spectral analysis revealed that oscillations occurred with a periodicity of 12.0 ± 0.7 min. The Ca 2+ response was dependent on influx of extracellular Ca2+ via the Ltype voltage-gated Ca2+ channels as well as IP3-dependent release of Ca2+ from IP3-R gated internal Ca2+ stores. Production of the p ro-inflammatory cytokines interleukin (IL)-6 and IL-8 was linked to the toxin-induced Ca 2+ oscillation, and this occurred most probably via frequency-modulated activation of the transcription factor NF-kappaB.

Pyelonephritis is more common in infants as compared to adults and the clinical manifestations are often more severe. Using renal cortical tissue from rats of different ages, we investigated whether the innate immune response shows developmental differences that could explain the increased risk of renal scarring in the young. Infant and pubertal tissue showed similar cytokine responses when Ca2+ oscillations were induced by HlyA, as well as when cells were exposed to bacterial LPS. Thus, the higher susceptibility of the young kidney can most probably be explained by anatomical factors rather than developmental regulation of the innate immune response mechanisms.

We identified a novel vesicle-dependent mechanism for activation and export of ClyA from bacteria. ClyA is activated and oligomerized in outer-membrane vesicles (OMVs), where the toxin forms pore-assemblies protruding out from the OMVs. The activated form of ClyA (ClyA+omv) displayed an increased hemolytic and cytolytic effect on erythrocytes and epithelial cells. In addition, sub-lytic concentrations of CIyA+Omv induced intracellular Ca2+ oscillations in epithelial cells similar to the HlyA-induced response. However, the ClyA+omvinduced response was somewhat slower, with a periodicity of 22.9 ± 0.9 min. No effects on the IL-8 production could be related to Ca2+ oscillations of this specific periodicity. Investigations of the immunostimulatory properties of the ClyA protein in TLR4-positive and -negative epithelial cell lines revealed that the ClyA toxin per se is not immuno-stimulatory, while LPS and other proteins present in the outer membrane of the OMVs are recognized by the cell thus eliciting a proinflammatory response.

In conclusion, we have presented a novel feature of the E. coli pore-forming toxins HlyA and ClyA, which suggest that bacterial factors can modulate eukaryotic gene expression by induction of oscillating second messenger response in epithelial cells.

List of scientific papers

I. Uhlen P, Laestadius A, Jahnukainen T, Soderblom T, Backhed F, Celsi G, Brismar H, Normark S, Aperia A, Richter-Dahlfors A (2000). Alpha-haemolysin of uropathogenic E. coli induces Ca2+ oscillations in renal epithelial cells. Nature. 405(6787): 694-7.
https://pubmed.ncbi.nlm.nih.gov/10864327

II. Laestadius A, Soderblom T, Aperia A, Richter-Dahlfors A (2003). Developmental aspects of Escherichia coli-induced innate responses in rat renal epithelial cells. Pediatr Res. 54(4): 536-41. Epub 2003 Jul 02
https://pubmed.ncbi.nlm.nih.gov/12840157

III. Wai SN, Lindmark B, Soderblom T, Takade A, Westermark M, Oscarsson J, Jass J, Richter-Dahlfors A, Mizunoe Y, Uhlin BE (2003). Vesicle-mediated export and assembly of pore-forming oligomers of the enterobacterial ClyA cytotoxin. Cell. 115(1): 25-35.
https://pubmed.ncbi.nlm.nih.gov/14532000

IV. Soderblom T, Wai SN, Uhlén P, Aperia A, Uhlin BE, Richter-Dahlfors A (2004). The ClyA toxin induces Ca2+ signaling when activated in outermembrane vesicles. [Manuscript]