Dual effects of Escherichia coli alpha-hemolysin on rat renal proximal tubule cells.
BACKGROUND
alpha-Hemolysin (HlyA) producing Escherichia coli is a common cause of pyelonephritis and subsequent renal scarring. Recent studies have suggested that toxin secreted from HlyA E. coli may not only have a lytic effect, but also may activate a calcium signaling pathway in renal tubule cells. A dose dependent study was performed on the interaction between HlyA E. coli secretions and rat renal proximal tubule (PT) cells with regards to calcium signaling and cell morphology. The site of interaction between HlyA secretion and PT cells was examined by using an antagonist to a common binding motif in bacterial proteins.


METHODS
Supernatant from an overnight culture of HlyA was freshly prepared for each experiment. Renal PT cells from infant rats were cultured for three days and exposed for 30 minutes to four hours to supernatant or purified HlyA. Effects on cell morphology were studied semiquantitatively with light microscopy. Intracellular calcium was measured ratiometrically in the presence or absence of drugs.


RESULTS
Renal PT cells incubated with low doses of HlyA supernatant responded within five minutes with calcium oscillations. Morphology appeared unchanged after four hours of incubation. In contrast, high doses of HlyA caused a sustained increase in intracellular calcium and majority of cells were lysed within four hours. Calcium oscillations caused by lower doses of HlyA supernatant were highly regular and slow in the 10 to 12 minute range. Oscillations were abolished by 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX), indicating that HlyA is interacting with a QPB/LAOBP-motif.


CONCLUSIONS
HlyA secreted from uropathogenic E. coli exerts a dual action on renal PT cells. Sublytical concentrations induce a response that may serve as a host defense, while high concentrations cause irreversible cell damage. The data emphasize the importance of high diuresis in urinary tract infection.
Laestadius
Asa
A
Department of Woman and Child Health, and Microbiology and Tumor Biology Centre, Karolinska Institutet, Stockholm, Sweden. asa.laestadius@ks.se
Richter-Dahlfors
Agneta
A
Aperia
Anita
A
D018750:6-Cyano-7-nitroquinoxaline-2,3-dione / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects; D002478:Cells, Cultured; D016291:Dizocilpine Maleate / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D029968:Escherichia coli Proteins / Q000633:toxicity*; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D015870:Gene Expression; D006460:Hemolysin Proteins / Q000633:toxicity*; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D008464:Mecamylamine / Q000494:pharmacology; D018733:Nicotinic Antagonists / Q000494:pharmacology; D011704:Pyelonephritis / Q000473:pathology; D051381:Rats; D017207:Rats, Sprague-Dawley; D018091:Receptors, AMPA / Q000235:genetics
D029968:Escherichia coli Proteins; D018691:Excitatory Amino Acid Antagonists; D006460:Hemolysin Proteins; C036507:Hlya protein, E coli; D018733:Nicotinic Antagonists; D018091:Receptors, AMPA; D008464:Mecamylamine; D016291:Dizocilpine Maleate; D018750:6-Cyano-7-nitroquinoxaline-2,3-dione; D002118:Calcium