Prasadarao Nemani
Department of Pediatrics and Surgery,
Childrens Hospital Los Angeles and
University of Southern California School of Medicine,
Los Angeles, CA 90027.
Abstract:
Escherichia coli K1 is the most common cause of meningitis in premature infants and the second most common cause in term neonates. Ineffectiveness of antibiotic therapy over the last few decades and the emergence of antibiotic resistant E. coli strains make neonatal E. coli K1 meningitis the most serious brain disease. Incomplete understanding of the mechanisms involved at every step of the pathogenesis is attributed to our inefficiency to develop new methods of treatment and prevention. For example, it is eminent that the disruption of the blood-brain barrier (BBB) during E. coli K1 meningitis leads to alterations in the brain’s microenvironment and contributes to neurological sequelae of this disease.
Our previous studies have established that outer membrane protein A (OmpA) of E. coli interacts with gp96; a receptor specifically expresses on HBMEC to invade and disrupts the TJs. We demonstrate that three small domains in the extracellular loops of OmpA are critical to interact with gp96 to bind and invade HBMEC. The importance of OmpA-gp96 interaction is further supported by our findings that 1) E. coli strains that either lack OmpA or express non-functional OmpA do not induce meningitis in a newborn mouse model and 2) Mice in which gp96 expression was suppressed were resistant to E. coli infection. Intriguingly, OmpA interaction with gp96 triggers the production of nitric oxide (NO) via inducible nitric oxide (iNOS) activation and thereby enhances the expression of the receptor to invade more efficiently.
Novel computer modeling methods were developed and utilized to study the interaction of OmpA and gp96, and to identify small molecule inhibitors that prevent the E. coli invasion of HBMEC. Two million compounds from a drug database have been screened for their binding to gp96. Fifteen compounds with high binding affinities were selected for further analysis. Of these, four compounds: PNG-1, PNG-2, MSR-7 and MSR-15 have shown significant inhibition of E. coli invasion of HBMEC at the range of IC50 7-10 mM. The interaction of these compounds in various configurations has been simulated and appears to bind to the N-terminal region of gp96. The compounds exhibited neither anti-bacterial nor cytotoxic effects up to 24 h in tissue culture. In addition, PNG-2 and MSR-15 were given orally at a dose of 10 mg/kg body weight to newborn mice or rats prior to E. coli infection and at every 24 h post-infection, which showed protection against E. coli infection. MSR-4, used as a control showed no such inhibition. These results indicate that interfering with the OmpA-gp96 interaction will prevent the occurrence of meningitis in newborn mice.
Of note, pre-treatment with the small molecules that bind gp96 also reduced the bacteremia levels by day 7 post-infection in newborn mice. Our in vitro studies revealed that neutrophils pre-treated with the compounds showed greater anti-microbial activity by increasing the expression of CR3 and TLR4. Taken together these data indicate that gp96 antagonists identified in this study prevent the binding of E. coli to gp96 on neutrophils as well as on HBMEC and thereby the occurrence of meningitis. Studies are in progress to improve the efficacy of these small molecules for preventing the neonatal meningitis by E. coli K1.
