Khalifa, M. M. | bioRxiv Science

bioRxiv2019

Rapid and highly sensitive detection of pyocyanin biomarker in different Pseudomonas aeruginosa infections using gold nanoparticles modified sensor

Successful antibiotic treatment of infections relies on accurate and rapid identification of the infectious agents. Pseudomonas aeruginosa is implicated in a wide range of human infections that almost complicated and become life threating especially in immunocompromised and critically ill patients. Conventional microbiological methods take more than 3 days to obtain accurate results. Pyocyanin is a distinctive electroactive biomarker for Pseudomonas aeruginosa. Here, we have developed a rapid diagnostic (polyaniline) PANI gold nanoparticles (Au NPs) modified indium tin oxide (ITO) electrode that showed 100% sensitivity for pyocyanin in culture of Pseudomonas aeruginosa clinical isolates and high selectivity for pyocyanin at low concentration when measured in the presence of other substances like ascorbic acid, uric acid, and glucose as interferences. The constructed electrode was characterized using scanning electron microscopy and cyclic voltammetry. The determined linear range for pyocyanin detection was from 238 {micro}M to 1.9 {micro}M with a detection limit of 500 nM. Compared to the screen-printed electrode used before, the constructed electrode showed a 4-fold enhanced performance.

biochemistry↗

bioRxiv2018

Enzymatic and structural characterization of the Naegleria fowleri glucokinase

Infection with the free-living amoeba Naegleria fowleri leads to life-threatening primary amoebic meningoencephalitis. Efficacious treatment options for these infections are limited and the mortality rate is very high (~98%). Parasite metabolism may provide suitable targets for therapeutic design. Like most other organisms, glucose metabolism is critical for parasite viability, being required for growth in culture. The genome of the parasite encodes a single glucose phosphorylating enzyme, a glucokinase (Glck). The products of this enzyme are required for both glycolysis and the pentose phosphate pathway. The N. fowleri Glck (NfGlck) shares limited (25%) amino acid identity with the mammalian host enzyme (HsGlck), suggesting that parasite-specific inhibitors with anti-amoeba activity could be generated. Following heterologous expression, NfGlck was found to have a limited hexose substrate range, with greatest activity observed with glucose. The enzyme had apparent Km values of 42.5 {+/-} 7.3 M and 141.6 {+/-} 9.9 M for glucose and ATP, respectively. The NfGlck structure was determined and refined to 2.2 [A] resolution, revealing that the enzyme shares greatest structural similarity with the Trypanosoma cruzi Glck. These similarities include binding modes and binding environments for substrates. To identify inhibitors of NfGlck, we screened a small collection of inhibitors of glucose phosphorylating enzymes and identified several small molecules with IC50 values < 1 M that may prove useful as hit chemotypes for further lead and therapeutic development against N. fowleri.

biochemistry↗