A.C. Matin

Abstract:

Uropathogenic Escherichia coli (UPEC) causes urinary tract infections, e.g., cystitis, which are treated by gentamicin. The protein ss, encoded by the rpoS gene, controls E. coli general resistance. We discovered that rpoS deletion renders UPEC more sensitive to Gm and other bactericidal antibiotics, and proteomic analysis suggested a weakened antioxidant defense as the reason. Reactive oxygen species (ROS) detectors (psfiA gene reporter and appropriate chemicals) indicated greater ROS generation by Gm in the mutant. Gm treatment along with an antioxidant, or under anaerobic conditions (that prevent ROS formation), decreased drug lethality. Treating UPEC infection of mice bladder corroborated these findings in vivo. Thus, oxidative stress produced by insufficient quenching of metabolic ROS accounted for greater sensitivity of the mutant. E. coli strains missing antioxidant proteins also generated greater ROS and were also more sensitive to Gm. These lacked the ROS quencher proteins, (e.g., SodA/SodB; KatE/SodA), or the pentose phosphate pathway proteins, which provide NADPH (e.g., Zwf/Gnd; TalA) required by the quenchers. We have recently made similar findings with norfloxacin. Use of a microfluidic device indicated that the results applied to a single cell level. Gm is known to kill bacteria by inhibiting protein synthesis, but UPEC has developed resistance to this mode of killing. Therefore, these findings provide a timely means of restoring Gm effectiveness by curbing antioxidant proteins. Using bioinformatic approaches, we have identified several small molecules that inhibit these proteins and can enhance Gm effectiveness. In space flights, astronauts often suffer from cystitis. Bacterial gene regulation can differ in normal vs. microgravity (MG) experienced during space flights. However, the “EcAMSat” Stanford/NASA mission showed that ss-controls Gm resistance also in MG. EcAMSat employed a free- flying “nanosatellite” equipped with a technically sophisticated microfluidic system for autonomous determination of UPEC sensitivity to Gm and its telemetric transmission in real time during space flight to Earth. Bacterial multidrug resistance (MDR), such as the one regulated by the emrRAB operon and the EmrR protein is a major public health problem. Its activation is due to alteration in the EmrR protein conformation, which too can be prevented by small molecules and bioinformatic approaches that we have pursued. Another such pump along with penetration barriers contribute to bacterial biofilm antibiotic resistance. Several collaborators contributed to this work; they will be identified in the presentation.