The Study On Mechanism Of Nitrite Induced Tolerance To Aminoglycoside Antibiotics In Bacteria

Nitrite（usually sodium nitrite）is a chemical molecule full of stories.As a bacteriostatic agent,nitrite has been used in food preservation over a thousand years;in the past 60 years,nitrite has triggered unprecedented health concerns since it was regarded as a carcinogen;the fearful carcinogenicity of nitrite has been fading in the latest researches（in the recent 10 years）,and instead its antibacterial effect has been re-drawn attention.At present the combination of nitrite and other antibiotics combating drug-resistant bacteria becomes one of the research hotspots.However,the progress remains limited and controversial:some reports stated that nitrite could play a cooperative role with certain antibiotics,while others suggested an antagonistic effect of nitrite.In order to explain these contradictory findings,this thesis identified the inhibitory targets of nitrite in a number of model bacteria,and largly unravelled the molecular mechanism for nitrite inhibition.There are two pathways by which nitrite exerts its inhibitory physiological effects in organisms:nitric oxide（NO）-dependent pathway and NO-independent pathway.This study firstly demonstrated that the bacteriostatic ability of nitrite was not related to NO,that is,the function of nitrite is achieved through a NO-independent pathway;secondly,this study confirmed that the antagonistic effect usually occurs when nitrite is used in combination with aminoglycosides but not other antibiotics,that is,nitrite is able to induce the resistance of bacteria to aminoglycosides;finally,the molecular mechanism underlying the antagonistic effect of nitrite was systematically analyzed in this study,which is also the focus of this thesis.This study employed Gram-negative bacterium Shewanella oneidensis as the research model because the previous studies of this laboratory have determined that the cell target of nitrite in S.oneidensis is heme-copper oxidase（HCO）cbb₃.Mutants that only possess cbb₃ are highly sensitive to nitrite,from which this thesis stems.In order to eliminate the interference of the different physiological backgrounds of various species,we compared the effects of nitrite on various HCOs in the same genetic background,by expressing HCOs from Escherichia coli（bo₃）,Staphylococcus aureus（aa₃）and S.oneidensis（caa₃）in the S.oneidensis cbb₃-deficient mutant.Although caa₃,bo₃ and aa₃ exhibit certain differences in their sensitivity to nitrite,all of these HCOs are extremely sensitive to nitrite compared to other types of oxidases,indicating that they are the primary targets of nitrite.This thesis further clarifies that the antagonistic effect of nitrite on aminoglycosides depends on the proton motive force（PMF）,rather than the“general respiration”（oxygen consumption rate,hereafter referred to as respiration）.Antibiotic susceptibility experiments suggest that HCOs-deficient strains have significantly increased tolerance to aminoglycosides but not other antibiotics.Although the ability of other oxidases to support cell respiration is similar to that of HCOs,their effecacy to produce PMF is substantially insufficient.As a result,PMF is low in HCOs-deficient strains,which in turn reduce the uptake rate of aminoglycosides,leading to elevated tolerance.In brief,by attacking the HCOs in the respiratory chain,nitrite modulates bacterial tolerance to aminoglycosides.The investigations conducted in this thesis suggest that nitrite is a promising antibacterial drug targeting HCOs,but cautions should be taken when used with other antibiotics,especially aminoglycosides.