| Popis: |
Compounds that exert nitrosative and oxidative stress play key roles in the innate immune response and microbicidal activity. Survival and recovery after exposure to stresses encountered in the macrophage from nitric oxide, partially reduced oxygen species, hypochlorous acid and other chemicals may lead to infection and pathogenesis. This project aims to mimic some aspects of the environment encountered by pathogenic bacteria inside macrophages using E. coli as a model and study the response of stress- related genes after adding these stresses individually and/or simultaneously. Experiments were done to determine the effect of combinations of these three reagents on the growth and viability of E coli MG1655 and hmp mutant strains. The results showed that combined stresses caused a significant impact on the growth and viability of both strains. This study investigated the ability of E coli MG1655 wild-type and a mutant defective in the flavohaemoglobin (hmp) to recover from these stresses. The results indicated that both strains recovered when the three stresses were removed and the cells were transferred to fresh medium. RT-PCR data confirmed that DETA NONOate causes significant up-regulation of hmp (encoding the NO- detoxifying flavohaemoglobin) and norV (encoding an NO reductase), while H2O2 causes up-regulation of katG (catalase), ahpC (alkylhydroperoxide) and sodA (manganese superoxide dismutase). Expression of nemA was previously implicated in HOCl responses, and was also affected by this reagent. In addition, RT-PCR showed differential expression of genes in response to oxidative and nitrosative stresses when they were added simultaneously, compared to when they were added individually. Cells were treated with DETA NONOate (slow NO releaser) alone or NOC-5 plus NOC-7 (fast NO releasers) showed an increase in the expression of all tested oxidative and nitrosative stress-related genes, dependent on treatment time. Surprisingly, the nemA gene was also up-regulated in response to NO donors. Oxidative stress-related genes were highly up-regulated when cells were treated to combined stresses for 10 or 25 min relative to the individual stresses. On the other hand, treating cells with NO first, followed with H2O2 I impaired the expression of norV. The hmp was highly up-regulated when cells were treated with combined stresses. Checkerboard studies were used to determine fractional inhibitory concentrations and revealed that NO in combination with cefotaxime, gentamicin and polymyxin B showed additive effects against multidrug-resistant uropathogenic E. coli (EC958). Moreover, the combination of NO and doxycycline showed antagonistic effects against E.coli MG1655, hmp mutant and EC958 strains. This study has contributed to understanding interaction between antibiotics and other antimicrobial agent when used simultaneously or sequentially. Since bacterial assault by multiple stress reagents is a hallmark of the macrophage environment, this work may contribute to an understanding of bacterial survival mechanism in infection and disease. |
