Gentamicin毒殺奇異變形桿菌之機制探討

Abstract

Proteus mirabilis為革蘭氏陰性的腸內菌，一般在健康人類腸道屬於正常菌叢，不過在長期使用導尿管之病患身上，則會造成伺機性感染，嚴重甚至可能導致腎臟病、肺炎等併發症。 臨床上使用之gentamicin屬於aminoglycoside 類抗生素，常用於治療敗血症、嚴重尿道感染及院內呼吸道感染，但長期使用下會導致聽力受損及腎臟功能傷害等副作用。Gentamicin作用機制主要是當藥物進入細菌體內後，會和細菌的30S核醣體結合，導致核醣體發生mistranslation，進而造成細胞內蛋白質生成受到抑制，最終導致細菌死亡。然而，近期在E. coli的研究指出aminoglycoside藥物的作用機制不單只是和核醣體結合，會透過CpxA cross-talk至ArcA，進而影響細菌體內的代謝調控，導致hydroxyl radical等氧化壓力的大量產生而造成細菌死亡。 本篇論文首先利用跳躍子突變法 (transposon mutagenesis) 的方式，篩選出11株對gentamicin (Gm) 感受性改變的P. mirabilis突變株，其中10株對Gm感受性下降 (Gm-resistant突變株：cpxA、ispA、ubiD、pepP、nrpU、PMI1554、PMI2851、mutL、mutS和PMI0674)，MIC數值皆比野生株高2 ~ 4倍；1株對Gm感受性增加 (Gm-susceptible突變株：speA)，MIC數值比野生株低2倍。進一步發現當處理Gm後，Gm-resistant突變株生長情況和野生株並無差異或略為下降，且hydroxyl radical幾乎不會增加；而Gm-susceptible突變株生長會下降，hydroxyl radical則會增加。此外，若在Gm處理下額外添加抗氧化劑glutathione或iron chelator後，可顯著降低Gm誘發hydroxyl radical的生成。由此可證明，當P. mirabilis在Gm處理後，可能透過hydroxyl radical的生成導致細菌死亡。 在這些對Gm感受性改變的突變株中，我們也看見two-component system中的CpxA參與其中。為釐清Gm殺菌是否透過ArcA，故將arcA進行knockout，發現MIC數值比野生株高2倍，且hydroxyl radical生成較低，因此推測在P. mirabilis處理Gm後，也是循著CpxA-ArcA的殺菌途徑。 接著以real-time PCR方式測定，發現野生株在Gm處理後，ahpC、oxyR、sodA及sodB表現量上升，顯示Gm的確會造成氧化壓力增加；同時，acnA表現量上升，顯示Gm可透過影響TCA cycle和電子傳遞鏈表現，導致細菌內氧化壓力增加。然而這些指標在目前收集之10株P. mirabilis臨床菌株中並未觀察到顯著差異。此外，在Gm處理後cpxR的表現量會增加，同時在cpxA突變株中cpxR有過度表現的情況，由此推測CpxR可能對抵抗Gm扮演某種程度的角色尚待釐清。 另一方面，ubiquinone生合成相關之ispA和ubiD基因也參與其中，與能量生成有關。藉由電子傳遞鏈抑制劑CCCP來降低細菌體內能量的生成，發現野生株對Gm的抗性增加了2倍，而ispA和ubiD突變株則並無改變，由此可間接推測在P. mirabilis中Gm的吸收是需要能量的。 唯一挑到對Gm感受性增加的speA基因，參與細菌體內polyamine合成途徑。首先我們也發現speA突變株對於H2O2的感受性增加，進一步利用reporter assay得知oxyR的表現量比野生株略低。因此推測polyamine可透過影響OxyR的表現來影響對氧化壓力的抵抗性，進而影響到對Gm的感受性。 最後在毒力因子分析方面，cpxA突變株swarming較野生株緩慢；ispA和ubiD突變株的生長速度較為緩慢，連帶也使得swarming的速度較慢；cpxA和ispA突變株在haemolysin cycle中比野生株緩慢；speA的突變株swarming能力下降，同時haemolysin activity也較低。ispA、speA突變株之outer membrane protein以及其他菌株在swimming、biofilm的結果中，皆和野生株並無顯著差異。 總結，本研究證實了在P. mirabilis中，gentamicin可經由CpxA-ArcA途徑，透過hydroxyl radical的生成，而達到殺菌之功效。此外，透過降低細菌體內能量的生成以及polyamine含量的高低可協助細菌抵抗gentamicin殺菌。

Proteus mirabilis is a facultative Gram-negative bacterium and a member of the Enterobacteriaceae family. It commonly causes urinary tract infection (UTI), and leads to kidney disease, pneumonia and septicemia in individuals with long-term catheterization or with structural or functional abnormalities in the urinary tract. Gentamicin (Gm) is a kind of aminoglycoside antibiotics, which is commonly used in treatment of septicemia, severe urinary tract infections and nosocomial respiratory infections. It potentially damages the ears and kidney once given for a long period of time. Gentamicin could directly target the 30S subunit of ribosome, leading to protein mistranslation and cell death. Recently, studies in E. coli have suggested that the mechanism of aminoglycoside-lethality is a function of more than ribosome inhibition and may be due to disorders of multiple cellular metabolic targets through CpxA-ArcA pathway, triggering oxidative stress and cell death. In order to know the mechanisms of gentamicin killing in P. mirabilis, we performed transposon mutagenesis and 11 mutants were identified. Ten mutants were 2 to 4-fold more resistant (Gm-resistant mutants: cpxA、ispA、ubiD、pepP、nrpU、PMI1554、PMI2851、mutL、mutS and PMI0674), and one mutant was 2-fold more sensitive (Gm-susceptible mutant: speA) than the wild-type. We observed that in the present of gentamicin, Gm-resistant mutants had higher survival rates and less hydroxyl radical formation than wild-type. On the contrary, Gm-susceptible mutants had lower survival rates and more hydroxyl radical formation. We also observed that addition of glutathione or iron chelator significantly reduced hydroxyl radical formation in Gm-exposed strains, confirming that gentamicin killing in P. mirabilis is through hydroxyl radical formation. Knowing that CpxA, the sensor kinase of Cpx two-component system, is involved in gentamicin killing, we knockout arcA gene to determine the role of ArcA, another member of two-component system, in gentamicin killing. We observed that arcA mutant was 2-fold more resistant to Gm than wild-type and had less hydroxyl radical formation. These results imply that both CpxA and ArcA are associated with gentamicin killing. Moreover, we used real-time PCR to examine expression of oxidative stress-related genes following Gm treatment. We observed that the expression of ahpC, oxyR, sodA and sodB genes were activated after gentamicin treatment, indicating that cellular oxidative stress responses were triggered by gentamicin exposure. We also observed that the expression of acnA gene was activated, and nuoF was inhibited slightly, indicating that gentamicin-mediated disorders in TCA cycle and electron transfer chain may lead to oxidative stress. However, the expression of these oxidative stress-related genes showed no significant difference in the 10 clinical isolates of P. mirabilis. Besides, cpxR gene was activated after gentamicin treatment and overexpression of cpxR gene in cpxA mutant was observed, implying the protective role of CpxR in the present of gentamicin. We found ispA and ubiD genes which were related to ubiquinone biosynthesis and cellular energy production are also involved in Gm susceptibility. By addition of CCCP, an electron transfer chain inhibitor, we observed 2-fold more resistant to Gm in wild-type but not in ispA and ubiD mutants, suggesting that the gentamicin uptake is energy-dependent in P. mirabilis. The only Gm-susceptible mutant is speA gene, which is involved in polyamine biosynthesis. We observed that speA mutant was more sensitive to H2O2 than wild-type. We also found that the expression of oxyR gene (a regulator for H2O2 stress) in speA mutant was decreased by using reporter assay, suggested that polyamine may protect P. mirabilis from oxidative stress possibly through OxyR pathway. Finally, we assayed the virulence factors of these mutants. We found that cpxA mutant exhibited decreased swarming mobility. Due to the growth defect in ispA and ubiD mutants, decreased swarming mobility was found. In haemolysin assay, cpxA and ispA mutant had delayed cycle of haemolysin expression. Besides, speA mutant exhibited decreased swarming mobility and decreased haemolysin ability. No significant difference of swimming, biofilm formation and outer membrane proteins was observed between wild-type and the mutants. In conclusion, we found that gentamicin may affect multiple metabolic targets in P. mirabilis through CpxA-ArcA pathway, triggering hydroxyl radical formation and leading to cell death. In addition, we found that diminished energy production and the presence of polyamines may protect cells from gentamicin killing.