定量蛋白质组学分析ClpS在分枝杆菌耐药中的功能

ClpS是原核生物蛋白质降解复合物ClpAPS的重要组成成分,它可以识别某些特定的氨基酸序列并将其呈递给ClpAP以促进其降解。同时,ClpS也抑制了其他蛋白质底物的降解。本研究通过在耻垢分枝杆菌中过度表达ClpS,发现所构建的重组菌株提高了利福平的抗药性。应用定量蛋白质组学技术,我们系统地分析了过度表达ClpS对于细菌蛋白质组的影响,并推测出细菌抗利福平的分子机制:ClpS促进稳态的调整、促进药物沉降以及加速药物代谢。本研究首次通过改变细菌降解复合物的相关蛋白的表达增加细菌的抗药性,并证明蛋白质组学技术是细菌的抗药性研究以及耐药株筛选的重要工具。     

Evaluation of the differences between biofilm and planktonic Brucella abortus via metabolomics and proteomics

Functional & Integrative Genomics - This study analyzed the difference between biofilm and planktonic Brucella abortus using metabolomics and proteomics. Brucella abortus was cultured in...     

Investigation of biomarkers of bile tolerance in Lactobacillus casei using comparative proteomics

The identification of cell determinants involved in probiotic features is a challenge in current probiotic research. In this work, markers of bile tolerance in Lactobacillus casei were investigated using comparative proteomics. Six L. casei strains were classified on the basis of their ability to grow in the presence of bile salts in vitro. Constitutive differences between whole cell proteomes of the most tolerant strain (L. casei Rosell-215), the most sensitive one (L. casei ATCC 334), and a moderately tolerant strain (L. casei DN-114 001) were investigated. The ascertained subproteome was further studied for the six strains in both standard and bile stressing conditions. Focus was on proteins whose expression levels were correlated with observed levels of bile tolerance in vitro, particularly those previously reported to be involved in the bile tolerance process of lactobacilli. Analysis revealed that 12 proteins involved in membrane modification (NagA, NagB, and RmlC), cell protection and detoxification (ClpL and OpuA), as well as central metabolism (Eno, GndA, Pgm, Pta, Pyk, Rp1l, and ThRS) were likely to be key determinants of bile tolerance in L. casei and may serve as potential biomarkers for phenotyping or screening purposes. The approach used enabled the correlation of expression levels of particular proteins with a specific probiotic trait.     

Glucose Transport in Brucella abortus

Brucella abortus British strain 19 transported glucose with an apparent K(m) of 0.16 mM and an apparent V(max) of 250 nmol per min per mg of N. The only common glucose analogue transported was 2-deoxyglucose (2-DOG), with an apparent K(i) of 0.73 mM. Alpha- or beta-methyl glucosides and 3-O-methylglucose were not transported. Transport was linear for 70 to 90 s, depending on the concentration of substrate used. 2-Deoxyglucose was transported as the free sugar and was not further metabolized once inside the cell. There was no glucose phosphoenolpyruvate phosphotransferase system (PEP-PTS) present, and there were no inhibitors present in Brucella cell-free extract that inhibited the Escherichia coli glucose PEP-PTS. N-Ethylmaleimide (NEM) and p-chloromercuribenzoate (pCMB) completely inhibited transport of glucose and 2-DOG. Glutathione, dithiothreitol, and beta-mercaptoethanol reversed the effects of pCMB but not of NEM. A pH optimum of 7.2 and a temperature optimum of 37 to 45 C were observed for both K(m) and V(max). The glucose transport system appeared to be constitutive for glucose transport in cells grown on fructose, galactose, erythritol, or glucose. The electron transfer inhibitors carbonyl cyanide, m-chlorophenylhydrazone, NaN(3), 2,4-dinitrophenol, and KCN inhibited 2-DOG transport to a greater extent than did the metabolic energy inhibitors NaAsO(4), iodoacetate, KF, and 2-heptyl-4-hydroxyquinoline-N-oxide. Dicyclohexylcarbodiimide, an inhibitor of membrane-bound adenosine triphosphatases, inhibited transport by 100%.     

Investigation of doxorubicin resistance in MCF-7 breast cancer cells using shot-gun comparative proteomics with proteolytic 18O labeling

A shot-gun comparative proteomic investigation utilizing proteolytic 18O labeling has been carried out on a drug susceptible MCF-7 human breast cancer cell line and a related cell line that is resistant to doxorubicin. The proteolytic 18O labeling method has been further refined and optimized for application to a protein fraction stemming from the cytosol of the breast cancer cells. The comparative investigation revealed several proteins with altered expression levels in the doxorubicin resistant line. These altered proteins are considered for a possible role in doxorubicin resistance.     

Periplasmic protein EipA determines envelope stress resistance and virulence in Brucella abortus

Molecular components of the Brucella abortus cell envelope play a major role in its ability to infect, colonize and survive inside mammalian host cells. In this study, we have defined a role for a conserved gene of unknown function in B. abortus envelope stress resistance and infection. Expression of this gene, which we name eipA, is directly activated by the essential cell cycle regulator, CtrA. eipA encodes a soluble periplasmic protein that adopts an unusual eight‐stranded β‐barrel fold. Deletion of eipA attenuates replication and survival in macrophage and mouse infection models, and results in sensitivity to treatments that compromise the cell envelope integrity. Transposon disruption of genes required for LPS O‐polysaccharide biosynthesis is synthetically lethal with eipA deletion. This genetic connection between O‐polysaccharide and eipA is corroborated by our discovery that eipA is essential in Brucella ovis, a naturally rough species that harbors mutations in several genes required for O‐polysaccharide production. Conditional depletion of eipA expression in B. ovis results in a cell chaining phenotype, providing evidence that eipA directly or indirectly influences cell division in Brucella. We conclude that EipA is a molecular determinant of Brucella virulence that functions to maintain cell envelope integrity and influences cell division.     

Experimental Brucella abortus infection in wolves

Four juvenile male wolves (Canis lupus) each received an oral dose of 1.6-1.7 x 10(12) colony-forming units of Brucella abortus biovar 1 isolated from a bison (Bison bison) in Wood Buffalo National Park (Canada), and two others served as negative controls. Infected wolves did not show clinical signs of disease but did develop high Brucella antibody titers. Small numbers of B. abortus were excreted sporadically in feces until day 50 postinoculation (PI). Very small numbers of the bacterium were isolated from urine of only one wolf late on the same day that it was infected, and very small numbers of colonies of B. abortus were obtained from buccal swabs of three wolves for up to 48 hr PI. Two infected wolves euthanized 6 mo after the start of the experiment had no lesions, and colonies of B. abortus were isolated from thymus and most major lymph nodes. The other two infected wolves euthanized 12 mo after the start of the experiment had no lesions, and smaller numbers of brucellae were recovered from fewer lymph nodes compared with the wolves killed 6 mo earlier. The sporadic excretion of very small numbers of brucellae by the wolves was insignificant when compared with the infective dose for cattle. Brucella abortus, brucellosis, Canis lupus, pathogenesis, serology, wolf.     

Antibiotic persistence of intracellular Brucella abortus

BackgroundHuman brucellosis caused by the facultative intracellular pathogen Brucella spp. is an endemic bacterial zoonosis manifesting as acute or chronic infections with high morbidity. Treatment typically involves a combination therapy of two antibiotics for several weeks to months, but despite this harsh treatment relapses occur at a rate of 5–15%. Although poor compliance and reinfection may account for a fraction of the observed relapse cases, it is apparent that the properties of the infectious agent itself may play a decisive role in this phenomenon.Methodology/Principal findingsWe used B. abortus carrying a dual reporter in a macrophage infection model to gain a better understanding of the efficacy of recommended therapies in cellulo. For this we used automated fluorescent microscopy as a prime read-out and developed specific CellProfiler pipelines to score infected macrophages at the population and the single cell level. Combining microscopy of constitutive and induced reporters with classical CFU determination, we quantified the protective nature of the Brucella intracellular lifestyle to various antibiotics and the ability of B. abortus to persist in cellulo despite harsh antibiotic treatments.Conclusion/SignificanceWe demonstrate that treatment of infected macrophages with antibiotics at recommended concentrations fails to fully prevent growth and persistence of B. abortus in cellulo, which may be explained by a protective nature of the intracellular niche(s). Moreover, we show the presence of bona fide intracellular persisters upon antibiotic treatment, which are metabolically active and retain the full infectious potential, therefore constituting a plausible reservoir for reinfection and relapse. In conclusion, our results highlight the need to extend the spectrum of models to test new antimicrobial therapies for brucellosis to better reflect the in vivo infection environment, and to develop therapeutic approaches targeting the persister subpopulation.     

Use of Mitomycin C to Distinguish Brucella abortus Field Strains from the Vaccinal Brucella abortus Strain 19

Field strains of Brucella abortus were resistant to mitomycin C, whereas strain 19 was sensitive; therefore, the antibiotic was used to distinguish B. abortus strain 19 from other strains.