投稿分類 微生物

主委發表種類: 壁報

投稿標題(中): 結核分枝桿菌在不同熱滅活方法的生物安全性和蛋白質組譜表現

投稿標題(英): Biosafety and proteome profiles of distinct heat-inactivation methods for Mycobacterium tuberculosis

投稿摘要: 前言：Inactivation of Mycobacterium tuberculosis is an important step to guarantee the biosafety for subsequent MTB identification and related research, notably in endemic area with minimal resources. However, certain biomolecules might be denatured or hydrolyzed because of the harsh inactivation process, and a standardized protocol is yet to be determined. We evaluated distinct heating conditions to report its inactivation efficiency, and performed downstream mass spectrometry-based MTB identification and proteomics study. Our results pointed out an inactivation approach with reliable biosafety and protein preservation for application in basic and clinical MTB studies.

方法：120 specimens of the MTB H37Rv strain were proportionally assigned to six heat-inactivation methods in a thermoblock at 80 and 95 °C for 20, 30, and 90 min, and bacterial growth was followed-up for 12 weeks. Further protein extraction and a matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) MS assay demonstrated adequate scores for bacterial identification (≥1.7). Protein abundances were calculated using the normalized spectral protein intensity (label free quantification (LFQ) intensity). Only proteins identified in at least two samples were included in the analysis. Statistical analyses that compared the difference between each heat-inactivation time group were performed using an unpaired t-test. Quantified proteins were subjected to functional analyses using Gene Ontology (GO; release 1 June 2019) for annotations of biological processes, molecular functions, and cellular components. Annotations with p＜0.05 were considered significant.

討論：Our study identified a relatively lower number of proteins following the LC/MS-MS assay in all three groups, compared to previous findings, which may have resulted from the lack of a pre-fractionation approach to reduce the sample complexity . Generally, this step requires a sufficient amount of material, and selection of an appropriate method depends on the study purpose. For an in-depth protein characterization of MTB, a more-rigorous method should be employed to improve the identification and quantification of more peptides and proteins, including those with lower abundances. In addition, we only examined the laboratory-grown H37Rv species. Future studies should aim to observe the biosafety of the heat-inactivation method and protein preservation efficiency on other MTB strains, including clinical specimens.

結論：In summary, we report an approach to inactivate MTB by heat inactivation in a thermoblock. Respective heating to 80 and 95 °C for 20, 30, and 90 min resulted in the absence of bacterial growth for 12 weeks of observation. Further investigation indicated that heat inactivation of MTB at both 80 °C for 90 min or 95 °C for 30 min was efficient for species identification with MALDI-TOF MS assays.
We evaluated distinct heat-inactivation methods and showed that each method gave satisfying identification scores (of ＞1.7). Among the six conditions, heating at 80 °C for 90 min and at 95 °C for 30 min consistently produced higher identification scores (of ＞2.0) and similar MALDI-TOF MS profiles by using PCA anlaysis . A high-throughput LC/MS-MS analysis further revealed comparable protein expressions among three different heating periods, confirming that the heat-inactivation protocol is safe and reliable for preserving protein components of mycobacteria.

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