Construction of fluorescence-labeled <italic style="font-style: italic">Burkholderia pseudomallei</italic> and its interaction with host cells

Jiangao CHEN; Chenglong RAO; Wenzheng LIU; Pan WU; Xuran WANG; Dongqi NAN; Wenbo YANG; Xuhu MAO

doi:10.16016/j.2097-0927.202204009

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Construction of fluorescence-labeled Burkholderia pseudomallei and its interaction with host cells

Laboratory Medicine | 更新时间：2026-01-09

- Construction of fluorescence-labeled Burkholderia pseudomallei and its interaction with host cells
- Journal of Army Medical University Vol. 44, Issue 20, Pages: 2105-2112(2022)
- 作者机构：
  
  400038 重庆, 陆军军医大学(第三军医大学)药学与检验医学系临床微生物与免疫学教研室
- 作者简介：
- 基金信息：
  
  the General Program of National Natural Science Foundation of China(82272350);the Technological Innovation Capability Improvement Project of Army Medical University(2021XJS07)
- DOI：10.16016/j.2097-0927.202204009
  CLC： Q935;R-331;R378.99
- Received：01 April 2022，
  
  Revised：2022-06-24，
  
  Published：30 October 2022
- 稿件说明：
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Jiangao CHEN, Chenglong RAO, Wenzheng LIU, et al. Construction of fluorescence-labeled Burkholderia pseudomallei and its interaction with host cells[J]. Journal of Army Medical University, 2022, 44(20): 2105-2112.
DOI：

Jiangao CHEN, Chenglong RAO, Wenzheng LIU, et al. Construction of fluorescence-labeled Burkholderia pseudomallei and its interaction with host cells[J]. Journal of Army Medical University, 2022, 44(20): 2105-2112. DOI： 10.16016/j.2097-0927.202204009.

摘要

目的

构建类鼻疽菌(

Burkholderia pseudomallei

，

B. pseudomallei

)的荧光示踪载体，获得自发荧光的类鼻疽菌株，并对其与宿主相互作用的感染过程进行初步应用研究。

方法

采用基因重组技术，将绿色、红色和蓝色荧光蛋白(green/red/blue fluorescent protein，GFP/RFP/BFP)基因分别重组到原核表达载体pUCP28T上，构建类鼻疽菌组成型荧光表达载体，并通过体外细菌培养观察外源性荧光蛋白的表达对细菌生长能力的影响，以及荧光标记细菌遗传稳定性的检测。通过炎症因子IL-8的检测评价荧光标记细菌对宿主细胞感染能力的变化。利用活细胞工作站观察类鼻疽菌被宿主细胞自噬体包裹的动态过程。

结果

构建了三种荧光标记的类鼻疽菌示踪载体，并且能在体外传代20代内稳定表达，且外源性荧光蛋白的表达不影响类鼻疽菌的生长和对宿主细胞的感染。同时，利用高分辨率活细胞成像系统观察到红色荧光标记的类鼻疽菌与宿主细胞GFP-LC3共定位的动态变化过程。

结论

本文成功构建了三种荧光标记的类鼻疽菌，以期为类鼻疽菌致病机制的研究、抗菌药物的筛选提供重要实用工具。

Abstract

Objective

To construct the fluorescence-labeled

Burkholderia pseudomallei

(

B. pseudomallei

) to study the pathogenesis of the interaction between the bacteria and host cells.

Methods

Gene recombination technique was carried out to recombine the green fluorescent protein (GFP)

red fluorescent protein (RFP) and blue fluorescent protein (BFP) genes into the prokaryotic expression vector pUCP28T

respectively

in order to obtain the fluorescence-labeled

B. pseudomallei

. Furthermore

the biological characteristics of fluorescence-labeled bacteria were detected in bacterial culture

in vitro

and inflammatory factor IL-8 was detected to evaluate the changes in the ability of fluorescence-labeled bacteria infecting host cells. Then

the dynamic process of fluorescent-labeled

B. pseudomallei

being enveloped by host cell autophagosome was observed with aid of living cell workstation.

Results

Three fluorescence-labeled

B. pseudomallei

were constructed respectively

and were stably expressed in 20 passages

in vitro

. The expression of exogenous fluorescent proteins did not affect the growth of

B. pseudomallei

and infection of host cells. Meanwhile

the dynamic changes of co-localization between RFP-labeled

B. pseudomallei

and GFP-LC3 were observed by live cell imaging system.

Conclusion

Three different fluorescence-labeled

B. pseudomallei

are successfully constructed. And they are important practical tools for the pathogenesis of the bacteria and the screening of antimicrobial agents of melioidosis.

关键词

Keywords

references

WIERSINGA W J, VIRK H S, TORRES A G, et al . Melioidosis[J ] . Nat Rev Dis Primers, 2018, 4: 17107. DOI: 10.1038/nrdp.2017.107.

Wiersinga WJ, Currie BJ, Peacock SJ: Melioidosis[J]. N Engl J Med 2012, 367(11): 1035-1044. DOI: 10.1056/NEJMra1204699.

GASSIEP I, ARMSTRONG M, NORTON R. Human melioidosis[J]. Clin Microbiol Rev, 2020, 33(2): e00006-e00019. DOI: 10.1128/CMR.00006-19.

BIRNIE E, VIRK H S, SAVELKOEL J, et al . Global burden of melioidosis in 2015: a systematic review and data synthesis[J ] . Lancet Infect Dis, 2019, 19(8): 892-902. DOI: 10.1016/S1473-3099(19)30157-4.

FANG Y, HUANG Y, LI Q, et al . First genome sequence of a Burkholderia pseudomallei isolate in China, strain BPC006, obtained from a melioidosis patient in Hainan[J ] . J Bacteriol, 2012, 194(23): 6604-6605. DOI: 10.1128/jb.01577-12.

WILLCOCKS S J, DENMAN C C, ATKINS H S, et al . Intracellular replication of the well-armed pathogen Burkholderia pseudomallei[J ] . Curr Opin Microbiol, 2016, 29: 94-103. DOI: 10.1016/j.mib.2015.11.007.

ALLWOOD E M, DEVENISH R J, PRESCOTT M, et al . Strategies for intracellular survival of Burkholderia pseudomallei[J ] . Front Microbiol, 2011, 2: 170. DOI: 10.3389/fmicb.2011.00170.

STONE J K, DESHAZER D, BRETT P J, et al . Melioidosis: molecular aspects of pathogenesis[J ] . Expert Rev Anti Infect Ther, 2014, 12(12): 1487-1499. DOI: 10.1586/14787210.2014.970634.

TANG M L, HU Z Q, RAO C L, et al . Burkholderia pseudomallei interferes with host lipid metabolism via NR1D2-mediated PNPLA2/ATGL suppression to block autophagy-dependent inhibition of infection[J ] . Autophagy, 2021, 17(8): 1918-1933. DOI: 10.1080/15548627.2020.1801270.

HU Z Q, RAO C L, TANG M L, et al . Rab32 GTPase, as a direct target of miR-30b/c, controls the intracellular survival of Burkholderia pseudomallei by regulating phagosome maturation[J ] . PLoS Pathog, 2019, 15(6): e1007879. DOI: 10.1371/journal.ppat.1007879.

HU Y L, WEN Z H, LIU S, et al . Ibrutinib suppresses intracellular Mycobacterium tuberculosis growth by inducing macrophage autophagy[J ] . J Infect, 2020, 80(6): e19-e26. DOI: 10.1016/j.jinf.2020.03.003.

XU Y, ZHOU P, CHENG S, et al . A bacterial effector reveals the V-ATPase-ATG16L1 axis that initiates xenophagy[J ] . Cell, 2019, 178(3): 552-566. e20. DOI: 10.1016/j.cell.2019.06.007.

GLUECK N K, O'BRIEN K M, SEGUIN D C, et al . Legionella pneumophila LegC7 effector protein drives aberrant endoplasmic Reticulum: endosome contacts in yeast[J ] . Traffic, 2021, 22(8): 284-302. DOI: 10.1111/tra.12807.

CHALFIE M, TU Y, EUSKIRCHEN G, et al . Green fluorescent protein as a marker for gene expression[J ] . Science, 1994, 263(5148): 802-805. DOI: 10.1126/science.8303295.

JOSENHANS C, FRIEDRICH S, SUERBAUM S. Green fluorescent protein as a novel marker and reporter system in Helicobacter sp[J]. FEMS Microbiol Lett, 1998, 161(2): 263-273. DOI: 10.1111/j.1574-6968.1998.tb12957.x.

CODY C W, PRASHER D C, WESTLER W M, et al . Chemical structure of the hexapeptide chromophore of the Aequorea green-fluorescent protein[J ] . Biochemistry, 1993, 32(5): 1212-1218. DOI: 10.1021/bi00056a003.

CHUA K L, CHAN Y Y, GAN Y H. Flagella are virulence determinants of Burkholderia pseudomallei[J]. Infect Immun, 2003, 71(4): 1622-1629. DOI: 10.1128/IAI.71.4.1622-1629.2003.

WONG J, CHEN Y H, GAN Y H. Host cytosolic glutathione sensing by a membrane histidine kinase activates the type VI secretion system in an intracellular bacterium[J]. Cell Host Microbe, 2015, 18(1): 38-48. DOI: 10.1016/j.chom.2015.06.002.

DERETIC V. Autophagy in inflammation, infection, and immunometabolism[J]. Immunity, 2021, 54(3): 437-453. DOI: 10.1016/j.immuni.2021.01.018.

DERETIC V, SAITOH T, AKIRA S. Autophagy in infection, inflammation and immunity[J]. Nat Rev Immunol, 2013, 13(10): 722-737. DOI: 10.1038/nri3532.

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⁰

Construction of fluorescence-labeled Burkholderia pseudomallei and its interaction with host cells

DOI：10.16016/j.2097-0927.202204009

摘要

Abstract

关键词

Keywords

references