Identification of novel genes essential for Brucella abortus to establish infection by signature-tagged mutagenesis
Introduction
Brucella is a genus of Gram-negative facultative intracellular pathogens, causing brucellosis, an important bacterial zoonosis worldwide (Boschiroli et al., 2001; von Bargen et al., 2012). In contrast to other pathogenic bacteria, Brucella has no classic virulence factors, such as exotoxins, cytolysins, capsules, fimbria or endotoxic lipopolysaccharide (LPS) (Seleem et al., 2008). A key aspect of Brucella virulence is the ability to replicate within host cells. Upon invasion of the host cell, Brucella uses several sophisticated strategies for intracellular survival, including resistance to the bactericidal effects of phagocytes, inhibition of host cell apoptosis, adaption to acidified membrane-bound vesicles and prevention of phagosome-lysosome fusion (Martirosyan et al., 2011). Together, these properties endow Brucella with the ability to establish infection in preferred hosts. Hence, it is very important to identify key genes necessary for Brucella infection and to further elucidate the mechanisms of Brucella pathogenesis.
To date, several key factors of Brucella virulence have been identified, which include LPS (Lapaque et al., 2005), the type IV secretion system (T4SS) (Celli et al., 2003; O’Callaghan et al., 1999) and the two-component regulatory system BvrS/BvrR (Sola-Landa et al., 1998). Besides, many other components are reportedly involved in Brucella virulence, including cyclic β-1,2-glucans, superoxide dismutase, catalase, urease and cytochrome oxidase (Seleem et al., 2008). So far, based on a platform of the Brucella Bioinformatics Portal, 245 genes involved in Brucella virulence have been collected in a database (Xiang et al., 2006). However, with the development of molecular genetic techniques, more and more genes associated with Brucella virulence continue to be discovered, thereby offering further insight into Brucella pathogenesis.
In this study, a PCR-based signature-tagged mutagenesis technique was used to identify attenuated mutants in a mouse model of Brucella infection. Finally, 53 genes were identified as necessary for Brucella infection, including 34 that are newly reported in this study, thereby providing further insight into Brucella pathogenesis.
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Ethics statement
This study was performed in strict accordance with the guidelines of the Care and Use of Laboratory Animals of the Institutional Animal Care and Use Committee of Shanghai Veterinary Research Institute, the Chinese Academy of Agricultural Sciences (CAAS). Mice (SLAC Experimental Animal Inc., Shanghai, China) were housed in cages with ad libitum access to food and water under biosafety conditions. All animal handling procedures were approved by the Committee on the Ethics of Animal Experiments of
Virulence was similar between the NA-resistant and WT B. abortus strains
To construct a mutagenesis library of B. abortus, the bacterial strain requires an antibiotic resistant marker to screen for mutants. According to a previous report by Kim et al. (2003), NA resistance is easily induced in Brucella. A NA-resistant strain of B. abortus S2308 was successfully constructed in this study. The WT B. abortus strain S2308 was inoculated into TSB with 5% FBS and NA, and a gradually increasing concentration of antibiotics. Brucella was grown in TSB containing 50 μg/mL of
Discussion
Brucella virulence is associated with genes involved in energy production and metabolism. In this study, the pyc (BAB1_1791), pyk (BAB1_1761) and ppdk (BAB1_0525) genes associated with pyruvate metabolism were identified, suggesting that the node of pyruvate catabolism may play an important role in Brucella virulence. Pyc is a ligase that irreversibly catalyses carboxylation of pyruvate to form oxaloacetate, which is important to carbon metabolism of the cyclic pyruvate-tricarboxylic acid
Conflict of interest
The authors declare no conflict of interest.
Acknowledgements
This work was supported by funds from the National Natural Science Foundation of China (31602070), the Shanghai Sailing Program (16YF1414600), the Scientific and Technical Innovation Project of the Chinese Academy of Agricultural Sciences (SHVRI-ASTIP-2014-8) and the National Key Research and Development Program of China (2018YFD0500500).
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