Elsevier

Veterinary Microbiology

Volume 230, March 2019, Pages 130-137
Veterinary Microbiology

Identification of novel genes essential for Brucella abortus to establish infection by signature-tagged mutagenesis

https://doi.org/10.1016/j.vetmic.2019.02.005Get rights and content

Highlights

  • A PCR-based STM technique was used to identify Brucella virulence-related genes.

  • 34 genes were newly identified as Brucella virulence-related genes.

  • pyruvate carboxylase (pyc) plays an important role in Brucella infection in a mouse model.

Abstract

Brucella is a facultative intracellular bacterium, causing brucellosis, an important zoonosis worldwide. Brucella has no classic virulence factors, thus virulence is dependent on invasion of host cells and subsequent intracellular replication. Identification of key genes involved in Brucella virulence is important to further elucidate its pathogenesis. In this study, signature-tagged mutagenesis was used to identify novel genes involved in B. abortus infection in a mouse model. In total 3600 mutants were obtained, of which 56 were identified as attenuated mutants. Furthermore, 53 genes were identified to be inactivated by transposon insertion, including 19 genes previously reported to be essential for Brucella virulence and 34 others that were newly identified in this study. These genes were catalogued into 16 functional classifications, except for three that were not cited in the Clusters of Orthologous Groups database. Bioinformatics analysis revealed that energy production and conversion, amino acid transport and metabolism, as well as inorganic ion transport and metabolism were predominant functional classifications, suggesting that genes involved in these functions were crucial for Brucella virulence. In addition, the function of the identified pyruvate carboxylase (pyc) gene in bacterial virulence was confirmed using an allelic replacement pyc mutant and a mouse model. These findings provide novel genetic information associated with Brucella infection.

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.

Section snippets

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).

References (28)

  • S. Endley et al.

    Interruption of the cydB locus in Brucella abortus attenuates intracellular survival and virulence in the mouse model of infection

    J. Bacteriol.

    (2001)
  • V. Haine et al.

    Systematic targeted mutagenesis of Brucella melitensis 16M reveals a major role for GntR regulators in the control of virulence

    Infect. Immun.

    (2005)
  • P.C. Hong et al.

    Identification of genes required for chronic persistence of Brucella abortus in mice

    Infect. Immun.

    (2000)
  • G. Kikuchi

    The glycine cleavage system: composition, reaction mechanism, and physiological significance

    Mol. Cell. Biochem.

    (1973)
  • Cited by (8)

    • The novel LysR-family transcriptional regulator BvtR is involved in the resistance of Brucella abortus to nitrosative stress, detergents and virulence through the genetic regulation of diverse pathways

      2022, Veterinary Microbiology
      Citation Excerpt :

      Thus, the identification of virulence-related factors in Brucella has long been a research focus. Previous studies have widely used the method of disrupting putative virulence genes to study their effects on infection in cell lines or mouse models (Foulongne et al., 2000; Kohler et al., 2002; Tian et al., 2019b). The identification of novel virulence genes may provide new insights into Brucella pathogenesis.

    • Genomic insights into Brucella

      2021, Infection, Genetics and Evolution
      Citation Excerpt :

      Also, similar studies should be extended to other Brucella species and their respective host animals. Recently, Tian et al. (2019) have used STM to identify attenuated mutants of B. abortus in a mouse model. They have used 3600 tagged mutants and identified 56 attenuated mutants representing insertional inactivation in 53 genes.

    • Quantification of Brucella abortus population structure in a natural host

      2021, Proceedings of the National Academy of Sciences of the United States of America
    View all citing articles on Scopus
    View full text