Role of aspartate ammonia-lyase in Pasteurella multocida

Zui Wang,Xiaozhong Wang,Chen Wang,Qin Lu,Huabin Shao,Lina Liu,Qingping Luo,Li Li,Peng Liu

doi:10.1186/s12866-020-02049-2

Abstract

BackgroundPasteurella multocida is responsible for a highly infectious and contagious disease in birds, leading to heavy economic losses in the chicken industry. However, the pathogenesis of this disease is poorly understood. We recently identified an aspartate ammonia-lyase (aspA) in P. multocida that was significantly upregulated under iron-restricted conditions, the protein of which could effectively protect chicken flocks against P. multocida. However, the functions of this gene remain unclear. In the present study, we constructed aspA mutant strain △aspA::kan and complementary strain C△aspA::kan to investigate the function of aspA in detail.ResultDeletion of the aspA gene in P. multocida resulted in a significant reduction in bacterial growth in LB (Luria-Bertani) and MH (Mueller-Hinton) media, which was rescued by supplementation with 20 mM fumarate. The mutant strain △aspA::kan showed significantly growth defects in anaerobic conditions and acid medium, compared with the wild-type strain. Moreover, growth of △aspA::kan was more seriously impaired than that of the wild-type strain under iron-restricted conditions, and this growth recovered after supplementation with iron ions. AspA transcription was negatively regulated by iron conditions, as demonstrated by quantitative reverse transcription-polymerase chain reaction. Although competitive index assay showed the wild-type strain outcompetes the aspA mutant strain and △aspA::kan was significantly more efficient at producing biofilms than the wild-type strain, there was no significant difference in virulence between the mutant and the wild-type strains.ConclusionThese results demonstrate that aspA is required for bacterial growth in complex medium, and under anaerobic, acid, and iron-limited conditions.

Highlights

Pasteurella multocida is responsible for a highly infectious and contagious disease in birds, leading to heavy economic losses in the chicken industry
The aspartate ammonia-lyase (aspA) gene was significantly upregulated under iron-restricted conditions in various bacteria including C. jejuni [11], A. pleuropneumoniae [12], Edwardsiella ictaluri [13], and P. multocida [14], suggesting that aspA might be related to iron acquisition
To further characterize the aspA mutant, the expressions of aspA were detected in the parent strain C48–1 and complementary strain C△aspA::kan but not in the mutant strain △aspA::kan or control strain △aspA::kan (Fig. 1c), indicating that the aspA gene was successfully deleted from C48–1

Summary

Introduction

Pasteurella multocida is responsible for a highly infectious and contagious disease in birds, leading to heavy economic losses in the chicken industry. Pasteurella multocida is a capsulated, Gram-negative facultative anaerobic bacterium responsible for fowl cholera in poultry, leading to great economic losses in commercial layer flocks and local chicken breeds [1]. Once a Aspartate ammonia-lyase (aspA) has been identified in various other Gram-negative bacteria, including Salmonella enterica [4], Actinobacillus pleuropneumoniae [5] and Escherichia coli [6]. This enzyme is involved in catalyzing the reversible conversion of L-aspartate to form fumarate and release ammonia [7], and plays a vital role in the production of L-aspartate [8]. An iron-restricted environment often triggers the expression of virulence factors in pathogens [15], indicating the need to determine if aspA is an important virulence factor in P. multocida

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