Either fadD1 or fadD2, Which Encode acyl-CoA Synthetase, Is Essential for the Survival of Haemophilus parasuis SC096.

Saixiang Feng,Chenggang Xu,Huiying Fan,Kaijie Yang,Ming Liao,Haihong Wang

doi:10.3389/fcimb.2017.00072

Abstract

In Haemophilus parasuis, the genes HAPS_0217 and HAPS_1695 are predicted to encode long-chain fatty acid-CoA ligases (FACSs). These proteins contain ATP/AMP signature motifs and FACS conserved motifs that are homologous to those in Escherichia coli FadD (EcFadD). In this study, we demonstrate that HAPS_0217 and HAPS_1695 can functionally replace EcFadD in the E. coli fadD mutant JW1794, and were thus designated fadD1 and fadD2, respectively. An evaluation of kinetic parameters indicated that FadD1 and FadD2 have a substrate preference for long-chain fatty acids. Moreover, FadD2 exhibited substrate inhibition in the presence of high concentrations of oleic acid. Single mutants of each of the fadD genes were easily constructed, whereas double mutants were not. These results were further confirmed using genomic site-directed mutagenesis, which supported the idea that H. parasuis requires either fadD1 or fadD2 for survival. The fadD1 mutant exhibited slower growth than the wild-type strain SC096, and its complementation resulted in a restored phenotype. The wild-type strain did not grow on chemically defined medium without the addition of oleic acid, indicating that lipids are a vital nutrient for this bacterium. Additionally, strains with a disrupted fadD1 gene also exhibited increased sensitivity to quinolone antibiotics, including levofloxacin, enrofloxacin, ciprofloxacin and nalidixic acid.

Highlights

Haemophilus parasuis is a pathogenic bacterium of the upper respiratory tract in conventional pigs and the etiological agent of Glässer’s disease, which is characterized by fibrinous polyserositis, arthritis, and meningitis
An alignment of the FadD homolog revealed that HpFadD1 was 65% identical to E. coli FadD and that the ATP/AMP and fatty acid-CoA ligases (FACSs) motifs were conserved between them (Figure 1C)
HpFadD2 shared no similarity with Escherichia coli FadD (EcFadD) when analyzed using BLAST but was 22% identical to E. coli FadK, a short-chain acylCoA synthetase (Morgan-Kiss and Cronan, 2004)

Summary

Introduction

Haemophilus parasuis is a pathogenic bacterium of the upper respiratory tract in conventional pigs and the etiological agent of Glässer’s disease, which is characterized by fibrinous polyserositis, arthritis, and meningitis. Previous differential expression studies showed that exogenous fatty acid utilization enzyme FadD was a potential virulence factor in H. parasuis (Hill et al, 2003; Metcalf and MacInnes, 2007). Fatty acids are important metabolic intermediates as well as major components of phospholipids, which are essential for membrane formation fadD Is Essential for Haemophilus parasuis in pathogenic bacteria (Zhang and Rock, 2008a). Because fatty acid biosynthesis is vital and energetically expensive, most pathogens use and incorporate extracellular fatty acids into their phospholipid membrane (Yao and Rock, 2015)

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