AUTHOR=Martínez-Alcantar Lorena , Orozco Gabriela , Díaz-Pérez Alma Laura , Villegas Javier , Reyes-De la Cruz Homero , García-Pineda Ernesto , Campos-García Jesús 

TITLE=Participation of Acyl-Coenzyme A Synthetase FadD4 of Pseudomonas aeruginosa PAO1 in Acyclic Terpene/Fatty Acid Assimilation and Virulence by Lipid A Modification

JOURNAL=Frontiers in Microbiology

VOLUME=12

YEAR=2021

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2021.785112

DOI=10.3389/fmicb.2021.785112

ISSN=1664-302X

ABSTRACT=<p>The pathogenic bacterium <italic>Pseudomonas aeruginosa</italic> possesses high metabolic versatility, with its effectiveness to cause infections likely due to its well-regulated genetic content. <italic>P. aeruginosa</italic> PAO1 has at least six <italic>fadD</italic> paralogous genes, which have been implicated in fatty acid (FA) degradation and pathogenicity. In this study, we used mutagenesis and a functional approach in <italic>P. aeruginosa</italic> PAO1 to determine the roles of the <italic>fadD4</italic> gene in acyclic terpene (AT) and FA assimilation and on pathogenicity. The results indicate that <italic>fadD4</italic> encodes a terpenoyl-CoA synthetase utilized for AT and FA assimilation. Additionally, mutations in <italic>fadD</italic> paralogs led to the modification of the quorum-sensing <italic>las</italic>/<italic>rhl</italic> systems, as well as the content of virulence factors pyocyanin, biofilm, rhamnolipids, lipopolysaccharides (LPS), and polyhydroxyalkanoates. In a <italic>Caenorhabditis elegans in vivo</italic> pathogenicity model, culture supernatants from the 24-h-grown <italic>fadD4</italic> single mutant increased lethality compared to the PAO1 wild-type (WT) strain; however, the double mutants <italic>fadD1/fadD2</italic>, <italic>fadD1/fadD4</italic>, and <italic>fadD2/fadD4</italic> and single mutant <italic>fadD2</italic> increased worm survival. A correlation analysis indicated an interaction between worm death by the PAO1 strain, the <italic>fadD4</italic> mutation, and the virulence factor LPS. Fatty acid methyl ester (FAME) analysis of LPS revealed that a proportion of the LPS and FA on lipid A were modified by the <italic>fadD4</italic> mutation, suggesting that FadD4 is also involved in the synthesis/degradation and modification of the lipid A component of LPS. LPS isolated from the <italic>fadD4</italic> mutant and double mutants <italic>fadD1/fadD4</italic> and <italic>fadD2/fadD4</italic> showed a differential behavior to induce an increase in body temperature in rats injected with LPS compared to the WT strain or from the <italic>fadD1</italic> and <italic>fadD2</italic> mutants. In agreement, LPS isolated from the <italic>fadD4</italic> mutant and double mutants <italic>fadD1/fadD2</italic> and <italic>fadD2/fadD4</italic> increased the induction of IL-8 in rat sera, but IL1-β cytokine levels decreased in the double mutants <italic>fadD1/fadD2</italic> and <italic>fadD1/fadD4</italic>. The results indicate that the <italic>fadD</italic> genes are implicated in the degree of pathogenicity of <italic>P. aeruginosa</italic> PAO1 induced by LPS-lipid A, suggesting that FadD4 contributes to the removal of acyl-linked FA from LPS, rendering modification in its immunogenic response associated to Toll-like receptor TLR4. The genetic redundancy of <italic>fadD</italic> is important for bacterial adaptability and pathogenicity over the host.</p>