The Fatty Acid Regulator FadR Influences the Expression of the Virulence Cascade in the El Tor Biotype of Vibrio cholerae by Modulating the Levels of ToxT via Two Different Mechanisms

Abstract

FadR is a master regulator of fatty acid (FA) metabolism that coordinates the pathways of FA degradation and biosynthesis in enteric bacteria. We show here that a ΔfadR mutation in the El Tor biotype of Vibrio cholerae prevents the expression of the virulence cascade by influencing both the transcription and the posttranslational regulation of the master virulence regulator ToxT. FadR is a transcriptional regulator that represses the expression of genes involved in FA degradation, activates the expression of genes involved in unsaturated FA (UFA) biosynthesis, and also activates the expression of two operons involved in saturated FA (SFA) biosynthesis. Since FadR does not bind directly to the toxT promoter, we determined whether the regulation of any of its target genes indirectly influenced ToxT. This was accomplished by individually inserting a double point mutation into the FadRbinding site in the promoter of each target gene, thereby preventing their activation or repression. Although preventing FadR-mediated activation of fabA, which encodes the enzyme that carries out the first step in UFA biosynthesis, did not significantly influence either the transcription or the translation of ToxT, it reduced its levels and prevented virulence gene expression. In the mutant strain unable to carry out FadRmediated activation of fabA, expressing fabA ectopically restored the levels of ToxT and virulence gene expression. Taken together, the results presented here indicate that V. cholerae FadR influences the virulence cascade in the El Tor biotype by modulating the levels of ToxT via two different mechanisms.IMPORTANCE Fatty acids (FAs) play important roles in membrane lipid homeostasis and energy metabolism in all organisms. In Vibrio cholerae, the causative agent of the acute intestinal disease cholera, they also influence virulence by binding into an N-terminal pocket of the master virulence regulator, ToxT, and modulating its activity. FadR is a transcription factor that coordinately controls the pathways of FA degradation and biosynthesis in enteric bacteria. This study identifies a new link between FA metabolism and virulence in the El Tor biotype by showing that FadR influences both the transcription and posttranslational regulation of the master virulence regulator ToxT by two distinct mechanisms.

Switching fatty acid metabolism by an RNA-controlled feed forward loop

“…S6 G and H). Of note, FarS only acts to down-regulate the existing vc1740 and vc2231 mRNAs, while transcription of these genes is simultaneously repressed by FadR (52). Together, both factors (transcriptional and posttranscriptional regulation) allow FadE repression when fatty acids become scarce.…”

Structural and Functional Characterization of the FadR Regulatory Protein from Vibrio alginolyticus

“…The two major virulence determinants of Vibrio cholerae referred to toxin-coregulated pilus (TCP) (Taylor et al, 1987 ) and cholera toxin (CT) (Matson et al, 2007 ), which are coordinated by two independent activators ToxR, a transmembrane regulatory protein (Miller et al, 1987 ), and ToxT, a member of AraC family transcription factors (Champion et al, 1997 ). It seems true that the regulated expression of fatty acid metabolism is implicated into Vibrio pathogenicity in that (i) inactivation of fadR impairs virulence of V. vulnificus in mice (Brown and Gulig, 2008 ); (ii) the disruption of fadD gene encoding a long-chain fatty acyl coenzyme A ligase affected production of virulence factors, CtxAB and TcpA (Ray et al, 2011 ); and (iii) unsaturated fatty acids from bile interfered with bacterial mobility via remodeling bacterial membrane structure (Giles et al, 2011 ) and inhibited the expression of virulence factors (CtxAB and TcpA) by decreased DNA-binding activity of ToxT in V. cholerae (Brown and Gulig, 2008 ; Giles et al, 2011 ; Yang et al, 2013 ; Kovacikova et al, 2017 ). More recently, Kovacikova et al ( 2017 ) proposed that the V. cholerae FadR controls the expression of the virulence cascade by indirect-regulation of ToxT via two different mechanisms (Kovacikova et al, 2017 ).…”

“…It seems true that the regulated expression of fatty acid metabolism is implicated into Vibrio pathogenicity in that (i) inactivation of fadR impairs virulence of V. vulnificus in mice (Brown and Gulig, 2008 ); (ii) the disruption of fadD gene encoding a long-chain fatty acyl coenzyme A ligase affected production of virulence factors, CtxAB and TcpA (Ray et al, 2011 ); and (iii) unsaturated fatty acids from bile interfered with bacterial mobility via remodeling bacterial membrane structure (Giles et al, 2011 ) and inhibited the expression of virulence factors (CtxAB and TcpA) by decreased DNA-binding activity of ToxT in V. cholerae (Brown and Gulig, 2008 ; Giles et al, 2011 ; Yang et al, 2013 ; Kovacikova et al, 2017 ). More recently, Kovacikova et al ( 2017 ) proposed that the V. cholerae FadR controls the expression of the virulence cascade by indirect-regulation of ToxT via two different mechanisms (Kovacikova et al, 2017 ). In contrast to the E. coli FadR paradigm, its Vibrio homologs with an insert of 40 aa exhibit stronger ability of binding LC acyl-CoA thioesters (Heidelberg et al, 2000 ; Iram and Cronan, 2005 ).…”

Redox, amino acid, and fatty acid metabolism intersect with bacterial virulence in the gut

“…LCFAs influence the virulence of the enteric pathogens Vibrio cholerae and Salmonella enterica serovar Typhimurium. In V. cholera, FadR indirectly activates the expression of the master virulence regulator toxT, which activates the expression of cholera toxin and toxin-coregulated pilus (14). FadR also participates in ToxT regulation by activating the expression of fabA, encoding an enzyme required for unsaturated fatty acid (UFA) synthesis.…”