Nicotinic acid modulates Legionella pneumophila gene expression and induces virulence traits

Abstract

In response to environmental fluctuations or stresses, bacteria can activate transcriptional and phenotypic programs to coordinate an adaptive response. The intracellular pathogen Legionella pneumophila converts from a noninfectious replicative form to an infectious transmissive form when the bacterium encounters alterations in either amino acid concentrations or fatty acid biosynthesis. Here, we report that L. pneumophila differentiation is also triggered by nicotinic acid, a precursor of the central metabolite NAD(+). In particular, when replicative L. pneumophila are treated with 5 mM nicotinic acid, the bacteria induce numerous transmissive-phase phenotypes, including motility, cytotoxicity toward macrophages, sodium sensitivity, and lysosome avoidance. Transcriptional profile analysis determined that nicotinic acid induces the expression of a panel of genes characteristic of transmissive-phase L. pneumophila. Moreover, an additional 213 genes specific to nicotinic acid treatment were altered. Although nearly 25% of these genes lack an assigned function, the gene most highly induced by nicotinic acid treatment encodes a putative major facilitator superfamily transporter, Lpg0273. Indeed, lpg0273 protects L. pneumophila from toxic concentrations of nicotinic acid as judged by analyzing the growth of the corresponding mutant. The broad utility of the nicotinic acid pathway to couple central metabolism and cell fate is underscored by this small metabolite's modulation of gene expression by diverse microbes, including Candida glabrata, Bordetella pertussis, Escherichia coli, and L. pneumophila.

Fig 1

Nicotinic acid treatment triggers growth inhibition and premature motility. E-phase cultures of WT L. pneumophila carrying the flaA-GFP gene reporter were supplemented with water (H₂O) or 5 mM nicotinic acid (NA). At the times indicated, samples of each culture were collected to quantify bacterial growth as determined by optical density (A) and expression of the transmission gene flaA by relative fluorescence (B). Shown are representative graphs from three or more independent experiments. At 6 and 9 h posttreatment, the optical densities and fluorescence levels were statistically different between E-phase cultures treated with water and E-phase bacteria treated with 5 mM NA (P < 0.05).

Fig 2

Nicotinic acid treatment induces WT L. pneumophila to express multiple transmissive-phase traits. (A) The extent of lysosome evasion after a 2-h infection of macrophages with L. pneumophila at an MOI of 1 was quantified as the percentage of intracellular bacteria that were intact, observed by fluorescence microscopy. Displayed are the mean results from duplicate samples in three independent experiments. Error bars indicate standard deviations (SD), and asterisks designate significant differences (P < 0.01) compared to the results for the E-phase water control. (B) Macrophage viability after 1-h infection of macrophages at the multiplicities of infection (MOI) shown was quantified with PE-phase (triangles) or E-phase cultures supplemented with water (H₂O) or nicotinic acid (NA) using the colorimetric dye alamarBlue. The values plotted represent the means ± SD for triplicate samples determined in one of three similar experiments. At similar MOIs, E-phase cultures supplemented with NA were significantly different than E-phase cultures supplemented with water (P < 0.05). E-phase cultures treated with NA were not statistically different than PE-phase bacteria (P > 0.05). (C) The percentage of sodium-resistant bacteria was measured by plating cultures on medium with or without 100 mM NaCl and then calculating CFU as described previously (41). Values shown = mean [(E + NA or PE control)/(E + H₂O) × 100]. Error bars represent SD from duplicate samples in three independent experiments; for E-phase + water results, the error bar is too small to be visible. Asterisks denote statistically significant differences (P < 0.01) compared to the results for the E-phase water control.

Fig 3

LetA/LetS is dispensable for growth inhibition but required for full induction of motility in response to nicotinic acid treatment. WT (circles), letA (triangles) or letS (squares) L. pneumophila cells containing the flaA-GFP gene reporter were cultured to E phase and then supplemented with water (closed symbols) or 10 mM nicotinic acid (NA; open symbols). At the times indicated, samples were collected and growth was analyzed as OD₆₀₀ (A) and flaA expression as relative fluorescence (B). Similar fluorescence patterns were observed in three independent experiments using two different fluorometers. At time points after 0 h, the OD₆₀₀ values for strains treated with NA were significantly different than those for strains treated with water (P < 0.05).

Fig 4

Comparison of genes regulated in the PE-phase and nicotinic acid-treated L. pneumophila. Genes with higher levels of transcriptional activation (black bars) and repression (white bars) than those of E-phase control cells were identified by microarray analysis for the following conditions: Total, genes that responded to either the PE phase or 3-h nicotinic acid treatment (NA); Common, genes that responded to both the PE phase and NA; PE specific, genes that responded to PE but not NA; NA specific, genes that responded to NA but not PE.

Fig 5

Region of the L. pneumophila chromosome that encodes lpg0273 and proposed structure of Lpg0273. (A) Chromosomal locus of lpg0273. Arrows indicate orientation of the genes. Sizes (bp) of the genes are indicated above, and the predicted function of each gene is listed below. Red indicates genes with significantly elevated expression levels when treated with 5 mM nicotinic acid. Gray indicates genes that are not affected by a 3-h nicotinic acid treatment. (B) Structure of Lpg0273 and ligand binding site (blue) predicted using 3DLigandSite prediction software (71), showing helices typical of MFS transporter proteins.

Fig 6

The lpg0273 MFS gene confers resistance to growth inhibition by nicotinic acid. WT or lpg0273 mutant L. pneumophila carrying plasmid-born lpg0273 (plpg0273) or the parent vector (pGent) were cultured in AYET plus 200 μM IPTG and supplemented with H₂O or 5 mM (A) or 2.5 mM (B) nicotinic acid (NA), and the optical density was recorded by a Bioscreen growth curve analyzer at the times shown. Displayed are means ± SD calculated from triplicate wells in a single experiment that is representative of at least three independent experiments.