Blocking Phosphatidylcholine Utilization in <i>Pseudomonas aeruginosa</i>, via Mutagenesis of Fatty Acid, Glycerol and Choline Degradation Pathways, Confirms the Importance of This Nutrient Source <i>In Vivo</i>

<div><p><i>Pseudomonas aeruginosa</i> can grow to very high-cell-density (HCD) during infection of the cystic fibrosis (CF) lung. Phosphatidylcholine (PC), the major component of lung surfactant, has been hypothesized to support HCD growth of <i>P. aeruginosa in vivo.</i> The phosphorylcholine headgroup, a glycerol molecule, and two long-chain fatty acids (FAs) are released by enzymatic cleavage of PC by bacterial phospholipase C and lipases. Three different bacterial pathways, the choline, glycerol, and fatty acid degradation pathways, are then involved in the degradation of these PC components. Here, we identified five potential FA degradation (Fad) related <i>fadBA</i>-operons (<i>fadBA1-5</i>, each encoding 3-hydroxyacyl-CoA dehydrogenase and acyl-CoA thiolase). Through mutagenesis and growth analyses, we showed that three (<i>fadBA145</i>) of the five <i>fadBA</i>-operons are dominant in medium-chain and long-chain Fad. The triple <i>fadBA145</i> mutant also showed reduced ability to degrade PC <i>in vitro</i>. We have previously shown that by partially blocking Fad, via mutagenesis of <i>fadBA5</i> and <i>fadD</i>s, we could significantly reduce the ability of <i>P. aeruginosa</i> to replicate on FA and PC <i>in vitro</i>, as well as in the mouse lung. However, no studies have assessed the ability of mutants, defective in choline and/or glycerol degradation in conjunction with Fad, to grow on PC or <i>in vivo</i>. Hence, we constructed additional mutants (Δ<i>fadBA145</i>Δ<i>glpD</i>, Δ<i>fadBA145</i>Δ<i>betAB</i>, and Δ<i>fadBA145</i>Δ<i>betAB</i>Δ<i>glpD</i>) significantly defective in the ability to degrade FA, choline, and glycerol and, therefore, PC. The analysis of these mutants in the BALB/c mouse lung infection model showed significant inability to utilize PC <i>in vitro</i>, resulted in decreased replication fitness and competitiveness <i>in vivo</i> compared to the complement strain, although there was little to no variation in typical virulence factor production (e.g., hemolysin, lipase, and protease levels). This further supports the hypothesis that lung surfactant PC serves as an important nutrient for <i>P. aeruginosa</i> during CF lung infection.</p></div

Characteristics of Argentinean HIV-1 primary isolates and reference strains.

<p>DRC: Democratic DRC: Republic of Congo, na: not applicable; nd: not done.</p

Relative fitness difference (W_D) between Argentinean subtype B strains a) B271, b) B563 c) B735 d) B872, e) B958 and Subtype C strains C674 (Argentina), C5, C8, C9 (reference strains).

<p>Competitions were performed in PHA/IL2 treated PBMCs as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092084#pone-0092084-g003" target="_blank">Figure 3</a>. The fitness difference shown on the Y axis ranges from 0.01 to 100. Bars represent the fitness of the isolates shown on the Y-axis.</p

Comparison of intra- and intersubtype fitness of R5 HIV-1 subtype B and various BF recombinants from Argentina.

<p>Mean relative fitness values were determined for each HIV-1 isolate from pairwise competitions between isolates of the same subtype (mean intrasubtype relative fitness) and of different subtypes (mean intersubtype relative fitness). The mean intrasubtype and intersubtype fitness values for each HIV-1 isolate were then plotted as the <i>x</i> and <i>y</i> coordinates. Subtype B viruses are shown as black filled squares while BF recombinants are indicated by a red filled square. The mean fitness of subtype B and BF are very similar and also indicated as red and black filled squares respectively. The insert figure represents the fitness of HIV-1 subtype C relative to group M strains as described in a previous study (Abraha et al, 2009). Subtype C strains (red squares and triangles) have low mean relative fitness and cluster together (orange oval shade) while group M strains (green dots and tetragon) are more fit and cluster together as well (green oval shade).</p

Bacterial strains used in this study^a.

a<p>For strains constructed in this study, please see text for further details.</p>b<p>Please use Lab ID for requesting strains.</p

Comparing the relative replicative fitness difference (W_D) of syncytium inducing (SI) HIV-1 Argentinean subtype B (a) B552, (b) B775 and (c) reference subtype F, 93BR020 against BF Argentinean recombinants (BF134; BF559; BF819, BF027, BF555 and BF640) in direct competitions using PHA/IL2 PBMCs.

<p>Bars represent the fitness of the viruses shown on the Y-axis.</p

Changes in the prevalence of HIV-1 subtypes and recombinant forms during the past ten years in Buenos Aires, Argentina (white) and Rio Grande do Sol, Brazil (gray).

<p>Rio do Sol is located in the most southern part of Brazil while Buenos Aires is in north eastern Argentina. Both regions are located close to each other and share a common characteristic in that they harbor dominant and evolving recombinant forms. The prevalence numbers shown were obtained by combining the subtype distribution from published reports originating in these 2 regions. Only samples with a known sampling date were included in the analyses.</p

Plasmids used in this study^a.

a<p>For plasmids constructed in this study, please see text for further details.</p>b<p>Please use Lab ID for requesting plasmids.</p

Comparing the relative replicative fitness difference (W_D) of HIV-1 sub-subtype F1 reference strains; (a) CA20 and (b) V1850 and Argentinean subtype B (B271, B524, B542, B563, B735 and B872) in direct competitions using PHA/IL2 PBMCs.

<p>Similarly, subtype F (c) CA20 and (d) V1850 were competed against BFs (BF116, BF118, BF119, BF303, BF456, BF549, BF714 and BF955). Bars represent the relative fitness of the viruses shown on the Y-axis.</p

Analysis of viral tropism.

<p>Tropism of each viral isolate was assessed by infecting U87.CD4 cells expressing either CCR5 or CXCR4, in parallel, with each viral stock and by detection of syncytia on MT2 cell line. To further confirm viral tropism, V3-loop sequence of the gp120 protein of each sample was also evaluated in terms of net amino acid charge (net charge), the presence of positively charged amino acids at codons 11 and/or 25 (bold) of V3-loop (bold italics; HXB2 gp160 positions 306 and 322, respectively) (11/25 rule), and the two major bioinformatics algorithms Position-Specific Scoring Matrix (PSSM) and geno2pheno [coreceptor] (g2p). The 20 CCR5, 4 CXCR4-tropic and 4 dual/mixed-tropic isolates (o) are shown on the table. Sample identifications are represented as follows: subtype B (<i>bold italic</i>), C (normal) and BF (bold). All sequences were obtained from the original PBMC of patients.</p><p>For net charge, 11/25 rule, PSSM and g2p all possible amino acidic combinations according to amino acid change due to base ambiguities were evaluated. R5-X4: sample B563’s FPR value fell between the two cutoffs, evidencing the presence of X4 variants as well. R5-X4: for sample BF134o three out of four possible amino acidic combinations according to amino acid change due to base ambiguities were R5.</p