Major Transcriptome Changes Accompany the Growth of Pseudomonas aeruginosa in Blood from Patients with Severe Thermal Injuries.

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that causes serious infections in immunocompromised hosts including severely burned patients. After multiplying within the burn wound, P. aeruginosa translocate into the bloodstream causing bacterial sepsis frequently leading to organ dysfunction and septic shock. Although the pathogenesis of P. aeruginosa infection of thermally-injured wounds has been extensively analyzed, little is known regarding the ability of P. aeruginosa to adapt and survive within the blood of severely burned patients during systemic infection. To identify such adaptations, transcriptome analyses (RNA-seq) were conducted on P. aeruginosa strain PA14 that was grown in whole blood from a healthy volunteer or three severely burned patients. Compared with growth in blood from healthy volunteers, growth of PA14 in the blood from severely burned patients significantly altered the expression of 2596 genes, with expression of 1060 genes enhanced, while that of 1536 genes was reduced. Genes whose expression was significantly reduced included genes related to quorum sensing, quorum sensing-controlled virulence factors and transport of heme, phosphate, and phosphonate. Genes whose expression was significantly enhanced were related to the type III secretion system, the pyochelin iron-acquisition system, flagellum synthesis, and pyocyanin production. We confirmed changes in expression of many of these genes using qRT-PCR. Although severe burns altered the levels of different blood components in each patient, the growth of PA14 in their blood produced similar changes in the expression of each gene. These results suggest that, in response to changes in the blood of severely burned patients and as part of its survival strategy, P. aeruginosa enhances the expression of certain virulence genes and reduces the expression of others

Comparison of T3SS gene expression in LB broth with 5% albumin to LB Broth.

<p>Comparison of T3SS gene expression in LB broth with 5% albumin to LB Broth.</p

Growth of PA14 in whole blood from a severely burned patient significantly repressed expression of QS and QS-controlled genes.

<p>RNA extracted from PA14 grown in whole blood from Patient 3 or the healthy volunteer was examined in qRT-PCR as described in Methods. <b>A.</b> Level of expression of <i>lasI</i>, <i>lasR</i>, and <i>lasB</i>. <b>B.</b> Level of expression of <i>rhlI</i>, <i>rhlR</i>, and <i>rhlA</i>. Values in A and B represent the average of triplicate PCR experiments conducted on three independently obtained RNA preparations ±SEM; *<i>P</i> <0.05, **<i>P</i><0.01, ****<i>P</i> <0.0001.</p

Characteristics of the three burn patients.

<p>Characteristics of the three burn patients.</p

Growth of PA14 in whole blood from a severely burned patient significantly enhanced expression of the T3SS genes <i>exsA</i>, <i>pcrV</i>, and <i>popB</i>.

<p>RNA extracted from PA14 grown in whole blood from Patient 2 or the healthy volunteer was examined in qRT-PCR as described in Methods. Values represent the average of triplicate PCR experiments conducted on three independently obtained RNA preparations ±SEM; **<i>P</i><0.01, *** <i>P</i> <0.001, ****<i>P</i> <0.0001.</p

Severe burn injury alters the level of blood constituents.

<p>Severe burn injury alters the level of blood constituents.</p

Differentially expressed genes of the phosphate regulon (Pho).

<p>Differentially expressed genes of the phosphate regulon (Pho).</p

Color scale map of the T3SS genes that are either upregulated.

<p>Expression of genes in PA14 grown in whole blood from the three severely burned patients was compared with expression of these genes when PA14 was grown in whole blood from the a healthy volunteer. Upregulated genes are indicated in shades of blue. Genes within operons, whether all genes of the operon are differentially expressed or not, are indicated in shades of yellow on the left. Single genes are not shaded. Operon groupings and functions for PA14 genes were obtained from the MGH-ParaBioSys:NHLBI Program for Genomic Applications, Massachusetts General Hospital and Harvard Medical School, Boston, MA (<a href="http://pga.mgh.harvard.edu" target="_blank">http://pga.mgh.harvard.edu</a>; accessed 10Nov2015) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149229#pone.0149229.ref045" target="_blank">45</a>] made available by the <i>Pseudomonas Genome Database</i> (<a href="http://www.pseudomonas.com/" target="_blank">http://www.pseudomonas.com/</a>; accessed 10Nov2015) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149229#pone.0149229.ref044" target="_blank">44</a>].</p

Color scale map of upregulated or downregulated iron acquisition genes.

<p>Expression of genes in PA14 grown in whole blood from the three severely burned patients was compared with expression of these genes when PA14 was grown in whole blood from a healthy volunteer (control). Upregulated genes are indicated in shades of blue while downregulated genes are indicated in shades of red. Genes within operons, whether all genes of the operon are differentially expressed or not, are indicated in shades of yellow on the left. Single genes are not shaded. Operon groupings and gene product functions for PA14 genes were obtained from the MGH-ParaBioSys:NHLBI Program for Genomic Applications, Massachusetts General Hospital and Harvard Medical School, Boston, MA (<a href="http://pga.mgh.harvard.edu" target="_blank">http://pga.mgh.harvard.edu</a>; accessed 10Nov2015) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149229#pone.0149229.ref045" target="_blank">45</a>] made available by the <i>Pseudomonas Genome Database</i> (<a href="http://www.pseudomonas.com/" target="_blank">http://www.pseudomonas.com/</a>; accessed 10Nov2015) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149229#pone.0149229.ref044" target="_blank">44</a>].</p

QS and QS-controlled genes differentially expressed in PA14 grown in whole blood from severely burned patients.

<p>QS and QS-controlled genes differentially expressed in PA14 grown in whole blood from severely burned patients.</p