Isolation and characterization of HepP: a virulence-related Pseudomonas aeruginosa heparinase

Abstract Background Pseudomonas aeruginosa is an opportunistic pathogen that causes serious infections in immunocompromised hosts including severely burned patients. In burn patients, P. aeruginosa infection often leads to septic shock and death. Despite numerous studies, the influence of severe thermal injuries on the pathogenesis of P. aeruginosa during systemic infection is not known. Through RNA-seq analysis, we recently showed that the growth of P. aeruginosa strain UCBPP-PA14 (PA14) in whole blood obtained from severely burned patients significantly altered the expression of the PA14 transcriptome when compared with its growth in blood from healthy volunteers. The expression of PA14_23430 and the adjacent gene, PA14_23420, was enhanced by seven- to eightfold under these conditions. Results Quantitative real-time PCR analysis confirmed the enhancement of expression of both PA14_23420 and PA14_23430 by growth of PA14 in blood from severely burned patients. Computer analysis revealed that PA14_23430 (hepP) encodes a potential heparinase while PA14_23420 (zbdP) codes for a putative zinc-binding dehydrogenase. This analysis further suggested that the two genes form an operon with zbdP first. Presence of the operon was confirmed by RT-PCR experiments. We characterized hepP and its protein product HepP. hepP was cloned from PA14 by PCR and overexpressed in E. coli. The recombinant protein (rHepP) was purified using nickel column chromatography. Heparinase assays using commercially available heparinase as a positive control, revealed that rHepP exhibits heparinase activity. Mutation of hepP resulted in delay of pellicle formation at the air-liquid interface by PA14 under static growth conditions. Biofilm formation by PA14ΔhepP was also significantly reduced. In the Caenorhabditis elegans model of slow killing, mutation of hepP resulted in a significantly lower rate of killing than that of the parent strain PA14. Conclusions Changes within the blood of severely burned patients significantly induced expression of hepP in PA14. The heparinase encoded by hepP is a potential virulence factor for PA14 as HepP influences pellicle formation as well as biofilm development by PA14 and the protein is required for full virulence in the C. elegans model of slow killing

Additional file 1: of Isolation and characterization of HepP: a virulence-related Pseudomonas aeruginosa heparinase

Figure S1. Characteristics of HepP and its homology to other proteins. Figure S2. Amino acid sequence of the 78-kDa predicted protein encoded by zbdP. Figure S3. Nucleotide sequences of zbdP and hepP showing locations of primers used in experiments to analyze these two genes. Figure S4. Mutation of hepP or zbdP does not significantly alter the growth of PA14 in vitro. Figure S5. Detailed experiment to confirm transcription of hepP in PA14ΔzbdP. Table S1. Primers used to compare virulence factor gene transcription in PA14, PA14ΔhepP and PA14ΔzbdP. (PDF 1972 kb

During bacteremia, Pseudomonas aeruginosa PAO1 adapts by altering the expression of numerous virulence genes including those involved in quorum sensing.

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that produces numerous virulence factors and causes serious infections in trauma patients and patients with severe burns. We previously showed that the growth of P. aeruginosa in blood from severely burned or trauma patients altered the expression of numerous genes. However, the specific influence of whole blood from healthy volunteers on P. aeruginosa gene expression is not known. Transcriptome analysis of P. aeruginosa grown for 4 h in blood from healthy volunteers compared to that when grown in laboratory medium revealed that the expression of 1085 genes was significantly altered. Quorum sensing (QS), QS-related, and pyochelin synthesis genes were downregulated, while genes of the type III secretion system and those for pyoverdine synthesis were upregulated. The observed effect on the QS and QS-related genes was shown to reside within serum fraction: growth of PAO1 in the presence of 10% human serum from healthy volunteers significantly reduced the expression of QS and QS-regulated genes at 2 and 4 h of growth but significantly enhanced their expression at 8 h. Additionally, the production of QS-regulated virulence factors, including LasA and pyocyanin, was also influenced by the presence of human serum. Serum fractionation experiments revealed that part of the observed effect resides within the serum fraction containing <10-kDa proteins. Growth in serum reduced the production of many PAO1 outer membrane proteins but enhanced the production of others including OprF, a protein previously shown to play a role in the regulation of QS gene expression. These results suggest that factor(s) within human serum: 1) impact P. aeruginosa pathogenesis by influencing the expression of different genes; 2) differentially regulate the expression of QS and QS-related genes in a growth phase- or time-dependent mechanism; and 3) manipulate the production of P. aeruginosa outer membrane proteins

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

Severe burn injury alters the level of blood constituents.

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

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

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