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Isolation and characterization of HepP: a virulence-related Pseudomonas aeruginosa heparinase
Nyaradzo Dzvova1, Jane A Colmer-Hamood1,2, John A Griswold3
1Department of Immunology and Molecular Microbiology, Texas Tech University Health Sciences Center, 3601 4th St. Mail Stop 6591, Lubbock, TX, 79430, USA.
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.
Insights
Severe thermal injuries increase Pseudomonas aeruginosa heparinase (hepP) expression, a key virulence factor impacting biofilm formation and host killing. This study reveals hepP
Area of Science:
- Microbiology
- Infectious Diseases
- Biochemistry
Background:
- Pseudomonas aeruginosa (PA14) is an opportunistic pathogen causing severe infections in immunocompromised individuals, particularly burn patients.
- The impact of severe thermal injuries on PA14 pathogenesis during systemic infection remains largely unknown.
- PA14 gene expression significantly alters in whole blood from burn patients compared to healthy volunteers.
Purpose of the Study:
- To investigate the influence of severe thermal injuries on PA14 pathogenesis.
- To characterize the function and role of the PA14_23430 (hepP) gene and its encoded heparinase in PA14 virulence.
Main Methods:
- RNA-sequencing (RNA-seq) analysis of PA14 grown in blood from burn patients versus healthy volunteers.
- Quantitative real-time PCR and RT-PCR to confirm gene expression and operon structure.
- Molecular cloning, protein expression, purification, and enzymatic assays for the heparinase (HepP).
- Genetic mutation studies (hepP deletion) to assess effects on pellicle and biofilm formation.
- In vivo virulence assessment using the Caenorhabditis elegans slow-killing model.
Main Results:
- Growth in burn patient blood significantly enhanced expression of PA14_23420 (zbdP) and PA14_23430 (hepP), forming an operon.
- Recombinant HepP protein exhibited significant heparinase activity.
- Mutation of hepP impaired pellicle formation, reduced biofilm development, and attenuated virulence in the C. elegans model.
Conclusions:
- Expression of the heparinase gene (hepP) in PA14 is significantly induced by changes in burn patient blood.
- HepP is a crucial virulence factor for PA14, influencing biofilm formation and contributing to host mortality.
- The heparinase encoded by hepP is essential for full PA14 virulence in a model of systemic infection.
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