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 the...
| Main Authors: | , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
BMC
2017-12-01
|
| Series: | BMC Microbiology |
| Subjects: | |
| Online Access: | http://link.springer.com/article/10.1186/s12866-017-1141-0 |
| _version_ | 1811265279082102784 |
|---|---|
| author | Nyaradzo Dzvova Jane A. Colmer-Hamood John A. Griswold Abdul N. Hamood |
| author_facet | Nyaradzo Dzvova Jane A. Colmer-Hamood John A. Griswold Abdul N. Hamood |
| author_sort | Nyaradzo Dzvova |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-12T20:20:52Z |
| format | Article |
| id | doaj.art-be8ce8289f5e4b06bcf781e9ba427509 |
| institution | Directory Open Access Journal |
| issn | 1471-2180 |
| language | English |
| last_indexed | 2024-04-12T20:20:52Z |
| publishDate | 2017-12-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Microbiology |
| spelling | doaj.art-be8ce8289f5e4b06bcf781e9ba4275092022-12-22T03:18:00ZengBMCBMC Microbiology1471-21802017-12-0117111610.1186/s12866-017-1141-0Isolation and characterization of HepP: a virulence-related Pseudomonas aeruginosa heparinaseNyaradzo Dzvova0Jane A. Colmer-Hamood1John A. Griswold2Abdul N. Hamood3Department of Immunology and Molecular Microbiology, Texas Tech University Health Sciences CenterDepartment of Immunology and Molecular Microbiology, Texas Tech University Health Sciences CenterDepartment of Surgery, Texas Tech University Health Sciences CenterDepartment of Immunology and Molecular Microbiology, Texas Tech University Health Sciences CenterAbstract 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.http://link.springer.com/article/10.1186/s12866-017-1141-0Pseudomonas aeruginosaHeparinaseVirulencePellicleBiofilmC. elegans killing model |
| spellingShingle | Nyaradzo Dzvova Jane A. Colmer-Hamood John A. Griswold Abdul N. Hamood Isolation and characterization of HepP: a virulence-related Pseudomonas aeruginosa heparinase BMC Microbiology Pseudomonas aeruginosa Heparinase Virulence Pellicle Biofilm C. elegans killing model |
| title | Isolation and characterization of HepP: a virulence-related Pseudomonas aeruginosa heparinase |
| title_full | Isolation and characterization of HepP: a virulence-related Pseudomonas aeruginosa heparinase |
| title_fullStr | Isolation and characterization of HepP: a virulence-related Pseudomonas aeruginosa heparinase |
| title_full_unstemmed | Isolation and characterization of HepP: a virulence-related Pseudomonas aeruginosa heparinase |
| title_short | Isolation and characterization of HepP: a virulence-related Pseudomonas aeruginosa heparinase |
| title_sort | isolation and characterization of hepp a virulence related pseudomonas aeruginosa heparinase |
| topic | Pseudomonas aeruginosa Heparinase Virulence Pellicle Biofilm C. elegans killing model |
| url | http://link.springer.com/article/10.1186/s12866-017-1141-0 |
| work_keys_str_mv | AT nyaradzodzvova isolationandcharacterizationofheppavirulencerelatedpseudomonasaeruginosaheparinase AT janeacolmerhamood isolationandcharacterizationofheppavirulencerelatedpseudomonasaeruginosaheparinase AT johnagriswold isolationandcharacterizationofheppavirulencerelatedpseudomonasaeruginosaheparinase AT abdulnhamood isolationandcharacterizationofheppavirulencerelatedpseudomonasaeruginosaheparinase |