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.ResultsQuantitative 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.ConclusionsChanges 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.Electronic supplementary materialThe online version of this article (10.1186/s12866-017-1141-0) contains supplementary material, which is available to authorized users.

Pseudomonas donghuensis HYS virulence towards Caenorhabditis elegans is regulated by the Cbr/Crc system

“…donghuensis HYS killed worms in 5 days, and the average LT 50 value was 3.25 ± 0.15 days (n = 3). This virulence is stronger than that of PA14 or PAO1 previously reported 17,18 . Repellence of worms by P .…”

Role of heparinase in the gastrointestinal dysfunction of sepsis (Review)

“…HPA expression was increased in animal models of septic lung and kidney injury, suggesting that HPA may be involved in the early onset of sepsis (66). An additional study demonstrated that expression of the gene coding for HPA (hepP) plays an important role in a mouse model of sepsis induced by Pseudomonas aeruginosa, and that mutations in the hepP gene enhance survival in infected mice (67)(68)(69). A previous in vivo study determined that patients with sepsis induced by gram-negative bacteria exhibited a pronounced increase in plasma HPA activity (70).…”

Pseudomonas donghuensis HYS gtrA/B/II Gene Cluster Contributes to Its Pathogenicity toward Caenorhabditis elegans

“…Pseudomonas , a genus of obligate aerobic Gram-negative bacilli, belongs to the class of Gammaproteobacteria [ 3 , 4 , 5 , 6 ]. This genus contains many pathogen species, including P. aeruginosa , P. putida , P. fluorescens and P. syringae [ 7 , 8 , 9 ]. P. donghuensis exhibits antifungal and plant-promoting activities [ 10 , 11 ].…”