Uropathogenic specific protein gene, highly distributed in extraintestinal uropathogenic Escherichia coli, encodes a new member of H-N-H nuclease superfamily
© Zaw et al.; licensee BioMed Central Ltd. 2013
Received: 24 April 2013
Accepted: 5 June 2013
Published: 10 June 2013
The uropathogenic specific protein (Usp) and three OrfU proteins (OrfU1, OrfU2 and OrfU3) are encoded in the putative small pathogenicity island which is closely associated with Uropathogenic Escherichia coli. Although homology search revealed that Usp and OrfUs have a homology with nuclease-type bacteriocins, which possess H-N-H nuclease motif, and immunity proteins respectively, the molecular activity of these proteins was never investigated. In this study, we try to over-express Usp in E. coli, purify Usp and characterize its molecular activity.
Recombinant Usp protein was expressed in E. coli BL21(DE3) cells together with 6× Histidine tagged OrfU1 (OrfU1-His) protein, and purified with affinity chromatography using Ni2+ chelating agarose. The nuclease activity of the purified Usp was examined in vitro by using plasmid DNA as a substrate. The importance of H-N-H motif in nuclease activity of Usp was examined by site-directed mutagenesis study.
We revealed that pET expression vector encoding Usp alone could not be maintained in E. coli BL21(DE3), and insertion of the orfUs as well as usp in the constructed plasmid diminished the toxic effect, suggesting that co-expressed OrfUs masked the activity of Usp. To purify Usp protein, we employed the expression vector encoding untagged Usp together with OrfU1-His. A tight complex formation could be observed between Usp and OrfU1-His, which allowed the purification of Usp in a single chromatographic step: binding of Usp/OrfU1-His complex to Ni2+ chelating agarose followed by elution of Usp from the complex with denaturing reagent. The purified free Usp was found to have the nuclease activity, and the activity was constitutively higher than Usp/OrfU1-His complex. H-N-H motif, which is found in various types of nucleases including a subfamily of nuclease-type bacteriocin, had been identified in the C-terminal region of Usp. Site-directed mutagenesis study showed that the H-N-H motif in Usp is indispensable for its nuclease activity.
This is the first evidence of the molecular activity of the new member of H-N-H superfamily and lays the foundation for the biological characterization of Usp and its inhibitor protein, OrfUs.
KeywordsUropathogenic Escherichia coli Pathogenicity island Uropathogenic specific protein Non-specific nuclease H-N-H superfamily
Urinary tract infections (UTI) are one of the most common infections in human and therefore an important health problem, resulting in 8.2 million physician visits, 1.7 million emergency department visits and 366,000 hospitalizations with an annual projected cost of more than $3.4 billion during the year 2000 in the United States. Uropathogenic Escherichia coli (UPEC) are responsible for 80 - 90% of community-acquired UTIs and 40% of nosocomial UTIs[2, 3]. UPEC possesses a diverse array of virulence and fitness factors. Adherence factors such as type 1, P, F fimbriae and Dr family adhesin help the UPEC to attach to uroepithelium and establish infection. The UPEC strains also possess an iron uptake system which enables it to survive under iron limiting host environments. UPEC also produces toxins such as alpha-hemolysin and cytotoxic necrotizing factor 1 which can inflict direct damage on the urinary tract tissues[6, 7].
The uropathogenic specific protein (usp) gene was discovered in the UPEC strain Z42 isolated from a prostatitis patient when looking for homologues of the Vibrio cholerae zot gene in UPEC. The usp gene was predicted to encode a 346 amino acid protein designated as uropathogenic specific protein (Usp). Located downstream of the usp gene were three small open reading frames (designated as orfU1, orfU2 and orfU3) putatively encoding 98, 97 and 96 amino acid proteins known as OrfU1, OrfU2, and OrfU3 proteins respectively. Although no function has been assigned to Usp, the usp gene was reported to be more frequently associated with UPEC strains than fecal E. coli isolates, and enhance the infectious potential of E. coli strains in mouse pyelonephritis model, suggesting that Usp may play a role in UPEC pathogenesis.
The possibility of Usp to be a bacteriocin was suggested by A. H. A. Parret and R. De Mot, based on sequence homology analysis. In bacteriocin-producing bacteria, immunity protein which inhibits the killing activity of bacteriocin is co-synthesized to protect the producing cell from suicide. The immunity proteins directly bind to the bacteriocins and form a considerably tight complex, which remain stable even after it is released from producing bacterial cells into the environment. The homology analysis revealed that whereas Usp has a homology with nuclease-type bacteriocins such as S-type pyocin produced in Pseudomonas aeruginosa and E group colicins produced in E. coli, OrfUs have a homology with immunity proteins for these bacteriocins. Most nuclease-type bacteriocins possess three functional domains: receptor recognition domain, translocation domain and nuclease domain, which are respectively responsible for recognition of specific receptor protein on target cell membrane, translocation of the protein into target cell and degradation of chromosomal DNA of target cell. The receptor protein varies for each bacteriocin. For example, colicin E7 and E9 of E. coli bind to the BtuB on E. coli membrane, and pyocin S1, S2 and S3 of P. aeruginosa bind to ferripyoverdine receptor on P. aeruginosa membrane. These specific recognitions contribute to the creation of narrow and species specific killing spectrum of each bacteriocin.
The H-N-H motif is known as a divalent metal ion binding, nucleic acid cleavage-module consisting of 30 to 40 amino acid residues. This motif could be observed in various types of nucleases represented by nuclease-type bacteriocins[13, 14] and intron-encoded homing endonucleases. The C-terminal region of Usp shows the homology to H-N-H motif. In this study, to investigate the molecular activity of Usp, we constructed an E. coli strain overproducing Usp, developed the purification method for Usp, and examined nuclease activity of the purified protein and importance of H-N-H motif in the activity of Usp. The purification method described in this study provides a useful material for further analysis of molecular and biological activity of Usp.
Construction of E. coli strain overproducing recombinant Usp
Plasmids used in this study
Proteins encoded on the plasmids
Usp, OrfU1, OrfU2, OrfU3-His
Usp, OrfU1, OrfU2-His
H314A/H315A mutant of Usp, OrfU1-His
N330A mutant of Usp, OrfU1-His
H339A mutant of Usp, OrfU1-His
H314A/H315A mutant of Usp-His
N330A mutant of Usp-His
H339A mutant of Usp-His
Purification of recombinant free Usp
Nuclease activity of recombinant Usp
Significance of H-N-H motif in nuclease activity of Usp
Although usp gene could also be detected in non-UPEC isolates, usp-positive strains are predominant in UTI isolates[21, 22]. In addition, based on its ability to confer infectious potential to non-pathogenic E. coli, Usp is thought to be an important factor responsible for UPEC infection. But, the mode of action or molecular activity of Usp has never been investigated, because purification method for Usp protein was not established. In this study, we constructed recombinant E. coli strain in which Usp was over-expressed. Based on its homology with nuclease-type bacteriocins, Usp is thought to be a nuclease protein. The over-expression of nuclease proteins, either of eukaryotic or prokaryotic origin, in E. coli cells is problematic due to cellular toxicity of these proteins. It is impossible to over-express these proteins in E. coli without employing certain strategies to reduce their toxicity, and co-expression with inhibitor proteins is one of the frequently used strategies[16–18, 23, 24]. In this study, we chose OrfUs as the inhibitor protein for Usp because OrfUs have a high homology with immunity proteins for nuclease-type bacteriocins, and the co-expression method successfully reduced the cellular toxicity of Usp. Three types of orfUs (orfU1, orfU2 and orfU3) had been identified in the downstream region of usp. Moreover, usp had been divided into two subtypes based on its sequence, designated as uspI and uspII. The putative pathogenicity islands consisting of usp and orfUs (PAIusp) contains one usp and two or three orfUs, and have been classified into four subtypes according to their sequential patterns[21, 22, 25]. Comparison of these PAIusp revealed that uspI is closely linked with immediately downstream orfU1 whilst uspII is linked to orfU2. On the basis of this observation, it had been speculated that OrfU1 is responsible for immunity against UspI whereas OrfU2 is responsible for UspII. The usp gene used in this study was cloned from the UPEC strain possessing uspI gene. The results obtained in this study indicate that OrfU1 could provide immunity against UspI in the absence of OrfU2 and 3. Although we have not examined capability of OrfU2 and 3 as an immunity protein, we have revealed that OrfU2 could not bind to UspI in the presence of OrfU1 (data not shown).
The H-N-H motif, the importance of which in the nuclease activity of Usp has been revealed in this study, can be found in numerous nucleases other than bacteriocins. The most well known member of this H-N-H nuclease superfamily is the homing endonuclease, which catalyze intron and intein mobility. Moreover, the H-N-H motif is found in a range of nucleases which participate in various biological processes including recombination, programmed DNA rearrangement during differentiation, and phage packing. The motif can also be observed in the active center of some restriction enzymes. These nucleases are produced in wide-range of hosts including bacteria, viruses and eukaryotes. Although several of these nucleases are not well-characterized, all the characterized nucleases with H-N-H motif other than bacteriocins are site-specific endonucleases whereas bacteriocins are non-specific endonucleases. In this study, we revealed that Usp is a non-specific nuclease similar to bacteriocins. Sequence homology between Usp and nuclease-type bacteriocins, including colicins produced by E. coli, have been previously suggested. Generally, nuclease-type bacteriocins can be divided into three domains: a nuclease domain, a translocation domain and a receptor recognition domain. Sequence alignment revealed that, in addition to the nuclease domain in which H-N-H motif was conserved, Usp has a region homologous to the translocation domain. However, Usp lacks the region homologous to the receptor recognition domain. A receptor recognition domain of bacteriocins is required for binding to specific membrane receptor on target bacterial cells. It is considered that these specific interactions between receptor recognition domains and membrane receptors regulate the narrow killing spectrum of bacteriocins. It is known that the narrow killing spectrum of bacteriocins is usually restricted to the strains of the same or closely related species[28, 29]. Although we demonstrated that Usp has a non-specific nuclease activity similar to known nuclease-type bacteriocins including colicins, we had not yet found susceptible E. coli strain which was killed by purified free Usp or Usp/OrfU1 complex (data not shown). Based on the sequence homology, it has been assumed that Usp and colicins, both of them produced in E. coli, share the bacteriocin activity, but the killing spectrum of these proteins might differ considerably.
Within the nuclease-type bacteriocins, the difference in ability to degrade RNA was reported. Colicin E9 was reported to cleave short ssRNA whereas colicin E2 did not have RNase activity against phage RNA[20, 30], although both of them belong to H-N-H nuclease superfamily. Therefore, we examined RNase activity of free Usp, but we could not detect any RNase activity against short ssRNA at least under the same condition as DNase activity assay (Additional file1: Figure S1).
In this study, we could successfully establish a purification method for recombinant Usp which possesses non-specific nuclease activity. The H-N-H motif conserved in the C-terminal region of the Usp was indispensable for its nuclease activity, indicating Usp is the new family member of H-N-H nuclease superfamily. Although Usp is considered as an important virulence factor of UPEC infection based on the previous result of mouse UTI model, the role of Usp in UTI has not been investigated. In addition, there is a possibility that Usp also participates in infections outside of urinary tract because usp can detected in some non-uropathogenic E. coli isolates. The purified protein obtained in this study would facilitate an analysis of biological activity and role of Usp during E. coli infections.
ECOS competent E. coli DH5α cells (NIPPON GENE CO., LTD.) were used for construction and maintenance of recombinant plasmids. ECOS competent E. coli BL21(DE3) cells (NIPPON GENE CO., LTD.) were used for expression of recombinant proteins. UPEC strain Z42 was used as source of usp and orfU genes.
Construction of the plasmids encoding Usp
Primers used in this study
5′-gttaagaaattccagatagc tgc tgtagttgctatagaacatgg-3′
5′-ccatgttctatagcaactacagc agc tatctggaatttcttaac-3′
Purification of Usp/OrfU1-His complex and free Usp
The ECOS competent E. coli BL21(DE3) (NIPPON GENE CO., LTD.) was used as the host strain for expression of recombinant Usp/OrfU1-His complex. The transformed E. coli BL21(DE3) was cultured in LB broth supplemented with kanamycin (25 μg/ml) and glucose (1%) at 30°C overnight with vigorous shaking (pre-cultivation). The pre-culture was inoculated into fresh LB broth supplemented with kanamycin (25 μg/ml) and glucose (1%), and further incubation was done at 30°C for 3 h before induction of recombinant protein expression by addition of IPTG (0.1 mM). Six hour after induction, the bacterial cells were harvested by centrifugation, resuspended in the binding buffer (20 mM Tris, 0.5 M NaCl, 5 mM imidazole, pH 7.5), and disrupted by sonication. The cleared cell lysate obtained after the centrifugation of the cell sonicate at 18,000×g for 30 minutes was applied onto the column filled with Ni-NTA agarose (QIAGEN) which was equilibrated with the binding buffer. The column was sequentially washed with the binding buffer, the washing buffer (20 mM Tris, 0.5 M NaCl, 60 mM imidazole, pH 7.5), and the binding buffer again. In the case of Usp/OrfU1-His complex purification, the complex was eluted by the elution buffer (20 mM Tris, 0.5 M NaCl, 250 mM imidazole, pH 7.5). On the other hand, the free Usp was eluted with the Guanidine-containing buffer (20 mM Tris, 0.5 M NaCl, 6 M guanidine-HCl, pH 7.5). The eluted fraction was further incubated with Ni-NTA agarose for 1 h with rotation to remove residual OrfU1-His. Obtained Usp/OrfU1-His complex or free Usp was dialyzed against a potassium phosphate buffer (0.02 M KH2PO4, 0.03 M K2HPO4, pH 7.0) to remove excess salts or guanidine HCl before performing the nuclease activity assay. The over-expressed proteins and purified proteins were analyzed by MALDI-TOF MS with the view to confirm the identity of each protein by database search. The MALDI-TOF/TOF MS was performed on an AutoFlex II TOF/TOF mass spectrometer (Bruker Daltonics) in accordance with the manufacturer’s instructions. The data acquisition, processing and database search was performed on instrument-specific software: FlexControl, FlexAnalysis, BioTools connected to Masccot search engine with NCBI nr database.
Determination of protein concentration
Concentrations of purified free Usps were determined by Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad) by using bovine serum albumin as a standard. For concentration measurement of Usp in purified Usp/OrfU1-His complex, purified Usp/OrfU1-His complex was separated with sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie Brilliant Blue (CBB) staining, and the band intensities of Usp was determined. And the concentration of Usp in Usp/OrfU1-His complex was calculated from standard curve representing the relationship between free Usp concentration and band intensities. Determination of band intensities was performed by image processing and analysis software ImageJ (http://imagej.nih.gov/ij/).
Analytical gel filtration
Gel filtration chromatography was used to determine the molecular weight of free Usp. The purified free Usp and 5 standard proteins from Gel Filtration LMW Calibration Kit (GE Healthcare Life Sciences) were dialyzed against sodium phosphate buffer (0.05 M NaH2PO4, 0.05 M Na2HPO4, 0.2 M NaCl pH 8.0), and separated on the Superdex 75 10/300 GL Column (GE Healthcare Life Sciences) equilibrated with the same buffer. The calibration curve was drawn by plotting of elution volume and LogMr of 5 standard proteins, and molecular weight of each peak obtained from purified free Usp sample was determined from the curve.
Construction of Usp mutants
Site-directed mutagenesis was carried out by the methods described in QuickChange Lightening site-directed mutagenesis kit (Agilent Technologies). Purified pUSP/ORF1 or pUSP plasmids were used as the template. Mutagenic primers used were HH314AAF and HH314AAR for H314A/H315A mutant; N330AF and N330AR for N330A mutant and H339AF and H339AR for H339A mutant. The sequences of these primer are shown in Table 2. The DNA sequence of the products were confirmed by DNA sequencing. The sequence analysis was done at Hokkaido System Science Co., Ltd. with the ABI PRISM 3130 Genetic Analyzer using the BigDye Terminator v3.1 kit (Applied Biosystems). Expression and purification of these mutant proteins were done following the same procedures as wild-type Usp.
Nuclease activity assay
Linearized pUC18 plasmid, which was digested by Sal I, was incubated with wild-type or mutant Usps in reaction buffer (0.05 M Tris, 80 mM NaCl, 10 mM MgSO4, pH 8.0) at 37°C. After incubating for desired time, reactions were stopped by addition of 6× Loading Buffer (TaKaRa Bio. Inc.), and products were separated by electrophoresis through 1.0% (w/v) agarose gel and the DNA was visualized by ethidium bromide staining. For quantitative analysis, the extent of degradation was quantified by densitometric measurement of substrate DNA with Image J.
Urinary tract infection
Uropathogenic Escherichia coli
Uropathogenic specific protein
Sodium dodecyl sulphate-polyacrylamide gel electrophoresis
Coomassie Brilliant Blue.
This study was supported in part by JSPS KAKENHI Grant Number 20590436, 22591803.
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