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Uropathogenic specific protein: epidemiologic marker of uropathogenic Escherichia coli as well as non-specific DNase.

Introduction

Urinary tract infections (UTI) are one of the most common infections in human. (1) Uropathogenic Escherichia coli (UPEC) are the most common bacteria causing uncomplicated UTIs. (2) UPEC possesses a diverse array of virulence and fitness factors. Adherence factors such as type 1, P, S fimbriae and Dr family adhesins help the UPEC to attach to uroepithelium and establish infection. (3) The UPEC strains also possess iron uptake systems which enable it to survive under iron limiting host environments. (4) UPEC also produces toxins such as alpha-hemolysin and cytotoxic necrotizing factor 1 which can inflict direct damage on the urinary tract tissues. (5,6) This review article focuses on the gene encoding uropathogenic specific protein (usp) which is widely distributed in UPEC strains.

The 1038 bp open reading frame (ORF) encoding 346 amino acid protein was discovered in UPEC strain Z42 isolated from a prostatitis patient when searching for homologues of zonula occludens toxin among UPEC strains. (7) The zonula occludens toxin is produced by Vibrio cholerae and was demonstrated to act on zonula occludens, a component of tight junction between small intestinal mucosal cells, thereby increasing the intestinal permeability. (8) Because this ORF was frequently associated with UPEC strains, it was thought to encode the previously unknown virulence factor of UPEC and designated as uropathogenic specific protein gene (usp).

The usp gene is located on previously unknown pathogenicity island of UPEC

The usp gene is predicted to code for 346 amino acid protein designated as uropathogenic specific protein (Usp). The predicted molecular weight of Usp is 38.659 kDa. The downstream region of usp gene is occupied by 3 small ORFs (orfU1, orfU2, orfU3) putatively encoding 98, 97 and 96 amino acid proteins designated as OrfU1, OrfU2 and OrfU3 respectively. (7) The orfU1-3 are transcribed in the same direction as the usp gene. Comparison of the DNA fragment containing usp gene and orfU1-3 from UPEC strain Z42 with whole genome sequence of E. coli strain K12 showed that the 4167 bp fragment carrying usp gene and orfU1-3 occupies the 270 bp intergenic region between aroP and pdhR genes in E. coli strain K12. (7) The intergenic region between aroP and usp contains the sequence characteristic of the Tn3 family transposon and the direct repeats of 4 base pairs are present on each side of this fragment, which is similar to the target size duplication at the insertion sequence insertion site. (9) In addition, the G+C content of this DNA fragment is different from that of surrounding region. These features suggest that this DNA fragment is a horizontally acquired transposon-like element (9) and together with the frequent association of the usp gene with UPEC strains, imply that this DNA fragment could be the previously unrecognized pathogenicity island (PAI) of UPEC. (7) This pathogenicity island is distinct from the pathogenicity islands previously reported in UPEC strains and was designated as PAIusp.

Structural and sequence diversity of PAIusp

Nakano et al. performed the sequencing analysis of PAIusp in seven representative usp gene-positive E. coli strains (one from cystitis case, four from prostatitis case, one from pyelonephritis and one from stool). (9) It was found that usp gene has DNA sequence heterogeneity starting from 230 bp before 3' end. Based on this sequence variation usp gene can be classified into 2 types--uspI and uspII. The sequence variation in 3' end of uspI and uspII gene results in 26 differences in amino acid sequences (Figure 1). In addition, structural diversity also exists in region downstream of usp gene containing orfUs. The number of orfUs and position of orfU in relation to each other differ from strain to strain. Interestingly, PAIusp in E. coli strains isolated from UTI cases has two or three orfUs whereas PAIusp in fecal E. coli strain has only one orfU. (9)

Prevalence of usp gene in extraintestinal pathogenic E. coli strains including uropathogenic E. coli strains

The usp gene is designated as such because it is more frequently associated with UPEC strains than fecal E. coli strains. (7) When 378 UPEC strains and 50 E. coli strains from stools of the healthy individuals were examined with colony hybridization test using the usp gene-specific DNA probe, most of the UPEC strains (84%) was positive for usp gene whereas only 24% of the fecal E. coli strains hybridized with the probe. When the prevalence of usp gene in the E. coli isolates from urine and feces of companion animals (dogs and cats) was examined, the pattern of distribution of usp gene in urinary and fecal E. coli isolates of these animals is same as that observed in human E. coli isolates. (11) UPEC strains are associated with limited number of O serotypes such as O1, O2, O4, O6, O16, O18, O22, O25 and O75. The presence of usp gene is significantly associated with all common serotypes of UPEC. (12)

As mentioned earlier, usp gene can be divided into 2 variants--uspI and uspII having sequence variation in 3' end. Depending on the usp variants and sequential arrangement of orfUs, PAIusp can be classified into 4 subtypes (Figure 2). (13) When PAIusp in usp-positive UPEC strains isolated in Japan was subtyped using a polymerase chain reaction (PCR) method, type IIa was found to be most common (42.4%) followed by type Ia, type Ib and type IIb (Table 1). It was also revealed that 96.6% of E. coli strains belonging to phylogenetic group B2 possess usp gene and 94.9% of usp-positive E. coli strains belong to phylogenetic group B2. However, the prevalence of usp gene is comparatively low in E. coli strains of other phylogenetic group: 11.4% in phylogenetic group A, 13.8% in phylogenetic group B1 and 24.3% in phylogenetic group D (13). Extraintestinal pathogenic Escherichia coli (ExPEC) strains including E. coli strains isolated from UTI were reported to be frequently associated with phylogenetic group B2. (14,15,16,17,18)

Recently E. coli strains belonging to sequence type 131 (as determined by multilocus sequence typing) has emerged as the globally disseminated cause of multi-drug resistant extraintestinal infections including UTIs. Interestingly, it was found that usp gene was almost always detected in E. coli strains of sequence type 131. (19,20,21,22)

Despite the high prevalence of usp gene in UPEC strains, the usp gene can also be detected in E. coli strains isolated from patients with sepsis and bacteremia. (23,24) However, the prevalence of usp gene is significantly lower in these E. coli isolates than in urinary E. coli isolates. A study conducted in Sweden monitored 130 healthy infants during the first year of life with regular stool culture and examined the E. coli isolated from stool for the presence of usp gene. It was revealed that the carriage of usp gene is significantly associated with the E. coli strains persisting in intestinal microbiota for more than one year. (25) These studies suggest that Usp may also have role in pathogenesis of E. coli infection outside urinary tract.

Virulence of usp gene in vivo

The virulence of usp gene was examined in vivo using mouse pyelonephritis model. The plasmid vector carrying usp gene was found to enhance the infectivity of host E. coli cells in mouse pyelonephritis model whereas the vector containing orfU1-3 and the vector lacking both usp gene and orfU1-3 did not exhibit this effect, suggesting that Usp may contribute to the causation of UTI and could be an important virulent determinant of UPEC. (12)

Homology with nuclease-type bacteriocins

Sequence homology analysis has revealed that Usp shares homology with nuclease-type bacteriocins such as colicin E9 and pyocin AP41. In addition, OrfUs proteins encoded by 3 small ORFs downstream of usp gene have homology with immunity proteins for these bacteriocins. (10) Based on sequence homology, it is thought that Usp is a nuclease-type bacteriocin and OrfU proteins are immunity proteins for Usp. Bacteriocins are defined as bactericidal peptides or proteins produced by bacteria and are different from traditional antibiotics in that bacteriocins are active against the bacteria closely related to the producing strain. (26) Nuclease-type bacteriocins kill the target bacterial cells by degradation of their nucleic acid. Nuclease-type bacteriocins are produced together with immunity proteins, which inhibit the bactericidal activity of bacteriocin preventing the bacteriocin producing cell from killing by its own bacteriocin. (27,28) As there are more than one orfU downstream of usp gene, it was speculated that the orfU immediately downstream of usp gene protects the Usp producing cell from bacteriocin activity of Usp and additional orfUs are responsible for conferring immunity towards other bacteriocins and provide advantage when competing with other strains. (10) As the result, the strains possessing 3 orfUs are resistant to more bacteriocins and more prevalent than those possessing 2 orfUs. In agreement with this speculation, it has been reported that UPEC strains containing three orfUs are more common than those containing two orfUs. (13) Moreover, orfU1 lies immediately downstream of uspI while orfU2 lies immediately downstream of uspII, suggesting that OrfU1 confer immunity towards UspI and OrU2 is responsible for immunity against UspII. (10) Amino acid sequence variation in 3' end of UspI and UspII determines which orfU should be immediately downstream.

Usp is a non-specific nuclease belonging to H-N-H nuclease superfamily

Zaw et al. overexpressed Usp in E. coli, purified it and characterized its activity. Initial attempt to overexpress Usp in E. coli was unsuccessful because the pET vector encoding Usp could not be maintained in E. coli BL21(DE3) due to cellular toxicity of Usp. (29) As mentioned earlier, it is thought that Usp is a nuclease-type bacteriocin and OrfU proteins are immunity proteins for Usp based on sequence homology. (9) When recombinant colicins were overexpressed in E. coli, their cognate immunity proteins were expressed together with colicins to protect the host E. coli cell from cellular toxicity of overexpressed colicin. (30,31) Similarly, the pET vector encoding Usp together with OrfU allowed the overexpression of Usp in E. coli BL21(DE3), suggesting that OrfU could mask the cellular toxicity of Usp. (29) In addition, co-purification of Usp and OrfU1 was possible, indicating that there was tight complex formation between Usp and OrfU1. To purify Usp, Usp complexed with 6xhistidine tagged OrfU1 (OrfU1-His) was bound to [Ni.sup.2+]-chelating agarose and Usp was separated from OrfU1-His bound to the agarose using denaturing reagent. (29)

Purified Usp was found to have DNase activity and the DNase activity of Usp/OrfU1 complex was significantly lower than that of Usp suggesting that OrfU1 could reduce the DNase activity of Usp (Figure 3). (29) The C terminal region of Usp contains the sequence homologous with H-NH motif. (10) The H-N-H motif is the 30-33 amino acids consensus sequence containing two pairs of conserved histidines surrounding a conserved asparagine and found in various nucleases including E-group DNase colicins such as colicin E7 (32), and E9 (33) and homing endonucleases (34). Site-directed mutagenesis of the conserved residues in H-N-H motif in Usp showed that H-N-H motif was important for DNase activity of Usp, indicating that Usp is a member of H-N-H nuclease superfamily. (29)

Future recommendations

Although the usp gene was discovered more than ten years ago, there are many questions that need to be answered. Usp shares homology with nuclease-type bacteriocins. However, there has been no report demonstrating the bacteriocin activity of Usp. Zaw et al. demonstrated that Usp is a protein having DNase activity whose expression is toxic to E. coli host cell and is not possible unless the OrfU proteins are expressed together. (29) Future study on usp should investigate the expression of Usp in UPEC strains. It can be suggested that the expression of Usp in UPEC strain is tightly regulated and will be elusive to detect. As the usp gene is predicted to encode a virulence factor of UPEC, it would be interesting to see if UPEC express Usp during the course of UTI, which could be investigated using experimental animal UTI model. Another way of detecting the expression of Usp in UTI would be to develop an ELISA system based on purified Usp and screen the blood of UTI patients for presence of specific antibodies against Usp. Previous studies have demonstrated that UPEC can multiply or persist in quiescent state after invading the uroepithelial cells, (35) so it is interesting whether Usp is expressed while UPEC exist intracellularly.

Zaw et al. characterized the activity of UspI from UPEC strain Z42. However, uspII gene is more common than uspI gene suggesting that strains possessing uspII has competitive advantage compared with those possessing uspI gene. So the activity of UspII needs to be characterized.

Kurazono et al. reported that the amino acid sequence of Usp contains a putative cleavage site for signal peptidase between serine 24 residues and alanine 25 residues and therefore is a secreted protein. (7) However, experimental evidence that Usp is secreted into the extracellular medium is lacking.

In nucleotide sequence databases, ORFs of differing lengths from various UPEC strains such as UTI89 and 536 are annotated as usp gene. For example, a 1782 bp ORF encoding 593 amino acid protein in complete genome sequence of UPEC strain UTI89 (Accession number CP000243) is annotated as usp gene although this ORF starts with the codon coding for amino acid other than methionine. Parret and De Mot also anticipated that Usp may be a 600 amino acid protein containing N terminal extension homologous with hemolysin coregulated pilus produced by Vibrio cholerae (10) Future study should seek to purify native Usp from wild-type UPEC strains to clarify whether Usp is 346 amino acid protein as described by Kurazono et al. or 600 amino acid protein as anticipated by Parret and De Mot.

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List of abbreviations

UTI: Urinary tract infection; UPEC: Uropathogenic Escherichia coli; Usp: Uropathogenic specific protein; PAI: Pathogenicity island; ORF: Open reading frame; PCR: Polymerase chain reaction.

Conflict of Interest: The authors have no conflict of interest to declare.

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Myo Thura Zaw [1], Yun Mei Lai [2], Zaw Lin [2] *

[1] Division of Food Hygiene, Department of Animal and Food Hygiene, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, 080-8555, Japan

[2] Pathological and Medical Diagnostics Department, School of Medicine, Universiti Malaysia Sabah, Kota Kinabalu, 88400, Sabah, Malaysia

* Corresponding author: Dr. Zaw Lin Email: zawlin@ums.edu.my
Table 1: Prevalence of PAIusp subtypes in urinary E. coli
Isolates (13)

Presence or absence of usp genes/
PAIusp subtypes                        Number of UPEC isolates (%)

Number of usp-positive UPEC isolates   320 (84.9)
  Type Ia                              107 (28.4)
  Type Ib                               37 (9.8)
  Type IIa                             160 (42.4)
  Type IIb                              10 (2.7)
  Non-typed                              6 (1.6)
Number of usp-negative UPEC isolates    57 (15.1)
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Author:Zaw, Myo Thura; Lai, Yun Mei; Lin Zaw
Publication:International Journal of Collaborative Research on Internal Medicine & Public Health (IJCRIMPH)
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Date:Nov 1, 2013
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