T. Zahraei Salehi, Department of Microbiology, Faculty of Veterinary Medicine, University of Tehran, Qareeb Street, Azadi Av. PO Box: 14155-6453, Tehran, Iran. E-mail: firstname.lastname@example.org
Aims: To identify, clone and sequence the iss (increased serum survival) gene from E. coli strain χ1378 isolated from Iranian poultry and to predict its protein product, Iss.
Methods and Results: The iss gene from E. coli strain χ1378 was amplified and cloned into the pTZ57R/T vector and sequenced. From the DNA sequence, the Iss predictive protein was evaluated using bioinformatics. Iss from strain χ1378 had 100% identity with other E. coli serotypes and isolates from different origins and also 98% identity with E. coli O157:H7 Iss protein. Phylogenetic analysis showed no significant different phylogenic groups among E. coli strains.
Conclusions: The strong association of predicted Iss protein among different E. coli strains suggests that it could be a good antigen to control and detect avian pathogenic E. coli (APEC).
Significance and Impact of the study: Because the exact pathogenesis and the role of virulence factors are unknown, the Iss protein could be used as a target for vaccination in the future, but further research is required.
Colibacillosis is a disease caused by avian pathogenic Escherichia coli (APEC) (La Ragione and Woodward 2002; Vandekerchove et al. 2004). This disease results in significant morbidity and mortality, which cause multimillion dollar annual losses in all facets of the world’s poultry industry (Barnes et al. 2008). Approaches to preventing and controlling APEC infection include improved hygiene, vaccination, the use of competitive exclusion products and antimicrobial chemotherapy (La Ragione et al. 2001, 2004; Gomis et al. 2003). However, these management approaches are largely ineffective. One reason for this is that APEC isolates are becoming more resistant to antimicrobial agents (Johnson et al. 2004, 2005). Furthermore, the use of vaccination to control APEC infection is difficult because the exact mechanism of pathogenesis and the role of virulence factors are unclear and also because of the extensive gene diversity in E. coli (Mokady et al. 2005) (La Ragione and Woodward 2002). Evidence in the literature shows that resistance to complement-mediated lysis and opsonophagocytosis play an important role in APEC virulence (Vidotto et al. 1990; Nolan et al. 1992, 2003). In general, the complement resistance to E. coli has been related to several structural factors, including the K1-antigen capsule, LPS (lipopolysaccharide) and certain outer membrane proteins including TraT and Iss (Nolan et al. 2003). Compared with LPS and the K1 capsule, Iss is reported to play a subtle role in resistance of APEC to serum (Mellata et al. 2003). Iss is a plasmid-encoded outer membrane protein (OMP) (Binns et al. 1979). In addition, many E. coli strains have the iss gene within their chromosomes (Johnson et al. 2008). While Iss has been associated with ColV plasmids, it has been found on other Col plasmids (Johnson et al. 2006). The role of Iss in serum resistance and virulence has been confirmed (Sukupolvi et al. 1987; Wooley et al. 1993). Some studies have identified iss as a derivative of the bacteriophage lambda gene known as bor and showed a significant homology between Iss and Bor proteins (Chuba et al. 1989; Waters and Crosa 1991).The iss gene occurs more frequently in APEC than in strains isolated apparently from healthy birds, and it has been reported in different serotypes (Pfaff-McDonough et al. 2000; McPeake et al. 2005; Rodriguez-Siek et al. 2005). The iss gene could be used as a target antigen to apply vaccination; however, there are some differences among the iss genes previously reported (Chuba et al. 1989; Horne et al. 2000; Lynne et al. 2006). In the present study, we were aiming to clone sequence and characterize the iss gene from E. coli strain χ1378 isolated from systemic colibacillosis in Iran (Zahraei Salehi et al. 2004; Nayeri Fasaei et al. 2009) and to compare the predicted protein sequence with previously submitted sequences in the GenBank database.
Material and methods
Bacterial strains and plasmids
A virulent wild-type avian E. coli (laboratory designation E. coli strain χ1378) isolated from a chicken with systemic colibacillosis in Iran was used as the source of iss. This strain was characterized by biochemical tests and identified as O78:K80 serotype (MAST serotyping kit; MAST Group Ltd, Merseyside, UK). The virulence of the bacterial strain was assayed by chicken embryo lethality test (Gibbs et al. 2003). E. coli DH5α (Fermentas, Vilnius, Lithuania) was used as the host strain for plasmid transformation. The plasmid pTZ57R/T (Fermentas) was used to clone the amplified fragment carrying iss. Luria-Bertani (LB) broth and agar (Difco) were used to grow the bacterial strains. Ampicillin (100 mg l−1) was added when required.
Preparation of chromosomal and plasmid DNA
Chromosomal and plasmid DNA from E. coli strain χ1378 were extracted using the Wizard genomic DNA purification kit (Promega, USA) and Qiagen Plasmid midi purification kit (Qiagen Inc., USA), respectively, as recommended by the manufactures.
The iss gene sequence of E. coli O78:K80 was amplified by using iss F: 5′-GTGGCGAAAACTAGTAAAACAGC-3′ and iss R: 5′-CGCCTCGGGGTGGATAA-3′ as previously described (Horne et al. 2000). The PCR was carried out on a total volume of 25 μl containing 3 μl of template DNA and each of the primers at 30 μmol l−1. The amplification programme used was 94°C for 4 min, 30 cycles of 94°C for 1 min, 58°C for 1 min, 72°C for 1 min and 72°C for 5 min. PCR were repeated at least three times. The amplified DNA was visualized in 1% agarose gels stained with ethidium bromide (25 μg ml−1). A 100-bp ladder (Fermentas) was used as the standard. The PCR product was purified from the agarose gel using a Gel extraction kit (Fermentas) then confirmed by agarose gel. The amplicons were dissolved in TE buffer.
The amplified iss fragments were cloned into the pTZ57R/T vector following the protocol provided by the manufacturer with T4 ligase (Ins T/A clone PCR Product Cloning Kit; Fermentas). Briefly, the ligation mixture included 3 μl of vector pTZ57R/T, 10 μl of purified PCR product and 1 μl T4 ligase. The mixture was incubated at room temperature (22°C) for 1 h. A 2·5 μl aliquot of the ligation mixture was directly used for chemical transformation to compete E. coli DH5α as indicated in the manufacturer’s instructions (INS T/A clone PCR Product Cloning Kit; Fermentas). Transformed cells were cultured on LB agar with ampicillin. Transformants were confirmed by PCR with iss primers as described earlier. The plasmid was extracted from the transformed cells, and the iss insert was further confirmed using M13 primers (F: 5′-GTAAAACGACGGCCAGT-3′, R: 5′-CAGGAAACAGCTATGAC-3′) and iss primers. In addition, some plasmid was digested with restriction enzymes EcoRI and BamHI (Fermentas) as described in the manufacturer’s protocol.
Plasmid clones of iss were sequenced using ABI PRISMcc BigDye™ Terminator Cycle Sequencing Kits. The samples were read using an ABI PRISMcc 3730XL automated sequencer (Macrogen Inc., Seoul, South Korea). Assembly accuracy was measured by forward and reverse read-pair concordance of individual plasmid subclones. Sequencing was performed in triplicate.
DNA and protein sequence alignment and analysis were performed with Bio edit, molecular evolutionary genetics analysis software, ver. 4·0 (Mega4), and the basic local alignment search tool software (Blast) (Altschul et al. 1997). A phylogenetic tree was generated by comparing the predicted amino acid sequence of the E. coli O78:K80 strain χ1378 (this study) iss gene with those of related sequences of other iss genes, and bor lambda phage sequences obtained from the GenBank database by UPGMA (1000 replicates) using Mega4. Bootstrap values are indicated at branch positions.
Amplification of iss gene carried out on plasmid DNA resulted in a band with the expected size (760 bp), while in the case of chromosomal DNA no band was detected. Cloning vector pTZ57R/T constructed the correct plasmid with the iss gene detected in E. coli strain χ1378 with the correct orientation. The iss gene sequence and flanking regions (760-bp fragment) were submitted to GenBank with the assigned accession number FJ416147. Comparison of the iss gene from strain χ1378 with previous iss sequences from other serotypes and isolates of E. coli submitted to the database indicated that E. coli strain χ1378 (gb|ACJ37387) was 100% identical to 20 previously published protein sequences of Iss from poultry. Three E. coli strains with accession numbers ABB95737, ABC25991 and ABB95738 are 98% identical with our sequence (Fig. 1). Lambda Bor protein was included in this analysis because of the high homology between Iss and lambda Bor protein (CAA393117) (Chuba et al. 1989). Comparison with bor lambda sequence showed an 86·3% identity. Strain χ1378 is 98% and 97% identical to E. coli O157:H7 strain EC 869 (ZP 02815438) and E. coli alberti TW 07627 (ZP 02902639) Iss protein, respectively. In addition, it is 87% identical to Iss from E. coli of mammalian origin (P19592). Phylogenetic analysis shows no significant different phylogenic groups among the different strains (Fig. 2).
The purpose of this study was to identify, clone and sequence the iss gene and to compare the predicted protein sequence with previous submitted sequences in the GenBank database. The current study confirms the similarity between the predicted Iss protein from E. coli strain χ1378 and those from different E. coli serotypes and isolates of different origins. Therefore, we can conclude that iss is a conserved gene. This observation is consistent with previous findings that iss is located in a conserved portion of some plasmids (Johnson et al. 2006). Johnson et al. (2008) showed different iss alleles and that many extra pathogenic E. coli have iss within their chromosome, while in the present study, we did not detect iss gene in chromosomal DNA. The iss gene has been reported in different serogroups of APEC and occurs more frequently in poultry colibacillosis than in healthy bird (Pfaff-McDonough et al. 2000; McPeake et al. 2005; Rodriguez-Siek et al. 2005). Different serotypes of E. coli such as O78, O2 and O1 have been associated with avian colibacillosis, but in the majority of flocks, the E. coli strains isolated belonged almost exclusively to the O78 serotype (Vandekerchove et al. 2004), so it seems that Iss protein from strain χ1378 could be used as a marker for the control and detection of APEC strains in many flocks. It could also be suitable for other serotypes as the iss gene has a conserved sequence.
Some studies report that the distribution of virulence factors in APEC strains is independent of the host, and that APEC could pose a zoonotic risk via horizontal gene transfer between strains (Ron 2006; Johnson et al. 2008; Mellata et al. 2009). Furthermore, our results showed a high homology between iss genes from strains of poultry origin and E. coli O157:H7 strain EC869. Therefore, the iss gene may be transferred between human and avian strains, thus increasing the zoonotic risk of APEC strains.
The phylogenetic tree shows that all Iss sequences are closely related and have evolved from a common ancestor. Some observed differences between strains may be caused by point mutation and environmental conditions. The phylogenic groups show that Iss present in strains of different origins is very similar (in E. coli from not only poultry origin but also other hosts). Moreover, Iss is located on the surface of E. coli, and it has been reported to stimulate an immunoprotective response against colibacillosis (Lynne et al. 2006, 2007). These findings indicate that the iss gene and its protein product, Iss, could be used to detect and control APEC infection in different parts of the world, although further research is required. Overall, the iss gene from E. coli (O78:K80) strain χ1378 isolated from Iranian poultry does not differ from previous reported genes, so the Iss protein of our strain could be a good antigen to be used for the detection and control of APEC. Currently, efforts are being made in our laboratory to express and purify the Iss protein.
We are grateful to the Iranian Ministry of Science, Research and Technology, the Research Council of the University of Tehran and the Research Council of the Faculty of Veterinary Medicine of the University of Tehran for financial support under project no. 7504002/6/5. The authors are also grateful to Mr M.M. Ghaffari and Mr I. Ashrafi for excellent technical assistance.