The pap Operon of Avian Pathogenic Escherichia coli Strain O1:K1 Is Located on a Novel Pathogenicity Island
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感染与免疫杂志 2006年第1期
Department of Veterinary Microbiology and Preventive Medicine, College of Veterinary Medicine, Iowa State University, 1802 Elwood Drive, VMRI #2, Iowa State University, Ames, Iowa 50011
ABSTRACT
We have identified a 56-kb pathogenicity island (PAI) in avian pathogenic Escherichia coli strain O1:K1 (APEC-O1). This PAI, termed PAI IAPEC-O1, is integrated adjacent to the 3' end of the pheV tRNA gene. It carries putative virulence genes of APEC (pap operon), other E. coli genes (tia and ireA), and a 1.5-kb region unique to APEC-O1. The kps gene cluster required for the biosynthesis of polysialic acid capsule was mapped to a location immediately downstream of this PAI.
TEXT
Colibacillosis is one of the principal causes of morbidity and mortality of poultry and, as such, it is an economic threat to the poultry industry worldwide (3). A variety of factors, including pilus adhesins (F1 and P pili), nonpilus adhesins (temperature-sensitive hemagglutinins and curli), serum resistance traits (certain outer membrane proteins, K1 capsule, and smooth lipopolysaccharide layers), iron acquisition systems (aerobactin, yersiniabactin, and salmochelin), and vacuolating autotransporter toxin (Vat) have been recognized as putative virulence markers of avian pathogenic Escherichia coli (APEC) (3, 7, 11, 16, 17, 18, 19, 27). Among these, P pili, encoded by the pap (pyelonephritis-associated pili) gene cluster, have been studied extensively. Reportedly, P pili are important at a latter stage of infection leading to multiple organ system damage and septicemia (7, 18, 19).
Many of the virulence genes of gram-negative and certain gram-positive bacteria are contained within pathogenicity islands (PAIs) (10, 24). PAIs are horizontally acquired, large genomic regions of 20 to 200 kb, which carry genes encoding one or more virulence factors (15). They are present in pathogenic bacteria but absent from nonpathogenic members of the same species (15). PAIs differ from the rest of the genome by their G+C content and codon usage (15). Additionally, they are flanked by small directly repeated DNA sequences, often associated with tRNA genes, carry cryptic or functional genes encoding mobility factors, such as integrases, transposases, or parts of insertion elements, and are often unstable (9, 10, 15, 24). To date, only one PAI (VAT-PAI), of about 22 kb, has been identified on the APEC chromosome (16). This PAI carries a gene that encodes Vat, among others. In the present study, the complete sequence and genetic organization of a second APEC PAI, termed PAI IAPEC-O1, are described.
Identification of a PAI in APEC-O1. APEC strain O1:K1 (APEC-O1) is a highly virulent strain of APEC isolated from a chicken with colisepticemia. Previous studies carried out in our laboratory indicated that this strain contained many of the genes associated with APEC virulence, some of which are known to be plasmid-encoded (GenBank accession number AY545598) (14, 21, 22). We recently performed suppression subtractive hybridization (PCR-Select genome subtraction kit; Clontech, BD Biosciences, Palo Alto, CA), comparing APEC-O1 (O1:K1) isolated from a chicken with colibacillosis to an avian fecal Escherichia coli (AFEC-O120) isolated from an apparently healthy chicken in order to identify APEC-specific regions that might contribute to the pathogenesis of colibacillosis (unpublished data). One of the subtracted fragments (HB4; GenBank accession number DQ105563) showed 99% sequence homology to a PAI of Shiga toxin-producing E. coli strain 4797/97, suggesting that a novel PAI might be present in APEC-O1. In the present study, we employed genome walking (Universal genome walking kit; Clontech) with the subtracted fragment HB4 and fosmid cloning (EpiFOS fosmid library production kit; Epicenter, Madison, WI) to discover whether HB4 is located on a PAI. Sequence analysis and homology comparisons carried out using the BLAST (Basic Local Alignment Search Tool) program of the National Center for Biotechnology Information and software packages from DNAStar (Lasergene, Madison, WI) revealed the presence of a 56-kb fragment in APEC-O1 that possesses all of the characteristics of a PAI (Fig. 1). The sequence of this PAI, termed PAI IAPEC-O1, has been deposited in GenBank.
Genetic characteristics of the PAI in APEC-O1. PAI IAPEC-O1 is associated with the tRNA-encoding gene pheV located at 64 min on the E. coli K-12 MG1655 chromosomal map. It also contains a truncated copy of the pheV gene 55,867 bp downstream of the intact copy of pheV gene. The functional and truncated pheV genes are located at the 5' and 3' ends, respectively, of the 56-kb region and contribute to the 17-bp direct repeats flanking the PAI IAPEC-O1. Such tRNA-encoding genes often provide target sites for integration of foreign DNA. The overall G+C content of the island is 46.5%, whereas the average G+C content of the E. coli K-12 genome is 50.8%. This discrepancy in G+C content suggests that this particular stretch of DNA does not belong to the E. coli backbone and is foreign. The G+C content of the open reading frames (ORFs) within the PAI ranges from 34.3% to 58.7% (Table 1 and Fig. 2). A comparison of codon usage tables with a Z-test demonstrated that several codons occur with markedly different frequencies in PAI IAPEC-O1 and in the E. coli K-12 chromosome. At least one codon for the amino acids tyrosine, leucine, histidine, glutamine, isoleucine, asparagine, lysine, valine, serine, cysteine, proline, arginine, threonine, glycine, and alanine had a relative synonymous codon usage value of greater than one.
As listed in Table 1, PAI IAPEC-O1 contains several mobile genetic elements demonstrating the possible contribution of horizontal gene transfer to the evolution of PAI IAPEC-O1. Of the mobile genetic elements present, the P4-like bacteriophage-integrase-encoding gene (orf1), located immediately downstream of the pheV gene, might have served as the integration site for this newly discovered PAI. Additionally, copies of cryptic insertion sequences (IS629 and IS2) and transposases are also present in this piece of DNA (Table 1).
The entire PAI contains 54 ORFs and consists of genes already known to be associated with virulence of APEC, genes not previously reported in APEC, genes encoding hypothetical proteins, and a region unique to APEC-O1. The region unique to APEC-O1 is 1.5 kb in size and contains one ORF coding for a protein of 511 amino acids. Interestingly, the complete pap operon (papI to papG) that encodes the P pilus (4, 29), the ireA gene that encodes a siderophore receptor protein of extraintestinal E. coli (23), and the tia invasion determinant gene of enterotoxigenic E. coli (ETEC) (8) reside on PAI IAPEC-O1. The genes involved in the phosphoglycerate transport system (pgtA to pgtP) (25) are also found on this 56-kb DNA stretch. In addition to the known determinants, several genes that code for hypothetical proteins are also found within PAI IAPEC-O1. The kps gene cluster involved in capsular biosynthesis is located immediately downstream of the right junction of the PAI (Fig. 1).
Genetic similarities of PAI IAPEC-O1 to PAIs of other gram-negative bacteria. It is interesting that the bacteriophage P4-like integrase gene serves as the main integrase gene in almost all pheV tRNA-associated PAIs of bacteria (1, 6), including PAI IAPEC-O1. Also, phe-tRNA sites are common insertion sites for PAIs in many different bacteria, including uropathogenic E. coli (UPEC), APEC, enteropathogenic E. coli, enterohemorrhagic E. coli, Yersinia pseudotuberculosis, and Shigella flexneri (1, 6, 21, 25, 26). The PAI described in this report also exhibits marked similarities to certain chromosomal regions of UPEC strains CFT073, J96, and 536, particularly with regard to certain PAIs of these strains. Both PAI IAPEC-O1 and PAI V536 contain a truncated copy of the pheV gene at the 3' end of the island, forming the 17-bp direct repeats, and both possess the pgt gene cluster that encodes the phosphoglycerate transport system (25). Interestingly, PAI II96, PAI V536, and PAI IAPEC-O1 contain a kps gene cluster immediately downstream of the truncated copy of pheV tRNA gene (25). In PAI V536, the kps gene cluster also is considered to be a part of the PAI. However, we did not include it within the PAI sequence, since the kps genes are located outside the 17-bp direct repeats at the 3' terminus of the PAI. Rather, it seems to be associated with the PAI insertion site within the K-12 genome. Additionally, a particular UPEC K1 strain also contains the genes involved in capsular biosynthesis on a 20-kb stretch of DNA located close to the pheV-tRNA gene (5); it is uncertain whether this stretch of DNA actually is part of a PAI. Resembling PAI II96 and PAI ICFT073, PAI IAPEC-O1 also contains the complete pap operon (4, 26, 29). Previous studies indicated that PAI ICFT073, carrying only 9-bp direct repeats flanking the island, is more stable than PAIs possessing long direct repeats (4). Hence, it can be speculated that PAI IAPEC-O1, having 17-bp direct repeats, is less stable within the chromosome than PAI ICFTO73. Similar to ireA in PAI IAPEC-O1, certain UPEC PAIs contain genes (e.g., the yersiniabactin-encoding gene in PAI IVCFT073) involved in iron acquisition (1, 28). Sequence similarities observed between UPEC and APEC are quite fascinating, since they strongly support the recent arguments that APEC is a possible food-borne source of UPEC (12, 13, 20, 22) and that APEC and UPEC genomes have distinct similarities (22).
It has been amply reported that P pili are important determinants of APEC virulence (3, 7, 18, 19). They are not important for initial colonization but are involved in a latter stage of infection leading to polyserositis and septicemia (18, 19). PAI IAPEC-O1's pap operon, except for papA, shows high sequence homology to that of UPEC strain CFT073 (29). The papC gene contained truncated papCa and papCb genes and showed 98% sequence identity with the papCa and papCb genes of PAI II of UPEC strain CFT073 (Table 1). Interestingly, the papA gene showed greater homology (99%) to the papA gene of porcine septicemia E. coli strain 4787 than it did to UPEC papA genes (GenBank accession numbers L07420 and AAA92574). In addition to this well-studied pap operon, other genes known to code for virulence factors in several other E. coli strains were revealed in the sequence analysis of PAI IAPEC-O1. Two such examples are the ireA and tia genes. The ireA gene was first reported in an extraintestinal E. coli isolate that causes sepsis in humans (23). The protein encoded by ireA, the putative siderophore receptor IreA, is involved in iron acquisition and is believed to be a urovirulence factor (23). On the other hand, the tia gene of ETEC shares significant homology with hraI from porcine ETEC and limited homology with the ail locus from Yersinia spp. and encodes a 25-kDa outer membrane protein, the Tia invasion determinant protein. Tia plays a role in adherence of ETEC to epithelial cells and subsequent invasion (8). A recent study indicated that APEC contains adhesins that have not yet been identified (2), and Tia may be one such candidate. However, the role of ireA and tia in APEC pathogenesis is yet to be determined (such research is in progress).
Acquisition of PAIs is a means by which relatively avirulent bacterial strains may evolve into virulent forms. Interestingly, PAI IAPEC-O1, which bears the pap operon and the ireA gene, has been integrated into a chromosomal location close to the kps gene cluster. This has provided an opportunity for APEC-O1 to form one single virulence gene cluster, harboring the genes encoding a colonization factor (P pilus), an iron acquisition system (siderophore receptor protein), and serum resistance (capsule). Therefore, APEC-O1 may be more successful in establishing severe disease in a given host than an APEC which does not contain this particular stretch of genes. APEC-O1, which contains numerous virulence genes, including the newly discovered PAI, may behave as a primary pathogen (such research is in progress).
In conclusion, we have identified and completely sequenced a novel PAI of APEC-O1, which is located at the pheV tRNA gene. This 56-kb PAI, termed PAI IAPEC-O1, carries the complete pap operon and a few putative virulence genes of other E. coli strains that had been hitherto unknown as APEC strains. The kps gene cluster also is located immediately after the right junction of the PAI. Further studies are needed to determine the role of newly identified putative virulence genes and genes with unknown functions as virulence markers of APEC to strengthen the current understanding of mechanisms underlying the pathogenesis of avian colibacillosis.
Nucleotide sequence accession number. The sequence of PAI IAPEC-O1 has been deposited in GenBank under accession number DQ095216.
ACKNOWLEDGMENTS
This work was supported by the Roy J. Carver Charitable Trust, Iowa State University’s Biotechnology Council, Provost’s office, and CVM Dean’s office.
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ABSTRACT
We have identified a 56-kb pathogenicity island (PAI) in avian pathogenic Escherichia coli strain O1:K1 (APEC-O1). This PAI, termed PAI IAPEC-O1, is integrated adjacent to the 3' end of the pheV tRNA gene. It carries putative virulence genes of APEC (pap operon), other E. coli genes (tia and ireA), and a 1.5-kb region unique to APEC-O1. The kps gene cluster required for the biosynthesis of polysialic acid capsule was mapped to a location immediately downstream of this PAI.
TEXT
Colibacillosis is one of the principal causes of morbidity and mortality of poultry and, as such, it is an economic threat to the poultry industry worldwide (3). A variety of factors, including pilus adhesins (F1 and P pili), nonpilus adhesins (temperature-sensitive hemagglutinins and curli), serum resistance traits (certain outer membrane proteins, K1 capsule, and smooth lipopolysaccharide layers), iron acquisition systems (aerobactin, yersiniabactin, and salmochelin), and vacuolating autotransporter toxin (Vat) have been recognized as putative virulence markers of avian pathogenic Escherichia coli (APEC) (3, 7, 11, 16, 17, 18, 19, 27). Among these, P pili, encoded by the pap (pyelonephritis-associated pili) gene cluster, have been studied extensively. Reportedly, P pili are important at a latter stage of infection leading to multiple organ system damage and septicemia (7, 18, 19).
Many of the virulence genes of gram-negative and certain gram-positive bacteria are contained within pathogenicity islands (PAIs) (10, 24). PAIs are horizontally acquired, large genomic regions of 20 to 200 kb, which carry genes encoding one or more virulence factors (15). They are present in pathogenic bacteria but absent from nonpathogenic members of the same species (15). PAIs differ from the rest of the genome by their G+C content and codon usage (15). Additionally, they are flanked by small directly repeated DNA sequences, often associated with tRNA genes, carry cryptic or functional genes encoding mobility factors, such as integrases, transposases, or parts of insertion elements, and are often unstable (9, 10, 15, 24). To date, only one PAI (VAT-PAI), of about 22 kb, has been identified on the APEC chromosome (16). This PAI carries a gene that encodes Vat, among others. In the present study, the complete sequence and genetic organization of a second APEC PAI, termed PAI IAPEC-O1, are described.
Identification of a PAI in APEC-O1. APEC strain O1:K1 (APEC-O1) is a highly virulent strain of APEC isolated from a chicken with colisepticemia. Previous studies carried out in our laboratory indicated that this strain contained many of the genes associated with APEC virulence, some of which are known to be plasmid-encoded (GenBank accession number AY545598) (14, 21, 22). We recently performed suppression subtractive hybridization (PCR-Select genome subtraction kit; Clontech, BD Biosciences, Palo Alto, CA), comparing APEC-O1 (O1:K1) isolated from a chicken with colibacillosis to an avian fecal Escherichia coli (AFEC-O120) isolated from an apparently healthy chicken in order to identify APEC-specific regions that might contribute to the pathogenesis of colibacillosis (unpublished data). One of the subtracted fragments (HB4; GenBank accession number DQ105563) showed 99% sequence homology to a PAI of Shiga toxin-producing E. coli strain 4797/97, suggesting that a novel PAI might be present in APEC-O1. In the present study, we employed genome walking (Universal genome walking kit; Clontech) with the subtracted fragment HB4 and fosmid cloning (EpiFOS fosmid library production kit; Epicenter, Madison, WI) to discover whether HB4 is located on a PAI. Sequence analysis and homology comparisons carried out using the BLAST (Basic Local Alignment Search Tool) program of the National Center for Biotechnology Information and software packages from DNAStar (Lasergene, Madison, WI) revealed the presence of a 56-kb fragment in APEC-O1 that possesses all of the characteristics of a PAI (Fig. 1). The sequence of this PAI, termed PAI IAPEC-O1, has been deposited in GenBank.
Genetic characteristics of the PAI in APEC-O1. PAI IAPEC-O1 is associated with the tRNA-encoding gene pheV located at 64 min on the E. coli K-12 MG1655 chromosomal map. It also contains a truncated copy of the pheV gene 55,867 bp downstream of the intact copy of pheV gene. The functional and truncated pheV genes are located at the 5' and 3' ends, respectively, of the 56-kb region and contribute to the 17-bp direct repeats flanking the PAI IAPEC-O1. Such tRNA-encoding genes often provide target sites for integration of foreign DNA. The overall G+C content of the island is 46.5%, whereas the average G+C content of the E. coli K-12 genome is 50.8%. This discrepancy in G+C content suggests that this particular stretch of DNA does not belong to the E. coli backbone and is foreign. The G+C content of the open reading frames (ORFs) within the PAI ranges from 34.3% to 58.7% (Table 1 and Fig. 2). A comparison of codon usage tables with a Z-test demonstrated that several codons occur with markedly different frequencies in PAI IAPEC-O1 and in the E. coli K-12 chromosome. At least one codon for the amino acids tyrosine, leucine, histidine, glutamine, isoleucine, asparagine, lysine, valine, serine, cysteine, proline, arginine, threonine, glycine, and alanine had a relative synonymous codon usage value of greater than one.
As listed in Table 1, PAI IAPEC-O1 contains several mobile genetic elements demonstrating the possible contribution of horizontal gene transfer to the evolution of PAI IAPEC-O1. Of the mobile genetic elements present, the P4-like bacteriophage-integrase-encoding gene (orf1), located immediately downstream of the pheV gene, might have served as the integration site for this newly discovered PAI. Additionally, copies of cryptic insertion sequences (IS629 and IS2) and transposases are also present in this piece of DNA (Table 1).
The entire PAI contains 54 ORFs and consists of genes already known to be associated with virulence of APEC, genes not previously reported in APEC, genes encoding hypothetical proteins, and a region unique to APEC-O1. The region unique to APEC-O1 is 1.5 kb in size and contains one ORF coding for a protein of 511 amino acids. Interestingly, the complete pap operon (papI to papG) that encodes the P pilus (4, 29), the ireA gene that encodes a siderophore receptor protein of extraintestinal E. coli (23), and the tia invasion determinant gene of enterotoxigenic E. coli (ETEC) (8) reside on PAI IAPEC-O1. The genes involved in the phosphoglycerate transport system (pgtA to pgtP) (25) are also found on this 56-kb DNA stretch. In addition to the known determinants, several genes that code for hypothetical proteins are also found within PAI IAPEC-O1. The kps gene cluster involved in capsular biosynthesis is located immediately downstream of the right junction of the PAI (Fig. 1).
Genetic similarities of PAI IAPEC-O1 to PAIs of other gram-negative bacteria. It is interesting that the bacteriophage P4-like integrase gene serves as the main integrase gene in almost all pheV tRNA-associated PAIs of bacteria (1, 6), including PAI IAPEC-O1. Also, phe-tRNA sites are common insertion sites for PAIs in many different bacteria, including uropathogenic E. coli (UPEC), APEC, enteropathogenic E. coli, enterohemorrhagic E. coli, Yersinia pseudotuberculosis, and Shigella flexneri (1, 6, 21, 25, 26). The PAI described in this report also exhibits marked similarities to certain chromosomal regions of UPEC strains CFT073, J96, and 536, particularly with regard to certain PAIs of these strains. Both PAI IAPEC-O1 and PAI V536 contain a truncated copy of the pheV gene at the 3' end of the island, forming the 17-bp direct repeats, and both possess the pgt gene cluster that encodes the phosphoglycerate transport system (25). Interestingly, PAI II96, PAI V536, and PAI IAPEC-O1 contain a kps gene cluster immediately downstream of the truncated copy of pheV tRNA gene (25). In PAI V536, the kps gene cluster also is considered to be a part of the PAI. However, we did not include it within the PAI sequence, since the kps genes are located outside the 17-bp direct repeats at the 3' terminus of the PAI. Rather, it seems to be associated with the PAI insertion site within the K-12 genome. Additionally, a particular UPEC K1 strain also contains the genes involved in capsular biosynthesis on a 20-kb stretch of DNA located close to the pheV-tRNA gene (5); it is uncertain whether this stretch of DNA actually is part of a PAI. Resembling PAI II96 and PAI ICFT073, PAI IAPEC-O1 also contains the complete pap operon (4, 26, 29). Previous studies indicated that PAI ICFT073, carrying only 9-bp direct repeats flanking the island, is more stable than PAIs possessing long direct repeats (4). Hence, it can be speculated that PAI IAPEC-O1, having 17-bp direct repeats, is less stable within the chromosome than PAI ICFTO73. Similar to ireA in PAI IAPEC-O1, certain UPEC PAIs contain genes (e.g., the yersiniabactin-encoding gene in PAI IVCFT073) involved in iron acquisition (1, 28). Sequence similarities observed between UPEC and APEC are quite fascinating, since they strongly support the recent arguments that APEC is a possible food-borne source of UPEC (12, 13, 20, 22) and that APEC and UPEC genomes have distinct similarities (22).
It has been amply reported that P pili are important determinants of APEC virulence (3, 7, 18, 19). They are not important for initial colonization but are involved in a latter stage of infection leading to polyserositis and septicemia (18, 19). PAI IAPEC-O1's pap operon, except for papA, shows high sequence homology to that of UPEC strain CFT073 (29). The papC gene contained truncated papCa and papCb genes and showed 98% sequence identity with the papCa and papCb genes of PAI II of UPEC strain CFT073 (Table 1). Interestingly, the papA gene showed greater homology (99%) to the papA gene of porcine septicemia E. coli strain 4787 than it did to UPEC papA genes (GenBank accession numbers L07420 and AAA92574). In addition to this well-studied pap operon, other genes known to code for virulence factors in several other E. coli strains were revealed in the sequence analysis of PAI IAPEC-O1. Two such examples are the ireA and tia genes. The ireA gene was first reported in an extraintestinal E. coli isolate that causes sepsis in humans (23). The protein encoded by ireA, the putative siderophore receptor IreA, is involved in iron acquisition and is believed to be a urovirulence factor (23). On the other hand, the tia gene of ETEC shares significant homology with hraI from porcine ETEC and limited homology with the ail locus from Yersinia spp. and encodes a 25-kDa outer membrane protein, the Tia invasion determinant protein. Tia plays a role in adherence of ETEC to epithelial cells and subsequent invasion (8). A recent study indicated that APEC contains adhesins that have not yet been identified (2), and Tia may be one such candidate. However, the role of ireA and tia in APEC pathogenesis is yet to be determined (such research is in progress).
Acquisition of PAIs is a means by which relatively avirulent bacterial strains may evolve into virulent forms. Interestingly, PAI IAPEC-O1, which bears the pap operon and the ireA gene, has been integrated into a chromosomal location close to the kps gene cluster. This has provided an opportunity for APEC-O1 to form one single virulence gene cluster, harboring the genes encoding a colonization factor (P pilus), an iron acquisition system (siderophore receptor protein), and serum resistance (capsule). Therefore, APEC-O1 may be more successful in establishing severe disease in a given host than an APEC which does not contain this particular stretch of genes. APEC-O1, which contains numerous virulence genes, including the newly discovered PAI, may behave as a primary pathogen (such research is in progress).
In conclusion, we have identified and completely sequenced a novel PAI of APEC-O1, which is located at the pheV tRNA gene. This 56-kb PAI, termed PAI IAPEC-O1, carries the complete pap operon and a few putative virulence genes of other E. coli strains that had been hitherto unknown as APEC strains. The kps gene cluster also is located immediately after the right junction of the PAI. Further studies are needed to determine the role of newly identified putative virulence genes and genes with unknown functions as virulence markers of APEC to strengthen the current understanding of mechanisms underlying the pathogenesis of avian colibacillosis.
Nucleotide sequence accession number. The sequence of PAI IAPEC-O1 has been deposited in GenBank under accession number DQ095216.
ACKNOWLEDGMENTS
This work was supported by the Roy J. Carver Charitable Trust, Iowa State University’s Biotechnology Council, Provost’s office, and CVM Dean’s office.
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