Mutational and transcriptional analyses of an avian pathogenic Escherichia coli ColV plasmid
© Skyberg et al; licensee BioMed Central Ltd. 2008
Received: 16 July 2007
Accepted: 29 January 2008
Published: 29 January 2008
Previously we described a 184-kb ColV plasmid, pAPEC-O2-ColV, that contributed to the ability of an E. coli to kill avian embryos, grow in human urine, and colonize the murine kidney. Here, the roles of several genes encoded by this plasmid in virulence were assessed using mutational and transcriptional analyses.
Genes chosen for deletion were iss, tsh, iutA, iroN, sitA, and cvaB. In addition, a 35-kb region of the plasmid, containing iss, tsh, and the ColV and iro operons, along with a 15-kb region containing both the aerobactin and sit operons, were deleted. Mutants were compared to the wild-type (APEC O2) for lethality to chick embryos and growth in human urine. Expression of the targeted genes was also assessed under these same conditions using RT-PCR
No significant differences between the mutants and the wild-type in these phenotypic traits were detected. However, genes encoding known or predicted iron transport systems were up-regulated during growth in human urine, as compared to growth in LB broth, while iss, hlyF, and iroN were strongly up-regulated in chick embryos.
While no difference was observed between the mutant strains and their wild-type parent in the phenotypic traits assayed, we reasoned that some compensatory virulence mechanism, insensitivity of the virulence assays, or other factor could have obscured changes in the virulence of the mutants. Indeed we found several of these genes to be up-regulated in human urine and/or in the chick embryo, suggesting that certain genes linked to ColV plasmids are involved in the establishment of avian extraintestinal infection.
A 184-kb ColV plasmid, known as pAPEC-O2-ColV, was sequenced and analyzed . In addition to regions devoted to plasmid transfer, maintenance, and replication, pAPEC-O2-ColV was found to contain a 94-kb putative pathogenicity island (PAI), containing hlyF, ompT, iss, tsh, the ColV operon, and several genes encoding known or predicted iron transport systems. The iron-related systems included those encoding aerobactin and salmochelin, and the sit ABC transport system. Additionally, pAPEC-O2-ColV contained a putative iron transport system novel to APEC called eit and another putative ABC transport system known as ets. This plasmid was transmissible by conjugation from the donor, avian pathogenic Escherichia coli (APEC) O2, to recipient strains, and it was found to co-transfer with a large R plasmid known as pAPEC-O2-R . When the role of APEC O2's plasmids in virulence was investigated, it was found that acquisition of these plasmids resulted in an enhancement in the recipient's ability to kill avian embryos, grow in human urine, and colonize the murine kidney . It was thought that the increase in virulence was likely due to acquisition of pAPEC-O2-ColV .
Also, a study of the distribution of genes of pAPEC-O2-ColV's putative PAI in APEC and avian fecal commensal E. coli (AFEC) isolates revealed that a portion of this PAI was highly conserved among APEC and that these conserved genes occurred much more often in APEC than in commensal strains [4, 5]. This conserved portion, which occurred in most of the APEC examined, included sit, an iron/manganese transport system [6, 7]; salmochelin and aerobactin, both siderophore iron acquisition systems [8, 9]; ets, a putative ABC transport system ; hlyF, an avian hemolysin ; iss, the increased serum survival gene ; ompT, an outer membrane protease ; the RepFIB replicon; and the 5' end of the ColV operon [1, 4]. The variable portion of this PAI contained the 5' end of the ColV operon; tsh, the temperature sensitive hemagglutinin gene [13, 14]; and the eit operon . The split between conserved and variable portions occurred within the cvaB gene of the ColV operon, with the 5' end of cvaB and many of its upstream genes occurring significantly more often among APEC than the 3' end of cvaB and many of its downstream genes [1, 4]. Genes of these plasmid-linked PAIs occur widely among APEC isolated from different parts of the world [5, 15–20] various avian host species [5, 15, 20] and different syndromes [5, 21]. These observations suggest that these plasmid-linked PAIs, especially their conserved portions, might be a defining characteristic of the APEC pathotype  that could be exploited in colibacillosis control. Indeed, protocols for rapid characterization of APEC based on detection of certain virulence genes, including some from this cluster, show promise [22, 23].
While the data from these epidemiological studies are useful at identifying genes of interest and have been widely used to characterize avian E. coli[5, 15, 22, 23], they are no substitute for an in-depth study of the contributions to virulence of individual genes. Such studies typically require comparisons of wild-type and mutant strains, differing in a single trait of interest, for their abilities to cause disease in animal models . Here, we sought to determine the contributions of certain genes of pAPEC-O2-ColV's PAI to E. coli's ability to kill embryos and grow in human urine using mutational analysis. When differences in virulence between the mutants and the wild-type were not detected, follow-up studies to determine if these same genes are differentially expressed in APEC O2 during infection were undertaken.
Results and Discussion
Large conjugative ColV plasmids and the genes they carry are found in a much higher proportion of E. coli incriminated in cases of avian colibacillosis than in E. coli isolated from the feces of apparently healthy birds [5, 25]. In addition, it has been shown that ColV plasmids may mediate avian E. coli virulence and are often implicated in cases of human extraintestinal disease [3, 26–28]. In a previous study we found that transfer of a large ColV plasmid, pAPEC-O2-ColV (along with a co-transferring R plasmid) conferred upon a recipient strain enhanced abilities to kill chick embryos, grow in human urine, and cause urinary tract infection (UTI) in mice . In the present study, we sought to determine what regions of this ColV plasmid contributed to these traits.
To do this, we mutated several genes localized to pAPEC-O2-ColV. The genes chosen for mutagenesis, iss, tsh, cvaB, iutA, iroN, and sitA, have been found to be epidemiologically associated with APEC [1, 5]. In addition tsh and iroN, have been shown to contribute to the virulence of APEC [28, 29], while virulence of E. coli K-12 was shown to be enhanced after the acquisition of an iss encoding plasmid . The expected mutations were verified by PCR protocols  targeting the deleted gene and the new kanamycin resistance (kanR) cassette junction fragment. To ensure that the mutants were truly isogenic, we examined the genotype of the mutant strains for over 40 other virulence associated genes and allelic variants using published protocols [5, 12, 23]. In no instance did we find a loss of a gene other than the one which was targeted. This observation suggests that the method of mutagenesis used in this study may have a higher fidelity than does suicide vector driven allelic exchange which has been found to produce secondary mutations at a high rate in extraintestinal pathogenic E. coli.
Embryo Lethality of APEC O2 along with its Mutant Derivatives
Embryo Lethality %
In view of these results, we considered the possibility that chromosomal genes found in APEC O2 may compensate for the function(s) of the lost plasmid genes. For example, many of the genes targeted for deletion in APEC O2 are associated with iron acquisition or complement resistance. Since APEC O2 is known to contain at least two other chromosomal operons (yersinabactin and enterobactin) involved in the acquisition of iron , overexpression of chromosomal iron acquisition loci might compensate for the loss of certain plasmid-linked iron acquisition operons. In addition, others have shown that transfer of a ColV plasmid into a K-12 recipient was accompanied by a concomitant increase in the K-12 strain's ability to resist the bactericidal effects of serum complement . However, other studies have demonstrated that when a native host was cured of the ColV plasmid, its ability to survive in serum was not affected , suggesting that chromosomal loci may compensate for the lost plasmid genes which conferred complement resistance. Similarly, in our work we did not find any of the mutant derivatives of APEC O2 to be attenuated in their ability to resist complement or to grow under low iron conditions (data not shown).
To further investigate whether chromosomal genes found in APEC O2 were compensating for the deleted plasmid genes, we transferred the mutated pAPEC-O2-ColV derivatives generated above into the AFEC strain NC via conjugation. NC was previously used as a recipient for pAPEC-O2-ColV and was found to have enhanced abilities to kill chick embryos and grow in human urine upon receipt of the plasmids of APEC O2 . The virulence of NC derivatives containing pAPEC-O2-ColV was then compared to that of NC with an intact version of pAPEC-O2-ColV (NC/pAPEC-O2). Again, no differences were found. Knowing that others have found that some of these same genes and regions, such as tsh and the iro operon [29, 34], contribute to the virulence of other APEC, we then reasoned that our virulence assays might be too insensitive to detect subtle changes in virulence caused by the mutations. Indeed, bacterial virulence, as measured with the embryo lethality assay, has been shown to have only a moderate correlation to results of assays done in three-week old chickens . In addition, we and others  have not observed a correlation between the infectious dose of a strain and its lethality to chick embryos which would permit comparisons of virulence based on LD50 determinations.
Therefore, in an effort to increase the sensitivity of our chick embryo model, we infected 16 day-old chick embryos (the age at which chick embryos are most resistant to E. coli infection ) via the chorioallantoic route with an equivalent mixture of wildtype and mutant strains (~500 CFUs of each). Three days after infection the surviving embryos (generally > 80% of those infected) were killed by chilling at -20 C for two hours, after which brains, hearts, and livers were collected sterilely. Organ homogenates were cultured quantitatively on agar with or without antibiotics to determine the relative proportions of the strains. The results of these mixed infection experiments partially mimicked our previous embryo lethality data . Strain NC/pAPEC-O2 generally outcompeted strain NC, and no marked differences in organ colonization was seen with any of the mutant strains in relation to the APEC O2 (data not shown). However, we were able to recover E. coli from the internal of organs of only a very small (< 15%) proportion of the embryos. This is not entirely surprising as the systemic spread of E. coli is not thought to be required for embryo death , however it would indicate that mixed infections in 16 day-old chick embryos may have marginal utility in assessing E. coli virulence.
The results of this expression analysis are intriguing and suggest that genes localized to ColV plasmids, such as pAPEC-O2-ColV, are involved in the pathogenesis of colibacillosis. The lack of confirmatory results from the mutational analyses illustrates the complexities of such studies, especially where multiple alternative mechanisms may compensate for the deleted genes/operons.
Taken altogether, the results presented here suggest several plausible reasons for an inability to detect significant attenuation in the mutants examined in this study. One possibility is that the embryo lethality assay lacks the sensitivity to detect changes owed to the mutated genes. This is a distinct possibility, as acquisition of pAPEC-O2-ColV by an avirulent recipient confers the ability to kill chick embryos  while none of the isogenic mutants created in this study and involving genes and/or regions of pAPEC-O2-ColV were attenuated in the chick embryo model of infection. A second possibility is that none of the deleted genes in this study actually contribute to the abilities of APEC O2 to kill chick embryos or grow in human urine, although this seems unlikely based on the literature, which suggests that several of these genes including tsh and iroN[28, 29, 39], do contribute to the virulence of ExPEC. In addition an association of iss with the virulence of APEC in a respiratory model of infection has been postulated , however a direct role for iss in virulence was not demonstrated. In accordance with previous findings, we found that several of these genes were strongly up-regulated during growth of APEC O2 in these models, suggesting that these genes play at least some role in urine growth and infection. To better pinpoint the contributions of these targeted genes and ColV plasmids to APEC virulence, more sensitive virulence assays and use of functional genomics and proteomics approaches examining the total APEC genome will be needed.
The differences seen in the results of the mutational and transcriptional analyses in this study underscore the need to use multiple approaches in ascertaining genes' contributions to disease.
While the data presented here suggests roles for iss, hlyF, and iroN during E. coli-caused septicemia, a more comprehensive analysis of these plasmids is necessary to better understand their nature, and such analysis must also include genes of unknown function found on pAPEC-O2-ColV and other similar plasmids.
Media and bacterial strains
Phenotypic Characteristics of Strains/Plasmids Used
Strain or Plasmid
Reference and/or Source
Virulent to chick embryos; contains pAPEC-O2-ColV and pAPEC-O2-R
Diseased Chicken [1–3]
ColV plasmid of APEC O2; contains iss, tsh, and traT genes, along with the aerobactin, ColV, sit and iro operons
R plasmid of APEC O2; encodes resistance to multiple antimicrobial agents
Relatively avirulent to chick embryos and lacks iss, tsh, and the aerobactin, ColV, and iro operons
Healthy Chicken 
Transconjugant derivative of NC containing pAPEC-O2-ColV and pAPEC-O2-R; has enhanced abilities to kill chick embryos and grow in human urine relative to strain NC
ampR ; temperature sensitive plasmid which expresses the λ Red recombinase proteins when induced by arabinose
camR derivative of pKD46
Primers used in Mutagenesis*
Primer Seq. (5'-3')
Embryo lethality assay
APEC O2 and its mutant derivatives were assessed for lethality in chicken embryos by inoculation of overnight washed bacterial cultures (~500 colony forming units (CFU)) into the allantoic cavity of 12-day old embryonated, specific-pathogen-free eggs . Phosphate buffered saline (PBS) inoculated and uninoculated embryos were used as controls. Embryo deaths were recorded for four days. Differences in embryo lethality between the strains were evaluated for statistical significance using a z-test for the equality of two binomial proportions. P-values of less than 0.05 were considered statistically significant .
Growth in human urine
APEC O2 and its mutant derivatives were compared by their ability to grow in human urine. The assay was performed as described elsewhere . Only urine from healthy, antibiotic-free volunteers, who reported never having experienced a UTI, was used for study. Prior to the study, urine from five volunteers was collected, individually filter sterilized with 0.2 um filters, pooled, and stored at -20 C. On the day before the assay was run, the strains to be tested were grown overnight in 2 ml of Luria Bertani (LB) broth. The next day the cell density was estimated by spectrophotometry, and cultures were diluted in PBS prior to inoculation (100 μl of inoculum into 4.9 ml of urine) to achieve an approximate starting concentration of 102 to 103 CFUs per ml, which was confirmed by viable counts. This concentration of bacteria was chosen as a starting point since it represents the lower end of what is considered a significant indicator of UTI in symptomatic young women . Mixtures were incubated at 37 C with shaking, and aliquots of these urine cultures were removed at set time intervals for use in determining viable counts.
Chick embryos were inoculated via the allantoic cavity with APEC O2. Two days later, 12 viable infected embryos were removed from their eggs, and the livers were excised and pooled together in 20 volumes of RNALater (Ambion/Applied Biosystems, Austin, TX). RNA was extracted from these pooled liver samples using Tri Reagent (Ambion), treated with Turbo DNAse (Ambion), followed by phenol/chloroform extraction, and resuspension in distilled water. For in vitro isolation, a single colony APEC O2 was inoculated into 3 ml LB Broth or urine and grown at 37° with shaking until the cells were in early- to mid-exponential growth phase (A600 of approximately 0.3). Cells were than pelleted by centrifugation, resuspended immediately in RNALater, and the RNA isolated and purified as described above.
Primers used in RT-PCR studies.
Predicted amplicon size
Glyceraldehyde 3-phosphate dehydrogenase A (reference)
CAT CGT TTC CAA CGC TTC CT
ACC TTC GAT GAT GCC GAA GTT
Iron/manganese transport gene
TAC GAT CCG GCA AAT GCA CAA ACC
TGG TGA CCA TCC ATC GCT GAT TCT
Aerobactin receptor gene
TCT GAT AAG AGC GTG GTG GCG AAT
AGC ACG TTG AAG TTC ACT CCG GTA
Avian hemolysin gene
AAC TTT GGC GGT TTA GGC ATT CCG
TGA CAT ACT GGC AAT GAG CCG TCA
Putative ABC transport gene
ATT ACG AAC AGC GAG TGC TGG AGT
ATA CGT ACT GCA CCA TGC CGG TAA
Increased serum survival gene
GCC GCT CTG GCA ATG CTT ATT ACA
TCC TTT GGT GTT ACT GCT GTC GGT
Salmochelin receptor gene
TTC ACC TGG GAA GAT TAC CAC GCA
ATA TAT GCG CCT GAA GCG GTT TGC
ColV structural gene
CGG GCA ATT TGT TGC AGG AGG AAT
ACC GGA TGG AGA CAT TGC AGG ATT
Temperature-sensitive hemagglutinin gene
TAC TGA ACC AGC AGG CGG ACA ATA
TTT ACC TGC CGC TCA TCA GTC AGT
This study was supported by a grant from the Healthy Livestock Initiative Competitive Grants Program. Salary support for JAS was provided though North Dakota State University's Presidential Doctoral Scholar Program. This work was done in partial fulfillment of the requirements for JAS' Ph.D. degree in Molecular Pathogenesis at the same institution.
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