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Infection and Immunity, August 2006, p. 4900-4909, Vol. 74, No. 8
0019-9567/06/$08.00+0     doi:10.1128/IAI.00412-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

The BarA-UvrY Two-Component System Regulates Virulence in Avian Pathogenic Escherichia coli O78:K80:H9

Christopher D. Herren,1,{dagger} Arindam Mitra,1,2,{dagger} Senthil Kumar Palaniyandi,1,{dagger} Adam Coleman,1,3 Subbiah Elankumaran,1 and Suman Mukhopadhyay1,4*

Virginia-Maryland Regional College of Veterinary Medicine,1 Department of Animal and Avian Sciences,2 Department of Biochemistry,3 Center for Biosystems Research, University of Maryland Biotechnology Institute, University of Maryland at College Park, College Park, Maryland4

Received 14 March 2006/ Returned for modification 17 April 2006/ Accepted 22 May 2006


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ABSTRACT
 
The BarA-UvrY two-component system (TCS) in Escherichia coli is known to regulate a number of phenotypic traits. Both in vitro and in vivo assays, including the chicken embryo lethality assay, showed that this TCS regulates virulence in avian pathogenic E. coli (APEC) serotype O78:K80:H9. A number of virulence determinants, such as the abilities to adhere, invade, persist within tissues, survive within macrophages, and resist bactericidal effects of serum complement, were compromised in mutants lacking either the barA or uvrY gene. The reduced virulence was attributed to down regulation of type 1 and Pap fimbriae, reduced exopolysaccharide production, and increased susceptibility to oxidative stress. Our results indicate that BarA-UvrY regulates virulence properties in APEC and that the chicken embryo lethality assay can be used as a surrogate model to determine virulence determinants and their regulation in APEC strains.


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TEXT
 
Avian pathogenic Escherichia coli (APEC) infection causes avian colibacillosis, a complex syndrome characterized by air sacculitis, pericarditis, peritonitis, salpingitis, polyserositis, septicemia, synovitis, osteomyelitis, and yolk sac infection (19, 21). Cellulitis caused by APEC is the second leading cause of condemnation of broiler chickens and costs the U.S. poultry industry an estimated 40 million dollars per year (43). Most often, APEC strains infect chickens, turkeys, ducks, and other avian species through fecal dust via the respiratory tract. Isolates reported in some studies predominantly belong to the O1, O2, and O78 serogroups, other serotypes predominate in other studies, and often, untypeable isolates predominate (7, 19-22).

While many of the genes involved in APEC virulence have been identified, global regulation of the virulence of APEC, including large virulence plasmids, has only now begun to be characterized (6, 9, 10, 21, 26, 31). From attachment and colonization to the host cells to systemic invasion, a complex regulatory network in E. coli exists; this network senses the environment and activates genes that are required for each step in the infection process (18). The infection process requires rapid adaptation to the host environment by alteration of gene expression and, as a result, of bacterial structures and processes (15, 51, 62). In addition to strain-specific virulence determinants, it is highly likely that conserved global regulator(s) controlling adaptive metabolic systems are at play.

E. coli utilizes several evolutionarily conserved sensory systems to sense and adapt to their ever-changing surroundings. Common features in these signal transduction processes are conserved families of histidine-aspartate kinases that assist the bacteria in environmental adaptation (18, 52). The BarA protein is one such membrane-associated sensor protein conserved in certain gram-negative pathogens (23, 35, 39, 54). In uropathogenic E. coli, barA transcription is induced upon contact with the uroepithelial cell surface and has been implicated in the metabolic switching between glycolytic and gluconeogenic carbon sources (45, 70). Additionally, E. coli barA mutants show impaired catalase expression, which therefore renders cells sensitive to oxidative stress (36, 37). In Salmonella enterica serovar Typhimurium, barA or uvrY mutants exhibit a significant reduction in their ability to invade cultured epithelial cells, due in part to regulation of the type III secretion system required for modulating eukaryotic cellular physiology for uptake of bacteria (3, 4, 29).

The UvrY protein is the cognate response regulator for BarA in E. coli, and orthologs of UvrY are present in Pseudomonas (gacA), Erwinia (expA), Vibrio (varA), and Salmonella (sirA) species within evolutionarily conserved regions of their respective genomes (17, 44, 46, 59). The S. enterica UvrY ortholog, SirA, binds to the promoters of the hilA, hilC, and csrB genes, and thus, regulates bacterial motility and host cell invasion during infection (17, 56, 59).

In E. coli, the BarA-UvrY two-component system (TCS) affects the activity of CsrA RNA-binding protein by regulating the expression of csrB and csrC untranslated regulatory RNA. The csrB and csrC RNA binds to CsrA protein and prevents it from binding to the 5' untranslated region of target mRNAs. CsrA controls carbon metabolism, flagellum biosynthesis, and biofilm formation (3, 57, 59, 66). These findings not only indicate an important, evolutionarily conserved role for the BarA-UvrY/SirA TCS in establishing early infection in pathogenic {gamma}-proteobacteria but suggest that BarA-UvrY TCS could be an important regulator in the pathogenesis of E. coli.

The purpose of this investigation was to test the hypothesis that the BarA-UvrY TCS is critical to the virulence of APEC O78:K80:H9. To that end, isogenic barA and uvrY mutants of APEC O78:K80:H9 strain {chi}7122 were constructed using {lambda} Red recombination as described previously (8) using the primers listed in Table 1. These mutants were tested for various attributes of virulence, including in vivo assays in developing chicken embryos and various in vitro assays to study the early steps in pathogenesis.


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  		TABLE 1. Primers used in this study

Mutation in barA or uvrY reduces virulence of E. coli O78:K80:H9 ({chi}7122) in vivo. The role of the BarA-UvrY TCS in the virulence of APEC strain {chi}7122 was determined using an chicken embryo lethality assay, in which live embryos were infected with a controlled amount of bacteria and were scored as alive or dead by movement of the embryo when held close to a bright light source (12, 16, 42, 69). Mutant strains of {chi}7122 were generated by {lambda} Red-mediated recombination, with modifications suggested for clinical isolates of E. coli (8, 32, 38) (Table 2). Complementation of the mutants was accomplished by expressing wild-type copies of either the barA or uvrY gene in pBR322 (Tables 1 and 2).


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  		TABLE 2. E. coli strains and plasmids used in this study

Bacterial strains were grown for a total of 48 h in LB medium in two subcultures without shaking at 37°C to facilitate pilus formation. The barA or uvrY mutants did not exhibit a growth defect, as determined by generation time in LB and tryptone broth at 37°C. Cells were pelleted by centrifugation and gently resuspended in phosphate-buffered saline (PBS). A 10-fold serial dilution of bacterial suspension was made in PBS (9.0 to 1.0 log10 CFU in 0.1 ml). Groups of 20 12-day-old specific-pathogen-free (SPF) chicken embryos were infected with 0.1 ml of each dilution through the allantoic cavity using an 18-gauge needle. The needle hole was sealed with adhesive cement. The eggs were then incubated at 37°C and examined every 12 h for 7 days. The first time each embryo was observed to be dead was recorded (lack of movement on candling). The highest dilution at which half of all the embryos died was considered the minimum lethal dose (MLD50). The MLD50 of wild-type E. coli O78:K80:H9 strain {chi}7122 was determined to be 4 ± 0.5 log10 CFU, while the MLD50s of prototrophic E. coli K-12 strains DH5{alpha} and HB101 were >9 ± 0.5 log10 CFU, and they were considered to be avirulent. The effects of various mutations and their complement were determined by inoculating 0.1 ml of ~5 x 103 CFU bacterial culture (final count in 0.1 ml determined by plating) into the allantoic cavities of a set of 20 12-day-old SPF chicken embryos. The experiment was repeated twice. Our results indicated that a mutation in either the barA or uvrY gene reduced the virulence of E. coli O78:K80:H9 strain {chi}7122 (Fig. 1). The reduction in virulence was significant (P < 0.05 between each set of eggs from two experiments by the paired t test), as 12 of 20 embryos and 16 of 20 embryos inoculated with the mutant strains were still alive after 5 days, while only 2 of 20 embryos survived for the wild-type strain. A plasmid-borne copy of the wild-type gene was capable of restoring virulence of the mutant strain; however, the complementation was not 100%. Tomenius et al. report that barA plasmid clones acquire mutations that result in poor complementation (60). It could also be due to the loss of plasmid from the bacterial strain in vivo, within the embryo, due to the absence of antibiotic selection. There was a 10% loss of plasmid-bearing colonies in the case of barA/p-barA complemented strain, as determined by dilution plating. A uvrY mutant was less virulent than a barA mutant, indicating that the transcription modulator, the UvrY protein, has a larger role in determining virulence. However, the reduction in virulence in the uvrY mutant was not similar to the level of avirulent E. coli K-12 strain, indicating as expected, that certain virulence determinants are independent of the BarA-UvrY regulatory system, and that the virulence of APEC is multifactorial in nature.


Figure 1
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  		FIG. 1. The BarA-UvrY two-component system regulates the virulence of E. coli O78:K80:H9 in chicken embryos. The lethality of E. coli K-12 DH5{alpha} (control), {chi}7122 (wild type [wt]), SM3000 (barA::kan) ({blacksquare}), SM3001 (uvrY::cm) ({blacktriangleup}), SM3002 (barA::kan uvrY::cm) (x), SM3003 (barA::kan) carrying pSM1 ({square}), and SM3004 (uvrY::cm) carrying pSM2 ({triangleup}) was determined by inoculating 0.1 ml of 5 x 10 3 CFU of bacteria into the allantoic cavity of a set of 20 12-day-old embryonated chicken eggs. Bacterial strains were grown in LB broth to an optical density at 600 nm of 1.5 at 37°C for 48 h without shaking. Cells were washed and gently resuspended in phosphate-buffered saline. Bacterial suspension in 0.1 ml of PBS was injected into the allantoic cavities of embryonated eggs using an 18-gauge needle, and the hole was subsequently sealed with glue. The eggs were incubated at 37°C and examined every 12 h for 7 days. The first time that each embryo was first observed to be dead (no movement on candling) was recorded. The results are the means of two such experiments.

The barA and uvrY mutants poorly colonize embryonic tissues and fail to persist within the liver and spleen. To further investigate the reason for the reduced virulence in either a barA or uvrY mutant, a set of eight 12-day-old embryos were infected with various strains. At 24 h and 48 h, a set of four embryos was harvested, and the bacterial load was determined in various tissues (Table 3). Although barA mutants were able to colonize the chorioallantoic membrane (CAM) and the liver (~3.0 ± 1.4 log10 CFU/mg of tissue), multiply in allantoic fluid (ALF) and amniotic fluid (~2.0 ± 1.0 log10 CFU/ml of fluid), similar to the wild-type strain, they were unable to persist in the lungs or spleen. The persistence in liver and lungs decreased 10-fold (or more) after 48 h of infection. The uvrY mutant could initially replicate in ALF and colonize CAM, liver, and lungs, but it failed to persist in these organs, particularly in the liver (~200-fold decrease) after 48 h of incubation. However, complementation of the uvrY mutant strain by a plasmid-borne copy of the wild-type uvrY gene (p-uvrY) (pSM2) restored colonization and persistence. These results, particularly the number of bacteria in the liver and spleen, indicate that UvrY may regulate virulence determinants required for systemic infection in the chicken embryo. Since the initial site of APEC infection is the lungs (air sacculitis), followed by a generalized infection (perihepatitis, pericarditis, or septicemia), our results indicate that a nonfunctional BarA-UvrY TCS may lead to poor colonization of the lung tissues and limit systemic invasion. Interestingly, unlike wild-type strains, embryos infected with mutants did not exhibit pericardial lesions, a characteristic of cellulitis-derived isolates (40). Our results showing the abilities of APEC strain {chi}7122 to colonize lungs, invade internal organs, and disseminate in allantoic and amniotic fluids of a 12-day-old embryos are essentially similar to that shown for the same APEC strain in 3.5-week-old chickens by Mellata et al. (33). Our results, therefore, suggest that 12-day-old SPF chicken embryos could serve as surrogate models for determining virulence.


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  		TABLE 3. Attributes of APEC strain {chi}7122 and various isogenic mutants to colonize 12-day-old chicken embryos, invade internal organs, and disseminate in allantoic and amniotic fluids after 24 h and 48 h of infection

Mutation in barA or uvrY reduces serum resistance, while aerobactin production is not altered. The poor survival of the mutants in vivo could be due to a reduced ability to resist the bactericidal effects of serum complement (Table 3). Resistance to serum has been associated with E. coli causing infections in poultry and extraintestinal infections in other species (9, 14, 40). Serum resistance is a multifactorial characteristic involving outer membrane proteins, lipopolysaccharide, type 1 fimbriae, capsule, and O antigen and production of aerobactin (28). APEC strains more often contain ColV plasmids that encode serum resistance (41). APEC O78:K80:H9 carries three plasmids, of which one is of ColV origin. A similar plasmid from an O2:K2 serotype has recently been sequenced and found to carry serum resistance genes (24). One likely explanation is that BarA-UvrY TCS regulates a plasmid-borne pathogenic trait, directly or indirectly via other regulators. It is known that this TCS regulates stationary-phase sigma factor RpoS (36), which in turn regulates plasmid-borne pathogenicity genes, such as the spvR gene in Salmonella (68). Alternately, since the BarA-UvrY TCS affects carbon metabolism by regulating RpoS and CsrA, it is likely that the sugar substrates necessary to produce core O-78 antigen may be affected, leading to reduced serum resistance, as reported previously (33). Also, it is likely that this TCS may regulate pilus expression, contributing to serum resistance. Such an effect in production of exopolysaccharide (EPS) and pili was observed in E. coli K-12 and is reported for the strain under study (see Table 6; A. Mitra, S. Acharya, I. Patel, N. Chakraborty, G. Purrinton-Herren, D. Colley, T. Cebula, and S. Mukhopadhyay, unpublished observation).


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  		TABLE 6. Mutation in barA or uvrY reduces attachment and survival of E. coli O78:K80:H9 {chi}7122 in chicken macrophages

The ability to scavenge iron is critical for survival and pathogenesis in vivo, under conditions of low free iron that limit bacterial growth. The aerobactin iron acquisition system of APEC appeared not to be affected by the BarA-UvrY TCS (Table 3). Thus, the inability of barA or uvrY mutants to persist in the internal organs of 12-day-old chicken embryos appears to be independent of iron acquisition.

Mutation in barA or uvrY reduces MRHA. To further understand the mechanistic basis for reduced embryo lethality in barA and uvrY mutants, we examined the effect(s) of these mutations on attachment. APEC strains adhere to chicken epithelial cells of the pharynx and trachea by type 1 fimbriae via D-mannose residues, but not in the deeper tissues (48). P fimbriae, which recognize globoseries and glycolipids, are responsible for colonization of lungs, air sacs, and internal organs but not peripheral tissues, such as trachea (48). Hemagglutination (HA) assays with chicken red blood cells under conditions that induce type 1 and P fimbriae indicated that the BarA-UvrY TCS may be regulating a mannose-resistant adherence (Table 4), either via P fimbriae or through other novel adhesins. There was a more than sixfold decrease in hemagglutination in the mutants (Table 4, log2 4 in the wild type versus log2 1 in mutants). We found similar results with human O+ P+ and guinea pig red blood cells (data not reported). The total HA could be restored by expressing p-uvrY in a uvrY strain, but not the mannose-resistant hemagglutination (MRHA) phenotype, suggesting that type 1 fimbriae also had a subordinate role in adhesion of these strains as reported earlier (12, 34). It is possible that the observed MRHA phenotype could also be a function of adhesins other than P fimbriae.


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  		TABLE 4. Mutation in barA and uvrY exhibits a reduction in mannose-resistant hemagglutination to chicken blood

The BarA-UvrY TCS is known to regulate the production of a basic polysaccharide, unbranched ß-1,6-N-acetyl-D-glucosamine (PGA), via CsrA (64). PGA has been shown to regulate adhesion to abiotic surfaces, but there is no report of its role in adhesion to biotic surfaces. Curli is another adhesin that has been implicated in binding to various biotic surfaces and is a known virulence determinant of APEC strains (50). Expression of curli is regulated by stationary-phase sigma factor RpoS (49), which, in turn, is regulated by BarA-UvrY TCS (36). Other factors that could contribute to the HA phenotype are capsular polysaccharides and outer membrane proteins (11). Thus, the reduction in HA in the absence of the BarA-UvrY TCS could be a reflection of global down regulation of several of these factors.

Mutation in barA or uvrY reduces adherence and invasion to cultured chicken embryo fibroblasts. To understand why mutant strains of {chi}7122 cause a decrease in embryo lethality and reduction in HA, we assayed for the possible effects barA and uvrY might have on the initial attachment phase of bacteria to a cultured chicken embryo fibroblast line, DF-1 (55). For APEC to cause colibacillosis, bacterial cells must be able to invade epithelial cells and move through the host fibroblasts that make up the connective tissue. In vivo, the uvrY mutant colonized the chorioallantoic membrane, an epithelium, 100-fold more poorly than the wild-type strain did (Table 3). Since we did not have a transformed chicken epithelial cell line, we assayed the abilities of barA and uvrY mutant strains and uvrY-complemented strain of APEC to adhere to and invade chicken fibroblasts (Table 5). Deletion of either the barA or uvrY gene in strain {chi}7122 reduced bacterial attachment to fibroblasts by 100-fold (~2 log10 CFU/ml difference) of the wild type, respectively (Table 5). These less adherent phenotypes could be complemented to wild-type {chi}7122 levels when the respective gene was provided in trans. Complementation was best achieved in the uvrY/p-uvrY complemented strain (Table 5 and Fig. 2A to D). About 16% of the adherent APEC could invade DF-1 cells, as indicated by their ability to resist gentamicin treatment after 8 h of initial infection (Table 5). However, a mutation in either barA or uvrY (especially uvrY), lead to almost 100-fold reduction in invasiveness of these mutants that were adhering to DF-1 cells. The invasiveness could be restored to near wild-type levels in the uvrY/p-uvrY complemented strain. These results indicate that the BarA-UvrY TCS, either directly or indirectly, regulates a number of bacterial determinants responsible for attachment and invasion of APEC strains. The BarA-SirA TCS has been shown to be required for full virulence in S. enterica because of its effects on the type III secretion system (4, 30). This TCS has also been implicated in regulating invasiveness in Salmonella by regulating pathogenicity island I genes through the master regulator HilA (30). Whether such regulation of yet unknown APEC-specific pathogenicity island/type III secretion system operates in E. coli O78:K80:H9 strain is currently under investigation.


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  		TABLE 5. Mutation in barA or uvrY reduces attachment and invasion of E. coli O78:K80:H9 strain {chi}7122 to chicken embryo fibroblasts


Figure 2
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  		FIG. 2. Mutation in uvrY reduces attachment to cultured chicken fibroblasts and macrophages. The monolayers of cultured cells over glass coverslips were incubated with bacteria (grown under static conditions) with a multiplicity of infection of 10. As described in Table 5, footnote c, the unattached bacteria were washed, stained with Hema-3 stain (Fischer Scientific, Middletown, VA), and visualized using a Spot RT camera attached to a Olympus BH-2 microscope with a 100x objective lens. Attachment of E. coli {chi}7122 (wild type [wt]), uvrY mutant, and uvrY mutant complemented with a plasmid-borne copy of the wild-type uvrY gene (p-uvrY). (A to D) Attachment to DF-1 chicken fibroblasts. Arrows indicate surface-attached bacteria. (E to H) Attachment to HD11 chicken macrophages. Panels F and H show attached bacteria, and panel G shows bacteria that are mostly engulfed (indicated by arrows).

Mutation in barA or uvrY reduces survival within chicken macrophages. The most common form of APEC infection in poultry is characterized by initial respiratory tract colonization followed by a systemic spread to other parts of the body. Avian air sacs do not have cellular defense mechanisms and depend initially on the influx of heterophils followed by macrophages as a cellular defense (34, 61). APEC strains are known to be associated in vivo with macrophages of the air sacs and lungs, while nonpathogenic strains were observed to lack these attributes (34). Moreover, the pathogenic APEC strains are more resistant to killing by chicken macrophages in vitro than the less-pathogenic strains are (33, 47). Therefore, using HD-11 chicken macrophage line, we examined the effects of mutations of barA and uvrY on bacterial survival within cultured macrophages.

Internalized fractions of bacteria surviving within cultured HD-11 macrophages were enumerated by the standard gentamicin protection assay (13). Briefly, HD-11 cells were infected with the APEC strain(s) and incubated for a total of 2 h for adhesion prior to treatment of the infected cells with gentamicin to kill any external bacteria. Subsequently, the gentamicin-treated mixture was incubated for another 6 hours, before internalized bacteria were enumerated by dilution plating (13). There appeared to be no differences in adhesion (Table 6). However, mutation in barA reduced APEC survival within chicken macrophages by 1,000-fold compared to that of the wild type, and the uvrY mutant survived but at a level 104-fold less than that of the wild type (Table 6). Although there was not much of an adherence defect, the difference in survival of internalized bacteria, compared to wild type, is significant enough (P < 0.05 in all experiments) to be independent of adhesion. The mutant bacteria appeared to be engulfed quickly by the macrophages, while the wild-type or uvrY/p-uvrY complemented bacteria appeared to resist engulfment (Fig. 2F, H versus G, and the arrows indicating engulfed bacteria). A probable reason for this phenotype could be the lack of catalase activity in the mutant strains to counteract the oxidative onslaught of hydrogen peroxide within the macrophages. As expected, mutant colonies produced less catalase as determined by bubbling upon addition of 10 µl of 1% hydrogen\ peroxide on individual colonies grown on a LB plate. Exopolysaccharide and pili are also known to help APEC better survive within macrophages. It is also possible that mutation in uvrY leads to enhanced engulfment and destruction of the bacterium due to a reduction in pili and reduced EPS expression.

Mutations in barA or uvrY increase susceptibility to oxidative stress and reduce expression of exopolysaccharide and pili. E. coli K-12, deficient in BarA and UvrY, has been shown to be impaired in catalase production and very sensitive to oxidative stress (36, 46). This phenotype is caused by the effect the BarA-UvrY TCS has on the expression of the stationary-phase sigma factor, RpoS, and the resultant regulation of stress responses (36). Indeed, this was true for the APEC strain {chi}7122 strain (Table 7). The barA or uvrY mutants exhibited a greater zone of inhibition of bacterial growth in the presence of 1% hydrogen peroxide (42 mm for the uvrY mutant compared to 32 mm for the wild-type strain [Table 7]).


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  		TABLE 7. Mutation in barA and uvrY genes in APEC strain {chi}7122 leads to lower pilus expression, exopolysaccharide production, and increased susceptibility to oxidative stressa

Increased serum resistance, failure to persist in the liver and macrophages, and poor attachment to cultured cells indicated a possible role of exopolysaccharide and pili (27, 28). The BarA-UvrY TCS regulates carbon metabolism that provides substrates for synthesis of capsules, EPS, and surface antigens (A. Mitra and S. Mukhopadhyay, unpublished observation). The system is also known to indirectly regulate synthesis of a neutral unbranched polymer ß-1,6-N-acetyl-D-glucosamine, encoded by the pga operon of E. coli (65). However, our studies indicate that BarA-UvrY also has an effect on the production of acidic EPS and capsules in E. coli K-12 (Mitra et al., submitted). Capsules contribute to the observed resistance to oxidative stress, survival within a phagosome, and inflammatory response. We found that disruption of BarA-UvrY led to a reduced uronic acid production (approximately twofold [Table 7]). Uronic acids are common constituents of bacterial EPS and a much more specific indicator of EPS (5). This may partly explain why an uvrY mutant fails to establish a systemic infection as observed in Table 3 or shows accelerated engulfment by macrophages.

Mellata et al. have demonstrated that possible regulation of type 1 and P fimbriae can provide protection for APEC from the bactericidal effects of phagocytes (34). As stated previously, type 1 pili are responsible for the initial stages of infection, while Pap contributes to tissue invasion in APEC (48). To further understand whether the defect in adhesion and bactericidal effect was due to decreased pilus expression, we examined the mRNA levels of pilus genes by quantitative real-time PCR in 24-h static cultures (Table 7). Since the genome of strain {chi}7122 has not yet been sequenced, we used published sequence from uropathogenic E. coli CFT073 to design primers for papA and fimA genes encoding the major Pap and type 1 structural proteins. The level of fimA expression was approximately twofold down regulated, and the level of papA expression was approximately threefold down regulated in either a barA or uvrY mutant (Table 7). These results support our phenotypic observations and suggest a new role of regulation of surface adhesins by the BarA-UvrY TCS. However, whether this regulation is direct or indirect is yet to be determined.

Conclusions. This and other studies continue to dissect the virulence of APEC and to distinguish them from other extraintestinal pathogenic E. coli strains (26, 31, 53). While considerable work has been done to identify the virulence factors, little is known of their regulation. Large-scale genomic screenings have identified potential regulators (6, 10, 31), but only the Pst system has been recently examined in detail and shown to affect virulence in APEC (26). We now show that in addition to the Pst system/Pho regulon, there is also a BarA-UvrY regulon that controls virulence in APEC. It is interesting to note that a barA or uvrY mutant negatively affects the transcription of the rpoS gene, encoding the stationary-phase sigma factor RpoS (36). Decreased levels of RpoS result in down regulation of pstS transcription (58), which in turn governs the expression of the entire pst operon (1). In S. enterica, barA-sirA and pstS are known to affect expression of hilA, which is a regulator of the Salmonella pathogenicity island I-encoded type III secretion apparatus involved in bacterial invasion of epithelial cells (2, 25). However, there appears to be other factors that are, directly or indirectly, regulated by the BarA-UvrY TCS, including pili (type 1 and P) and exopolysaccharide that contribute to virulence in APEC. Whether this regulation is direct or indirect or a combination remains to be determined.

Using a chicken embryo lethality assay, we have shown that BarA-UvrY TCS regulates virulence factors in APEC serotype O78:K80:H9. A combination of virulence determinants, such as the abilities to adhere, invade, and survive within antigen-presenting cells, such as macrophages, and the ability to resist the bactericidal effect of serum complement are compromised in mutants lacking either barA and uvrY genes. The ability to resist the bactericidal effects of complement and persist within macrophages provides a survival advantage to APEC strains by potentiating efficient replication while abrogating elimination by the host immune responses. This was evident in our chicken embryo lethality assay, where isogenic mutant strains were rapidly eliminated from the livers and spleens of the infected embryos, while the wild-type APEC strain persisted within tissues, causing mortality. Our results also indicate that 12-day-old SPF chicken embryos can be used as a model to determine the initial virulence properties of APEC strains conveniently, since the mortality and colonization results of the wild-type strain are similar to those of 3.5-week-old chickens (33). Our results, therefore, suggest that the BarA-UvrY TCS may be a global regulator of APEC virulence.


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ACKNOWLEDGMENTS
 
We thank Roy Curtis III for providing the parent APEC strains; T. Romeo, J. Johnson, and L. K. Nolan for various strains and plasmids; and I. Dryburgh-Barry for assistance throughout the project. We are grateful to Siba Samal for his advice and critical analysis of our experimental design.

This work was supported by USDA-NRI-CSREES Competitive Grant 2004-35204-14749, USDA-Animal Health 2002-1106-0195318, and a Maryland Agriculture Experimental Station grant to S.M.


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FOOTNOTES
 
* Corresponding author. Mailing address: Virginia-Maryland Regional College of Veterinary Medicine, University of Maryland at College Park, 8075 Greenmead Drive, College Park, MD 20742-3711. Phone: (301) 314-6812. Fax: (301) 314-6855. E-mail: smukhopa{at}umd.edu. Back

Editor: F. C. Fang

{dagger} These authors made equal contributions to this study. Back


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Infection and Immunity, August 2006, p. 4900-4909, Vol. 74, No. 8
0019-9567/06/$08.00+0     doi:10.1128/IAI.00412-06
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