IbeR Facilitates Stress-Resistance, Invasion and Pathogenicity of Avian Pathogenic Escherichia coli

Systemic infections by avian pathogenic Escherichia coli (APEC) are economically devastating to poultry industries worldwide. IbeR, located on genomic island GimA, was shown to serve as an RpoS-like regulator in rpoS gene mutation neonatal meningitis E. coli (NMEC) RS218. However, the role of IbeR in pathogenicity of APEC carrying active RpoS has not yet been investigated. We showed that the APEC IbeR could elicit antibodies in infected ducks, suggesting that IbeR might be involved in APEC pathogenicity. To investigate the function of IbeR in APEC pathogenesis, mutant and complementation strains were constructed and characterized. Inactivation of ibeR led to attenuated virulence and reduced invasion capacity towards DF-1 cells, brains and cerebrospinal fluid (CSF) in vitro and in vivo. Bactericidal assays demonstrated that the mutant strain had impaired resistance to environmental stress and specific pathogen-free (SPF) chicken serum. These virulence-related phenotypes were restored by genetic complementation. Quantitative real-time reverse transcription PCR revealed that IbeR controlled expression of stress-resistance genes and virulence genes, which might led to the associated virulence phenotype.


Introduction
Extraintestinal pathogenic E. coli (ExPEC) strains have been implicated in a range of infections in humans and animals such as neonatal meningitis, urinary tract infections, pneumonia, and septicemia. ExPEC is currently categorized as newborn meningitis E. coli (NMEC), uropathogenic E. coli (UPEC), avian pathogenic E. coli (APEC), and septicemia-associated E. coli based on the original host and clinical symptoms [1][2][3][4]. ExPEC possess a range of similar virulence factors such as the aerobactin iron transport system, Ibe proteins (IbeA, IbeB, IbeC), the K1 capsule, and types 1 and P fimbriae [3,[5][6][7][8][9][10]. Mounting evidence shows that poultry can be a vehicle or a reservoir for E. coli capable of causing urinary tract infections and newborn meningitis [11]. Thus, studying the zoonotic potential of APEC is necessary.
The genetic island of meningitic E. coli that contains ibeA (GimA) has been identified and shown to contribute to NMEC invasion of brain microvascular endothelial cells [12][13][14]. GimA is present in NMEC and APEC and has 15 genes that form 4 operons. The last operon (ibeRAT) of GimA encodes IbeR, IbeA, and IbeT, which contributes to E. coli K1 invasion of host cells [12][13]15]. The roles of ibeA and ibeT in the invasion process of infection were reported [16][17][18][19]. Previous studies suggest that IbeR is an RpoS-like regulator of stationaryphase gene expression related to stress-resistance in NMEC strain RS218, which carries a lossof-function mutation rpoS gene [20]. However, the role of IbeR in the virulence of APEC with active RpoS has yet not been investigated.
In this study, IbeR from APEC DE205B was characterized. The ibeR and ibeR-ibeA mutant and complementary strains were constructed. The effects of IbeR on the virulence, invasion capacity, environment stress-resistance, specific pathogen-free (SPF) chicken serum resistance and gene expressions were evaluated to understand the precise function of IbeR in APEC pathogenicity.

Bacterial resistance to environmental stress and SPF chicken serum
Bacterial resistance to environmental stress was determined as described previously with some modifications [20]. Bacteria were suspended in PBS and diluted to 5 × 10 7 colony forming units (CFUs)/mL. For alkali resistance, the bacterial suspension was diluted 1:10 in 100 mM Tris, pH 10.0 and incubated at 37°C for 30 min. For acid resistance, one-tenth volume of the bacterial suspension was added to LB (pH 4.0) or LB (pH 5.0) and incubated at 37°C for 20 min. For high osmolarity stress, bacteria were mixed with an equal volume of 4.8 M NaCl and incubated at 37°C for 1 h. For oxidative stress, bacteria were treated with 10 mM H 2 O 2 at 37°C for 30 min. After stress exposure, bacteria were diluted in PBS and plated on LB agar. Survival was calculated as the ratio of bacterial number under stress to the bacteria number under nonstress. Survival was compared to DE205B.
Bactericidal assays were in a 96-well plates as described previously with some modifications [28][29]. Briefly, SPF chicken serum was diluted to 5%, 12.5%, 25%, 50%, and 100% in PBS. Bacteria were added at different dilutions and incubated at 37°C for 30 min. Bacteria were counted by plating on LB agar. Heat-inactivated SPF serum was used as control.

Construction of gene mutant and complementation strains
The isogenic mutants ΔibeR and ΔibeRibeA were constructed using the lambda red recombinase method [26]. A kanamycin resistance cassette was PCR amplified with primers WSH109F and WSH110R (Table 2) and transformed into strain DE205B containing plasmid pKD46. Mutants were screened and confirmed by PCR and sequenced using primers k1 and k2 [26] in combination with primers WSH107F and WSH33R. The kanamycin resistance cassette was cured by transforming with plasmid pCP20 and selecting for a kanamycin-sensitive mutant strain, which were designated as ΔibeR or ΔibeRibeA.
For complementation studies, the ibeR operon including its putative promoters was amplified using primers WSH130F and WSH131R and the fragment was subcloned into the pUC18 vector. The resulting plasmid pUC18-ibeR and control vector pUC18 were transformed into mutant strain ΔibeR to generate strains CΔibeR and PΔibeR, respectively. To detect the effect of IbeR on growth rate, the growth kinetics of each strain were determined.

Bacterial invasion assays
Bacterial invasion assays were performed as described previously [16]. Chicken embryo fibroblast DF-1 cell monolayers were washed with Dulbecco's modified Eagle's medium (DMEM) without fetal bovine serum and cells were infected with bacteria at a multiplicity of infection (MOI) of 100 for 2 h, 37°C under 5% CO 2 . Extracellular bacteria were eliminated by adding DMEM containing gentamicin (100 μg/mL). Monolayers were washed and lysed with 0.5% Triton X-100. Released bacteria were counted by plating on LB agar plates. Negative control wells containing DF-1 cells only were used in all experiments. Assays were performed three times.

Virulence test
To determine the virulence of wild-type, mutant, and complementation strains, 7-day-old ducks were inoculated intratracheally with bacterial suspensions at 10 7 CFUs. Bacterial CFUs in the injected inoculums were confirmed by plating on LB agar. Negative controls were injected with PBS. Mortality was monitored until 7 days post infection. The 50% lethal dose (LD 50 ) was determined using mouse models as described previously [16,[21][22][23]. Imprinting control region (ICR) mice, 8 weeks old, were inoculated intraperitoneally with 0.2 mL bacterial suspension at different CFUs. Bacterial CFUs in the injected inoculum were confirmed by plating on LB agar. Negative controls were injected with PBS. Mortality was monitored until 7 days post infection. LD 50 results were calculated using the method by Reed and Muench [30].

Counting of bacteria in organs during systemic infection in a rat neonatal meningitis model
Animal systemic infection experiments determined the colonization and invasion capabilities of each strain as described previously [16,[21][22][23]. Groups of six 8-week-old ICR mice were infected intraperitoneally with 2.0 × 10 6 CFUs of bacteria. At 24 h post infection, mice were euthanized and dissected. Organs were homogenized and diluted homogenate was plated onto LB agar to determine the number of bacteria colonizing organs.
The capacities to enter the central nervous system was determined for each strain in a neonatal rat model as described previously with some modifications [31][32]. SPF Sprague-Dawley rat pups, 5 days old, were infected intraperitoneally with a bacterial suspension containing 10 7 CFUs. At 18 h after bacterial inoculation, blood were obtained by tail veins. The rat were then killed, and cerebrospinal fluid (CSF) was immediately obtained by cisternal puncture.  Numbers of bacteria in samples were determined by plating 10-fold serial dilutions onto LB agar. Bacterial penetration across the blood-brain barrier was defined as a positive culture.

Ethics Statement
All animal experimental protocols were carried out in accordance with guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care International. The animal study protocol was approved by the Animal Care and Use Committee of Nanjing Agricultural University (SYXK(SU)2011-0036), China.

Quantitative real-time reverse transcription PCR
The RNA was isolated from bacteria using E.Z.N.A. Bacterial RNA kits (Omega Bio-Tek, Beijing, China) according to the manufacturer's instructions. Contaminating DNA was removed using RNase-free DNaseI (TaKaRa), and cDNA synthesis was performed using PrimeScript RT reagent kits (TaKaRa) according to the manufacturer's protocol. Quantitative real-time reverse transcription PCR (qRT-PCR) was performed to determine transcription of virulence genes using SYBR Premix Ex Taq (TaKaRa) and gene-specific primers ( Table 2). Relative gene expression was normalized to the expression of the housekeeping gene dnaE using the ΔΔCt method [33]. PCR efficiency (> 90%) for each gene was verified via standard dilution curves.

Statistical analysis
Statistical analysis for in vitro and in vivo experiments used GraphPad Software package (GraphPad Software, La Jolla, CA, USA). One-way ANOVA was used for analysis of invasion assay in vitro data. Two-way ANOVA was performed on qRT-PCR results. Animal infection study analysis was performed using the nonparametric Mann-Whitney U-Test. Statistical significance was established at p < 0.05.

Deletion of IbeR does not affect growth kinetics and motility of APEC
The ibeR gene from APEC DE205B was first sequenced and submitted to Genbank (Accession No: JQ767181.1). The ibeR gene of APEC DE205B was 1950 bp, which was 99% identical to those of APEC O1, NMEC strains RS218 and IHE3034. Based on the sequence, mutant strains ΔibeR, ΔibeRibeA were generated as described previously [26]. For genetic complementation, the recombinant plasmid pUC18-ibeR was transformed into the mutant strain ΔibeR yielding the complementation strain CΔibeR. No significant growth defect was observed among them during growth in LB medium (data not shown). ΔibeR migration on swarming agar plates was similar to the parental strain, indicating that motility was not affected by IbeR (data not shown).

IbeR is expressed and triggers antibody production in ducks
The expression of IbeR in wild-type, mutant, and complementation strains was compared by SDS-PAGE. No differences in protein patterns between the wild-type and mutant strains were detected (data not shown). Immunoblotting was performed with anti-IbeR serum, showing expected protein bands for IbeR from strains DE205B and CΔibeR. However, no IbeR protein was detected from mutant strain ΔibeR and PΔibeR (Fig. 1). These results indicated that IbeR was expressed under laboratory conditions and verified the construction of the ibeR mutant strain.
To determine whether IbeR was expressed and triggered antibody production during infection, immunized anti-DE205B serum was raised. Purified IbeR protein was transferred to membranes and anti-DE205B serum was used as a primary antibody. The results showed that incubation with anti-DE205B led to detected bands of purified IbeR protein, indicating that IbeR elicited an antibody response during infection.

IbeR is involved in bacterial resistance to environmental stress and serum
The role of IbeR in bacterial resistance to environmental stresses including alkali endurance (pH 10 for 30 min), acid endurance (acetic acid, pH 4.0 and pH 5.0 for 20 min) and high osmolarity challenge (2.4 M NaCl for 1 h) were determined. In all experiments, survival of wild-type strain DE205B was higher than the mutant strain ΔibeR (Fig. 2A), indicating that IbeR was required for stress-resistance. Previous study showed that GimA and IbeA paly a role in H 2 O 2 stress-   [34]. Thus, the resistance to H 2 O 2 stress of each strain was determined. The results showed that mutant strains ΔibeR, ΔibeA, ΔibeRibeA were more sensitive to H 2 O 2 killing than the wild type strain DE205B. Moreover, the resistance to H 2 O 2 was restored for the complementation strains (Fig. 2B). Thus, our results indicated that the deletion of ibeR was responsible for the lower resistance to H 2 O 2 and other environmental stresses of the mutant strain ΔibeR.
Resistance to serum provides APEC infection and virulence advantages. Bactericidal assays revealed that the mutant strain ΔibeR had lower resistance than the wild-type strain DE205B to SPF chicken serum (p < 0.05). Resistance was restored in the complementation strain (Fig. 2C). These results indicated that IbeR was involved in bacterial serum resistance.
Previous studies indicated that mice and ducks can be used as models to study APEC virulence [16,[21][22][23]. Thus, the LD 50 of each strain was evaluated in a mouse model. LD 50 values were 3.2 × 10 6 CFU/mouse for ΔibeR and 5.0 × 10 5 CFU/mouse for DE205B (Table 3). Moreover, the LD 50 of the complementation strain CΔibeR was partially restored (1.2 × 10 6 CFU/ mouse). These results suggested that IbeR was an important virulence factor in APEC strains.

IbeR involvement in APEC invasion of DF-1 cells
The role of IbeR in adhesion and invasion of APEC to avian cell lines was determined. The adhesion capacity of mutant strain ΔibeR was similar to the wild-type strain DE205B and the complementation strain CΔibeR, indicating that IbeR did not affect APEC adhesion of DF-1 cells (data not shown). A significant reduction of 35% was detected in invasion of the mutant strain ΔibeR compared with DE205B (p < 0.01) (Fig. 4). Invasion capacity was restored in complementation strain CΔibeR, with a significant difference compared to strains ΔibeR (p < 0.05). Similar to the results of the virulence test, the double-mutant strain ΔibeRibeA showed decreased ability to invade host cells compared to wild-type and single mutant strains (Fig. 4). Thus, we assumed that IbeR was involved in the invasion of APEC into DF-1 cells.

IbeR facilitates invasion of APEC during systemic infection and in a rat neonatal meningitis model
To determine the effect of IbeR in vivo, systemic infection experiments were performed. Bacteria were recovered from blood, brains, lungs, livers and spleens of infected mice at 24 h post ΔibeR was significantly lower than DE205B (* p < 0.05). The complementation strain CΔibeR recovered the most resistance. (B) Sensitivity to oxidants of DE205B and its ΔibeR and ΔibeA derivatives. Bacterial suspensions were treated with 10 mM H 2 O 2 at 37°C for 30 min. After stress exposure, bacteria were diluted in PBS and plated on LB agar. The data were expressed as survival relative to wild-type strain DE205B. Mutant strains ΔibeR, ΔibeA, ΔibeRibeA were more sensitive to H 2 O 2 killing than the wild type strain DE205B (** p < 0.01; *** p < 0.001). Moreover, the resistance to H 2 O 2 was restored for the complementation strains. (C) Resistance to SPF chicken serum. Bacteria were incubated at 37°C with SPF chicken serum at different dilutions, and counted at 30 min. Mutant strain ΔibeR showed significantly reduced resistance to SPF chicken serum compared to DE205B (* p < 0.05). The error bars indicate standard deviations. inoculation. Recovered ΔibeR compared to wild-type strain DE205B was reduced 1.6-fold in blood, 7.7-fold in brain, 3.4-fold in lung, 4.7-fold in liver, and 1.1-fold in spleen (Fig. 5A). Colonization and invasion capacities in brain and liver were significantly decreased (p < 0.05). Recovered complementation strains in organs were restored with differences between strains DE205B and CΔibeR that were not significant (p > 0.05). Furthermore, the complementation strain CΔibeR showed significantly increased invasion capacity in brain compared to the  mutant strain ΔibeR (p < 0.05). These results indicated that IbeR was involved in invasion of the brain of APEC strain DE205B during systemic infection. IbeR involvement in invasion of host cells was validated in the rat neonatal meningitis model. Using previous methods [31][32], bacteria were recovered from the blood and CSF of infected mice. When mice were infected with ΔibeR, a distinct reduction in numbers of bacterial recovered in CSF was observed compared to DE205B (p < 0.05) (Fig. 5B). The invasion capacity of the complementation strain in CSF was restored. Although the recovered bacteria in blood were reduced and restored for strains ΔibeR and CΔibeR, it was not significantly different from the wild-type strain DE205B. These results indicated that IbeR was involved in APEC systemic infection and facilitated invasion into the brain.

Expression profile of genes involved in resistance and virulence
To identify metabolic defaults that was responsible for the decreased resistance and attenuated virulence, the expression levels of range of genes involved in resistance and virulence were analyzed by qRT-PCR for various strains. The results showed that the expression of lpdA, tufB, gapA, aphC, ibeA, ibeB and fimC were significantly upregulated in the mutant strain ΔibeR. The mRNA levels were moderately decreased in the mutant strain ΔibeR by 0.23 for ompA, 0.26 for aatA, 0.56 for iucD and 0.26 for luxS genes (p < 0.01). However, the expression of genes involved in oxidative stress response katE, sodC, osmC were significantly reduced in the mutant strain ΔibeR (Fig. 6).
Previous study showed that IbeR acted as an RpoS-like regulator in NMEC strain RS218, which carries a loss-of-function mutation in rpoS gene [20]. Moreover, RpoS is a potential  regulator for the expression of some of the genes described above. Therefore, we analyzed whether the deletion of ibeR had any influence on the transcript of rpoS. Our result indicated that transcription of rpoS was not effeced by disruption of IbeR (Fig. 6).

Discussion
Systemic infections by APEC are economically devastating to the poultry industry worldwide. APEC shares virulence traits with other ExPEC strains (NMEC and UPEC), such as a GimA genomic island. The GimA island consists of 15 genes, some of which are predicted to encode proteins involved in carbon source metabolism and stress-resistance. IbeR, located in GimA, contributes to bacteria stress-resistance in the stationary-phase (SP) in RpoS-negative strain NMEC RS218 [20]. However, the role of IbeR in the strains with active RpoS function is still unknown. Thus, the ibeR mutant of APEC DE205B was constructed and characterized. Our results indicated that IbeR acted as a regulator controlling gene expression critical for stress-resistance, and also regulating the virulence genes (ompA, aatA, iucD, luxS) for full virulence in the APEC strain with active RpoS.
Expressed APEC IbeR elicited antibodies in infected ducks. Moreover, IbeR controlled gene expression critical for stress-resistance, cell survival and virulence. Thus, we propose that mutation of IbeR results in a decrease in APEC virulence. Animal experiments showed that ibeR mutant virulence was decreased compared to the parent strain in duck and mouse models. The complementation strain recovered bacterial virulence. Moreover, the mutant strain did not exhibit a growth defect. Thus, we concluded that IbeR was necessary for full APEC DE205B virulence.
Microbial pathogenicity is a complex phenomenon encompassing diverse mechanisms. However pathogenic organisms use several common strategies such as colonization and invasion to sustain themselves and overcome host barriers. We determined IbeR influence on the virulence and infection of APEC in vivo and in vitro. Adhesion assays indicated that IbeR was not involved in adhesion of APEC to DF-1 cells. Invasion capacity in vitro and in vivo of the mutant strain ΔibeR was significantly reduced compared to wild-type strain. Moreover, invasion capacity was restored in complementation strains. These data indicated that IbeR mediated APEC invasion and infection.
APEC infects poultry by initial respiratory tract colonization followed by systemic spread. Resistance to the bactericidal effects and the capacity of APEC strains to cause septicemia and mortality are correlated [35][36]. The capacity to resist serum and environmental stress is an advantage in APEC infection. During infection, the lung environment presents a high oxygen tension, which could lead to a higher rate of production of reactive oxygen species. The bactericidal assays demonstrated that resistance to environmental stress and SPF chicken serum were impaired in the mutant strain ΔibeR (Fig. 3). To determine the metabolic defaults responsible for these phenomenons, we measured the effects of the ibeR deletion on the expression of genes involved in the environmental resistance. It has been reported that RpoS regulates katE, sodC, osmC, and yfcG gene expression and OxyR regulates ahpC genes [34]. Previous study proposed that IbeR acted as a functional equivalent of RpoS in RS218 that presents a loss-offunction mutation in the rpoS gene [20]. Our results demonstrated that the rpoS was not affected by the ibeR mutation. Moreover, the motility, a phenotype linked to RpoS, was not changed in the ibeR mutant. Thus, IbeR was resposible for the modification of gene expression and reduced resistance in APEC DE205B carring a active RpoS.
We also measured the effects of the ibeR deletion on the expression of virulence genes. The results showed that virulence genes associated with adhesion and invasion (aatA and ompA) [22,37], iron acquisition (iucD) [11], and quorum sensing (luxS) [38], were significantly decreased in the mutant strain ΔibeR compared with DE205B (p < 0.05). This expression pattern might be responsible for the reduction of invasion capacities and attenuated virulence of mutant strain ΔibeR. However, the downstream gene of ibeR, invasion-associated gene ibeA, was significantly upregulated in mutant strain ΔibeR. In this study, lambda red recombinase method was used for the construction of mutant strains, which was used to create nonpolar gene deletion [26]. Moreover, the scar of FRT site did not contain promoter sequence. Then, a ibeR-ibeA mutant strain was constructed and characterized. Similar to invasion phenotypes of other double (ΔibeA/ΔibeB, ΔompA/ΔibeB) and triple knockouts [39], our experiments revealed that invasion capacity and virulence of the mutant strain ΔibeRibeA were reduced compared to wild-type and single-gene mutant strains. Thus, the reason for increased expression of ibeA might be to compensate during invasion for the deletion of IbeR.
In summary, our results demonstrated that IbeR acted as a regulator controlled gene expression critical for stress-resistance genes and virulence genes, which led to impaired resistance to environmental stress and reduced invasion capacity and defective virulence in the active RpoS APEC strain DE205B. The substrate interact with IbeR should to be identified and deserves further study in the future.
Supporting Information S1