Microbiological Research 171 (2015) 39–44
Contents lists available at ScienceDirect
Microbiological Research journal homepage: www.elsevier.com/locate/micres
Functional analysis of the uropathogenic Escherichia coli R049 gene Dongjing Yang ∗ , Jie Dong, Xu Su, Wei Zhang, Li Zhang, Li Li, Likun Lv, Liru Guo Tianjin Centers for Disease Control and Prevention, Tianjin 300011, China
a r t i c l e
i n f o
Article history: Received 19 September 2014 Received in revised form 2 January 2015 Accepted 3 January 2015 Available online 7 January 2015 Keywords: Uropathogenic Escherichia coli Gene expression profile KEGG pathway analysis GO analysis
a b s t r a c t The objective of this study was to determine the function of the novel uropathogenic Escherichia coli (UPEC) gene R049 during host infection. We infected the urinary tracts of mice with E. coli UPEC132 or the R049 deletion mutant UPEC132R049.The mouse kidneys were harvested at 4 and 8 h post-infection and screened for differentially expressed genes by microarray analysis. We identified 379 and 515 differentially expressed genes at 4 and 8 h post-infection, respectively. Thirtyfour of these genes were associated with inflammatory and immune signaling pathways, including those related to mitogen-activated protein kinase signaling, leukocyte transendothelial migration, cytokine–cytokine receptor interaction, Toll-like receptor signaling, and apoptosis. Protein binding (GO 0005515) was the most prevalent molecular function in the Gene Ontology terms related to differentially expressed genes. In conclusion, R049 expression in UPEC132 is related to the early innate immune and inflammatory responses in UPEC-infected hosts. This work lays the foundation for further research on anti-infective immunity against UPEC. © 2015 Elsevier GmbH. All rights reserved.
Introduction Urinary tract infections (UTIs), including pyelonephritis (infection of the kidney) and cystitis (infection of the bladder), are very common and present a serious health problem. Worldwide, an estimated 150 million patients with UTIs visit hospitals, and the treatment costs are staggering. Uropathogenic Escherichia coli (UPEC) is the major causal pathogen in UTIs (Stamm and Hooton, 1993). UPEC virulence factors, including iron uptake systems, adhesins, and cytotoxins such as enterobactin, P fimbriae, type 1 fimbriae, Dr fimbriae, hemolysin, and cytotoxic necrotizing factor 1, contribute to UPEC invasion and colonization of the host. In particular, type 1, P, and Dr fimbriae are the most important UPEC virulence factors, mediating both bacterial adherence to and invasion of host cells. Type 1 fimbriae are highly conserved among UPEC strains (Gunther et al., 2002), and the expression of P fimbriae is often associated with pyelonephritic UPEC isolates (Lane and Mobley, 2007), while Dr fimbriae play a key role in the process of biofilm formation when UPEC invade the urinary system (Zalewska et al., 2009). In addition, molecular epidemiology data indicated that Dr adhesins are closely associated with cystitis (Arthur et al., 1989).
∗ Corresponding author at: Tianjin Centers for Disease Control and Prevention, 6 Huayue Road, HeDong District, Tianjin 300011, China. Tel.: +86 22 24333437; fax: +86 22 24333490. E-mail address: [email protected] (D. Yang). http://dx.doi.org/10.1016/j.micres.2015.01.002 0944-5013/© 2015 Elsevier GmbH. All rights reserved.
The UPEC132 strain has been isolated from the urine specimen of a patient with acute pyelonephritis in Tianjin. In UPEC132, host adhesion and invasion are mostly mediated by P fimbriae, whereas type 1 fimbriae have not been found in this strain (Ge et al., 2009). UPEC132 P fimbriae contain major structural protein papA of F13 type (Chen et al., 2000) and a specific adhesion protein papG type II localized at the distal tip of P fimbriae (Zheng et al., 2002). On the surface of target cells, particularly in the kidney, papG recognizes globosylceramide antigens of the human blood group P. The R049 gene was identified in UPEC132 by suppression subtractive hybridization (Zhang et al., 2007), and its 1311-nucleotide sequence has been deposited in GenBank (No. EF488001.2). We have found that R049 is present in the genomes of 40% of domestic UPEC strains (Ge et al., 2008b). In UPEC132, R049 ORF encodes an outer membrane protein of 47 kDa that exerts immunoprotective effects in animals challenged with a homologous UPEC strain (Ge et al., 2008a; Zhang et al., 2011). To investigate functional characteristic of the novel R049 gene, we have knocked out the R049 sequence from UPEC132 genome via Red recombination and have constructed the deletion mutant UPEC132R049 (Yang et al., 2012). In this study, we infected the urinary tracts of mice with E. coli UPEC132 wild-type strain or the R049 deletion mutant UPEC132R049. The mouse kidneys were harvested at 4 and 8 h post-infection, and the genes differentially expressed in the renal tissue were screened using microarray analysis. Based on the KEGG (Kyoto Encyclopedia of Genes and Genomes) signaling
40
D. Yang et al. / Microbiological Research 171 (2015) 39–44
pathways and GO (Gene Ontology) molecular functions, we identified the genes differentially expressed in the inflammatory and immunoregulatory pathways of the kidneys infected with the R049 deletion mutant, as compared to those in the wild-type strain. The differentially expressed genes were further validated by quantitative real-time PCR. Materials and methods Materials UPEC132 is a clinical UPEC isolate maintained in our laboratory. UPEC132R049 has been constructed in our laboratory. Experimental animals included 60 specific pathogen-free (SPF) female BALB/c mice (7–8 weeks old, weighing 16–18 g) purchased from Vital River Laboratories (Beijing, China). Peptone and yeast extract were purchased from Oxford (Basingstoke, UK), chloral hydrate was obtained from Sigma (St. Louis, MO, USA), the RNeasy Plus Kit was from QIAGEN (Dusseldorf, Germany), and the SYBR premix Ex Taq kit was purchased from Takara (Dalian, China). UPEC infection via the urinary tract UPEC132 and UPEC132R049 were inoculated in Luria-Bertani (LB) liquid medium and incubated with shaking for 18 h at 37 ◦ C; 1% of the bacterial suspension was re-inoculated into fresh LB medium and cultured for 3 h to an estimated 1 × 109 CFU/mL. Eightweek-old BALB/c mice were randomly divided into the UPEC132, UPEC132R049, and phosphate-buffered saline (PBS) groups (20 mice/group); chloral hydrate was intraperitoneally injected at 350 mg/kg. After the animals were placed under general anesthesia, 50 L of the prepared bacterial suspension (1 × 109 CFU/mL) was transurethrally inserted for about 1.5 cm, slowly injected, and held for 30 s before removal (Hung et al., 2009). PBS controls were injected with 0.01 M PBS, pH 7.2. Colony counting of mouse kidney tissue At 4 and 8 h post infection, 10 mice per group were sacrificed and dissected; the kidneys were harvested and quickly placed in liquid nitrogen. After weighing, one kidney was placed in 2 mL sterile PBS and homogenized, and aliquots of 1:10 serial dilutions were spread on MacConkey agar to determine the infectious load (CFU/g of kidney). All data are presented as log10 (CFU/g kidney). The study was approved by the Experimental Animal Ethics Committee of Tianjin Centers for Disease Control and Prevention, and all experiments were performed according to the National Institute of Health Guidelines for the Care and Use of Laboratory Animals. Microarray analysis The Roche NimbleGen 12*135K mouse expression profile chip (catalog number 05543797001) was used in this study; hybridization and analysis were performed under contract at CapitalBio Corporation (Beijing, China). Two mice were randomly selected in each group after 4 h and 8 h post-infection; one kidney was removed, and 100 mg of renal tissue was grinded and used for total RNA extraction with the RNeasy Plus kit. RNA was prepared as the test sample for the gene expression profile chip, which was tested by Beijing CapitalBio. A difference in gene expression level of more than 2-fold was considered as upregulation and less than 0.5-fold as downregulation, according to the thresholds recommended by CapitalBio for the screening of differentially expressed genes (Mills and Gordon, 2001).
Bioinformatics analysis of the gene expression profile The genes found to be differentially expressed using the gene expression profile chip were analyzed with the Molecular Annotation System 3.0 (www.capitalbio.com), the KEGG pathways website (http://www.genome.jp/kegg/), and the GO website (http://www.geneontology.org). Verification by quantitative real-time PCR Three differentially expressed genes Dusp8, Ccl7, and Ccl1 were analyzed; the primers are listed in Table S1. The kidney RNA was reverse-transcribed to cDNA using the RT reagent kit; cDNA was then used as a template in real-time quantitative PCR with the SYBR premix ExTaq kit according to the manufacturer’s instructions. The data were analyzed by the relative quantitation method with glyceraldehyde-3-phosphate dehydrogenase (Gapdh) as the reference gene. Each experiment was performed in triplicate and calculations were made using the ABI7500 instrument. Statistical analysis Statistical Program for Social Sciences (SPSS; SPSS Inc., Chicago, IL, USA) 11.5 software was used to perform analysis of variance and t-tests. The data have been expressed as the mean ± standard deviation; P < 0.05 was considered statistically significant. Results Bacterial counts in the mouse kidneys Table 1 shows the kidney colonization by the UPEC132 and UPEC132R049 strains at 4 and 8 h post-infection expressed in terms of log10 (CFU/g kidney). The difference between the two groups at 4 h post-infection was not statistically significant (P > 0.05). The values for UPEC 132 were lower than those for UPEC132R049 at 8 h post-infection (P < 0.05), and no colonies were obtained for the kidneys of the PBS group. The results demonstrate that kidney colonization by the UPEC132 wild-type strain was less than that by the UPEC132R049 mutant strain at 8 h post-infection. Gene expression profiles in the mouse kidney after the infection The data on the gene expression profiling were submitted to Gene Expression Omnibus (GEO; accession number GSE63502). The comparison of gene expression profiles in the mouse kidneys at 4 h and 8 h post-infection with UPEC132 or UPElC132R049 or injection with PBS is shown in Fig. 1. We found that UPEC infection markedly affected gene expression in the mouse kidneys by downregulating a significant number of genes, especially at 8 h post-infection. Compared to the control (PBS-injected) mice, the number of differentially expressed genes in the mouse kidneys infected with UPElC132R049 was more than that in the kidneys infected with UPEC132 wild-type strain. KEGG pathway analysis The Molecular Annotation System 3.0 (MAS) was used for the KEGG pathway analysis of the kidney expression profiles at 4 and 8 h post-infection. The results showed that 34 differentially expressed genes were associated with host inflammation and immune signaling pathways, involving a total of five signaling cascades; among these genes, Il1b, Pik3cd, and Ifnar2 participated in multiple signaling pathways (Table 2). There were 14 differently expressed genes involved in the mitogen-activated protein kinase
D. Yang et al. / Microbiological Research 171 (2015) 39–44
41
Table 1 UPEC colonization levels in the mouse kidney. Time (h)
4 8
UPEC132
UPEC132R049
n
Mean ± SD
n
Mean ± SD
10 9
5.42 ± 0.96 5.57 ± 0.81
10 8
5.71 ± 0.85 6.43 ± 0.79
t
P
0.72 2.21
0.48 0.04
Mice were transurethrally infected with the UPEC132 and UPEC132R049 respectively. The kidneys were harvested and the infectious load was determined at 4 h and 8 h post infection. All data are presented as mean log10 CFU/g kidney ± standard deviation.
(MAPK) signaling pathways, leukocyte transendothelial migration, and cytokine–cytokine receptor interaction at 4 h post-infection. Compared to the expression in the UPEC132R049 group, 2 genes were upregulated and 12 were downregulated in the UPEC132 group. Eight hours after the infection, 23 genes were differently expressed, including those involved in the MAPK signaling pathway, leukocyte transendothelial migration, cytokine–cytokine receptor interaction, Toll-like receptor (TLR) signaling pathway, and apoptosis; 18 of these genes were upregulated and 5 were downregulated. MAS 3.0 showed statistical significance for each KEGG signaling pathway (P < 0.005).
Verification by quantitative real-time PCR Dusp8, Cldn1, and Ccl7 transcription was quantified by the relative standard curve method. At 4 h post-infection, Dusp8 and Cldn1 levels were higher and at 8 h post-infection, the Cldn1 level was still higher and the Ccl7 level was lower in the kidneys of the UPEC132R049-infected mice than in the UPEC132-infected animals (Fig. 2). The real-time PCR data were consistent with the microarray results, thus confirming the accuracy of the microarray assessment (Table S2). Discussion
GO functional analysis Thirty-four differentially expressed genes presented in Table 2 were analyzed by GO; 20 GO terms were involved in the enrichment analysis (Fig. S1). The major five terms were cellular process, physiological process, biological regulation, regulation of biological process, and developmental process; their relative involvement (represented as the percentage in the pie graph) was 15.10%, 12.02%, 11.22%, 10.66% and 5.96%, respectively. In the molecular function analysis (Fig. S2), the protein network of the differentially expressed genes demonstrated that protein binding (GO 0005515) was the most significant of the GO terms: all other GO terms and proteins were, directly or indirectly, linked to GO 0005515. These data indicate that protein binding was the key molecular function of the genes differentially expressed in the infected mouse kidneys.
In this study, we used microarray analysis to characterize gene expression in the mouse kidney after the infection with the UPEC132 wild-type and UPEC132R049 mutant strains. Our goal was to explore the functional role of the R049 gene in the UPEC pathogenesis and the early events of host immune response. The NimbleGen microarray detected 44,170 genes, each of which was represented by three probes and could be statistically analyzed by the average signal calculated for each gene; the 60-bp oligonucleotide probes provided a better signal-to-noise ratio, sensitivity, and specificity, thus ensuring the reliability and accuracy of the analysis. The MAPK signaling is an important pathway in the eukaryotic signal transduction network, regulating gene expression and functional activities. The NOD-like receptors (NLRs), similar to NOD1 and NOD2, recruit and activate RICK (RIP2) via oligomerization,
Fig. 1. Gene expression profiles of mice with urinary tract infection. Gene expression was compared in the mouse kidneys at 4 h and 8 h post-infection with UPEC132 or UPElC132R049 strains or injection with PBS by microarray analysis using the Roche NimbleGen 12*135K mouse expression profile chip. (A) UPEC132 vs. UPElC132R049 group; (B) UPEC132 vs. PBS group; (C) UPElC132R049 vs. PBS group.
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D. Yang et al. / Microbiological Research 171 (2015) 39–44
Table 2 KEGG pathway analysis of differentially expressed genes. Ratios of UPEC132 group to R049 mutant group
Contents lists available at ScienceDirect
Microbiological Research journal homepage: www.elsevier.com/locate/micres
Functional analysis of the uropathogenic Escherichia coli R049 gene Dongjing Yang ∗ , Jie Dong, Xu Su, Wei Zhang, Li Zhang, Li Li, Likun Lv, Liru Guo Tianjin Centers for Disease Control and Prevention, Tianjin 300011, China
a r t i c l e
i n f o
Article history: Received 19 September 2014 Received in revised form 2 January 2015 Accepted 3 January 2015 Available online 7 January 2015 Keywords: Uropathogenic Escherichia coli Gene expression profile KEGG pathway analysis GO analysis
a b s t r a c t The objective of this study was to determine the function of the novel uropathogenic Escherichia coli (UPEC) gene R049 during host infection. We infected the urinary tracts of mice with E. coli UPEC132 or the R049 deletion mutant UPEC132R049.The mouse kidneys were harvested at 4 and 8 h post-infection and screened for differentially expressed genes by microarray analysis. We identified 379 and 515 differentially expressed genes at 4 and 8 h post-infection, respectively. Thirtyfour of these genes were associated with inflammatory and immune signaling pathways, including those related to mitogen-activated protein kinase signaling, leukocyte transendothelial migration, cytokine–cytokine receptor interaction, Toll-like receptor signaling, and apoptosis. Protein binding (GO 0005515) was the most prevalent molecular function in the Gene Ontology terms related to differentially expressed genes. In conclusion, R049 expression in UPEC132 is related to the early innate immune and inflammatory responses in UPEC-infected hosts. This work lays the foundation for further research on anti-infective immunity against UPEC. © 2015 Elsevier GmbH. All rights reserved.
Introduction Urinary tract infections (UTIs), including pyelonephritis (infection of the kidney) and cystitis (infection of the bladder), are very common and present a serious health problem. Worldwide, an estimated 150 million patients with UTIs visit hospitals, and the treatment costs are staggering. Uropathogenic Escherichia coli (UPEC) is the major causal pathogen in UTIs (Stamm and Hooton, 1993). UPEC virulence factors, including iron uptake systems, adhesins, and cytotoxins such as enterobactin, P fimbriae, type 1 fimbriae, Dr fimbriae, hemolysin, and cytotoxic necrotizing factor 1, contribute to UPEC invasion and colonization of the host. In particular, type 1, P, and Dr fimbriae are the most important UPEC virulence factors, mediating both bacterial adherence to and invasion of host cells. Type 1 fimbriae are highly conserved among UPEC strains (Gunther et al., 2002), and the expression of P fimbriae is often associated with pyelonephritic UPEC isolates (Lane and Mobley, 2007), while Dr fimbriae play a key role in the process of biofilm formation when UPEC invade the urinary system (Zalewska et al., 2009). In addition, molecular epidemiology data indicated that Dr adhesins are closely associated with cystitis (Arthur et al., 1989).
∗ Corresponding author at: Tianjin Centers for Disease Control and Prevention, 6 Huayue Road, HeDong District, Tianjin 300011, China. Tel.: +86 22 24333437; fax: +86 22 24333490. E-mail address: [email protected] (D. Yang). http://dx.doi.org/10.1016/j.micres.2015.01.002 0944-5013/© 2015 Elsevier GmbH. All rights reserved.
The UPEC132 strain has been isolated from the urine specimen of a patient with acute pyelonephritis in Tianjin. In UPEC132, host adhesion and invasion are mostly mediated by P fimbriae, whereas type 1 fimbriae have not been found in this strain (Ge et al., 2009). UPEC132 P fimbriae contain major structural protein papA of F13 type (Chen et al., 2000) and a specific adhesion protein papG type II localized at the distal tip of P fimbriae (Zheng et al., 2002). On the surface of target cells, particularly in the kidney, papG recognizes globosylceramide antigens of the human blood group P. The R049 gene was identified in UPEC132 by suppression subtractive hybridization (Zhang et al., 2007), and its 1311-nucleotide sequence has been deposited in GenBank (No. EF488001.2). We have found that R049 is present in the genomes of 40% of domestic UPEC strains (Ge et al., 2008b). In UPEC132, R049 ORF encodes an outer membrane protein of 47 kDa that exerts immunoprotective effects in animals challenged with a homologous UPEC strain (Ge et al., 2008a; Zhang et al., 2011). To investigate functional characteristic of the novel R049 gene, we have knocked out the R049 sequence from UPEC132 genome via Red recombination and have constructed the deletion mutant UPEC132R049 (Yang et al., 2012). In this study, we infected the urinary tracts of mice with E. coli UPEC132 wild-type strain or the R049 deletion mutant UPEC132R049. The mouse kidneys were harvested at 4 and 8 h post-infection, and the genes differentially expressed in the renal tissue were screened using microarray analysis. Based on the KEGG (Kyoto Encyclopedia of Genes and Genomes) signaling
40
D. Yang et al. / Microbiological Research 171 (2015) 39–44
pathways and GO (Gene Ontology) molecular functions, we identified the genes differentially expressed in the inflammatory and immunoregulatory pathways of the kidneys infected with the R049 deletion mutant, as compared to those in the wild-type strain. The differentially expressed genes were further validated by quantitative real-time PCR. Materials and methods Materials UPEC132 is a clinical UPEC isolate maintained in our laboratory. UPEC132R049 has been constructed in our laboratory. Experimental animals included 60 specific pathogen-free (SPF) female BALB/c mice (7–8 weeks old, weighing 16–18 g) purchased from Vital River Laboratories (Beijing, China). Peptone and yeast extract were purchased from Oxford (Basingstoke, UK), chloral hydrate was obtained from Sigma (St. Louis, MO, USA), the RNeasy Plus Kit was from QIAGEN (Dusseldorf, Germany), and the SYBR premix Ex Taq kit was purchased from Takara (Dalian, China). UPEC infection via the urinary tract UPEC132 and UPEC132R049 were inoculated in Luria-Bertani (LB) liquid medium and incubated with shaking for 18 h at 37 ◦ C; 1% of the bacterial suspension was re-inoculated into fresh LB medium and cultured for 3 h to an estimated 1 × 109 CFU/mL. Eightweek-old BALB/c mice were randomly divided into the UPEC132, UPEC132R049, and phosphate-buffered saline (PBS) groups (20 mice/group); chloral hydrate was intraperitoneally injected at 350 mg/kg. After the animals were placed under general anesthesia, 50 L of the prepared bacterial suspension (1 × 109 CFU/mL) was transurethrally inserted for about 1.5 cm, slowly injected, and held for 30 s before removal (Hung et al., 2009). PBS controls were injected with 0.01 M PBS, pH 7.2. Colony counting of mouse kidney tissue At 4 and 8 h post infection, 10 mice per group were sacrificed and dissected; the kidneys were harvested and quickly placed in liquid nitrogen. After weighing, one kidney was placed in 2 mL sterile PBS and homogenized, and aliquots of 1:10 serial dilutions were spread on MacConkey agar to determine the infectious load (CFU/g of kidney). All data are presented as log10 (CFU/g kidney). The study was approved by the Experimental Animal Ethics Committee of Tianjin Centers for Disease Control and Prevention, and all experiments were performed according to the National Institute of Health Guidelines for the Care and Use of Laboratory Animals. Microarray analysis The Roche NimbleGen 12*135K mouse expression profile chip (catalog number 05543797001) was used in this study; hybridization and analysis were performed under contract at CapitalBio Corporation (Beijing, China). Two mice were randomly selected in each group after 4 h and 8 h post-infection; one kidney was removed, and 100 mg of renal tissue was grinded and used for total RNA extraction with the RNeasy Plus kit. RNA was prepared as the test sample for the gene expression profile chip, which was tested by Beijing CapitalBio. A difference in gene expression level of more than 2-fold was considered as upregulation and less than 0.5-fold as downregulation, according to the thresholds recommended by CapitalBio for the screening of differentially expressed genes (Mills and Gordon, 2001).
Bioinformatics analysis of the gene expression profile The genes found to be differentially expressed using the gene expression profile chip were analyzed with the Molecular Annotation System 3.0 (www.capitalbio.com), the KEGG pathways website (http://www.genome.jp/kegg/), and the GO website (http://www.geneontology.org). Verification by quantitative real-time PCR Three differentially expressed genes Dusp8, Ccl7, and Ccl1 were analyzed; the primers are listed in Table S1. The kidney RNA was reverse-transcribed to cDNA using the RT reagent kit; cDNA was then used as a template in real-time quantitative PCR with the SYBR premix ExTaq kit according to the manufacturer’s instructions. The data were analyzed by the relative quantitation method with glyceraldehyde-3-phosphate dehydrogenase (Gapdh) as the reference gene. Each experiment was performed in triplicate and calculations were made using the ABI7500 instrument. Statistical analysis Statistical Program for Social Sciences (SPSS; SPSS Inc., Chicago, IL, USA) 11.5 software was used to perform analysis of variance and t-tests. The data have been expressed as the mean ± standard deviation; P < 0.05 was considered statistically significant. Results Bacterial counts in the mouse kidneys Table 1 shows the kidney colonization by the UPEC132 and UPEC132R049 strains at 4 and 8 h post-infection expressed in terms of log10 (CFU/g kidney). The difference between the two groups at 4 h post-infection was not statistically significant (P > 0.05). The values for UPEC 132 were lower than those for UPEC132R049 at 8 h post-infection (P < 0.05), and no colonies were obtained for the kidneys of the PBS group. The results demonstrate that kidney colonization by the UPEC132 wild-type strain was less than that by the UPEC132R049 mutant strain at 8 h post-infection. Gene expression profiles in the mouse kidney after the infection The data on the gene expression profiling were submitted to Gene Expression Omnibus (GEO; accession number GSE63502). The comparison of gene expression profiles in the mouse kidneys at 4 h and 8 h post-infection with UPEC132 or UPElC132R049 or injection with PBS is shown in Fig. 1. We found that UPEC infection markedly affected gene expression in the mouse kidneys by downregulating a significant number of genes, especially at 8 h post-infection. Compared to the control (PBS-injected) mice, the number of differentially expressed genes in the mouse kidneys infected with UPElC132R049 was more than that in the kidneys infected with UPEC132 wild-type strain. KEGG pathway analysis The Molecular Annotation System 3.0 (MAS) was used for the KEGG pathway analysis of the kidney expression profiles at 4 and 8 h post-infection. The results showed that 34 differentially expressed genes were associated with host inflammation and immune signaling pathways, involving a total of five signaling cascades; among these genes, Il1b, Pik3cd, and Ifnar2 participated in multiple signaling pathways (Table 2). There were 14 differently expressed genes involved in the mitogen-activated protein kinase
D. Yang et al. / Microbiological Research 171 (2015) 39–44
41
Table 1 UPEC colonization levels in the mouse kidney. Time (h)
4 8
UPEC132
UPEC132R049
n
Mean ± SD
n
Mean ± SD
10 9
5.42 ± 0.96 5.57 ± 0.81
10 8
5.71 ± 0.85 6.43 ± 0.79
t
P
0.72 2.21
0.48 0.04
Mice were transurethrally infected with the UPEC132 and UPEC132R049 respectively. The kidneys were harvested and the infectious load was determined at 4 h and 8 h post infection. All data are presented as mean log10 CFU/g kidney ± standard deviation.
(MAPK) signaling pathways, leukocyte transendothelial migration, and cytokine–cytokine receptor interaction at 4 h post-infection. Compared to the expression in the UPEC132R049 group, 2 genes were upregulated and 12 were downregulated in the UPEC132 group. Eight hours after the infection, 23 genes were differently expressed, including those involved in the MAPK signaling pathway, leukocyte transendothelial migration, cytokine–cytokine receptor interaction, Toll-like receptor (TLR) signaling pathway, and apoptosis; 18 of these genes were upregulated and 5 were downregulated. MAS 3.0 showed statistical significance for each KEGG signaling pathway (P < 0.005).
Verification by quantitative real-time PCR Dusp8, Cldn1, and Ccl7 transcription was quantified by the relative standard curve method. At 4 h post-infection, Dusp8 and Cldn1 levels were higher and at 8 h post-infection, the Cldn1 level was still higher and the Ccl7 level was lower in the kidneys of the UPEC132R049-infected mice than in the UPEC132-infected animals (Fig. 2). The real-time PCR data were consistent with the microarray results, thus confirming the accuracy of the microarray assessment (Table S2). Discussion
GO functional analysis Thirty-four differentially expressed genes presented in Table 2 were analyzed by GO; 20 GO terms were involved in the enrichment analysis (Fig. S1). The major five terms were cellular process, physiological process, biological regulation, regulation of biological process, and developmental process; their relative involvement (represented as the percentage in the pie graph) was 15.10%, 12.02%, 11.22%, 10.66% and 5.96%, respectively. In the molecular function analysis (Fig. S2), the protein network of the differentially expressed genes demonstrated that protein binding (GO 0005515) was the most significant of the GO terms: all other GO terms and proteins were, directly or indirectly, linked to GO 0005515. These data indicate that protein binding was the key molecular function of the genes differentially expressed in the infected mouse kidneys.
In this study, we used microarray analysis to characterize gene expression in the mouse kidney after the infection with the UPEC132 wild-type and UPEC132R049 mutant strains. Our goal was to explore the functional role of the R049 gene in the UPEC pathogenesis and the early events of host immune response. The NimbleGen microarray detected 44,170 genes, each of which was represented by three probes and could be statistically analyzed by the average signal calculated for each gene; the 60-bp oligonucleotide probes provided a better signal-to-noise ratio, sensitivity, and specificity, thus ensuring the reliability and accuracy of the analysis. The MAPK signaling is an important pathway in the eukaryotic signal transduction network, regulating gene expression and functional activities. The NOD-like receptors (NLRs), similar to NOD1 and NOD2, recruit and activate RICK (RIP2) via oligomerization,
Fig. 1. Gene expression profiles of mice with urinary tract infection. Gene expression was compared in the mouse kidneys at 4 h and 8 h post-infection with UPEC132 or UPElC132R049 strains or injection with PBS by microarray analysis using the Roche NimbleGen 12*135K mouse expression profile chip. (A) UPEC132 vs. UPElC132R049 group; (B) UPEC132 vs. PBS group; (C) UPElC132R049 vs. PBS group.
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D. Yang et al. / Microbiological Research 171 (2015) 39–44
Table 2 KEGG pathway analysis of differentially expressed genes. Ratios of UPEC132 group to R049 mutant group
