Microbiological Research 171 (2015) 39–44
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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
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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
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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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Table 2 KEGG pathway analysis of differentially expressed genes. Ratios of UPEC132 group to R049 mutant group<
Signaling pathway
Gene
GenBank accession number
MAPK signaling pathway (P = 4.80E−24)
Gadd45b Dusp14 Dusp5 Dusp10 Dusp8 Mapk9 Map3k6 Nr4a1 Myc Fgf9 Rasgrp1 Fas
BC023815 AK166840 NM 001085390 BC025066 BC052705 AK043169 AK014061 BC004770 AK133952 AK158584 BC057120 AK086933
0.49 0.49 0.48 0.44 0.3 0.48 2.42 0.27
Vav1 Pik3r1 Cldn3 Cldn4 Cldn19 Cldn1 Cldn14 Ncf1 Bcar1
AK151214 BC026146 BC012650 BC132376 BC115827 BC002003 BC132183 BC055836 U28151
2.52 0.45 0.45 0.37 0.34 0.28
Cytokine–cytokine receptor interaction (P = 4.50E−18)
Il1b Ifnar2 Ccr1 Cxcl10 Cxcl5 Ccl12 Ccl11 Ccl7 Ccl2
BC011437 BC071225 BC011092 BC030067 BC024392 BC027520 BC027521 BC061126 BC145867
4.8 2.2 4.0 3.7 13.7 20.0 5.5 24.4 3.5
Toll-like receptor signaling pathway (P = 3.30E−13)
Il1b Cxcl10 Pik3cd Ifnar2
BC011437 BC030067 AK040867 BC071225
4.8 3.7 2.6 2.2
Apoptosis (P = 2.80E−11)
Cflar Il1b Irak3 Pik3cd
AK152515 BC011437 BC120829 AK040867
2.77 4.8 3.95 2.6
4h
Leukocyte transendothelial migration (P = 1.70E−19)
8h
2.4 0.4 2 2 2.3 0.2
0.44 0.11 2.24 0.48
The 34 differentially expressed genes in the mouse kidneys infected with UPEC132 and UPElC132R049 at 4 h and 8 h post-infection were analyzed using MAS. The 34 differentially expressed genes were involved in five signaling pathways which were associated with host inflammation and immune response. The Il1b, Pik3cd, and Ifnar2 genes participated in multiple signaling pathways.
Fig. 2. Relative expression of the Dusp8, Cldn1, and Ccl7 genes. Transcriptional levels of the Dusp8, Cldn1, and Ccl7 genes differentially expressed in the kidneys of the UPEC132-infected (white bars) and UPEC132R049-infected (black bars) mice were quantified by real-time PCR at 4 h (Dusp8 and Cldn1) and 8 h (Cldn1 and Ccl7) post-infection. The data were analyzed by the relative quantization method with glyceraldehyde-3-phosphate dehydrogenase (Gapdh) as the reference gene. The data are expressed as the mean ± statistical significance of triplicate experiments. * Indicates P < 0.05.
when NLRs, as the pattern recognition receptors recognize the ligand structure of bacteria (Girardin et al., 2003; Inohara et al., 2003). Then, RICK activates the NF-B and MAPK signaling pathways of p38, ERK, and JNK (Abbott et al., 2007; Park et al., 2007), which regulate cell proliferation, differentiation, transformation, and apoptosis. In this study, we analyzed 12 differentially expressed genes, including Erk, Jnk/Sap, and Erk5 in the MAPK signaling network; Erk showed the most substantial difference. Fgf9 and Rasgrp1 were upregulated, while the negative regulatory factors Dusp14, Dusp5, Dusp10, and Dusp8 were downregulated, enhancing downstream expression of Myc. Compared with the R049 mutant, the UPEC132 wild-type strain induced higher levels of MAPK expression. These results indicate significant changes in the pattern recognition receptors of the R049 mutant, which weakened the ability of UPEC to induce host inflammatory and innate immune responses. In the pathway of cytokine–cytokine receptor interactions (Table 2), Ccl2, Ccl7, Ccl11, Ccl12, Cxcl10, Cxcl5, and Ccr1 represent chemokines and their receptors, lfnar2 encodes interferon family receptor, and Il1b belongs to the interleukin-1 (IL-1) family; all of them were upregulated in the kidneys infected with the wild-type UPEC compared to those infected with the mutant strain. IL-1 (Il1b) is an important factor in the innate immune
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response and inflammation: it stimulates the release of TNF-␣, IL-6, IL-8, and other inflammatory mediators, activates T and B lymphocytes, increases the expression of adhesion molecules, and stimulates the production and release of the acute phase protein (Guan et al., 1997). IL-1 is activated by inflammasome NLRP3; recently, it has been shown that inflammasome-activated IL-1 promotes neutrophil chemotaxis and bacterial clearance (Fremond et al., 2007; Miller et al., 2007; Mishra et al. 2010), suggesting activation of host innate immune responses. The FTT0584 and FTT0748 genes, which are involved in Francisella tularensis pathogenicity, inhibit host innate immunity by restricting IL-1 production (Weiss et al., 2007). The transcription of Il1b was downregulated 4.8-fold in the R049 deletion mutant compared to the wild-type UPEC132 strain, indicating that R049 deletion affected the inflammasome recognition and weakened subsequent innate immune responses; however, further studies are needed to validate this effect. IL-1 increases permeability of the intestinal epithelium through the NF-B pathway and promotes proinflammatory reactions (Al-Sadi et al., 2010). By analyzing the leukocyte transendothelial migration pathway, we found that the tight-junction protein genes Cldn3, Cldn4, Cldn19, Cldn1, and Cldn14 were upregulated in the mutant-infected kidneys. Some cytokines such as TNF-␣ and IL-13 could downregulate the expression of the tight-junction protein, increasing the epithelial paracellular permeability (Prasad et al., 2005; Tedelind et al., 2003) and leading to the shift of bacterial antigens and inflammatory factors and activation of immune responses. We hypothesize that downregulation of IL-1 levels by the R049 mutant can induce the expression of the tight-junction proteins, thus decreasing the permeability of renal epithelial tissue. We will test this hypothesis in future studies. Acute inflammation and neutrophil accumulation mediated by TLRs constitute the main natural defense system against urinary tract infections (Cirl et al., 2008). The chemokine Cxcl10 and proinflammatory cytokine Il1b genes are also involved in the TLR signaling and apoptosis pathways (Table 2). All the differentially expressed genes of these pathways, Cxcl10, Il1b, Pik3cd, Ifnar2, Cfla, and Irak3, were upregulated in the mouse kidneys by the R049 mutant. Pik3cd is a member of the PI3K family, which transmits the transcriptional activation signal of the PI3K-Akt pathway to the transcription factor NF-B; in turn, NF-B translocates to the nucleus and induces the expression of proinflammatory cytokines, including IL-6 and IL-1 (Deane and Fruman, 2004; Koyasu, 2003; Lawrence, 2009). Interferon (IFN) receptor 1 and IFN alpha/beta receptor 2 precursor (IFNAR2) form the type I IFN receptor which, upon IFN binding, activates the JAK-STAT pathway (Haan et al., 2006; Severa et al., 2006; Yeh and Pellegrini, 1999), inducing the expression of proinflammatory cytokines and chemokines and promoting inflammatory responses (Rauch et al., 2013). The upregulation of Pik3cd, Ifnar2, Cxcl10, and Il1b, the members of the TLR pathway, indicates that UPEC132 induces and activates the TLRmediated signaling more potently than UPEC132R049. CFLAR and IRAK3 inhibit apoptosis via CASP8, IRAK1, and IRAK4 (Hoogerwerf et al., 2012; Hx and He, 2013; van der Windt et al., 2012). In this study, Cflar and Irak3 were upregulated 2.77 and 3.95-fold, respectively, in the wild-type UPEC132 versus the UPEC132R049 mutant, suggesting that the apoptosis pathway was inhibited in the wild-type strain-infected renal epithelial cells by UPEC132. It should be noted that, for unknown reasons, the results for mouse renal biopsy tissues did not differ between 4 and 8 h after infection. We used microarray analysis to determine differences in gene expression in the mouse kidneys infected with the UPEC132 and UPEC132R049 strains and to explore the effect of UPEC on the host immune response. We also assessed the kidney colonization by UPEC not only to ensure the suitability of kidney
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specimens used in the expression profile chip but also to compare infectivity between the two strains. It was found that kidney colonization by the R049 deletion mutant 8 h post-infection was higher than that by the wild-type strain. Our data indicate that the deletion of the outer membrane protein R049 had no influence on the UPEC pathogenicity. Our analysis suggests that R049 influences early host immune reactions during UPEC infection in BALB/c mice. This protein may enhance early inflammatory responses in mice against UPEC infection through activation of the MAPK signaling, leukocyte transendothelial migration, cytokine–cytokine receptor interaction, TLR signaling, and apoptosis. These results lay the foundation for further research on the role of R049 in the early host immune and inflammatory responses. Conflict of interest None. Acknowledgement This work was supported by the National Nature Science Foundation of China (Grant No. 81000707). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.micres. 2015.01.002. References Abbott DW, Yang Y, Hutti JE, Madhavarapu S, Kelliher MA, Cantley LC. Coordinated regulation of Toll-like receptor and NOD2 signaling by K63-linked polyubiquitin chains. Mol Cell Biol 2007;27:6012–25. Al-Sadi R, Ye D, Said HM, Ma TY. IL-1beta-induced increase in intestinal epithelial tight junction permeability is mediated by MEKK-1 activation of canonical NFkappaB pathway. Am J Pathol 2010;177:2310–22. Arthur M, Johnson CE, Rubin RH, Arbeit RD, Campanelli C, Kim C, et al. Molecular epidemiology of adhesin and hemolysin virulence factors among uropathogenic Escherichia coli. Infect Immun 1989;57:303–13. Chen JY, Kang J, Li FT. Cloning and sequencing of papA gene from uropathogenic Escherichia coli. Chin J Microbiol Immunol 2000;20:189–92. Cirl C, Wieser A, Yadav M, Duerr S, Schubert S, Fischer H, et al. Subversion of Tolllike receptor signaling by a unique family of bacterial Toll/interleukin-1 receptor domain-containing proteins. Nat Med 2008;14:399–406. Deane JA, Fruman DA. Phosphoinositide 3-kinase: diverse roles in immune cell activation. Annu Rev Immunol 2004;22:563–98. Fremond CM, Togbe D, Doz E, Rose S, Vasseur V, Maillet I. IL-1 receptor-mediated signal is an essential component of MyD88-dependent innate response to Mycobacterium tuberculosis infection. J Immunol 2007;179:1178–89. Ge X, Zhang YM, Chen JY, Lin X, Zhao FL. The immunoprotective effects of new gene R049 of uropathogenic Escherichia coli. Chin J Microbiol Immunol 2008a;28:712–6. Ge X, Chen JY, Zhang YM, Gao YT, Hou M, He JD. Clustering distribution of the specific fragment R049 of uropathogenic Escherichia coli. Chin J Microbiol Immunol 2008b;28:777–80. Ge X, Dong J, Chen JY, Yao P, Gu C, Yang DJ. Comparison of infection of different cell lines by uropathogenic Escherichia coli. Chin Sci Bull 2009;54:2662–8. Girardin SE, Boneca IG, Carneiro LA, Antignac A, Jéhanno M, Viala J. Nod1 detects a unique muropeptide from gram-negative bacterial peptidoglycan. Science 2003;300:1584–7. Guan Z, Baier LD, Morrison AR. p38 mitogen-activated protein kinase downregulates nitric oxide and up regulates prostaglandin E2 biosynthesis stimulated by interleukin-1 beta. J Biol Chem 1997;272:8083–9. Gunther NW, Snyder JA, Lockatell V, Blomfield I, Johnson DE, Mobley HL. Assessment of virulence of uropathogenic Escherichia coli type 1 fimbrial mutants in which the invertible element is phase-locked on or off. Infect Immun 2002;70:3344–54. Haan C, Kreis S, Margue C, Behrmann I. Jaks and cytokine receptors: an intimate relationship. Biochem Pharmacol 2006;72:1538–46. Hoogerwerf JJ, van der Windt GJ, Blok DC, Hoogendijk AJ, De Vos AF, van ‘t Veer CF, et al. Interleukin-1 receptor-associated kinase M-deficient mice demonstrate an improved host defense during gram-negative pneumonia. Mol Med 2012;18:1067–75.
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