RESUMO
Urinary tract infection (UTI) commonly afflicts people with diabetes. This augmented infection risk is partly due to deregulated insulin receptor (IR) signaling in the kidney collecting duct. The collecting duct is composed of intercalated cells (ICs) and principal cells (PCs). Evidence suggests that ICs contribute to UTI defenses. Here, we interrogate how IR deletion in ICs impacts antibacterial defenses against uropathogenic Escherichia coli. We also explore how IR deletion affects immune responses in neighboring PCs with intact IR expression. To accomplish this objective, we profile the transcriptomes of IC and PC populations enriched from kidneys of wild-type and IC-specific IR knock-out mice that have increased UTI susceptibility. Transcriptomic analysis demonstrates that IR deletion suppresses IC-integrated stress responses and innate immune defenses. To define how IR shapes these immune defenses, we employ murine and human kidney cultures. When challenged with bacteria, murine ICs and human kidney cells with deregulated IR signaling cannot engage central components of the integrated stress response-including activating transcriptional factor 4 (ATF4). Silencing ATF4 impairs NFkB activation and promotes infection. In turn, NFkB silencing augments infection and suppresses antimicrobial peptide expression. In diabetic mice and people with diabetes, collecting duct cells show reduced IR expression, impaired integrated stress response engagement, and compromised immunity. Collectively, these translational data illustrate how IR orchestrates collecting duct antibacterial responses and the communication between ICs and PCs.
Assuntos
Camundongos Knockout , Receptor de Insulina , Infecções Urinárias , Escherichia coli Uropatogênica , Animais , Humanos , Camundongos , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/metabolismo , Infecções por Escherichia coli/microbiologia , Imunidade Inata , Rim/metabolismo , Túbulos Renais Coletores/metabolismo , Camundongos Endogâmicos C57BL , Receptor de Insulina/metabolismo , Transdução de Sinais , Infecções Urinárias/microbiologia , Infecções Urinárias/metabolismo , Infecções Urinárias/imunologia , Escherichia coli Uropatogênica/imunologiaRESUMO
Given the high frequency of urinary tract infections (UTIs) and their recurrence, there is keen interest in developing effective UTI vaccines. Currently, most vaccine studies, including those in humans, involve parenteral vaccination aimed at evoking and sustaining elevated levels of systemic antibody directed at the uropathogens. In view of recent reports of aberrant Th2-biased bladder immune responses to infection, we hypothesized that immunizing mice intravesically with antigens from uropathogenic Escherichia coli (UPEC) combined with a Th1-skewing adjuvant could correct this defect and promote protection against UTIs. Here we report that compared with mice immunized subcutaneously with this vaccine combination, intravesically immunized mice were markedly more protected from UTIs because of their distinctive ability to recruit Th1 cells into the bladder. This mode of vaccination was effective even in mice that experienced multiple UTIs and displayed pronounced aberrant bladder immune responses. Thus, intravesical vaccination with one or more UPEC antigens to induce bladder Th1 responses represents a superior strategy to combat UTIs, especially in UTI-prone subjects.
Assuntos
Infecções por Escherichia coli , Vacinas contra Escherichia coli/farmacologia , Células Th1/imunologia , Bexiga Urinária/imunologia , Infecções Urinárias , Escherichia coli Uropatogênica/imunologia , Animais , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/prevenção & controle , Vacinas contra Escherichia coli/imunologia , Camundongos , Camundongos Knockout , Infecções Urinárias/imunologia , Infecções Urinárias/prevenção & controleRESUMO
TcpC is a virulence factor of uropathogenic E. coli (UPEC). It was found that TIR domain of TcpC impedes TLR signaling by direct association with MyD88. It has been a long-standing question whether bacterial pathogens have evolved a mechanism to manipulate MyD88 degradation by ubiquitin-proteasome pathway. Here, we show that TcpC is a MyD88-targeted E3 ubiquitin ligase. Kidney macrophages from mice with pyelonephritis induced by TcpC-secreting UPEC showed significantly decreased MyD88 protein levels. Recombinant TcpC (rTcpC) dose-dependently inhibited protein but not mRNA levels of MyD88 in macrophages. Moreover, rTcpC significantly promoted MyD88 ubiquitination and accumulation in proteasomes in macrophages. Cys12 and Trp106 in TcpC are crucial amino acids in maintaining its E3 activity. Therefore, TcpC blocks TLR signaling pathway by degradation of MyD88 through ubiquitin-proteasome system. Our findings provide not only a novel biochemical mechanism underlying TcpC-medicated immune evasion, but also the first example that bacterial pathogens inhibit MyD88-mediated signaling pathway by virulence factors that function as E3 ubiquitin ligase.
Assuntos
Proteínas de Escherichia coli/metabolismo , Fator 88 de Diferenciação Mieloide/metabolismo , Transdução de Sinais/fisiologia , Escherichia coli Uropatogênica/patogenicidade , Fatores de Virulência/metabolismo , Animais , Linhagem Celular , Feminino , Humanos , Evasão da Resposta Imune/fisiologia , Macrófagos , Camundongos , Camundongos Endogâmicos C57BL , Pielonefrite/imunologia , Pielonefrite/microbiologia , Receptores Toll-Like/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Escherichia coli Uropatogênica/imunologia , Escherichia coli Uropatogênica/metabolismo , Virulência/fisiologiaRESUMO
Neutrophils play a crucial role in immune defense against and clearance of uropathogenic Escherichia coli (UPEC)-mediated urinary tract infection, the most common bacterial infection in healthy humans. CD300a is an inhibitory receptor that binds phosphatidylserine and phosphatidylethanolamine, presented on the membranes of apoptotic cells. CD300a binding to phosphatidylserine and phosphatidylethanolamine, also known as the "eat me" signal, mediates immune tolerance to dying cells. Here, we demonstrate for the first time that CD300a plays an important role in the neutrophil-mediated immune response to UPEC-induced urinary tract infection. We show that CD300a-deficient neutrophils have impaired phagocytic abilities and despite their increased accumulation at the site of infection, they are unable to reduce bacterial burden in the bladder, which results in significant exacerbation of infection and worse host outcome. Finally, we demonstrate that UPEC's pore forming toxin α-hemolysin induces upregulation of the CD300a ligand on infected bladder epithelial cells, signaling to neutrophils to be cleared.
Assuntos
Infecções por Escherichia coli/prevenção & controle , Neutrófilos/imunologia , Receptores Imunológicos/deficiência , Receptores Imunológicos/imunologia , Infecções Urinárias/imunologia , Escherichia coli Uropatogênica/imunologia , Animais , Apoptose/imunologia , Infecções por Escherichia coli/imunologia , Proteínas de Escherichia coli/metabolismo , Feminino , Técnicas de Inativação de Genes , Proteínas Hemolisinas/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Fagocitose/genética , Fagocitose/imunologia , Fosfatidiletanolaminas/metabolismo , Fosfatidilserinas/metabolismo , Receptores Imunológicos/genética , Bexiga Urinária/imunologia , Bexiga Urinária/microbiologia , Bexiga Urinária/patologia , Infecções Urinárias/microbiologia , Escherichia coli Uropatogênica/crescimento & desenvolvimentoRESUMO
The lower urinary tract's virtually inevitable exposure to external microbial pathogens warrants efficient tissue-specialized defenses to maintain sterility. The observation that the bladder can become chronically infected in combination with clinical observations that antibody responses after bladder infections are not detectable suggest defects in the formation of adaptive immunity and immunological memory. We have identified a broadly immunosuppressive transcriptional program specific to the bladder, but not the kidney, during infection of the urinary tract that is dependent on tissue-resident mast cells (MCs). This involves localized production of interleukin-10 and results in suppressed humoral and cell-mediated responses and bacterial persistence. Therefore, in addition to the previously described role of MCs orchestrating the early innate immunity during bladder infection, they subsequently play a tissue-specific immunosuppressive role. These findings may explain the prevalent recurrence of bladder infections and suggest the bladder as a site exhibiting an intrinsic degree of MC-maintained immune privilege.
Assuntos
Cistite/patologia , Células Dendríticas/patologia , Tolerância Imunológica , Interleucina-10/imunologia , Mastócitos/patologia , Bexiga Urinária/patologia , Escherichia coli Uropatogênica/imunologia , Animais , Doença Crônica , Cistite/imunologia , Cistite/microbiologia , Células Dendríticas/imunologia , Células Dendríticas/microbiologia , Feminino , Expressão Gênica , Humanos , Imunidade Celular , Imunidade Humoral , Imunidade Inata , Memória Imunológica , Interleucina-10/biossíntese , Rim/imunologia , Rim/microbiologia , Rim/patologia , Mastócitos/imunologia , Mastócitos/microbiologia , Camundongos , Especificidade de Órgãos , Pielonefrite/imunologia , Pielonefrite/microbiologia , Pielonefrite/patologia , Transcrição Gênica/imunologia , Bexiga Urinária/imunologia , Bexiga Urinária/microbiologiaRESUMO
Bacterial prostatitis affects 1% of men, with increased incidence in the elderly. Acute bacterial prostatitis frequently progresses to chronicity, marked by recurrent episodes interspersed with asymptomatic periods of variable duration. Antibiotic treatment is standard of care; however, dissemination of antimicrobially resistant uropathogens threatens therapy efficacy. Thus, development of nonantibiotic-based approaches to treat chronic disease is a priority. Currently, why chronic prostatitis arises is unclear, as the immune response to prostate infection is incompletely understood. As 80% of prostatitis cases are caused by Gram-negative uropathogenic Escherichia coli (UPEC) or Gram-positive Enterococcus faecalis, we used a mouse transurethral instillation model to address the hypothesis that an innate immune response fails to develop following prostate infection with these uropathogens, leading to chronic disease. Surprisingly, infection induced robust proinflammatory cytokine expression and myeloid cell infiltration. Following a second infection, cytokine responses and innate cell infiltration were largely comparable to primary infection. Characteristic of memory responses, more lymphoid cells infiltrated the prostate in a second infection compared with a first, suggesting that adaptive immunity develops to eliminate the pathogens. Unexpectedly, bacterial burden in prostates challenged with either UPEC or E. faecalis was equal or greater than primary infection despite that a protective adaptive response to UPEC infection was evident in the bladder of the same animals. Our findings support that chronic or recurrent prostatitis develops despite strong innate immune responses and may be the result of a failure to develop immune memory to infection, pointing to actionable targets for immunotherapy.
Assuntos
Infecções por Escherichia coli/imunologia , Infecções por Bactérias Gram-Positivas/imunologia , Prostatite/imunologia , Infecções Urinárias/imunologia , Animais , Doença Crônica , Citocinas/metabolismo , Modelos Animais de Doenças , Enterococcus faecalis/imunologia , Enterococcus faecalis/patogenicidade , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/terapia , Infecções por Bactérias Gram-Positivas/microbiologia , Infecções por Bactérias Gram-Positivas/terapia , Humanos , Imunidade Inata , Memória Imunológica , Imunoterapia , Masculino , Camundongos , Próstata/imunologia , Próstata/microbiologia , Prostatite/microbiologia , Prostatite/terapia , Recidiva , Bexiga Urinária/imunologia , Bexiga Urinária/microbiologia , Infecções Urinárias/microbiologia , Infecções Urinárias/terapia , Escherichia coli Uropatogênica/imunologia , Escherichia coli Uropatogênica/patogenicidadeRESUMO
Toll-like receptor (TLR)-inducible zinc toxicity is a recently described macrophage antimicrobial response used against bacterial pathogens. Here we investigated deployment of this pathway against uropathogenic Escherichia coli (UPEC), the major cause of urinary tract infections. Primary human macrophages subjected EC958, a representative strain of the globally disseminated multidrug-resistant UPEC ST131 clone, to zinc stress. We therefore used transposon-directed insertion site sequencing to identify the complete set of UPEC genes conferring protection against zinc toxicity. Surprisingly, zinc-susceptible EC958 mutants were not compromised for intramacrophage survival, whereas corresponding mutants in the nonpathogenic E. coli K-12 strain MG1655 displayed significantly reduced intracellular bacterial loads within human macrophages. To investigate whether the intramacrophage zinc stress response of EC958 reflected the response of only a subpopulation of bacteria, we generated and validated reporter systems as highly specific sensors of zinc stress. Using these tools we show that, in contrast to MG1655, the majority of intramacrophage EC958 evades the zinc toxicity response, enabling survival within these cells. In addition, EC958 has a higher tolerance to zinc than MG1655, with this likely being important for survival of the minor subset of UPEC cells exposed to innate immune-mediated zinc stress. Indeed, analysis of zinc stress reporter strains and zinc-sensitive mutants in an intraperitoneal challenge model in mice revealed that EC958 employs both evasion and resistance against zinc toxicity, enabling its dissemination to the liver and spleen. We thus demonstrate that a pathogen of global significance uses multiple mechanisms to effectively subvert innate immune-mediated zinc poisoning for systemic spread.
Assuntos
Imunidade Inata/efeitos dos fármacos , Escherichia coli Uropatogênica/efeitos dos fármacos , Escherichia coli Uropatogênica/imunologia , Escherichia coli Uropatogênica/metabolismo , Zinco/toxicidade , Transportadores de Cassetes de Ligação de ATP/genética , Adenosina Trifosfatases/genética , Animais , Carga Bacteriana , Proteínas de Bactérias/genética , Elementos de DNA Transponíveis , Modelos Animais de Doenças , Farmacorresistência Bacteriana Múltipla/genética , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Fatores de Transcrição/genética , Infecções Urinárias/microbiologia , Escherichia coli Uropatogênica/genéticaRESUMO
Most uropathogenic Escherichia coli (UPEC) express type-1 fimbriae (T1F), a key virulence factor for urinary tract infection (UTI) in mice. Evidence that conclusively associates this pilus with uropathogenesis in humans has, however, been difficult to obtain. We used an experimental porcine model of cystitis to assess the role of T1F in larger mammals more closely related to humans. Thirty-one pigs were infected with UPEC strain UTI89 or its T1F deficient mutant, UTI89ΔfimH, at inoculum titres of 102 to 108 colony forming units per millilitre. Urine and blood samples were collected and analysed 7 and 14 days post-inoculation, and whole bladders were removed at day 14 and analysed for uroepithelium-associated UPEC. All animals were consistently infected and reached high urine titres independent of inoculum titre. UTI89ΔfimH successfully colonized the bladders of 1/6 pigs compared to 6/6 for the wild-type strain. Intracellular UPEC were detectable in low numbers in whole bladder explants. In conclusion, low doses of UPEC are able to establish robust infections in pigs, similar to what is presumed in humans. T1F are critical for UPEC to surpass initial bottlenecks during infection but may be dispensable once infection is established. While supporting the conclusions from mice studies regarding a general importance of T1F in successfully infecting the host, the porcine UTI models' natural high, more human-like, susceptibility to infection, allowed us to demonstrate a pivotal role of T1F in initial establishment of infection upon a realistic low-inoculum introduction of UPEC in the bladder.
Assuntos
Cistite/microbiologia , Infecções por Escherichia coli/microbiologia , Fímbrias Bacterianas/metabolismo , Infecções Urinárias/microbiologia , Escherichia coli Uropatogênica/patogenicidade , Fatores de Virulência/metabolismo , Animais , Anticorpos Antibacterianos/sangue , Carga Bacteriana , Contagem de Colônia Microbiana , Modelos Animais de Doenças , Fímbrias Bacterianas/genética , Fímbrias Bacterianas/imunologia , Gentamicinas/farmacologia , Viabilidade Microbiana/efeitos dos fármacos , Mutação , Suínos , Bexiga Urinária/microbiologia , Escherichia coli Uropatogênica/efeitos dos fármacos , Escherichia coli Uropatogênica/genética , Escherichia coli Uropatogênica/imunologia , Fatores de Virulência/genéticaRESUMO
Acute pyelonephritis is frequently associated with metabolic acidosis. We previously reported that metabolic acidosis stimulates expression of hypoxia-inducible factor (HIF)-1α-induced target genes such as stromal derived factor-1 and cathelicidin, an antimicrobial peptide. Since the collecting duct (CD) plays a pivotal role in regulating acid-base homeostasis and is the first nephron segment encountered by an ascending microbial infection, we examined the contribution of HIF-1α to innate immune responses elicited by acid loading of an M-1 immortalized mouse CD cell line. Acid loading of confluent M-1 cells was achieved by culture in pH 6.8 medium supplemented with 5-(N-ethyl-N-isopropyl)-amiloride to block Na+/H+ exchange activity for 24 h. Acid loading induced antimicrobial peptide [cathelicidin and ß-defensin (Defb2 and Defb26)] mRNA expression and M-1 cell resistance to uropathogenic Escherichia coli infection to an extent similar to that obtained by inhibition of HIF prolyl hydroxylases, which promote HIF-1α protein degradation. The effect of acid loading on M-1 cell resistance to uropathogenic E. coli infection was reduced by inhibition of HIF-1α (PX-478), and, in combination with prolyl hydroxylase inhibitors, acidosis did not confer additional resistance. Thus, metabolic stress of acidosis triggers HIF-1α-dependent innate immune responses in CD (M-1) cells. Whether pharmacological stabilization of HIF prevents or ameliorates pyelonephritis in vivo warrants further investigation.
Assuntos
Acidose/metabolismo , Peptídeos Catiônicos Antimicrobianos/metabolismo , Infecções por Escherichia coli/prevenção & controle , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Túbulos Renais Coletores/metabolismo , Infecções Urinárias/prevenção & controle , Escherichia coli Uropatogênica/patogenicidade , Acidose/imunologia , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Linhagem Celular , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/metabolismo , Infecções por Escherichia coli/microbiologia , Interações Hospedeiro-Patógeno , Imunidade Inata , Túbulos Renais Coletores/imunologia , Túbulos Renais Coletores/microbiologia , Camundongos , Prolil Hidroxilases/metabolismo , Estabilidade Proteica , Transdução de Sinais , Regulação para Cima , Infecções Urinárias/imunologia , Infecções Urinárias/metabolismo , Infecções Urinárias/microbiologia , Escherichia coli Uropatogênica/imunologia , beta-Defensinas/metabolismo , CatelicidinasRESUMO
Urinary tract infections (UTI) are extremely common and can be highly recurrent, with 1-2% of women suffering from six or more recurrent episodes per year. The high incidence of recurrent UTI, including recurrent infections caused by the same bacterial strain that caused the first infection, suggests that at least some women do not mount a protective adaptive immune response to UTI. Here we observed in a mouse model of cystitis (bladder infection) that infection with two different clinical uropathogenic Escherichia coli (UPEC) isolates, UTI89 or CFT073, resulted in different kinetics of bacterial clearance and different susceptibility to same-strain recurrent infection. UTI89 and CFT073 both caused infections that persisted for at least two weeks in similar proportions of mice, but whereas UTI89 infections could persist indefinitely, CFT073 infections began to clear two weeks after inoculation and were uniformly cleared within eight weeks. Mice with a history of CFT073 cystitis lasting four weeks were protected against recurrent CFT073 infection after antibiotic therapy, but were not protected against challenge with UTI89. In contrast, mice with a history of UTI89 cystitis lasting four weeks were highly susceptible to challenge infection with either strain after antibiotic treatment. We found that depletion of CD4+ and CD8+ T cell subsets impaired the ability of the host to clear CFT073 infections and rendered mice with a history of CFT073 cystitis lasting four weeks susceptible to recurrent CFT073 cystitis upon challenge. Our findings demonstrate the complex interplay between the broad genetic diversity of UPEC and the host innate and adaptive immune responses during UTI. A better understanding of these host-pathogen interactions is urgently needed for effective drug and vaccine development in the era of increasing antibiotic resistance.
Assuntos
Cistite/imunologia , Suscetibilidade a Doenças/imunologia , Infecções por Escherichia coli/imunologia , Interações Hospedeiro-Patógeno/imunologia , Escherichia coli Uropatogênica/imunologia , Animais , Camundongos , Escherichia coli Uropatogênica/genéticaRESUMO
Both the C3a/C3aR and C5a/C5aR1 axes are regarded as important pathways for inducing and regulating inflammatory responses. It is well documented that the C5a/C5aR1 axis is a potent inflammatory mediator in the pathogenesis of many clinic disorders. However, our understanding of the role of the C3a/C3aR axis in renal disorders remains limited. Contrary to the C5a/C5aR axis, we now show that the C3a/C3aR axis has a protective role in uropathogenic Escherichia coli (UPEC)-induced renal injury. C3aR-/- mice were found to develop severe renal pathology compared to wild type mice, a pathology characterized by intense tissue damage and an increased bacterial load within the kidney. This was associated with an overwhelming production of pro-inflammatory mediators and increased neutrophil infiltration in the kidney. Bone marrow chimera experiments found that tissue damage and bacterial load were significantly reduced in C3aR-/- mice that received bone marrow from wild type mice, compared with that in mice re-populated with bone marrow from C3aR-/- mice. This supports a critical role for C3aR on myeloid cells in the pathological process. Pharmacological treatment of mice with a C3aR agonist reduced both the extent of tissue injury and bacterial load. Mechanistic analyses indicated that the C3a/C3aR axis downregulates the lipopolysaccharide-induced pro-inflammatory responses in macrophages and facilitates the phagocytosis of UPEC by phagocytes. Thus, our findings clearly demonstrate a protective role of the C3a/C3aR axis in UPEC-induced renal injury, conferred by the suppression of pro-inflammatory responses and enhanced phagocytosis by macrophages.
Assuntos
Complemento C3a/metabolismo , Infecções por Escherichia coli/imunologia , Receptores de Complemento/metabolismo , Transdução de Sinais/imunologia , Infecções Urinárias/imunologia , Escherichia coli Uropatogênica/imunologia , Animais , Modelos Animais de Doenças , Resistência à Doença/imunologia , Infecções por Escherichia coli/diagnóstico , Infecções por Escherichia coli/microbiologia , Feminino , Humanos , Camundongos , Camundongos Knockout , Receptores de Complemento/genética , Índice de Gravidade de Doença , Infecções Urinárias/diagnóstico , Infecções Urinárias/microbiologiaRESUMO
The NLRP3 inflammasome, IL-1ß release and pyroptosis (cell lysis) have recently been proposed to be essential for the progression of urinary tract infection (UTI) and elimination of intracellular bacterial niches. However, the effects of IL-1R antagonist (IL-1RA) on immune responses during UTI, except for its ability to disrupt IL-1ß signalling, are not well understood. The aim of this study was to investigate the role of IL-1RA in UPEC colonization of bladder epithelial cells and the subsequent host inflammatory response. Human bladder epithelial cells (5637) and CRISPR/Cas9 generated NLRP3 and caspase-1 knockdown cells and IL-1RA knockout cells were stimulated with the UPEC isolate CFT073. The results showed that the UPEC virulence factor α-hemolysin is essential for IL-1RA release, and that the inflammasome-associated proteins caspase-1 and NLRP3 affect the release of IL-1RA. IL-1RA deficient cells showed a reduced adherence and invasion by CFT073 compared to wild-type cells, suggesting that IL-1RA may oppose mechanisms that protects against bacterial colonization. A targeted protein analysis of inflammation-related proteins showed that the basal expression of 23 proteins and the UPEC-induced expression of 10 proteins were significantly altered in IL-1RA deficient bladder epithelial cells compared to Cas9 control cells. This suggests that IL-1RA has a broad effect on the inflammatory response in bladder epithelial cells.
Assuntos
Células Epiteliais/imunologia , Infecções por Escherichia coli/imunologia , Inflamassomos/imunologia , Proteína Antagonista do Receptor de Interleucina 1/imunologia , Bexiga Urinária/imunologia , Infecções Urinárias/imunologia , Escherichia coli Uropatogênica/imunologia , Linhagem Celular , Células Epiteliais/microbiologia , Infecções por Escherichia coli/patologia , Humanos , Bexiga Urinária/microbiologia , Bexiga Urinária/patologia , Infecções Urinárias/microbiologia , Infecções Urinárias/patologiaRESUMO
Surfactant protein A (SP-A) is a multifunctional host defense collectin that was first identified as a component of pulmonary surfactant. Although SP-A is also expressed in various tissues, including the urinary tract, its innate immune functions in nonpulmonary tissues are poorly understood. In this study, we demonstrated that adherence of uropathogenic Escherichia coli (UPEC) to the bladder was enhanced in SP-A-deficient mice, which suggests that SP-A plays an important role in innate immunity against UPEC. To understand the innate immune functions of SP-A in detail, we performed in vitro experiments. SP-A directly bound to UPEC in a Ca2+-dependent manner, but it did not agglutinate UPEC. Our results suggest that a bouquet-like arrangement seems unsuitable to agglutinate UPEC. Meanwhile, SP-A inhibited growth of UPEC in human urine. Furthermore, the binding of SP-A to UPEC decreased the adherence of bacteria to urothelial cells. These results indicate that direct action of SP-A on UPEC is important in host defense against UPEC. Additionally, adhesion of UPEC to urothelial cells was decreased when the cells were preincubated with SP-A. Adhesion of UPEC to urothelial cells is achieved via interaction between FimH, an adhesin located at bacterial pili, and uroplakin Ia, a glycoprotein expressed on the urothelium. SP-A directly bound to uroplakin Ia and competed with FimH for uroplakin Ia binding. These results lead us to conclude that SP-A plays important roles in host defense against UPEC.
Assuntos
Infecções por Escherichia coli/imunologia , Proteína A Associada a Surfactante Pulmonar/imunologia , Infecções Urinárias/imunologia , Animais , Proliferação de Células , Humanos , Imunidade Inata/imunologia , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infecções Urinárias/microbiologia , Escherichia coli Uropatogênica/imunologiaRESUMO
Uropathogenic Escherichia coli (UPEC) is the primary cause of uncomplicated urinary tract infections (UTIs). Whereas most infections are isolated cases, 1 in 40 women experience recurrent UTIs. The rise in antibiotic resistance has complicated the management of chronic UTIs and necessitates new preventative strategies. Currently, no UTI vaccines are approved for use in the United States, and the development of a highly effective vaccine remains elusive. Here, we have pursued a strategy for eliciting protective immunity by vaccinating with small molecules required for pathogenesis, rather than proteins or peptides. Small iron-chelating molecules called siderophores were selected as antigens to vaccinate against UTI for this vaccine strategy. These pathogen-associated stealth siderophores evade host immune defenses and enhance bacterial virulence. Previous animal studies revealed that vaccination with siderophore receptor proteins protects against UTI. The poor solubility of these integral outer-membrane proteins in aqueous solutions limits their practical utility. Because their cognate siderophores are water soluble, we hypothesized that these bacterial-derived small molecules are prime vaccine candidates. To test this hypothesis, we immunized mice with siderophores conjugated to an immunogenic carrier protein. The siderophore-protein conjugates elicited an adaptive immune response that targeted bacterial stealth siderophores and protected against UTI. Our study has identified additional antigens suitable for a multicomponent UTI vaccine and highlights the potential use of bacterial-derived small molecules as antigens in vaccine therapies.
Assuntos
Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/prevenção & controle , Sideróforos/imunologia , Infecções Urinárias/imunologia , Infecções Urinárias/prevenção & controle , Escherichia coli Uropatogênica/imunologia , Vacinas Conjugadas/imunologia , Animais , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/patologia , Feminino , Inflamação/patologia , Camundongos , Infecções Urinárias/microbiologia , Infecções Urinárias/patologia , VacinaçãoRESUMO
BACKGROUND AND AIM: Urinary tract infection (UTI) is the second most frequent infection in human, and uropathogenic Escherichia coli is its most common cause. Although antibiotics are the standard treatment for UTI, they can cause harmful effects on gut microbiome and increase the rate of existing drug-resistant bacteria, which make the vaccine research reasonable. This study was conducted to construct a Killed but Metabolically Active (KBMA) E. coli strain, and to determine its characteristics as a possible vaccine candidate for UTI, which will be evaluated in further investigations. METHODS: The uvrB gene of uvrABC excision repair system of E.coli was deleted to construct a ΔuvrB mutant, lacking repairing system of intercross linkages between DNA strands. To construct KBMA strain, the ΔuvrB mutant was PUVA-treated, using different doses of 8-methoxypsoralen (8-MOP) followed by different doses of ultraviolet A (UVA) irradiation (365â¯nm), until the optimal doses of each were achieved. Then, different characteristics of the PUVA-treated E. coli (with the optimal doses) were assessed, using cell counting, colony formation assay, MTT and XTT assays, fluorescent staining, and flow cytometry. RESULTS: PUVA treatment's optimal dose for E. coli isolates was 150â¯ng/ml 8-MOP plus 1000 mj/cm2 UVA. While the PUVA-treated isolates had a significant decrease in cell counting, the fluorescent dying of the un-grown parts of the culture plates revealed living bacteria with bizarre shapes. Meanwhile, MTT and XTT assays demonstrated the metabolic activity of these bacteria and flow cytometry confirmed their aliveness. CONCLUSION: These PUVA-treated bacteria, with metabolic activity and proliferation inability, seem to be good enough to be tested in vitro and in vivo as a candidate for vaccine against UTI. Therefore it seems the first step toward development of a vaccine candidate is successfully done. The immunogenicity and protectivity of these treated bacteria is under evaluation.
Assuntos
Infecções por Escherichia coli/prevenção & controle , Vacinas contra Escherichia coli/isolamento & purificação , Infecções Urinárias/prevenção & controle , Escherichia coli Uropatogênica/imunologia , DNA Helicases/deficiência , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli , Vacinas contra Escherichia coli/imunologia , Deleção de Genes , Metoxaleno/metabolismo , Viabilidade Microbiana/efeitos da radiação , Fármacos Fotossensibilizantes/metabolismo , Raios Ultravioleta , Infecções Urinárias/microbiologia , Vacinas de Produtos Inativados/imunologia , Vacinas de Produtos Inativados/isolamento & purificaçãoRESUMO
Innate-immunity-related genes in humans are activated during urinary tract infections (UTIs) caused by pathogenic strains of Escherichia coli but are suppressed by commensals. Epigenetic mechanisms play a pivotal role in the regulation of gene expression in response to environmental stimuli. To determine whether epigenetic mechanisms can explain the different behaviors of pathogenic and commensal bacteria, we infected larvae of the greater wax moth, Galleria mellonella, a widely used model insect host, with a uropathogenic E. coli (UPEC) strain that causes symptomatic UTIs in humans or a commensal-like strain that causes asymptomatic bacteriuria (ABU). Infection with the UPEC strain (CFT073) was more lethal to larvae than infection with the attenuated ABU strain (83972) due to the recognition of each strain by different Toll-like receptors, ultimately leading to differential DNA/RNA methylation and histone acetylation. We used next-generation sequencing and reverse transcription (RT)-PCR to correlate epigenetic changes with the induction of innate-immunity-related genes. Transcriptomic analysis of G. mellonella larvae infected with E. coli strains CFT073 and 83972 revealed strain-specific variations in the class and expression levels of genes encoding antimicrobial peptides, cytokines, and enzymes controlling DNA methylation and histone acetylation. Our results provide evidence for the differential epigenetic regulation of transcriptional reprogramming by UPEC and ABU strains of E. coli in G. mellonella larvae, which may be relevant to understanding the different behaviors of these bacterial strains in the human urinary tract.
Assuntos
Epigênese Genética , Escherichia coli/imunologia , Escherichia coli/fisiologia , Imunidade Inata/genética , Lepidópteros/imunologia , Escherichia coli Uropatogênica/imunologia , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/metabolismo , Metilação de DNA , DNA Bacteriano/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Lepidópteros/metabolismo , Lepidópteros/microbiologia , Reação em Cadeia da Polimerase , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismo , Escherichia coli Uropatogênica/genética , Escherichia coli Uropatogênica/patogenicidade , Virulência , Fatores de Virulência/genéticaRESUMO
Uropathogenic E. coli (UPEC) is the primary cause of urinary tract infections (UTI) affecting approximately 150 million people worldwide. Here, we revealed the importance of transcriptional regulator hypoxia-inducible factor-1 α subunit (HIF-1α) in innate defense against UPEC-mediated UTI. The effects of AKB-4924, a HIF-1α stabilizing agent, were studied using human uroepithelial cells (5637) and a murine UTI model. UPEC adherence and invasion were significantly reduced in 5637 cells when HIF-1α protein was allowed to accumulate. Uroepithelial cells treated with AKB-4924 also experienced reduced cell death and exfoliation upon UPEC challenge. In vivo, fewer UPEC were recovered from the urine, bladders and kidneys of mice treated transurethrally with AKB-4924, whereas increased bacteria were recovered from bladders of mice with a HIF-1α deletion. Bladders and kidneys of AKB-4924 treated mice developed less inflammation as evidenced by decreased pro-inflammatory cytokine release and neutrophil activity. AKB-4924 impairs infection in uroepithelial cells and bladders, and could be correlated with enhanced production of nitric oxide and antimicrobial peptides cathelicidin and ß-defensin-2. We conclude that HIF-1α transcriptional regulation plays a key role in defense of the urinary tract against UPEC infection, and that pharmacological HIF-1α boosting could be explored further as an adjunctive therapy strategy for serious or recurrent UTI.
Assuntos
Infecções por Escherichia coli/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Imunidade Inata , Infecções Urinárias/metabolismo , Escherichia coli Uropatogênica/imunologia , Urotélio/metabolismo , Administração Intravesical , Animais , Antibacterianos/administração & dosagem , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Peptídeos Catiônicos Antimicrobianos/agonistas , Peptídeos Catiônicos Antimicrobianos/metabolismo , Aderência Bacteriana/efeitos dos fármacos , Linhagem Celular , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/prevenção & controle , Feminino , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/agonistas , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Imunidade Inata/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico/agonistas , Óxido Nítrico/metabolismo , Piperazinas/administração & dosagem , Piperazinas/farmacologia , Piperazinas/uso terapêutico , Estabilidade Proteica/efeitos dos fármacos , Piridonas/administração & dosagem , Piridonas/farmacologia , Piridonas/uso terapêutico , RNA Mensageiro/metabolismo , Infecções Urinárias/imunologia , Infecções Urinárias/microbiologia , Infecções Urinárias/prevenção & controle , Escherichia coli Uropatogênica/efeitos dos fármacos , Urotélio/efeitos dos fármacos , Urotélio/imunologia , Urotélio/microbiologiaRESUMO
OBJECTIVE: This is a random blinded placebo controlled murine experimental model to study the effects of Cantharis 6 CH, a homeopathic medicine, on E coli-induced cystitis. METHODS: 24 adult susceptible female BALB/c mice were inoculated with E coli - UPEC O4:K-:H5 by a transurethral catheter. Cantharis 6cH or vehicle (placebo) was offered to mice by free access into the drinking water (1:100), during 24 h after infection. Spleen, bladder and kidneys were processed for quantitative histopathology after immunohistochemistry, using anti-CD3, CD79, MIF, NK and VEGF antibodies; the cytokines present in the bladder washing fluid were measured using a LUMINEX-Magpix KIT. Mann-Whitney and Fisher exact test were used as statistical analysis. RESULTS: Cantharis 6 CH increased IL12p40, IFN-γ and decreased IL10 concentrations in the bladder fluid (p⩽0.05); in the bladder mucosa, it increased the ratio between B and T lymphocytes (31%) and between B lymphocytes and MIF+ macrophages (57%, p⩽0.05). In the pelvis, instead, it decreased the B/T cells ratio (41%, p⩽0.05) and increased the M1/M2 macrophage ratio (42%, p⩽0.05). No differences were seen in the kidney and spleen analysis. CONCLUSION: The inverted balance of inflammatory cells and cytokines in bladder and pelvis mucosa shows specific local immune modulation induced by Cantharis 6cH.
Assuntos
Cistite/tratamento farmacológico , Infecções por Escherichia coli/tratamento farmacológico , Materia Medica/farmacologia , Infecções Urinárias/tratamento farmacológico , Escherichia coli Uropatogênica/imunologia , Animais , Cistite/imunologia , Cistite/microbiologia , Cistite/patologia , Citocinas/imunologia , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/patologia , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Infecções Urinárias/imunologia , Infecções Urinárias/patologiaRESUMO
Uropathogenic Escherichia coli (UPEC) are among the most prevalent agents of urinary tract infections (UTIs). Antibiotic resistance reaches the need for alternative treatment approaches such as vaccination against UTIs. There is no ideal vaccine against UTIs, thus there is a need to evaluate different targets of uropathogens against UTIs. Ferric scavenger receptor FyuA in UPEC has the properties of an ideal vaccine candidate against UTIs. In the present study, the prevalence of FyuA among UPEC isolates, its immunogenicity with and without alum adjuvant, and its efficacy against experimental UTI were assessed. Totally, fyuA gene was present in 77% of the UPEC isolates tested. Alignments of FyuA exhibited a high degree of conservation among different submitted UPEC isolates in GenBank. The bioinformatics studies showed the high confidence value and stability of the FyuA structure. SDS-PAGE and Western blot confirmed the purification of FyuA with high yield by nickel resins. Mice vaccinated subcutaneously with the FyuA induced a significantly higher humoral response (total IgG, IgG1 and IgG2a) than control mice that alum enhanced these responses. The FuyA alone showed the ability to reduce the colonization of UPEC in bladder and kidney of mice as compared to the control group. But the addition of alum to FyuA increased the protection level against UPEC in these organs. Since, FyuA induced significant IgG1 (Th2) and IgG2a (Th1) responses and protected the mice against experimental UTI, it could be a promising target against UPEC infections.
Assuntos
Infecções por Escherichia coli/prevenção & controle , Proteínas de Escherichia coli/imunologia , Vacinas contra Escherichia coli/imunologia , Receptores de Superfície Celular/imunologia , Infecções Urinárias/prevenção & controle , Escherichia coli Uropatogênica/imunologia , Adjuvantes Imunológicos/administração & dosagem , Compostos de Alúmen/administração & dosagem , Animais , Anticorpos Antibacterianos/sangue , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/genética , Vacinas contra Escherichia coli/administração & dosagem , Imunoglobulina G/sangue , Injeções Subcutâneas , Rim/microbiologia , Camundongos , Receptores de Superfície Celular/genética , Resultado do Tratamento , Bexiga Urinária/microbiologia , Infecções Urinárias/microbiologia , Escherichia coli Uropatogênica/genéticaRESUMO
Urinary tract infections (UTIs) are among the most common bacterial infections worldwide. Humans evolved various immune-dependent and independent defense mechanisms, while pathogens evolved multiple virulence factors to fight back. This article summarizes recent findings regarding the arms race between hosts and pathogens in UTIs. It was recently reported that macrophage subsets regulate neutrophil-mediated defense in primary UTIs but seem to subvert adaptive immunity upon re-infection. Moreover, some bacterial strains can survive inside macrophages, leading to recurrent infections. Inflammasome activation results in infected host cell death and pathogen release, facilitating the removal of intracellular bacteria. As a counteraction, some bacteria evolved mechanisms to disrupt inflammasome activation. Mucosal-associated invariant T cells are further effectors that can lyse infected epithelial cells and release intracellular bacteria. Once released, the bacteria are phagocytosed by neutrophils. However, some bacteria can inhibit neutrophil migration and deprive neutrophils of nutrients. Furthermore, the complement system, considered generally bactericidal, is exploited by the bacteria for cellular invasion. Another weapon against UTI is antimicrobial peptides, e.g. ribonuclease 7, but its production is inhibited by certain bacterial strains. Thus the arms race in UTI is ongoing, and knowing the enemy's methods can help in developing new drugs to win the race.