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1.
PLoS Pathog ; 19(5): e1011388, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-37167325

RESUMO

There is a growing consensus that a significant proportion of recurrent urinary tract infections are linked to the persistence of uropathogens within the urinary tract and their re-emergence upon the conclusion of antibiotic treatment. Studies in mice and human have revealed that uropathogenic Escherichia coli (UPEC) can persist in bladder epithelial cells (BECs) even after the apparent resolution of the infection. Here, we found that, following the entry of UPEC into RAB27b+ fusiform vesicles in BECs, some bacteria escaped into the cytoplasmic compartment via a mechanism involving hemolysin A (HlyA). However, these UPEC were immediately recaptured within LC3A/B+ autophagosomes that matured into LAMP1+ autolysosomes. Thereafter, HlyA+ UPEC-containing lysosomes failed to acidify, which is an essential step for bacterial elimination. This lack of acidification was related to the inability of bacteria-harboring compartments to recruit V-ATPase proton pumps, which was attributed to the defragmentation of cytosolic microtubules by HlyA. The persistence of UPEC within LAMP1+ compartments in BECs appears to be directly linked to HlyA. Thus, through intravesicular instillation of microtubule stabilizer, this host defense response can be co-opted to reduce intracellular bacterial burden following UTIs in the bladder potentially preventing recurrence.


Assuntos
Infecções por Escherichia coli , Infecções Urinárias , Escherichia coli Uropatogênica , Animais , Camundongos , Humanos , Bexiga Urinária/microbiologia , Escherichia coli Uropatogênica/fisiologia , Proteínas Hemolisinas , Infecções por Escherichia coli/microbiologia , Infecções Urinárias/microbiologia , Células Epiteliais/microbiologia , Lisossomos/patologia , Concentração de Íons de Hidrogênio
2.
Elife ; 112022 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-35881547

RESUMO

A key attribute of persistent or recurring bacterial infections is the ability of the pathogen to evade the host's immune response. Many Enterobacteriaceae express type 1 pili, a pre-adapted virulence trait, to invade host epithelial cells and establish persistent infections. However, the molecular mechanisms and strategies by which bacteria actively circumvent the immune response of the host remain poorly understood. Here, we identified CD14, the major co-receptor for lipopolysaccharide detection, on mouse dendritic cells (DCs) as a binding partner of FimH, the protein located at the tip of the type 1 pilus of Escherichia coli. The FimH amino acids involved in CD14 binding are highly conserved across pathogenic and non-pathogenic strains. Binding of the pathogenic strain CFT073 to CD14 reduced DC migration by overactivation of integrins and blunted expression of co-stimulatory molecules by overactivating the NFAT (nuclear factor of activated T-cells) pathway, both rate-limiting factors of T cell activation. This response was binary at the single-cell level, but averaged in larger populations exposed to both piliated and non-piliated pathogens, presumably via the exchange of immunomodulatory cytokines. While defining an active molecular mechanism of immune evasion by pathogens, the interaction between FimH and CD14 represents a potential target to interfere with persistent and recurrent infections, such as urinary tract infections or Crohn's disease.


Assuntos
Infecções por Escherichia coli , Escherichia coli Uropatogênica , Adesinas de Escherichia coli/química , Adesinas de Escherichia coli/genética , Adesinas de Escherichia coli/metabolismo , Animais , Infecções por Escherichia coli/microbiologia , Proteínas de Fímbrias/metabolismo , Fímbrias Bacterianas/metabolismo , Imunidade , Camundongos , Escherichia coli Uropatogênica/fisiologia
3.
Cell Rep ; 39(4): 110758, 2022 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-35476986

RESUMO

Urinary tract infections (UTIs) are a cause for alarm given the high rates of treatment failure. In a recent issue of Cell Reports, Pang et al. uncovered dueling molecular machinery at the host-pathogen interface in response to phosphate that points to new anti-infective strategies against UTIs.


Assuntos
Infecções por Escherichia coli , Infecções Urinárias , Escherichia coli Uropatogênica , Feminino , Humanos , Masculino , Escherichia coli Uropatogênica/fisiologia , Urotélio
4.
Sci Rep ; 12(1): 486, 2022 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-35017565

RESUMO

Uropathogenic Escherichia coli (UPEC) may undergo a cyclic cascade of morphological alterations that are believed to enhance the potential of UPEC to evade host responses and re-infect host cell. However, knowledge on the pathogenic potential and host activation properties of UPEC during the morphological switch is limited. Microarray analysis was performed on mRNA isolated from human bladder epithelial cells (HBEP) after exposure to three different morphological states of UPEC (normal coliform, filamentous form and reverted form). Cells stimulated with filamentous bacteria showed the lowest number of significant gene alterations, although the number of enriched gene ontology classes was high suggesting diverse effects on many different classes of host genes. The normal coliform was in general superior in stimulating transcriptional activity in HBEP cells compared to the filamentous and reverted form. Top-scored gene entities activated by all three morphological states included IL17C, TNFAIP6, TNF, IL20, CXCL2, CXCL3, IL6 and CXCL8. The number of significantly changed canonical pathways was lower in HBEP cells stimulated with the reverted form (32 pathways), than in cells stimulated with the coliform (83 pathways) or filamentous bacteria (138 pathways). A host cell invasion assay showed that filamentous bacteria were unable to invade bladder cells, and that the number of intracellular bacteria was markedly lower in cells infected with the reverted form compared to the coliform. In conclusion, the morphological state of UPEC has major impact on the host bladder response both when evaluating the number and the identity of altered host genes and pathways.


Assuntos
Células Epiteliais/metabolismo , Infecções por Escherichia coli/genética , Transcrição Gênica , Bexiga Urinária/microbiologia , Escherichia coli Uropatogênica/fisiologia , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Linhagem Celular , Quimiocina CXCL2/genética , Quimiocina CXCL2/metabolismo , Quimiocinas CXC/genética , Quimiocinas CXC/metabolismo , Células Epiteliais/microbiologia , Infecções por Escherichia coli/metabolismo , Infecções por Escherichia coli/microbiologia , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Bexiga Urinária/metabolismo , Escherichia coli Uropatogênica/crescimento & desenvolvimento
5.
Appl Biochem Biotechnol ; 194(1): 504-516, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-34618338

RESUMO

Nanotechnology involves the synthesis of nanoparticles that have been used in the therapeutic application for treating diseases. In this present study, we have adopted the synthesis of myconanoparticles from the extracellular extract of endophytic fungi Penicillium sclerotiorum (PsNps) and validated its antibacterial potential against antibiotic-resistant uropathogenic E. coli and ATCC (25,922) strain of Escherichia coli. Endophytic fungi were isolated from the healthy leaves of Tamarindus indica. The genomic DNA from endophytic fungi was isolated and the ITS region was amplified by polymerase chain reaction (PCR) using universal fungal primers ITS1 and ITS4 and sequenced for the identification of endophytic fungal isolates. Penicillium sclerotiorum extract was used for the synthesis of silver nanoparticles (PsNps) and was characterized by UV-vis spectroscopy, Fourier transform infrared spectroscopy (FTIR), zeta potential, FE-SEM, and Energy dispersive X-ray analysis (EDAX). Antibacterial activity of PsNps was tested against the antibiotic-resistant uropathogenic E. coli and ATCC (25,922) strain of E. coli. Further experiments were carried out to explore the potential of PsNps in regulating the CTX-M-15 gene. The antimicrobial activity showed that the PsNps inhibited growth, biofilm formation in both the strains of E. coli. The expression of the gene encoding CTX-M-15 was downregulated in a resistant strain of uropathogenic E. coli. Our results suggest that the PsNps could be used as an alternative source for antibiotics. Thus, further studies can be conducted to prove the in vivo potential of PsNps and can be formulated for commercialization.


Assuntos
Biofilmes/efeitos dos fármacos , Nanopartículas/química , Penicillium/química , Prata , Escherichia coli Uropatogênica/fisiologia , Biofilmes/crescimento & desenvolvimento , Prata/química , Prata/farmacologia
6.
Cell Rep ; 36(3): 109351, 2021 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-34289360

RESUMO

Recurrence of uropathogenic Escherichia coli (UPEC) infections has been attributed to reactivation of quiescent intracellular reservoirs (QIRs) in deep layers of the bladder wall. QIRs are thought to arise late during infection following dispersal of bacteria from intracellular bacterial communities (IBCs) in superficial umbrella cells. Here, we track the formation of QIR-like bacteria in a bladder organoid model that recapitulates the stratified uroepithelium within a volume suitable for high-resolution live-cell imaging. Bacteria injected into the organoid lumen enter umbrella-like cells and proliferate to form IBC-like bodies. In parallel, single bacteria penetrate deeper layers of the organoid wall, where they localize within or between uroepithelial cells. These "solitary" bacteria evade killing by antibiotics and neutrophils and are morphologically distinct from bacteria in IBCs. We conclude that bacteria with QIR-like properties may arise at early stages of infection, independent of IBC formation and rupture.


Assuntos
Antibacterianos/farmacologia , Modelos Biológicos , Neutrófilos/patologia , Organoides/microbiologia , Bexiga Urinária/microbiologia , Escherichia coli Uropatogênica/fisiologia , Animais , Diferenciação Celular/efeitos dos fármacos , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/patologia , Feminino , Humanos , Imageamento Tridimensional , Camundongos Endogâmicos C57BL , Viabilidade Microbiana/efeitos dos fármacos , Movimento , Neutrófilos/efeitos dos fármacos , Organoides/efeitos dos fármacos , Organoides/ultraestrutura , Bexiga Urinária/patologia , Escherichia coli Uropatogênica/efeitos dos fármacos , Escherichia coli Uropatogênica/crescimento & desenvolvimento , Escherichia coli Uropatogênica/ultraestrutura
7.
PLoS Pathog ; 17(5): e1009617, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-34043736

RESUMO

Urinary tract infections (UTIs) are a common bacterial infectious disease in humans, and strains of uropathogenic Escherichia coli (UPEC) are the most frequent cause of UTIs. During infection, UPEC must cope with a variety of stressful conditions in the urinary tract. Here, we demonstrate that the small RNA (sRNA) RyfA of UPEC strains is required for resistance to oxidative and osmotic stresses. Transcriptomic analysis of the ryfA mutant showed changes in expression of genes associated with general stress responses, metabolism, biofilm formation and genes coding for cell surface proteins. Inactivation of ryfA in UPEC strain CFT073 decreased urinary tract colonization in mice and the ryfA mutant also had reduced production of type 1 and P fimbriae (pili), adhesins which are known to be important for UTI. Furthermore, loss of ryfA also reduced UPEC survival in human macrophages. Thus, ryfA plays a key regulatory role in UPEC adaptation to stress, which contributes to UTI and survival in macrophages.


Assuntos
Biofilmes/crescimento & desenvolvimento , Infecções por Escherichia coli/microbiologia , Pequeno RNA não Traduzido/genética , Infecções Urinárias/microbiologia , Escherichia coli Uropatogênica/genética , Adaptação Fisiológica , Adesinas Bacterianas/genética , Adesinas Bacterianas/metabolismo , Animais , Fímbrias Bacterianas/metabolismo , Perfilação da Expressão Gênica , Humanos , Macrófagos/microbiologia , Camundongos , Osmorregulação , Estresse Oxidativo , RNA Bacteriano/genética , Deleção de Sequência , Escherichia coli Uropatogênica/crescimento & desenvolvimento , Escherichia coli Uropatogênica/fisiologia , Virulência
8.
PLoS Pathog ; 17(5): e1009553, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-34015044

RESUMO

Bacterial infection results in a veritable cascade of host responses, both local and systemic. To study the initial stages of host-pathogen interaction in living tissue we use spatially-temporally controlled in vivo models. Using this approach, we show here that within 4 h of a uropathogenic Escherichia coli (UPEC) infection in the kidney, an IFNγ response is triggered in the spleen. This rapid infection-mediated inter-organ communication was found to be transmitted via nerve signalling. Bacterial expression of the toxin α-hemolysin directly and indirectly activated sensory neurons, which were identified in the basement membrane of renal tubules. Nerve activation was transmitted via the splenic nerve, inducing upregulation of IFNγ in the marginal zones of the spleen that led to increasing concentrations of IFNγ in the circulation. We found that IFNγ modulated the inflammatory signalling generated by renal epithelia cells in response to UPEC infection. This demonstrates a new concept in the host response to kidney infection; the role of nerves in sensing infection and rapidly triggering a systemic response which can modulate inflammation at the site of infection. The interplay between the nervous and immune systems is an exciting, developing field with the appealing prospect of non-pharmaceutical interventions. Our study identifies an important role for systemic neuro-immune communication in modulating inflammation during the very first hours of a local bacterial infection in vivo.


Assuntos
Infecções por Escherichia coli/complicações , Interações Hospedeiro-Patógeno , Inflamação/patologia , Interferon gama/metabolismo , Rim/microbiologia , Neuroimunomodulação , Baço/metabolismo , Animais , Células Epiteliais/microbiologia , Inflamação/etiologia , Inflamação/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley , Escherichia coli Uropatogênica/fisiologia
9.
NPJ Biofilms Microbiomes ; 7(1): 35, 2021 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-33863914

RESUMO

Nutrient gradients in biofilms cause bacteria to organize into metabolically versatile communities capable of withstanding threats from external agents including bacteriophages, phagocytes, and antibiotics. We previously determined that oxygen availability spatially organizes respiration in uropathogenic Escherichia coli biofilms, and that the high-affinity respiratory quinol oxidase cytochrome bd is necessary for extracellular matrix production and biofilm development. In this study we investigate the physiologic consequences of cytochrome bd deficiency in biofilms and determine that loss of cytochrome bd induces a biofilm-specific increase in expression of general diffusion porins, leading to elevated outer membrane permeability. In addition, loss of cytochrome bd impedes the proton mediated efflux of noxious chemicals by diminishing respiratory flux. As a result, loss of cytochrome bd enhances cellular accumulation of noxious chemicals and increases biofilm susceptibility to antibiotics. These results identify an undescribed link between E. coli biofilm respiration and stress tolerance, while suggesting the possibility of inhibiting cytochrome bd as an antibiofilm therapeutic approach.


Assuntos
Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Grupo dos Citocromos b/genética , Farmacorresistência Bacteriana , Complexo de Proteínas da Cadeia de Transporte de Elétrons/genética , Proteínas de Escherichia coli/genética , Oxirredutases/genética , Escherichia coli Uropatogênica/fisiologia , Alelos , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Biofilmes/crescimento & desenvolvimento , Grupo dos Citocromos b/metabolismo , Relação Dose-Resposta a Droga , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Proteínas de Escherichia coli/metabolismo , Técnicas de Silenciamento de Genes , Testes de Sensibilidade Microbiana , Oxirredutases/metabolismo , Plâncton/efeitos dos fármacos , Plâncton/genética , Escherichia coli Uropatogênica/efeitos dos fármacos
10.
Immunology ; 164(1): 3-14, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-33763853

RESUMO

Urinary tract infections (UTI) are among the most prevalent infectious diseases and the most common cause of nosocomial infections, worldwide. Uropathogenic E. coli (UPEC) are responsible for approximately 80% of all UTI, which most commonly affect the bladder. UPEC colonize the urinary tract by ascension of the urethra, followed by cell invasion, and proliferation inside and outside urothelial cells, thereby causing symptomatic infections and quiescent intracellular reservoirs that may lead to recurrence. Sugars, or glycans, are key molecules for host-pathogen interactions, and UTI are no exception. Surface glycans regulate many of the events associated with UPEC adhesion and infection, as well as induction of the host immune response. While the bacterial protein FimH binds mannose-containing host glycoproteins to initiate infection and UPEC-secreted polysaccharides block immune mechanisms to favour intracellular replication, host glycans on the urothelial surface and on secreted glycoproteins prevent or limit infection by inhibiting UPEC adhesion. Given the importance of glycans during UTI, here we review the glycobiology of UPEC infection to highlight fundamental sugar-mediated processes of immunological interest for their potential clinical applications. Interdisciplinary approaches incorporating glycomics and infection biology may help to develop novel non-antibiotic-based therapeutic strategies for bacterial infections as the spread of antimicrobial-resistant uropathogens is currently threatening modern healthcare systems.


Assuntos
Polissacarídeos/metabolismo , Sistema Urinário/imunologia , Escherichia coli Uropatogênica/fisiologia , Animais , Infecções por Escherichia coli , Glicômica , Interações Hospedeiro-Patógeno , Humanos , Polissacarídeos/imunologia , Infecções Urinárias , Virulência
11.
Front Immunol ; 12: 582858, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33679734

RESUMO

The structural and functional destruction of the blood-testis barrier (BTB) following uropathogenic E. coli (UPEC) infection may be a critical component of the pathologic progress of orchitis. Recent findings indicate that the mammalian target of the rapamycin (mTOR)-signaling pathway is implicated in the regulation of BTB assembly and restructuring. To explore the mechanisms underlying BTB damage induced by UPEC infection, we analyzed BTB integrity and the involvement of the mTOR-signaling pathway using in vivo and in vitro UPEC-infection models. We initially confirmed that soluble virulent factors secreted from UPEC trigger a stress response in Sertoli cells and disturb adjacent cell junctions via down-regulation of junctional proteins, including occludin, zonula occludens-1 (ZO-1), F-actin, connexin-43 (CX-43), ß-catenin, and N-cadherin. The BTB was ultimately disrupted in UPEC-infected rat testes, and blood samples from UPEC-induced orchitis in these animals were positive for anti-sperm antibodies. Furthermore, we herein also demonstrated that mTOR complex 1 (mTORC1) over-activation and mTORC2 suppression contributed to the disturbance in the balance between BTB "opening" and "closing." More importantly, rapamycin (a specific mTORC1 inhibitor) significantly restored the expression of cell-junction proteins and exerted a protective effect on the BTB during UPEC infection. We further confirmed that short-term treatment with rapamycin did not aggravate spermatogenic degeneration in infected rats. Collectively, this study showed an association between abnormal activation of the mTOR-signaling pathway and BTB impairment during UPEC-induced orchitis, which may provide new insights into a potential treatment strategy for testicular infection.


Assuntos
Barreira Hematotesticular/imunologia , Infecções por Escherichia coli/imunologia , Alvo Mecanístico do Complexo 1 de Rapamicina/imunologia , Alvo Mecanístico do Complexo 2 de Rapamicina/imunologia , Infecções Urinárias/imunologia , Escherichia coli Uropatogênica/imunologia , Animais , Barreira Hematotesticular/metabolismo , Células Cultivadas , Infecções por Escherichia coli/metabolismo , Infecções por Escherichia coli/microbiologia , Humanos , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Orquite/imunologia , Orquite/metabolismo , Orquite/microbiologia , Ratos Sprague-Dawley , Células de Sertoli/imunologia , Células de Sertoli/metabolismo , Células de Sertoli/microbiologia , Espermatogênese/imunologia , Testículo/imunologia , Testículo/metabolismo , Proteínas de Junções Íntimas/imunologia , Proteínas de Junções Íntimas/metabolismo , Infecções Urinárias/metabolismo , Infecções Urinárias/microbiologia , Escherichia coli Uropatogênica/fisiologia
12.
J Ethnopharmacol ; 274: 114053, 2021 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-33746003

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Phaseaoli pericarpium (bean pods) is a pharmacopeial plant material traditionally used as a diuretic and antidiabetic agents. Diuretic activity of pod extracts was reported first in 1608. Since then Phaseoli pericarpium tea figures in many textbooks as medicinal plant material used by patients. AIM OF THE STUDY: Despite the traditional use of extracts from Phaseolium vulgaris pericarp, limited information is available on bioactivity, chemical composition, and bioavailability of such preparations. The following study aimed to investigate the phytochemical composition, the in vitro permeability of selected extract's constituents over the Caco-2 permeation system, and potential antivirulence activity against uropathogenic Escherichia coli of a hydroalcoholic Phaseoli pericarpium extract (PPX) in vitro to support its traditional use as a remedy used in urinary tract infections. MATERIAL AND METHODS: The chemical composition of the extract PPX [ethanol:water 7:3 (v/v)] investigated by using UHPLC-DAD-MSn and subsequent dereplication. The permeability of compounds present in PPX was evaluated using the Caco-2 monolayer permeation system. The influence of PPX on uropathogenic E. coli (UPEC) strain NU14 proliferation and against the bacterial adhesion to T24 epithelial cells was determined by turbidimetric assay and flow cytometry, respectively. The influence of the extract on the mitochondrial activity of T24 host cells was monitored by MTT assay. RESULTS: LC-MSn investigation and dereplication, indicated PPX extract to be dominated by a variety of flavonoids, with rutin as a major compound, and soyasaponin derivatives. Rutin, selected soyasaponins and fatty acids were shown to permeate the Caco-2 monolayer system, indicating potential bioavailability following oral intake. The extract did not influence the viability of T24 cells after 1.5h incubation at 2 mg/mL and UPEC. PPX significantly reduced the bacterial adhesion of UPEC to human bladder cells in a concentration-dependent manner (0.5-2 mg/mL). Detailed investigations by different incubation protocols indicated that PPX seems to interact with T24 cells, which subsequently leads to reduced recognition and adhesion of UPEC to the host cell membrane. CONCLUSIONS: PPX is characterised by the presence of flavonoids (e.g. rutin) and saponins, from which selected compounds might be bioavailable after oral application, as indicated by the Caco-2 permeation experiments. Rutin and some saponins can be considered as potentially bioavailable after the oral intake. The concentration-dependent inhibition of bacterial adhesion of UPEC to T24 cells justifies the traditional use of Phaseoli pericarpium in the prevention and treatment of urinary tract infections.


Assuntos
Aderência Bacteriana/efeitos dos fármacos , Phaseolus , Extratos Vegetais/farmacologia , Escherichia coli Uropatogênica/efeitos dos fármacos , Linhagem Celular , Células Epiteliais/metabolismo , Etanol/química , Flavonoides/análise , Flavonoides/farmacologia , Humanos , Permeabilidade/efeitos dos fármacos , Compostos Fitoquímicos/análise , Compostos Fitoquímicos/farmacologia , Extratos Vegetais/química , Saponinas/análise , Saponinas/farmacologia , Sementes/química , Solventes/química , Escherichia coli Uropatogênica/fisiologia , Água/química
13.
Microbiol Res ; 246: 126709, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33578264

RESUMO

Uropathogenic Escherichia coli (UPEC) is a major cause of urinary tract infections (UTI). UPEC persister bacteria play crucial roles in clinical treatment failure and relapse. Although DNA methylation is known to regulate gene expression, its role in persister formation has not been investigated. Here, we show that Δdam (adenine methylase) mutant from UPEC strain UTI89 had significant defect in persister formation and complementation of the Δdam mutant restored this defect. Using PacBio sequencing of methylome and RNA sequencing of Δdam, we defined, for the first time, the role of Dam in persister formation. We found that Δdam mutation had an overwhelming effect on demethylation of the genome and the demethylation sites affected expression of genes involved in broad transcriptional and metabolic processes. Using comparative COG analysis of methylome and transcriptome, we demonstrate that Dam mediates persister formation through transcriptional control, cell motility, DNA repair and metabolite transport processes. These findings provide the first evidence and molecular basis for DNA methylation mediated persister formation and implicate Dam DNA methylation as a potential drug target for persister bacteria.


Assuntos
Adaptação Fisiológica , Adenina/metabolismo , Metilação de DNA , Metiltransferases/genética , Escherichia coli Uropatogênica/fisiologia , Reparo do DNA , DNA Bacteriano , Epigenoma , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Humanos , Mutação , Análise de Sequência de RNA , Estresse Fisiológico , Transcriptoma , Infecções Urinárias/microbiologia , Virulência
14.
Infect Immun ; 89(4)2021 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-33468577

RESUMO

Uropathogenic Escherichia coli (UPEC), the primary etiologic agent of urinary tract infections (UTIs), encounters a restrictive population bottleneck within the female mammalian bladder. Its genetic diversity is restricted during establishment of cystitis because successful UPEC must invade superficial bladder epithelial cells prior to forming clonal intracellular bacterial communities (IBCs). In this study, we aimed to understand UPEC population dynamics during ascending pyelonephritis, namely, formation of kidney bacterial communities (KBCs) in the renal tubular lumen and nucleation of renal abscesses. We inoculated the bladders of both male and female C3H/HeN mice, a background which features vesicoureteral reflux; we have previously shown that in this model, males develop severe, high-titer pyelonephritis and renal abscesses much more frequently than females. Mice were infected with 40 isogenic, PCR-tagged ("barcoded") UPEC strains, and tags remaining in bladder and kidneys were ascertained at intervals following infection. In contrast to females, males maintained a majority of strains within both the bladder and kidneys throughout the course of infection, indicating only a modest host-imposed bottleneck on overall population diversity during successful renal infection. Moreover, the diverse population in the infected male kidneys obscured any restrictive bottleneck in the male bladder. Finally, using RNA in situ hybridization following mixed infections with isogenic UPEC bearing distinct markers, we found that despite their extracellular location (in the urinary space), KBCs are clonal in origin. This finding indicates that even with bulk reflux of infected bladder urine into the renal pelvis, successful ascension of UPEC to establish the tubular niche is an uncommon event.


Assuntos
Infecções por Escherichia coli/microbiologia , Nefrite/microbiologia , Infecções Urinárias/microbiologia , Escherichia coli Uropatogênica/fisiologia , Animais , Modelos Animais de Doenças , Feminino , Regulação Bacteriana da Expressão Gênica , Humanos , Masculino , Camundongos , Dinâmica Populacional , Fatores Sexuais
15.
Immunobiology ; 226(1): 152020, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33246308

RESUMO

The bladder epithelial cells elicit robust innate immune responses against urinary tract infections (UTIs) for preventing the bacterial colonization. Physiological fluctuations in circulating estrogen levels in women increase the susceptibility to UTI pathogenesis, often resulting in adverse health outcomes. Dr adhesin bearing Escherichia coli (Dr E. coli) cause recurrent UTIs in menopausal women and acute pyelonephritis in pregnant women. Dr E. coli bind to epithelial cells via host innate immune receptor CD55, under hormonal influence. The role of estrogens or estrogen receptors (ERs) in regulating the innate immune responses in the bladder are poorly understood. In the current study, we investigated the role of ERα, ERß and GPR30 in modulating the innate immune responses against Dr E. coli induced UTI using human bladder epithelial carcinoma 5637 cells (HBEC). Both ERα and ERß agonist treatment in bladder cells induced a protection against Dr E. coli invasion via upregulation of TNFα and downregulation of CD55 and IL10, and these effects were reversed by action of ERα and ERß antagoinsts. In contrast, the agonist-mediated activation of GPR30 led to an increased bacterial colonization due to suppression of innate immune factors in the bladder cells, and these effects were reversed by the antagonist-mediated suppression of GPR30. Further, siRNA-mediated ERα knockdown in the bladder cells reversed the protection against bacterial invasion observed in the ERα positive bladder cells, by modulating the gene expression of TNFα, CD55 and IL10, thus confirming the protective role of ERα. We demonstrate for the first time a protective role of nuclear ERs, ERα and ERß but not of membrane ER, GPR30 against Dr E. coli invasion in HBEC 5637 cells. These findings have many clinical implications and suggest that ERs may serve as potential drug targets towards developing novel therapeutics for regulating local innate immunity and treating UTIs.


Assuntos
Células Epiteliais/imunologia , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/metabolismo , Neoplasias da Bexiga Urinária/imunologia , Bexiga Urinária/imunologia , Bexiga Urinária/patologia , Infecções Urinárias/metabolismo , Escherichia coli Uropatogênica/fisiologia , Adesinas de Escherichia coli/metabolismo , Animais , Proliferação de Células , Células Cultivadas , Suscetibilidade a Doenças , Receptor alfa de Estrogênio/genética , Receptor beta de Estrogênio/genética , Estrogênios/metabolismo , Feminino , Humanos , Imunidade Inata , Menopausa , Camundongos , Terapia de Alvo Molecular , Gravidez , RNA Interferente Pequeno/genética , Receptores de Estrogênio , Receptores Acoplados a Proteínas G
16.
Libyan J Med ; 16(1): 1845444, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33170767

RESUMO

Biofilm is an important virulent marker attributed to the development of urinary tract infections (UTIs) by uropathogenic E. coli (UPEC). Drug-resistant and biofilm-producing UPEC are highly problematic causing catheter-associated or recurrent UTIs with significant morbidity and mortality. The aim of the current study was to investigate the prevalence of biofilm formation and phylogenetic groups in drug-resistant UPEC to predict their ability to cause disease. This prospective study was conducted at the Department of Microbiology, University of Karachi from January to June 2019. A total of 50 highly drug-resistant UPEC were selected for this study. UPEC isolates were screened to form biofilm by Congo-red agar (CRA) and microtiter plate (MTP) technique. The representative biofilm-producing isolates were analysed by scanning electron microscopy (SEM) monitoring. Phylogenetic analysis was done by PCR method based on two preserved genes; chuA, yjaA and TspE4-C2 DNA fragment. On CRA 34 (68%) UPEC were slime producers, while on MTP 20 (40%) were strong biofilm producers, 19 (38%) moderate and 11 (22%) were low to negligible biofilm producers. Molecular typing confirmed that phylogenetic group B2 was prevalent in drug resistant UPEC strains. Pathogenic strains belonged to phylogenetic group B2 and D were found to have greater biofilm forming ability as compare to non-pathogenic commensal strains that belonged to phylogenetic group A. Our results indicate that biofilm formation vary in drug resistant UPEC belonged to different phylogenetic groups. This study indicates possible link between in vitro biofilm formation and phylogenetic groups of UPEC, therefore this knowledge might be helpful to predict the pathogenic potential of UPEC and help design strategies for controlling UTIs.


Assuntos
Biofilmes/crescimento & desenvolvimento , Farmacorresistência Bacteriana/genética , Infecções por Escherichia coli/microbiologia , Infecções Urinárias/microbiologia , Escherichia coli Uropatogênica/fisiologia , Humanos , Filogenia , Estudos Prospectivos , Escherichia coli Uropatogênica/genética
17.
J Vis Exp ; (166)2020 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-33346201

RESUMO

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of UTI in male and female mice have illustrated the procedures for bacterial inoculation and enumeration in urine and tissues. During an initial bladder infection in C57BL/6 mice, UPEC establish latent reservoirs inside bladder epithelial cells that persist following clearance of UPEC bacteriuria. This model builds on these studies to examine rUTI caused by the emergence of UPEC from within latent bladder reservoirs. The urogenital bacterium Gardnerella vaginalis is used as the trigger of rUTI in this model because it is frequently present in the urogenital tracts of women, especially in the context of vaginal dysbiosis that has been associated with UTI. In addition, a method for in situ bladder fixation followed by scanning electron microscopy (SEM) analysis of bladder tissue is also described, with potential application to other studies involving the bladder.


Assuntos
Infecções por Escherichia coli/microbiologia , Gardnerella vaginalis/fisiologia , Bexiga Urinária/microbiologia , Bexiga Urinária/patologia , Infecções Urinárias/microbiologia , Animais , Modelos Animais de Doenças , Reservatórios de Doenças/microbiologia , Infecções por Escherichia coli/patologia , Infecções por Escherichia coli/urina , Feminino , Camundongos Endogâmicos C57BL , Recidiva , Espectrofotometria , Bexiga Urinária/ultraestrutura , Infecções Urinárias/patologia , Infecções Urinárias/urina , Urina/citologia , Escherichia coli Uropatogênica/fisiologia
18.
Front Immunol ; 11: 570872, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33193351

RESUMO

Reproductive tract infections contribute to the development of testicular inflammatory lesions, leading to male infertility. Previous research shows that the activation of the NLRP3 inflammasome in orchitis promotes the secretion and maturation of IL-1ß and, thus, decreases male fertility. The calcium-sensing receptor (CaSR) is closely related to the secretion of proinflammatory cytokines. An increase in the CaSR level promotes the assembly and activation of the NLRP3 inflammasome. However, the role of CaSRs in orchitis is unknown. We first constructed a uropathogenic Escherichia Coli (UPEC) rat orchitis model and then detected the expression of CaSR and NLRP3 inflammatory pathway proteins in testicular macrophages (TM) through RT-PCR and WB, calcium levels in TM through flow cytometry, and proinflammatory factor IL-1ß through ELISA. In addition, testosterone levels in the serum samples were detected using liquid chromatography-mass spectrometry (LC-MS). Here, we show that CaSR upregulation after infection in TM in a rat model of UPEC induces the activation of the NLRP3 inflammasome pathway and thereby enhances IL-1ß secretion and reduces the testosterone level in the blood. Moreover, CaSR inhibitors can alleviate inflammatory impairment. After UPEC challenge in vitro, CaSR promoted NLRP3 expression and released IL-1ß cleaved from TM into the supernatant. Overall, elevated CaSR levels in TM in testes with UPEC-induced orchitis may impair testosterone synthesis through the activation of the NLRP3 pathway and PK2 is an upstream regulatory protein of CaSR. Our research further shows the underlying mechanisms of inflammation-related male infertility and provides anti-inflammatory therapeutic targets for male infertility.


Assuntos
Infecções por Escherichia coli/imunologia , Hormônios Gastrointestinais/metabolismo , Inflamassomos/metabolismo , Macrófagos/imunologia , Neuropeptídeos/metabolismo , Orquite/imunologia , Testículo/imunologia , Escherichia coli Uropatogênica/fisiologia , Animais , Cálcio/metabolismo , Modelos Animais de Doenças , Hormônios Gastrointestinais/genética , Interleucina-1beta/metabolismo , Masculino , Camundongos , Neuropeptídeos/genética , Células RAW 264.7 , Ratos , Ratos Wistar , Receptores de Detecção de Cálcio/metabolismo , Transdução de Sinais
19.
FASEB J ; 34(11): 14572-14587, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32901999

RESUMO

Urinary tract infections (UTI) frequently progress to chronicity in infected individuals but the mechanisms of pathogenesis underlying chronic UTI are not well understood. We examined the role of interleukin (IL)-17A in UTI because this cytokine promotes innate defense against uropathogenic Escherichia coli (UPEC). Analysis of UPEC persistence and pyelonephritis in mice deficient in IL-17A revealed that UPEC CFT073 caused infection at a rate higher than the multidrug resistant strain EC958. Il17a-/- mice exhibited pyelonephritis with kidney bacterial burdens higher than those of wild-type (WT) mice. Synthesis of IL-17A in the bladder reflected a combination of γδ-T and TH 17 cell responses. Analysis of circulating inflammatory mediators at 24h postinoculation identified predictors of progression to chronicity, including IL-6 and monocyte chemoattractant protein-1 (MCP-1). Histological analysis identified infiltrating populations of neutrophils, NK cells, and γδ T cells in the bladder, whereas neutrophils predominated in the kidney. Analysis of the contribution of flagella to chronicity using hyper-flagellated and fliC-deficient UPEC in WT and Il17a-/- mice revealed that, in a host that is deficient for the production of IL-17A, flagella contribute to bacterial persistence. These findings show a role for IL-17A in defense against chronic UTI and a contribution of flagella to the pathogenesis of infection.


Assuntos
Flagelos/metabolismo , Imunidade Inata , Interleucina-17/metabolismo , Subpopulações de Linfócitos T/imunologia , Infecções Urinárias/imunologia , Escherichia coli Uropatogênica/patogenicidade , Animais , Quimiocina CCL2/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Feminino , Flagelos/genética , Flagelina/genética , Flagelina/metabolismo , Interações Hospedeiro-Patógeno , Interleucina-17/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Bexiga Urinária/citologia , Bexiga Urinária/imunologia , Bexiga Urinária/microbiologia , Infecções Urinárias/genética , Infecções Urinárias/microbiologia , Escherichia coli Uropatogênica/genética , Escherichia coli Uropatogênica/fisiologia
20.
Infect Immun ; 88(9)2020 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-32540870

RESUMO

Uropathogenic Escherichia coli (UPEC) is the leading cause of urinary tract infections. These bacteria undertake a multistage infection cycle involving invasion of and proliferation within urinary tract epithelial cells, leading to the rupture of the host cell and dispersal of the bacteria, some of which have a highly filamentous morphology. Here, we established a microfluidics-based model of UPEC infection of immortalized human bladder epithelial cells that recapitulates the main stages of bacterial morphological changes during the acute infection cycle in vivo and allows the development and fate of individual cells to be monitored in real time by fluorescence microscopy. The UPEC-infected bladder cells remained alive and mobile in nonconfluent monolayers during the development of intracellular bacterial communities (IBCs). Switching from a flow of growth medium to human urine resulted in immobilization of both uninfected and infected bladder cells. Some IBCs continued to develop and then released many highly filamentous bacteria via an extrusion-like process, whereas other IBCs showed strong UPEC proliferation, and yet no filamentation was detected. The filamentation response was dependent on the weak acidity of human urine and required component(s) in a low molecular-mass (<3,000 Da) fraction from a mildly dehydrated donor. The developmental fate for bacteria therefore appears to be controlled by multiple factors that act at the level of the whole IBC, suggesting that variable local environments or stochastic differentiation pathways influence IBC developmental fates during infection.


Assuntos
Células Epiteliais/microbiologia , Técnicas Analíticas Microfluídicas , Escherichia coli Uropatogênica/patogenicidade , Escherichia coli Uropatogênica/ultraestrutura , Linhagem Celular Transformada , Movimento Celular , Proliferação de Células , Células Epiteliais/patologia , Células Epiteliais/ultraestrutura , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/patologia , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Microscopia de Fluorescência , Modelos Biológicos , Reologia , Bexiga Urinária/microbiologia , Bexiga Urinária/patologia , Infecções Urinárias/microbiologia , Infecções Urinárias/patologia , Escherichia coli Uropatogênica/fisiologia , Urotélio/microbiologia , Urotélio/patologia
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