Genetic requirements for uropathogenic E. coli proliferation in the bladder cell infection cycle.

Uropathogenic Escherichia coli (UPEC) requires an adaptable physiology to survive the wide range of environments experienced in the host, including gut and urinary tract surfaces. To identify UPEC genes required during intracellular infection, we developed a transposon-directed insertion-site sequencing approach for cellular infection models and searched for genes in a library of ~20,000 UTI89 transposon-insertion mutants that are specifically required at the distinct stages of infection of cultured bladder epithelial cells. Some of the bacterial functional requirements apparent in host bladder cell growth overlapped with those for M9-glycerol, notably nutrient utilization, polysaccharide and macromolecule precursor biosynthesis, and cell envelope stress tolerance. Two genes implicated in the intracellular bladder cell infection stage were confirmed through independent gene deletion studies: neuC (sialic acid capsule biosynthesis) and hisF (histidine biosynthesis). Distinct sets of UPEC genes were also implicated in bacterial dispersal, where UPEC erupts from bladder cells in highly filamentous or motile forms upon exposure to human urine, and during recovery from infection in a rich medium. We confirm that the dedD gene linked to septal peptidoglycan remodeling is required during UPEC dispersal from human bladder cells and may help stabilize cell division or the cell wall during envelope stress created by host cells. Our findings support a view that the host intracellular environment and infection cycle are multi-nutrient limited and create stress that demands an array of biosynthetic, cell envelope integrity, and biofilm-related functions of UPEC. IMPORTANCE: Urinary tract infections (UTIs) are one of the most frequent infections worldwide. Uropathogenic Escherichia coli (UPEC), which accounts for ~80% of UTIs, must rapidly adapt to highly variable host environments, such as the gut, bladder sub-surface, and urine. In this study, we searched for UPEC genes required for bacterial growth and survival throughout the cellular infection cycle. Genes required for de novo synthesis of biomolecules and cell envelope integrity appeared to be important, and other genes were also implicated in bacterial dispersal and recovery from infection of cultured bladder cells. With further studies of individual gene function, their potential as therapeutic targets may be realized. This study expands knowledge of the UTI cycle and establishes an approach to genome-wide functional analyses of stage-resolved microbial infections.

Multispecies bacterial invasion of human host cells.

Urinary tract infection (UTI), one of the most common bacterial infections worldwide, is a typical example of an infection that is often polymicrobial in nature. While the overall infection course is known on a macroscale, bacterial behavior is not fully understood at the cellular level and bacterial pathophysiology during multispecies infection is not well characterized. Here, using clinically relevant bacteria, human epithelial bladder cells and human urine, we establish co-infection models combined with high resolution imaging to compare single- and multi-species bladder cell invasion events in three common uropathogens: uropathogenic Escherichia coli (UPEC), Klebsiella pneumoniae and Enterococcus faecalis. While all three species invaded the bladder cells, under flow conditions the Gram-positive E. faecalis was significantly less invasive compared to the Gram-negative UPEC and K. pneumoniae. When introduced simultaneously during an infection experiment, all three bacterial species sometimes invaded the same bladder cell, at differing frequencies suggesting complex interactions between bacterial species and bladder cells. Inside host cells, we observed encasement of E. faecalis colonies specifically by UPEC. During subsequent dispersal from the host cells, only the Gram-negative bacteria underwent infection-related filamentation (IRF). Taken together, our data suggest that bacterial multispecies invasions of single bladder cells are frequent and support earlier studies showing intraspecies cooperation on a biochemical level during UTI.

Uropathogenic Escherichia coli Subverts Host Autophagic Defenses by Stalling Preautophagosomal Structures to Escape Lysosome Exocytosis.

Urinary tract infections are primarily caused by uropathogenic Escherichia coli (UPEC). UPEC infects bladder epithelial cells (BECs) via fusiform vesicles and escapes into the cytosol by disrupting fusiform vesicle membrane using outer membrane phospholipase PldA, and establishes biofilm-like intracellular bacterial communities (IBCs) for protection from host immune clearance. Cytosolic UPEC is captured by autophagy to form autophagosomes, then transported to lysosomes, triggering the spontaneous exocytosis of lysosomes. The mechanism by which UPEC evades autophagy to recognize and form IBCs remains unclear. Here, we demonstrate that by inhibiting autophagic flux, UPEC PldA reduces the lysosome exocytosis of BECs. By reducing intracellular phosphatidylinositol 3-phosphate levels, UPEC PldA increases the accumulation of NDP52 granules and decreases the targeting of NDP52 to autophagy, hence stalling preautophagosome structures. Thus, our results uncover a critical role for PldA to inhibit autophagic flux, favoring UPEC escapes from lysosome exocytosis, thereby contributing to acute urinary tract infection.

α-Hemolysin promotes uropathogenic E. coli persistence in bladder epithelial cells via abrogating bacteria-harboring lysosome acidification.

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.

Transcriptional alterations in bladder epithelial cells in response to infection with different morphological states of uropathogenic Escherichia coli.

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.

The RyfA small RNA regulates oxidative and osmotic stress responses and virulence in uropathogenic Escherichia coli.

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.

UPEC kidney infection triggers neuro-immune communication leading to modulation of local renal inflammation by splenic IFNγ.

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.

Antiadhesive activity of hydroethanolic extract from bean pods of Phaseolus vulgaris (common bean) against uropathogenic E. coli and permeability of its constituents through Caco-2 cells monolayer.

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.

Sex Differences in Population Dynamics during Formation of Kidney Bacterial Communities by Uropathogenic Escherichia coli.

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.

Estrogen receptors in human bladder cells regulate innate cytokine responses to differentially modulate uropathogenic E. coli colonization.

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.

Prokineticin 2 via Calcium-Sensing Receptor Activated NLRP3 Inflammasome Pathway in the Testicular Macrophages of Uropathogenic Escherichia coli-Induced Orchitis.

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.

Distinct Morphological Fates of Uropathogenic Escherichia coli Intracellular Bacterial Communities: Dependency on Urine Composition and pH.

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.

The oxidative fumarase FumC is a key contributor for E. coli fitness under iron-limitation and during UTI.

The energy required for a bacterium to grow and colonize the host is generated by metabolic and respiratory functions of the cell. Proton motive force, produced by these processes, drives cellular mechanisms including redox balance, membrane potential, motility, acid resistance, and the import and export of substrates. Previously, disruption of succinate dehydrogenase (sdhB) and fumarate reductase (frdA) within the oxidative and reductive tricarboxylic acid (TCA) pathways in uropathogenic E. coli (UPEC) CFT073 indicated that the oxidative, but not the reductive TCA pathway, is required for fitness in the urinary tract. Those findings led to the hypothesis that fumA and fumC encoding fumarase enzymes of the oxidative TCA cycle would be required for UPEC colonization, while fumB of the reductive TCA pathway would be dispensable. However, only UPEC strains lacking fumC had a fitness defect during experimental urinary tract infection (UTI). To further characterize the role of respiration in UPEC during UTI, additional mutants disrupting both the oxidative and reductive TCA pathways were constructed. We found that knock-out of frdA in the sdhB mutant strain background ameliorated the fitness defect observed in the bladder and kidneys for the sdhB mutant strain and results in a fitness advantage in the bladder during experimental UTI. The fitness defect was restored in the sdhBfrdA double mutant by complementation with frdABCD. Taken together, we demonstrate that it is not the oxidative or reductive pathway that is important for UPEC fitness per se, but rather only the oxidative TCA enzyme FumC. This fumarase lacks an iron-sulfur cluster and is required for UPEC fitness during UTI, most likely acting as a counter measure against exogenous stressors, especially in the iron-limited bladder niche.

Matrix stiffness regulates endosomal escape of uropathogenic E. coli.

Uropathogenic E. coli (UPEC) infection in vivo is characterized by invasion of bladder umbrella epithelial cells followed by endosomal escape and proliferation in the cytoplasm to form intracellular bacterial communities. By contrast, UPEC infection in tissue culture models results in bacteria being trapped within Lamp1-positive endosomes where proliferation is limited. Pharmacological disruption of the actin cytoskeleton has been shown to facilitate UPEC endosomal escape in vitro and extracellular matrix stiffness is a well-characterized physiological regulator of actin dynamics; therefore, we hypothesized that substrate stiffness may play a role in UPEC endosomal escape. Using functionalized polyacrylamide substrates, we found that at physiological stiffness, UPEC escaped the endosome and proliferated rapidly in the cytoplasm of bladder epithelial cells. Dissection of the cytoskeletal signaling pathway demonstrated that inhibition of the Rho GTPase RhoB or its effector PRK1 was sufficient to increase cytoplasmic bacterial growth and that RhoB protein level was significantly reduced at physiological stiffness. Our data suggest that tissue stiffness is a critical regulator of intracellular bacterial growth. Due to the ease of doing genetic and pharmacological manipulations in cell culture, this model system may provide a useful tool for performing mechanistic studies on the intracellular life cycle of uropathogens.

Activation of the NLRP3 Inflammasome Pathway by Prokineticin 2 in Testicular Macrophages of Uropathogenic Escherichia coli- Induced Orchitis.

Infections of the reproductive tract are known to contribute to testicular inflammatory impairment, leading to an increase of pro-inflammatory cytokines such as IL-1ß, and a decline in sperm quality. Prokineticin 2 (PK2), a secretory protein, is closely associated with the secretion of pro-inflammatory cytokines in inflamed tissue. It was reported that increased PK2 is related to the upregulation of IL-1ß, but the underlying mechanism remains elusive. Here, we illustrated that PK2 was upregulated in testicular macrophages (TM) in a rat model of uropathogenic Escherichia coli (UPEC) infection, which induced the activation of the NLRP3 inflammasome pathway to boost IL-1ß secretion. Administration of PK2 inhibitor alleviated the inflammatory damage and suppressed IL-1ß secretion. Moreover, PK2 promoted NLRP3 expression and the release of cleaved IL-1ß from TM to the supernatants after the challenge with UPEC in vitro. IL-1ß in the supernatants affected Leydig cells by suppressing the expression of genes encoding for the enzymes P450scc and P450c17, which are involved in testosterone production. Overall, we revealed that increased PK2 levels in TM in UPEC-induced orchitis may impair testosterone synthesis via the activation of the NLRP3 pathway. Our study provides a new insight into the mechanisms underlying inflammation-associated male infertility and suggests an anti-inflammatory therapeutic target for male infertility.

Orthosipon stamineus extract exerts inhibition of bacterial adhesion and chaperon-usher system of uropathogenic Escherichia coli-a transcriptomic study.

Specific recognition and bacterial adhesion to host cells by uropathogenic Escherichia coli (UPEC) are the first steps towards infection of epithelial tissue of the human urogenital system. Therefore, targeting of UPEC virulence factors, relevant for adhesion, is a promising approach for prevention of recurrent urinary tract infections (UTI). A fully characterized plant-derived aqueous extract from the leaves of Orthosiphon stamineus (OWE), a plant traditionally used in clinical practice in Europe and Asia for UTI, has been shown to exert strong antiadhesive effects under in vitro and in vivo conditions. For improved understanding of the underlying mechanisms, transcriptome analysis of OWE-treated UPEC strain UTI89 by Illumina sequencing and cross-validation of these data by qPCR indicated significant downregulation of bacterial adhesins (curli, type 1-, F1C-, and P fimbriae) and of the chaperone-mediated protein folding/unfolding and pilus assembly process; in contrast, flagellar and motility-related genes were upregulated. We conclude that OWE transforms the sessile lifestyle of bacteria into a motile one and therefore disables bacterial attachment to the host cell. Additionally, the extract inhibited gene expression of multiple iron-acquisition systems (ent, fep, feo, fhu, chu, sit, ybt). The present study explains the antiadhesive and anti-infective effect of the plant extract by pinpointing specific biochemical and molecular targets.

Antiadhesive phthalides from Apium graveolens fruits against uropathogenic E. coli.

ETHNOPHARMACOLOGICAL RELEVANCE: Fruits of Apium graveolens (celery) are used traditionally in Persian and European medicine for the treatment of uncomplicated urinary tract infections. AIM OF THE STUDY: The study aimed at identifying potential antiadhesive compounds from celery extracts to provide strategies for improved standardization of the herbal material. MATERIALS AND METHODS: Decoction, hydroalcoholic and acetone extracts were prepared from celery fruits. Bioassay-guided fractionation was performed by Fast Centrifugal Partition Chromatography and preparative HPLC, followed by LC-MS and NMR investigations for structure elucidation. The antiadhesive activity of extracts, fractions and purified compounds was assessed by flow cytometry, evaluating the adhesion of fluorescent-labelled uropathogenic bacteria (UPEC NU14) to T24 bladder cells; mannose served as positive control. Influence of the extract on gene expression of selected adhesins and fitness genes was monitored by qPCR. RESULTS: Concentration-dependent antiadhesive activity was found for the hydroalcoholic and even more for the acetone extract AE (IC50 85 µg/mL) from celery fruits. Bioassay-guided fractionation revealed the presence of the phthalides senkyunolide (1, inactive) and sedanenolide (2, IC50 790 µM). 2 is assessed as the main antiadhesive compound, which accounts for 4.0% in the water extract, for 18% in the hydroethanolic extract and for 71% in AE. Additionally a similar phthalide, Z-ligustilide (5), was shown to exert an IC50 of 611 µM. Furthermore, AE caused a significant upregulation of fimH and sfaG in free floating, non-attached UPEC and significantly down-regulated these genes in adherent bacteria. CONCLUSIONS: Phthalides were identified as the main active compounds in polar and semi-polar extracts, which exert strong antiadhesive activity against uropathogenic E. coli. The current findings support the traditional use in phytotherapy for urinary tract infections and provide a base for standardization of the herbal material.

Inefficient N2-Like Neutrophils Are Promoted by Androgens During Infection.

Neutrophils are major effectors of acute inflammation against infection and tissue damage, with ability to adapt their phenotype according to the microenvironment. Although sex hormones regulate adaptive immune cells, which explains sex differences in immunity and infection, little information is available about the effects of androgens on neutrophils. We therefore aimed to examine neutrophil recruitment and plasticity in androgen-dependent and -independent sites under androgen manipulation. By using a bacterial model of prostate inflammation, we showed that neutrophil recruitment was higher in testosterone-treated rats, with neutrophil accumulation being positively correlated to serum levels of testosterone and associated to stronger inflammatory signs and tissue damage. Testosterone also promoted LPS-induced neutrophil recruitment to the prostate, peritoneum, and liver sinusoids, as revealed by histopathology, flow cytometry, and intravital microscopy. Strikingly, neutrophils in presence of testosterone exhibited an impaired bactericidal ability and a reduced myeloperoxidase activity. This inefficient cellular profile was accompanied by high expression of the anti-inflammatory cytokines IL10 and TGFß1, which is compatible with the "N2-like" neutrophil phenotype previously reported in the tumor microenvironment. These data reveal an intriguing role for testosterone promoting inefficient, anti-inflammatory neutrophils that prolong bacterial inflammation, generating a pathogenic environment for several conditions. However, these immunomodulatory properties might be beneficially exploited in autoimmune and other non-bacterial diseases.

Cytotoxic Necrotizing Factor 1 Downregulates CD36 Transcription in Macrophages to Induce Inflammation During Acute Urinary Tract Infections.

Urinary tract infections (UTIs) caused by uropathogenic Escherichia coli (UPEC) induce cystitis, pyelonephritis, and can cause kidney scarring and failure if inflammation is not under control. The detailed effects of cytotoxic necrotizing factor 1 (CNF1), the key UPEC toxin, on the pathogenicity of UPEC remain unclear. CD36 is an important scavenger receptor, responsible for pathogen and apoptotic cell clearance, and plays an essential role in host immune defense and homeostasis. Regulation of CD36 by bacterial toxins has not been reported. In this study, using a pyelonephritis mouse model, CNF1 was observed to contribute to increasing neutrophils and bacterial titers in infected bladder and kidney tissues, resulting in severe inflammation and tissue damage. CD36 expression in macrophages was found to be decreased by CNF1 in vitro and in vivo. We demonstrated that CNF1 attenuated CD36 transcription by decreasing expressions of its upstream transcription factors LXRß and C/EBPα and their recruitment to the CD36 promotor. In addition, Cdc42 was found to be involved in CNF1-mediated downregulation of LXRß. Our study investigated the pathogenesis of cnf1-carrying UPEC, which affected host innate immune defenses and homeostasis through regulation of CD36 in macrophages during acute UTIs.

A broad-spectrum antibiotic, DCAP, reduces uropathogenic Escherichia coli infection and enhances vorinostat anticancer activity by modulating autophagy.

The cellular recycling pathway of autophagy plays a fundamental role in adaptive responses to nutrient deprivation and other forms of stress under physiological and pathological conditions. However, autophagy can also be a double-edge sword during certain bacterial infections (such as urinary tract infections) and in cancer, where it can be hijacked by the pathogens and cancer cells, respectively, to promote their own survival. Thus, autophagy modulation can potentially have multiple effects in multiple contexts and this property can be leveraged to improve outcomes. In this report, we identify that a broad-spectrum antibiotic, 2-((3-(3, 6-dichloro-9H-carbazol-9-yl)-2-hydroxypropyl) amino)-2-(hydroxymethyl) propane-1, 3-diol (DCAP) modulates autophagy. We employed combined biochemical, fluorescence microscopy and correlative light electron microscopy approaches to demonstrate that DCAP treatment blocks autophagy at the late stages by preventing autophagolysosome maturation and interrupting the autophagic flux. We further show that, DCAP significantly reduces UPEC infection in urinary tract epithelial cells via inhibition of autophagy. Finally, we reveal that DCAP enhances the anticancer activity of the histone acetyltransferase (HDAC) inhibitor, vorinostat, which has been reported to increase susceptibility to bacterial infections as a common adverse effect. Collectively, our data support the concept that DCAP represents a valuable chemical scaffold for the development of an innovative class of bactericidal autophagy inhibitors for treatment of urinary tract infections and/or for adjuvant therapy in cancer treatment.