Petra Lüthje

Uropathogenic Escherichia coli Modulates Immune Responses and Its Curli Fimbriae Interact with the Antimicrobial Peptide LL-37

Bacterial growth in multicellular communities, or biofilms, offers many potential advantages over single-cell growth, including resistance to antimicrobial factors. Here we describe the interaction between the biofilm-promoting components curli fimbriae and cellulose of uropathogenic E. coli and the endogenous antimicrobial defense in the urinary tract. We also demonstrate the impact of this interplay on the pathogenesis of urinary tract infections. Our results suggest that curli and cellulose exhibit differential and complementary functions. Both of these biofilm components were expressed by a high proportion of clinical E. coli isolates. Curli promoted adherence to epithelial cells and resistance against the human antimicrobial peptide LL-37, but also increased the induction of the proinflammatory cytokine IL-8. Cellulose production, on the other hand, reduced immune induction and hence delayed bacterial elimination from the kidneys. Interestingly, LL-37 inhibited curli formation by preventing the polymerization of the major curli subunit, CsgA. Thus, even relatively low concentrations of LL-37 inhibited curli-mediated biofilm formation in vitro. Taken together, our data demonstrate that biofilm components are involved in the pathogenesis of urinary tract infections by E. coli and can be a target of local immune defense mechanisms.

Vitamin D Induction of the Human Antimicrobial Peptide Cathelicidin in the Urinary Bladder

The urinary tract is frequently being exposed to potential pathogens and rapid defence mechanisms are therefore needed. Cathelicidin, a human antimicrobial peptide is expressed and secreted by bladder epithelial cells and protects the urinary tract from infection. Here we show that vitamin D can induce cathelicidin in the urinary bladder. We analyzed bladder tissue from postmenopausal women for expression of cathelicidin, before and after a three-month period of supplementation with 25-hydroxyvitamin D3 (25D3). Cell culture experiments were performed to elucidate the mechanisms for cathelicidin induction. We observed that, vitamin D per se did not up-regulate cathelicidin in serum or in bladder tissue of the women in this study. However, when the bladder biopsies were infected with uropathogenic E. coli (UPEC), a significant increase in cathelicidin expression was observed after 25D3 supplementation. This observation was confirmed in human bladder cell lines, even though here, cathelicidin induction occurred irrespectively of infection. Vitamin D treated bladder cells exerted an increased antibacterial effect against UPEC and colocalization to cathelicidin indicated the relevance of this peptide. In the light of the rapidly growing problem of resistance to common urinary tract antibiotics, we suggest that vitamin D may be a potential complement in the prevention of UTI.

Estrogen Supports Urothelial Defense Mechanisms

Estrogen supports urothelial defense against infection by increasing the expression of antimicrobial peptides and by protecting epithelial integrity. Urothelial Defense Tug-of-War It is well known that urinary tract infections (UTIs) are common in women, but which women are at more risk of UTIs is still far from certain, with some studies suggesting that it’s younger women, and others showing evidence of increased risk after menopause. Now, a study by Lüthje and coauthors suggests that both of those views may be partially correct and demonstrates the mechanisms for each. To understand how estrogen contributes to UTI pathogenesis, the authors examined cells from the urothelium (bladder lining) of menstruating women and older, postmenopausal subjects, studying the postmenopausal women before and after a 2-week period of estrogen supplementation. Then, to determine the mechanistic basis for their observations, they studied urothelial cells in an estrogen-depleted mouse model and in vitro. Thus, they demonstrated that estrogen has a protective effect on the urothelium, stimulating production of antimicrobial peptides and strengthening the attachment between urothelial cells. At the same time, the authors found that estrogen also increases the amount of bacteria taken up inside the urothelial cells. On the basis of the findings of Lüthje et al., one can conclude that young women may experience a greater incidence of UTIs because the high-estrogen environment increases the risk of bacterial invasion of the urothelium. Conversely, postmenopausal women may have more difficulty fighting off the infections because of their decreased production of antimicrobial peptides and diminished integrity of the urothelial lining. Additional studies of human and animal subjects will be needed to learn more about the molecular mechanism of estrogen’s effects on the urothelium and understand what determines the balance between its pro- and anti-UTI effects. However, this line of research does advance us closer to understanding and eventually helping prevent the problem of recurrent UTIs in women. Epidemiological data imply a role of estrogen in the pathogenesis of urinary tract infections (UTIs), although the underlying mechanisms are not well understood. However, it is thought that estrogen supplementation after menopause decreases the risk of recurrent infections. We sought to investigate the influence of estrogen on host-pathogen interactions and the consequences for UTI pathogenesis. We analyzed urothelial cells from menstruating and postmenopausal women before and after a 2-week period of estrogen supplementation, and also studied the influence of estradiol during Escherichia coli UTI in a mouse infection model. Important findings were confirmed in two human urothelial cell lines. We identified two epithelial defense mechanisms modulated by estrogen. Estrogen induced the expression of antimicrobial peptides, thereby enhancing the antimicrobial capacity of the urothelium and restricting bacterial multiplication. In addition, estrogen promoted the expression and redistribution of cell-cell contact–associated proteins, thereby strengthening the epithelial integrity and preventing excessive loss of superficial cells during infection. These two effects together may prevent bacteria from reaching deeper layers of the urinary tract epithelium and developing reservoirs that can serve as a source for recurrent infections. Thus, this study presents some underlying mechanisms for the beneficial effect of estradiol after menopause and supports the application of estrogen in postmenopausal women suffering from recurrent UTI.

Estrogenic action on innate defense mechanisms in the urinary tract

Clinical data suggest an impact of estrogen on the pathogenesis of urinary tract infections (UTI). In particular, women after menopause often suffer from recurrent UTI, characterized by at least three acute UTI episodes within a year. Aside from bacterial factors promoting persistence within the urinary bladder, the low estrogen levels induce structural and chemical changes in the urogenital tract which facilitate UTI. Increased residual urine volume and changes in the vaginal microflora are well documented risk factors. Local supplementation with estrogen can at least partly reverse these changes. Treatment allows the re-establishment of a lactobacilli-dominated vaginal microflora and improves epithelial differentiation and integrity in the urogenital tract. This estrogenic effect on the epithelium is marked by an increased production of antimicrobial peptides and a tighter intercellular connection, preventing bacteria from reaching cells where they can hide and later emerge and cause a new infection. Estrogen in the dosages and applications used to date is considered safe for the endometrium in the majority of women. Based on the actions and safety of estrogen, local supplementation thus offers a treatment option for postmenopausal women suffering from recurrent UTI.

Virulence Factors of Uropathogenic E. coli and Their Interaction with the Host

Urinary tract infections (UTIs) belong to the most common infectious diseases worldwide. The most frequently isolated pathogen from uncomplicated UTIs is Escherichia coli. To establish infection in the urinary tract, E. coli has to overcome several defence strategies of the host, including the urine flow, exfoliation of urothelial cells, endogenous antimicrobial factors and invading neutrophils. Thus, uropathogenic E. coli (UPEC) harbour a number of virulence and fitness factors enabling the bacterium to resist and overcome these different defence mechanisms. There is no particular factor which allows the identification of UPEC among the commensal faecal flora apart from the ability to enter the urinary tract and cause an infection. Many of potential virulence or fitness factors occur moreover with high redundancy. Fimbriae are inevitable for adherence to and invasion into the host cells; the type 1 pilus is an established virulence factor in UPEC and indispensable for successful infection of the urinary tract. Flagella and toxins promote bacterial dissemination, while different iron-acquisition systems allow bacterial survival in the iron-limited environment of the urinary tract. The immune response to UPEC is primarily mediated by toll-like receptors recognising lipopolysaccharide, flagella and other structures on the bacterial surface. UPEC have the capacity to subvert this immune response of the host by means of actively impacting on pro-inflammatory signalling pathways, or by physical masking of immunogenic structures. The large repertoire of bacterial virulence and fitness factors in combination with host-related differences results in a complex interaction between host and pathogen in the urinary tract.

Ag43 promotes persistence of uropathogenic Escherichia coli isolates in the urinary tract.

Uropathogenic E scherichia coli (UPEC) harbors an arsenal of virulence factors which enable it to infect the host. Adhesion factors of E. coli , facilitating the initial colonization of epithelia and eventually leading to invasion of the host cell, have been described extensively elsewhere ([2][1

Markers of innate immune activity in patients with type 1 and type 2 diabetes mellitus and the effect of the anti‐oxidant coenzyme Q10 on inflammatory activity

Major long‐term complications in patients with diabetes are related to oxidative stress, caused by the hyperglycaemia characteristic for diabetes mellitus. The anti‐oxidant coenzyme Q10 (CoQ10) has therefore been proposed as a beneficial supplement to diabetes treatment. Apart from its anti‐oxidative function, CoQ10 appears to modulate immune functions by largely unknown mechanisms. The aim of this study was therefore to investigate the effect of CoQ10 on antimicrobial peptides and natural killer (NK) cells, both innate immune components implicated in the pathogenesis of diabetes and diabetes‐associated long‐term complications such as cardiovascular disease. We determined serum levels of antimicrobial peptides and the phenotype of NK cells isolated from peripheral blood of patients with type 1 (T1DM) or type 2 diabetes mellitus (T2DM) and from healthy controls. In addition, the same parameters were determined in diabetic patients after a 12‐week period of CoQ10 supplementation. Two antimicrobial peptides, the human cathelicidin antimicrobial peptide (CAMP) and the human beta defensin 1 (hBD1), were reduced in serum from patients with T1DM. This defect was not reversible by CoQ10 supplementation. In contrast, CoQ10 reduced the levels of circulating hBD2 in these patients and induced changes in subset distribution and activation markers in peripheral NK cells. The results of the present study open up novel approaches in the prevention of long‐term complications associated to T1DM, although further investigations are needed.

Yersinia enterocolitica-mediated degradation of neutrophil extracellular traps (NETs).

Neutrophil extracellular trap (NET) formation is described as a tool of the innate host defence to fight against invading pathogens. Fibre-like DNA structures associated with proteins such as histones, cell-specific enzymes and antimicrobial peptides are released, thereby entrapping invading pathogens. It has been reported that several bacteria are able to degrade NETs by nucleases and thus evade the NET-mediated entrapment. Here we studied the ability of three different Yersinia serotypes to induce and degrade NETs. We found that the common Yersinia enterocolitica serotypes O:3, O:8 and O:9 were able to induce NETs in human blood-derived neutrophils during the first hour of co-incubation. At later time points, the NET amount was reduced, suggesting that degradation of NETs has occurred. This was confirmed by NET degradation assays with phorbol-myristate-acetate-pre-stimulated neutrophils. In addition, we found that the Yersinia supernatants were able to degrade purified plasmid DNA. The absence of Ca(2+) and Mg(2+) ions, but not that of a protease inhibitor cocktail, completely abolished NET degradation. We therefore postulate that Y. enterocolitica produces Ca(2+)/Mg(2+)-dependent NET-degrading nucleases as shown for some Gram-positive pathogens.

Control of pathogen growth and biofilm formation using a urinary catheter that releases antimicrobial nitrogen oxides.

Antibacterial nitrogen oxides including nitric oxide are formed from nitrite under acidic conditions. In a continuous-flow model of the urinary bladder we used the retention cuff of an all-silicone Foley catheter as a depot for export of nitrogen oxides. The cuff was filled with sodium nitrite (50mM) and an acidic buffer solution (pH 3.6) and the growth of nine common uropathogens in the surrounding artificial urine was measured along with biofilm formation on the catheter surface. In experiments with control catheters (NaCl) bacteria grew readily and biofilm developed within hours in five of nine strains. In contrast, with test catheters bacterial counts were markedly reduced and biofilm formation by Pseudomonas aeruginosa, Klebsiella pneumoniae, and Enterobacter cloace was prevented, whereas Escherichia coli and Staphylococcus aureus were unaffected. We conclude that antibacterial nitrogen oxides generated in the retention cuff of a urinary catheter diffuse into urine and prevent the growth of urinary pathogens and biofilm formation. Although promising, future studies will reveal if this novel approach can be clinically useful for the prevention of catheter-associated urinary tract infections.

Controlled Evaluation of the New BacT/Alert Virtuo Blood Culture System for Detection and Time to Detection of Bacteria and Yeasts

ABSTRACT We compared the newly approved BacT/Alert Virtuo blood culture system to the BacT/Alert 3D system using 115 clinical bacterial and fungal isolates in 784 simulated blood culture bottles. The time to detection was reduced by roughly 20% in the Virtuo system (P < 0.0001) while the detection rate did not differ.