Polysaccharide Capsule and Sialic Acid-Mediated Regulation Promote Biofilm-Like Intracellular Bacterial Communities during Cystitis

Uropathogenic Escherichia coli (UPEC) is the leading cause of urinary tract infections (UTIs). A murine UTI model has revealed an infection cascade whereby UPEC undergoes cycles of invasion of the bladder epithelium, intracellular proliferation in polysaccharide-containing biofilm-like masses called intracellular bacterial communities (IBC), and then dispersal into the bladder lumen to initiate further rounds of epithelial colonization and invasion. We predicted that the UPEC K1 polysaccharide capsule is a key constituent of the IBC matrix. Compared to prototypic E. coli K1 strain UTI89, a capsule assembly mutant had a fitness defect in functionally TLR4⁺ and TLR4⁻ mice, suggesting a protective role of capsule in inflamed and noninflamed hosts. K1 capsule assembly and synthesis mutants had dramatically reduced IBC formation, demonstrating the common requirement for K1 polysaccharide in IBC development. The capsule assembly mutant appeared dispersed in the cytoplasm of the bladder epithelial cells and failed to undergo high-density intracellular replication during later stages of infection, when the wild-type strain continued to form serial generations of IBC. Deletion of the sialic acid regulator gene nanR partially restored IBC formation in the capsule assembly mutant. These data suggest that capsule is necessary for efficient IBC formation and that aberrant sialic acid accumulation, resulting from disruption of K1 capsule assembly, produces a NanR-mediated defect in intracellular proliferation and IBC development. Together, these data demonstrate the complex but important roles of UPEC polysaccharide encapsulation and sialic acid signaling in multiple stages of UTI pathogenesis.Uropathogenic Escherichia coli (UPEC) is the leading cause of urinary tract infection (UTI), and health care costs for UTI exceed $1.5 billion per year in the United States alone (22). Most UTIs occur in the bladder (cystitis), but more-invasive infections lead to infection of the kidneys (pyelonephritis) and dissemination to the bloodstream (urosepsis) and central nervous system (meningitis). UPEC that expresses K1 capsule, a linear α2-8-linked sialic acid homopolymer, is commonly associated with each of these infections (29, 36). Cystitis is a common syndrome for the treatment of which antibiotics are widely administered. However, recurrent UTI often occurs despite appropriate antibiotic therapy. In addition, a disturbing trend toward increasingly antibiotic-resistant UPEC has been occurring over the past decade (11, 30). Thus, defining new targets for therapy through elucidation of the molecular basis for cystitis, as for other aspects of UTIs, has become increasingly important.UPEC produces cystitis through complex interactions with the host. In mouse models, UPEC adheres to the bladder epithelium by type 1 pili. UPEC invades the epithelium through caveolae/lipid raft-mediated and clathrin-mediated endocytic pathways (20, 21, 40). In addition, UPEC has been shown to enter the bladder epithelium by cyclic AMP (cAMP)-responsive fusiform vesicles where elevations in intraepithelial cAMP can result in exocytosis of the vesicles and expulsion of the bacteria into the bladder lumen (7). To subvert the host response, however, UPEC has been shown in mouse models to escape into the cytoplasm of the infected cell and replicate in biomasses called intracellular bacterial communities (IBC) (2, 40, 43). The presence of IBC has been shown in numerous different mouse strains, as well as in urine sediment from humans experiencing acute and recurring UTIs (23, 53). IBC expand to fill the infected host epithelial cell. The IBC is transient in nature, and soon after its maturation, the bacterial community disperses, exiting the infected cell to invade naïve epithelium. Prior work has shown that the cycle of colonization, invasion, IBC formation, and dispersal is cyclical, producing multiple generations of IBC (31). Failure to initiate or complete IBC formation has been shown to attenuate cystitis (32, 33, 72).The formation of IBC has similarities to that of biofilm communities previously modeled on abiotic surfaces. Bacteria embedded in the IBC have cell-associated appendages such as type 1 pili and antigen 43 (3). The matrix also stains by periodic acid Schiff, suggesting the presence of polysaccharides (3). However, the specific type of polysaccharide present in the IBC matrix is not known. The majority of UPEC isolates produce group 2 capsules, such as the prototype K1 (29, 52). In addition, the UPEC genome encodes colanic acid, β-1,6-N-acetyl-d-glucosamine, and cellulose, all of which have known roles in biofilm formation in vitro (16, 17, 69). We sought to determine if the common K1 polysaccharide contributes to IBC formation.Like other bacterial polysaccharide capsules, K1 capsule has two classical roles in defense against host innate immunity: (i) inhibition of phagocytosis by granulocytes/monocytes (70) and (ii) serum resistance (47). More-recent studies have demonstrated that K capsules may be involved in nonspecific adherence and also in biofilm formation on artificial surfaces (48, 67). Like that of other group 2 capsules, the biogenesis of the K1 capsule requires three genetic regions located at a shared locus for their synthesis, assembly, and exportation (71). Region II gene products (neu) direct the synthesis and modification of the primary monosaccharides used in capsule assembly, thus determining the actual capsule antigen (K) type. Region I (kps) and III (kpsMT) gene products are involved in capsule assembly and export (9, 58, 71).Although epidemiological studies suggest that UPEC is widely encapsulated, with K1 being a leading capsular subtype, few details are known about the complete molecular roles of encapsulation during the pathogenesis of E. coli UTI. Several prior studies have revealed that strains with capsule mutations have reduced fitness, but the specific molecular defects in pathogenesis were not demonstrated (6, 46). Similarly, a recent study found that mutation of K2 capsule synthesis in UPEC strain CFT073, a urosepsis blood isolate, resulted in decreased survival in the mouse urinary tract (10). The capsule mutant strain was most attenuated in the kidneys and urine; however, complementation of the capsule synthesis mutant resulted in a significant increase in the number of bacteria recovered from the bladder, suggesting a role for the K2 capsule as a virulence factor in upper and lower UTIs. These studies argue that polysaccharide capsules are important virulence factors during UTI. However, the precise function of capsule in the pathogenic cascade has not been previously studied. Furthermore, previous studies used non-K1 pyelonephritis and sepsis isolates as prototypic strains where specific evaluation of the molecular pathogenesis of cystitis may be better performed using clinical cystitis isolates, particularly carrying the prevalent K1 capsule type.In our present study, we sought to elucidate the role of the K1 capsule of prototypic cystitis isolate UTI89 (12, 44) during cystitis. We hypothesized that the polysialic acid K1 capsule may not only protect UPEC from innate immunity but also form an IBC matrix component, facilitating aggregation of the bacterial communities, which in turn preclude infiltration of host inflammatory mediators and environmental stressors. We discovered that K1 capsule is an important virulence determinant in functionally TLR4⁺ and TLR4⁻ mice and that the K1 capsule is significant in the maintenance of IBC morphology. We further demonstrated that IBC formation was partially restored in a capsule assembly mutant by abrogation of NanR-sialic acid regulation, suggesting a unique role for sialic acid signaling during intracellular UPEC growth. Together, these data suggest that both K1 capsule and sialic acid-dependent regulation have novel roles in the intracellular pathogenesis of cystitis caused by UPEC K1.     

Blood acylpeptide hydrolase activity is a sensitive marker for exposure to some organophosphate toxicants.

Acylpeptide hydrolase (APH) unblocks N-acetyl peptides. It is a major serine hydrolase in rat blood, brain, and liver detected by derivatization with (3)H-diisopropyl fluorophosphate (DFP) or a biotinylated fluorophosphonate. Although APH does not appear to be a primary target of acute poisoning by organophosphorus (OP) compounds, the inhibitor specificity of this secondary target is largely unknown. This study fills the gap and emphasizes blood APH as a potential marker of OP exposure. The most potent in vitro inhibitors for human erythrocyte and mouse brain APH are DFP (IC(50) 11-17 nM), chlorpyrifos oxon (IC(50) 21-71 nM), dichlorvos (IC(50) 230-560 nM), naled (IC(50) 370-870 nM), and their analogs with modified alkyl substituents. (3)H-diisopropyl fluorophosphate is a potent inhibitor of mouse blood and brain APH in vivo (ED(50) 0.09-0.2 mg/kg and 0.02-0.03 mg/l for ip and vapor exposure, respectively). Mouse blood and brain APH and blood butyrylcholinesterase (BChE) are of similar sensitivity to DFP in vitro and in vivo (ip and vapor exposure), but APH inhibition is much more persistent in vivo (still >80% inhibition after 4 days). The inhibitory potency of OP pesticides in vivo in mice varies from APH selective (dichlorvos, naled, and trichlorfon), to APH and BChE selective (profenofos and tribufos), to ChE selective or nonselective (many commercial insecticides). Sarin administered ip at a lethal dose to guinea pigs inhibits blood acetylcholinesterase and BChE completely but erythrocyte APH only partially. Blood APH activity is therefore a sensitive marker for exposure to some but not all OP pesticides and chemical warfare agents.     

Activity and expression of nitric oxide synthase in the hypertrophied rat bladder and the effect of nitric oxide on bladder smooth muscle growth

PURPOSE: We investigated the expression and activity of nitric oxide synthase (NOS) and the localization of cyclic guanosine monophosphate (cGMP) in hypertrophied rat bladder. We also examined whether nitric oxide (NO) has a growth inhibitory effect in bladder smooth muscle cells. MATERIALS AND METHODS: The urethra was partly ligated and the bladder was removed 3 days, 3 or 6 weeks after obstruction. NOS activity was determined as the conversion of L-[14C]citrulline from L-[14C]arginine (Amersham Life Science, Solna, Sweden). Neuronal NOS (nNOS) expression was studied with Western blot analysis and immunohistochemistry. The expression of inducible NOS (iNOS) and cGMP was evaluated by immunohistochemistry. The effect of NO on isolated bladder smooth muscle cell growth was assessed as protein and DNA synthesis by [3H]-leucine and [3H]-thymidine (NEN Life Science Products, Zaventem, Belgium) incorporation, respectively. RESULTS: Ca independent iNOS activity increased after short-term obstruction. Immunohistochemical studies in obstructed bladders demonstrated iNOS expression primarily in urothelial and inflammatory cells. Ca dependent nNOS activity decreased after obstruction, as confirmed by Western blot analysis. The cGMP immunoreactive cells were mainly found within the serosal layer of obstructed bladders. The NO donor DETA-NONOate (Alexis Biochemicals, Lausen, Switzerland) (300 microM.) reduced [3H]-leucine and [ H]-thymidine incorporation by a mean of 29% +/- 2% and 95% +/- 2%, respectively, in cultured bladder smooth muscle cells. CONCLUSIONS: Bladder obstruction caused a small increase in iNOS activity and a decrease in nNOS activity. NO was found to have a growth inhibitory effect in bladder smooth muscle cells, suggesting that changes in NOS activity may influence the progress of bladder hypertrophy.     

Adrenal medullary implants in the dopamine-denervated rat striatum. I. Acute catecholamine levels in grafts and host caudate as determined by HPLC-electrochemistry and fluorescence histochemical image analysis

Rats with unilateral 6-hydroxydopamine-induced degeneration of the left nigrostriatal dopamine system were given intrastriatal implants of one cortex-free adrenal medulla divided into 4 pieces. Two pieces were placed in the center of the anterior part of the denervated caudate and two pieces in a more posterior position in lateral caudate. The distribution of catecholamines (CA) in grafts and host brain was studied 2, 100 and 400 min after grafting by HPLC-electrochemistry and Falck-Hillarp fluorescence histochemistry combined with computer-aided image analysis. Two minutes after implantation the chromaffin tissue grafts contained large amounts of adrenaline (A) and noradrenaline (NA) and small amounts of dopamine (DA). The chromaffin cells had a relatively normal fluorescence histochemical appearance. From the grafts, CA had spread into the surrounding host brain tissue where high levels of A and NA and low levels of DA were now found in the denervated host striatum. Fluorescence histochemistry and image analysis showed the CA to have spread 1-1.5 mm in all directions from the grafts. The CA concentrations decreased almost linearly with increasing distance from the grafts. At 100 min after implantation approximately a third of the chromaffin cells were still strongly fluorescent while the rest of the cells were very weakly fluorescent or non-fluorescent. The amounts of A, NA and DA in the host brain had decreased considerably, while the size of the fluorescent halo around the grafts had not diminished. At 400 min after grafting, only scattered cells in the chromaffin implants were strongly fluorescent and the surrounding host striatum contained low amounts of CA. It is concluded that intrastriatal adrenal medullary implants acutely release or leak large amounts of CA into surrounding host brain tissue. Taken together with results from the accompanying paper these data show that the grafts can maintain CA levels in host striatum high enough to elicit strong rotational responses during approximately 200 min.