The structure of the PapD-PapGII pilin complex reveals an open and flexible P5 pocket

P pili are hairlike polymeric structures that mediate binding of uropathogenic Escherichia coli to the surface of the kidney via the PapG adhesin at their tips. PapG is composed of two domains: a lectin domain at the tip of the pilus followed by a pilin domain that comprises the initial polymerizing subunit of the 1,000-plus-subunit heteropolymeric pilus fiber. Prior to assembly, periplasmic pilin domains bind to a chaperone, PapD. PapD mediates donor strand complementation, in which a beta strand of PapD temporarily completes the pilin domain's fold, preventing premature, nonproductive interactions with other pilin subunits and facilitating subunit folding. Chaperone-subunit complexes are delivered to the outer membrane usher where donor strand exchange (DSE) replaces PapD's donated beta strand with an amino-terminal extension on the next incoming pilin subunit. This occurs via a zip-in-zip-out mechanism that initiates at a relatively accessible hydrophobic space termed the P5 pocket on the terminally incorporated pilus subunit. Here, we solve the structure of PapD in complex with the pilin domain of isoform II of PapG (PapGIIp). Our data revealed that PapGIIp adopts an immunoglobulin fold with a missing seventh strand, complemented in parallel by the G1 PapD strand, typical of pilin subunits. Comparisons with other chaperone-pilin complexes indicated that the interactive surfaces are highly conserved. Interestingly, the PapGIIp P5 pocket was in an open conformation, which, as molecular dynamics simulations revealed, switches between an open and a closed conformation due to the flexibility of the surrounding loops. Our study reveals the structural details of the DSE mechanism

Bacteria-Induced Uroplakin Signaling Mediates Bladder Response to Infection

Urinary tract infections are the second most common infectious disease in humans and are predominantly caused by uropathogenic E. coli (UPEC). A majority of UPEC isolates express the type 1 pilus adhesin, FimH, and cell culture and murine studies demonstrate that FimH is involved in invasion and apoptosis of urothelial cells. FimH initiates bladder pathology by binding to the uroplakin receptor complex, but the subsequent events mediating pathogenesis have not been fully characterized. We report a hitherto undiscovered signaling role for the UPIIIa protein, the only major uroplakin with a potential cytoplasmic signaling domain, in bacterial invasion and apoptosis. In response to FimH adhesin binding, the UPIIIa cytoplasmic tail undergoes phosphorylation on a specific threonine residue by casein kinase II, followed by an elevation of intracellular calcium. Pharmacological inhibition of these signaling events abrogates bacterial invasion and urothelial apoptosis in vitro and in vivo. Our studies suggest that bacteria-induced UPIIIa signaling is a critical mediator of bladder responses to insult by uropathogenic E. coli

Targeting deficiencies in the TLR5 mediated vaginal response to treat female recurrent urinary tract infection

Abstract The identification of the host defence peptides as target effectors in the innate defence of the uro-genital tract creates new translational possibilities for immunomodulatory therapies, specifically vaginal therapies to treat women suffering from rUTI, particularly those carrying the TLR5_C1174T SNP. Urinary tract infections (UTIs) are a microbial disease reported worldwide. Women are particularly susceptible with many suffering debilitating recurrent (r) infections. Treatment is by antibiotics, but such therapy is linked to antibiotic resistance and re-infection. This study explored the innate protective mechanisms of the urogenital tract with the aim of boosting such defences therapeutically. Modelling UTIs in vitro, human vaginal and bladder epithelial cells were challenged with uropathogenic Escherichia coli (CFT073) and microbial PAMPs including flagellin, LPS and peptidoglycan. Flagellin functioning via the TLR5/NFκB pathway was identified as the key UPEC virulence factor causing a significant increase (P < 0.05) in the production of the host-defence peptide (HDP), BD2. BD2-depleted urine samples from bladder infected mice supported increased UPEC growth, strengthening the significance of the HDPs in protecting the urogenital tissues from infection. Clinically, vaginal-douche BD2 concentrations were reduced (p < 0.05) in women suffering rUTIs, compared to age-matched healthy controls with concentrations further decreased (p < 0.05) in a TLR5392Stop SNP rUTI subgroup. Topical vaginal estrogen treatment increased (p < 0.001) BD2 concentrations in all women, including those carrying the SNP. These data identify therapeutic and antibiotic sparing roles for vaginal immunomodulatory agents that specifically target HDP induction, facilitate bacterial killing and disrupt the UPEC infection cycle

Metabolic profiles in five high-producing Swedish dairy herds with a history of abomasal displacement and ketosis

<p>Abstract</p> <p>Background</p> <p>Body condition score and blood profiles have been used to monitor management and herd health in dairy cows. The aim of this study was to examine BCS and extended metabolic profiles, reflecting both energy metabolism and liver status around calving in high-producing herds with a high incidence of abomasal displacement and ketosis and to evaluate if such profiles can be used at herd level to pinpoint specific herd problems.</p> <p>Methods</p> <p>Body condition score and metabolic profiles around calving in five high-producing herds with high incidences of abomasal displacement and ketosis were assessed using linear mixed models (94 cows, 326 examinations). Cows were examined and blood sampled every three weeks from four weeks ante partum (ap) to nine weeks postpartum (pp). Blood parameters studied were glucose, fructosamine, non-esterified fatty acids (NEFA), insulin, β-hydroxybutyrate, aspartate aminotransferase, glutamate dehydrogenase, haptoglobin and cholesterol.</p> <p>Results</p> <p>All herds had overconditioned dry cows that lost body condition substantially the first 4–6 weeks pp. Two herds had elevated levels of NEFA ap and three herds had elevated levels pp. One herd had low levels of insulin ap and low levels of cholesterol pp. Haptoglobin was detected pp in all herds and its usefulness is discussed.</p> <p>Conclusion</p> <p>NEFA was the parameter that most closely reflected the body condition losses while these losses were not seen in glucose and fructosamine levels. Insulin and cholesterol were potentially useful in herd profiles but need further investigation. Increased glutamate dehydrogenase suggested liver cell damage in all herds.</p

Long-term survival after initial hospital admission for peripheral arterial disease in the lower extremities

ABSTRACT: Background As the population ages, peripheral arterial disease (PAD) in the lower extremities will become a larger public health problem. Awareness in patients as well clinicians of the high risk of morbidity and mortality is important but seems currently low. Insights in absolute mortality risks following admission for PAD in the lower extremities can be useful to improve awareness as they are easy to interpret. Methods A nationwide cohort of 4,158 patients with an initial admission for PAD in the lower extremities was identified through linkage of the national hospital and population register in 1997 and 2000. Results Over 60% of 4,158 patients were men. 28 days, 1 year and 5 year mortality risk were 2.4%, 10.3% and 31.0% for men and 3.5%, 10.4% and 27.4% for women. Coronary heart disease and stroke were frequent cause of death. Five years mortality risk was higher for men compared to women (HR 1.36, 95% CI 1.21-1.53). Conclusions Our findings demonstrate that, 5 year mortality risk is high, especially in men and comparable to that of patients admitted for acute myocardial infarction or ischemic stroke. Though, in general population the awareness of the severity of PAD in the lower extremities is significantly lower than that for any other cardiovascular disease and it seems that cardiovascular risk factor management for prevention in PAD patients is very modes

Control and Manipulation of Pathogens with an Optical Trap for Live Cell Imaging of Intercellular Interactions

The application of live cell imaging allows direct visualization of the dynamic interactions between cells of the immune system. Some preliminary observations challenge long-held beliefs about immune responses to microorganisms; however, the lack of spatial and temporal control between the phagocytic cell and microbe has rendered focused observations into the initial interactions of host response to pathogens difficult. This paper outlines a method that advances live cell imaging by integrating a spinning disk confocal microscope with an optical trap, also known as an optical tweezer, in order to provide exquisite spatial and temporal control of pathogenic organisms and place them in proximity to host cells, as determined by the operator. Polymeric beads and live, pathogenic organisms (Candida albicans and Aspergillus fumigatus) were optically trapped using non-destructive forces and moved adjacent to living cells, which subsequently phagocytosed the trapped particle. High resolution, transmitted light and fluorescence-based movies established the ability to observe early events of phagocytosis in living cells. To demonstrate the broad applicability of this method to immunological studies, anti-CD3 polymeric beads were also trapped and manipulated to form synapses with T cells in vivo, and time-lapse imaging of synapse formation was also obtained. By providing a method to exert fine control of live pathogens with respect to immune cells, cellular interactions can be captured by fluorescence microscopy with minimal perturbation to cells and can yield powerful insight into early responses of innate and adaptive immunity.National Institute of Biomedical Imaging and Bioengineering (U.S.) (grant T32EB006348)Massachusetts General Hospital (Department of Medicine Internal Funds)Center for Computational and Integrative Biology (Development fund)Center for Computational and Integrative Biology (AI062773)Center for Computational and Integrative Biology (grant AI062773)Center for Computational and Integrative Biology (grant DK83756)Center for Computational and Integrative Biology (grant DK 043351)National Institute of Allergy and Infectious Diseases (U.S.)National Institutes of Health (U.S.) (grant AI057999

Horizontal gene transfer in Histophilus somni and its role in the evolution of pathogenic strain 2336, as determined by comparative genomic analyses

<p>Abstract</p> <p>Background</p> <p>Pneumonia and myocarditis are the most commonly reported diseases due to <it>Histophilus somni</it>, an opportunistic pathogen of the reproductive and respiratory tracts of cattle. Thus far only a few genes involved in metabolic and virulence functions have been identified and characterized in <it>H. somni </it>using traditional methods. Analyses of the genome sequences of several <it>Pasteurellaceae </it>species have provided insights into their biology and evolution. In view of the economic and ecological importance of <it>H. somni</it>, the genome sequence of pneumonia strain 2336 has been determined and compared to that of commensal strain 129Pt and other members of the <it>Pasteurellaceae</it>.</p> <p>Results</p> <p>The chromosome of strain 2336 (2,263,857 bp) contained 1,980 protein coding genes, whereas the chromosome of strain 129Pt (2,007,700 bp) contained only 1,792 protein coding genes. Although the chromosomes of the two strains differ in size, their average GC content, gene density (total number of genes predicted on the chromosome), and percentage of sequence (number of genes) that encodes proteins were similar. The chromosomes of these strains also contained a number of discrete prophage regions and genomic islands. One of the genomic islands in strain 2336 contained genes putatively involved in copper, zinc, and tetracycline resistance. Using the genome sequence data and comparative analyses with other members of the <it>Pasteurellaceae</it>, several <it>H. somni </it>genes that may encode proteins involved in virulence (<it>e.g</it>., filamentous haemaggutinins, adhesins, and polysaccharide biosynthesis/modification enzymes) were identified. The two strains contained a total of 17 ORFs that encode putative glycosyltransferases and some of these ORFs had characteristic simple sequence repeats within them. Most of the genes/loci common to both the strains were located in different regions of the two chromosomes and occurred in opposite orientations, indicating genome rearrangement since their divergence from a common ancestor.</p> <p>Conclusions</p> <p>Since the genome of strain 129Pt was ~256,000 bp smaller than that of strain 2336, these genomes provide yet another paradigm for studying evolutionary gene loss and/or gain in regard to virulence repertoire and pathogenic ability. Analyses of the complete genome sequences revealed that bacteriophage- and transposon-mediated horizontal gene transfer had occurred at several loci in the chromosomes of strains 2336 and 129Pt. It appears that these mobile genetic elements have played a major role in creating genomic diversity and phenotypic variability among the two <it>H. somni </it>strains.</p

Consensus guidelines for the use and interpretation of angiogenesis assays

The formation of new blood vessels, or angiogenesis, is a complex process that plays important roles in growth and development, tissue and organ regeneration, as well as numerous pathological conditions. Angiogenesis undergoes multiple discrete steps that can be individually evaluated and quantified by a large number of bioassays. These independent assessments hold advantages but also have limitations. This article describes in vivo, ex vivo, and in vitro bioassays that are available for the evaluation of angiogenesis and highlights critical aspects that are relevant for their execution and proper interpretation. As such, this collaborative work is the first edition of consensus guidelines on angiogenesis bioassays to serve for current and future reference