Temperature Control of Fimbriation Circuit Switch in Uropathogenic Escherichia coli: Quantitative Analysis via Automated Model Abstraction

Uropathogenic Escherichia coli (UPEC) represent the predominant cause of urinary tract infections (UTIs). A key UPEC molecular virulence mechanism is type 1 fimbriae, whose expression is controlled by the orientation of an invertible chromosomal DNA element—the fim switch. Temperature has been shown to act as a major regulator of fim switching behavior and is overall an important indicator as well as functional feature of many urologic diseases, including UPEC host-pathogen interaction dynamics. Given this panoptic physiological role of temperature during UTI progression and notable empirical challenges to its direct in vivo studies, in silico modeling of corresponding biochemical and biophysical mechanisms essential to UPEC pathogenicity may significantly aid our understanding of the underlying disease processes. However, rigorous computational analysis of biological systems, such as fim switch temperature control circuit, has hereto presented a notoriously demanding problem due to both the substantial complexity of the gene regulatory networks involved as well as their often characteristically discrete and stochastic dynamics. To address these issues, we have developed an approach that enables automated multiscale abstraction of biological system descriptions based on reaction kinetics. Implemented as a computational tool, this method has allowed us to efficiently analyze the modular organization and behavior of the E. coli fimbriation switch circuit at different temperature settings, thus facilitating new insights into this mode of UPEC molecular virulence regulation. In particular, our results suggest that, with respect to its role in shutting down fimbriae expression, the primary function of FimB recombinase may be to effect a controlled down-regulation (rather than increase) of the ON-to-OFF fim switching rate via temperature-dependent suppression of competing dynamics mediated by recombinase FimE. Our computational analysis further implies that this down-regulation mechanism could be particularly significant inside the host environment, thus potentially contributing further understanding toward the development of novel therapeutic approaches to UPEC-caused UTIs

Type 1 Fimbriae, a Colonization Factor of Uropathogenic Escherichia coli, Are Controlled by the Metabolic Sensor CRP-cAMP

Type 1 fimbriae are a crucial factor for the virulence of uropathogenic Escherichia coli during the first steps of infection by mediating adhesion to epithelial cells. They are also required for the consequent colonization of the tissues and for invasion of the uroepithelium. Here, we studied the role of the specialized signal transduction system CRP-cAMP in the regulation of type 1 fimbriation. Although initially discovered by regulating carbohydrate metabolism, the CRP-cAMP complex controls a major regulatory network in Gram-negative bacteria, including a broad subset of genes spread into different functional categories of the cell. Our results indicate that CRP-cAMP plays a dual role in type 1 fimbriation, affecting both the phase variation process and fimA promoter activity, with an overall repressive outcome on fimbriation. The dissection of the regulatory pathway let us conclude that CRP-cAMP negatively affects FimB-mediated recombination by an indirect mechanism that requires DNA gyrase activity. Moreover, the underlying studies revealed that CRP-cAMP controls the expression of another global regulator in Gram-negative bacteria, the leucine-responsive protein Lrp. CRP-cAMP-mediated repression is limiting the switch from the non-fimbriated to the fimbriated state. Consistently, a drop in the intracellular concentration of cAMP due to altered physiological conditions (e.g. growth in presence of glucose) increases the percentage of fimbriated cells in the bacterial population. We also provide evidence that the repression of type 1 fimbriae by CRP-cAMP occurs during fast growth conditions (logarithmic phase) and is alleviated during slow growth (stationary phase), which is consistent with an involvement of type 1 fimbriae in the adaptation to stress conditions by promoting biofilm growth or entry into host cells. Our work suggests that the metabolic sensor CRP-cAMP plays a role in coupling the expression of type 1 fimbriae to environmental conditions, thereby also affecting subsequent attachment and colonization of host tissues

A Review of Dietary Prevention of Human Papillomavirus-Related Infection of the Cervix and Cervical Intraepithelial Neoplasia

The natural history of cervical cancer suggests that prevention can be achieved by modification of the host's immune system through a nutrient-mediated program. This study reviews the preventive role of dietary intake on cervical intraepithelial neoplasia (CIN) induced by human papillomavirus (HPV). Electronic databases were searched using relevant keywords such as, but not limited to, human papillomavirus infection, cervical intraepithelial neoplasia, lifestyle factors, nutrients intake, and diet. High consumption of fruit and vegetables appears to be protective against CIN. The findings also highlight the possibility of consuming high levels of specific nutrients, vitamins, and minerals, and retaining sufficient level of these elements in the body, especially those with high antioxidants and antiviral properties, to prevent progression of transient and persistent HPV infections to high-grade CIN 2 and 3 (including in situ cervical cancer). The protective effect is not significant for high-risk HPV persistent infections and invasive cervical cancer. Although it appears that intake of specific nutrients, vitamins, and minerals may be good in CIN prevention, there is lack of evidence from controlled trial to confirm this. Health professionals shall focus on implementation of a balanced-diet prevention strategy at an early stage for cervical cancer prevention

Signature-Tagged Mutagenesis in a Chicken Infection Model Leads to the Identification of a Novel Avian Pathogenic Escherichia coli Fimbrial Adhesin

The extraintestinal pathogen, avian pathogenic E. coli (APEC), known to cause systemic infections in chickens, is responsible for large economic losses in the poultry industry worldwide. In order to identify genes involved in the early essential stages of pathogenesis, namely adhesion and colonization, Signature-tagged mutagenesis (STM) was applied to a previously established lung colonization model of infection by generating and screening a total of 1,800 mutants of an APEC strain IMT5155 (O2:K1:H5; Sequence type complex 95). The study led to the identification of new genes of interest, including two adhesins, one of which coded for a novel APEC fimbrial adhesin (Yqi) not described for its role in APEC pathogenesis to date. Its gene product has been temporarily designated ExPEC Adhesin I (EA/I) until the adhesin-specific receptor is identified. Deletion of the ExPEC adhesin I gene resulted in reduced colonization ability by APEC strain IMT5155 both in vitro and in vivo. Furthermore, complementation of the adhesin gene restored its ability to colonize epithelial cells in vitro. The ExPEC adhesin I protein was successfully expressed in vitro. Electron microscopy of an afimbriate strain E. coli AAEC189 over-expressed with the putative EA/I gene cluster revealed short fimbrial-like appendages protruding out of the bacterial outer membrane. We observed that this adhesin coding gene yqi is prevalent among extraintestinal pathogenic E. coli (ExPEC) isolates, including APEC (54.4%), uropathogenic E. coli (UPEC) (65.9%) and newborn meningitic E. coli (NMEC) (60.0%), and absent in all of the 153 intestinal pathogenic E. coli strains tested, thereby validating the designation of the adhesin as ExPEC Adhesin I. In addition, prevalence of EA/I was most frequently associated with the B2 group of the EcoR classification and ST95 complex of the multi locus sequence typing (MLST) scheme, with evidence of a positive selection within this highly pathogenic complex. This is the first report of the newly identified and functionally characterized ExPEC adhesin I and its significant role during APEC infection in chickens

Drinking in transition: trends in alcohol consumption in Russia 1994-2004

BACKGROUND: Heavy alcohol consumption is widespread in Russia, but studying changes in drinking during the transition from Communism has been hampered previously by the lack of frequent data. This paper uses 1-2 yearly panel data, comparing consumption trends with the rapid concurrent changes in economic variables (notably around the "Rouble crisis", shortly preceding the 1998 survey round), and mortality. METHODS: Data were from 9 rounds (1994-2004) of the 38-centre Russia Longitudinal Monitoring Survey. Respondents aged over 18 were included (>7,000 per round). Trends were measured in alcohol frequency, quantity per occasion (by beverage type) and 2 measures of potentially hazardous consumption: (i) frequent, heavy spirit drinking (≥80 g per occasion of vodka or samogon and >weekly) (ii) consuming samogon (cheap home-distilled spirit). Trends in consumption, mean household income and national mortality rates (in the same and subsequent 2 years) were compared. Finally, in a subsample of individual male respondents present in both the 1996 and 1998 rounds (before and after the financial crash), determinants of changes in harmful consumption were studied using logistic regression. RESULTS: Frequent, heavy spirit drinking (>80 g each time, ≥weekly) was widespread amongst men (12-17%) throughout, especially in the middle aged and less educated; with the exception of a significant, temporary drop to 10% in 1998. From 1996-2000, samogon drinking more than doubled, from 6% to 16% of males; despite a decline, levels were significantly higher in 2004 than 1996 in both sexes. Amongst women, frequent heavy spirit drinking rose non-significantly to more than 1% during the study. Heavy frequent male drinking and mortality in the same year were correlated in lower educated males, but not in women. Individual logistic regression in a male subsample showed that between 1996 and1998, those who lost their employment were more likely to cease frequent, heavy drinking; however, men who commenced drinking samogon in 1998 were more likely to be rural residents, materially poor, very heavy drinkers or pessimistic about their finances. These changes were unexplained by losses to follow-up. CONCLUSIONS: Sudden economic decline in late 1990s Russia was associated with a sharp, temporary fall in heavy drinking, and a gradual and persistent increase in home distilled spirit consumption, with the latter more common amongst disadvantaged groups. The correlation between heavy drinking and national mortality in lower educated men is interesting, but the timing of RLMS surveys late in the calendar year, and the absence of any correlation between drinking and the subsequent year's mortality, makes these data hard to interpret. Potential study limitations include difficulty in measuring multiple beverages consumed per occasion, and not specifically recording "surrogate" (non-beverage) alcohols

Prenatal Hypoxic-Ischemic Insult Changes the Distribution and Number of NADPH-Diaphorase Cells in the Cerebellum

Astrogliosis, oligodendroglial death and motor deficits have been observed in the offspring of female rats that had their uterine arteries clamped at the 18th gestational day. Since nitric oxide has important roles in several inflammatory and developmental events, here we evaluated NADPH-diaphorase (NADPH-d) distribution in the cerebellum of rats submitted to this hypoxia-ischemia (HI) model. At postnatal (P) day 9, Purkinje cells of SHAM and non-manipulated (NM) animals showed NADPH-d+ labeling both in the cell body and dendritic arborization in folia 1 to 8, while HI animals presented a weaker labeling in both cellular structures. NADPH-d+ labeling in the molecular (ML), and in both the external and internal granular layer, was unaffected by HI at this age. At P23, labeling in Purkinje cells was absent in all three groups. Ectopic NADPH-d+ cells in the ML of folia 1 to 4 and folium 10 were present exclusively in HI animals. This labeling pattern was maintained up to P90 in folium 10. In the cerebellar white matter (WM), at P9 and P23, microglial (ED1+) NADPH-d+ cells, were observed in all groups. At P23, only HI animals presented NADPH-d labeling in the cell body and processes of reactive astrocytes (GFAP+). At P9 and P23, the number of NADPH-d+ cells in the WM was higher in HI animals than in SHAM and NM ones. At P45 and at P90 no NADPH-d+ cells were observed in the WM of the three groups. Our results indicate that HI insults lead to long-lasting alterations in nitric oxide synthase expression in the cerebellum. Such alterations in cerebellar differentiation might explain, at least in part, the motor deficits that are commonly observed in this model

Intervening with Urinary Tract Infections Using Anti-Adhesives Based on the Crystal Structure of the FimH–Oligomannose-3 Complex

Escherichia coli strains adhere to the normally sterile human uroepithelium using type 1 pili, that are long, hairy surface organelles exposing a mannose-binding FimH adhesin at the tip. A small percentage of adhered bacteria can successfully invade bladder cells, presumably via pathways mediated by the high-mannosylated uroplakin-Ia and alpha3beta1 integrins found throughout the uroepithelium. Invaded bacteria replicate and mature into dense, biofilm-like inclusions in preparation of fluxing and of infection of neighbouring cells, being the major cause of the troublesome recurrent urinary tract infections.We demonstrate that alpha-D-mannose based inhibitors of FimH not only block bacterial adhesion on uroepithelial cells but also antagonize invasion and biofilm formation. Heptyl alpha-D-mannose prevents binding of type 1-piliated E. coli to the human bladder cell line 5637 and reduces both adhesion and invasion of the UTI89 cystitis isolate instilled in mouse bladder via catheterization. Heptyl alpha-D-mannose also specifically inhibited biofilm formation at micromolar concentrations. The structural basis of the great inhibitory potential of alkyl and aryl alpha-D-mannosides was elucidated in the crystal structure of the FimH receptor-binding domain in complex with oligomannose-3. FimH interacts with Man alpha1,3Man beta1,4GlcNAc beta1,4GlcNAc in an extended binding site. The interactions along the alpha1,3 glycosidic bond and the first beta1,4 linkage to the chitobiose unit are conserved with those of FimH with butyl alpha-D-mannose. The strong stacking of the central mannose with the aromatic ring of Tyr48 is congruent with the high affinity found for synthetic inhibitors in which this mannose is substituted for by an aromatic group.The potential of ligand-based design of antagonists of urinary tract infections is ruled by the structural mimicry of natural epitopes and extends into blocking of bacterial invasion, intracellular growth and capacity to fluxing and of recurrence of the infection

Lysogeny with Shiga Toxin 2-Encoding Bacteriophages Represses Type III Secretion in Enterohemorrhagic Escherichia coli

Lytic or lysogenic infections by bacteriophages drive the evolution of enteric bacteria. Enterohemorrhagic Escherichia coli (EHEC) have recently emerged as a significant zoonotic infection of humans with the main serotypes carried by ruminants. Typical EHEC strains are defined by the expression of a type III secretion (T3S) system, the production of Shiga toxins (Stx) and association with specific clinical symptoms. The genes for Stx are present on lambdoid bacteriophages integrated into the E. coli genome. Phage type (PT) 21/28 is the most prevalent strain type linked with human EHEC infections in the United Kingdom and is more likely to be associated with cattle shedding high levels of the organism than PT32 strains. In this study we have demonstrated that the majority (90%) of PT 21/28 strains contain both Stx2 and Stx2c phages, irrespective of source. This is in contrast to PT 32 strains for which only a minority of strains contain both Stx2 and 2c phages (28%). PT21/28 strains had a lower median level of T3S compared to PT32 strains and so the relationship between Stx phage lysogeny and T3S was investigated. Deletion of Stx2 phages from EHEC strains increased the level of T3S whereas lysogeny decreased T3S. This regulation was confirmed in an E. coli K12 background transduced with a marked Stx2 phage followed by measurement of a T3S reporter controlled by induced levels of the LEE-encoded regulator (Ler). The presence of an integrated Stx2 phage was shown to repress Ler induction of LEE1 and this regulation involved the CII phage regulator. This repression could be relieved by ectopic expression of a cognate CI regulator. A model is proposed in which Stx2-encoding bacteriophages regulate T3S to co-ordinate epithelial cell colonisation that is promoted by Stx and secreted effector proteins

AD51B in Familial Breast Cancer

Common variation on 14q24.1, close to RAD51B, has been associated with breast cancer: rs999737 and rs2588809 with the risk of female breast cancer and rs1314913 with the risk of male breast cancer. The aim of this study was to investigate the role of RAD51B variants in breast cancer predisposition, particularly in the context of familial breast cancer in Finland. We sequenced the coding region of RAD51B in 168 Finnish breast cancer patients from the Helsinki region for identification of possible recurrent founder mutations. In addition, we studied the known rs999737, rs2588809, and rs1314913 SNPs and RAD51B haplotypes in 44,791 breast cancer cases and 43,583 controls from 40 studies participating in the Breast Cancer Association Consortium (BCAC) that were genotyped on a custom chip (iCOGS). We identified one putatively pathogenic missense mutation c.541C>T among the Finnish cancer patients and subsequently genotyped the mutation in additional breast cancer cases (n = 5259) and population controls (n = 3586) from Finland and Belarus. No significant association with breast cancer risk was seen in the meta-analysis of the Finnish datasets or in the large BCAC dataset. The association with previously identified risk variants rs999737, rs2588809, and rs1314913 was replicated among all breast cancer cases and also among familial cases in the BCAC dataset. The most significant association was observed for the haplotype carrying the risk-alleles of all the three SNPs both among all cases (odds ratio (OR): 1.15, 95% confidence interval (CI): 1.11–1.19, P = 8.88 x 10−16) and among familial cases (OR: 1.24, 95% CI: 1.16–1.32, P = 6.19 x 10−11), compared to the haplotype with the respective protective alleles. Our results suggest that loss-of-function mutations in RAD51B are rare, but common variation at the RAD51B region is significantly associated with familial breast cancer risk