RfaH Suppresses Small RNA MicA Inhibition of fimB Expression in Escherichia coli K-12

The phase variation (reversible on-off switching) of the type 1 fimbrial adhesin of Escherichia coli involves a DNA inversion catalyzed by FimB (switching in either direction) or FimE (on-to-off switching). Here, we demonstrate that RfaH activates expression of a FimB-LacZ protein fusion while having a modest inhibitory effect on a comparable fimB-lacZ operon construct and on a FimE-LacZ protein fusion, indicating that RfaH selectively controls fimB expression at the posttranscriptional level. Further work demonstrates that loss of RfaH enables small RNA (sRNA) MicA inhibition of fimB expression even in the absence of exogenous inducing stress. This effect is explained by induction of σE , and hence MicA, in the absence of RfaH. Additional work con- firms that the procaine-dependent induction of micA requires OmpR, as reported previously (A. Coornaert et al., Mol. Microbiol. 76:467–479, 2010, doi:10.1111/j.1365-2958.2010.07115.x), but also demonstrates that RfaH inhibition of fimB transcription is enhanced by procaine independently of OmpR. While the effect of procaine on fimB transcription is shown to be independent of RcsB, it was found to require SlyA, another known regulator of fimB transcription. These results demonstrate a complex role for RfaH as a regulator of fimB expression

The pancreatic zymogen granule membrane protein, GP2, binds Escherichia coli type 1 Fimbriae

<p>Abstract</p> <p>Background</p> <p>GP2 is the major membrane protein present in the pancreatic zymogen granule, and is cleaved and released into the pancreatic duct along with exocrine secretions. The function of GP2 is unknown. GP2's amino acid sequence is most similar to that of uromodulin, which is secreted by the kidney. Recent studies have demonstrated uromodulin binding to bacterial Type 1 fimbria. The fimbriae serve as adhesins to host receptors. The present study examines whether GP2 also shares similar binding properties to bacteria with Type 1 fimbria. Commensal and pathogenic bacteria, including E. coli and Salmonella, express type 1 fimbria.</p> <p>Methods</p> <p>An <it>in vitro </it>binding assay was used to assay the binding of recombinant GP2 to defined strains of <it>E. coli </it>that differ in their expression of Type 1 fimbria or its subunit protein, FimH. Studies were also performed to determine whether GP2 binding is dependent on the presence of mannose residues, which is a known determinant for FimH binding.</p> <p>Results</p> <p>GP2 binds <it>E. coli </it>that express Type 1 fimbria. Binding is dependent on GP2 glycosylation, and specifically the presence of mannose residues.</p> <p>Conclusion</p> <p>GP2 binds to Type 1 fimbria, a bacterial adhesin that is commonly expressed by members of the <it>Enterobacteriacae </it>family.</p

Voice, autonomy and utopian desire in participatory film-making with young refugees

This article is a reflection on what reflexive documentary scholars call the ‘moral dimension’ (Nash 2012: 318) of a participatory filmmaking project with refugee young people, who wanted to make a film to support other new young arrivals in the process of making home in Scotland. In the first part, we highlight some of the challenges of collaborating with refugee young people, in light of the often de-humanising representations of refugees in mainstream media and the danger of the triple conflation of authenticity-voice-pain in academic narratives about refugees. In the second part, we show how honouring young people’s desire to convey the hopeful aspects of making home, emerged as a key pedagogical strategy to affirm their expert position and encourage their participation in the project. Revisiting key moments of learning and interaction, we demonstrate how young people’s process of ‘finding a voice’ in moment-by-moment filmmaking practice was not a linear, developmental process towards ‘pure’ individual empowerment and singular artistic expression. Their participation in shaping their visual (self-)representation in the final film, was embedded in the dialogical process and pragmatic requirements of a collaborative film production, in which voice, autonomy and teacher authority were negotiated on a moment-by-moment basis. We conclude that it is vital for a reflexive practice and research to not gloss over the moral dilemmas in the name of progressive ideals, for example, when representations are co-created by project filmmakers/educators, but embrace these deliberations as part of the ‘fascinating collaborative matrix’ (Chambers 2019: 29) of participatory filmmaking

Lrp Acts as Both a Positive and Negative Regulator for Type 1 Fimbriae Production in Salmonella enterica Serovar Typhimurium

Leucine-responsive regulatory protein (Lrp) is known to be an indirect activator of type 1 fimbriae synthesis in Salmonella enterica serovar Typhimurium via direct regulation of FimZ, a direct positive regulator for type 1 fimbriae production. Using RT-PCR, we have shown previously that fimA transcription is dramatically impaired in both lrp-deletion (Δlrp) and constitutive-lrp expression (lrpC) mutant strains. In this work, we used chromosomal PfimA-lacZ fusions and yeast agglutination assays to confirm and extend our previous results. Direct binding of Lrp to PfimA was shown by an electrophoretic mobility shift assay (EMSA) and DNA footprinting assay. Site-directed mutagenesis revealed that the Lrp-binding motifs in PfimA play a role in both activation and repression of type 1 fimbriae production. Overproduction of Lrp also abrogates fimZ expression. EMSA data showed that Lrp and FimZ proteins independently bind to PfimA without competitive exclusion. In addition, both Lrp and FimZ binding to PfimA caused a hyper retardation (supershift) of the DNA-protein complex compared to the shift when each protein was present alone. Nutrition-dependent cellular Lrp levels closely correlated with the amount of type 1 fimbriae production. These observations suggest that Lrp plays important roles in type 1 fimbriation by acting as both a positive and negative regulator and its effect depends, at least in part, on the cellular concentration of Lrp in response to the nutritional environment

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

Id4 promotes the elimination of the pro-activation factor ascl1 to maintain quiescence of adult hippocampal stem cells

Quiescence is essential for the long-term maintenance of adult stem cells but how stem cells maintain quiescence is poorly understood. Here we show that neural stem cells in the adult mouse hippocampus actively transcribe the pro-activation factor Ascl1 regardless of their activated or quiescent states. We found that the inhibitor of DNA binding protein Id4 is enriched in quiescent neural stem cells and that elimination of Id4 results in abnormal accumulation of Ascl1 protein and premature stem cell activation. Accordingly, Id4 and other Id proteins promote elimination of Ascl1 protein in neural stem cell cultures. Id4 sequesters Ascl1 heterodimerisation partner E47, promoting Ascl1 protein degradation and stem cell quiescence. Our results highlight the importance of non-transcriptional mechanisms for the maintenance of neural stem cell quiescence and reveal a role for Id4 as a quiescence-inducing factor, in contrast with its role of promoting the proliferation of embryonic neural progenitors

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

Influences of club connectedness among young adults in Western Australian community-based sports clubs

Background: Along with physical benefits, community-based sport provides opportunities to enhance connectedness, an important protective factor of social and emotional health. However, young Australians participating in sport have been found to drink alcohol at higher levels than their non-sporting peers, and many clubs serve unhealthy food and beverages. This study explored the association between the dependent variable, level of alcohol consumption (AUDIT-C) and connectedness to club and other health behaviours among young people aged 18-30 years who play club sport in Western Australia. Methods: An online cross sectional survey measured levels of alcohol consumption (AUDIT-C), alcohol-related harm, connectedness (including volunteering and team cohesion), mental wellbeing, healthy food options and club sponsorship among young adults aged 18-30 years involved in sports clubs in Western Australia (n = 242). Relationships and association between the dependent variable (AUDIT-C) and independent variables were assessed. Results: Male sportspeople were more likely to drink alcohol at high-risk levels than females (p <.001), and respondents belonging to a club that received alcohol-related sponsorship were more likely to drink at high-risk levels (p =.019). Females were significantly more likely to want healthy food and beverage options provided at their clubs (p = 0.011). When all factors were considered team cohesion (p = 0.02), alcohol expectations (p = <.001), occurrences of experienced alcohol-related harm (p = <.001) and length of club membership (p = 0.18) were significant predictors of high-risk AUDIT-C (R 2 =.34, adjusted R 2 =.33, F (4, 156) = 20.43, p = <.001). High-risk AUDIT-C and club connectedness predicted strong team cohesion (R 2 =.39, adjusted R 2 =.39, F (2, 166) = 53.74, p = <.001). Conclusions: Findings from this study may inform policy and practice to enhance healthy behaviours among young adults participating in community sports clubs in Australia and other countries

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