Knowledge, perceptions and practices on antimicrobial resistance in humans and animals in Wakiso district, Uganda: A cross sectional study.

Despite increasing evidence on antimicrobial resistance (AMR), there is limited literature on antimicrobial access and use in humans and animals in community settings globally. This study assessed knowledge and perceptions of AMR, as well as practices relating to the use of antimicrobials in humans and animals in Wakiso district, Uganda. This was a cross-sectional study among 418 participants that employed quantitative data collection methods. A structured questionnaire that included questions on knowledge, perceptions, practices related to AMR, and perceptions on access to antimicrobials in humans and animals was used. Data was analysed in STATA version 10. The majority of participants 63.6% (266/418) had heard about AMR mainly from family and friends 57.5% (153/266), and most 70.8% (296/418) were aware that resistant microorganisms cause infections that are difficult to treat. Most participants 62.7% (262/418) thought that they should complete the full dose of antimicrobials when on treatment. However, on the last occasion of antimicrobial use, 13.0% (44/338) revealed that they did not complete the full course of treatment. Participants who were single (APR = 1.12, C.I = 1.03-1.12, p-value = 0.01) and earning between 91 and 290 USD on average per month (APR = 1.12, C.I = 1.02-1.23, p-value = 0.02) were more likely to have completed a given antimicrobial course as compared to those who were married/cohabiting and earned less than 15 USD respectively. The majority of participants 60% (251/418) owned animals, and 81.3% (204/251) reported using antimicrobials mainly for prevention 61.3% (125/204) or treatment of sick animals 70.6% (144/204). Among the participants, 57.4% (117/204) reported not having sold or consumed animal products within a week after exposure to antimicrobials. Interventions to prevent AMR should adopt a One Health approach to address the gap in knowledge and practices relating to the use of antimicrobials in humans and animals.

High incidence of antibiotic resistance amongst isolates of Helicobacter pylori collected in Nottingham, UK, between 2001 and 2018.

Introduction. Helicobacter pylori is the leading cause of peptic ulcers and gastric cancer. The most common treatment regimens use combinations of two or three antibiotics and a proton pump inhibitor (PPI) to suppress stomach acid. The World Health Organization designated clarithromycin-resistant H. pylori as a high priority pathogen for drug development, due to increasing antibiotic resistance globally.Hypothesis/Gap Statement. There is no routine surveillance of H. pylori primary antimicrobial sensitivities in the UK, and published data are lacking.Aim. This study aimed to characterize antimicrobial sensitivities of isolates collected in Nottingham, UK, between 2001 and 2018.Methodology. Gastric biopsy samples were collected, with informed written consent and ethics approval, from 162 patients attending the Queen's Medical Centre in Nottingham for an upper GI tract endoscopy. Antibiotic sensitivity was assessed using E-Tests and a more cost-effective disc diffusion test.Results. The clarithromycin, amoxicillin and levofloxacin disc diffusion tests provided identical results to E-Tests on a subset of 30 isolates. Disparities were observed in the metronidazole test results, however. In total, 241 isolates from 162 patients were tested using at least one method. Of all isolates, 28 % were resistant to clarithromycin, 62 % to metronidazole and 3 % to amoxicillin, which are used in first-line therapies. For those antibiotics used in second- and third-line therapies, 4 % were resistant to levofloxacin and none of the isolates were resistant to tetracycline. Resistance to more than one antibiotic was found in 27 % of isolates. The frequency of patients with a clarithromycin-resistant strain increased dramatically over time: from 16 % between 2001 and 2005 to 40 % between 2011 and 2018 (P=0.011). For the same time periods, there was also an increase in those with a metronidazole-resistant strain (from 58 to 78 %; P=0.05). The frequencies of clarithromycin and metronidazole resistance were higher in isolates from patients who had previously received eradication therapy, compared to those who had not (40 % versus 77 %, and 80 % versus 92 %, respectively). Of 79 pairs of isolates from the antrum and corpus regions of the same patient's stomach, only six had differences in their antimicrobial susceptibility profiles.Conclusion. Although there was high and increasing resistance to clarithromycin and metronidazole, there was no resistance to tetracycline and the frequencies of amoxicillin and levofloxacin resistance were very low.

Antimicrobial stewardship in private pharmacies in Wakiso district, Uganda: a qualitative study.

BACKGROUND: Private pharmacies are the first point of contact for the public regarding acquisition of medicines and other pharmaceuticals in many low- and middle-income countries including Uganda. Most antimicrobial stewardship (AMS) programmes in Uganda have targeted pharmacies in public health facilities, with little known about private pharmacies. This study explored knowledge and practices related to AMS in private pharmacies in Wakiso district, central Uganda. METHODS: This was a qualitative study that involved 31 in-depth interviews to explore AMS among retail private pharmacy staff including pharmacists, pharmacy technicians/dispensers, and nurses. Participants were asked about antimicrobial resistance (AMR) and AMS practices at their pharmacy. The audio-recorded interviews were transcribed verbatim and imported to NVivo 2020 (QSR International) for thematic analysis. RESULTS: Five major themes emerged from the study: commonly sold antimicrobials; knowledge on AMR and AMS; potential contributors to AMR; practices related to AMS; and challenges to AMS. The commonly sold antimicrobials in the pharmacies with or without prescriptions were oral azithromycin, Ampiclox® (ampicillin and cloxacillin), amoxicillin, ciprofloxacin, Septrin® (co-trimoxazole), metronidazole, Flucamox® (amoxicillin and flucloxacillin), Augmentin® (amoxicillin and clavulanic acid), cephalexin, doxycycline, and chloramphenicol. Participants had heard about AMR but not AMS, although only a few correctly defined AMR. Lack of knowledge among health workers and local communities; the overuse, misuse, and abuse of antimicrobials such as non-adherence to dosage; self-medication; and purchase of drugs without prescription were identified as potential accelerators to the emergence of AMR. Current practices related to AMS in private pharmacies were limited to meetings, antimicrobial dispensing, providing client advice, record keeping, and monitoring of drugs. Cost of healthcare, client satisfaction and retention, outdated guidelines, and the business orientation of pharmacies were the main challenges related to AMS. CONCLUSION: There was poor knowledge of AMR and AMS, and limited AMS practices in private pharmacies. Private pharmacies have the potential to contribute to Uganda's fight against AMR if motivated and equipped with adequate knowledge to enhance their practices related to AMS.

Characterization of a Helicobacter pylori strain with high biofilm-forming ability.

Introduction. Helicobacter pylori is highly polymorphic, and some strains are much more likely to cause disease than others. Biofilm formation can help bacteria to survive antibiotic treatment, immune attack and other stresses, promoting persistent infection.Hypothesis/Gap Statement. We hypothesized that H. pylori isolates from patients with more severe H. pylori-associated disease would be better at forming biofilms than isolates from patients with less severe disease.Aim. We initially aimed to determine whether or not the biofilm-forming ability of H. pylori isolates was associated with disease in the UK-based patients from whom the bacteria were isolated.Methodology. Biofilm-forming ability of H. pylori isolates was determined using a crystal violet assay on glass coverslips. The complete genome sequence of strain 444A was generated by hybrid assembly of Nanopore MinION and Illumina MiSeq data.Results. Although we found no associations between biofilm-forming ability of H. pylori and disease severity in patients, we discovered that strain 444A had particularly high biofilm-forming ability. This strain had been isolated from a patient with gastric ulcer disease and moderate to severe scores for H. pylori-induced histopathology. Analysis of the genome of the high biofilm-forming H. pylori strain 444A revealed that it possesses numerous biofilm- and virulence-associated genes and a small cryptic plasmid encoding a type II toxin-antitoxin system.Conclusion. There is substantial variation in biofilm-forming ability in H. pylori, but this was not significantly associated with disease severity in our study. We identified and characterized an interesting strain with high biofilm-forming ability, including generation and analysis of the complete genome.

Genomic diversity of Helicobacter pylori populations from different regions of the human stomach.

Individuals infected with Helicobacter pylori harbor unique and diverse populations of quasispecies, but diversity between and within different regions of the human stomach and the process of bacterial adaptation to each location are not yet well understood. We applied whole-genome deep sequencing to characterize the within- and between-stomach region genetic diversity of H. pylori populations from paired antrum and corpus biopsies of 15 patients, along with single biopsies from one region of an additional 3 patients, by scanning allelic diversity. We combined population deep sequencing with more conventional sequencing of multiple H. pylori single colony isolates from individual biopsies to generate a unique dataset. Single colony isolates were used to validate the scanning allelic diversity pipelines. We detected extensive population allelic diversity within the different regions of each patient's stomach. Diversity was most commonly found within non-coding, hypothetical, outer membrane, restriction modification system, virulence, lipopolysaccharide biosynthesis, efflux systems, and chemotaxis-associated genes. Antrum and corpus populations from the same patient grouped together phylogenetically, indicating that most patients were initially infected with a single strain, which then diversified. Single colonies from the antrum and corpus of the same patients grouped into distinct clades, suggesting mechanisms for within-location adaptation across multiple H. pylori isolates from different patients. The comparisons made available by combined sequencing and analysis of isolates and populations enabled comprehensive analysis of the genetic changes associated with H. pylori diversification and stomach region adaptation.

Access, use and disposal of antimicrobials among humans and animals in Wakiso district, Uganda: a qualitative study.

BACKGROUND: Inappropriate use of antimicrobials in both humans and animals is a key driver of antimicrobial resistance (AMR). In addition, human behaviours such as poor disposal of antimicrobials in the environment can increase their exposure to microbes which can impact on humans and animals. However, evidence on access, use and disposal of antimicrobials for humans and animals at community level in Uganda is limited. This study therefore explored access, use and disposal of antimicrobials among humans and animals in Wakiso district, Uganda. METHODS: A qualitative study was conducted that involved focus group discussions (FGDs) and key informant interviews (KIIs). Participants of the FGDs were community health workers (CHWs) and farmers involved in animal husbandry, while key informants included: officials from the Ministry of Health; Ministry of Agriculture, Animal Industry and Fisheries; human and animal health professionals; district health officials; and members of the national AMR surveillance committee. Twelve FGDs were held (8 for CHWs and 4 for farmers) while 15 KIIs were conducted. Thematic analysis in NVivo (version 12) was performed. RESULTS: Five main themes emerged from the study: access to antimicrobials in humans; access to antimicrobials in animals; use of antimicrobials in humans; use of antimicrobials in animals; and disposal of antimicrobials. Community members mainly accessed antimicrobials for humans from public health facilities such as government health centres, as well as private facilities, including drug shops and clinics. Antimicrobials for animals were obtained from veterinary practitioners and drug shops (both for humans and veterinary). Examples of inappropriate use of antimicrobials in both humans and animals was evident, such as sharing antibiotics among household members, and giving human-prescribed antimicrobials to food-producing animals as growth promoters. While some CHWs returned unused antimicrobials to public health facilities for proper disposal, community members mainly disposed of antimicrobials with general household waste including dumping in rubbish pits. CONCLUSIONS: There is a need to increase awareness among the population on proper access, use and disposal of antimicrobials for both humans and animals. Development of a drug disposal system at community level would facilitate improved waste management of antimicrobials. Together, these measures would help prevent the rate of progression of AMR in communities.

A One Health Approach to Strengthening Antimicrobial Stewardship in Wakiso District, Uganda.

Antimicrobial stewardship (AMS), as one of the global strategies to promote responsible use of antimicrobials to prevent antimicrobial resistance (AMR), remains poor in many low-and middle-income countries (LMICs). We implemented a project aimed at strengthening AMS in Wakiso district, Uganda using a One Health approach. A total of 86 health practitioners (HPs), including animal health workers, and 227 community health workers (CHWs) participated in training workshops, and over 300 pupils from primary schools were sensitized on AMR, AMS, and infection prevention and control (IPC). We further established two multidisciplinary online communities of practice (CoPs) for health professionals and students, with a current membership of 321 and 162, respectively. In addition, a Medicine and Therapeutics Committee (MTC) was set up at Entebbe Regional Referral Hospital. The project evaluation, conducted three months after training, revealed that the majority of the HPs (92.2%) and CHWs (90.3%) reported enhanced practices, including improved hand washing (57.3% and 81.0%, respectively). In addition, 51.5% of the HPs reported a reduction in the quantity of unnecessary antibiotics given per patient. This project demonstrates that AMS interventions using a One Health approach can promote understanding of the prudent use of antimicrobials and improve practices at health facilities and in communities.

Protective effects of Helicobacter pylori membrane vesicles against stress and antimicrobial agents.

Outer-membrane vesicles (OMVs) produced by Helicobacter pylori deliver bacterial components to host cells, provide a mechanism for stabilization of secreted components and may allow the bacteria to exert 'long-range' effects in the gastric niche, promoting persistence. In addition to their well-characterized host cell interactions, membrane vesicles improve stress survival in other bacterial species, and are constitutively produced by both pathogenic and non-pathogenic bacteria. We aimed to determine whether OMVs could improve H. pylori survival of a range of stressors. The effects of purified OMVs on the resistance of H. pylori to a range of environmental and antimicrobial stresses were determined using growth curves and survival assays. Addition of purified OMVs to H. pylori cultures provided dose-dependent protection against hydrogen peroxide-mediated killing. Supplementation with OMVs also partially protected H. pylori against the bactericidal effects of the antibiotics clarithromycin and levofloxacin, but not against amoxicillin nor metronidazole. Addition of purified OMVs allowed H. pylori to grow in the presence of inhibitory concentrations of the antimicrobial peptide LL-37. In the presence of 50 µg OMVs ml-1, significantly enhanced H. pylori growth was observed at higher LL-37 concentrations compared with lower LL-37 concentrations, suggesting that OMV-LL-37 interactions might facilitate release of growth-promoting nutrients. Taken together, these data indicate that production of membrane vesicles could help H. pylori to survive exposure to antibiotics and host antimicrobial defences during infection.

Sodium Taurocholate Stimulates Campylobacter jejuni Outer Membrane Vesicle Production via Down-Regulation of the Maintenance of Lipid Asymmetry Pathway.

Campylobacter jejuni outer membrane vesicles (OMVs) contain numerous virulence-associated proteins including the cytolethal distending toxin and three serine proteases. As C. jejuni lacks the classical virulence-associated secretion systems of other enteric pathogens that deliver effectors directly into target cells, OMVs may have a particularly important role in virulence. C. jejuni OMV production is stimulated by the presence of physiological concentrations of the bile salt sodium taurocholate (ST) through an unknown mechanism. The maintenance of lipid asymmetry (MLA) pathway has been implicated in a novel mechanism for OMV biogenesis, open to regulation by host signals. In this study we investigated the role of the MLA pathway in C. jejuni OMV biogenesis with ST as a potential regulator. OMV production was quantified by analyzing protein and lipid concentrations of OMV preparations and OMV particle counts produced by nanoparticle tracking analysis. Mutation of mlaA which encodes the outer membrane component of the MLA pathway significantly increased OMV production compared to the wild-type strain. Detergent sensitivity and membrane permeability assays confirmed the increased OMV production was not due to changes in membrane stability. The presence of 0.2% (w/v) ST increased wild-type OMV production and reduced OMV size, but did not further stimulate mlaA mutant OMV production or significantly alter mlaA mutant OMV size. qRT-PCR analysis demonstrated that the presence of ST decreased expression of both mlaA and mlaC in C. jejuni wild-type strains 11168 and 488. Collectively the data in this study suggests C. jejuni can regulate OMV production in response to host gut signals through changes in expression of the MLA pathway. As the gut bile composition is dependent on both diet and the microbiota, this study highlights the potential importance of diet and lifestyle factors on the varying disease presentations associated with gut pathogen infection.

Co-expression and purification of the RadA recombinase with the RadB paralog from Haloferax volcanii yields heteromeric ring-like structures.

The study of archaeal proteins and the processes to which they contribute poses particular challenges due to the often extreme environments in which they function. DNA recombination, replication and repair proteins of the halophilic euryarchaeon, Haloferax volcanii (Hvo) are of particular interest as they tend to resemble eukaryotic counterparts in both structure and activity, and genetic tools are available to facilitate their analysis. In the present study, we show using bioinformatics approaches that the Hvo RecA-like protein RadA is structurally similar to other recombinases although is distinguished by a unique acidic insertion loop. To facilitate expression of Hvo RadA a co-expression approach was used, providing its lone paralog, RadB, as a binding partner. At present, structural and biochemical characterization of Hvo RadA is lacking. Here, we describe for the first time co-expression of Hvo RadA with RadB and purification of these proteins as a complex under in vitro conditions. Purification procedures were performed under high salt concentration (>1 M sodium chloride) to maintain the solubility of the proteins. Quantitative densitometry analysis of the co-expressed and co-purified RadAB complex estimated the ratio of RadA to RadB to be 4 : 1, which suggests that the proteins interact with a specific stoichiometry. Based on a combination of analyses, including size exclusion chromatography, Western blot and electron microscopy observations, we suggest that RadA multimerizes into a ring-like structure in the absence of DNA and nucleoside co-factor.

Helicobacter pylori-Mediated Protection from Allergy Is Associated with IL-10-Secreting Peripheral Blood Regulatory T Cells.

Helicobacter pylori infections are usually established in early childhood and continuously stimulate immunity, including T-helper 1 (Th1), Th17, and regulatory T-cell (Treg) responses, throughout life. Although known to be the major cause of peptic ulcer disease and gastric cancer, disease occurs in a minority of those who are infected. Recently, there has been much interest in beneficial effects arising from infection with this pathogen. Published data robustly show that the infection is protective against asthma in mouse models. Epidemiological studies show that H. pylori is inversely associated with human allergy and asthma, but there is a paucity of mechanistic data to explain this. Since Th1 and Treg responses are reported to protect against allergic responses, we investigated if there were links between the human systemic Th1 and Treg response to H. pylori and allergen-specific IgE levels. The human cytokine and T-cell responses were examined using peripheral blood mononuclear cells (PBMCs) from 49 infected and 58 uninfected adult patients. Concentrations of total and allergen-specific plasma IgE were determined by ELISA and ImmunoCAP assays. These responses were analyzed according to major virulence factor genotypes of the patients' colonizing H. pylori strains. An in vitro assay was employed, using PBMCs from infected and uninfected donors, to determine the role of Treg cytokines in the suppression of IgE. Significantly higher frequencies of IL-10-secreting CD4(+)CD25(hi) Tregs, but not H. pylori-specific Th1 cells, were present in the peripheral blood of infected patients. Total and allergen-specific IgE concentrations were lower when there was a strong Treg response, and blocking IL-10 in vitro dramatically restored IgE responses. IgE concentrations were also significantly lower when patients were infected with CagA(+) strains or those expressing the more active i1 form of VacA. The systemic IL-10(+) Treg response is therefore likely to play a role in H. pylori-mediated protection against allergy in humans.

Differential inflammatory response to Helicobacter pylori infection: etiology and clinical outcomes.

The bacterial pathogen Helicobacter pylori commonly colonizes the human gastric mucosa during early childhood and persists throughout life. The organism has evolved multiple mechanisms for evading clearance by the immune system and, despite inducing inflammation in the stomach, the majority of infections are asymptomatic. H. pylori is the leading cause of peptic ulcer disease and gastric cancer. However, disease outcomes are related to the pattern and severity of chronic inflammation in the gastric mucosa, which in turn is influenced by both bacterial and host factors. Despite over 2 decades of intensive research, there remains an incomplete understanding of the circumstances leading to disease development, due to the fascinating complexity of the host-pathogen interactions. There is accumulating data concerning the virulence factors associated with increased risk of disease, and the majority of these have pro-inflammatory activities. Despite this, only a small proportion of those infected with virulent strains develop disease. Several H. pylori virulence factors have multiple effects on different cell types, including the induction of pro- and anti-inflammatory, immune stimulatory, and immune modulatory responses. The expression of multiple virulence factors is also often linked, making it difficult to assess the meaning of their effects in isolation. Overall, H. pylori is thought to usually modulate inflammation and limit acute damage to the mucosa, enabling the bacteria to persist. If this delicate balance is disturbed, disease may then develop.

Expression of the Helicobacter pylori virulence factor vacuolating cytotoxin A (vacA) is influenced by a potential stem-loop structure in the 5' untranslated region of the transcript.

The vacuolating cytotoxin, VacA, is an important virulence factor secreted by the gastric pathogen Helicobacter pylori. Certain vacA genotypes are strongly associated with disease risk, but the association is not absolute. The factors determining vacA gene expression are not fully understood, and the mechanisms of its regulation are elusive. We have identified a potential mRNA stem-loop forming structure in the 5' untranslated region (UTR) of the vacA transcript. Using site-directed mutagenesis, we found that disruption of the stem-loop structure reduced steady-state mRNA levels between two- and sixfold (P = 0.0005) and decreased mRNA half-life compared with wild type (P = 0.03). This led to a marked reduction in VacA protein levels and overall toxin activity. Additionally, during stressful environmental conditions of acid pH or high environmental salt concentrations, when general transcription of vacA was decreased or increased respectively, the stabilising effects of the stem-loop were even more pronounced. Our results suggest that the stem-loop structure in the vacA 5' UTR is an important determinant of vacA expression through stabilisation of the vacA mRNA transcript and that the stabilising effect is of particular importance during conditions of environmental stress.

A Fluorescence Resonance Energy Transfer Assay For Monitoring α- Synclein Aggregation in a Caenorhabditis Elegans Model For Parkinson's Disease.

The aggregation of α-synuclein (Syn or S) to form insoluble fibrils is important in the pathogenesis of Parkinson's disease, but key risk factors remain ill-defined. We have developed Fluorescence Resonance Energy Transfer (FRET)-based assays for α-synuclein aggregation, using Green Fluorescent Protein variants Cerulean (C) or Venus (V), fused to each other (CV, VC) or to human synuclein (SC, SV etc). Bacterially expressed proteins were purified to homogeneity, and C-terminal fusions SC and SV largely retained their ability to aggregate in vitro. FRET signals from mixtures of SC and SV were used to monitor aggregation. These fusion genes were linked to the C. elegans unc-54 myosin promoter to generate integrated transgenic strains. Increased FRET signals, indicative of S aggregation, were observed following treatment of unc-54::SC + unc-54::SV double transgenic worms with low concentrations of mercury or chlorpyrifos, or with RNAi against hsp-70 and hip-1. Opposite changes in Yellow Fluorescent Protein (YFP) fluorescence in an unc-54::SV strain (NL5901) are likely to reflect FRET from Yellow Fluorescent Protein to aggregates of Syn fusion protein. This could provide the basis for a high throughput screening assay, which could be used for studying the effects of toxic chemicals and environmental pollutants on the aggregation of proteins such as Syn in vivo.

Characterization of outer membrane vesicles from a neonatal meningitic strain of Cronobacter sakazakii.

Cronobacter sakazakii is associated with severe and often fatal cases of infant meningitis and necrotizing enterocolitis. The form of meningitis differs from that due to Neisseria meningitidis and Streptococcus spp., in that it is highly invasive and destructive towards human brain cells. However, there is relatively little understanding of the cytopathogenic interaction of C. sakazakii with host cells which results in stimulation of an inflammatory immune response. The production of Cronobacter outer membrane vesicles (OMV) and their potential pathogenic functions have not yet been elucidated. This study is the first to show that C. sakazakii produce OMV, which may play a role in the activation of cytopathogenic and host cell responses on human intestinal epithelial cells. Cronobacter sakazakii strain 767 was used which had been isolated from a fatal outbreak of neonatal meningitis and necrotizing enterocolitis. Cronobacter sakazakii OMV were internalized by Caco-2 cells, increased cell proliferation and stimulated the host's innate proinflammatory response without inducing overt toxicity. A total of 18 OMV-associated proteins were identified by mass spectrometry and their potential pathogenicity roles were evaluated. Collectively, these data indicate that C. sakazakii OMV could play a role in pathogenesis by delivering bacterial toxins into host epithelial cells, driving proliferative and proinflammatory responses.

A role for the vacuolating cytotoxin, VacA, in colonization and Helicobacter pylori-induced metaplasia in the stomach.

Carriage of Helicobacter pylori strains producing more active (s1/i1) forms of VacA is strongly associated with gastric adenocarcinoma. To our knowledge, we are the first to determine effects of different polymorphic forms of VacA on inflammation and metaplasia in the mouse stomach. Bacteria producing the less active s2/i2 form of VacA colonized mice more efficiently than mutants null for VacA or producing more active forms of it, providing the first evidence of a positive role for the minimally active s2/i2 toxin. Strains producing more active toxin forms induced more severe and extensive metaplasia and inflammation in the mouse stomach than strains producing weakly active (s2/i2) toxin. We also examined the association in humans, controlling for cagPAI status. In human gastric biopsy specimens, the vacA i1 allele was strongly associated with precancerous intestinal metaplasia, with almost complete absence of intestinal metaplasia in subjects infected with i2-type strains, even in a vacA s1, cagA(+) background.

Helicobacter pylori membrane vesicles stimulate innate pro- and anti-inflammatory responses and induce apoptosis in Jurkat T cells.

Persistent Helicobacter pylori infection induces chronic inflammation in the human gastric mucosa, which is associated with development of peptic ulceration, gastric atrophy, and gastric adenocarcinoma. It has been postulated that secretion of immunomodulatory molecules by H. pylori facilitates bacterial persistence, and membrane vesicles (MV), which have the potential to cross the gastric epithelial barrier, may mediate delivery of these molecules to host immune cells. However, bacterial MV effects on human immune cells remain largely uncharacterized to date. In the present study, we investigated the immunomodulatory effects of H. pylori MV with and without the vacuolating cytotoxin, VacA, which inhibits human T cell activity. We show a high degree of variability in the toxin content of vesicles between two H. pylori strains (SS1 and 60190). Vesicles from the more toxigenic 60190 strain contain more VacA (s1i1 type) than vesicles from the SS1 strain (s2i2 VacA), but engineering the SS1 strain to produce s1i1 VacA did not increase the toxin content of its vesicles. Vesicles from all strains tested, including a 60190 isogenic mutant null for VacA, strongly induced interleukin-10 (IL-10) and IL-6 production by human peripheral blood mononuclear cells independently of the infection status of the donor. Finally, we show that H. pylori MV induce T cell apoptosis and that this is enhanced by, but not completely dependent on, the carriage of VacA. Together, these findings suggest a role for H. pylori MV in the stimulation of innate pro- and anti-inflammatory responses and in the suppression of T cell immunity.

The UmuC subunit of the E. coli DNA polymerase V shows a unique interaction with the ß-clamp processivity factor.

BACKGROUND: Strict regulation of replisome components is essential to ensure the accurate transmission of the genome to the next generation. The sliding clamp processivity factors play a central role in this regulation, interacting with both DNA polymerases and multiple DNA processing and repair proteins. Clamp binding partners share a common peptide binding motif, the nature of which is essentially conserved from phage through to humans. Given the degree of conservation of these motifs, much research effort has focussed on understanding how the temporal and spatial regulation of multiple clamp binding partners is managed. The bacterial sliding clamps have come under scrutiny as potential targets for rational drug design and comprehensive understanding of the structural basis of their interactions is crucial for success. RESULTS: In this study we describe the crystal structure of a complex of the E. coli ß-clamp with a 12-mer peptide from the UmuC protein. UmuC is the catalytic subunit of the translesion DNA polymerase, Pol V (UmuD'2C). Due to its potentially mutagenic action, Pol V is tightly regulated in the cell to limit access to the replication fork. Atypically for the translesion polymerases, both bacterial and eukaryotic, Pol V is heterotrimeric and its ß-clamp binding motif (³57QLNLF³6¹) is internal to the protein, rather than at the more usual C-terminal position. Our structure shows that the UmuC peptide follows the overall disposition of previously characterised structures with respect to the highly conserved glutamine residue. Despite good agreement with the consensus ß-clamp binding motif, distinct variation is shown within the hydrophobic binding pocket. While UmuC Leu-360 interacts as noted in other structures, Phe-361 does not penetrate the pocket at all, sitting above the surface. CONCLUSION: Although the ß-clamp binding motif of UmuC conforms to the consensus sequence, variation in its mode of clamp binding is observed compared to related structures, presumably dictated by the proximal aspartate residues that act as linker to the poorly characterised, unique C-terminal domain of UmuC. Additionally, interactions between Asn-359 of UmuC and Arg-152 on the clamp surface may compensate for the reduced interaction of Phe-361.

Effects of α-synuclein overexpression in transgenic Caenorhabditis elegans strains.

The neural protein α-synuclein aggregates both in vivo and in vitro to form insoluble fibrils that are involved in Parkinson's disease pathogenesis. We have generated α-synuclein/fluorescent-protein fusion constructs overexpressed in muscle cells of the nematode, Caenorhabdtis elegans. Green Fluorescent Protein (GFP) variants, Cerulean (C) or Venus (V), were fused to the C-terminus of human α-synuclein (S); the resultant fusion genes were designated SV and SC, plus a CV fusion as well as S, C and V singly. The aggregation behavior of the purified fusion proteins (expressed in E. coli) will be described elsewhere. These constructs were fused to a C. elegans unc-54 myosin promoter, and integrated transgenic lines generated by microinjection, λ-irradiation, and outcrossing of fluorescent progeny. All transgenic lines expressing α- synuclein showed significant reductions (p <0.05) in lifespan, motility and pharyngeal pumping, as compared to wildtype worms or lines expressing CFP and/or YFP only. We showed that CFP and YFP labels colocalised in granular inclusions throughout the body wall in transgenic lines expressing both SC and SV fusions (SC+SV), whereas SV+C worms displayed YFP-labelled inclusions on a diffuse CFP background. These findings implied that the α-synuclein moieties of these fusion proteins still aggregated together in vivo, whereas CFP or YFP moieties alone did not. This in turn suggested that Foerster Resonanace Energy Transfer (FRET) between CFP and YFP labels in α-synuclein aggregates could allow the extent of aggregation to be quantified. Accordingly, we also showed that net FRET signals increased 2- fold between L4 and adult SC+SV worms.

Rings in the extreme: PCNA interactions and adaptations in the archaea.

Biochemical and structural analysis of archaeal proteins has enabled us to gain great insight into many eukaryotic processes, simultaneously offering fascinating glimpses into the adaptation and evolution of proteins at the extremes of life. The archaeal PCNAs, central to DNA replication and repair, are no exception. Characterisation of the proteins alone, and in complex with both peptides and protein binding partners, has demonstrated the diversity and subtlety in the regulatory role of these sliding clamps. Equally, studies have provided valuable detailed insight into the adaptation of protein interactions and mechanisms that are necessary for life in extreme environments.