Conformational and aggregation properties of a pegylated alanine-rich polypeptide

The conformational and aggregation behavior of PEG conjugates of an alanine-rich polypeptide (PEG-c17H6) were investigated and compared to that of the polypeptide equipped with a deca-histidine tag (17H6). These polypeptides serve as simple and stimuli-responsive models for the aggregation behavior of helix-rich proteins, as our previous studies have shown that the helical 17H6 self-associates at acidic pH and converts to β-sheet structures at elevated temperature under acidic conditions. In the work here, we show that PEG-c17H6 also adopts a helical structure at ambient/subambient temperatures, at both neutral and acidic pH. The thermal denaturation behavior of 17H6 and PEG-c17H6 is similar at neutral pH, where the alanine-rich domain has no self-association tendency. At acidic pH and elevated temperature, however, PEGylation slows β-sheet formation of c17H6, and reduces the apparent cooperativity of thermally induced unfolding. Transmission electron microscopy of PEG-c17H6 conjugates incubated at elevated temperatures showed fibrils with widths of ∼20-30 nm, wider than those observed for fibrils of 17H6. These results suggest that PEGylation reduces β-sheet aggregation in these polypeptides by interfering, only after unfolding of the native helical structure, with interprotein conformational changes needed to form β-sheet aggregates.National Center for Research Resources (NCRR); Center for Neutron Science (U.S. Dept. of Commerce

From science to practice: Bringing innovations to agronomy and forestry

The challenge of the work presented here is to make innovative research output in the agronomy and forestry domain accessible to end-users, so that it can be practically applied. We have developed an approach that consists of three key-elements: an ontology with domain knowledge, a set of documents that have been annotated and meta-annotated, and a system (ask-Valerie) that is based on a dialogue to represent the interaction between end user and system.<br/> We show that the dialogue-metaphor is a good way of modelling the interaction between user and system. The system helps the user in formulating his question and in answering it in a useful way. Meta-annotations of key-paragraphs in the document-base turn out to be relevant in assessing in one glance what the content of a document is. <br/> End-users are very enthusiastic about the possibilities that ask-Valerie offers them in translating scientific results to their own situation

Mode and dynamics of vanA-type vancomycin resistance dissemination in Dutch hospitals

Background Enterococcus faecium is a commensal of the gastrointestinal tract of animals and humans but also a causative agent of hospital-acquired infections. Resistance against glycopeptides and to vancomycin has motivated the inclusion of E. faecium in the WHO global priority list. Vancomycin resistance can be conferred by the vanA gene cluster on the transposon Tn1546, which is frequently present in plasmids. The vanA gene cluster can be disseminated clonally but also horizontally either by plasmid dissemination or by Tn1546 transposition between different genomic locations. Methods We performed a retrospective study of the genomic epidemiology of 309 vancomycin-resistant E. faecium (VRE) isolates across 32 Dutch hospitals (2012-2015). Genomic information regarding clonality and Tn1546 characterization was extracted using hierBAPS sequence clusters (SC) and TETyper, respectively. Plasmids were predicted using gplas in combination with a network approach based on shared k-mer content. Next, we conducted a pairwise comparison between isolates sharing a potential epidemiological link to elucidate whether clonal, plasmid, or Tn1546 spread accounted for vanA-type resistance dissemination. Results On average, we estimated that 59% of VRE cases with a potential epidemiological link were unrelated which was defined as VRE pairs with a distinct Tn1546 variant. Clonal dissemination accounted for 32% cases in which the same SC and Tn1546 variants were identified. Horizontal plasmid dissemination accounted for 7% of VRE cases, in which we observed VRE pairs belonging to a distinct SC but carrying an identical plasmid and Tn1546 variant. In 2% of cases, we observed the same Tn1546 variant in distinct SC and plasmid types which could be explained by mixed and consecutive events of clonal and plasmid dissemination. Conclusions In related VRE cases, the dissemination of the vanA gene cluster in Dutch hospitals between 2012 and 2015 was dominated by clonal spread. However, we also identified outbreak settings with high frequencies of plasmid dissemination in which the spread of resistance was mainly driven by horizontal gene transfer (HGT). This study demonstrates the feasibility of distinguishing between modes of dissemination with short-read data and provides a novel assessment to estimate the relative contribution of nested genomic elements in the dissemination of vanA-type resistance.Peer reviewe

Cirrhosis-induced defects in innate pulmonary defenses against Streptococcus pneumoniae

<p>Abstract</p> <p>Background</p> <p>The risk of mortality from pneumonia caused by <it>Streptococcus pneumoniae </it>is increased in patients with cirrhosis. However, the specific pneumococcal virulence factors and host immune defects responsible for this finding have not been clearly established. This study used a cirrhotic rat model of pneumococcal pneumonia to identify defect(s) in innate pulmonary defenses in the cirrhotic host and to determine the impact of the pneumococcal toxin pneumolysin on these defenses in the setting of severe cirrhosis.</p> <p>Results</p> <p>No cirrhosis-associated defects in mucociliary clearance of pneumococci were found in these studies, but early intrapulmonary killing of the organisms before the arrival of neutrophils was significantly impaired. This defect was exacerbated by pneumolysin production in cirrhotic but not in control rats. Neutrophil-mediated killing of a particularly virulent type 3 pneumococcal strain also was significantly diminished within the lungs of cirrhotic rats with ascites. Levels of lysozyme and complement component C3 were both significantly reduced in bronchoalveolar lavage fluid from cirrhotic rats. Finally, complement deposition was reduced on the surface of pneumococci recovered from the lungs of cirrhotic rats in comparison to organisms recovered from the lungs of control animals.</p> <p>Conclusion</p> <p>Increased mortality from pneumococcal pneumonia in this cirrhotic host is related to defects in both early pre-neutrophil- and later neutrophil-mediated pulmonary killing of the organisms. The fact that pneumolysin production impaired pre-neutrophil-mediated pneumococcal killing in cirrhotic but not control rats suggests that pneumolysin may be particularly detrimental to this defense mechanism in the severely cirrhotic host. The decrease in neutrophil-mediated killing of pneumococci within the lungs of the cirrhotic host is related to insufficient deposition of host proteins such as complement C3 on their surfaces. Pneumolysin likely plays a role in complement consumption within the lungs. Our studies, however, were unable to determine whether pneumolysin more negatively impacted this defense mechanism in cirrhotic than in control rats. These findings contribute to our understanding of the defects in innate pulmonary defenses that lead to increased mortality from pneumococcal pneumonia in the severely cirrhotic host. They also suggest that pneumolysin may be a particularly potent pneumococcal virulence factor in the setting of cirrhosis.</p

Dynamics of ampicillin-resistant Enterococcus faecium clones colonizing hospitalized patients: data from a prospective observational study

<p>Abstract</p> <p>Background</p> <p>Little is known about the dynamics of colonizing <it>Enterococcus faecium </it>clones during hospitalization, invasive infection and after discharge.</p> <p>Methods</p> <p>In a prospective observational study we compared intestinal <it>E. faecium </it>colonization in three patient cohorts: 1) Patients from the Hematology Unit at the University Hospital Basel (UHBS), Switzerland, were investigated by weekly rectal swabs (RS) during hospitalization (group 1a, n = 33) and monthly after discharge (group 1b, n = 21). 2) Patients from the Intensive Care Unit (ICU) at the University Medical Center Utrecht, the Netherlands (group 2, n = 25) were swabbed weekly. 3) Patients with invasive <it>E. faecium </it>infection at UHBS were swabbed at the time of infection (group 3, n = 22). From each RS five colonies with typical <it>E</it>. <it>faecium </it>morphology were picked. Species identification was confirmed by PCR and ampicillin-resistant <it>E. faecium </it>(ARE) isolates were typed using Multiple Locus Variable Number Tandem Repeat Analysis (MLVA). The Simpson's Index of Diversity (SID) was calculated.</p> <p>Results</p> <p>Out of 558 ARE isolates from 354 RS, MT159 was the most prevalent clone (54%, 100%, 52% and 83% of ARE in groups 1a, 1b, 2 and 3, respectively). Among hematological inpatients 13 (40%) had ARE. During hospitalization, the SID of MLVA-typed ARE decreased from 0.745 [95%CI 0.657-0.833] in week 1 to 0.513 [95%CI 0.388-0.637] in week 3. After discharge the only detected ARE was MT159 in 3 patients. In the ICU (group 2) almost all patients (84%) were colonized with ARE. The SID increased significantly from 0.373 [95%CI 0.175-0.572] at week 1 to a maximum of 0.808 [95%CI 0.768-0.849] at week 3 due to acquisition of multiple ARE clones. All 16 patients with invasive ARE were colonized with the same MLVA clone (<it>p </it>< 0.001).</p> <p>Conclusions</p> <p>In hospitalized high-risk patients MT159 is the most frequent colonizer and cause of invasive <it>E. faecium </it>infections. During hospitalization, ASE are quickly replaced by ARE. Diversity of ARE increases on units with possible cross-transmission such as ICUs. After hospitalization ARE are lost with the exception of MT159. In invasive infections, the invasive clone is the predominant gut colonizer.</p

Genomic rearrangements uncovered by genome-wide co-evolution analysis of a major nosocomial pathogen, Enterococcus faecium

Enterococcus faecium is a gut commensal of the gastro-digestive tract, but also known as nosocomial pathogen among hospitalized patients. Population genetics based on whole-genome sequencing has revealed that E. faecium strains from hospitalized patients form a distinct Glade, designated Glade A1, and that plasmids are major contributors to the emergence of nosocomial E. faecium. Here we further explored the adaptive evolution of E faecium using a genome-wide co-evolution study (GWES) to identify co-evolving single-nucleotide polymorphisms (SNPs). We identified three genomic regions harbouring large numbers of SNPs in tight linkage that are not proximal to each other based on the completely assembled chromosome of the Glade A1 reference hospital isolate AUS0004. Close examination of these regions revealed that they are located at the borders of four different types of large-scale genomic rearrangements, insertion sites of two different genomic islands and an IS30-like transposon. In non-Glade A1 isolates, these regions are adjacent to each other and they lack the insertions of the genomic islands and IS30-like transposon. Additionally, among the Glade A1 isolates there is one group of pet isolates lacking the genomic rearrangement and insertion of the genomic islands, suggesting a distinct evolutionary trajectory. In silico analysis of the biological functions of the genes encoded in three regions revealed a common link to a stress response. This suggests that these rearrangements may reflect adaptation to the stringent conditions in the hospital environment, such as antibiotics and detergents, to which bacteria are exposed. In conclusion, to our knowledge, this is the first study using GWES to identify genomic rearrangements, suggesting that there is considerable untapped potential to unravel hidden evolutionary signals from population genomic data.Peer reviewe

mlplasmids : a user-friendly tool to predict plasmid- and chromosome-derived sequences for single species

Assembly of bacterial short-read whole-genome sequencing data frequently results in hundreds of contigs for which the origin, plasmid or chromosome, is unclear. Complete genomes resolved by long-read sequencing can be used to generate and label short-read contigs. These were used to train several popular machine learning methods to classify the origin of contigs from Enterococcus faecium, Klebsiella pneumoniae and Escherichia colt using pentamer frequencies. We selected support-vector machine (SVM) models as the best classifier for all three bacterial species (Fl-score E. faecium=0.92, F1-score K. pneumoniae=0.90, F1-score E. coli=0.76), which outperformed other existing plasmid prediction tools using a benchmarking set of isolates. We demonstrated the scalability of our models by accurately predicting the plasmidome of a large collection of 1644 E. faecium isolates and illustrate its applicability by predicting the location of antibiotic-resistance genes in all three species. The SVM classifiers are publicly available as an R package and graphical-user interface called 'mlplasmids'. We anticipate that this tool may significantly facilitate research on the dissemination of plasmids encoding antibiotic resistance and/or contributing to host adaptation.Peer reviewe

Plasmids Shaped the Recent Emergence of the Major Nosocomial Pathogen Enterococcus faecium

Enterococcus faecium is a gut commensal of humans and animals but is also listed on the WHO global priority list of multidrug-resistant pathogens. Many of its antibiotic resistance traits reside on plasmids and have the potential to be disseminated by horizontal gene transfer. Here, we present the first comprehensive population-wide analysis of the pan-plasmidome of a clinically important bacterium, by whole-genome sequence analysis of 1,644 isolates from hospital, commensal, and animal sources of E. faecium. Long-read sequencing on a selection of isolates resulted in the completion of 305 plasmids that exhibited high levels of sequence modularity. We further investigated the entirety of all plasmids of each isolate (plasmidome) using a combination of short-read sequencing and machine-learning classifiers. Clustering of the plasmid sequences unraveled different E. faecium populations with a clear association with hospitalized patient isolates, suggesting different optimal configurations of plasmids in the hospital environment. The characterization of these populations allowed us to identify common mechanisms of plasmid stabilization such as toxin-antitoxin systems and genes exclusively present in particular plasmidome populations exemplified by copper resistance, phosphotransferase systems, or bacteriocin genes potentially involved in niche adaptation. Based on the distribution of k-mer distances between isolates, we concluded that plasmidomes rather than chromosomes are most informative for source specificity of E. faecium. IMPORTANCE Enterococcus faecium is one of the most frequent nosocomial pathogens of hospital-acquired infections. E. faecium has gained resistance against most commonly available antibiotics, most notably, against ampicillin, gentamicin, and vancomycin, which renders infections difficult to treat. Many antibiotic resistance traits, in particular, vancomycin resistance, can be encoded in autonomous and extrachromosomal elements called plasmids. These sequences can be disseminated to other isolates by horizontal gene transfer and confer novel mechanisms to source specificity. In our study, we elucidated the total plasmid content, referred to as the plasmidome, of 1,644 E. faecium isolates by using short- and long-read whole-genome technologies with the combination of a machine-learning classifier. This was fundamental to investigate the full collection of plasmid sequences present in our collection (pan-plasmidome) and to observe the potential transfer of plasmid sequences between E. faecium hosts. We observed that E. faecium isolates from hospitalized patients carried a larger number of plasmid sequences compared to that from other sources, and they elucidated different configurations of plasmidome populations in the hospital environment. We assessed the contribution of different genomic components and observed that plasmid sequences have the highest contribution to source specificity. Our study suggests that E. faecium plasmids are regulated by complex ecological constraints rather than physical interaction between hosts.Peer reviewe

Mode and dynamics of vanA-type vancomycin resistance dissemination in Dutch hospitals

Abstract Background Enterococcus faecium is a commensal of the gastrointestinal tract of animals and humans but also a causative agent of hospital-acquired infections. Resistance against glycopeptides and to vancomycin has motivated the inclusion of E. faecium in the WHO global priority list. Vancomycin resistance can be conferred by the vanA gene cluster on the transposon Tn1546, which is frequently present in plasmids. The vanA gene cluster can be disseminated clonally but also horizontally either by plasmid dissemination or by Tn1546 transposition between different genomic locations. Methods We performed a retrospective study of the genomic epidemiology of 309 vancomycin-resistant E. faecium (VRE) isolates across 32 Dutch hospitals (2012–2015). Genomic information regarding clonality and Tn1546 characterization was extracted using hierBAPS sequence clusters (SC) and TETyper, respectively. Plasmids were predicted using gplas in combination with a network approach based on shared k-mer content. Next, we conducted a pairwise comparison between isolates sharing a potential epidemiological link to elucidate whether clonal, plasmid, or Tn1546 spread accounted for vanA-type resistance dissemination. Results On average, we estimated that 59% of VRE cases with a potential epidemiological link were unrelated which was defined as VRE pairs with a distinct Tn1546 variant. Clonal dissemination accounted for 32% cases in which the same SC and Tn1546 variants were identified. Horizontal plasmid dissemination accounted for 7% of VRE cases, in which we observed VRE pairs belonging to a distinct SC but carrying an identical plasmid and Tn1546 variant. In 2% of cases, we observed the same Tn1546 variant in distinct SC and plasmid types which could be explained by mixed and consecutive events of clonal and plasmid dissemination. Conclusions In related VRE cases, the dissemination of the vanA gene cluster in Dutch hospitals between 2012 and 2015 was dominated by clonal spread. However, we also identified outbreak settings with high frequencies of plasmid dissemination in which the spread of resistance was mainly driven by horizontal gene transfer (HGT). This study demonstrates the feasibility of distinguishing between modes of dissemination with short-read data and provides a novel assessment to estimate the relative contribution of nested genomic elements in the dissemination of vanA-type resistance