A general protein O-glycosylation system within the Burkholderia cepacia complex is involved in motility and virulence.

Bacteria of the Burkholderia cepacia complex (Bcc) are pathogens of humans, plants, and animals. Burkholderia cenocepacia is one of the most common Bcc species infecting cystic fibrosis (CF) patients and its carriage is associated with poor prognosis. In this study, we characterized a general O-linked protein glycosylation system in B. cenocepacia K56-2. The PglLBc O-oligosaccharyltransferase (O-OTase), encoded by the cloned gene bcal0960, was shown to be capable of transferring a heptasaccharide from the Campylobacter jejuni N-glycosylation system to a Neisseria meningitides-derived acceptor protein in an Escherichia coli background, indicating that the enzyme has relaxed specificities for both the sugar donor and protein acceptor. In B cenocepacia K56-2, PglLBc is responsible for the glycosylation of 23 proteins involved in diverse cellular processes. Mass spectrometry analysis revealed that these proteins are modified with a trisaccharide HexNAc-HexNAc-Hex, which is unrelated to the O-antigen biosynthetic process. The glycosylation sites that were identified existed within regions of low complexity, rich in serine, alanine, and proline. Disruption of bcal0960 abolished glycosylation and resulted in reduced swimming motility and attenuated virulence towards both plant and insect model organisms. This study demonstrates the first example of post-translational modification in Bcc with implications for pathogenesis.

Characterization of the type III secretion associated low calcium response genes of Vibrio parahaemolyticus RIMD2210633.

Vibrio parahaemolyticus is a significant human pathogen associated with gastroenteritis. Two V. parahaemolyticus type 3 secretion systems, T3SS-1 and T3SS-2, secrete effector proteins and have been implicated in host-cell cytotoxicity and enterotoxicity, respectively. Vibrio parahaemolyticus T3SS-1 substrates have been identified, although many predicted substrates (based on homologies) remain undetected in secreted fractions and therefore uncharacterized. We have experimentally developed and optimized a secretion assay protocol allowing for reliable and reproducible detection of V. parahaemolyticus T3SS-1 secreted proteins within culture supernatants. The presence of magnesium and absence of calcium were critical factors in promoting type III secretion of protein substrates. Proteomic approaches identified known V. parahaemolyticus secreted effectors in addition to previously unidentified proteins. Isogenic mutants in putative low calcium response genes were generated, and experiments further implicated the genes in secretion and V. parahaemolyticus-mediated host-cell cytotoxicity during infection. These approaches should be valuable towards future detailed genetic and biochemical analyses of T3SS-1 in V. parahaemolyticus.

Antimicrobial activity in the egg wax of the tick Amblyomma hebraeum (Acari: Ixodidae) is associated with free fatty acids C16:1 and C18:2.

Untreated eggs of the tick Amblyomma hebraeum Koch (Acari: Ixodidae) exhibited antimicrobial activity (AMA) against Gram-negative but not Gram-positive bacteria; eggs denuded of wax by solvent extraction showed no AMA. The unfractionated egg wax extract, however, showed AMA against Gram-positive but not Gram-negative bacteria, as also shown by Arrieta et al. (Exp Appl Acarol 39: 297-313, 2006). In this study we partitioned the egg wax into various fractions, using a variety of techniques, analyzed their compositions, and tested them for AMA. The crude aqueous extract exhibited AMA. However, although more than 30 metabolites were identified in this extract by nuclear magnetic resonance analysis, none of them seemed likely to be responsible for the observed AMA. In the crude organic extract, cholesterol esters were the most abundant lipids, but were devoid of AMA. Fatty acids (FAs), with chain lengths between C13 and C26 were the next most abundant lipids. After lipid fractionation and gas chromatography/mass spectroscopy, free FAs, especially C16:1 and C18:2, accounted for most of the AMA in the organic extract. The material responsible for AMA in the crude aqueous extract remains unidentified. No AMA was detected in the intracellular contents of untreated eggs.

Study protocol for resolution of organ injury in acute pancreatitis (RESORP): an observational prospective cohort study.

INTRODUCTION: Survivors of acute pancreatitis (AP) have shorter overall survival and increased incidence of new-onset cardiovascular, respiratory, liver and renal disease, diabetes mellitus and cancer compared with the general population, but the mechanisms that explain this are yet to be elucidated. Our aim is to characterise the precise nature and extent of organ dysfunction following an episode of AP. METHODS AND ANALYSIS: This is an observational prospective cohort study in a single centre comprising a University hospital with an acute and emergency receiving unit and clinical research facility. Participants will be adult patient admitted with AP. Participants will undergo assessment at recruitment, 3 months and 3 years. At each time point, multiple biochemical and/or physiological assessments to measure cardiovascular, respiratory, liver, renal and cognitive function, diabetes mellitus and quality of life. Recruitment was from 30 November 2017 to 31 May 2020; last follow-up measurements is due on 31 May 2023. The primary outcome measure is the incidence of new-onset type 3c diabetes mellitus during follow-up. Secondary outcome measures include: quality of life analyses (SF-36, Gastrointestinal Quality of Life Index); montreal cognitive assessment; organ system physiological performance; multiomics predictors of AP severity, detection of premature cellular senescence. In a nested cohort within the main cohort, individuals may also consent to multiparameter MRI scan, echocardiography, pulmonary function testing, cardiopulmonary exercise testing and pulse-wave analysis. ETHICS AND DISSEMINATION: This study has received the following approvals: UK IRAS Number 178615; South-east Scotland Research Ethics Committee number 16/SS/0065. Results will be made available to AP survivors, caregivers, funders and other researchers. Publications will be open-access. TRIAL REGISTRATION NUMBERS: ClinicalTrials.gov Registry (NCT03342716) and ISRCTN50581876; Pre-results.

Genome Sequence of Megasphaera cerevisiae NSB1, a Bacterium Isolated from a Canning Line and Able To Grow in Beer with High Alcohol Content.

The genome sequence of the brewery isolate Megasphaera cerevisiae NSB1 was determined. Strain NSB1 tolerates 5% (vol/vol) alcohol, which is higher than previously reported for M. cerevisiae The NSB1 genome will help elucidate genetics required for alcohol tolerance and niche adaptation of this Gram-negative beer-spoilage bacterium.

Common duckweed (Lemna minor) is a versatile high-throughput infection model for the Burkholderia cepacia complex and other pathogenic bacteria.

Members of the Burkholderia cepacia complex (Bcc) have emerged in recent decades as problematic pulmonary pathogens of cystic fibrosis (CF) patients, with severe infections progressing to acute necrotizing pneumonia and sepsis. This study presents evidence that Lemna minor (Common duckweed) is useful as a plant model for the Bcc infectious process, and has potential as a model system for bacterial pathogenesis in general. To investigate the relationship between Bcc virulence in duckweed and Galleria mellonella (Greater wax moth) larvae, a previously established Bcc infection model, a duckweed survival assay was developed and used to determine LD50 values. A strong correlation (R(2) = 0.81) was found between the strains' virulence ranks in the two infection models, suggesting conserved pathways in these vastly different hosts. To broaden the application of the duckweed model, enteropathogenic Escherichia coli (EPEC) and five isogenic mutants with previously established LD50 values in the larval model were tested against duckweed, and a strong correlation (R(2) = 0.93) was found between their raw LD50 values. Potential virulence factors in B. cenocepacia K56-2 were identified using a high-throughput screen against single duckweed plants. In addition to the previously characterized antifungal compound (AFC) cluster genes, several uncharacterized genes were discovered including a novel lysR regulator, a histidine biosynthesis gene hisG, and a gene located near the gene encoding the recently characterized virulence factor SuhB(Bc). Finally, to demonstrate the utility of this model in therapeutic applications, duckweed was rescued from Bcc infection by treating with bacteriophage at 6-h intervals. It was observed that phage application became ineffective at a timepoint that coincided with a sharp increase in bacterial invasion of plant tissue. These results indicate that common duckweed can serve as an effective infection model for the investigation of bacterial virulence factors and therapeutic strategies to combat them.

A Burkholderia cepacia complex non-ribosomal peptide-synthesized toxin is hemolytic and required for full virulence.

Members of the Burkholderia cepacia complex (Bcc) have recently gained notoriety as significant bacterial pathogens due to their extreme levels of antibiotic resistance, their transmissibility in clinics, their persistence in bacteriostatic solutions, and their intracellular survival capabilities. As pathogens, the Bcc are known to elaborate a number of virulence factors including proteases, lipases and other exoproducts, as well as a number of secretion system associated effectors. Through random and directed mutagenesis studies, we have identified a Bcc gene cluster capable of expressing a toxin that is both hemolytic and required for full Bcc virulence. The Bcc toxin is synthesized via a non-ribosomal peptide synthetase mechanism, and appears to be related to the previously identified antifungal compound burkholdine or occidiofungin. Further testing shows mutations to this gene cluster cause a significant reduction in both hemolysis and Galleria mellonella mortality. Mutation to a glycosyltransferase gene putatively responsible for a structural-functional toxin variant causes only partial reduction in hemolysis. Molecular screening identifies the Bcc species containing this gene cluster, of which several strains produce hemolytic activity.