A general protein O-glycosylation machinery conserved in Burkholderia species improves bacterial fitness and elicits glycan immunogenicity in humans.

The Burkholderia genus encompasses many Gram-negative bacteria living in the rhizosphere. Some Burkholderia species can cause life-threatening human infections, highlighting the need for clinical interventions targeting specific lipopolysaccharide proteins. Burkholderia cenocepacia O-linked protein glycosylation has been reported, but the chemical structure of the O-glycan and the machinery required for its biosynthesis are unknown and could reveal potential therapeutic targets. Here, using bioinformatics approaches, gene-knockout mutants, purified recombinant proteins, LC-MS-based analyses of O-glycans, and NMR-based structural analyses, we identified a B. cenocepacia O-glycosylation (ogc) gene cluster necessary for synthesis, assembly, and membrane translocation of a lipid-linked O-glycan, as well as its structure, which consists of a ß-Gal-(1,3)-α-GalNAc-(1,3)-ß-GalNAc trisaccharide. We demonstrate that the ogc cluster is conserved in the Burkholderia genus, and we confirm the production of glycoproteins with similar glycans in the Burkholderia species: B. thailandensis, B. gladioli, and B. pseudomallei Furthermore, we show that absence of protein O-glycosylation severely affects bacterial fitness and accelerates bacterial clearance in a Galleria mellonella larva infection model. Finally, our experiments revealed that patients infected with B. cenocepacia, Burkholderia multivorans, B. pseudomallei, or Burkholderia mallei develop O-glycan-specific antibodies. Together, these results highlight the importance of general protein O-glycosylation in the biology of the Burkholderia genus and its potential as a target for inhibition or immunotherapy approaches to control Burkholderia infections.

The LpxL acyltransferase is required for normal growth and penta-acylation of lipid A in Burkholderia cenocepacia.

Lipid A anchors the lipopolysaccharide (LPS) to the outer membrane and is usually composed of a hexa-acylated diglucosamine backbone. Burkholderia cenocepacia, an opportunistic pathogen, produces a mixture of tetra- and penta-acylated lipid A. "Late" acyltransferases add secondary acyl chains to lipid A after the incorporation of four primary acyl chains to the diglucosamine backbone. Here, we report that B. cenocepacia has only one late acyltransferase, LpxL (BCAL0508), which adds a myristoyl chain to the 2' position of lipid A resulting in penta-acylated lipid A. We also identified PagL (BCAL0788), which acts as an outer membrane lipase by removing the primary ß-hydroxymyristate (3-OH-C14:0) chain at the 3 position, leading to tetra-acylated lipid A. Unlike PagL, LpxL depletion caused reduced cell growth and defects in cell morphology, both of which were suppressed by overexpressing the LPS flippase MsbA (BCAL2408), suggesting that lipid A molecules lacking the fifth acyl chain contributed by LpxL are not good substrates for the flippase. We also show that intracellular B. cenocepacia within macrophages produced more penta-acylated lipid A, suggesting lipid A penta-acylation in B. cenocepacia is required not only for bacterial growth and morphology but also for adaptation to intracellular lifestyle.

The Burkholderia cenocepacia peptidoglycan-associated lipoprotein is involved in epithelial cell attachment and elicitation of inflammation.

The Burkholderia cepacia complex (Bcc) is a group of Gram-negative opportunistic pathogens causing infections in people with cystic fibrosis (CF). Bcc is highly antibiotic resistant, making conventional antibiotic treatment problematic. The identification of novel targets for anti-virulence therapies should improve therapeutic options for infected CF patients. We previously identified that the peptidoglycan-associated lipoprotein (Pal) was immunogenic in Bcc infected CF patients; however, its role in Bcc pathogenesis is unknown. The virulence of a pal deletion mutant (Δpal) in Galleria mellonella was 88-fold reduced (p < .001) compared to wild type. The lipopolysaccharide profiles of wild type and Δpal were identical, indicating no involvement of Pal in O-antigen transport. However, Δpal was more susceptible to polymyxin B. Structural elucidation by X-ray crystallography and calorimetry demonstrated that Pal binds peptidoglycan fragments. Δpal showed a 1.5-fold reduced stimulation of IL-8 in CF epithelial cells relative to wild type (p < .001), demonstrating that Pal is a significant driver of inflammation. The Δpal mutant had reduced binding to CFBE41o- cells, but adhesion of Pal-expressing recombinant E. coli to CFBE41o- cells was enhanced compared to wild-type E. coli (p < .0001), confirming that Pal plays a direct role in host cell attachment. Overall, Bcc Pal mediates host cell attachment and stimulation of cytokine secretion, contributing to Bcc pathogenesis.

ArnT proteins that catalyze the glycosylation of lipopolysaccharide share common features with bacterial N-oligosaccharyltransferases.

ArnT is a glycosyltransferase that catalyzes the addition of 4-amino-4-deoxy-l-arabinose (l-Ara4N) to the lipid A moiety of the lipopolysaccharide. This is a critical modification enabling bacteria to resist killing by antimicrobial peptides. ArnT is an integral inner membrane protein consisting of 13 predicted transmembrane helices and a large periplasmic C-terminal domain. We report here the identification of a functional motif with a canonical consensus sequence DEXRYAX(5)MX(3)GXWX(9)YFEKPX(4)W spanning the first periplasmic loop, which is highly conserved in all ArnT proteins examined. Site-directed mutagenesis demonstrated the contribution of this motif in ArnT function, suggesting that these proteins have a common mechanism. We also demonstrate that the Burkholderia cenocepacia and Salmonella enterica serovar Typhimurium ArnT C-terminal domain is required for polymyxin B resistance in vivo. Deletion of the C-terminal domain in B. cenocepacia ArnT resulted in a protein with significantly reduced in vitro binding to a lipid A fluorescent substrate and unable to catalyze lipid A modification with l-Ara4N. An in silico predicted structural model of ArnT strongly resembled the tertiary structure of Campylobacter lari PglB, a bacterial oligosaccharyltransferase involved in protein N-glycosylation. Therefore, distantly related oligosaccharyltransferases from ArnT and PglB families operating on lipid and polypeptide substrates, respectively, share unexpected structural similarity that could not be predicted from direct amino acid sequence comparisons. We propose that lipid A and protein glycosylation enzymes share a conserved catalytic mechanism despite their evolutionary divergence.

Exposure Patterns Driving Ebola Transmission in West Africa: A Retrospective Observational Study.

BACKGROUND: The ongoing West African Ebola epidemic began in December 2013 in Guinea, probably from a single zoonotic introduction. As a result of ineffective initial control efforts, an Ebola outbreak of unprecedented scale emerged. As of 4 May 2015, it had resulted in more than 19,000 probable and confirmed Ebola cases, mainly in Guinea (3,529), Liberia (5,343), and Sierra Leone (10,746). Here, we present analyses of data collected during the outbreak identifying drivers of transmission and highlighting areas where control could be improved. METHODS AND FINDINGS: Over 19,000 confirmed and probable Ebola cases were reported in West Africa by 4 May 2015. Individuals with confirmed or probable Ebola ("cases") were asked if they had exposure to other potential Ebola cases ("potential source contacts") in a funeral or non-funeral context prior to becoming ill. We performed retrospective analyses of a case line-list, collated from national databases of case investigation forms that have been reported to WHO. These analyses were initially performed to assist WHO's response during the epidemic, and have been updated for publication. We analysed data from 3,529 cases in Guinea, 5,343 in Liberia, and 10,746 in Sierra Leone; exposures were reported by 33% of cases. The proportion of cases reporting a funeral exposure decreased over time. We found a positive correlation (r = 0.35, p < 0.001) between this proportion in a given district for a given month and the within-district transmission intensity, quantified by the estimated reproduction number (R). We also found a negative correlation (r = -0.37, p < 0.001) between R and the district proportion of hospitalised cases admitted within ≤4 days of symptom onset. These two proportions were not correlated, suggesting that reduced funeral attendance and faster hospitalisation independently influenced local transmission intensity. We were able to identify 14% of potential source contacts as cases in the case line-list. Linking cases to the contacts who potentially infected them provided information on the transmission network. This revealed a high degree of heterogeneity in inferred transmissions, with only 20% of cases accounting for at least 73% of new infections, a phenomenon often called super-spreading. Multivariable regression models allowed us to identify predictors of being named as a potential source contact. These were similar for funeral and non-funeral contacts: severe symptoms, death, non-hospitalisation, older age, and travelling prior to symptom onset. Non-funeral exposures were strongly peaked around the death of the contact. There was evidence that hospitalisation reduced but did not eliminate onward exposures. We found that Ebola treatment units were better than other health care facilities at preventing exposure from hospitalised and deceased individuals. The principal limitation of our analysis is limited data quality, with cases not being entered into the database, cases not reporting exposures, or data being entered incorrectly (especially dates, and possible misclassifications). CONCLUSIONS: Achieving elimination of Ebola is challenging, partly because of super-spreading. Safe funeral practices and fast hospitalisation contributed to the containment of this Ebola epidemic. Continued real-time data capture, reporting, and analysis are vital to track transmission patterns, inform resource deployment, and thus hasten and maintain elimination of the virus from the human population.

A Burkholderia cenocepacia MurJ (MviN) homolog is essential for cell wall peptidoglycan synthesis and bacterial viability.

The cell wall peptidoglycan (PG) of Burkholderia cenocepacia, an opportunistic pathogen, has not yet been characterized. However, the B. cenocepacia genome contains homologs of genes encoding PG biosynthetic functions in other bacteria. PG biosynthesis involves the formation of the undecaprenyl-pyrophosphate-linked N-acetyl glucosamine-N-acetyl muramic acid-pentapeptide, known as lipid II, which is built on the cytosolic face of the cell membrane. Lipid II is then translocated across the membrane and its glycopeptide moiety becomes incorporated into the growing cell wall mesh; this translocation step is critical to PG synthesis. We have investigated candidate flippase homologs of the MurJ family in B. cenocepacia. Our results show that BCAL2764, herein referred to as murJBc, is indispensable for viability. Viable B. cenocepacia could only be obtained through a conditional mutagenesis strategy by placing murJBc under the control of a rhamnose-inducible promoter. Under rhamnose depletion, the conditional strain stopped growing and individual cells displayed morphological abnormalities consistent with a defect in PG synthesis. Bacterial cells unable to express MurJBc underwent cell lysis, while partial MurJBc depletion sensitized the mutant to the action of ß-lactam antibiotics. Depletion of MurJBc caused accumulation of PG precursors consistent with the notion that this protein plays a role in lipid II flipping to the periplasmic compartment. Reciprocal complementation experiments of conditional murJ mutants in B. cenocepacia and Escherichia coli with plasmids expressing MurJ from each strain indicated that MurJBc and MurJEc are functional homologs. Together, our results are consistent with the notion that MurJBc is a PG lipid II flippase in B. cenocepacia.

Lead, Mercury, and Cadmium Concentrations in Blood Products Transfused to Neonates: Elimination Not Just Mitigation.

Lead (Pb), mercury (Hg), and cadmium (Cd) are identified as potent developmental neurotoxicants. Neonates are the main group receiving multiple blood transfusions. The exposure of neonates to these heavy metals (HMs) can occur through blood transfusions. This study aimed to determine the concentrations of lead (Pb), mercury (Hg), and cadmium (Cd) in various blood products (plasma, platelets, packed red blood cells (pRBCs), and whole blood (WB)) to explore the probability of concurrent exposure of these HMs and to identify the metal load per transfusion with risk assessment. Residual bloods from blood bank bags were collected after neonatal transfusion. Pb, Hg, and Cd concentrations were determined in 120 samples of blood products by inductively coupled plasma mass spectrometry (ICP-MS). Pb and Cd levels were over the normal levels in 19.2 and 5.9% of all blood units, respectively. In 35 and 0.8% of blood units, the Pb and Cd concentrations, respectively, were higher than that recommended for transfusions in premature neonates. The anticipated safe value was surpassed by 2.5% for Cd of all transfusions, primarily because of WB. However, Hg was detected only in 5.8% of all samples and their concentrations were within the normal range. The concurrent neonatal exposure to Pb, Hg, and Cd was statistically significant. Hazard quotients of Hg and Cr were >1 and Pb cancer risk was 2.41 × 10-4. To the best of our knowledge, this study is the first report examining Pb, Hg, and Cd in blood products other than WB and pRBCs using ICP-MS. This study demonstrated the exposure of neonates to Pb, Hg, and Cd during transfusion with a considerable amount of Pb. It confirms the significant concurrent exposure to the three HMs, which maximize their potential developmental neurotoxicity with a high probability of developing non-carcinogenic and carcinogenic health effects.

Anti-inflammatory effects of cannabidiol against lipopolysaccharides in cardiac sodium channels.

BACKGROUND: Sepsis, caused by a dysregulated response to infections, can lead to cardiac arrhythmias. However, the mechanisms underlying sepsis-induced inflammation, and how inflammation provokes cardiac arrhythmias, are not well understood. We hypothesized that cannabidiol (CBD) may ameliorate lipopolysaccharide (LPS)-induced cardiotoxicity, via Toll-like receptors (TLR4) and cardiac sodium channels (NaV 1.5). METHODS AND RESULTS: We incubated human immune cells (THP-1 macrophages) with LPS for 24 h, then extracted the THP-1 incubation media. ELISA assays showed that LPS (1 or 5 µg·ml-1 ), in a concentration-dependent manner, or MPLA (TLR4 agonist, 5 µg·ml-1 ) stimulated the THP-1 cells to release inflammatory cytokines (TNF-α and IL-6). Prior incubation (4 h) with CBD (5 µM) or C34 (TLR4 antagonist: 5 µg·ml-1 ) inhibited LPS and MPLA-induced release of both IL-6 and TNF-α. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) were subsequently incubated for 24 h in the media extracted from THP-1 cells incubated with LPS, MPLA alone, or in combination with CBD or C34. Voltage-clamp experiments showed a right shift in the voltage dependence of NaV 1.5 activation, steady state fast inactivation (SSFI), increased persistent current and prolonged in silico action potential duration in hiSPC-CMs incubated in the LPS or MPLA-THP-1 media. Co-incubation with CBD or C34 rescued the biophysical dysfunction caused by LPS and MPLA. CONCLUSION: Our results suggest that CBD may protect against sepsis-induced inflammation and subsequent arrhythmias through (i) inhibition of the release of inflammatory cytokines, antioxidant and anti-apoptotic effects and/or (ii) a direct effect on NaV 1.5.

Recurrent Fever with Oral Lesions in Egyptian Children: A Familial Mediterranean Fever Diagnosis Not to Be Missed.

OBJECTIVES: the aim of this study was to describe the genetic and clinical features of familial Mediterranean fever (FMF) in a group of Egyptian children. MATERIALS AND METHODS: This cross-sectional observational study included 65 children diagnosed with FMF according to the (Eurofever/PRINTO) classification criteria. The complete blood count (CBC), and acute phase reactants such as Serum amyloid A (SAA), and C-reactive protein (CRP) were all measured during the febrile episode. Mutation analysis for the MEFV gene was carried out for all subjects. RESULTS: A total of 65 patients with FMF were included in the study. The first clinical manifestation was recurrent fever in all patients. Recurrent oral lesions accompanied fever in 63% of cases, abdominal pain in 31%, and musculoskeletal pain in 6%. The mean SAA level was 162.5 ± 85.78 mg/L. MEFV mutations were detected in 56 patients (86%). Among these patients, 6 (10.7%) were homozygous, while 44 (78.6%) were heterozygous. The most frequently observed mutation was E148Q 24 (37.5%), followed by M694I 18 (32.1%), and V726A 13 (20.3%). Half of the patients with oral lesions were E148Q positive, however abdominal pain was found to be higher in the patients with the M694I mutation. CONCLUSION: Recurrent fever with oral lesions could be an important atypical presentation of FMF in Egyptian children that should not be ignored and/or missed.

A glycoengineered antigen exploiting a conserved protein O-glycosylation pathway in the Burkholderia genus for detection of glanders infections.

We recently described a protein O-glycosylation pathway conserved in all species of the Burkholderia genus that results in the synthesis and incorporation of a trisaccharide glycan to membrane-exported proteins. Here, we exploited this system to construct and evaluate a diagnostic tool for glanders. Burkholderia mallei, the causative agent of glanders, is a highly infectious and fatal zoonotic pathogen that infects horses, mules, donkeys, and occasionally humans. A highly sensitive and specific diagnostic tool is crucial for the control, elimination, and eradication of B. mallei infections. We constructed plasmids carrying synthetic genes encoding a modified, previously unannotated Burkholderia glycoprotein containing three glycosylation sequons fused to the cholera toxin B-subunit. The resulting proteins were glycosylated in the B. cenocepacia K56-2 parental strain, but not in glycosylation-deficient mutants, as determined by SDS-PAGE and fluorescent lectin blots. One of these glycoproteins was used as an antigen in ELISA and western blots to screen a panel of serum samples collected from glanders-infected and healthy horses, which were previously investigated by complement fixation test and indirect ELISA based on a semi-purified fraction of B. mallei. We show that ELISA and western blot assays based on our glycoprotein antigen provide 100% specificity, with a sensitivity greater than 88%. The glycoprotein antigen was recognized by serum samples collected from patients infected with B. pseudomallei, B. mallei, B. multivorans, and B. cenocepacia. Our results indicate that protein O-glycosylation in Burkholderia can be exploited as a biomarker for diagnosis of Burkholderia-associated infections.

Electrocatalytic redox neutral [3 + 2] annulation of N-cyclopropylanilines and alkenes.

Although synthetic organic electrochemistry (EC) has advanced significantly, net redox neutral electrosynthesis is quite rare. Two approaches have been employed to achieve this type of electrosynthesis. One relies on turnover of the product by the reactant in a chain mechanism. The other involves both oxidation on the anode and reduction on the cathode in which the radical cation or the radical anion of the product has to migrate between two electrodes. Herein, a home-built electrochemistry/mass spectrometry (EC/MS) platform was used to generate an N-cyclopropylaniline radical cation electrochemically and to monitor its reactivity toward alkenes by mass spectrometry (MS), which led to the discovery of a new redox neutral reaction of intermolecular [3 + 2] annulation of N-cyclopropylanilines and alkenes to provide an aniline-substituted 5-membered carbocycle via direct electrolysis (yield up to 81%). A chain mechanism, involving the regeneration of the substrate radical cation and the formation of the neutral product, is shown to be responsible for promoting such a redox neutral annulation reaction, as supported by experimental evidence of EC/MS.

Membrane permeabilization of colistin toward pan-drug resistant Gram-negative isolates.

Pan-drug resistant Gram-negative bacteria, being resistant to most available antibiotics, represent a huge threat to the medical community. Colistin is considered the last therapeutic option for patients in hospital settings. Thus, we were concerned in this study to demonstrate the membrane permeabilizing activity of colistin focusing on investigating its efficiency toward those pan-drug resistant isolates which represent a critical situation. We determined the killing dynamics of colistin against pan-drug resistant isolates. The permeability alteration was confirmed by different techniques as: leakage, electron microscopy and construction of an artificial membrane model; liposomes. Moreover, selectivity of colistin against microbial cells was also elucidated. Colistin was proved to be rapid bactericidal against pan-drug resistant isolates. It interacts with the outer bacterial membrane leading to deformation of its outline, pore formation, leakage of internal contents, cell lysis and finally death. Furthermore, variations in membrane composition of eukaryotic and microbial cells provide a key for colistin selectivity toward bacterial cells. Colistin selectively alters membrane permeability of pan-drug resistant isolates which leads to cell lysis. Colistin was proved to be an efficient last line treatment for pan-drug resistant infections which are hard to treat.

Burkholderia cenocepacia Lipopolysaccharide Modification and Flagellin Glycosylation Affect Virulence but Not Innate Immune Recognition in Plants.

UNLABELLED: Burkholderia cenocepacia causes opportunistic infections in plants, insects, animals, and humans, suggesting that "virulence" depends on the host and its innate susceptibility to infection. We hypothesized that modifications in key bacterial molecules recognized by the innate immune system modulate host responses to B. cenocepacia. Indeed, modification of lipopolysaccharide (LPS) with 4-amino-4-deoxy-L-arabinose and flagellin glycosylation attenuates B. cenocepacia infection in Arabidopsis thaliana and Galleria mellonella insect larvae. However, B. cenocepacia LPS and flagellin triggered rapid bursts of nitric oxide and reactive oxygen species in A. thaliana leading to activation of the PR-1 defense gene. These responses were drastically reduced in plants with fls2 (flagellin FLS2 host receptor kinase), Atnoa1 (nitric oxide-associated protein 1), and dnd1-1 (reduced production of nitric oxide) null mutations. Together, our results indicate that LPS modification and flagellin glycosylation do not affect recognition by plant receptors but are required for bacteria to establish overt infection. IMPORTANCE: Virulence and pathogenicity are properties ascribed to microbes, which actually require careful consideration of the host. Using the term "pathogen" to define a microbe without considering its host has recently been debated, since the microbe's capacity to establish a niche in a given host is a critical feature associated with infection. Opportunistic bacteria are a perfect example of microbes whose ability to cause disease is intimately related to the host's ability to recognize and respond to the infection. Here, we use the opportunistic bacterium Burkholderia cenocepacia and the host plant Arabidopsis thaliana to investigate the role of bacterial surface molecules, namely, lipopolysaccharide and flagellin, in contributing to infection and also in eliciting a host response. We reveal that both molecules can be modified by glycosylation, and although the modifications are critical for the bacteria to establish an infection, they do not impact the host's ability to recognize the pathogen.

Isolation and characterization of a heavy metal-resistant, thermophilic esterase from a Red Sea brine pool.

The Red Sea Atlantis II brine pool is an extreme environment that displays multiple harsh conditions such as high temperature, high salinity and high concentrations of multiple, toxic heavy metals. The survival of microbes in such an environment by utilizing resistant enzymes makes them an excellent source of extremophilic enzymes. We constructed a fosmid metagenomic library using DNA isolated from the deepest and most secluded layer of this pool. We report the isolation and biochemical characterization of an unusual esterase: EstATII. EstATII is thermophilic (optimum temperature, 65°C), halotolerant (maintains its activity in up to 4.5 M NaCl) and maintains at least 60% of its activity in the presence of a wide spectrum of heavy metals. The combination of biochemical characteristics of the Red Sea Atlantis II brine pool esterase, i.e., halotolerance, thermophilicity and resistance to heavy metals, makes it a potentially useful biocatalyst.

Membrane permeabilization of colistin toward pan-drug resistant Gram-negative isolates

Pan-drug resistant Gram-negative bacteria, being resistant to most available antibiotics, represent a huge threat to the medical community. Colistin is considered the last therapeutic option for patients in hospital settings. Thus, we were concerned in this study to demonstrate the membrane permeabilizing activity of colistin focusing on investigating its efficiency toward those pan-drug resistant isolates which represent a critical situation. We determined the killing dynamics of colistin against pan-drug resistant isolates. The permeability alteration was confirmed by different techniques as: leakage, electron microscopy and construction of an artificial membrane model; liposomes. Moreover, selectivity of colistin against microbial cells was also elucidated. Colistin was proved to be rapid bactericidal against pan-drug resistant isolates. It interacts with the outer bacterial membrane leading to deformation of its outline, pore formation, leakage of internal contents, cell lysis and finally death. Furthermore, variations in membrane composition of eukaryotic and microbial cells provide a key for colistin selectivity toward bacterial cells. Colistin selectively alters membrane permeability of pan-drug resistant isolates which leads to cell lysis. Colistin was proved to be an efficient last line treatment for pan-drug resistant infections which are hard to treat.(AU)

Membrane permeabilization of colistin toward pan-drug resistant Gram-negative isolates

Abstract Pan-drug resistant Gram-negative bacteria, being resistant to most available antibiotics, represent a huge threat to the medical community. Colistin is considered the last therapeutic option for patients in hospital settings. Thus, we were concerned in this study to demonstrate the membrane permeabilizing activity of colistin focusing on investigating its efficiency toward those pan-drug resistant isolates which represent a critical situation. We determined the killing dynamics of colistin against pan-drug resistant isolates. The permeability alteration was confirmed by different techniques as: leakage, electron microscopy and construction of an artificial membrane model; liposomes. Moreover, selectivity of colistin against microbial cells was also elucidated. Colistin was proved to be rapid bactericidal against pan-drug resistant isolates. It interacts with the outer bacterial membrane leading to deformation of its outline, pore formation, leakage of internal contents, cell lysis and finally death. Furthermore, variations in membrane composition of eukaryotic and microbial cells provide a key for colistin selectivity toward bacterial cells. Colistin selectively alters membrane permeability of pan-drug resistant isolates which leads to cell lysis. Colistin was proved to be an efficient last line treatment for pan-drug resistant infections which are hard to treat.