scholarly journals Shortening the Lipid A Acyl Chains of Bordetella pertussis Enables Depletion of Lipopolysaccharide Endotoxic Activity

Vaccines ◽  
2020 ◽  
Vol 8 (4) ◽  
pp. 594
Author(s):  
Jesús Arenas ◽  
Elder Pupo ◽  
Coen Phielix ◽  
Dionne David ◽  
Afshin Zariri ◽  
...  
Keyword(s):  
Bordetella Pertussis ◽  
Lipid A ◽  
Whooping Cough ◽  
Acyl Chain ◽  
In Vitro Assays ◽  
Whole Cell ◽  
Whole Cells ◽  
Acyl Chain Length ◽  
Acyl Chains

Whooping cough, or pertussis, is an acute respiratory infectious disease caused by the Gram-negative bacterium Bordetella pertussis. Whole-cell vaccines, which were introduced in the fifties of the previous century and proved to be effective, showed considerable reactogenicity and were replaced by subunit vaccines around the turn of the century. However, there is a considerable increase in the number of cases in industrialized countries. A possible strategy to improve vaccine-induced protection is the development of new, non-toxic, whole-cell pertussis vaccines. The reactogenicity of whole-cell pertussis vaccines is, to a large extent, derived from the lipid A moiety of the lipopolysaccharides (LPS) of the bacteria. Here, we engineered B. pertussis strains with altered lipid A structures by expressing genes for the acyltransferases LpxA, LpxD, and LpxL from other bacteria resulting in altered acyl-chain length at various positions. Whole cells and extracted LPS from the strains with shorter acyl chains showed reduced or no activation of the human Toll-like receptor 4 in HEK-Blue reporter cells, whilst a longer acyl chain increased activation. Pyrogenicity studies in rabbits confirmed the in vitro assays. These findings pave the way for the development of a new generation of whole-cell pertussis vaccines with acceptable side effects.

scholarly journals Acyl-chain selectivity of the 85 kDa phospholipase A2 and of the release process in intact macrophages

1994 ◽  
Vol 301 (2) ◽  
pp. 455-458 ◽  
Author(s):  
R Sundler ◽  
D Winstedt ◽  
J Wijkander
Keyword(s):  
Phospholipase A2 ◽  
Acyl Chain ◽  
Calcium Ionophore ◽  
Release Process ◽  
In Vitro System ◽  
Acyl Chain Length ◽  
Mouse Macrophages ◽  
J774 Cells ◽  
Acyl Chains

The selectivity of the intracellular 85 kDa phospholipase A2 (PLA2-85) towards fatty acids closely related to arachidonic acid has been investigated, using purified PLA2-85 from J774 cells and mixed phospholipids, dually acyl-chain-labelled in the sn-2 position. In parallel experiments, we assessed the acyl-chain selectivity of the release process in intact, dually labelled, peritoneal mouse macrophages responding to either calcium ionophore or zymosan beads in the presence of indomethacin and BSA. The results obtained in the two systems were very similar, which supports previous evidence that PLA2-85 is responsible for stimulus-induced release of eicosanoid precursor in mouse macrophages. In the in vitro system, PLA2-85 was found to exhibit a moderate selectivity towards C20 acyl chains differing in double-bond structure, while the sensitivity to acyl-chain length was more pronounced. Together with previous data, these results demonstrate a striking preference for C20 over either C18 or C22 unsaturated acyl chains.

scholarly journals Substrate specificity of the pyrophosphohydrolase LpxH determines the asymmetry of Bordetella pertussis lipid A

2019 ◽  
Vol 294 (20) ◽  
pp. 7982-7989 ◽  
Author(s):  
Jesús Arenas ◽  
Elder Pupo ◽  
Eline de Jonge ◽  
Jesús Pérez-Ortega ◽  
Joerg Schaarschmidt ◽  
...  
Keyword(s):  
Chain Length ◽  
Bordetella Pertussis ◽  
Lipid A ◽  
Whooping Cough ◽  
Acyl Chain ◽  
Biosynthesis Pathway ◽  
Hydrophobic Moiety ◽  
Chain Length Specificity ◽  
Lipid A Biosynthesis ◽  
The Asymmetry

Lipopolysaccharides are anchored to the outer membrane of Gram-negative bacteria by a hydrophobic moiety known as lipid A, which potently activates the host innate immune response. Lipid A of Bordetella pertussis, the causative agent of whooping cough, displays unusual structural asymmetry with respect to the length of the acyl chains at the 3 and 3′ positions, which are 3OH-C10 and 3OH-C14 chains, respectively. Both chains are attached by the acyltransferase LpxA, the first enzyme in the lipid A biosynthesis pathway, which, in B. pertussis, has limited chain length specificity. However, this only partially explains the strict asymmetry of lipid A. In attempts to modulate the endotoxicity of B. pertussis lipid A, here we expressed the gene encoding LpxA from Neisseria meningitidis, which specifically attaches 3OH-C12 chains, in B. pertussis. This expression was lethal, suggesting that one of the downstream enzymes in the lipid A biosynthesis pathway in B. pertussis cannot handle precursors with a 3OH-C12 chain. We considered that the UDP-diacylglucosamine pyrophosphohydrolase LpxH could be responsible for this defect as well as for the asymmetry of B. pertussis lipid A. Expression of meningococcal LpxH in B. pertussis indeed resulted in new symmetric lipid A species with 3OH-C10 or 3OH-C14 chains at both the 3 and 3′ positions, as revealed by MS analysis. Furthermore, co-expression of meningococcal lpxH and lpxA resulted in viable cells that incorporated 3OH-C12 chains in B. pertussis lipid A. We conclude that the asymmetry of B. pertussis lipid A is determined by the acyl chain length specificity of LpxH.

scholarly journals Bordetella pertussis Lipid A Glucosamine Modification Confers Resistance to Cationic Antimicrobial Peptides and Increases Resistance to Outer Membrane Perturbation

2014 ◽  
Vol 58 (8) ◽  
pp. 4931-4934 ◽  
Author(s):  
Nita R. Shah ◽  
Robert E. W. Hancock ◽  
Rachel C. Fernandez
Keyword(s):  
Immune System ◽  
Antimicrobial Peptides ◽  
Outer Membrane ◽  
Bordetella Pertussis ◽  
Lipid A ◽  
Whooping Cough ◽  
Human Immune System ◽  
Cationic Antimicrobial Peptides ◽  
Content Type ◽  
Membrane Perturbation

ABSTRACTBordetella pertussis, the causative agent of whooping cough, has many strategies for evading the human immune system. Lipopolysaccharide (LPS) is an important Gram-negative bacterial surface structure that activates the immune system via Toll-like receptor 4 and enables susceptibility to cationic antimicrobial peptides (CAMPs). We show modification of the lipid A region of LPS with glucosamine increased resistance to numerous CAMPs, including LL-37. Furthermore, we demonstrate that this glucosamine modification increased resistance to outer membrane perturbation.

In vitro innate immune cell based models to assess whole cell Bordetella pertussis vaccine quality: A proof of principle

Biologicals ◽  
2015 ◽  
Vol 43 (2) ◽  
pp. 100-109 ◽  
Author(s):  
M.E. Hoonakker ◽  
L.M. Verhagen ◽  
C.F.M. Hendriksen ◽  
C.A.C.M. van Els ◽  
R.J. Vandebriel ◽  
...  
Keyword(s):  
Pertussis Vaccine ◽  
Bordetella Pertussis ◽  
Immune Cell ◽  
Innate Immune ◽  
Innate Immune Cell ◽  
Whole Cell ◽  
Proof Of Principle

scholarly journals Synthesis, Antifungal Activity and Structure-Activity Relationships of Vanillin Oxime-N-O-Alkanoates

2012 ◽  
Vol 7 (12) ◽  
pp. 1934578X1200701 ◽  
Author(s):  
Vivek Ahluwalia ◽  
Nandini Garg ◽  
Birendra Kumar ◽  
Suresh Walia ◽  
Om P. Sati
Keyword(s):  
Antifungal Activity ◽  
Chain Length ◽  
Acyl Chain ◽  
Sclerotium Rolfsii ◽  
Hydroxylamine Hydrochloride ◽  
Macrophomina Phaseolina ◽  
Phytopathogenic Fungi ◽  
Acyl Chlorides ◽  
Acyl Chain Length

Vanillin oxime- N-O-alkanoates were synthesized following reaction of vanillin with hydroxylamine hydrochloride, followed by reaction of the resultant oxime with acyl chlorides. The structures of the compounds were confirmed by IR, 1H, 13C NMR and mass spectral data. The test compounds were evaluated for their in vitro antifungal activity against three phytopathogenic fungi Macrophomina phaseolina, Rhizoctonia solani and Sclerotium rolfsii by the poisoned food technique. The moderate antifungal activity of vanillin was slightly increased following its conversion to vanillin oxime, but significantly increased after conversion of the oxime to oxime- N-O-alkanoates. While vanillin oxime- N-O-dodecanoate with an EC50 value 73.1 μg/mL was most active against M. phaseolina, vanillin oxime- N-O-nonanoate with EC50 of value 66.7 μg/mL was most active against R. solani. The activity increased with increases in the acyl chain length and was maximal with an acyl chain length of nine carbons.

scholarly journals Genetic Basis and Physiological Effects of Lipid A Hydroxylation in Pseudomonas aeruginosa PAO1

Pathogens ◽  
2019 ◽  
Vol 8 (4) ◽  
pp. 291 ◽  
Author(s):  
Alessandra Lo Sciuto ◽  
Matteo Cervoni ◽  
Roberta Stefanelli ◽  
Maria Concetta Spinnato ◽  
Alessandra Di Giamberardino ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Outer Membrane ◽  
Human Blood ◽  
Genetic Basis ◽  
Lipid A ◽  
Acyl Chain ◽  
Membrane Integrity ◽  
Physiological Role ◽  
Infection Model ◽  
Acyl Chains

Modifications of the lipid A moiety of lipopolysaccharide influence the physicochemical properties of the outer membrane of Gram-negative bacteria. Some bacteria produce lipid A with a single hydroxylated secondary acyl chain. This hydroxylation is catalyzed by the dioxygenase LpxO, and is important for resistance to cationic antimicrobial peptides (e.g., polymyxins), survival in human blood, and pathogenicity in animal models. The lipid A of the human pathogen Pseudomonas aeruginosa can be hydroxylated in both secondary acyl chains, but the genetic basis and physiological role of these hydroxylations are still unknown. Through the generation of single and double deletion mutants in the lpxO1 and lpxO2 homologs of P. aeruginosa PAO1 and lipid A analysis by mass spectrometry, we demonstrate that both LpxO1 and LpxO2 are responsible for lipid A hydroxylation, likely acting on different secondary acyl chains. Lipid A hydroxylation does not appear to affect in vitro growth, cell wall stability, and resistance to human blood or antibiotics in P. aeruginosa. In contrast, it is required for infectivity in the Galleria mellonella infection model, without relevantly affecting in vivo persistence. Overall, these findings suggest a role for lipid A hydroxylation in P. aeruginosa virulence that could not be directly related to outer membrane integrity.

Optimization of Phosphatidylserine-Modified Titanium Surfaces for Enhanced Osteoblast Response

2006 ◽  
Author(s):  
Neera Satsangi ◽  
Arpan Satsangi ◽  
Joo L. Ong ◽  
Rajiv V. Satsangi
Keyword(s):  
Alkaline Phosphatase ◽  
Protein Production ◽  
Specific Activity ◽  
Acyl Chain ◽  
Fatty Acyl ◽  
Separate Experiment ◽  
Calcium Deposition ◽  
Fatty Acyl Chain ◽  
Acyl Chain Length

This report is part of a continued effort to evaluate the in vitro osteoblast responses on different phospholipid coatings on Titanium (Ti) implant materials. It has been established that, among analogous phopholipids, the Ti surfaces coated with calcium phosphate (CaP) complex of phosphatidylserine induce the best calcium deposition and osteoblast growth and metabolism. This communication describes an effort to optimize the chemical structure of phosphatidylserine at its position−1 and −2, as Ti surface coating relative to enhancement in osteoblast differentiation and growth in culture. Four synthetic phosphatidylserine analogs with varying fatty acyl chain length and unsaturation were converted to CaP complex, coated on Ti discs, and the osteoblast progenitor cells were cultured on them for up to 14 days to study their differentiation, growth and biochemistry as marked by the expression of alkaline phosphatase specific activity and protein production. In a separate experiment, the topography of the glass surface (glass Petri-dishes) coated the analogous phosphatidylserines, after immersion in simulated body fluid, was examined by scanning electron microscopy (SEM). The presence of calcium and phosphate ions in this deposit was also confirmed. The inclusion of unsaturation in fatty acyl chain in phosphatidylserine enhanced the Total protein production (TPP) as well as the alkaline phosphatase (ALP) specific activity.

scholarly journals The Adenylate Cyclase (CyaA) Toxin from Bordetella pertussis Has No Detectable Phospholipase A (PLA) Activity In Vitro

Toxins ◽  
2019 ◽  
Vol 11 (2) ◽  
pp. 111 ◽  
Author(s):  
Alexis Voegele ◽  
Mirko Sadi ◽  
Dorothée Raoux-Barbot ◽  
Thibaut Douché ◽  
Mariette Matondo ◽  
...  
Keyword(s):  
Adenylate Cyclase ◽  
Bordetella Pertussis ◽  
Whooping Cough ◽  
Adenyl Cyclase ◽  
Phospholipase A ◽  
Eukaryotic Cells ◽  
Experimental Conditions ◽  
Cyclase Domain ◽  
Charged Membranes

The adenylate cyclase (CyaA) toxin produced in Bordetella pertussis is the causative agent of whooping cough. CyaA exhibits the remarkable capacity to translocate its N-terminal adenyl cyclase domain (ACD) directly across the plasma membrane into the cytosol of eukaryotic cells. Once translocated, calmodulin binds and activates ACD, leading to a burst of cAMP that intoxicates the target cell. Previously, Gonzalez-Bullon et al. reported that CyaA exhibits a phospholipase A activity that could destabilize the membrane to facilitate ACD membrane translocation. However, Bumba and collaborators lately reported that they could not replicate these results. To clarify this controversy, we assayed the putative PLA activity of two CyaA samples purified in two different laboratories by using two distinct fluorescent probes reporting either PLA2 or both PLA1 and PLA2 activities, as well as in various experimental conditions (i.e., neutral or negatively charged membranes in different buffers.) However, we could not detect any PLA activity in these CyaA batches. Thus, our data independently confirm that CyaA does not possess any PLA activity.

scholarly journals The RNA Chaperone Hfq Is Required for Virulence of Bordetella pertussis

2013 ◽  
Vol 81 (11) ◽  
pp. 4081-4090 ◽  
Author(s):  
Ilona Bibova ◽  
Karolina Skopova ◽  
Jiri Masin ◽  
Ondrej Cerny ◽  
David Hot ◽  
...  
Keyword(s):  
Bordetella Pertussis ◽  
Posttranscriptional Regulation ◽  
Noncoding Rna ◽  
Whooping Cough ◽  
Lethal Dose ◽  
Rna Chaperone ◽  
Content Type ◽  
Small Noncoding Rna ◽  
Mrna Targets

ABSTRACTBordetella pertussisis a Gram-negative pathogen causing the human respiratory disease called pertussis or whooping cough. Here we examined the role of the RNA chaperone Hfq inB. pertussisvirulence. Hfq mediates interactions between small regulatory RNAs and their mRNA targets and thus plays an important role in posttranscriptional regulation of many cellular processes in bacteria, including production of virulence factors. We characterized anhfqdeletion mutant (Δhfq) ofB. pertussis18323 and show that the Δhfqstrain produces decreased amounts of the adenylate cyclase toxin that plays a central role inB. pertussisvirulence. Production of pertussis toxin and filamentous hemagglutinin was affected to a lesser extent.In vitro, the ability of the Δhfqstrain to survive within macrophages was significantly reduced compared to that of the wild-type (wt) strain. The virulence of the Δhfqstrain in the mouse respiratory model of infection was attenuated, with its capacity to colonize mouse lungs being strongly reduced and its 50% lethal dose value being increased by one order of magnitude over that of the wt strain. In mixed-infection experiments, the Δhfqstrain was then clearly outcompeted by the wt strain. This requirement for Hfq suggests involvement of small noncoding RNA regulation inB. pertussisvirulence.

scholarly journals The effect of the antigen which elicits the bactericidal antibody and of the mouse-protective antiǵen on the growth of Bordetella pertussis in the mouse brain

1975 ◽  
Vol 74 (1) ◽  
pp. 71-83 ◽  
Author(s):  
Jean M. Dolby ◽  
J. P. Ackers ◽  
D. E. Dolby
Keyword(s):  
Bordetella Pertussis ◽  
Protective Antigen ◽  
Passive Immunity ◽  
Cell Vaccine ◽  
Potency Assay ◽  
Whole Cell ◽  
Protective Antibody ◽  
Bactericidal Antibody

SUMMARYThe effect of antigens of Bordetella pertussis and their antibodies on brain infections by B. pertussis in mice are suppression of an infection immediately, so that the initial 90 % loss due to leakage from the brain is maintained or the numbers of bacteria are reduced even further, sometimes with complete sterilization particularly after a small lethal challenge of 10 LD 50 (mechanism 1), and a delayed antibacterial activity in vivo which does not begin until 3 days after challenge (mechanism 2). The first, immediate reaction is over in 2–3 days; the second is maintained from 3–4 days onwards, and results in elimination of the bacteria and protection of mice.The parts played in vivo in overcoming infection in these two ways by two antigens and their respective antibodies have been investigated. These antigens are a lipopolysaccharide capable of eliciting an antibody which is bactericidal in vitro in the presence of complement called the ‘bactericidal antigen’, and the mouse protective antigen.Considering first passive immunity, bactericidal antibody elicited by isolated antigen, and of high titre in vitro, is only very weakly active by mechanism (1) in vivo. Brains are seldom sterilized and mice not therefore protected. Antisera to whole cell vaccines whether they contain the ‘bactericidal antigen’ or not, or the protective antigen or not can more easily reduce infections by mechanism (1), eliminating small lethal challenges in some mice which are protected. A passive, intracerebrally protective antibody (PIPA) different from other known antibodies, has been postulated to account for this. Antisera to whole cell vaccine which is protective as denned in the potency assay, can, in additon to this, protect mice by mechanism (2) not only against 10 LD 50 but also 100 LD 50 challenge, and is the only antibody which can do this.These antibodies have been investigated by injecting them with the challenging organisms. The antibody effects described above are given by antisera stimulated by several injections and also by the concentrated serum immunoglobulins of once vaccinated mice. The antibody, which is bactericidal in vitro only, is in the 7 S globulin fraction of the serum of once vaccinated mice. The protective antibody capable of overcoming small and large challenges is in the 19 S and 11 S globulins. The antibody, PIPA, protecting against small lethal challenges only is in the fraction A2 containing mainly 11 S globulin.In active immunization experiments the suppression of infection which immediately follows intracerebral vaccination, but which only lasts 2–3 days (mechanism 1), is not dependent on either ‘bactericidal’ or protective antigens but on a component present in all our whole cell vaccines. Vaccines which also had protective antigen eliminated the remaining infection at 4–6 days after challenge by mechanism (2).As in passive immunity, only the protective antigen can completely overcome 100 LD 50. Suppression of a small, lethal, intracerebral infection given 14 days after intraperitoneal vaccination by mechanism (1) may however be correlated with protective antigen.

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