scholarly journals Binding of surfactant protein A to the lipid A moiety of bacterial lipopolysaccharides

1994 ◽  
Vol 303 (2) ◽  
pp. 407-411 ◽  
Author(s):  
J F Van Iwaarden ◽  
J C Pikaar ◽  
J Storm ◽  
E Brouwer ◽  
J Verhoef ◽  
...  
Keyword(s):  
Lipid A ◽  
Magnetic Beads ◽  
Protein A ◽  
Surfactant Protein ◽  
Biologically Active ◽  
Surfactant Protein A ◽  
Carbohydrate Binding ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
E Coli

Surfactant protein A (SP-A) enhances the phagocytosis of opsonized and non-opsonized bacteria by alveolar macrophages, but it is not known with which component of the bacterial surface it associates. We investigated the interaction of SP-A with lipopolysaccharides (LPS), which are important biologically active constituents of the outer membranes of Gram-negative bacteria. Flow cytometry was used to study the binding of fluorescein isothiocyanate-labelled SP-A either to LPS of various chain lengths coupled to magnetic beads or to Gram-negative bacteria. The binding of SP-A to LPS-coated beads was saturable, both time- and concentration-dependent, and required both Ca2+ and Na+. SP-A bound to the lipid A moiety of LPS and to LPS from either the Re-mutant of Salmonella minnesota or the J5-mutant of Escherichia coli. In contrast, it did not bind to O111 LPS of E. coli, suggesting that SP-A binds only to rough LPS. The binding of SP-A to LPS was not affected by mannan and heparin or by deglycosylation of the SP-A, indicating that the carbohydrate-binding domain and the carbohydrate moiety of SP-A are not involved in its interaction with LPS. We also observed saturable and concentration-dependent binding of SP-A to the live J5 mutant of whole E. coli, but not to its O111 mutant. In addition, Re LPS aggregated in the presence of SP-A, Ca2+ and Na+. We conclude that SP-A associates with LPS via the lipid A moiety of rough LPS and may be involved in the anti-bacterial defences of the lung.

Surfactant protein A enhances the degradation of LPS-induced TLR4 in primary alveolar macrophages involving Rab7, β-arrestin2, and mTORC1

2021 ◽  
Author(s):  
Katja Freundt ◽  
Christian Herzmann ◽  
Dominika Biedziak ◽  
Claudia Scheffzük ◽  
Karoline I. Gaede ◽  
...  
Keyword(s):  
Immune Responses ◽  
Host Defense ◽  
Alveolar Macrophages ◽  
Protein A ◽  
Surfactant Protein ◽  
Surfactant Protein A ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Lps Challenge ◽  
Tract Infections

Respiratory infections by Gram-negative bacteria are a major cause of global morbidity and mortality. Alveolar macrophages (AMs) play a central role in maintaining lung immune homeostasis and host defense by sensing pathogens via pattern recognition receptors (PRR). The PRR Toll-like receptor (TLR) 4 is a key sensor of lipopolysaccharide (LPS) from Gram-negative bacteria. Pulmonary surfactant is the natural microenvironment of AMs. Surfactant protein A (SP-A), a multifunctional host defense collectin, controls LPS-induced pro-inflammatory immune responses at the organismal and cellular level via distinct mechanisms. We found that SP-A post-transcriptionally restricts LPS-induced TLR4 protein expression in primary AMs from healthy humans, rats, wild-type and SP-A -/- mice by further decreasing cycloheximide-reduced TLR4 protein translation and enhances the co-localization of TLR4 with the late endosome/lysosome. Both effects as well as the SP-A-mediated inhibition of LPS-induced TNFα release are counteracted by pharmacological inhibition of the small GTPase Rab7. SP-A-enhanced Rab7 expression requires β-arrestin2 and, in β-arrestin2 -/- AMs and after intratracheal LPS challenge of β-arrestin2 -/- mice, SP-A fails to enhance TLR4/lysosome co-localization and degradation of LPS-induced TLR4. In SP-A -/- mice, TLR4 levels are increased after pulmonary LPS challenge. SP-A-induced activation of mechanistic target of rapamycin complex 1 (mTORC1) kinase requires β-arrestin2 and is critically involved in degradation of LPS-induced TLR4. The data suggest that SP-A post-translationally limits LPS-induced TLR4 expression in primary AMs by lysosomal degradation comprising Rab7, β-arrestin2, and mTORC1. This study may indicate a potential role of SP-A-based therapeutic interventions in unrestricted TLR4-driven immune responses to lower respiratory tract infections caused by Gram-negative bacteria.

Diacylglycerol kinase A is essential for polymyxin resistance provided by EptA, MCR-1 and other lipid A phosphoethanolamine transferases.

2021 ◽  
Author(s):  
Alexandria B. Purcell ◽  
Bradley J. Voss ◽  
M. Stephen Trent
Keyword(s):  
Lipid A ◽  
Cell Envelope ◽  
Diacylglycerol Kinase ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
E Coli ◽  
Severe Reduction ◽  
Bacterial Fitness ◽  
Phosphoethanolamine Transferases ◽  
Kinase A

Gram-negative bacteria utilize glycerophospholipids (GPLs) as phospho-form donors to modify various surface structures. These modifications play important roles in bacterial fitness in diverse environments influencing cell motility, recognition by the host during infection, and antimicrobial resistance. A well-known example is the modification of the lipid A component of lipopolysaccharide by the phosphoethanolamine (pEtN) transferase EptA that utilizes phosphatidyethanoalmine (PE) as the phospho-form donor. Addition of pEtN to lipid A promotes resistance to cationic antimicrobial peptides (CAMPs), including the polymyxin antibiotics like colistin. A consequence of pEtN modification is the production of diacylglycerol (DAG) that must be recycled back into GPL synthesis via the diacylglycerol kinase A (DgkA). DgkA phosphorylates DAG forming phosphatidic acid, the precursor for GPL synthesis. Here we report that deletion of dgkA in polymyxin-resistant E. coli results in a severe reduction of pEtN modification and loss of antibiotic resistance. We demonstrate that inhibition of EptA is regulated post-transcriptionally and is not due to EptA degradation during DAG accumulation. We also show that the inhibition of lipid A modification by DAG is a conserved feature of different Gram-negative pEtN transferases. Altogether, our data suggests that inhibition of EptA activity during DAG accumulation likely prevents disruption of GPL synthesis helping to maintain cell envelope homeostasis.

scholarly journals Endotoxin protein: a B-cell mitogen and polyclonal activator of C3H/HeJ lymphocytes.

1976 ◽  
Vol 144 (3) ◽  
pp. 821-827 ◽  
Author(s):  
B M Sultzer ◽  
G W Goodman
Keyword(s):  
B Cell ◽  
Lipid A ◽  
Protein A ◽  
Cell Wall Protein ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Lipopolysaccharide Endotoxin ◽  
Polyclonal Activator ◽  
A Cell ◽  
Cell Mitogen

A cell wall protein that is ordinarily complexed to the lipopolysaccharide endotoxin in gram-negative bacteria has been separated by the use of aqueous phenol. The protein is active as a B-cell mitogen and polyclonal activator of murine lymphocytes including the C3H/HeJ strain which is a nonresponder to lipoplysaccharide or lipid A.

Review: Structural determinants of pattern recognition by lung collectins

2010 ◽  
Vol 16 (3) ◽  
pp. 143-150 ◽  
Author(s):  
Barbara A. Seaton ◽  
Erika C. Crouch ◽  
Francis X. McCormack ◽  
James F. Head ◽  
Kevan L. Hartshorn ◽  
...  
Keyword(s):  
Lipid A ◽  
Protein A ◽  
Structural Similarity ◽  
Acute Phase Reactant ◽  
Surfactant Protein ◽  
Surfactant Protein A ◽  
Surfactant Protein D ◽  
Structural Determinants ◽  
Calcium Dependent ◽  
Anionic Phospholipids

Host defense roles for the lung collectins, surfactant protein A (SP-A) and surfactant protein D (SP-D), were first suspected in the 1980s when molecular characterization revealed their sequence homology to the acute phase reactant of serum, mannose-binding lectin. Surfactant protein A and SP-D have since been shown to play diverse and important roles in innate immunity and pulmonary homeostasis. Their location in surfactant ideally positions them to interact with air-space pathogens. Despite extensive structural similarity, the two proteins show many functional differences and considerable divergence in their interactions with microbial surface components, surfactant lipids, and other ligands. Recent crystallographic studies have provided many new insights relating to these observed differences. Although both proteins can participate in calcium-dependent interactions with sugars and other polyols, they display significant differences in the spatial orientation, charge, and hydrophobicity of their binding surfaces. Surfactant protein D appears particularly adapted to interactions with complex carbohydrates and anionic phospholipids, such as phosphatidylinositol. By contrast, SP-A shows features consistent with its preference for lipid ligands, including lipid A and the major surfactant lipid, dipalmitoylphosphatidylcholine. Current research suggests that structural biology approaches will help to elucidate the molecular basis of pulmonary collectin—ligand recognition and facilitate development of new therapeutics based upon SP-A and SP-D.

scholarly journals Nonclonal Emergence of Colistin Resistance Associated with Mutations in the BasRS Two-Component System in Escherichia coli Bloodstream Isolates

mSphere ◽  
2020 ◽  
Vol 5 (2) ◽  
Author(s):  
Axel B. Janssen ◽  
Toby L. Bartholomew ◽  
Natalia P. Marciszewska ◽  
Marc J. M. Bonten ◽  
Rob J. L. Willems ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Lipid A ◽  
Gram Negative Bacteria ◽  
Colistin Resistance ◽  
Anionic Lipid ◽  
Gram Negative ◽  
Content Type ◽  
E Coli ◽  
Two Component ◽  
Resistant Strains

ABSTRACT Infections by multidrug-resistant Gram-negative bacteria are increasingly common, prompting the renewed interest in the use of colistin. Colistin specifically targets Gram-negative bacteria by interacting with the anionic lipid A moieties of lipopolysaccharides, leading to membrane destabilization and cell death. Here, we aimed to uncover the mechanisms of colistin resistance in nine colistin-resistant Escherichia coli strains and one Escherichia albertii strain. These were the only colistin-resistant strains of 1,140 bloodstream Escherichia isolates collected in a tertiary hospital over a 10-year period (2006 to 2015). Core-genome phylogenetic analysis showed that each patient was colonized by a unique strain, suggesting that colistin resistance was acquired independently in each strain. All colistin-resistant strains had lipid A that was modified with phosphoethanolamine. In addition, two E. coli strains had hepta-acylated lipid A species, containing an additional palmitate compared to the canonical hexa-acylated E. coli lipid A. One E. coli strain carried the mobile colistin resistance (mcr) gene mcr-1.1 on an IncX4-type plasmid. Through construction of chromosomal transgene integration mutants, we experimentally determined that mutations in basRS, encoding a two-component signal transduction system, contributed to colistin resistance in four strains. We confirmed these observations by reversing the mutations in basRS to the sequences found in reference strains, resulting in loss of colistin resistance. While the mcr genes have become a widely studied mechanism of colistin resistance in E. coli, sequence variation in basRS is another, potentially more prevalent but relatively underexplored, cause of colistin resistance in this important nosocomial pathogen. IMPORTANCE Multidrug resistance among Gram-negative bacteria has led to the use of colistin as a last-resort drug. The cationic colistin kills Gram-negative bacteria through electrostatic interaction with the anionic lipid A moiety of lipopolysaccharides. Due to increased use in clinical and agricultural settings, colistin resistance has recently started to emerge. In this study, we used a combination of whole-genome sequence analysis and experimental validation to characterize the mechanisms through which Escherichia coli strains from bloodstream infections can develop colistin resistance. We found no evidence of direct transfer of colistin-resistant isolates between patients. The lipid A of all isolates was modified by the addition of phosphoethanolamine. In four isolates, colistin resistance was experimentally verified to be caused by mutations in the basRS genes, encoding a two-component regulatory system. Our data show that chromosomal mutations are an important cause of colistin resistance among clinical E. coli isolates.

scholarly journals Internalization of surfactant protein A (SP-A) into lamellar bodies of rat alveolar type II cells in vitro.

1993 ◽  
Vol 41 (1) ◽  
pp. 57-70 ◽  
Author(s):  
M Kalina ◽  
F X McCormack ◽  
H Crowley ◽  
D R Voelker ◽  
R J Mason
Keyword(s):  
Protein A ◽  
Fluid Phase ◽  
Surfactant Protein ◽  
Biologically Active ◽  
Surfactant Protein A ◽  
Alveolar Type ◽  
Type Ii Cells ◽  
Type Ii ◽  
Lamellar Bodies ◽  
Alveolar Type Ii Cells

Pulmonary surfactant is thought to be internalized and processed for reuse by alveolar Type II cells. In the present study we followed the internalization and intracellular trafficking of purified surfactant protein A (SP-A) by primary cultures of alveolar Type II cells. Internalization of native rat SP-A was compared with that of recombinant rat and human SP-A isolated from a patient with alveolar proteinosis. All SP-A species were conjugated with colloidal gold for visualization by electron microscopy. The gold conjugates were biologically active, as demonstrated by inhibition of phospholipid secretion from alveolar Type II cells. The SP-A-gold conjugates were internalized to lamellar bodies (LB) via the endosomal system, which included both electron-lucent and -dense multivesicular bodies. Labeling of LB was time dependent, and after 7 hr 30-40% of these organelles were labeled. Alkylation of SP-A greatly reduced internalization, as did an excess of non-conjugated SP-A. No qualitative differences in uptake were observed with the three forms of SP-A. The percent of labeled LB was similar (30-40%) after 7 hr of internalization with the three species of SP-A. The recombinant SP-A produced using a baculovirus vector lacked hydroxyproline and had an altered oligosaccharide, but these features did not affect its internalization or the rate of LB labeling. Internalization of the gold-conjugated SP-A and endocytosis of the fluid-phase marker Lucifer Yellow were related to the shape of Type II cells. Both uptake of SP-A, which is receptor mediated, and fluid-phase endocytosis were found to be less active in the flattened than in the rounded cells. Therefore, cell shape and hence cytoskeletal organization may play an important role in SP-A recycling. However, it is possible that both morphology and decreased endocytosis are independent manifestations related to the loss of differentiated function of cultured Type II cells.

scholarly journals The lung lectin surfactant protein A aggregates phospholipid vesicles via a novel mechanism

1991 ◽  
Vol 275 (1) ◽  
pp. 273-276 ◽  
Author(s):  
H P Haagsman ◽  
R H Elfring ◽  
B L M van Buel ◽  
W F Voorhout
Keyword(s):  
Protein A ◽  
Surfactant Protein ◽  
Surfactant Protein A ◽  
Phospholipid Vesicles ◽  
Carbohydrate Binding ◽  
Membrane Interactions ◽  
Terminal Domain ◽  
Binding Domains ◽  
Extracellular Form

Surfactant protein A (SP-A), a lung-specific glycoprotein, consists of an N-terminal collagen-like domain and a C-terminal domain with a sequence similar to that of several Ca2(+)-dependent lectins. SP-A induces a rapid Ca2(+)-dependent aggregation of phospholipid vesicles. We report here that vesicle aggregation is mediated by Ca2(+)-induced interactions between carbohydrate-binding domains and oligosaccharide moieties of SP-A. This novel mechanism of membrane interactions may be relevant to the formation of the membrane lattice of tubular myelin, an extracellular form of surfactant.

scholarly journals LpxT-Dependent Phosphorylation of Lipid A in Escherichia coli Increases Resistance to Deoxycholate and Enhances Gut Colonization

2021 ◽  
Vol 12 ◽  
Author(s):  
Xudong Tian ◽  
Guillaume Manat ◽  
Elise Gasiorowski ◽  
Rodolphe Auger ◽  
Samia Hicham ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Surface ◽  
Lipid A ◽  
Negative Charge ◽  
Polymyxin B ◽  
Gram Negative Bacteria ◽  
Bacterial Cells ◽  
Gram Negative ◽  
E Coli ◽  
Gut Colonization

The cell surface of Gram-negative bacteria usually exhibits a net negative charge mostly conferred by lipopolysaccharides (LPS). This property sensitizes bacterial cells to cationic antimicrobial peptides, such as polymyxin B, by favoring their binding to the cell surface. Gram-negative bacteria can modify their surface to counteract these compounds such as the decoration of their LPS by positively charged groups. For example, in Escherichia coli and Salmonella, EptA and ArnT add amine-containing groups to the lipid A moiety. In contrast, LpxT enhances the net negative charge by catalyzing the synthesis of tri-phosphorylated lipid A, whose function is yet unknown. Here, we report that E. coli has the intrinsic ability to resist polymyxin B upon the simultaneous activation of the two component regulatory systems PhoPQ and PmrAB by intricate environmental cues. Among many LPS modifications, only EptA- and ArnT-dependent decorations were required for polymyxin B resistance. Conversely, the acquisition of polymyxin B resistance compromised the innate resistance of E. coli to deoxycholate, a major component of bile. The inhibition of LpxT by PmrR, under PmrAB-inducing conditions, specifically accounted for the acquired susceptibility to deoxycholate. We also report that the kinetics of intestinal colonization by the E. coli lpxT mutant was impaired as compared to wild-type in a mouse model of infection and that lpxT was upregulated at the temperature of the host. Together, these findings highlight an important function of LpxT and suggest that a tight equilibrium between EptA- and LpxT-dependent decorations, which occur at the same position of lipid A, is critical for the life style of E. coli.

scholarly journals Discovery of the Elusive UDP-Diacylglucosamine Hydrolase in the Lipid A Biosynthetic Pathway in Chlamydia trachomatis

mBio ◽  
2016 ◽  
Vol 7 (2) ◽  
Author(s):  
Hayley E. Young ◽  
Jinshi Zhao ◽  
Jeffrey R. Barker ◽  
Ziqiang Guan ◽  
Raphael H. Valdivia ◽  
...  
Keyword(s):  
Chlamydia Trachomatis ◽  
Biosynthetic Pathway ◽  
Lipid A ◽  
Genomic Library ◽  
Essential Gene ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
E Coli ◽  
Pyrophosphatase Activity

ABSTRACT Constitutive biosynthesis of lipid A via the Raetz pathway is essential for the viability and fitness of Gram-negative bacteria, including Chlamydia trachomatis . Although nearly all of the enzymes in the lipid A biosynthetic pathway are highly conserved across Gram-negative bacteria, the cleavage of the pyrophosphate group of UDP-2,3-diacyl-GlcN (UDP-DAGn) to form lipid X is carried out by two unrelated enzymes: LpxH in beta- and gammaproteobacteria and LpxI in alphaproteobacteria. The intracellular pathogen C. trachomatis lacks an ortholog for either of these two enzymes, and yet, it synthesizes lipid A and exhibits conservation of genes encoding other lipid A enzymes. Employing a complementation screen against a C. trachomatis genomic library using a conditional-lethal lpxH mutant Escherichia coli strain, we have identified an open reading frame (Ct461, renamed lpxG ) encoding a previously uncharacterized enzyme that complements the UDP-DAGn hydrolase function in E. coli and catalyzes the conversion of UDP-DAGn to lipid X in vitro . LpxG shows little sequence similarity to either LpxH or LpxI, highlighting LpxG as the founding member of a third class of UDP-DAGn hydrolases. Overexpression of LpxG results in toxic accumulation of lipid X and profoundly reduces the infectivity of C. trachomatis , validating LpxG as the long-sought-after UDP-DAGn pyrophosphatase in this prominent human pathogen. The complementation approach presented here overcomes the lack of suitable genetic tools for C. trachomatis and should be broadly applicable for the functional characterization of other essential C. trachomatis genes . IMPORTANCE Chlamydia trachomatis is a leading cause of infectious blindness and sexually transmitted disease. Due to the lack of robust genetic tools, the functions of many Chlamydia genes remain uncharacterized, including the essential gene encoding the UDP-DAGn pyrophosphatase activity for the biosynthesis of lipid A, the membrane anchor of lipooligosaccharide and the predominant lipid species of the outer leaflet of the bacterial outer membrane. We designed a complementation screen against the C. trachomatis genomic library using a conditional-lethal mutant of E. coli and identified the missing essential gene in the lipid A biosynthetic pathway, which we designated lpxG . We show that LpxG is a member of the calcineurin-like phosphatases and displays robust UDP-DAGn pyrophosphatase activity in vitro . Overexpression of LpxG in C. trachomatis leads to the accumulation of the predicted lipid intermediate and reduces bacterial infectivity, validating the in vivo function of LpxG and highlighting the importance of regulated lipid A biosynthesis in C. trachomatis .

11β-Hydroxysteroid dehydrogenase type 2 and the regulation of surfactant protein A by dexamethasone metabolites

2006 ◽  
Vol 290 (4) ◽  
pp. E653-E660 ◽  
Author(s):  
Mark R. Garbrecht ◽  
Thomas J. Schmidt ◽  
Zygmunt S. Krozowski ◽  
Jeanne M. Snyder
Keyword(s):  
Human Lung ◽  
Protein A ◽  
Fetal Lung ◽  
Hydroxysteroid Dehydrogenase ◽  
Surfactant Protein ◽  
Biologically Active ◽  
Surfactant Protein A ◽  
Human Adult ◽  
Sensitive Organ ◽  
Lung Epithelial

Glucocorticoid (GC) metabolism by the 11β-hydroxysteroid dehydrogenase (HSD) system is an important prereceptor regulator of GC action. The HSD enzymes catalyze the interconversion of the endogenous, biologically active GC cortisol and its inactive 11-dehydro metabolite cortisone. The role of the HSD enzymes in the metabolism of synthetic GCs, such as dexamethasone (Dex), is more complex. The human lung is a classic GC-sensitive organ; however, the roles of the HSD enzymes (HSD1 and HSD2) in the human lung are poorly understood. In the present study, we examined the expression of the HSD enzymes in human adult and fetal lung tissues and the human lung epithelial cell line NCI-H441. We observed that human adult and fetal lung tissues, as well as H441 cells, express HSD2 protein and that it is upregulated by Dex (10−7 M). By contrast, HSD1 protein was undetectable. We also show that the Dex-mediated regulation of surfactant protein A is attenuated by inhibition of HSD2 activity. Furthermore, we demonstrate that unlike the inactive, 11-dehydro metabolite of cortisol (i.e., cortisone), the 11-dehydro metabolite of Dex, 11-dehydro-Dex, competes for binding to the GC receptor (GR) in human lung epithelial cells and retains GR agonist activity. Together, these data suggest that differences exist in the biological activities of the metabolites of cortisol and Dex.

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