Editorial

6 pages, 240 KiB

Open AccessEditorial

Lipopolysaccharides: Regulated Biosynthesis and Structural Diversity

by Satish Raina

Int. J. Mol. Sci. 2023, 24(8), 7498; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms24087498 - 19 Apr 2023

Cited by 2 | Viewed by 1033

The cell envelope of Gram-negative bacteria contains two distinct membranes, an inner (IM) and an outer (OM) membrane, separated by the periplasm, a hydrophilic compartment that includes a thin layer of peptidoglycan [...] Full article

(This article belongs to the Special Issue Lipopolysaccharides)

Research

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11 pages, 1145 KiB

Open AccessArticle

Structural and Serological Characterization of the O Antigen of Proteus mirabilis Clinical Isolates Classified into a New Proteus Serogroup, O84

by Dominika Drzewiecka, Małgorzata Siwińska, Sof’ya N. Senchenkova, Evgeniya A. Levina, Alexander S. Shashkov and Yuriy A. Knirel

Int. J. Mol. Sci. 2023, 24(5), 4699; https://doi.org/10.3390/ijms24054699 - 28 Feb 2023

Cited by 2 | Viewed by 1366

Abstract

Two closely related Proteus mirabilis smooth strains, Kr1 and Ks20, were isolated from wound and skin samples, respectively, of two infected patients in central Poland. Serological tests, using the rabbit Kr1-specific antiserum, revealed that both strains presented the same O serotype. Their O [...] Read more.

Two closely related Proteus mirabilis smooth strains, Kr1 and Ks20, were isolated from wound and skin samples, respectively, of two infected patients in central Poland. Serological tests, using the rabbit Kr1-specific antiserum, revealed that both strains presented the same O serotype. Their O antigens are unique among the Proteus O serotypes, which had been described earlier, as they were not recognized in an enzyme-linked immunosorbent assay (ELISA) by a set of Proteus O1-O83 antisera. Additionally, the Kr1 antiserum did not react with O1-O83 lipopolysaccharides (LPSs). The O-specific polysaccharide (OPS, O antigen) of P. mirabilis Kr1 was obtained via the mild acid degradation of the LPSs, and its structure was established via a chemical analysis and one- and two-dimensional ¹H and ¹³C nuclear magnetic resonance (NMR) spectroscopy applied to both initial and O-deacetylated polysaccharides, where most β-2-acetamido-2-deoxyglucose (N-acetylglucosamine) (GlcNAc) residues are non-stoichiometrically O-acetylated at positions 3, 4, and 6 or 3 and 6, and a minority of α-GlcNAc residues are 6-O-acetylated. Based on the serological features and chemical data, P. mirabilis Kr1 and Ks20 were proposed as candidates to a new successive O-serogroup in the genus Proteus, O84, which is another example of new Proteus O serotypes identified lately among serologically differentiated Proteus bacilli infecting patients in central Poland. Full article

(This article belongs to the Special Issue Lipopolysaccharides)

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33 pages, 7112 KiB

Open AccessArticle

A New Factor LapD Is Required for the Regulation of LpxC Amounts and Lipopolysaccharide Trafficking

by Alicja Wieczorek, Anna Sendobra, Akshey Maniyeri, Magdalena Sugalska, Gracjana Klein and Satish Raina

Int. J. Mol. Sci. 2022, 23(17), 9706; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23179706 - 26 Aug 2022

Cited by 5 | Viewed by 2936

Abstract

Lipopolysaccharide (LPS) constitutes the major component of the outer membrane and is essential for bacteria, such as Escherichia coli. Recent work has revealed the essential roles of LapB and LapC proteins in regulating LPS amounts; although, if any additional partners are involved [...] Read more.

Lipopolysaccharide (LPS) constitutes the major component of the outer membrane and is essential for bacteria, such as Escherichia coli. Recent work has revealed the essential roles of LapB and LapC proteins in regulating LPS amounts; although, if any additional partners are involved is unknown. Examination of proteins co-purifying with LapB identified LapD as a new partner. The purification of LapD reveals that it forms a complex with several proteins involved in LPS and phospholipid biosynthesis, including FtsH-LapA/B and Fab enzymes. Loss of LapD causes a reduction in LpxC amounts and vancomycin sensitivity, which can be restored by mutations that stabilize LpxC (mutations in lapB, ftsH and lpxC genes), revealing that LapD acts upstream of LapB-FtsH in regulating LpxC amounts. Interestingly, LapD absence results in the substantial retention of LPS in the inner membranes and synthetic lethality when either the lauroyl or the myristoyl acyl transferase is absent, which can be overcome by single-amino acid suppressor mutations in LPS flippase MsbA, suggesting LPS translocation defects in ΔlapD bacteria. Several genes whose products are involved in cell envelope homeostasis, including clsA, waaC, tig and micA, become essential in LapD’s absence. Furthermore, the overproduction of acyl carrier protein AcpP or transcriptional factors DksA, SrrA can overcome certain defects of the LapD-lacking strain. Full article

(This article belongs to the Special Issue Lipopolysaccharides)

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14 pages, 1608 KiB

Open AccessArticle

Regulated Expression of lpxC Allows for Reduction of Endotoxicity in Bordetella pertussis

by Jesús Pérez-Ortega, Ria van Boxtel, Eline F. de Jonge and Jan Tommassen

Int. J. Mol. Sci. 2022, 23(14), 8027; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23148027 - 21 Jul 2022

Cited by 4 | Viewed by 2040

Abstract

The Gram-negative bacterium Bordetella pertussis is the causative agent of a respiratory infection known as whooping cough. Previously developed whole-cell pertussis vaccines were effective, but appeared to be too reactogenic mainly due to the presence of lipopolysaccharide (LPS, also known as endotoxin) in [...] Read more.

The Gram-negative bacterium Bordetella pertussis is the causative agent of a respiratory infection known as whooping cough. Previously developed whole-cell pertussis vaccines were effective, but appeared to be too reactogenic mainly due to the presence of lipopolysaccharide (LPS, also known as endotoxin) in the outer membrane (OM). Here, we investigated the possibility of reducing endotoxicity by modulating the LPS levels. The promoter of the lpxC gene, which encodes the first committed enzyme in LPS biosynthesis, was replaced by an isopropyl β-D-1-thiogalactopyranoside (IPTG)-inducible promoter. The IPTG was essential for growth, even when the construct was moved into a strain that should allow for the replacement of LPS in the outer leaflet of the OM with phospholipids by defective phospholipid transporter Mla and OM phospholipase A. LpxC depletion in the absence of IPTG resulted in morphological changes of the cells and in overproduction of outer-membrane vesicles (OMVs). The reduced amounts of LPS in whole-cell preparations and in isolated OMVs of LpxC-depleted cells resulted in lower activation of Toll-like receptor 4 in HEK-Blue reporter cells. We suggest that, besides lipid A engineering, also a reduction in LPS synthesis is an attractive strategy for the production of either whole-cell- or OMV-based vaccines, with reduced reactogenicity for B. pertussis and other Gram-negative bacteria. Full article

(This article belongs to the Special Issue Lipopolysaccharides)

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14 pages, 943 KiB

Open AccessArticle

Heterogenicity within the LPS Structure in Relation to the Chosen Genomic and Physiological Features of the Plant Pathogen Pectobacterium parmentieri

by Karolina Ossowska, Agata Motyka-Pomagruk, Natalia Kaczyńska, Agnieszka Kowalczyk, Wojciech Sledz, Ewa Lojkowska and Zbigniew Kaczyński

Int. J. Mol. Sci. 2022, 23(4), 2077; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23042077 - 14 Feb 2022

Cited by 7 | Viewed by 1995

Abstract

Pectobacterium parmentieri is a pectinolytic plant pathogenic bacterium causing high economic losses of cultivated plants. The highly devastating potential of this phytopathogen results from the efficient production of plant cell wall-degrading enzymes, i.e., pectinases, cellulases and proteases, in addition to the impact of [...] Read more.

Pectobacterium parmentieri is a pectinolytic plant pathogenic bacterium causing high economic losses of cultivated plants. The highly devastating potential of this phytopathogen results from the efficient production of plant cell wall-degrading enzymes, i.e., pectinases, cellulases and proteases, in addition to the impact of accessory virulence factors such as motility, siderophores, biofilm and lipopolysaccharide (LPS). LPS belongs to pathogen-associated molecular patterns (PAMPs) and plays an important role in plant colonization and interaction with the defense systems of the host. Therefore, we decided to investigate the heterogeneity of O-polysaccharides (OPS) of LPS of different strains of P. parmentieri, in search of an association between the selected genomic and phenotypic features of the strains that share an identical structure of the OPS molecule. In the current study, OPS were isolated from the LPS of two P. parmentieri strains obtained either in Finland in the 1980s (SCC3193) or in Poland in 2013 (IFB5432). The purified polysaccharides were analyzed by utilizing 1D and 2D NMR spectroscopy (¹H, DQF-COSY, TOCSY, ROESY, HSQC, HSQC-TOCSY and HMBC) in addition to chemical methods. Sugar and methylation analyses of native polysaccharides, absolute configuration assignment of constituent monosaccharides and NMR spectroscopy data revealed that these two P. parmentieri strains isolated in different countries possess the same structure of OPS with a very rare residue of 5,7-diamino-3,5,7,9-tetradeoxy-l-glycero-l-manno-non-2-ulosonic acid (pseudaminic acid) substituted in the position C-8: →3)-β-d-Galf-(1→3)-α-d-Galp-(1→8)-β-Pse4Ac5Ac7Ac-(2→6)-α-d-Glcp-(1→6)-β-d-Glcp-(1→. The previous study indicated that three other P. parmentieri strains, namely IFB5427, IFB5408 and IFB5443, exhibit a different OPS molecule than SCC3193 and IFB5432. The conducted biodiversity-oriented assays revealed that the P. parmentieri IFB5427 and IFB5408 strains possessing the same OPS structure yielded the highest genome-wide similarity, according to average nucleotide identity analyses, in addition to the greatest ability to macerate chicory tissue among the studied P. parmentieri strains. The current research demonstrated a novel OPS structure, characteristic of at least two P. parmentieri strains (SCC3193 and IFB5432), and discussed the observed heterogenicity in the OPS of P. parmentieri in a broad genomic and phenotype-related context. Full article

(This article belongs to the Special Issue Lipopolysaccharides)

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21 pages, 3536 KiB

Open AccessArticle

Remodeling of Lipid A in Pseudomonas syringae pv. phaseolicola In Vitro

by Tim Gerster, Michelle Wröbel, Casey E. Hofstaedter, Dominik Schwudke, Robert K. Ernst, Stefanie Ranf and Nicolas Gisch

Int. J. Mol. Sci. 2022, 23(4), 1996; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23041996 - 11 Feb 2022

Cited by 3 | Viewed by 2530

Abstract

Pseudomonas species infect a variety of organisms, including mammals and plants. Mammalian pathogens of the Pseudomonas family modify their lipid A during host entry to evade immune responses and to create an effective barrier against different environments, for example by removal of primary [...] Read more.

Pseudomonas species infect a variety of organisms, including mammals and plants. Mammalian pathogens of the Pseudomonas family modify their lipid A during host entry to evade immune responses and to create an effective barrier against different environments, for example by removal of primary acyl chains, addition of phosphoethanolamine (P-EtN) to primary phosphates, and hydroxylation of secondary acyl chains. For Pseudomonas syringae pv. phaseolicola (Pph) 1448A, an economically important pathogen of beans, we observed similar lipid A modifications by mass spectrometric analysis. Therefore, we investigated predicted proteomes of various plant-associated Pseudomonas spp. for putative lipid A-modifying proteins using the well-studied mammalian pathogen Pseudomonas aeruginosa as a reference. We generated isogenic mutant strains of candidate genes and analyzed their lipid A. We show that the function of PagL, LpxO, and EptA is generally conserved in Pph 1448A. PagL-mediated de-acylation occurs at the distal glucosamine, whereas LpxO hydroxylates the secondary acyl chain on the distal glucosamine. The addition of P-EtN catalyzed by EptA occurs at both phosphates of lipid A. Our study characterizes lipid A modifications in vitro and provides a useful set of mutant strains relevant for further functional studies on lipid A modifications in Pph 1448A. Full article

(This article belongs to the Special Issue Lipopolysaccharides)

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10 pages, 1725 KiB

Open AccessArticle

A Polymorphism of Bactericidal/Permeability-Increasing Protein Affects Its Neutralization Efficiency towards Lipopolysaccharide

by Katharina U. Ederer, Jonas M. Holzinger, Katharina T. Maier, Lisa Zeller, Maren Werner, Martina Toelge, André Gessner and Sigrid Bülow

Int. J. Mol. Sci. 2022, 23(3), 1324; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23031324 - 25 Jan 2022

Cited by 4 | Viewed by 2041

Abstract

Gram-negative sepsis driven by lipopolysaccharide (LPS) has detrimental outcomes, especially in neonates. The neutrophil-derived bactericidal/permeability-increasing protein (BPI) potently neutralizes LPS. Interestingly, polymorphism of the BPI gene at position 645 (rs4358188) corresponds to a favorable survival rate of these patients in the presence of [...] Read more.

Gram-negative sepsis driven by lipopolysaccharide (LPS) has detrimental outcomes, especially in neonates. The neutrophil-derived bactericidal/permeability-increasing protein (BPI) potently neutralizes LPS. Interestingly, polymorphism of the BPI gene at position 645 (rs4358188) corresponds to a favorable survival rate of these patients in the presence of at least one allele 645 A as opposed to 645 G. When we exploited the existing X-ray crystal structure, the corresponding amino acid at position 216 was revealed as surface exposed and proximal to the lipid-binding pocket in the N-terminal domain of BPI. Our further analysis predicted a shift in surface electrostatics by a positively charged lysine (BPI_216K) exchanging a negatively charged glutamic acid (BPI_216E). To investigate differences in interaction with LPS, we expressed both BPI variants recombinantly. The amino acid exchange neither affected affinity towards LPS nor altered bactericidal activity. However, when stimulating human peripheral blood mononuclear cells, BPI_216K exhibited a superior LPS-neutralizing capacity (IC₅₀ 12.0 ± 2.5 pM) as compared to BPI_216E (IC₅₀ 152.9 ± 113.4 pM, p = 0.0081) in respect to IL-6 secretion. In conclusion, we provide a functional correlate to a favorable outcome of sepsis in the presence of BPI_216K. Full article

(This article belongs to the Special Issue Lipopolysaccharides)

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22 pages, 5024 KiB

Open AccessArticle

Structure of Lipopolysaccharide from Liberibacter crescens Is Low Molecular Weight and Offers Insight into Candidatus Liberibacter Biology

by Ian M. Black, Christian Heiss, Mukesh Jain, Artur Muszyński, Russell W. Carlson, Dean W. Gabriel and Parastoo Azadi

Int. J. Mol. Sci. 2021, 22(20), 11240; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms222011240 - 18 Oct 2021

Cited by 6 | Viewed by 2829

Abstract

Huanglongbing (HLB) disease, also known as citrus greening disease, was first reported in the US in 2005. Since then, the disease has decimated the citrus industry in Florida, resulting in billions of dollars in crop losses and the destruction of thousands of acres [...] Read more.

Huanglongbing (HLB) disease, also known as citrus greening disease, was first reported in the US in 2005. Since then, the disease has decimated the citrus industry in Florida, resulting in billions of dollars in crop losses and the destruction of thousands of acres of citrus groves. The causative agent of citrus greening disease is the phloem limited pathogen Candidatus Liberibacter asiaticus. As it has not been cultured, very little is known about the structural biology of the organism. Liberibacter are part of the Rhizobiaceae family, which includes nitrogen-fixing symbionts of legumes as well as the Agrobacterium plant pathogens. To better understand the Liberibacter genus, a closely related culturable bacterium (Liberibacter crescens or Lcr) has attracted attention as a model organism for structural and functional genomics of Liberibacters. Given that the structure of lipopolysaccharides (LPS) from Gram-negative bacteria plays a crucial role in mediating host-pathogen interactions, we sought to characterize the LPS from Lcr. We found that the major lipid A component of the LPS consisted of a pentaacylated molecule with a β-6-GlcN disaccharide backbone lacking phosphate. The polysaccharide portion of the LPS was unusual compared to previously described members of the Rhizobiaceae family in that it contained ribofuranosyl residues. The LPS structure presented here allows us to extrapolate known LPS structure/function relationships to members of the Liberibacter genus which cannot yet be cultured. It also offers insights into the biology of the organism and how they manage to effectively attack citrus trees. Full article

(This article belongs to the Special Issue Lipopolysaccharides)

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Review

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28 pages, 23945 KiB

Open AccessReview

Checkpoints That Regulate Balanced Biosynthesis of Lipopolysaccharide and Its Essentiality in Escherichia coli

by Gracjana Klein, Alicja Wieczorek, Martyna Szuster and Satish Raina

Int. J. Mol. Sci. 2022, 23(1), 189; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23010189 - 24 Dec 2021

Cited by 10 | Viewed by 5121

Abstract

The outer membrane (OM) of Gram-negative bacteria, such as Escherichia coli, is essential for their viability. Lipopolysaccharide (LPS) constitutes the major component of OM, providing the permeability barrier, and a tight balance exists between LPS and phospholipids amounts as both of these [...] Read more.

The outer membrane (OM) of Gram-negative bacteria, such as Escherichia coli, is essential for their viability. Lipopolysaccharide (LPS) constitutes the major component of OM, providing the permeability barrier, and a tight balance exists between LPS and phospholipids amounts as both of these essential components use a common metabolic precursor. Hence, checkpoints are in place, right from the regulation of the first committed step in LPS biosynthesis mediated by LpxC through its turnover by FtsH and HslUV proteases in coordination with LPS assembly factors LapB and LapC. After the synthesis of LPS on the inner leaflet of the inner membrane (IM), LPS is flipped by the IM-located essential ATP-dependent transporter to the periplasmic face of IM, where it is picked up by the LPS transport complex spanning all three components of the cell envelope for its delivery to OM. MsbA exerts its intrinsic hydrocarbon ruler function as another checkpoint to transport hexa-acylated LPS as compared to underacylated LPS. Additional checkpoints in LPS assembly are: LapB-assisted coupling of LPS synthesis and translocation; cardiolipin presence when LPS is underacylated; the recruitment of RfaH transcriptional factor ensuring the transcription of LPS core biosynthetic genes; and the regulated incorporation of non-stoichiometric modifications, controlled by the stress-responsive RpoE sigma factor, small RNAs and two-component systems. Full article

(This article belongs to the Special Issue Lipopolysaccharides)

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