Modification of Lipooligosaccharide with Phosphoethanolamine by LptA in Neisseria meningitidis Enhances Meningococcal Adhesion to Human Endothelial and Epithelial Cells

ABSTRACT The lipooligosaccharide (LOS) of Neisseria meningitidis can be decorated with phosphoethanolamine (PEA) at the 4′ position of lipid A and at the O-3 and O-6 positions of the inner core of the heptose II residue. The biological role of PEA modification in N. meningitidis remains unclear. During the course of our studies to elucidate the pathogenicity of the ST-2032 (invasive) meningococcal clonal group, disruption of lptA, the gene that encodes the PEA transferase for 4′ lipid A, led to a approximately 10-fold decrease in N. meningitidis adhesion to four kinds of human endothelial and epithelial cell lines at an multiplicity of infection of 5,000. Complementation of the lptA gene in a ΔlptA mutant restored wild-type adherence. By matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analysis, PEA was lost from the lipid A of the ΔlptA mutant compared to that of the wild-type strain. The effect of LptA on meningococcal adhesion was independent of other adhesins such as pili, Opc, Opa, and PilC but was inhibited by the presence of capsule. These results indicate that modification of LOS with PEA by LptA enhances meningococcal adhesion to human endothelial and epithelial cells in unencapsulated N. meningitidis.

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Lipooligosaccharide Structure Contributes to Multiple Steps in the Virulence of Neisseria meningitidis

Infection and Immunity ◽

10.1128/iai.74.2.1360-1367.2006 ◽

2006 ◽

Vol 74 (2) ◽

pp. 1360-1367 ◽

Cited By ~ 44

Author(s):

Laura Plant ◽

Johanna Sundqvist ◽

Susu Zughaier ◽

Lena Lövkvist ◽

David S. Stephens ◽

...

Keyword(s):

Epithelial Cells ◽

Mouse Model ◽

Neisseria Meningitidis ◽

Type Strain ◽

Lipid A ◽

Wild Type Strain ◽

Inner Core ◽

Wild Type ◽

Proinflammatory Response

ABSTRACT Lipooligosaccharide (LOS) of Neisseria meningitidis has been implicated in meningococcal interaction with host epithelial cells and is a major factor contributing to the human proinflammatory response to meningococci. LOS mutants of the encapsulated N. meningitidis serogroup B strain NMB were used to further determine the importance of the LOS structure in in vitro adherence and invasion of human pharyngeal epithelial cells by meningococci and to study pathogenicity in a mouse (CD46 transgenic) model of meningococcal disease. The wild-type strain [NeuNAc-Galβ-GlcNAc-Galβ-Glcβ-Hep2 (GlcNAc, Glcα) 3-deoxy-d-manno-2-octulosonic acid (KDO2)-lipid A; 1,4′ bisphosphorylated], although poorly adherent, rapidly invaded an epithelial cell layer in vitro, survived and multiplied early in blood, reached the cerebrospinal fluid, and caused lethal disease in the mouse model. In contrast, the Hep2 (GlcNAc) KDO2-lipid A (pgm) mutant, which was highly adherent to cultured epithelial cells, caused significantly less bacteremia and mortality in the mouse model. The Hep2-KDO2-lipid A (rfaK) mutant was shown to be moderately adherent and to cause levels of bacteremia and mortality similar to those caused by the wild-type strain in the mouse model. The KDO2-lipid A (gmhB) mutant, which lacks the heptose disaccharide in the inner core of LOS, avidly attached to epithelial cells but was otherwise avirulent. Disease development correlated with expression of specific LOS structures and was associated with lower adherence but rapid meningococcal passage to and survival in the bloodstream, induction of proinflammatory cytokines, and the crossing of the blood-brain barrier. Taken together, the results of this study further define the importance of the LOS structure as a virulence component involved in multiple steps in the pathogenesis of N. meningitidis.

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Identification of Proteus mirabilisMutants with Increased Sensitivity to Antimicrobial Peptides

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.45.7.2030-2037.2001 ◽

2001 ◽

Vol 45 (7) ◽

pp. 2030-2037 ◽

Cited By ~ 79

Author(s):

Andrea J. McCoy ◽

Hongjian Liu ◽

Timothy J. Falla ◽

John S. Gunn

Keyword(s):

Antimicrobial Peptides ◽

Lipid A ◽

Bacterial Species ◽

Polymyxin B ◽

Mass Spectrometry Analysis ◽

Wild Type ◽

Swarming Motility ◽

Spectrometry Analysis ◽

O Antigen ◽

Flight Mass Spectrometry

ABSTRACT Antimicrobial peptides (APs) are important components of the innate defenses of animals, plants, and microorganisms. However, some bacterial pathogens are resistant to the action of APs. For example,Proteus mirabilis is highly resistant to the action of APs, such as polymyxin B (PM), protegrin, and the synthetic protegrin analog IB-367. To better understand this resistance, a transposon mutagenesis approach was used to generate P. mirabilismutants sensitive to APs. Four unique PM-sensitive mutants of P. mirabilis were identified (these mutants were >2 to >128 times more sensitive than the wild type). Two of these mutants were also sensitive to IB-367 (16 and 128 times more sensitive than the wild type). Lipopolysaccharide (LPS) profiles of the PM- and protegrin-sensitive mutants demonstrated marked differences in both the lipid A and O-antigen regions, while the PM-sensitive mutants appeared to have alterations of either lipid A or O antigen. Matrix-assisted laser desorption ionization–time of flight mass spectrometry analysis of the wild-type and PM-sensitive mutant lipid A showed species with one or two aminoarabinose groups, while lipid A from the PM- and protegrin-sensitive mutants was devoid of aminoarabinose. When the mutants were streaked on an agar-containing medium, the swarming motility of the PM- and protegrin-sensitive mutants was completely inhibited and the swarming motility of the mutants sensitive to only PM was markedly decreased. DNA sequence analysis of the mutagenized loci revealed similarities to an O-acetyltransferase (PM and protegrin sensitive) and ATP synthase and sap loci (PM sensitive). These data further support the role of LPS modifications as an elaborate mechanism in the resistance of certain bacterial species to APs and suggest that LPS surface charge alterations may play a role in P. mirabilis swarming motility.

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Polymyxin B Resistance in El Tor Vibrio cholerae Requires Lipid Acylation Catalyzed by MsbB

Journal of Bacteriology ◽

10.1128/jb.00023-10 ◽

2010 ◽

Vol 192 (8) ◽

pp. 2044-2052 ◽

Cited By ~ 31

Author(s):

Jyl S. Matson ◽

Hyun Ju Yoo ◽

Kristina Hakansson ◽

Victor J. DiRita

Keyword(s):

Antimicrobial Peptides ◽

Vibrio Cholerae ◽

Lipid A ◽

Polymyxin B ◽

Mass Spectrometry Analysis ◽

Peptide Antibiotic ◽

Wild Type ◽

Spectrometry Analysis ◽

Antimicrobial Peptide Resistance ◽

El Tor

ABSTRACTAntimicrobial peptides are critical for innate antibacterial defense. Both Gram-negative and Gram-positive microbes have mechanisms to alter their surfaces and resist killing by antimicrobial peptides. InVibrio cholerae, two natural epidemic biotypes, classical and El Tor, exhibit distinct phenotypes with respect to sensitivity to the peptide antibiotic polymyxin B: classical strains are sensitive and El Tor strains are relatively resistant. We carried out mutant screens of both biotypes, aiming to identify classicalV. choleraemutants resistant to polymyxin B and El TorV. choleraemutants sensitive to polymyxin B. Insertions in a gene annotatedmsbB(encoding a predicted lipid A secondary acyltransferase) answered both screens, implicating its activity in antimicrobial peptide resistance ofV. cholerae. Analysis of a defined mutation in the El Tor biotype demonstrated thatmsbBis required for resistance to all antimicrobial peptides tested. Mutation ofmsbBin a classical strain resulted in reduced resistance to several antimicrobial peptides but in no significant change in resistance to polymyxin B.msbBmutants of both biotypes showed decreased colonization of infant mice, with a more pronounced defect observed for the El Tor mutant. Mass spectrometry analysis showed that lipid A of themsbBmutant for both biotypes was underacylated compared to lipid A of the wild-type isolates, confirming that MsbB is a functional acyltransferase inV. cholerae.

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LPS Remodeling Triggers Formation of Outer Membrane Vesicles inSalmonella

mBio ◽

10.1128/mbio.00940-16 ◽

2016 ◽

Vol 7 (4) ◽

Cited By ~ 58

Author(s):

Wael Elhenawy ◽

Michael Bording-Jorgensen ◽

Ezequiel Valguarnera ◽

M. Florencia Haurat ◽

Eytan Wine ◽

...

Keyword(s):

Outer Membrane ◽

Molecular Mechanisms ◽

Lipid A ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Mass Spectrometry Analysis ◽

Membrane Remodeling ◽

Macrophage Infection ◽

Spectrometry Analysis

ABSTRACTOuter membrane vesicles (OMV) are proposed to mediate multiple functions during pathogenesis and symbiosis. However, the mechanisms responsible for OMV formation remain poorly understood. It has been shown in eukaryotic membranes that lipids with an inverted-cone shape favor the formation of positive membrane curvatures. Based on these studies, we formulated the hypothesis that lipid A deacylation might impose shape modifications that result in the curvature of the outer membrane (OM) and subsequent OMV formation. We tested the effect of lipid A remodeling on OMV biogenesis employingSalmonella entericaserovar Typhimurium as a model organism. Expression of the lipid A deacylase PagL resulted in increased vesiculation, without inducing an envelope stress response. Mass spectrometry analysis revealed profound differences in the patterns of lipid A in OM and OMV, with accumulation of deacylated lipid A forms exclusively in OMV. OMV biogenesis by intracellular bacteria upon macrophage infection was drastically reduced in apagLmutant strain. We propose a novel mechanism for OMV biogenesis requiring lipid A deacylation in the context of a multifactorial process that involves the orchestrated remodeling of the outer membrane.IMPORTANCEThe role of lipid remodeling in vesiculation is well documented in eukaryotes. Similarly, bacteria produce membrane-derived vesicles; however, the molecular mechanisms underlying their production are yet to be determined. In this work, we investigated the role of outer membrane remodeling in OMV biogenesis inS. Typhimurium. We showed that the expression of the lipid A deacylase PagL results in overvesiculation with deacylated lipid A accumulation exclusively in OMV. AnS. Typhimurium ΔpagLstrain showed a significant reduction in intracellular OMV secretion relative to the wild-type strain. Our results suggest a novel mechanism for OMV biogenesis that involves outer membrane remodeling through lipid A modification. Understanding how OMV are produced by bacteria is important to advance our understanding of the host-pathogen interactions.

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Role of Toll-Like Receptor 4 in Induction of Cell-Mediated Immunity and Resistance to Brucella abortus Infection in Mice

Infection and Immunity ◽

10.1128/iai.72.1.176-186.2004 ◽

2004 ◽

Vol 72 (1) ◽

pp. 176-186 ◽

Cited By ~ 81

Author(s):

Marco A. Campos ◽

Gracia M. S. Rosinha ◽

Igor C. Almeida ◽

Xirlene S. Salgueiro ◽

Bruce W. Jarvis ◽

...

Keyword(s):

Brucella Abortus ◽

Cho Cells ◽

Lipid A ◽

Mass Spectrometry Analysis ◽

Interleukin 12 ◽

Cell Mediated Immunity ◽

Adjuvant Activity ◽

Necrosis Factor Alpha ◽

Spectrometry Analysis

ABSTRACT Initial host defense to bacterial infection is executed by innate immunity, and therefore the main goal of this study was to examine the contribution of Toll-like receptors (TLRs) during Brucella abortus infection. CHO reporter cell lines transfected with CD14 and TLRs showed that B. abortus triggers both TLR2 and TLR4. In contrast, lipopolysaccharide (LPS) and lipid A derived from Brucella rough (R) and smooth (S) strains activate CHO cells only through TLR4. Consistently, macrophages from C3H/HePas mice exposed to R and S strains and their LPS produced higher levels of tumor necrosis factor alpha (TNF-α) and interleukin-12 compared to C3H/HeJ, a TLR4 mutant mouse. The essential role of TLR4 for induction of proinflammatory cytokines was confirmed with diphosphoryl lipid A from Rhodobacter sphaeroides. Furthermore, to determine the contribution of TLR2 and TLR4 in bacterial clearance, numbers of Brucella were monitored in the spleen of C3H/HeJ, C3H/HePas, TLR2 knockout, and wild-type mice at 1, 3, and 6 weeks following B. abortus infection. Interestingly, murine brucellosis was markedly exacerbated at weeks 3 and 6 after infection in animals that lacked functional TLR4 (C3H/HeJ) compared to C3H/HePas that paralleled the reduced gamma interferon production by this mouse strain. Finally, by mass spectrometry analysis we found dramatic differences on the lipid A profiles of R and S strains. In fact, S lipid A was shown to be more active to trigger TLR4 than R lipid A in CHO cells and more effective in inducing dendritic cell maturation. In conclusion, these results indicate that TLR4 plays a role in resistance to B. abortus infection and that S lipid A has potent adjuvant activity.

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The Structure of Neisseria meningitidis Lipid A Determines Outcome in Experimental Meningococcal Disease

Infection and Immunity ◽

10.1128/iai.01311-09 ◽

2010 ◽

Vol 78 (7) ◽

pp. 3177-3186 ◽

Cited By ~ 15

Author(s):

Floris Fransen ◽

Hendrik Jan Hamstra ◽

Claire J. Boog ◽

Jos P. van Putten ◽

Germie P. J. M. van den Dobbelsteen ◽

...

Keyword(s):

Neisseria Meningitidis ◽

Type Strain ◽

Lipid A ◽

Wild Type Strain ◽

Large Fraction ◽

Acyl Chain ◽

Wild Type ◽

Toll Like Receptor 4 ◽

Lipid A Structure

ABSTRACTLipopolysaccharide (LPS), a major component of the meningococcal outer membrane, is sensed by the host through activation of Toll-like receptor 4 (TLR4). Recently, we demonstrated that a surprisingly large fraction ofNeisseria meningitidisdisease isolates are lipid A mutants, due to inactivating mutations in thelpxL1gene. ThelpxL1mutants activate human TLR4 much less efficiently than wild-type bacteria, which may be advantageous by allowing them to escape from the innate immune system. Here we investigated the influence of lipid A structure on virulence in a mouse model of meningococcal sepsis. One limitation, however, is that murine TLR4 recognizeslpxL1mutant bacteria much better than human TLR4. We show that anlpxL2mutant, another lipid A mutant lacking an acyl chain at a different position, activates murine TLR4 less efficiently than thelpxL1mutant. Therefore, thelpxL2mutant in mice might be a better model for infections withlpxL1mutants in humans. Interestingly, we found that thelpxL2mutant is more virulent in mice than the wild-type strain, whereas thelpxL1mutant is actually much less virulent than the wild-type strain. These results demonstrate the crucial role ofN. meningitidislipid A structure in virulence.

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Structural Characterization of Act c 10.0101 and Pun g 1.0101—Allergens from the Non-Specific Lipid Transfer Protein Family

Molecules ◽

10.3390/molecules26020256 ◽

2021 ◽

Vol 26 (2) ◽

pp. 256

Author(s):

Andrea O’Malley ◽

Swanandi Pote ◽

Ivana Giangrieco ◽

Lisa Tuppo ◽

Anna Gawlicka-Chruszcz ◽

...

Keyword(s):

Immunoglobulin E ◽

Lipid Transfer Protein ◽

Mass Spectrometry Analysis ◽

Lipid Transfer ◽

Helical Structures ◽

Spectrometry Analysis ◽

X Ray Crystallography ◽

Transfer Protein ◽

Specific Lipid

(1) Background: Non-specific lipid transfer proteins (nsLTPs), which belong to the prolamin superfamily, are potent allergens. While the biological role of LTPs is still not well understood, it is known that these proteins bind lipids. Allergen nsLTPs are characterized by significant stability and resistance to digestion. (2) Methods: nsLTPs from gold kiwifruit (Act c 10.0101) and pomegranate (Pun g 1.0101) were isolated from their natural sources and structurally characterized using X-ray crystallography (3) Results: Both proteins crystallized and their crystal structures were determined. The proteins have a very similar overall fold with characteristic compact, mainly α-helical structures. The C-terminal sequence of Act c 10.0101 was updated based on our structural and mass spectrometry analysis. Information on proteins’ sequences and structures was used to estimate the risk of cross-reactive reactions between Act c 10.0101 or Pun g 1.0101 and other allergens from this family of proteins. (4) Conclusions: Structural studies indicate a conformational flexibility of allergens from the nsLTP family and suggest that immunoglobulin E binding to some surface regions of these allergens may depend on ligand binding. Both Act c 10.0101 and Pun g 1.0101 are likely to be involved in cross-reactive reactions involving other proteins from the nsLTP family.

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Expression of lysophosphatidic acid receptor 5 is necessary for the regulation of intestinal Na+/H+ exchanger 3 by lysophosphatidic acid in vivo

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00130.2018 ◽

2018 ◽

Vol 315 (4) ◽

pp. G433-G442 ◽

Cited By ~ 3

Author(s):

Kayte A. Jenkin ◽

Peijian He ◽

C. Chris Yun

Keyword(s):

Epithelial Cells ◽

Lysophosphatidic Acid ◽

Direct Evidence ◽

Intestinal Epithelial Cells ◽

Regulatory Role ◽

Intestinal Epithelial ◽

Fluid Absorption ◽

Wild Type

Lysophosphatidic acid (LPA) is a bioactive lipid molecule, which regulates a broad range of pathophysiological processes. Recent studies have demonstrated that LPA modulates electrolyte flux in the intestine, and its potential as an antidiarrheal agent has been suggested. Of six LPA receptors, LPA5 is highly expressed in the intestine. Recent studies by our group have demonstrated activation of Na+/H+ exchanger 3 (NHE3) by LPA5. However, much of what has been elucidated was achieved using colonic cell lines that were transfected to express LPA5. In the current study, we engineered a mouse that lacks LPA5 in intestinal epithelial cells, Lpar5ΔIEC, and investigated the role of LPA5 in NHE3 regulation and fluid absorption in vivo. The intestine of Lpar5ΔIEC mice appeared morphologically normal, and the stool frequency and fecal water content were unchanged compared with wild-type mice. Basal rates of NHE3 activity and fluid absorption and total NHE3 expression were not changed in Lpar5ΔIEC mice. However, LPA did not activate NHE3 activity or fluid absorption in Lpar5ΔIEC mice, providing direct evidence for the regulatory role of LPA5. NHE3 activation involves trafficking of NHE3 from the terminal web to microvilli, and this mobilization of NHE3 by LPA was abolished in Lpar5ΔIEC mice. Dysregulation of NHE3 was specific to LPA, and insulin and cholera toxin were able to stimulate and inhibit NHE3, respectively, in both wild-type and Lpar5ΔIEC mice. The current study for the first time demonstrates the necessity of LPA5 in LPA-mediated stimulation of NHE3 in vivo. NEW & NOTEWORTHY This study is the first to assess the role of LPA5 in NHE3 regulation and fluid absorption in vivo using a mouse that lacks LPA5 in intestinal epithelial cells, Lpar5ΔIEC. Basal rates of NHE3 activity and fluid absorption, and total NHE3 expression were not changed in Lpar5ΔIEC mice. However, LPA did not activate NHE3 activity or fluid absorption in Lpar5ΔIEC mice, providing direct evidence for the regulatory role of LPA5.

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