Characterization and heterologous expression of plasmalogen synthase MeHAD from Megasphaera elsdenii.

Plasmalogens (Pls) are vinyl-ether bond-containing glycerophospholipids or glycosyl diradyl glycerols, and are of great importance in the physiological functions and stability of cell membrane. Here, we identified and characterized that the plasmalogen synthase MeHAD from anaerobic Megasphaera elsdenii was responsible for vinyl-ether bond formation. Different from the 2-hydroxyacyl-CoA dehydratase (HAD) family plasmalogen synthase PlsA-PlsR which are encoded by two genes in Clostridium perfringens, the HAD homolog (MeHAD) encoded by a single gene MELS_0169 was found in M. elsdenii. By heterologous expression of the MeHAD gene into a nonplasmalogen-producing Escherichia coli strain, the expressed MeHAD was found to be located in the cell membrane region. Plasmalogens were detected in the recombinant strain using GC-MS and LC-MS, demonstrating that MeHAD was the key enzyme for plasmalogen synthesis. Moreover, the synthesized plasmalogens could enhance the oxidative stress-resistance and osmotic pressure-resistance of the recombinant strain, probably due to the ROS scavenging and decreased membrane permeability by the plasmalogens, respectively. The four-cysteine (Cys125, Cys164, Cys445 and Cys484) site-mutant of MeHAD, which were predicted binding to the [4Fe-4S] cluster, was unable to synthesize plasmalogens, indicating that the cysteines are important for the catalytic activity of MeHAD. Our results revealed the single gene encoded plasmalogen synthase in M. elsdenii and established a recombinant E. coli strain with plasmalogen production potential.

HexSDF Is Required for Synthesis of a Novel Glycolipid That Mediates Daptomycin and Bacitracin Resistance in C. difficile.

Clostridioides difficile is a Gram-positive opportunistic pathogen responsible for 250,000 hospital-associated infections, 12,000 hospital-associated deaths, and $1 billion in medical costs in the United States each year. There has been recent interest in using a daptomycin analog, surotomycin, to treat C. difficile infections. Daptomycin interacts with phosphatidylglycerol and lipid II to disrupt the membrane and halt peptidoglycan synthesis. C. difficile has an unusual lipid membrane composition, as it has no phosphatidylserine or phosphatidylethanolamine, and ~50% of its membrane is composed of glycolipids, including the unique C. difficile lipid aminohexosyl-hexosyldiradylglycerol (HNHDRG). We identified a two-component system (TCS), HexRK, that is required for C. difficile resistance to daptomycin. Using transcriptome sequencing (RNA-seq), we found that HexRK regulates expression of hexSDF, a three-gene operon of unknown function. Based on bioinformatic predictions, hexS encodes a monogalactosyldiacylglycerol synthase, hexD encodes a polysaccharide deacetylase, and hexF encodes an MprF-like flippase. Deletion of hexRK leads to a 4-fold decrease in daptomycin MIC, and that deletion of hexSDF leads to an 8- to 16-fold decrease in daptomycin MIC. The ΔhexSDF mutant is also 4-fold less resistant to bacitracin but no other cell wall-active antibiotics. Our data indicate that in the absence of HexSDF, the phospholipid membrane composition is altered. In wild-type (WT) C. difficile, the unique glycolipid HNHDRG makes up ~17% of the lipids in the membrane. However, in a ΔhexSDF mutant, HNHDRG is completely absent. While it is unclear how HNHDRG contributes to daptomycin resistance, the requirement for bacitracin resistance suggests it has a general role in cell membrane biogenesis. IMPORTANCE Clostridioides difficile is a major cause of hospital-acquired diarrhea and represents an urgent concern due to the prevalence of antibiotic resistance and the rate of recurrent infections. Little is understood about C. difficile membrane lipids, but a unique glycolipid, HNHDRG, has been previously identified in C. difficile and, currently, has not been identified in other organisms. Here, we show that HexSDF and HexRK are required for synthesis of HNHDRG and that production of HNHDRG impacts resistance to daptomycin and bacitracin.

Plasmalogens in bacteria, sixty years on.

The presence of plasmalogens in bacteria has been known for 60 years. The recent discovery of two genes encoding reductases that convert diacyl lipids to 1-alk-1'-enyl 2-acyl lipids has confirmed the derivation of plasmalogens from the corresponding diacyl lipids in bacteria. These genes are widely distributed in anaerobic and in some facultatively anaerobic bacteria. Plasmalogens evolved very early in the history of life on earth. Their persistence during eons of evolution suggests that they play a fundamental role in living organism. The phase behavior of plasmalogens and their conformation in membranes is discussed.

Cyclopropane Fatty Acids Are Important for Salmonella enterica Serovar Typhimurium Virulence.

A variety of eubacteria, plants, and protozoa can modify membrane lipids by cyclopropanation, which is reported to modulate membrane permeability and fluidity. The ability to cyclopropanate membrane lipids has been associated with resistance to oxidative stress in Mycobacterium tuberculosis, organic solvent stress in Escherichia coli, and acid stress in E. coli and Salmonella. In bacteria, the cfa gene encoding cyclopropane fatty acid (CFA) synthase is induced during the stationary phase of growth. In the present study, we constructed a cfa mutant of Salmonella enterica serovar Typhimurium 14028s (S. Typhimurium) and determined the contribution of CFA-modified lipids to stress resistance and virulence in mice. Cyclopropane fatty acid content was quantified in wild-type and cfa mutant S. Typhimurium. CFA levels in the cfa mutant were greatly reduced compared to CFA levels in the wild type, indicating that CFA synthase is the major enzyme responsible for cyclopropane modification of lipids in Salmonella. S. Typhimurium cfa mutants were more sensitive to extreme acid pH, the protonophore CCCP, and hydrogen peroxide compared to the wild type. In addition, cfa mutants exhibited reduced viability in murine macrophages and could be rescued by the addition of the NADPH phagocyte oxidase inhibitor diphenyleneiodonium (DPI) chloride. S. Typhimurium lacking cfa was also attenuated for virulence in mice. These observations indicate that CFA modification of lipids makes an important contribution to Salmonella virulence.

Lipid diversity in clostridia.

Studies of the lipidomes of twenty-one species of clostridia have revealed considerable diversity. Even among those species now defined as Clostridium sensu stricto, which are related to Clostridium butyricum, the type species, lipid analysis has shown that a number of distinct clades have characteristic polar lipids. All species of Clostridium sensu stricto have phosphatidylethanolamine, phosphatidylglycerol and cardiolipin which are present as all acyl or alk-1'-enyl acyl (plasmalogen) species. In addition, almost every clade has specialized polar lipids. For example, the group closely related to Clostridium beijerinckii and several other solventogenic species has glycerol acetals of plasmenylethanolamine, which protects the membrane bilayer arrangement when the lipids are highly unsaturated or in the presence of solvents. The group related to Clostridium novyi has aminoacyl-phosphatidylglycerol, which protects these pathogens from cationic antimicrobial peptides (CAMPs) of innate immunity. Clostridium botulinum species, which fall into several groups, align with these clades, and have the same specific lipids. This review will present the current state of knowledge on clostridial lipids.

Validation of a simplified procedure for convenient and rapid quantification of reduced and oxidized glutathione in human plasma by liquid chromatography tandem mass spectrometry analysis.

Endogenous glutathione (GSH) and glutathione disulfide (GSSG) status is highly sensitive to oxidative conditions and have broad application as a surrogate indicator of redox status in vivo. Established methods for GSH and GSSG quantification in whole blood display limited utility in human plasma, where GSH and GSSG levels are ~3-4 orders of magnitude below those observed in whole blood. This study presents simplified sample processing and analytical LC-MS/MS approaches exhibiting the sensitivity and accuracy required to measure GSH and GSSG concentrations in human plasma samples, which after 5-fold dilution to suppress matrix interferences range from 200 to 500 nm (GSH) and 5-30 nm (GSSG). The utility of the methods reported herein is demonstrated by assay performance and validation parameters which indicate good sensitivity [lower limits of quantitation of 4.99 nm (GSH) and 3.65 nm (GSSG), and high assay precision (intra-assay CVs 3.6 and 1.9%, and inter-assay CVs of 7.0 and 2.8% for GSH and GSSG, respectively). These methods also exhibited exceptional recovery of analyte-spiked plasma samples (98.0 ± 7.64% for GSH and 98.5 ± 12.7% for GSSG). Good sample stability at -80°C was evident for GSH for up to 55 weeks and GSSG for up to 46 weeks, with average CVs <15 and <10%, respectively.

Life without air.

An early exposure to lipid biochemistry in the laboratory of Konrad Bloch resulted in a fascination with the biosynthesis, structures, and functions of bacterial lipids. The discovery of plasmalogens (1-alk-1'-enyl, 2-acyl phospholipids) in anaerobic Gram-positive bacteria led to studies on the physical chemistry of these lipids and the cellular regulation of membrane lipid polymorphism in bacteria. Later studies in several laboratories showed that the formation of the alk-1-enyl ether bond involves an aerobic process in animal cells and thus is fundamentally different from that in anaerobic organisms. Our work provides evidence for an anaerobic process in which plasmalogens are formed from their corresponding diacyl lipids. Studies on the roles of phospholipases in Listeria monocytogenes revealed distinctions between its phospholipases and those previously discovered in other bacteria and showed how the Listeria enzymes are uniquely fitted to the intracellular lifestyle of this significant human pathogen.

Lipidomic Analysis of Clostridium cadaveris and Clostridium fallax.

The lipidomes of Clostridium fallax and Clostridium cadaveris were studied using thin-layer chromatography (TLC) and normal phase liquid chromatography/mass spectrometry (NPLC/MS). Both species contain diradylglycerol (DRG), monohexosyldiradylglycerol (MHDRG), monohexosyl monoacylglycerol (MHMAG), phosphatidylglycerol (PtdGro), and phosphatidylethanolamine (PtdEtn). DRG, MHDRG, PtdEtn, and PtdGro are present in both diacyl and alk-1-enyl acyl (plasmalogen) forms. Both species contain cardiolipin (Ptd2 Gro), which is present in tetraacyl, monoalkenyl-triacyl, and dialkenyl-diacyl forms. Both species contain small amounts of phosphatidylcholine (PtdCho). The presence of octadecadienoic (18:2) acyl chains in some PtdCho species indicates that they arise from the medium because no 18:2 is seen in the other lipids and clostridia generally lack the capacity to synthesize polyunsaturated fatty acids. The major lipidomic differences between these two species are that C. fallax contains a glycerolacetal of plasmenylethanolamine while C. cadaveris contains an ethanolamine-phosphate-modified diacylglycerol. The significance of these lipid compositions is discussed.

Caulobacter crescentus Adapts to Phosphate Starvation by Synthesizing Anionic Glycoglycerolipids and a Novel Glycosphingolipid.

Caulobacter crescentus adapts to phosphate starvation by elongating its cell body and a polar stalk structure. The stalk is an extension of the Gram-negative envelope containing inner and outer membranes as well as a peptidoglycan cell wall. Cellular elongation requires a 6- to 7-fold increase in membrane synthesis, yet phosphate limitation would preclude the incorporation of additional phospholipids. In the place of phospholipids, C. crescentus can synthesize several glycolipid species, including a novel glycosphingolipid (GSL-2). While glycosphingolipids are ubiquitous in eukaryotes, the presence of GSL-2 in C. crescentus is surprising since GSLs had previously been found only in Sphingomonas species, in which they play a role in outer membrane integrity. In this paper, we identify three proteins required for GSL-2 synthesis: CcbF catalyzes the first step in ceramide synthesis, while Sgt1 and Sgt2 sequentially glycosylate ceramides to produce GSL-2. Unlike in Sphingomonas, GSLs are nonessential in C. crescentus; however, the presence of ceramides does contribute to phage resistance and susceptibility to the cationic antimicrobial peptide polymyxin B. The identification of a novel lipid species specifically produced upon phosphate starvation suggests that bacteria may be able to synthesize a wider variety of lipids in response to stresses than previously observed. Uncovering these lipids and their functional relevance will provide greater insight into microbial physiology and environmental adaptation.IMPORTANCE Bacteria adapt to environmental changes in a variety of ways, including altering their cell shape. Caulobacter crescentus adapts to phosphate starvation by elongating its cell body and a polar stalk structure containing both inner and outer membranes. While we generally think of cellular membranes being composed largely of phospholipids, cellular elongation occurs when environmental phosphate, and therefore phospholipid synthesis, is limited. In order to adapt to these environmental constraints, C. crescentus synthesizes several glycolipid species, including a novel glycosphingolipid. This finding is significant because glycosphingolipids, while ubiquitous in eukaryotes, are extremely rare in bacteria. In this paper, we identify three proteins required for GSL-2 synthesis and demonstrate that they contribute to phage resistance. These findings suggest that bacteria may synthesize a wider variety of lipids in response to stresses than previously observed.

Enhancing TFEB-Mediated Cellular Degradation Pathways by the mTORC1 Inhibitor Quercetin.

Signaling pathways mediated by the mechanistic target of rapamycin (mTOR) play key roles in aging and age-related diseases. As a downstream protein of mTOR, transcription factor EB (TFEB) controls lysosome biogenesis and cellular trafficking, processes that are essential for the functions of phagocytic cells like the retinal pigment epithelium (RPE). In the current study, we show that a naturally occurring polyphenolic compound, quercetin, promoted TFEB nuclear translocation and enhanced its transcriptional activity in cultured RPE cells. Activated TFEB facilitated degradation of phagocytosed photoreceptor outer segments. Quercetin is a direct inhibitor of mTOR but did not influence the activity of Akt at the tested concentration range. Our data suggest that the dietary compound quercetin can have beneficial roles in neuronal tissues by improving the functions of the TFEB-lysosome axis and enhancing the capacities of cellular degradation and self-renewal.

Recognition of conserved antigens by Th17 cells provides broad protection against pulmonary Haemophilus influenzae infection.

Nontypeable Haemophilus influenzae (NTHi) is a major cause of community acquired pneumonia and exacerbation of chronic obstructive pulmonary disease. A current effort in NTHi vaccine development has focused on generating humoral responses and has been greatly impeded by antigenic variation among the numerous circulating NTHi strains. In this study, we showed that pulmonary immunization of mice with killed NTHi generated broad protection against lung infection by different strains. While passive transfer of immune antibodies protected only against the homologous strain, transfer of immune T cells conferred protection against both homologous and heterologous strains. Further characterization revealed a strong Th17 response that was cross-reactive with different NTHi strains. Responding Th17 cells recognized both cytosolic and membrane-associated antigens, while immune antibodies preferentially responded to surface antigens and were highly strain specific. We further identified several conserved proteins recognized by lung Th17 cells during NTHi infection. Two proteins yielding the strongest responses were tested as vaccine candidates by immunization of mice with purified proteins plus an adjuvant. Immunization induced antigen-specific Th17 cells that recognized different strains and, upon adoptive transfer, conferred protection. Furthermore, immunized mice were protected against challenge with not only NTHi strains but also a fully virulent, encapsulated strain. Together, these results show that the immune mechanism of cross-protection against pneumonia involves Th17 cells, which respond to a broad spectrum of antigens, including those that are highly conserved among NTHi strains. These mechanistic insights suggest that inclusion of Th17 antigens in subunit vaccines offers the advantage of inducing broad protection and complements the current antibody-based approaches.

Cytochrome c takes on plasmalogen catabolism.

Plasmalogens-phospholipids containing a characteristic vinyl ether group-are precursors of lipids important for cellular signaling such as arachidonic acid. Plasmalogen catabolism involves cleavage of the vinyl ether bond, but the identity of the corresponding enzyme that cleaves the sn-1 vinyl ether bond was unknown. New research shows that cytochrome c, with some help from another lipid, catalyzes the oxidative cleavage of this bond. This discovery, and the subsequent mechanistic dissection, provides exciting new directions for lipid signaling research.

The anaerobic biosynthesis of plasmalogens.

The biosynthesis of plasmalogens in anaerobic bacteria differs fundamentally from that in animal cells. Firstly, the formation of the alk-1'-enyl ether bond in animal cells is oxygen dependent. Secondly, the first step in plasmalogen formation in animal cells is an acylation of dihydroxyacetone phosphate, which has been ruled out as a precursor in anaerobes. In bacteria the alk-1'-enyl ether bond is formed after the fully formed acyl glycerolipids are synthesized. Evidence will be presented for the conversion of the sn-1 acyl-linked chain to an O-alk-1'-enyl ether by an as yet unknown mechanism.

Symbiont-derived sphingolipids modulate mucosal homeostasis and B cells in teleost fish.

Symbiotic bacteria and mucosal immunoglobulins have co-evolved for millions of years in vertebrate animals. Symbiotic bacteria products are known to modulate different aspects of the host immune system. We recently reported that Flectobacillus major is a predominant species that lives in the gill and skin mucosal surfaces of rainbow trout (Oncorhynchus mykiss). F. major is known to produce sphingolipids of a unique molecular structure. Here we propose a role for F. major and its sphingolipids in the regulation of B cell populations in rainbow trout, as well as an essential role for sphingolipids in trout mucosal homeostasis. We found that F. major-specific IgT titers are confined to the gill and skin mucus, whereas F. major-specific IgM titers are only detected in serum. Live F. major cells are able to stimulate sustained IgT expression and secretion in gills. F. major sphingolipids modulate the growth of trout total skin and gill symbiotic bacteria. In vivo systemic administration of F. major sphingolipids changes the proportion of IgT+ to IgM+ B cells in trout HK. These results demonstrate the key role of the symbiont F. major and its sphingolipids in mucosal homeostasis via the modulation of mucosal and systemic Igs and B cells.

Listeria ivanovii Infection in Mice: Restricted to the Liver and Lung with Limited Replication in the Spleen.

Listeria monocytogenes (LM) vectors have shown much promise in delivery of viral and tumor antigens for the development of vaccines. L. ivanovii (LI) is a closely related bacterium with a similar intracellular life cycle that may offer advantages over LM because it is not a human pathogen, but can infect other animal species. Recent studies show that recombinant LI expressing Mycobacterium tuberculosis antigens is effective in inducing protective immunity in mouse models, demonstrating the potential of LI as a live vaccine vector. However, a key barrier in the development of LI into a live vaccine vector is that its pathogenic and immunogenic characteristics have yet to be fully understood. Therefore, in this research, C57BL/6J mice were inoculated with LM or LI intravenously or intranasally, and bacterial loads, histopathologic changes, and cytokine production were determined at indicated days post inoculation. Results showed that after intravenous infection with LM or LI, bacteria were found proliferating in the liver, spleen, and lung. However, LI could only reach a heavy burden in the liver and its ability to multiply and to resist host immunity seemed limited in the spleen and lung. After intranasal inoculation with LI, bacteria were mainly localized in the lung and failed to infect liver or spleen, while LM could. In organs with heavy LI burden, lesions were isolated, localized and densely packed, compared to lesions caused by LM, which were invasive. In the liver of intravenously inoculated mice and lung of intranasally inoculate mice, LI was able to elicit comparable cytokine production with LM and cause less severe histopathologic damages, and thus could be considered as a vector for treating or preventing hepatic or pulmonary diseases.

The cellular lipids of Romboutsia.

We have examined the lipids of three isolates, Romboutsia lituseburensis, Romboutsia ilealis, and Romboutsia sp. strain FRIFI, of the newly described genus Romboutsia by two-dimensional thin-layer chromatography (2D-TLC) and by liquid chromatography/mass spectrometry (LC/MS). We have found three phospholipids, phosphatidylglycerol (PG), cardiolipin and phosphatidic acid in all three species. A fourth phospholipid, lysyl-PG, was found in R. lituseburensis and strain FRIFI. Polyprenyl-phosphates were identified in the lipid extracts of all three species. Three glycolipids, mono-, di- and tri-hexosyldiacylglycerol, were common to all three species. An additional glycolipid, tetrahexosyl-diacylglycerol was identified in strain FRIFI. Acylated trihexosyldiacylglycerol and acyl-tetrahexosydiacylglycerol were also found in R. ilealis and strain FRIFI. Remarkably, no alk-1-enyl ether lipids (plasmalogens) were present in Romboutsia as distinct from bacteria of the related genus Clostridium in which these ether lipids are common. We have compared the lipidome of Romboutsia with that recently described for Clostridium difficile, which has plasmalogens, no lysyl-PG, and no tetrahexosyl-diacylglycerol. According to 16S rRNA gene sequencing, Romboutsia spp. and C. difficile are closely related (>95% sequence identity).

Supplemental vitamin D increases serum cytokines in those with initially low 25-hydroxyvitamin D: a randomized, double blind, placebo-controlled study.

The purpose of this study was to determine if vitamin D status before supplementation influences the cytokine response after supplemental vitamin D. Forty-six reportedly healthy adults (mean(SD); age, 32(7) y; body mass index (BMI), 25.3(4.5) kg/m(2); serum 25-hydroxyvitamin D (25(OH)D), 34.8(12.2) ng/mL) were randomly assigned (double blind) to one of three groups: (1) placebo (n=15), or supplemental vitamin D (cholecalciferol) at (2) 4000 (n=14) or (3) 8000IU (n=17). Supplements were taken daily for 35days. Fasting blood samples were obtained before (Baseline, Bsl) and 35-days after (35-d) supplementation. Serum 25(OH)D, 1,25-dihydroxyvitamin D (1,25(OH)D), cytokines, and intact parathyroid hormone with calcium were measured in each blood sample. Supplemental vitamin D increased serum 25(OH)D (4000IU, ≈29%; 8000IU, ≈57%) and 1,25(OH)D (4000IU, ≈12%; 8000IU, ≈38%) without altering intact parathyroid hormone or calcium. The vitamin D metabolite increases in the supplemental vitamin D groups (n=31) were dependent on initial levels as serum 25(OH)D (r=-0.63, p<0.05) and 1,25(OH)D (r=-0.45, p<0.05) at Bsl correlated with their increases after supplementation. Supplemental vitamin D increased interferon (IFN)-γ and interleukin (IL)-10 in subjects that were vitamin D insufficient (serum 25(OH)D<29ng/mL) compared to sufficient (serum 25(OH)D⩾30ng/mL) at Bsl. We conclude that supplemental vitamin D increase a pro- and anti-inflammatory cytokine in those with initially low serum 25(OH)D.

Clostridium difficile contains plasmalogen species of phospholipids and glycolipids.

Analysis of the polar lipids of many pathogenic and non-pathogenic clostridia has revealed the presence of plasmalogens, alk-1'-enyl ether-containing phospholipids and glycolipids. An exception to this finding so far has been Clostridium difficile, an important human pathogen which is the cause of antibiotic-associated diarrhea and other more serious complications. We have examined the polar lipids of three strains of C. difficile by thin-layer chromatography and have found acid-labile polar lipids indicative of the presence of plasmalogens. The lipids from one of these strains were subjected to further analysis by liquid chromatography coupled to electrospray ionization-mass spectrometry (LC/ESI-MS), which revealed the presence of phosphatidylglycerol, cardiolipin, monohexosyldiradylglycerol, dihexosyldiradylglycerol, and two unusual glycolipids identified as an aminohexosyl-hexosyldiradylglycerol, and a trihexosyldiradylglycerol. High resolution tandem mass spectrometry determined that monohexosyldiradylglycerol, cardiolipin and phosphatidylglycerol contained significant amounts of plasmalogens. C. difficile thus joins the growing list of clostridia that have plasmalogens. Since plasmalogens in clostridia are formed by an anaerobic pathway distinct from those in animal cells, their formation represents a potential novel target for antibiotic action.