Sphingomonas Bacteria Research Articles

Sphingomonas bacteria could serve as an early bioindicator for the development of chlorantraniliprole resistance in Spodoptera frugiperda

The fall armyworm (Spodoptera frugiperda) was found to have invaded China in December 2018, and in just one year, crops in 26 provinces were heavily affected. Currently, the most effective method for emergency control of fulminant pests is to use of chemical pesticides. Recently, most fall armyworm populations in China were begining to exhibite low level resistance to chlorantraniliprole. At present, it is not possible to sensitively reflect the low level resistance of S. frugiperda by detecting target mutation and detoxification enzyme activity. In this study we found that 12 successive generations of screening with chlorantraniliprole caused S. frugiperda to develop low level resistance to this insecticide, and this phenotype was not attribute to genetic mutations in S. frugiperda, but rather to a marked increase in the relative amount of the symbiotic bacteria Sphingomonas. Using FISH and qPCR assays, we determined the amount of Sphingomonas in the gut of S. frugiperda and found Sphingomonas accumulation to be highest in the 3rd-instar larvae. Additionally, Sphingomonas was observed to provide a protective effect to against chlorantraniliprole stress to S. frugiperda. With the increase of the resistance to chlorantraniliprole, the abundance of bacteria also increased, we propose Sphingomonas monitoring could be adapted into an early warning index for the development of chlorantraniliprole resistance in S. frugiperda populations, such that timely measures can be taken to delay or prevent the widespread propagation of resistance to this highly useful agricultural chemical in S. frugiperda field populations.

Analysis of the Bacterial Community of Metal Scrap Using an Enrichment Culture Approach

Microbiologically influenced corrosion (MIC) of metal alloys is promoted by biofilms formed on metal surfaces. In the marine environment, MIC causes serious metal infrastructure problems, which lead to significant economic losses. In this study, we used an enrichment culture approach to examine the bacterial community that grows on metal surface at levels below the detection limit as a preliminary study for developing guidelines to prevent biofilm formation. An enrichment culture approach was employed to analyze the bacterial community on metal surface without biofilms and corrosion. Genomic DNA was extracted from culture sample after incubation in the enrichment culture with a metal piece, and then the V3–V4 variable regions of the bacterial 16S rRNA gene were amplified using the extracted genomic DNA as the template. Subsequently, using a next-generation sequencing approach, the amplified V3–V4 regions were sequenced, and the bacterial community was analyzed using the QIIME 2 microbiome bioinformatics platform. Using this enrichment culture approach, more than 80 bacterial genera were detected with Sphingomonas bacteria exhibiting the highest relative abundance (44%). These results demonstrated that this method could be useful for bacterial community analysis for bacteria below detection limits, and will serve as a basis for the development of the guidelines.

Chronic Drought Differentially Alters the Belowground Microbiome of Drought-Tolerant and Drought-Susceptible Genotypes of Populus trichocarpa

Populus trichocarpa is an ecologically important tree species and economically important biofeedstock. Belowground, P. trichocarpa interacts with diverse microorganisms in the rhizosphere and root endosphere. These plant–microbe interactions can bolster a variety of plant processes, ranging from pathogen suppression to drought tolerance, yet we know little about the impact of chronic drought stress on P. trichocarpa’s belowground microbiomes. To investigate the interactive effect of chronic drought on belowground microbial communities across genetically different P. trichocarpa hosts, we assessed archaeal/bacterial and fungal communities within the root endosphere, rhizosphere, and surrounding bulk soil of selected genotypes in a long-term drought experiment in Boardman, OR, U.S.A. We sequenced the 16S ribosomal RNA and internal transcribed spacer 2 gene region on samples collected from 16 distinct P. trichocarpa genotypes in plots with full or reduced irrigation. Eight of these genotypes have been previously identified as drought tolerant while the other eight genotypes were drought susceptible. Although reduced irrigation influenced the composition of every archaeal or bacterial microbiome compartment, fungal communities were only affected in the rhizosphere and bulk soil compartments. Drought-tolerant bacteria such as Actinobacteria were differentially abundant in reduced irrigation across all belowground microbiomes. Host drought tolerance influenced plant-associated microbiome compartments but had little impact on the bulk soil compartment. Drought-tolerant trees were enriched for potential growth-promoting microorganisms in the root endosphere and rhizosphere, including Sphingomonas bacteria and ectomycorrhizal fungi. Overall, associations of growth-promoting microbes in drought-resistant P. trichocarpa genotypes can be leveraged to improve biofeedstock productivity in regions prone to periodic drought.

Simultaneous Presence of Bacteriochlorophyll and Xanthorhodopsin Genes in a Freshwater Bacterium.

Photoheterotrophic bacteria represent an important part of aquatic microbial communities. There exist two fundamentally different light-harvesting systems: bacteriochlorophyll-containing reaction centers or rhodopsins. Here, we report a photoheterotrophic Sphingomonas strain isolated from an oligotrophic lake, which contains complete sets of genes for both rhodopsin-based and bacteriochlorophyll-based phototrophy. Interestingly, the identified genes were not expressed when cultured in liquid organic media. Using reverse transcription quantitative PCR (RT-qPCR), RNA sequencing, and bacteriochlorophyll a quantification, we document that bacteriochlorophyll synthesis was repressed by high concentrations of glucose or galactose in the medium. Coactivation of photosynthesis genes together with genes for TonB-dependent transporters suggests the utilization of light energy for nutrient import. The photosynthetic units were formed by ring-shaped light-harvesting complex 1 and reaction centers with bacteriochlorophyll a and spirilloxanthin as the main light-harvesting pigments. The identified rhodopsin gene belonged to the xanthorhodopsin family, but it lacks salinixanthin antenna. In contrast to bacteriochlorophyll, the expression of xanthorhodopsin remained minimal under all experimental conditions tested. Since the gene was found in the same operon as a histidine kinase, we propose that it might serve as a light sensor. Our results document that photoheterotrophic Sphingomonas bacteria use the energy of light under carbon-limited conditions, while under carbon-replete conditions, they cover all their metabolic needs through oxidative phosphorylation.IMPORTANCE Phototrophic organisms are key components of many natural environments. There exist two main phototrophic groups: species that collect light energy using various kinds of (bacterio)chlorophylls and species that utilize rhodopsins. Here, we present a freshwater bacterium Sphingomonas sp. strain AAP5 which contains genes for both light-harvesting systems. We show that bacteriochlorophyll-based reaction centers are repressed by light and/or glucose. On the other hand, the rhodopsin gene was not expressed significantly under any of the experimental conditions. This may indicate that rhodopsin in Sphingomonas may have other functions not linked to bioenergetics.

Decoding Wheat Endosphere-Rhizosphere Microbiomes in Rhizoctonia solani-Infested Soils Challenged by Streptomyces Biocontrol Agents.

The endosphere and the rhizosphere are pertinent milieus with microbial communities that perturb the agronomic traits of crop plants through beneficial or detrimental interactions. In this study, we challenged these communities by adding Streptomyces biocontrol strains to wheat seeds in soils with severe Rhizoctonia solani infestation. Wheat plants were grown in a glasshouse standardized system, and the bacterial and fungal microbiomes of 233 samples of wheat roots (endosphere) and rhizosphere soils were monitored for 20 weeks, from seed to mature plant stage. The results showed highly dynamic and diverse microbial communities that changed over time, with Sphingomonas bacteria and Aspergillus, Dipodascus, and Trichoderma fungi increasing over time. Application of biocontrol Streptomyces strains promoted plant growth and maturation of wheat heads and modulated the root microbiome, decreasing Paenibacillus and increasing other bacterial and fungal OTUs. The soils with the highest levels of R. solani had increased reads of Thanatephorus (Rhizoctonia anamorph) and increased root disease levels and increased Balneimonas, Massilia, Pseudomonas, and unclassified Micrococcaceae. As we enter the era of biologically sustainable agriculture, it may be possible to reduce and limit the effects of serious fungal infestations by promoting a beneficial microbiome through the application of biocontrol agents during different periods of plant development.

Ecological succession of the microbial communities of an air-conditioning cooling coil in the tropics.

Air-conditioning systems harbor microorganisms, potentially spreading them to indoor environments. While air and surfaces in air-conditioning systems are periodically sampled as potential sources of indoor microbes, little is known about the dynamics of cooling coil-associated communities and their effect on the downstream airflow. Here, we conducted a 4-week time series sampling to characterize the succession of an air-conditioning duct and cooling coil after cleaning. Using an universal primer pair targeting hypervariable regions of the 16S/18S ribosomal RNA, we observed a community succession for the condensed water, with the most abundant airborne taxon Agaricomycetes fungi dominating the initial phase and Sphingomonas bacteria becoming the most prevalent taxa toward the end of the experiment. Duplicate air samples collected upstream and downstream of the coil suggest that the system does not act as ecological filter or source/sink for specific microbial taxa during the duration of the experiment.

Statistical experimental design optimization of rhamsan gum production by Sphingomonas sp. CGMCC 6833.

Rhamsan gum is a type of water-soluble exopolysaccharide produced by species of Sphingomonas bacteria. The optimal fermentation medium for rhamsan gum production by Sphingomonas sp. CGMCC 6833 was explored definition. Single-factor experiments indicate that glucose, soybean meal, K(2)HPO(4) and MnSO(4) compose the optimal medium along with and initial pH 7.5. To discover ideal cultural conditions for rhamsan gum production in a shake flask culture, response surface methodology was employed, from which the following optimal ratio was derived: 5.38 g/L soybean meal, 5.71 g/L K(2)HPO(4) and 0.32 g/L MnSO(4). Under ideal fermentation rhamsan gum yield reached 19.58 g/L ± 1.23 g/L, 42.09% higher than that of the initial medium (13.78 g/L ± 1.38 g/L). Optimizing the fermentation medium results in enhanced rhamsan gum production.

In silico detection of virulence gene homologues in the human pathogen sphingomonas spp.

There is an ongoing debate about the clinical significance of Sphingomonas paucimobilis as a virulent bacterial pathogen. In the present study, we investigated the presence of different virulence factors and genes in Sphingomonas bacteria. We utilized phylogenetic, comparative genomics and bioinformatics analysis to investigate the potentiality of Sphingomonas bacteria as virulent pathogenic bacteria. The 16S ribosomal RNA gene (16S rDNA) phylogenetic tree showed that the closest bacterial taxon to Sphingomonas is Brucella with a bootstrap value of 87 followed by Helicobacter, Campylobacter, Pseudomonas, and then Legionella. Sphingomonas shared no virulence factors with Helicobacter or Campylobacter, despite their close phylogenic relationship. In spite of the phylogenetic divergence between Sphingomonas and Pseudomonas, they shared many major virulence factors, such as adherence, antiphagocytosis, iron uptake, proteases, and quorum sensing. In conclusion, Sphingomonas spp. contains several major virulence factors resembling Pseudomonas sp., Legionella sp., Brucella sp., and Bordetella sp. virulence factors. Similarity of virulence factors did not match phylogenetic relationships. These findings suggest horizontal gene transfer of virulence factors rather than sharing a common pathogenic ancestor. Sphingomonas spp. is potential virulent bacterial pathogen.

Biological control of winter wheat pathogens with the use of antagonistic Sphingomonas bacteria under greenhouse conditions

This paper describes, for the first time, the effect of bacteria of the genus Sphingomonas on healthiness of winter wheat. The effect of the application of Sphingomonas cell suspension on development of disease symptoms of powdery mildew and Fusarium head blight (FHB) of winter wheat cv. Bogatka was studied under greenhouse conditions. The abundance of populations of yeast and fungi producing mycelium as well as bacteria of the genus Azotobacter and pseudomonads was determined on wheat kernels. The biocontrol agent reduced the population size of Fusarium poae, and it contributed to better grain filling. The tested Sphingomonas isolate reduced the severity of flag leaf infection caused by pathogenic biotroph Blumeria graminis f. sp. tritici.

Intestinal Microbes Affect Phenotypes and Functions of Invariant Natural Killer T Cells in Mice

Invariant natural killer T (iNKT) cells undergo canonical, Vα14-Jα18 rearrangement of the T-cell receptor (TCR) in mice; this form of the TCR recognizes glycolipids presented by CD1d. iNKT cells mediate many different immune reactions. Their constitutive activated and memory phenotype and rapid initiation of effector functions after stimulation indicate previous antigen-specific stimulation. However, little is known about this process. We investigated whether symbiotic microbes can determine the activated phenotype and function of iNKT cells. We analyzed the numbers, phenotypes, and functions of iNKT cells in germ-free mice, germ-free mice reconstituted with specified bacteria, and mice housed in specific pathogen-free environments. Specific pathogen-free mice, obtained from different vendors, have different intestinal microbiota. iNKT cells isolated from these mice differed in TCR Vβ7 frequency and cytokine response to antigen, which depended on the environment. iNKT cells isolated from germ-free mice had a less mature phenotype and were hyporesponsive to activation with the antigen α-galactosylceramide. Intragastric exposure of germ-free mice to Sphingomonas bacteria, which carry iNKT cell antigens, fully established phenotypic maturity of iNKT cells. In contrast, reconstitution with Escherichia coli, which lack specific antigens for iNKT cells, did not affect the phenotype of iNKT cells. The effects of intestinal microbes on iNKT cell responsiveness did not require Toll-like receptor signals, which can activate iNKT cells independently of TCR stimulation. Intestinal microbes can affect iNKT cell phenotypes and functions in mice.

Impact of sugar stereochemistry on natural killer T cell stimulation by bacterial glycolipids

Natural killer T (NKT) cells recognize glycolipids produced by Sphingomonas bacteria, and these glycolipids contain C6-oxidized sugars, either glucuronic acid or galacturonic acid, linked to ceramides. Glycolipids with gluco stereochemistry are the most prevalent. Multiple studies have demonstrated that galactosylceramides are more potent stimulators of NKT cells than their glucose isomers. To determine if this stereoselectivity is retained in the context of the C6-oxidized sugars found in bacterial glycolipids, we prepared two sets of gluco and galacto-glycolipids oxidized at their C6 positions and compared their NKT stimulatory properties. In the context of carboxylic acid groups at C6, gluco stereochemistry gave the more potent responses. We also prepared bacterial glycolipids containing more complex ceramide groups to determine if these chains impact NKT cell responses.

Transcriptome and membrane fatty acid analyses reveal different strategies for responding to permeating and non-permeating solutes in the bacterium Sphingomonas wittichii

BackgroundSphingomonas wittichii strain RW1 can completely oxidize dibenzo-p-dioxins and dibenzofurans, which are persistent contaminants of soils and sediments. For successful application in soil bioremediation systems, strain RW1 must cope with fluctuations in water availability, or water potential. Thus far, however, little is known about the adaptive strategies used by Sphingomonas bacteria to respond to changes in water potential. To improve our understanding, strain RW1 was perturbed with either the cell-permeating solute sodium chloride or the non-permeating solute polyethylene glycol with a molecular weight of 8000 (PEG8000). These solutes are assumed to simulate the solute and matric components of the total water potential, respectively. The responses to these perturbations were then assessed and compared using a combination of growth assays, transcriptome profiling, and membrane fatty acid analyses.ResultsUnder conditions producing a similar decrease in water potential but without effect on growth rate, there was only a limited shared response to perturbation with sodium chloride or PEG8000. This shared response included the increased expression of genes involved with trehalose and exopolysaccharide biosynthesis and the reduced expression of genes involved with flagella biosynthesis. Mostly, the responses to perturbation with sodium chloride or PEG8000 were very different. Only sodium chloride triggered the increased expression of two ECF-type RNA polymerase sigma factors and the differential expression of many genes involved with outer membrane and amino acid metabolism. In contrast, only PEG8000 triggered the increased expression of a heat shock-type RNA polymerase sigma factor along with many genes involved with protein turnover and repair. Membrane fatty acid analyses further corroborated these differences. The degree of saturation of membrane fatty acids increased after perturbation with sodium chloride but had the opposite effect and decreased after perturbation with PEG8000.ConclusionsA combination of growth assays, transcriptome profiling, and membrane fatty acid analyses revealed that permeating and non-permeating solutes trigger different adaptive responses in strain RW1, suggesting these solutes affect cells in fundamentally different ways. Future work is now needed that connects these responses with the responses observed in more realistic scenarios of soil desiccation.

An optical microbial biosensor for detection of methyl parathion using Sphingomonas sp. immobilized on microplate as a reusable biocomponent

Organophosphorus pesticides such as methyl parathion have been widely used in the field of agriculture for insect pest control. These pesticides and their degradation products cause environmental pollution and ecological problem. With a view to monitor these pesticides biosensors are being developed. A bacterium Sphingomonas sp. from field soil has been isolated and identified in our laboratory that hydrolyzes the methyl parathion upto a chromophoric product, p-nitrophenol (PNP). PNP can be detected by electrochemical and colorimetric methods, which can be exploited to develop a biosensor for detection of the organophosphate pesticide. Whole cells of Sphingomonas bacteria were immobilized directly onto the surface of the wells of polystyrene microplates (96 wells) using glutaraldehyde as the cross-linker. SEM study confirmed the immobilization of Sphingomonas sp. Immobilized bacterial microplate was associated directly with the optical transducer, microplate reader. The microplate-based biosensor is having advantages as it has 96 reaction vessels and therefore it provides a convenient system for detecting multiple numbers of samples in a single platform. Detection range of the biosensor from the linear range was determined to be 4–80 μM methyl parathion. Cells-immobilized microplates were having reusability upto 75 reactions. Present study reports an innovative concept where the microplate can be used as immobilizing support for development of reusable microbial biocomponent.

A novel selective growth medium-PCR assay to isolate and detect Sphingomonas in environmental samples

Sphingomonas species can be found ubiquitously in the environment and can be frequently found in surface biofilms. Some Sphingomonas strains are well known for metabolizing complex organic pollutants but some are opportunistic human pathogens. Despite the importance of the Sphingomonas species, a reliable system to isolate this group of bacteria from the environment has not been developed. In this study, a combined streptomycin–piperacillin selective growth medium/polymerase chain reaction (PCR) detection approach is developed to isolate and identify the Sphingomonas bacteria. A total of 72 known Sphingomonas strains (including 21 different Sphingomonas species type strains) and 14 non- Sphingomonas species were tested using a new Sphingomonas-specific growth medium containing 100 and 50 µg/ml streptomycin and piperacillin, respectively. All the Sphingomonas strains showed positive growth on the selective medium and no growth was shown by the non- Sphingomonas species. In addition, two sets of PCR primers targeting the serine palmitoyltransferase gene ( spt), a crucial sphingolipid biosynthesis gene, were developed. With the exception of the Sphingomonas subarctica type strain, 71 of the 72 known Sphingomonas samples were amplified positively by either one or both of the spt-specific primers. None of the non- Sphingomonas bacteria were amplified by the spt primers. To verify the effectiveness of this novel approach for use in environmental screening applications the Sphingomonas selective medium was used to isolate 165 potential Sphingomonas isolates, including 101 yellow, 4 orange and 58 unpigmented isolates, from the influent water and biofilm samples of a pulp and paper mill in Northwestern Ontario. Screening of these isolates with the two Sphingomonas spt–PCR primer sets showed that 98% of the yellow isolates and 100% of the orange isolates were positive to the spt–PCR test. None of the unpigmented isolates was positive to the spt–PCR assay. The 16S rDNA of 17% of the spt + ve and − ve isolates were sequenced and analyzed. All of the yellow and orange pigmented isolates were Sphingomonas while none of the unpigmented isolates were Sphingomonas. REP-PCR was performed on 79 Sphingomonas samples randomly selected from the paper mill and hospital isolates and showed that a diverse group of Sphingomonas can be grown or isolated by our Sphingomonas selective growth medium. Therefore, by using the streptomycin–piperacillin selective growth medium in combination with the colour pigmentation and the positive spt–PCR reactions of the isolates, a diverse population of Sphingomonas strains can be isolated and identified from complex microbial communities with high accuracy.

Occurrence and diversity of nitrogen-fixingSphingomonasbacteria associated with rice plants grown in Brazil

So far, the occurrence of nitrogen-fixing Sphingomonas bacteria has been restricted to three strains of Sphingomonas azotifigens. In this work, a group of 46 Sphingomonas-like isolates, which originated from two rice varieties grown in two soils in Brazil, were characterized based on morphological, physiological and genetic analyses. The PCR genus specifically applied indicated that all 46 isolates belonged to the Sphingomonas genus and confirmed the results based on the yellow pigment of the colonies grown on potato agar medium and the BIOLOG data. It was also observed that 22 isolates are nitrogen-fixing bacteria as determined by the acetylene reduction method and confirmed by nifH gene detection. The genetic diversity based on the 16S rRNA analysis (amplified rDNA restriction analysis) showed that the isolates formed two distinct groups at a similarity value of 60%. Furthermore, five clusters at 60% similarity were observed with the 16S-23S intergenic space (ribosomal intergenic space analysis) analysis. Sequencing of the 16S rRNA gene and nifH fragments showed that most of the 22 nitrogen-fixing isolates formed clusters apart from that of the S. azotifigens. This is the first report on the occurrence of nitrogen-fixing Sphingomonas bacteria associated with rice grown in Brazil.

Liposomal glycosphingolipids activate natural killer T cell-mediated immune responses through the endosomal pathway

Natural killer T (NKT) cells recognize lipid antigens, such as glycosphingolipids (GSLs), via CD1d and contribute to host defense against various pathogens. Here, we demonstrate that GSLs isolated from Sphingomonas bacteria and inserted into liposomes (GSL-liposomes) enhance the activation of NKT cells and dendritic cells (DCs). GSL-liposomes remarkably enhanced the production of IFN-γ from splenocytes in vitro and this enhancement depended on the content of the pH-sensitive lipid dioleoyl-phosphoethanolamine (DOPE) in the liposomes. GSL-liposomes containing DOPE were clearly broken in late endosomes and this may facilitate effective loading of GSLs onto CD1 molecules. Treatment with GSL-liposomes also activated NKT cells and DCs in vivo. Collectively, our results strongly suggest that GSL-liposomes can effectively induce NKT cell-mediated immune responses and may be useful as an immune adjuvant for inducing protective immunity.

Natural Sphingomonas Glycolipids Vary Greatly in Their Ability to Activate Natural Killer T Cells

Mouse natural killer T (NKT) cells expressing an invariant T cell antigen receptor (TCR) recognize glycosphingolipids (GSLs) from Sphingomonas bacteria. The synthetic antigens previously tested, however, were designed to closely resemble the potent synthetic agonist alpha-galactosyl ceramide (alphaGalCer), which contains a monosaccharide and a C18:0 sphingosine lipid. Some Sphingomonas bacteria, however, also have oligosaccharide-containing GSLs, and they normally synthesize several GSLs with different sphingosine chains including one with a cyclopropyl ring-containing C21:0 (C21cycl) sphingosine. Here we studied the stimulation of NKT cells with synthetic GSL antigens containing natural tetrasaccharide sugars, or the C21cycl sphingosine. Our results indicate that there is a great degree of variability in the antigenic potency of different natural Sphingomonas glycolipids, with the C21cycl sphingosine having intermediate potency and the oligosaccharide-containing antigens exhibiting limited or no stimulatory capacity.

The antigenic activity of glycosphingolipids with tetrasaccharide sugars from Sphingomonas bacteria to natural killer T cells

Natural Killer T (NKT) cells, which express an invariant V(14 TCR (iNKT cells), regulate various immune responses by rapidly secreting cytokines upon stimulation with glycolipids presented by CD1d, a MHC class I‐like molecule. It has been shown a monosaccharide containing glycosphingolipid (GSL‐1) from Sphingomonas bacteria is a natural antigen for iNKT cells and mice lacking these cells have a reduced bacterial clearance. However Sphingomonas bacteria have tetrasaccharide containing glycolipids as well as monosaccharide‐containing ones. Here, we studied the stimulation of iNKT cells with two synthesized GSL‐4 antigens (4A and 4B) and a variant of GSL‐4A. We show that GSL‐4A but not 4B could stimulate iNKT hybridomas, although the response to 4A was much weaker than that to GSL‐1. There was no requirement for antigen endocytosis because GSL‐4A presentation by cells transfected with wild type CD1d was equivalent to cells expressing a CD1d with a cytoplasmic tail mutation (I/A), in which the mutant CD1d molecules cannot traffick to lysosomes. Using an antigen presenting cell‐free assay, we could confirm that lysosomal antigen processing was not necessary for GSL‐4A stimulation. Our data indicate that glycolipids containing more complex carbohydrates can be antigens for direct recognition by TCRs expressed by iNKT cells, although weaker than GSL‐1.

Sensing and recognition of bacteria by NKT cells (44.3)

Abstract Natural Killer T (NKT) cells are a conserved T lymphocyte subpopulation that participates in host defense against infections. NKT cell express an invariant TCR alpha chain and they recognize glycolipids presented by CD1d. We found that NKT cells also respond to IL-12 and IL-18 produced by APC that have been activated by TLR ligands. As a result, NKT cells rapidly produce IFN gamma that stimulates the innate immune response. By responding to activated APC, NKT cells can amplify the innate immunity to many different microbes. The invariant TCR expressed by mouse and human NKT cells recognizes glycosphingolipids with alpha linked sugars, which are unique to Sphingomonas bacteria. Mice that lack NKT cells have reduced Sphingomonas clearance. Mouse and human NKT cells with an invariant TCR also recognize glycosyl diacylglycerols from Borrelia burgdorferi, which causes Lyme disease. Interestingly, the response to these compounds was highly dependent on the nature of the aliphatic chains, with the addition of a single unsaturated bond having an enormous influence on antigenic potency. NKT cells are activated during B. burgdorferi infection. These data provide evidence that NKT cells recognize a new category of microbial glycolipids, diacylglycerols, which is more broadly distributed in pathogenic microbes, and they suggest that this TCR-mediated recognition provides protection from microbial pathogens.

Antigen receptor-dependent and -independent regulation of NKT patrolling and activation in liver sinusoids (91.7)

Abstract Natural killer T (NKT) cells are most abundant in liver and spleen and, recognize lipid antigen presented by CD1d. Utilizing intravital fluorescence microscopy, we previously demonstrated NKT patrolling in the liver sinusoids, meaning cell migration both with and against the blood flow in surveillance of antigen. Here, we report the relationship between patrolling and activation. First, both a Sphingomonas bacteria derived weak agonist, GalA-GSL at 2 μg/g, and a synthetic strong agonist, aGalCer at 10 ng/g, induced serum IL-4, CD69 upregualtion and acute NKT arrest. Surprisingly, a low dose of aGalCer (2 ng/g) did not significantly affect NKT patrolling, although a comparable biological response was elicited. Second, IL-12 and IL-18 synergized to activate NKT cells in the absence of exogenous antigen. These cytokines activated liver NKT cells and induced serum IFN- ? but not IL-4. NKT patrolling was not significantly affected. Therefore, glycolipid antigens can induce NKT cell activation and arrest, although the requirement for arrest may be restricted to weak agonists. Importantly, IL-12 and IL-18 activate NKT cells without affecting NKT patrolling thereby allowing these primed cells to continue to survey tissue for antigen. Current studies are aimed at understanding the hierarchy of antigen receptor versus cytokine driven activation and further defining their relationship to NKT cell patrolling.