In this study, we examined the relationship between c-di-GMP and its only known effector protein, PlzA, in Borrelia burgdorferi during the arthropod and mammalian phases of the enzootic cycle. Using a B. burgdorferi strain expressing a plzA point mutant (plzA-R145D) unable to bind c-di-GMP, we confirmed that the protective function of PlzA in ticks is c-di-GMP-dependent. Unlike ΔplzA spirochetes, which are severely attenuated in mice, the plzA-R145D strain was fully infectious, firmly establishing that PlzA serves a c-di-GMP-independent function in mammals. Contrary to prior reports, loss of PlzA did not affect expression of RpoS or RpoS-dependent genes, which are essential for transmission, mammalian host-adaptation and murine infection. To ascertain the nature of PlzA’s c-di-GMP-independent function(s), we employed infection models using (i) host-adapted mutant spirochetes for needle inoculation of immunocompetent mice and (ii) infection of scid mice with in vitro-grown organisms. Both approaches substantially restored ΔplzA infectivity, suggesting that PlzA enables B. burgdorferi to overcome an early bottleneck to infection. Furthermore, using a Borrelia strain expressing a heterologous, constitutively active diguanylate cyclase, we demonstrate that ‘ectopic’ production of c-di-GMP in mammals abrogates spirochete virulence and interferes with RpoS function at the post-translational level in a PlzA-dependent manner. Structural modeling and SAXS analysis of liganded- and unliganded-PlzA revealed marked conformational changes that underlie its biphasic functionality. This structural plasticity likely enables PlzA to serve as a c-di-GMP biosensor that in its respective liganded and unliganded states promote vector- and host-adaptation by the Lyme disease spirochete.

Necrotizing enterocolitis (NEC) is a devastating intestinal inflammatory disease of premature infants associated with gut bacterial dysbiosis. Using 16S rRNA-based methods, our laboratory identified an unclassified Enterobacteriaceae sequence (NEC_unk_OTU) with high abundance in NEC fecal samples. We aimed to identify this bacterium and determine its potential role in the disease. NCBI database searches for the 16S sequence, selective culture systems, biotyping and polymerase chain reaction were employed to refine classification of NEC_unk_OTU and identify toxinencoding genes from the index NEC case. Bacterial cytotoxin production was confirmed by mass spectrometry and apoptosis assays. Additional fecal samples from 9 NEC and 5 non-NEC controls were analyzed using similar methods and multi-locus sequence typing (MLST) was performed to investigate clonal relationships and define sequence types of the isolates. NEC_unk_OTU was identified as Klebsiella oxytoca, a pathobiont known to cause antibiotic-associated hemorrhagic colitis, but not previously linked to NEC. Including the index case, cytotoxin-producing strains of K. oxytoca were isolated from 6 of 10 subjects with NEC; in these, the K. oxytoca 16S sequence predominated the fecal microbiota. Cytotoxin-producing strains of K. oxytoca also were isolated from 4 of 5 controls; in these, however, the abundance of the corresponding 16S sequence was very low. MLST analysis of the toxin-positive isolates demonstrated no clonal relationships and similar genetic clustering between cases and controls. These results suggest cytotoxin-producing strains of K. oxytoca colonize a substantial proportion of premature infants. Some, perhaps many, cases of NEC may be precipitated by outgrowth of this opportunistic pathogen.

Porphyromonas gingivalis is a Gram-negative anaerobic periodontal microorganism strongly associated with tissue destructive processes in human periodontitis.  Following oral infection with P. gingivalis, the periodontal bone loss in mice is reported to require engagement of Toll-like receptor 2 (TLR2).   Serine-glycine lipodipeptide or glycine aminolipid classes of P. gingivalis engage human and mouse TLR2 but a novel lipid class reported here is considerably more potent in engaging TLR2 and the heterodimer receptor TLR2/TLR6.  The novel lipid class, termed Lipid 1256,  consists of a diacylated phosphoglycerol moiety linked to a serine-glycine lipodipeptide, previously termed Lipid 654.  Lipid 1256 is approximately 50-fold more potent in engaging TLR2 than the previously reported serine-glycine lipid classes.  Lipid 1256 also stimulates cytokine secretory responses from peripheral blood monocytes and is recovered in selected oral and intestinal Bacteroidetes organisms.  Therefore, these findings suggest that Lipid 1256 may be a microbial TLR2 ligand relevant to chronic periodontitis in humans.

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Furocoumarins are a class of photoactive compounds found in several plant species and may be responsible for the observed association between consumption of citrus products and the risk of skin cancer. Furocoumarin contents of several foods have been reported previously, but no comprehensive database of furocoumarin content of foods is currently available. Therefore, this study aimed to determine the distribution of furocoumarins in popularly consumed foods in the U.S. Samples of three varieties of each of 29 foods known or suspected to contain furocoumarins were purchased, prepared for analysis using a solid phase extraction method, and analyzed using UPLC-MS/MS for the presence of seven major furocoumarins. Most foods measured contained more than one furocoumarin, and some contained all seven of the furocoumarins examined. Total furocoumarin concentration was greatest in fresh parsley (23215 ng/g), grapefruits (21858 ng/g), lime juice (14580 ng/g), grapefruit juice (95341 ng/g), and limes (9151 ng/g). Bergamottin was found in the greatest proportion of foods sampled (23 of 29), followed by bergapten (19 of 29) and 6'7'-dihydroxybergamottin (16 of 29). These measurements will enable more accurate estimation of dietary furocoumarin exposure and will strengthen future epidemiological work investigating the relationships between furocoumarin intake and health outcomes.

To develop a comprehensive analytical method for photoactive furanocoumarins, grapefruit (whole, flesh, peel and juice) was extracted using QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) method. Seven furanocoumarins: bergaptol, psoralen, 8-methoxypsoralen, bergapten, 6',7'-dihydroxybergamottin (6',7'-DHB), epoxybergamottin and bergamottin were determined in grapefruit using UPLC-MS/MS. The concentrations of furanocoumarins in the plasma and urine of six healthy young adults before and after ingestion of grapefruit or grapefruit juice were also determined. Recovery rates of furanocoumarins by QuEChERS method from matrix spike sample and laboratory calibrate sample were 125.7 ± 25.4% and 105.7 ± 6.3%, respectively. Bergamottin and 6',7'-DHB were predominant compounds in grapefruit flesh, juice and plasma, while bergaptol and 6',7'-DHB were major compounds detected in the urine. The results demonstrated that bergamottin and 6',7'-DHB were metabolized to bergaptol. Overall, the analytical methods developed in the present study can be applied to the analysis of various furanocoumarins in plant sources and biological samples.

The serine-glycine dipeptide lipid classes, including lipid 430 and lipid 654, are produced by the periodontal pathogen Porphyromonas gingivalis and can be detected in lipid extracts of diseased periodontal tissues and teeth of humans. Both serine-glycine lipid classes were previously shown to engage human and mouse Toll-like receptor 2 (TLR2) and to inhibit mouse osteoblast differentiation and function through engagement of TLR2. It is not clear if other lipids related to serine-glycine lipids are also produced by P. gingivalis. The goal of this investigation was to determine whether P. gingivalis produces additional lipid classes similar to the serine-glycine lipids that possess biological properties. P. gingivalis (ATCC 33277) was grown in broth culture, and lipids were extracted and fractionated by high-performance liquid chromatography (HPLC). Lipids were separated using semipreparative HPLC, and specific lipid classes were identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and liquid chromatography-multiple reaction monitoring (LC-MRM) mass spectrometric approaches. Two glycine lipid classes were identified, termed lipid 567 and lipid 342, and these lipid classes are structurally related to the serine-glycine dipeptide lipids. Both glycine lipid classes were shown to promote TLR2-dependent tumor necrosis factor alpha (TNF-α) release from bone marrow macrophages, and both were shown to activate human embryonic kidney (HEK) cells through TLR2 and TLR6 but not TLR1. These results demonstrate that P. gingivalis synthesizes glycine lipids and that these lipids engage TLR2 similarly to the previously reported serine-glycine dipeptide lipids.

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