Endocrine disrupting chemicals (EDCs) impact female fertility, and their widespread presence demands deeper understanding of their effects on reproduction. In vitro maturation of bovine cumulus-oocyte complexes (COCs) serves as a relevant reproductive toxicology model aligned with the 3R principle. Given the link between lipid metabolism and COC quality, the possibility that lipid droplets (LDs) in cumulus cells (CCs) and overall COC lipid content are susceptible to EDC disruption was tested. COCs were exposed to diethylstilbestrol (DES; estrogen receptor agonist) and ketoconazole (KTZ; steroidogenesis inhibitor), during in vitro maturation. Neutral lipid dyes were used for LD visualization (count, size, area, and clustering) in CCs. Lipidomic analysis using LC-MS was conducted separately on oocytes and CCs. Of all tested DES concentrations (i.e., 10-9 M, 10-7 M, 10-5 M), 10-5 M DES reduced the total LD area per CC, associated with decreased LD count and increased size. KTZ at all tested concentrations (i.e., 10-8 M, 10-7 M, 10-6 M) reduced LD size and enhanced clustering. Lipidomic analysis revealed the selective depletion of cholesteryl esters in CCs exposed to 10-5 M DES or 10-6 M KTZ, with minimal changes in other neutral lipid classes. Thus EDCs can disrupt lipid storage and LD dynamics in bovine COCs. We discuss these new findings with our previous work: (i) DES-induced steroidogenesis while (ii) in contrast KTZ blocked steroidogenesis in CCs. Together, the data highlight the COC lipid metabolism as a novel EDC target, warranting further study due to potential implications for female fertility.

Viruses actively reprogram the metabolism of the host to ensure the availability of sufficient building blocks for virus replication and spreading. However, relatively little is known about how picornaviruses—a large family of small, non-enveloped positive-strand RNA viruses—modulate cellular metabolism for their own benefit. Here, we studied the modulation of host metabolism by coxsackievirus B3 (CVB3), a member of the enterovirus genus, and encephalomyocarditis virus (EMCV), a member of the cardiovirus genus, using steady-state as well as 13C-glucose tracing metabolomics. We demonstrate that both CVB3 and EMCV increase the levels of pyrimidine and purine metabolites and provide evidence that this increase is mediated through degradation of nucleic acids and nucleotide recycling, rather than upregulation of de novo synthesis. Finally, by integrating our metabolomics data with a previously acquired phosphoproteomics dataset of CVB3-infected cells, we identify alterations in phosphorylation status of key enzymes involved in nucleotide metabolism, providing insight into the regulation of nucleotide metabolism during infection.

Osteoarthritis causes progressive joint deterioration, severe morbidity, and reduced mobility in both humans and horses. Currently, osteoarthritis is diagnosed at late stages through clinical examination and radiographic imaging, hence it is challenging to address and provide timely therapeutic interventions to slow disease progression or ameliorate symptoms. Extracellular vesicles are cell-derived vesicles that play a key role in cell-to-cell communication and are potential sources for specific composite biomarker panel discovery. We here used a multi-omics strategy combining proteomics and phospholipidomics in an integral approach to identify composite biomarkers associated to purified extracellular vesicles from synovial fluid of healthy, mildly and severely osteoarthritic equine joints. Although the number of extracellular vesicles was unaffected by osteoarthritis, proteome profiling of extracellular vesicles by mass spectrometry identified 40 differentially expressed proteins (non-adjusted p < 0.05) in osteoarthritic joints associated with 7 significant canonical pathways in osteoarthritis. Moreover, pathway analysis unveiled changes in disease and molecular functions during osteoarthritis development. Phospholipidome profiling by mass spectrometry showed a relative increase in sphingomyelin and a decrease in phosphatidylcholine, phosphatidylinositol, and phosphatidylserine in extracellular vesicles derived from osteoarthritic joints compared to healthy joints. Unsupervised data integration revealed positive correlations between the proteome and the phospholipidome. Comprehensive analysis showed that some phospholipids and their related proteins increased as the severity of osteoarthritis progressed, while others decreased or remained stable. Altogether our data show interrelationships between synovial fluid extracellular vesicle-associated phospholipids and proteins responding to osteoarthritis pathology and which could be explored as potential composite diagnostic biomarkers of disease.

Obesity represents a significant health challenge, intricately linked to conditions such as type II diabetes, metabolic syndrome, and hepatic steatosis. Several existing obesity treatments exhibit limited efficacy, undesirable side effects or a limited capability to maintain therapeutics effects in the long-term. Recently, modulation Coenzyme Q (CoQ) metabolism has emerged as a promising target for treatment of metabolic syndrome. This potential intervention could involve the modulation of endogenous CoQ biosynthesis by the use of analogs of the precursor of its biosynthesis, such as β-resorcylic acid (β-RA). Here, we show that oral supplementation with β-RA, incorporated into the diet of diet-induced obese (DIO) mice, leads to substantial weight loss. The anti-obesity effects of β-RA are partially elucidated through the normalization of mitochondrial CoQ metabolism in white adipose tissue (WAT). Additionally, we identify an HFN4α/LXR-dependent transcriptomic activation of the hepatic lipid metabolism that contributes to the anti-obesity effects of β-RA. Consequently, β-RA mitigates WAT hypertrophy, prevents hepatic steatosis, counteracts metabolic abnormalities in WAT and liver, and enhances glucose homeostasis by reducing the insulin/glucagon ratio and plasma levels of gastric inhibitory peptide (GIP). Moreover, pharmacokinetic evaluation of β-RA supports its translational potential. Thus, β-RA emerges as an efficient, safe, and translatable therapeutic option for the treatment and/or prevention of obesity, metabolic dysfunction-associated steatotic liver disease (MASLD).

The molecular signature of cell-derived extracellular vesicles (EVs) from synovial fluid (SF) offers insights into the cells and molecular processes associated with joint disorders and can be exploited to define biomarkers. The EV-signature is determined by cargo molecules and the lesser-studied lipid bilayer. We here investigated the lipidome of SF-EVs in inflamed joints derived from Rheumatoid Arthritis (RA) and Spondyloarthritis (SpA) patients, two autoimmune-driven joint diseases, and compared these signatures to the lipid profile of equine SF-EVs obtained during induced acute synovitis. Since neutrophils are primary SF-infiltrating cells during these inflammatory joint diseases, we also analyzed how inflammatory stimuli alter the lipidomic profile of human and equine neutrophil-derived EVs (nEVs) in vitro and how these signatures relate to the lipidome signatures of SF-EVs from inflamed joints. We identified neutrophil stimulation intensity-dependent changes in the lipidomic profile of nEVs with elevated presence of dihexosylceramide (lactosylceramide), phosphatidylserine, and phosphatidylethanolamine ether-linked lipid classes in human nEVs upon full neutrophil activation. In horses, levels of monohexosylceramide (glucosylceramide) increased instead of dihexosylceramide, indicating species-specific differences. The lipid profiles of RA and SpA SF-EVs were relatively similar and showed a relative resemblance with stimulated human nEVs. Similarly, the lipidome of equine synovitis-derived SF-EVs closer resembled the one of stimulated equine nEVs. Hence, lipidome profiling can provide insights into the contribution of nEVs to the heterogeneous pool of SF-EVs, deepening our understanding of inflammatory joint diseases and revealing molecular changes in joint homeostasis, which can lead to the development of more precise disease diagnosis and treatment strategies.

Elevated concentrations of palmitate in serum of obese individuals can impair endothelial function, contributing to development of cardiovascular disease. Although several molecular mechanisms of palmitate-induced endothelial dysfunction have been proposed, there is no consensus on what signaling event is the initial trigger of detrimental palmitate effects. Here we report that inhibitors of ER stress or ceramid synthesis can rescue palmitate-induced autophagy impairment in macro- and microvascular endothelial cells. Furthermore, palmitate-induced cholesterol synthesis was reverted using these inhibitors. Similar to cell culture data, autophagy markers were increased in serum of obese individuals. Subsequent lipidomic analysis revealed that palmitate changed the composition of membrane phospholipids in endothelial cells and that these effects were not reverted upon application of above-mentioned inhibitors. However, ER stress inhibition in palmitate-treated cells enhanced the synthesis of trilglycerides and restored ceramide levels to control condition. Our results suggest that palmitate induces ER-stress presumably by shift in membrane architecture, leading to impaired synthesis of triglycerides and enhanced production of ceramides and cholesterol, which altogether enhances lipotoxicity of palmitate in endothelial cells.

Lecithin:retinol acyltransferase (LRAT) is the main enzyme producing retinyl esters (REs) in quiescent hepatic stellate cells (HSCs). When cultured on stiff plastic culture plates, quiescent HSCs activate and lose their RE stores in a process similar to that in the liver following tissue damage, leading to fibrosis. Here we validated HSC cultures in soft gels to study RE metabolism in stable quiescent HSCs and investigated RE synthesis and breakdown in activating HSCs.HSCs cultured in a soft gel maintained characteristics of quiescent HSCs, including the size, amount and composition of their characteristic large lipid droplets. Quiescent gel-cultured HSCs maintained high expression levels of Lrat and a RE storing phenotype with low levels of RE breakdown. Newly formed REs are highly enriched in retinyl palmitate (RP), similar to freshly isolated quiescent HSCs, which is associated with high LRAT activity. Comparison of these quiescent gel-cultured HSCs with activated plastic-cultured HSCs showed that although during early activation the total RE levels and RP-enrichment are reduced, levels of RE formation are maintained and mediated by LRAT. Loss of REs was caused by enhanced RE breakdown in activating HSCs. Upon prolonged culturing, activated HSCs have lost their LRAT activity and produce small amounts of REs by DGAT1. This study reveals unexpected dynamics in RE metabolism during early HSC activation, which might be important in liver disease as early stages are reversible. Soft gel cultures provide a promising model to study RE metabolism in quiescent HSCs, allowing detailed molecular investigations on the mechanisms for storage and release.

Arthritis, a diverse group of inflammatory joint disorders, poses great challenges in early diagnosis and targeted treatment. Timely intervention is imperative, yet conventional diagnostic methods are not able to detect subtle early symptoms. Hence, there is an urgent need for specific biomarkers that discriminate between different arthritis forms and for early diagnosis. The pursuit of such precise diagnostic tools has prompted a growing interest in extracellular vesicles (EVs). EVs, released by cells in a regulated fashion, are detectable in body fluids, including synovial fluid (SF), which fills the joint space. They provide insights into the intricate molecular landscapes of arthritis, and this has stimulated the search for minimally invasive EV-based diagnostics. As such, the analysis of EVs in SF has become a focus for identifying EV-based biomarkers for joint disease endotyping, prognosis, and progression. EVs are composed of a lipid bilayer and a wide variety of different cargo types, of which proteins and RNAs are widely investigated. In contrast, membrane lipids of EVs, especially the abundance, presence, or absence of specific lipids and their contribution to the biological activity of EVs, are largely overlooked in EV research. Furthermore, the identification of specific combinations of different EV components acting in concert in EVs can fuel the definition of composite biomarkers. We here provide a state-of-the-art overview of the knowledge on SF-derived EVs with emphasis on lipid analysis and we give an example of the added value of integrated proteomics and lipidomics analysis in the search for composite EV-associated biomarkers.

Inflammation is the hallmark of most joint disorders. However, the precise regulation of induction, perpetuation, and resolution of joint inflammation is not entirely understood. Since extracellular vesicles (EVs) are critical for intercellular communication, we aim to unveil their role in these processes. Here, we investigated the EVs' dynamics and phospholipidome profile from synovial fluid (SF) of healthy equine joints and from horses with lipopolysaccharide (LPS)-induced synovitis.LPS injection triggered a sharp increase of SF-EVs at 5-8 h post-injection, which started to decline at 24 h postinjection. Importantly, we identified significant changes in the lipid profile of SF-EVs after synovitis induction. Compared to healthy joint-derived SF-EVs (0 h), SF-EVs collected at 5, 24, and 48 h post-LPS injection were strongly increased in hexosylceramides. At the same time, phosphatidylserine, phosphatidylcholine, and sphingomyelin were decreased in SF-EVs at 5 h and 24 h post-LPS injection. Based on the lipid changes during acute inflammation, we composed specific lipid profiles associated with healthy and inflammatory state-derived SF-EVs. The sharp increase in SF-EVs during acute synovitis and the correlation of specific lipids with either healthy or inflamed states-derived SF-EVs are findings of potential interest for unveiling the role of SF-EVs in joint inflammation, as well as for the identification of EV-biomarkers of joint inflammation.

Lysophospholipids (LPLs) are crucial for regulating epithelial integrity and homeostasis in eukaryotes, however the effects of LPLs produced by bacteria on host cells is largely unknown. The membrane of the human bacterial pathogen Campylobacter jejuni is rich in LPLs. Although C. jejuni possesses several virulence factors, it lacks traditional virulence factors like type III secretion systems, present in most enteropathogens. Here, we provide evidence that membrane lipids lysophosphatidylethanolamines (lysoPEs) of C. jejuni are able to lyse erythrocytes and are toxic for HeLa and Caco-2 cells. Lactate dehydrogenase (LDH) release assays and confocal microscopy revealed that lysoPE permeabilizes the cells. LysoPE toxicity was partially rescued by oxidative stress inhibitors, indicating that intracellular reactive oxygen species may contribute to the cell damage. Our results show that especially the short-chain lysoPEs (C:14) which is abundantly present in the C. jejuni membrane may be considered as a novel virulence factor.

Purpose: One of the most common regenerative surgical treatments for osteochondral defects is osteochondral autologous transplantation (mosaicplasty). Osteochondral plugs are transferred from non-weightbearing areas to the sites to be treated, commonly weightbearing areas. In these procedures the topographical differences in biomechanical and biochemical characteristics of articular cartilage within the same joint are not taken into account. Nevertheless, matching of structural and mechanical characteristics between the harvest and the recipient site can be assumed to influence long-term outcomes in the repair of osteochondral defects.

Gross morphology of healthy and degenerated intervertebral discs (IVDs) is largely similar in horses as in dogs and humans. For further comparison, the biochemical composition and the histological and biochemical changes with age and degeneration were analyzed in 41 warmblood horses. From 33 horses, 139 discs and 2 fetal vertebral columns were evaluated and scored histologically. From 13 horses, 73 IVDs were assessed for hydration, DNA, glycosaminoglycans, total collagen, hydroxyl-lysyl-pyridinoline, hydroxylysine, and advanced glycation end-product (AGE) content. From 7 horses, 20 discs were assessed for aggrecan, fibronectin, and collagen type 1 and 2 content. Histologically, tearing of the nucleus pulposus (NP) and cervical annulus fibrosus (AF), and total histological score (tearing and vascular proliferation of the AF, and chondroid metaplasia, chondrocyte-like cell proliferation, presence of notochordal cells, matrix staining, and tearing of the NP) correlated with gross degeneration. Notochordal cells were not seen in IVDs of horses. Age and gross degeneration were positively correlated with AGEs and a fibrotic phenotype, explaining gross degenerative changes. In contrast to dogs and humans, there was no consistent difference in glycosaminoglycan content and hydration between AF and NP, nor decrease of these variables with age or degeneration. Hydroxylysine decrease and collagen 1 and AGEs increase were most prominent in the NP, suggesting degeneration started in the AP. In caudal cervical NPs, AGE deposition was significantly increased in grossly normal IVDs and total collagen significantly increased with age, suggesting increased biomechanical stress and likelihood for spinal disease in this part of the vertebral column.

The tumour suppressor PTEN is a negative regulator of the PI3K/AKT signalling pathway. Liver-specific deletion of Pten in mice results in the hyper-activation PI3K/AKT signalling accompanied by enhanced genome duplication (polyploidization), marked lipid accumulation (steatosis) and formation of hepatocellular carcinomas. However, it is unknown whether polyploidization in this model has an impact on the development of steatosis and the progression towards liver cancer. Here, we used a liver-specific conditional knockout approach to delete Pten in combination with deletion of E2f7/8, known key inducers of polyploidization. As expected, Pten deletion caused severe steatosis and liver tumours accompanied by enhanced polyploidization. Additional deletion of E2f7/8 inhibited polyploidization, alleviated Pten-induced steatosis without affecting lipid species composition and accelerated liver tumour progression. Global transcriptomic analysis showed that inhibition of polyploidization in Pten-deficient livers resulted in reduced expression of genes involved in energy metabolism, including PPAR-gamma signalling. However, we find no evidence that deregulated genes in Pten-deficient livers are direct transcriptional targets of E2F7/8, supporting that reduction in steatosis and progression towards liver cancer are likely consequences of inhibiting polyploidization. Lastly, flow cytometry and image analysis on isolated primary wildtype mouse hepatocytes provided further support that polyploid cells can accumulate more lipid droplets than diploid hepatocytes. Collectively, we show that polyploidization promotes steatosis and function as an important barrier against liver tumour progression in Pten-deficient livers.

Animal models continue to be used to investigate cartilage repair strategies. Adequate anaesthesia and pain management are essential in order to guarantee acceptable animal welfare as well as reproducible experimental results. This systematic review evaluates reporting of anaesthesia and pain management in surgical large animal models (horse, pig, dog, goat and sheep) of (osteo)chondral repair. Manuscripts published between 2015 and 2020 were included after a comprehensive search strategy. Data were evaluated using descriptive statistics and qualitative review. Out of 223 eligible studies, 220 studies contained incomplete information on anaesthetic and pain management. Pre-, intra- and post-operative analgesia were not mentioned in 68%, 94%, and 64% of manuscripts respectively. A total of 176 studies reported that animals underwent general anaesthesia during surgery. Surprisingly, 30% of these articles did not provide any detail on anaesthetic management, while 37% reported using inhalant, hypnotic or sedative drugs only, without mention of analgesics. Pain monitoring was not reported in 87% of manuscripts. The vast majority of preclinical large animal studies on cartilage repair did not meet veterinary clinical standards for anaesthesia and analgesia, and failed to report according to the ARRIVE international guidelines. In light of serious welfare, ethical and translational validity concerns, improvement is urgently needed.

There is an increasing interest in controlled release systems for local therapy in the treatment of human and equine joint diseases, aiming for optimal intra-articular concentrations with no systemic side effects. In this study, the intra-articular tolerability and suitability for local and sustained release of tacrolimus (FK506) from monospheres composed of [PDLA-PEG1000]-b-PLLA multiblock copolymers were investigated. Unloaded and tacrolimus-loaded (18.4 mg tacrolimus/joint) monospheres were injected into the joints of six healthy horses, with saline and hyaluronic acid (HA) in the contralateral joints as controls. Blood and synovial fluid were analysed for the tacrolimus concentration and biomarkers for inflammation and cartilage metabolism. After an initial burst release, sustained intra-articular tacrolimus concentrations (>20 ng/mL) were observed during the 42 days follow-up. Whole-blood tacrolimus levels were below the detectable level (<0.5 ng/mL). A transient inflammatory reaction was observed for all substances, evidenced by increases of the synovial fluid white blood cell count and total protein. Prostaglandin and glycosaminoglycan release were increased in joints injected with unloaded monospheres, which was mitigated by tacrolimus. Both tacrolimus-loaded monospheres and HA transiently increased the concentration of collagen II cleavage products (C2C). A histologic evaluation of the joints at the endpoint showed no pathological changes in any of the conditions. Together, these results indicate the good biocompatibility of intra-articular applied tacrolimus-loaded monospheres combined with prolonged local drug release while minimising the risk of systemic side effects. Further evaluation in a clinical setting is needed to determine if tacrolimus-loaded monospheres can be beneficial in the treatment of inflammatory joint diseases in humans and animals.

Classical in vitro fertilization (IVF) is still poorly successful in horses. This lack of success is thought to be due primarily to inadequate capacitation of stallion spermatozoa under in vitro conditions. In species in which IVF is successful, bicarbonate, calcium, and albumin are considered the key components that enable a gradual reorganization of the sperm plasma membrane that allows the spermatozoa to undergo an acrosome reaction and fertilize the oocyte. The aim of this work was to comprehensively examine contributors to stallion sperm capacitation by investigating bicarbonate-induced membrane remodelling steps, and elucidating the contribution of cAMP signalling to these events. In the presence of capacitating media containing bicarbonate, a significant increase in plasma membrane fluidity was readily detected using merocyanine 540 staining in the majority of viable spermatozoa within 15 min of bicarbonate exposure. Specific inhibition of soluble adenylyl cyclase (sAC) in the presence of bicarbonate by LRE1 significantly reduced the number of viable sperm with high membrane fluidity. This suggests a vital role for sAC-mediated cAMP production in the regulation of membrane fluidity. Cryo-electron tomography of viable cells with high membrane fluidity revealed a range of membrane remodelling intermediates, including destabilized membranes and zones with close apposition of the plasma membrane and the outer acrosomal membrane. However, lipidomic analysis of equivalent viable spermatozoa with high membrane fluidity demonstrated that this phenomenon was neither accompanied by a gross change in the phospholipid composition of stallion sperm membranes nor detectable sterol efflux (p > 0.05). After an early increase in membrane fluidity, a significant and cAMP-dependent increase in viable sperm with phosphatidylserine (PS), but not phosphatidylethanolamine (PE) exposure was noted. While the events observed partly resemble findings from the in vitro capacitation of sperm from other mammalian species, the lack of cholesterol removal appears to be an equine-specific phenomenon. This research will assist in the development of a defined medium for the capacitation of stallion sperm and will facilitate progress toward a functional IVF protocol for horse gametes.

Host defense peptides (HDPs), such as cathelicidins, are small, cationic, amphipathic peptides and represent an important part of the innate immune system. Most cathelicidins, including the porcine PMAP-36, are membrane active and disrupt the bacterial membrane. For example, a chicken cathelicidin, CATH-2, has been previously shown to disrupt both Escherichia coli membranes and to release, at sub-lethal concentrations, outer membrane vesicles (OMVs). Since OMVs are considered promising vaccine candidates, we sought to investigate the effect of sub-bactericidal concentrations of PMAP-36 on both OMV release by a porcine strain of Bordetella bronchiseptica and on the modulation of immune responses to OMVs. PMAP-36 treatment of bacteria resulted in a slight increase in OMV release. The characteristics of PMAP-36-induced OMVs were compared with those of spontaneously released OMVs and OMVs induced by heat treatment. The stability of both PMAP-36- and heat-induced OMVs was decreased compared to spontaneous OMVs, as shown by dynamic light scattering. Furthermore, treatment of bacteria with PMAP-36 or heat resulted in an increase in negatively charged phospholipids in the resulting OMVs. A large increase in lysophospholipid content was observed in heat-induced OMVs, which was at least partially due to the activity of the outer-membrane phospholipase A (OMPLA). Although PMAP-36 was detected in OMVs isolated from PMAP-36-treated bacteria, the immune response of porcine bone-marrow-derived macrophages to these OMVs was similar as those against spontaneous or heat-induced OMVs. Therefore, the effect of PMAP-36 addition after OMV isolation was investigated. This did decrease cytokine expression of OMV-stimulated macrophages. These results indicate that PMAP-36 is a promising molecule to attenuate undesirable immune responses, for instance in vaccines.

Host defense peptides (HDPs) are part of the innate immune system and constitute a first line of defense against invading pathogens. They possess antimicrobial activity against a broad spectrum of pathogens. However, pathogens have been known to adapt to hostile environments. Therefore, the bacterial response to treatment with HDPs was investigated. Previous observations suggested that sublethal concentrations of HDPs increase the release of outer membrane vesicles (OMVs) in Escherichia coli. First, the effects of sublethal treatment with HDPs CATH-2, PMAP36, and LL-37 on OMV release of several Gram-negative bacteria were analyzed. Treatment with PMAP-36 and CATH-2 induced release of OMVs, but treatment with LL-37 did not. The OMVs were further characterized with respect to morphological properties. The HDP-induced OMVs often had disc-like shapes. The beneficial effect of bacterial OMV release was studied by determining the susceptibility of E. coli toward HDPs in the presence of OMVs. The minimal bactericidal concentration was increased in the presence of OMVs. It is concluded that OMV release is a means of bacteria to dispose of HDP-affected membrane. Furthermore, OMVs act as a decoy for HDPs and thereby protect the bacterium. IMPORTANCE Antibiotic resistance is a pressing problem and estimated to be a leading cause of mortality by 2050. Antimicrobial peptides, also known as host defense peptides (HDPs), and HDP-derived antimicrobials have potent antimicrobial activity and high potential as alternatives to antibiotics due to low resistance development. Some resistance mechanisms have developed in bacteria, and complete understanding of bacterial defense against HDPs will aid their use in the clinic. This study provides insight into outer membrane vesicles (OMVs) as potential defense mechanisms against HDPs, which will allow anticipation of unforeseen resistance to HDPs in clinical use and possibly prevention of bacterial resistance by the means of OMVs.

In response to changes in their environment bacteria need to change both their protein and phospholipid repertoire to match environmental requirements, but the dynamics of bacterial phospholipid composition under different growth conditions is still largely unknown. In the present study, we investigated the phospholipidome of the bacterial pathogen Campylobacter jejuni. Transcription profiling on logarithmic and stationary phase grown cells of the microaerophilic human pathogen C. jejuni using RNA-seq revealed differential expression of putative phospholipid biosynthesis genes. By applying high-performance liquid chromatography tandem-mass spectrometry, we identified 203 phospholipid species representing the first determination of the phospholipidome of this pathogen. We identified nine different phospholipid classes carrying between one and three acyl chains. Phospholipidome analysis on bacteria of different ages (0-5 days) showed rapid changes in the ratio of phospholipids containing ethanolamine, or glycerol as phospholipid head group and in the number of cyclopropane bond containing fatty acids. Oxygen concentration influenced the percentage of lysophospholipids, and cyclo-propane bonds containing acyl chains. We show that large amounts of the phospholipids are lysophospholipids (30-45%), which mutant studies reveal are needed for normal C. jejuni motility at low oxygen conditions. C. jejuni possesses an unusual phospholipidome that is highly dynamic in response to environmental changes.