Genomics and Functional Genetics of the Zoonotic Pathogen Helicobacter Bizzozeronii

Helicobacter spp. are common causes of bacterial infection worldwide in both humans and animals.The canine Gram-negative Helicobacter bizzozeronii is one out of seven species in the group of H. heilmannii sensu lato (s. l.) also known as non-H. pylori gastric Helicobacter species (NPGHS) that are detected in 0.2-6% in gastric biopsies of human patients with gastritis. At present, H. bizzozeronii and H. felis are the only H. heilmannii s. l. species that have been successfully cultivated from human gastric biopsies. In contrast to human-to-human transmission described for H. pylori, the H. heilmannii sensu lato (s. l). are transmitted from animals to humans (zoonotic). To date, an understanding of the pathogenesis of non-pylori Helicobacter infections in humans and also of the mechanisms of their adaptation are lacking. In order to provide new insights into these topics, the present study focused on the molecular biology of H. bizzozeronii with particular interest in unravelling the bacterial features involved in host-jumping and adaptation to the human gastric mucosa. The study also investigated the molecular mechanisms of metronidazole resistance in H. bizzozeronii. Using comparative genome analysis between H. pylori and a human isolate of H. bizzozeronii CIII-1, we investigated several molecular genetic features that could explain the zoonotic nature of NPGHS. In particular, we propose the hypothesis that the high metabolic versatility and the ability to react to a range of environmental signals, factors which differentiate H. bizzozeronii and other species belonging to H. heilmannii s. l. from H. pylori, are the molecular basis of the of the zoonotic nature of H. heilmannii s. l. infection in humans. Further we characterized the biosynthesis of lipopolysaccharide (LPS) of H. bizzozeronii in order to understand the factors involved in host adaptation during host jump from dogs to humans. As a major Gram-negative cell surface component, LPS has the potential to interact with certain surface components of the host s cells during infection, which modifies the inflammatory response and promotes progress of chronic infection. Our results showed that H. bizzozeronii expresses sialyl-LPS (containing the sialyl-lactoseamine epitope), which is a common characteristic among canine and human H. bizzozeronii strains. The expression of sialyl-glycans undergoes phase- and antigen variation and may influence the adaptation process of H. bizzozeronii during the transmission from dogs to humans. Metronidazole, in combination with other antibiotics, is frequently used for the eradication of H. pylori and H. heilmannii s. l. infections but resistance to this antibiotic is a serious, increasing problem and a major cause of treatment failure. In this study, we performed comparative genomic analysis between antrum derived H. bizzozeronii populations that had been isolated from biopsy samples from a female Finnish patient who was suffering from chronic gastritis. The biopsies were taken three months before (T0) and six months after (T1) an unsuccessful eradication treatment. Our results that H. bizzozeronii had undergone genome diversification in order to colonize the patient s stomach successfully. We hypothesised that antimicrobial treatment induced a sudden decrease of H. bizzozeronii population size but effectively favoured the selection of a subpopulation, which subsequently acted as the founder of a new population that was characterized by higher numbers of fixed mutations. H. bizzozeronii isolates obtained after the treatment underwent a frame length extension of the oxygen-insensitive NAD(P)H-nitroreductase HBZC1_00960 (homologue of H. pylori RdxA) that is associated with the disruption of the C-terminal cysteine-containing conserved region (IACLXALGK). This disruption was the result of the extension (from C8 to C9) of a simple sequence cytosine repeat (SSCR) (homopolymeric tract) that is located in the 3 region of the rdxA gene. Further we investigated the role of rdxA gene in the molecular mechanisms of metronidazole in H. bizzozeronii. Our results showed that, in most of the in vitro spontaneous H. bizzozeronii metronidazole resistant mutants, the only observed mutation was that of the extension of the 3 SSCR of rdxA. Another major observation was that the MIC value of metronidazole for both spontaneous mutant isolates and an H. bizzozeronii ΔrdxA mutant strain were similar, which indicates that a loss of rdxA function is associated with the reduced susceptibility of H. bizzozeronii to metronidazole. Another key finding was that H. bizzozeronii acquires resistance to metronidazole at high mutation rates and that serial passages in vitro without selection induce reversibility of the resistant phenotype. These results suggest that the potential contingency nature of rdxA should be carefully considered if metronidazole is administered for the treatment of H. heilmannii-associated gastritis. The results of the present studies contribute substantially in the understanding of the zoonotic nature of non-H. pylori gastric Helicobacter species and of the molecular mechanisms behind successful colonization and acquired antimicrobial resistance. Further studies are required in order to elucidate the specific roles of characterized factors, such as polysaccharide lyase and Sialyl-LPS, in the development of gastritis and MALT lymphoma.Helicobakteerit (Helicobacter spp.) ovat mahasuolikanavassa kolonisoiva bakteerisuku, jossa on useita patogeenisia lajeja, jotka aiheuttavat sekä ihmisten että eläinten infektioita. H. bizzozeronii on koiran mahalaukun helikobakteerilaji (kuluu ns. mahalaukussa kolonisoituvaan non-H.pylori helikobakteeriryhmään; H. heilmannii sensu lato; s.l.). Tätä lajia eristetään myös 0.2-6% eriasteista gastriittia sairastavien ihmisten mahalaukun biopsianäytteistä. H. bizzozeronii ja toinen eläinten helikobakteerilaji H. felis ovat non-H.pylori lajeja, joita on myös onnistuttu eristämään viljelemällä potilaiden biopsianäytteistä. Näitä eläinten mahalaukun helikobakteereita voidaankin pitää zoonoottisina taudinaiheuttajina, jotka voivat tarttua koirista ihmisiin. Sen sijaan H. pylori tarttuu ainoastaan ihmisestä ihmiseen. Väitöskirjatutkimuksessa selvitettiin H. bizzoreroniin patogeenisuuteen liittyviä geneettisiä ominaisuuksia ja bakteerin adaptoitumista ihmispotilaaseen. Tavoitteena oli saada yleisemminkin tietoa ominaisuuksista, joita bakteeri tarvitsee, jotta se pystyy siirtymään eri lajien välillä (koira-ihminen). Menetelminä käytettiin laajasti molekyylibiologisia menetelmiä; sekä bakteerin genomitason analytiikkaa että bakteerin yksittäisten ominaisuuksien selvittämistä. Genomitutkimuksen avulla verrattiin ihmispotilaasta eristetyn H. bizzozeronii kannan (III) eroja ja samanlaisuutta H. pylorin kanssa sekä myös muihin eläinten mahalaukussa kolonisoituviin helikobakteerilajeihin, joista koko genomitason tietoa oli saatavilla. Analyysissä havaittiin, että, vaikka H.bizzozeroniin (Hb) ja H. pylorin genomit ovat samankokoisia, H.blla on geneettisesti paljon laajempi metabolinen kapasiteetti ja muita ominaisuuksia, joiden avulla se voi vastata erilaisiin ympäristön vasteisiin kuin Hplla. Bakteerisolun pinnan lipopolysakkaridilla (LPS) on merkitystä isännän ja bakteerin vuorovaikutuksessa, erityisesti kolonisoitumisen alkuvaiheessa, jossa muovaamalla inflammaatiovastetta edistetään kolonisoitumista ja kroonisen infektioprosessin etenemistä. Tulokset osoittivat että H. bizzozeroniin LPSn rakenne on sialyloitunut (sialyylilaktoosiamiiniepitooppi), joka oli tyypillinen myös muille eläinten mahalaukun helikobakteereille, mutta se puuttuu H. pylorista. Sialyyliglykaanien ilmenemistä infektion eri vaiheissa Hb säätelee geenitasolla faasivaihtelun avulla (ON/OFF). Täten LPSn rakenteen säätelyllä voi olla merkitystä infektion zoonoottisuuden kannalta. Kolmannessa tutkimuksessa verrattiin erään gastriittipotilaan biopsianäytteistä eri aikoina eristettyjen H. bizzozeronii populaatioiden genomitason diversiteettiä ja erityisesti sitä, miten epäonnistunut antibioottiterapia vaikutti populaatioihin. Tutkimukset osoittivat, että potilaan kanta oli metronidatsoliresistentti. Metronidatsoli kuului yhtenä antibioottina käytettyyn terapiaan. Biopsianäytteitä oli käytettävissä kolme kuukautta ennen terapiaa (T0) sekä kuusi kuukautta terapian jälkeen (T1). Tulokset osoittivat, että H. bizzozeroniin genomissa oli enemmän diversiteettiä näytteissä, jotka oli otettu ennen terapiaa, kuin näytteissä terapian jälkeen. Krooninen kolonisoituminen ja adaptaatio edellyttävät genomilta sopeutumista, johon bakteeri vastaa lisäämällä genomin diversiteettiä. Terapia todennäköisesti aiheutti äkillisen populaatiokoon vähenemisen ja samalla valikoitumisen, jonka seurauksena tietty subpopulaatio pääsi lisääntymään ja tuloksena saatiin bakteeripopulaatio, jossa oli vähemmän diversiteettiä kuin ennen terapiaa, ja jossa tietyt mutaatiot olivat muuttuneet vallitseviksi. Neljännessä työssä selvitettiin H. bizzozeroniin metronidatsoliresistenssin syntymekanismia. Terapian jälkeen eristetyissä isolaateissa havaittiin mutaatio, joka johti NAD(P)H- nitroreduktaasigeenin (HBZC1_00960; homologinen H. pylorin RdxAn kanssa) lukuraamin muutokseen geenin konservoituneessa C-terminaaliosassa (IACLXALGK). Lukuraamin muutos aiheutui sytosiinin (C) toistojakson (homopolymeerinen jakso) muuttumisesta (C8 -> C9). Tämän homopolymeerisen jakson avulla H. bizzozeronii säätelee RdxAn ilmenemistä. Tutkimuksessa selvitettiin edelleen, miten tämä vaihtelu vaikutti bakteerin metronidatsoliresistenssiin. Havaittiin, että isolaattien metronidatsoli MIC oli samalla tavalla korkea sekä spontaaneissa mutanteissa että in vitro ΔrdxA mutantissa, joka osoitti että rdxA geeni liittyy metronidatsoliresistenssiin. Edelleen tulokset osoittivat, että metronidatsoliresistenssin hankkimisen mutaatiofrekvenssi oli korkea, sekä, että isolaattien siirrostaminen in vitro ilman selektiopainetta, edisti resistentin fenotyypin korvautumista herkällä fenotyypillä. Tämä tulokset osoittavat, että metronidatsoli ei sovi H. heilmannii s.l. ryhmän bakteerien aiheuttamien infektioden hoitoon. Yhteenvetona voidaan todeta, että väitöskirjatyön tutkimukset tuovat merkittävästi uutta tietoa eläinten mahalaukun helikobakteerien (non-H.pylori gastric Helicobacter spp.) geneettisistä taudinaiheuttamisominaisuuksista, jotka liittyvät ihmisten gastriiteihin. Tästä aiheesta on hyvin vähän aikaisempia tutkimuksia. Tutkimuksessa saatiin myös uutta tietoa genomitason mekanismeista, jotka auttavat ymmärtämään toisaalta mahalukussa kolonisoivien helikobakteerien isäntäspesifisyyttä ja toisaalta eräisiin lajeihin liittyvää zoonottisuutta. Myös metronidatsoliresistenssin liittyminen rdxA geenin sytosiinin toistojakson pituuden vaihteluun on uusi mekanismi, joka laajentaa käsitystä resistenssimekanismien variaatiosta saman suvun eri lajien välillä. Jatkotutkimukset selvittävät, mikä merkitys väitöskirjatyössä kuvatuilla tekijöillä, kuten polysakkaridilyaasilla ja sialyloityneella LPSllä, on gastriitin ja MALTin lymfooman patogeneesissä

AFG3L2 deficiency impairs axonal transport of mitochondria dependent on ROS and tau levels

The m-AAA protease, present in the inner mitochondrial membrane facing the mitochondrial matrix, degrades misfolded polypeptides and processes substrates. AFG3L2 is a subunit of m-AAA protease. In humans, heterozygous missense mutations in AFG3L2 lead to Spinocerebellar Ataxia type 28 (SCA28) whereas homozygous mutations in AFG3L2 cause a severe recessive form of spastic-ataxia with early-onset and rapid progression (SPAX5). While depletion of AFG3L2 causes mitochondrial fragmentation in non-polarised cells, the mechanisms of neurodegeneration associated with mitochondrial dynamics and trafficking were not studied in AFG3L2 deficient neurons. We showed that depletion of AFG3L2 in murine primary cortical neurons leads to a selective defect of anterograde transport of mitochondria. The impaired anterograde transport defect was also observed upon concomitant depletion of AFG3L2 and OMA1 demonstrating that OMA1-mediated degradation of OPA1 (to inhibit mitochondrial fusion) was not the reason for mitochondrial transport defects. Anterograde transport defect of mitochondria in AFG3L2 depleted neurons could be rescued by antioxidants, N-acetyl cysteine (NAC) and vitamin E. Interestingly, we also observed a partial rescue in mitochondrial transport by depleting tau, a microtubule-associated protein. Hence, we conclude that neurons employ ROS to couple cytoskeletal modifications and mitochondrial transport

Efficacy and safety of SilverNovaTM skin cream in post-aesthetic skin procedures

Background: The objective of this study was to evaluate the efficacy and safety of SilverNovaTM skin cream composed of SilverSol® (a patented colloidal nano silver technology from American Biotech Labs, USA) with other skin rejuvenators (coconut oil, vitamin E, and hyaluronic acid), in post-aesthetic skin procedure subjects. Methods: In this prospective, interventional, open-label, multicentric study, subjects who undergone post aesthetic procedures were enrolled to receive the application of SilverNovaTM skin cream twice daily. The subjects received the topical application for seven days, after which they were followed up for the next seven days.  Endpoints were assessed at baseline and the end of the treatment (EOT). Primary endpoints were the investigator-assessed erythema, edema, dryness/scaling score, and the subject-assessed product tolerability in terms of burning, stinging, itching, and dryness/tightness. Results: A total of 60 subjects completed the study. There was a significant improvement in erythema, edema, and dryness/scaling score (p<0.0001). There was also significant improvement in burning, itching, and dryness/tightness scores. The physician global improvement assessment scale score was reduced from 3.3 to 1.38 (p<0.0001), showing the resolution of the symptom severity post-treatment with SilverNovaTM skin cream. Colorimetric characterization showed significant improvement in values at the end of the study, reflecting the improvement in skin color and erythema reduction. No adverse events were reported during the study. Conclusions: The application of SilverNovaTM cream in the immediate post procedure period significantly reduced the downtime of the treatment and improved patient acceptance of the treatment. This indicates that SilverNovaTM skin cream is a promising therapeutic option for managing the complications associated with aesthetic skin procedures without any safety concerns

Comparative genomics of Helicobacter pylori and the human-derived Helicobacter bizzozeronii CIII-1 strain reveal the molecular basis of the zoonotic nature of non-pylori gastric Helicobacter infections in humans

Peer reviewe

Microevolution of a zoonotic Helicobacter population colonizing the stomach of a human host before and after failed treatment

To investigate the microevolution of Helicobacter bizzozeronii in the human stomach, comparative genomics of antrum-derived populations, obtained 3 months before (T0) and 6 months after (T1) an unsuccessful eradication treatment, was performed. For each time point, the DNA of bacterial mass, representing the population diversity in three biopsies, was mixed in equal amounts and sequenced using Illumina technology. Polymorphic sites (PSs) were detected by mapping the reads against an isogenic reference genome, derived from a corpus isolate obtained at T0. The total numbers of PSs detected in the H. bizzozeronii population at T0 and T1 were 128 and 223, affecting 81 and 134 coding sequences, respectively. At T0 in 91.4% of the PSs the mutation appeared at a frequency of 50% or less. On the contrary, in the majority of the PSs observed in T1 (71.3%) the mutation had a frequency >75%. Although only a minority of mutations were fixed in the antrum-derived population at T0, a certain level of allelic variability, compared with the corpus-derived reference genome, was present and most likely arose as consequence of the long-term colonization of the patient. The treatment probably induced a sudden decrease of population size, selecting a subpopulation, which acted as founder for the new population at T1 characterized by a higher number of fixed mutations. These data demonstrate that genome plasticity is an important common prerequisite among gastric Helicobacter species for adaptation to the stomach environment allowing the bacterium to evolve rapidly once a selective pressure is applied.Peer reviewe

Sialylation of Helicobacter bizzozeronii lipopolysaccharides modulates Toll-like receptor (TLR) 2 mediated response

Abstract Sialic acid in lipopolysaccharides (LPS) of mucosal pathogens is known to be an important virulence factor. Few strains of Helicobacter pylori express sialyl-Lewis-X and we have reported that human and canine Helicobacter bizzozeronii strains express sialyl-lactoseamine in their LPS. However, the role of sialyation of Helicobacter LPS in the interaction with the host cells is still unknown. In this study H. bizzozeronii LPS is shown to activate the TLR2 in a dose and strain dependent manner in the in vitro HEK-293 cells model expressing TLR2, but not the cells expressing TLR4. These results indicate that TLR2 is the specific receptor for H. bizzozzeronii LPS, as previously described for H. pylori. To further explore the role of sialylation of H. bizzozeronii LPS on TLR2 response, H. bizzozeronii Δhbs2 mutant strains deficient in sialyltransferase activity were constructed by homologous recombination. LPS from H. bizzozeronii Δhbs2 strains enhanced the NF-ĸB induction via TLR2 compared to the respective wild types, leading to the conclusion that the sialylation of H. bizzozeronii LPS in wild-type strains may modulate host immune response

The mycotoxin phomoxanthone A disturbs the form and function of the inner mitochondrial membrane.

Mitochondria are cellular organelles with crucial functions in the generation and distribution of ATP, the buffering of cytosolic Ca2+ and the initiation of apoptosis. Compounds that interfere with these functions are termed mitochondrial toxins, many of which are derived from microbes, such as antimycin A, oligomycin A, and ionomycin. Here, we identify the mycotoxin phomoxanthone A (PXA), derived from the endophytic fungus Phomopsis longicolla, as a mitochondrial toxin. We show that PXA elicits a strong release of Ca2+ from the mitochondria but not from the ER. In addition, PXA depolarises the mitochondria similarly to protonophoric uncouplers such as CCCP, yet unlike these, it does not increase but rather inhibits cellular respiration and electron transport chain activity. The respiration-dependent mitochondrial network structure rapidly collapses into fragments upon PXA treatment. Surprisingly, this fragmentation is independent from the canonical mitochondrial fission and fusion mediators DRP1 and OPA1, and exclusively affects the inner mitochondrial membrane, leading to cristae disruption, release of pro-apoptotic proteins, and apoptosis. Taken together, our results suggest that PXA is a mitochondrial toxin with a novel mode of action that might prove a useful tool for the study of mitochondrial ion homoeostasis and membrane dynamics

NERNST: a genetically-encoded ratiometric non-destructive sensing tool to estimate NADP(H) redox status in bacterial, plant and animal systems

NADP(H) is a central metabolic hub providing reducing equivalents to multiple biosynthetic, regulatory and antioxidative pathways in all living organisms. While biosensors are available to determine NADP+ or NADPH levels in vivo, no probe exists to estimate the NADP(H) redox status, a determinant of the cell energy availability. We describe herein the design and characterization of a genetically-encoded ratiometric biosensor, termed NERNST, able to interact with NADP(H) and estimate E NADP(H). NERNST consists of a redox-sensitive green fluorescent protein (roGFP2) fused to an NADPH-thioredoxin reductase C module which selectively monitors NADP(H) redox states via oxido-reduction of the roGFP2 moiety. NERNST is functional in bacterial, plant and animal cells, and organelles such as chloroplasts and mitochondria. Using NERNST, we monitor NADP(H) dynamics during bacterial growth, environmental stresses in plants, metabolic challenges to mammalian cells, and wounding in zebrafish. NERNST estimates the NADP(H) redox poise in living organisms, with various potential applications in biochemical, biotechnological and biomedical research

NERNST: a genetically-encoded ratiometric non-destructive sensing tool to estimate NADP(H) redox status in bacterial, plant and animal systems

NADP(H) is a central metabolic hub providing reducing equivalents to multiple biosynthetic, regulatory and antioxidative pathways in all living organisms. While biosensors are available to determine NADP+ or NADPH levels in vivo, no probe exists to estimate the NADP(H) redox status, a determinant of the cell energy availability. We describe herein the design and characterization of a genetically-encoded ratiometric biosensor, termed NERNST, able to interact with NADP(H) and estimate ENADP(H). NERNST consists of a redox-sensitive green fluorescent protein (roGFP2) fused to an NADPH-thioredoxin reductase C module which selectively monitors NADP(H) redox states via oxidoreduction of the roGFP2 moiety. NERNST is functional in bacterial, plant and animal cells, and organelles such as chloroplasts and mitochondria. Using NERNST, we monitor NADP(H) dynamics during bacterial growth, environmental stresses in plants, metabolic challenges to mammalian cells, and wounding in zebrafish. NERNST estimates the NADP(H) redox poise in living organisms, with various potential applications in biochemical, biotechnological and biomedical research.Fil: Molinari, Pamela Estefanía. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET); Argentina.Fil: Krapp, Adriana. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET); Argentina.Fil: Weiner, Andrea María Julia. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET); Argentina.Fil: López, Melina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET); Argentina.Fil: Bustos Sanmamed, Pilar. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET); Argentina.Fil: Tevere, Evelyn. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET); Argentina.Fil: Calcaterra, Nora B. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET); Argentina.Fil: Carrillo, Néstor. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET); Argentina.Fil: Beyer, Hannes M. University of Düsseldorf. Institute of Synthetic Biology; Germany.Fil: Blomeier, Tim.University of Düsseldorf. Institute of Synthetic Biology; Germany.Fil: Zurbriggen, Matias D. University of Düsseldorf. Institute of Synthetic Biology; Germany.Fil: Kondadi, Arun Kumar. Heinrich-Heine-University Düsseldor. Medical Faculty and University Hospital Düsseldorf. Institute of Biochemistry and Molecular Biology I; Germany.Fil: Reichert, Andreas S. Heinrich-Heine-University Düsseldor. Medical Faculty and University Hospital Düsseldorf. Institute of Biochemistry and Molecular Biology I; Germany.Fil: Weber, Wilfried. University of Freiburg. Faculty of Biology and Signalling Research Centres BIOSS and CIBSS; Germany.Fil: Beller, Mathias. University of Düsseldorf. Institute of Mathematical Modeling of Biological Systems; Germany.Fil: Zurbriggen, Matias D. Cluster of Excellence on Plant Sciences; Germany.Fil: Weber, Wilfried. Saarland University. Leibniz Institute for New Materials and Department of Materials Sciences and Engineering; Germany

Sphingolipid subtypes differentially control proinsulin processing and systemic glucose homeostasis

Impaired proinsulin-to-insulin processing in pancreatic β-cells is a key defective step in both type 1 diabetes and type 2 diabetes (T2D) (refs. $^{1}$$^{,}$$^{2}$), but the mechanisms involved remain to be defined. Altered metabolism of sphingolipids (SLs) has been linked to development of obesity, type 1 diabetes and T2D (refs. $^{3-8}$); nonetheless, the role of specific SL species in β-cell function and demise is unclear. Here we define the lipid signature of T2D-associated β-cell failure, including an imbalance of specific very-long-chain SLs and long-chain SLs. β-cell-specific ablation of CerS2, the enzyme necessary for generation of very-long-chain SLs, selectively reduces insulin content, impairs insulin secretion and disturbs systemic glucose tolerance in multiple complementary models. In contrast, ablation of long-chain-SL-synthesizing enzymes has no effect on insulin content. By quantitatively defining the SL-protein interactome, we reveal that CerS2 ablation affects SL binding to several endoplasmic reticulum-Golgi transport proteins, including Tmed2, which we define as an endogenous regulator of the essential proinsulin processing enzyme Pcsk1. Our study uncovers roles for specific SL subtypes and SL-binding proteins in β-cell function and T2D-associated β-cell failure