Membrane lipid chemistry is remarkably different in archaea compared with bacteria and eukaryotes. In the evolutionary context, this is also termed the lipid divide and is reflected by distinct biosynthetic pathways. Contemporary organisms have almost without exception only one type of membrane lipid. During early membrane evolution, mixed membrane stages likely occurred, and it was hypothesized that the instability of such mixtures was the driving force for the lipid divide. To examine the compatibility between archaeal and bacterial lipids, the bacterium Escherichia coli has been engineered to contain both types of lipids with varying success. Only limited production of archaeal lipid archaetidylethanolamine was achieved. Here, we substantially increased its production in E. coli by overexpression of an archaeal phosphatidylserine synthase needed for ethanolamine headgroup attachment. Furthermore, we introduced a synthetic isoprenoid utilization pathway to increase the supply of isopentenyl-diphosphate and dimethylallyl diphosphate. This improved archaeal lipid production substantially. The archaeal phospholipids also served as a substrate for the E. coli cardiolipin synthase, resulting in archaeal and novel hybrid archaeal/bacterial cardiolipin species not seen in living organisms before. Growth of the E. coli strain with the mixed membrane shows an enhanced sensitivity to the inhibitor of fatty acid biosynthesis, cerulenin, indicating a critical dependence of the engineered E. coli strain on its native phospholipids.

Sgms1 encodes sphingomyelin synthase 1, an enzyme in the sphingosine-1-phosphate signalling pathway, and was previously reported to underlie hearing impairment in the mouse. A new mouse allele, Sgms1tm1a, unexpectedly showed normal Auditory Brainstem Response thresholds. We found that the Sgms1tm1a mutation led to incomplete knockdown of transcript to 20 % of normal values, which was enough to support normal hearing. The Sgms1tm1b allele was generated by knocking out exon 7, leading to a complete lack of detectable transcript in the inner ear. Sgms1tm1b homozygotes showed largely normal auditory brainstem response thresholds at first, followed by progressive loss of sensitivity until they showed severe impairment at 6 months old. The endocochlear potential was consistently reduced in Sgms1tm1b mutants at 3, 4 and 8 weeks old, to around 80 mV compared with around 120 mV in control littermates. The stria vascularis showed a characteristic irregularity of marginal cell surfaces and patchy loss of Kcnq1 expression at their apical membrane, and expression analysis of the lateral wall suggested that marginal cells were the most likely initial site of dysfunction in the mutants. Finally, significant association of auditory thresholds with DNA markers within and close to the human SGMS1 gene were found in the 1958 Birth Cohort, suggesting that SGMS1 variants may play a role in the range of hearing abilities in the human population.

Four tunicamycin class compounds, tunicamycin VII (1), tunicamycin VIII (2), corynetoxin U17a (3), and tunicamycin IX (4), were isolated from the culture broth of the marine-derived actinomycete Streptomyces sp. MBTG32. The strain was identified using the 16S rDNA sequencing technique, and the isolated strain was closely related to Streptomyces bacillaris. The structures of the isolated compounds were elucidated based on spectroscopic data and comparisons with previously reported NMR data. Compounds 1-4 showed potent antibacterial activities against Gram-positive bacteria, especially Staphylococcus aureus, with MIC values of 0.13-0.25 µg/mL. Through a recombinant enzyme assay and overexpression analysis, we found that the isolated compounds exerted potent inhibitory effects on S. aureus MurNAc-pentapeptide translocase (MraY), with IC50 values of 0.08-0.21 µg/mL. The present results support that the underlying mechanism of action of tunicamycins isolated from marine-derived Streptomyces sp. is also associated with the inhibition of MraY enzyme activity in S. aureus.

The lack of effective vaccines and the development of resistance to the current treatments highlight the urgent need for new anti-leishmanials. Sphingolipid metabolism has been proposed as a promising source of Leishmania-specific targets as these lipids are key structural components of the eukaryotic plasma membrane and are involved in distinct cellular events. Inositol phosphorylceramide (IPC) is the primary sphingolipid in the Leishmania species and is the product of a reaction mediated by IPC synthase (IPCS). The antihistamine clemastine fumarate has been identified as an inhibitor of IPCS in L. major and a potent anti-leishmanial in vivo. Here we sought to further examine the target of this compound in the more tractable species L. mexicana, using an approach combining genomic, proteomic, metabolomic and lipidomic technologies, with molecular and biochemical studies. While the data demonstrated that the response to clemastine fumarate was largely conserved, unexpected disturbances beyond sphingolipid metabolism were identified. Furthermore, while deletion of the gene encoding LmxIPCS had little impact in vitro, it did influence clemastine fumarate efficacy and, importantly, in vivo pathogenicity. Together, these data demonstrate that clemastine does inhibit LmxIPCS and cause associated metabolic disturbances, but its primary target may lie elsewhere.

Renal ischemia/reperfusion is a serious condition that not only causes acute kidney injury, a severe clinical syndrome with high mortality, but is also an inevitable part of kidney transplantation or other kidney surgeries. Alterations of oxygen levels during ischemia/reperfusion, namely hypoxia/reoxygenation, disrupt mitochondrial metabolism and induce structural changes that lead to cell death. A signature mitochondrial phospholipid, cardiolipin, with many vital roles in mitochondrial homeostasis, is one of the key players in hypoxia/reoxygenation-induced mitochondrial damage. In this study, we analyze the effect of hypoxia/reoxygenation on human renal proximal tubule epithelial cell (RPTEC) cardiolipins, as well as their metabolism and mitochondrial functions. RPTEC cells were placed in a hypoxic chamber with a 2% oxygen atmosphere for 24 h to induce hypoxia; then, they were replaced back into regular growth conditions for 24 h of reoxygenation. Surprisingly, after 24 h, hypoxia cardiolipin levels substantially increased and remained higher than control levels after 24 h of reoxygenation. This was explained by significantly elevated levels of cardiolipin synthase and lysocardiolipin acyltransferase 1 (LCLAT1) gene expression and protein levels. Meanwhile, hypoxia/reoxygenation decreased ADP-dependent mitochondrial respiration rates and oxidative phosphorylation capacity and increased reactive oxygen species generation. Our findings suggest that hypoxia/reoxygenation induces cardiolipin remodeling in response to reduced mitochondrial oxidative phosphorylation in a way that protects mitochondrial function.

MraY (phospho-N-acetylmuramoyl-pentapeptide-transferase) inhibitory natural products are attractive molecules as candidates for a new class of antibacterial agents to combat antimicrobial-resistant bacteria. Structural optimization of these natural products is required to improve their drug-like properties for therapeutic use. However, chemical modifications of these natural products are painstaking tasks due to complex synthetic processes, which is a bottleneck in advancing natural products to the clinic. Here, we develop a strategy for a comprehensive in situ evaluation of the build-up library, which enables us to streamline the preparation of the analogue library and directly assess its biological activities. We apply this approach to a series of MraY inhibitory natural products. Through construction and evaluation of the 686-compound library, we identify promising analogues that exhibit potent and broad-spectrum antibacterial activity against highly drug-resistant strains in vitro as well as in vivo in an acute thigh infection model. Structures of the MraY-analogue complexes reveal distinct interaction patterns, suggesting that these analogues represent MraY inhibitors with unique binding modes. We further demonstrate the generality of our strategy by applying it to tubulin-binding natural products to modulate their tubulin polymerization activities.

Ceramide (Cer) is synthesized de novo in the bilayer of the endoplasmic reticulum and transported to the cytosolic leaflet of the trans-Golgi apparatus for sphingomyelin (SM) synthesis. As the active site of SM synthase (SMS) is located on the luminal side of the Golgi membrane, Cer translocates to the lumen via transbilayer movement for SM synthesis. However, the mechanism of transbilayer movement is not fully understood. As the Cer-related translocases seem to localize near the SMS, the protein was identified using proximity-dependent biotin identification proteomics. Phospholipid scramblase 1 (PLSCR1), which is thought to act as a scramblase for phosphatidylserine and phosphatidylethanolamine, was identified as a protein proximal to the SMS isoforms SMS1 and SMS2. Although five isoforms of PLSCR have been reported in humans, only PLSCR1, PLSCR3, and PLSCR4 are expressed in HEK293T cells. Confocal microscopic analysis showed that PLSCR1 and PLSCR4 partially co-localized with p230, a trans-Golgi network marker, where SMS isoforms are localized. We established CRISPR/Cas9-mediated PLSCR1, PLSCR3, and PLSCR4 single-knockout cells and PLSCR1, 3, 4 triple knockout HEK293T cells. Liquid chromatography-tandem mass spectrometry revealed that the levels of species with distinct acyl chains in Cer and SM were not significantly different in single knockout cells or in the triple knockout cells compared to the wild-type cells. Our findings suggest that PLSCR1 is localized in the vicinity of SMS isoforms, however is not involved in the transbilayer movement of Cer for SM synthesis.

In this study, the progenitors of MCR-3, MCR-7 and MCR-5, namely NMCR-3, NMCR-4 and NMCR-5, were firstly discovered and indicating Aeromonas was a natural reservoir for MCR-3 and MCR-7. Furthermore, different evolutionary models for MCR-3, MCR-7 and MCR-5 were proposed.

Sphingomyelin synthase (SMS) is a sphingolipid-metabolizing enzyme involved in the de novo synthesis of sphingomyelin (SM) from ceramide (Cer). Recent studies have indicated that SMS is a key therapeutic target for metabolic diseases such as fatty liver, type 2 diabetes, atherosclerosis, and colorectal cancer. However, very few SMS inhibitors have been identified because of the limited sensitivity and selectivity of the current fluorescence-based screening assay. In this study, we developed a simple cell-based assay coupled with liquid chromatography/tandem mass spectrometry (LC-MS/MS) to screen for SMS inhibitors. HeLa cells stably expressing SMS1 or SMS2 were used for the screening. A non-fluorescent unnatural C6-Cer was used as a substrate for SMS to produce C6-SM. C6-Cer and C6-SM levels in the cells were monitored and quantified using LC-MS/MS. The activity of ginkgolic acid C15:1 (GA), a known SMS inhibitor, was measured. GA had half-maximal inhibitory concentrations of 5.5 μM and 3.6 μM for SMS1 and SMS2, respectively. To validate these findings, hSMS1 and hSMS2 proteins were optimized for molecular docking studies. In silico analyses were conducted to assess the interaction of GA with SMS1 and SMS2, and its binding affinity. This study offers an analytical approach for screening novel SMS inhibitors and provides in silico support for the experimental findings.

The degree of contamination of retail meat with colistin-resistant bacteria and its potential contribution to dissemination within communities remains to be determined. Thus, we aimed to elucidate the contamination status of colistin-resistance genes, indicative of colistin-resistant bacteria, in retail meats in Vietnam. In total, 46 chicken and 49 pork meats from stores in Vietnam and Japan were examined. Multiplex real-time polymerase chain reaction with TaqMan probes was performed for detecting mcr-1, mcr-3, and Escherichia coli 16S rRNA. Colistin-resistant bacteria in meats were isolated using selective media. The minimum inhibitory concentrations of colistin were determined using the broth microdilution method. The results showed that 70.7% of chicken meats in Vietnam were contaminated with both mcr-1 and mcr-3. Meanwhile, mcr-1 and mcr-3 were detected in 15.9% and 40.9% of pork meat, respectively. Only mcr-3 was detected in 40% of chicken in Japan. In addition, mcr-1-harboring E. coli and mcr-3-harboring Aeromonas were isolated from chicken meats in Vietnam. Some of these isolates showed colistin resistance. These results showed that most retail meats were highly contaminated with colistin-resistance genes. Notably, our results suggest that mcr-3 is more prevalent in the contaminated samples compared with mcr-1.