Genetic diversity and phylogenetic relationships of Clostridium perfringens strains isolated from mastitis and enteritis in Egyptian dairy farms.

BACKGROUND: Clostridium perfringens, a common environmental bacterium, is responsible for a variety of serious illnesses including food poisoning, digestive disorders, and soft tissue infections. Mastitis in lactating cattle and sudden death losses in baby calves are major problems for producers raising calves on dairy farms. The pathogenicity of this bacterium is largely mediated by its production of various toxins. RESULTS: The study revealed that Among the examined lactating animals with a history of mastitis, diarrheal baby calves, and acute sudden death cases in calves, C. perfringens was isolated in 23.5% (93/395) of the total tested samples. Eighteen isolates were obtained from mastitic milk, 59 from rectal swabs, and 16 from the intestinal contents of dead calves. Most of the recovered C. perfringens isolates (95.6%) were identified as type A by molecular toxinotyping, except for four isolates from sudden death cases (type C). Notably, C. perfringens was recovered in 100% of sudden death cases compared with 32.9% of rectal swabs and 9% of milk samples. This study analyzed the phylogeny of C. perfringens using the plc region and identified the plc region in five Egyptian bovine isolates (milk and fecal origins). Importantly, this finding expands the known data on C. perfringens phospholipase C beyond reference strains in GenBank from various animal and environmental sources. CONCLUSION: Phylogenetic analyses of nucleotide sequence data differentiated between strains of different origins. The plc sequences of Egyptian C. perfringens strains acquired in the present study differed from those reported globally and constituted a distinct genetic ancestor.

Unveiling the ocular battlefield: Insights into Pseudomonas aeruginosa virulence factors and their implications for multidrug resistance.

The research investigates the virulence factors of Pseudomonas aeruginosa (P. aeruginosa), a pathogen known for its ability to cause human infections by releasing various exoenzymes and virulence factors. Particularly relevant in ocular infections, where tissue degeneration can occur, even after bacterial growth has ceased due to the potential role of secreted proteins/enzymes. Clinical isolates of P. aeruginosa, both ocular (146) and non-ocular (54), were examined to determine the frequency and mechanism of virulence factors. Phenotypic characterization revealed the production of alginate, biofilm, phospholipase C, and alkaline protease, while genotypic testing using internal uniplex PCR identified the presence of Exo U, S, T, Y, and LasB genes. Results showed a significant prevalence of Exo U and Y genes in ocular isolates, a finding unique to Indian studies. Additionally, the study noted that ocular isolates often contained all four secretomes, suggesting a potential link between these factors and ocular infections. These findings contribute to understanding the pathogenesis of P. aeruginosa infections, particularly in ocular contexts, and highlights the importance of comprehensive virulence factor analysis in clinical settings.

Sphingosine induction of the Pseudomonas aeruginosa hemolytic phospholipase C/sphingomyelinase (PlcH).

Hemolytic phospholipase C, PlcH, is an important virulence factor for Pseudomonas aeruginosa. PlcH preferentially hydrolyzes sphingomyelin and phosphatidylcholine, and this hydrolysis activity drives tissue damage and inflammation and interferes with the oxidative burst of immune cells. Among other contributors, transcription of plcH was previously shown to be induced by phosphate starvation via PhoB and the choline metabolite, glycine betaine, via GbdR. Here, we show that sphingosine can induce plcH transcription and result in secreted PlcH enzyme activity. This induction is dependent on the sphingosine-sensing transcriptional regulator SphR. The SphR induction of plcH occurs from the promoter for the gene upstream of plcH that encodes the neutral ceramidase, CerN, and transcriptional readthrough of the cerN transcription terminator. Evidence for these conclusions came from mutation of the SphR binding site in the cerN promoter, mutation of the cerN terminator, enhancement of cerN termination by adding the rrnB terminator, and reverse transcriptase PCR (RT-PCR) showing that the intergenic region between cerN and plcH is made as RNA during sphingosine, but not choline, induction. We also observed that, like glycine betaine induction, sphingosine induction of plcH is under catabolite repression control, which likely explains why such induction was not seen in other studies using sphingosine in rich media. The addition of sphingosine as a novel inducer for PlcH points to the regulation of plcH transcription as a site for the integration of multiple host-derived signals. IMPORTANCE: PlcH is a secreted phospholipase C/sphingomyelinase that is important for the virulence of Pseudomonas aeruginosa. Here, we show that sphingosine, which presents itself or as a product of P. aeruginosa sphingomyelinase and ceramidase activity, leads to the induction of plcH transcription. This transcriptional induction occurs from the promoter of the upstream ceramidase gene generating a conditional operon. The transcript on which plcH resides, therefore, is different depending on which host molecule or condition leads to induction, and this may have implications for PlcH post-transcriptional regulation. This work also adds to our understanding of P. aeruginosa with host-derived sphingolipids.

Crystal Structure of Fungal Nonspecific Phospholipase C Unveils a Distinct Catalytic Mechanism.

Nonspecific phospholipase C (NPC) plays a pivotal role in hydrolyzing phospholipids, releasing diacylglycerol─an essential second messenger. Extensive research has elucidated the structure and function of bacterial and plant NPCs, but our understanding of their fungal counterparts remains limited. Here, we present the first crystal structure of a fungal NPC derived from Rasamsonia emersonii (RePLC), unraveling its distinguishable features divergent from other known phospholipase C. Remarkably, the structure of RePLC contains solely the phosphoesterase domain without the crucial C-terminal domain (CTD) found in plant NPCs, although CTD is important for their activity. Through a comparative analysis of structural features among NPCs from diverse species combined with structure-based mutation analyses and bioinformatics methods, we propose a potential molecular mechanism that may universally underlie the catalysis of phospholipid hydrolysis in fungal NPCs. Furthermore, our study sheds light on the captivating evolutionary trajectory of enzymes across diverse species.

Phosphatidylinositol-specific phospholipase C can decrease Müller cell viability and suppress its phagocytic activity by modulating PI3K/AKT signaling pathway.

Bacillus cereus endophthalmitis is a devastating eye infection that causes rapid blindness through the release of extracellular tissue-destructive exotoxins. The phagocytic and antibacterial functions of ocular cells are the keys to limiting ocular bacterial infections. In a previous study, we identified a new virulence gene, plcA-2 (different from the original plcA-1 gene), that was strongly associated with the plcA gene of Listeria monocytogenes. This plcA gene had been confirmed to play an important role in phagocytosis. However, how the Bc-phosphatidylinositol-specific phospholipase C (PI-PLC) proteins encoded by the plcA-1/2 genes affect phagocytes remains unclear in B. cereus endophthalmitis. Here, we found that the enzymatic activity of Bc-PI-PLC-A2 was approximately twofold higher than that of Bc-PI-PLC-A1, and both proteins inhibited the viability of Müller cells. In addition, PI-PLC proteins reduced phagocytosis of Müller cells by decreasing the phosphorylation levels of key proteins in the PI3K/AKT signaling pathway. In conclusion, we showed that PI-PLC proteins contribute to inhibit the viability of and suppress the phagocytosis of Müller cells, providing new insights into the pathogenic mechanism of B. cereus endophthalmitis.

Phosphatidylglycerol-specific phospholipase C from Amycolatopsis sp. NT115 strain: purification, characterization, and gene cloning.

Recently, phosphatidylglycerol (PG) focused on its important role in chloroplast photosynthesis, mitochondrial function of the sperm, an inhibitory effect on SARS-CoV-2 ability to infect naïve cells, and reducing lung inflammation caused by coronavirus disease 2019. To develop an enzymatic PG determination method as the high-throughput analysis of PG, a PG-specific phospholipase C (PG-PLC) was found in the culture supernatant of Amycolatopsis sp. NT115. PG-PLC (54 kDa by SDS-PAGE) achieved the maximal activity at pH 6.0 and 55 °C and was inhibited by detergents, such as Briji35, Tween 80, and sodium cholate, but not by EDTA and metal ions, except for Zn2+. The open reading frame of the PG-PLC gene consisted of 1620 bp encoding 515-amino-acid residues containing the preceding 25-amino-acid residues (Tat signal peptide sequence). The putative amino acid sequence of PG-PLC was highly similar to those of metallophosphoesterases; however, its substrate specificity was completely different from those of known PLCs.

Phospholipase C: Diverse functions in plant biotic stress resistance and fungal pathogenicity.

Phospholipase C (PLC) generates various second messenger molecules and mediates phospholipid hydrolysis. In recent years, the important roles of plant and fungal PLC in disease resistance and pathogenicity, respectively, have been determined. However, the roles of PLC in plants and fungi are unintegrated and relevant literature is disorganized. This makes it difficult for researchers to implement PLC-based strategies to improve disease resistance in plants. In this comprehensive review, we summarize the structure, classification, and phylogeny of the PLCs involved in plant biotic stress resistance and fungal pathogenicity. PLCs can be divided into two groups, nonspecific PLC (NPC) and phosphatidylinositol-specific PLC (PI-PLC), which present marked differences in phylogenetic evolution. The products of PLC genes in fungi play significant roles in physiological activity and pathogenesis, whereas those encoded by plant PLC genes mediate the immune response to fungi. This review provides a perspective for the future control of plant fungal diseases.

Cryptic mutations of PLC family members in brain disorders: recent discoveries and a deep-learning-based approach.

Phospholipase C (PLC) is an essential isozyme involved in the phosphoinositide signalling pathway, which maintains cellular homeostasis. Gain- and loss-of-function mutations in PLC affect enzymatic activity and are therefore associated with several disorders. Alternative splicing variants of PLC can interfere with complex signalling networks associated with oncogenic transformation and other diseases, including brain disorders. Cells and tissues with various mutations in PLC contribute different phosphoinositide signalling pathways and disease progression, however, identifying cryptic mutations in PLC remains challenging. Herein, we review both the mechanisms underlying PLC regulation of the phosphoinositide signalling pathway and the genetic variation of PLC in several brain disorders. In addition, we discuss the present challenges associated with the potential of deep-learning-based analysis for the identification of PLC mutations in brain disorders.

Roles of phosphatidylserine and phospholipase C in the activation of TOR complex 2 signaling in Saccharomyces cerevisiae.

Target of rapamycin (TOR) forms two distinct complexes, TORC1 and TORC2, to exert its essential functions in cellular growth and homeostasis. TORC1 signaling is regulated in response to nutrients such as amino acids and glucose; however, the mechanisms underlying the activation of TORC2 signaling are still poorly understood compared to those for TORC1 signaling. In the budding yeast Saccharomyces cerevisiae, TORC2 targets the protein kinases Ypk1 and Ypk2 (hereafter Ypk1/2), and Pkc1 for phosphorylation. Plasma membrane stress is known to activate TORC2-Ypk1/2 signaling. We have previously reported that methylglyoxal (MG), a metabolite derived from glycolysis, activates TORC2-Pkc1 signaling. In this study, we found that MG activates the TORC2-Ypk1/2 and TORC2-Pkc1 signaling, and that phosphatidylserine is involved in the activation of both signaling pathways. We also demonstrated that the Rho family GTPase Cdc42 contributes to the plasma membrane stress-induced activation of TORC2-Ypk1/2 signaling. Furthermore, we revealed that phosphatidylinositol-specific phospholipase C, Plc1, contributes to the activation of both TORC2-Ypk1/2 and TORC2-Pkc1 signaling.

The phosphoinositide-specific phospholipase C1 modulates flowering time and grain size in rice.

MAIN CONCLUSION: Preferential expression of OsPLC1 is detected at the heading stage of rice, OsPLC1 overexpression results in early flowering, increased-grain size and yield; however, opposing phenotypes produced in the osplc1 mutants. Abstract: The importance of phospholipase C (PLC) in plant development has been demonstrated in several studies. OsPLC1, a member of PI-PLC in rice, although its role in the response to salt stress of rice seedlings has been reported, its functions in the growth and development of rice is elusive. Here, we report that OsPLC1 expression could be detectable in various tissues throughout the developmental stages of rice, and the highest expression level of OsPLC1 was detected at the heading stage. OsPLC1 overexpression (OE) produced rice plants with early flowering, whereas OsPLC1 loss-of-function led to delay in flowering. The expression levels of subset genes, which are involved in the control of flowering time in rice, were altered in the plants of OE and osplc1. In addition, the enlargement of grain size was observed in OE plants, however, the reduction of grain size was noticed in osplc1 mutants. The increase in the grain size and the grain yield of OE lines were associated with the improvement of cell length and expression levels of a set of genes related to cell expansion, contrarily, the decrease in osplc1 mutant grain size and yield were linked to declined cell length and expression levels of related genes. No significant differences, in terms of the grain quality of mature seeds, were found in OE and osplc1 mutants, with compared to those in Nipponbare (Nip). In summary, our study suggests that OsPLC1 could modulate rice flowering time and grain size.

Phospholipase C is a novel regulator at the early stages of microspore embryogenesis in Nicotiana tabacum.

Microspore transfers the developmental fate into embryogenesis in vitro regulated by determinant factors of stress-induced. However, the key regulators of microspore embryogenesis (ME) are still largely undiscovered to reveal the mechanism of cell fate transition. Here, we report that Phospholipase C (PLC) is involved at the early stages of ME in Nicotiana tabacum. NtPLC2/3/4 are expressed at the initial stages of ME. The expression levels of NtPLC2/3 are transient activated after 3 days in culture, while the expression level of NtPLC4 maintains relatively stable. Inhibition of PLCs induces the decrease in NtPLC2/3/4 expression level and decline of ME yield. We confirm that lipids in microspore are degraded and then re-accumulate at first embryonic division stage. Inhibition of PLCs suppresses the lipids metabolism at the early stages of ME. Thus, we propose that PLCs-mediated lipid metabolism is a novel regulator at the early stages of ME.

Characterization of a novel thermostable phospholipase C from T. kodakarensis suitable for oil degumming.

The implementation of cleaner technologies that minimize environmental pollution caused by conventional industrial processes is an increasing global trend. Hence, traditionally used chemicals have been replaced by novel enzymatic alternatives in a wide variety of industrial-scale processes. Enzymatic oil degumming, the first step of the oil refining process, exploits the conversion catalyzed by phospholipases to remove vegetable crude oils' phospholipids. This enzymatic method reduces the gums' volume and increases the overall oil yield. A thermostable phospholipase would be highly advantageous for industrial oil degumming as oil treatment at higher temperatures would save energy and increase the recovery of oil by facilitating the mixing and gums removal. A thermostable phosphatidylcholine (PC) (and phosphatidylethanolamine (PE))-specific phospholipase C from Thermococcus kodakarensis (TkPLC) was studied and completely removed PC and PE from crude soybean oil at 80 °C. Due to these characteristics, TkPLC is an interesting promising candidate for industrial-scale enzymatic oil degumming at high temperatures. KEY POINTS: • A thermostable phospholipase C from T. kodakarensis (TkPLC) has been identified. • TkPLC was recombinantly produced in Pichia pastoris and successfully purified. • TkPLC completely hydrolyzed PC and PE in soybean oil degumming assays at 80 °C.

Involvement of Phospholipase C in Photosynthesis and Growth of Maize Seedlings.

Phospholipase C is an enzyme that catalyzes the hydrolysis of glycerophospholipids and can be classified as phosphoinositide-specific PLC (PI-PLC) and non-specific PLC (NPC), depending on its hydrolytic substrate. In maize, the function of phospholipase C has not been well characterized. In this study, the phospholipase C inhibitor neomycin sulfate (NS, 100 mM) was applied to maize seedlings to investigate the function of maize PLC. Under the treatment of neomycin sulfate, the growth and development of maize seedlings were impaired, and the leaves were gradually etiolated and wilted. The analysis of physiological and biochemical parameters revealed that inhibition of phospholipase C affected photosynthesis, photosynthetic pigment accumulation, carbon metabolism and the stability of the cell membrane. High-throughput RNA-seq was conducted, and differentially expressed genes (DEGS) were found significantly enriched in photosynthesis and carbon metabolism pathways. When phospholipase C activity was inhibited, the expression of genes related to photosynthetic pigment accumulation was decreased, which led to lowered chlorophyll. Most of the genes related to PSI, PSII and TCA cycles were down-regulated and the net photosynthesis was decreased. Meanwhile, genes related to starch and sucrose metabolism, the pentose phosphate pathway and the glycolysis/gluconeogenesis pathway were up-regulated, which explained the reduction of starch and total soluble sugar content in the leaves of maize seedlings. These findings suggest that phospholipase C plays a key role in photosynthesis and the growth and development of maize seedlings.

Rational engineering of phospholipase C from Bacillus cereus HSL3 for simultaneous thermostability and activity improvement.

As an essential enzyme for phospholipid degradation, phospholipase C (PLC) has been used for enzymatic degumming of vegetable oils and production of valuable phospholipid derivatives. In this study, rational engineering based on B-factor analysis and molecular dynamic simulation analysis were employed to rationally identify mutation candidates and a PLC double mutant F96R/Q153P was designed from Bacillus cereus HSL3. Compared to the wild-type PLC, F96R/Q153P exhibited significantly improved thermal properties, including higher temperature optima and better thermal stability. It showed the highest optimal reaction temperature (90 °C) reported so far. F96R/Q153P displayed 4.94 times kcat and 2.37 times kcat/Km as much as the wild-type, as well as improved substrate adaptability. Structural insights revealed that the mutations caused reduced proportion of random coil and constraint of certain loop fluctuations. These results demonstrated the great potential of knowledge-based rational design for improving the catalytic characteristics of industrial enzymes in the enzymatic degumming process.

Molecular Characterization of Phospholipase C in Infection Structure Differentiation Induced by Pear Fruit Surface Signals, Stress Responses, Secondary Metabolism, and Virulence of Alternaria alternata.

Fungal pathogens use plant surface physiochemical signals to trigger specific developmental processes. To assess the role of phospholipase C (PLC) in mediating plant stimuli sensing of Alternaria alternata, the function of three PLC genes was characterized by constructing ΔAaPLC mutants. Here we showed that fruit wax-coated surfaces significantly induced appressorium formation in A. alternata and mutants. Germination of ΔAaPLC mutants did not differ from the wild type. Deletion of AaPLC1 led to the decrease of appressorium formation and infected hyphae, but the degree of reduction varies between the different types of waxes, with the strongest response to pear wax. Appressorium formation and infected hyphae of the ΔAaPLC1 mutant on dewaxed onion epidermis mounted with pear wax (θ4) were reduced by 14.5 and 65.7% after 8 h incubation, while ΔAaPLC2 and ΔAaPLC3 formed the same infection hyphae as wild type. In addition, AaPLC1 mutation caused pleiotropic effects on fungal biological function, including growth deficiency, changes in stress tolerance, weakening of pathogenicity to the host, as well as destruction of mycotoxin synthesis. Both AaPLC2 and AaPLC3 genes were found to have some effects on stress response and mycotoxin production. Taken together, AaPLC genes differentially regulate the growth, stress response, pathogenicity, and secondary metabolism of A. alternata.

Identification and expression analysis of phospholipase C family genes between different male fertility accessions in pepper.

Phospholipase C (PLC) is one of the major lipid-hydrolyzing enzymes, involved in lipid-mediating signal pathway. PLCs have been found to play a significant role in the growth and development of plants. In this study, the genome-wide identification and characteristic analysis of CaPLC family genes in pepper were conducted and the expression of two CaPLC genes were investigated. The results showed that a total of 11 CaPLC family genes were systematically identified, which were distributed on five chromosomes and divided into two groups based on their evolutionary relevance. Some cis-elements responding to different hormones and stresses were screened in the promoters of CaPLC genes. Quantitative real-time PCR indicated that the expression of CaPIPLC1 and CaPIPLC5 in flowers were dozens of times higher than in other tissues. In addition, with the development of flower buds, the relative expressions of CaPIPLC1 and CaPIPLC5 gradually increased in fertile materials R1 and F1. However, no expression of CaPIPLC1 and CaPIPLC5 were detected at all developmental stages of cytoplasmic male sterile lines (CMS) compared with fertile accessions. The study revealed the number and characteristics of the CaPLC family genes, which supplied a basic and systematic understanding of CaPLC family. In addition, these findings provided new insights into the role of CaPLC genes in pollen development and fertility restoration in pepper.

DAF-2c signaling promotes taste avoidance after starvation in Caenorhabditis elegans by controlling distinct phospholipase C isozymes.

Previously, we reported that DAF-2c, an axonal insulin receptor isoform in Caenorhabditis elegans, acts in the ASER gustatory neuron to regulate taste avoidance learning, a process in which worms learn to avoid salt concentrations experienced during starvation. Here, we show that secretion of INS-1, an insulin-like peptide, after starvation conditioning is sufficient to drive taste avoidance via DAF-2c signaling. Starvation conditioning enhances the salt-triggered activity of AIA neurons, the main sites of INS-1 release, which potentially promotes feedback signaling to ASER to maintain DAF-2c activity during taste avoidance. Genetic studies suggest that DAF-2c-Akt signaling promotes high-salt avoidance via a decrease in PLCß activity. On the other hand, the DAF-2c pathway promotes low-salt avoidance via PLCε and putative Akt phosphorylation sites on PLCε are essential for taste avoidance. Our findings imply that animals disperse from the location at which they experience starvation by controlling distinct PLC isozymes via DAF-2c.

Mutations in phospholipase C eta-1 (PLCH1) are associated with holoprosencephaly.

BACKGROUND: Holoprosencephaly is a spectrum of developmental disorder of the embryonic forebrain in which there is failed or incomplete separation of the prosencephalon into two cerebral hemispheres. To date, dominant mutations in sonic hedgehog (SHH) pathway genes are the predominant Mendelian causes, and have marked interfamilial and intrafamilial phenotypical variabilities. METHODS: We describe two families in which offspring had holoprosencephaly spectrum and homozygous predicted-deleterious variants in phospholipase C eta-1 (PLCH1). Immunocytochemistry was used to examine the expression pattern of PLCH1 in human embryos. We used SHH as a marker of developmental stage and of early embryonic anatomy. RESULTS: In the first family, two siblings had congenital hydrocephalus, significant developmental delay and a monoventricle or fused thalami with a homozygous PLCH1 c.2065C>T, p.(Arg689*) variant. In the second family, two siblings had alobar holoprosencephaly and cyclopia with a homozygous PLCH1 c.4235delA, p.(Cys1079ValfsTer16) variant. All parents were healthy carriers, with no holoprosencephaly spectrum features. We found that the subcellular localisation of PLCH1 is cytoplasmic, but the p.(Cys1079ValfsTer16) variant was predominantly nuclear. Human embryo immunohistochemistry showed PLCH1 to be expressed in the notorcord, developing spinal cord (in a ventral to dorsal gradient), dorsal root ganglia, cerebellum and dermatomyosome, all tissues producing or responding to SHH. Furthermore, the embryonic subcellular localisation of PLCH1 was exclusively cytoplasmic, supporting protein mislocalisation contributing to the pathogenicity of the p.(Cys1079ValfsTer16) variant. CONCLUSION: Our data support the contention that PLCH1 has a role in prenatal mammalian neurodevelopment, and deleterious variants cause a clinically variable holoprosencephaly spectrum phenotype.

Phospholipase C (AoPLC2) regulates mycelial development, trap morphogenesis, and pathogenicity of the nematode-trapping fungus Arthrobotrys oligospora.

AIMS: Phospholipase C (PLC) is a hydrolase involved in signal transduction in eukaryotic cells. This study aimed to understand the function of PLC in the nematode-trapping fungus Arthrobotrys oligospora. METHODS AND RESULTS: Orthologous PLC (AoPLC2) of A. oligospora was functionally analysed using gene disruption and multi-phenotypic analysis. Disrupting Aoplc2 caused a deformation of partial hyphal cells (about 10%) and conidia (about 50%), decreased the number of nuclei in both conidia and hyphal cells, and increased the accumulation of lipid droplets. Meanwhile, the sporulation-related genes fluG and abaA were downregulated in ΔAoplc2 mutants than in the wild-type strain. Moreover, ΔAoplc2 mutants were more sensitive to osmotic stressors. Importantly, the number of traps, electron-dense bodies in traps, and nematicidal activity of ΔAoplc2 mutants were reduced, and the shape of the traps was deformed. In addition, AoPLC2 was involved in the biosynthesis of secondary metabolites in A. oligospora. CONCLUSIONS: AoPLC2 plays an important role in the development of hyphae, spores, and cell nuclei, responses to stress, formation of traps, and predation of nematodes in A. oligospora. SIGNIFICANCE AND IMPACT OF STUDY: This study reveals the various functions of phospholipase C and elucidates the regulation of trap morphogenesis in nematode-trapping fungi.

Sphingomyelin synthases 1 and 2 exhibit phosphatidylcholine phospholipase C activity.

Many studies have confirmed the enzymatic activity of a mammalian phosphatidylcholine (PC) phospholipase C (PLC) (PC-PLC), which produces diacylglycerol (DAG) and phosphocholine through the hydrolysis of PC in the absence of ceramide. However, the protein(s) responsible for this activity have never yet been identified. Based on the fact that tricyclodecan-9-yl-potassium xanthate can inhibit both PC-PLC and sphingomyelin synthase (SMS) activities, and SMS1 and SMS2 have a conserved catalytic domain that could mediate a nucleophilic attack on the phosphodiester bond of PC, we hypothesized that both SMS1 and SMS2 might have PC-PLC activity. In the present study, we found that purified recombinant SMS1 and SMS2 but not SMS-related protein have PC-PLC activity. Moreover, we prepared liver-specific Sms1/global Sms2 double-KO mice. We found that liver PC-PLC activity was significantly reduced and steady-state levels of PC and DAG in the liver were regulated by the deficiency, in comparison with control mice. Using adenovirus, we expressed Sms1 and Sms2 genes in the liver of the double-KO mice, respectively, and found that expressed SMS1 and SMS2 can hydrolyze PC to produce DAG and phosphocholine. Thus, SMS1 and SMS2 exhibit PC-PLC activity in vitro and in vivo.