Phosphatidylinositol-specific Phospholipase Research Articles

Phosphatidylinositol-specific phospholipase C-associated phospholipid metabolism mediates DcGLRs channel to promote calcium influx under CaCl2 treatment in shredded carrots during storage

The rapid activation of phosphatidylinositol-specific phospholipase C (PI-PLC) occurs early after the stimulation of biotic and abiotic stress in plants, which directly associated with the calcium channel-induced calcium ion (Ca2+) influx. Exogenous calcium chloride (CaCl2) mediates the calcium signaling transduction to promote the γ-aminobutyric acid accumulation and nutritional quality in shredded carrots whereas the generation mechanism remains uncertain. Therefore, the involvement of PI-PLC-associated phospholipid metabolism was investigated in present study. Our result revealed that CaCl2 treatment promoted the expression and activity of PI-PLC and increased the inositol 1,4,5-trisphosphate and hexakisphosphate content in shredded carrots. The transcripts of multi-glutamate receptor-like channels (DcGLRs), the glutamate and γ-aminobutyric acid (GABA) content, and Ca2+ influx were induced by CaCl2 treatment in shredded carrots during storage. However, PI-PLC inhibitor (U73122) treatment inhibited the activation of PI-PLC, the increase of many DcGLRs family genes expression levels, and Ca2+ influx. Moreover, the identification of DcPI-PLC4/6 and DcGLRs proteins, along with the analysis of characteristic domains such as PLCXc, PLCYc, C2 domain, transmembranous regions, and ligand binding domain, suggests their involvement in phospholipid catalysis and calcium transport in carrots. Furthermore, DcPI-PLC4/6 overexpression in tobacco leaves induced the Ca2+ influx by activating the expressions of NtGLRs and the accumulation of glutamate and GABA. These findings collectively indicate that CaCl2 treatment-induced PI-PLC activation influences DcGLRs expression levels to mediate cytosolic Ca2+ influx, thus, highlighting the “PI-PLC-GLRs-Ca2+” pathway in calcium signaling generation and GABA biosynthesis in shredded carrots.

A guide to selecting high-performing antibodies for PLC-gamma-2 for use in Western Blot, immunoprecipitation and immunofluorescence

Phosphatidylinositol-specific phospholipase C gamma 2 (PLC-gamma-2) is an enzyme that regulates the function of immune cells. PLC-gamma-2 has been implicated in neurodegenerative and autoimmune disorders, yet investigation of this protein has been limited by a lack of independently characterized antibodies. Here we have characterized eleven PLC-gamma-2 commercial antibodies for use in Western Blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. These studies are part of a larger, collaborative initiative seeking to address antibody reproducibility issues by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs.

Enzymatic activity of bone markers on Lithobates catesbeianus (Shaw, 1802) growth during the ossification process.

In order to better understand the ossification processes in anurans our study was carried out on tadpoles and adults of Lithobates catesbeianus. In this sense, we characterized the kinetic properties of alkaline phosphatase with p-nitrophenylphosphatase (pNPP) and pyrophosphate (PPi) and evaluated the activities of tartrate-resistant acid phosphatase and acid phosphatase. The enzyme extracts were obtained from tadpoles and adult femurs, which were divided into epiphysis and diaphysis. After homogenization, the samples were submitted to differential centrifugation to obtain cell membranes and, further, to phospholipase C (PIPLC) treatment, to remove membrane-bound proteins anchored by phosphatidylinositol. The average of specific activity for pNPP hydrolysis (at pH 10.5) by alkaline phosphatase released by phosphatidylinositol-specific phospholipase C (PIPLC) from Bacillus cereus among different bone regions at different animal ages was 1,142.57 U.mg-1, while for PPi hydrolysis (at pH 8.0), it was 1,433.82 U.mg-1. Among the compounds tested for enzymatic activity, the one that influenced the most was EDTA, with approximately 67% of inhibition for pNPPase activity and 77% for PPase activity. In the case of kinetic parameters, the enzyme showed a "Michaelian" behavior for pNPP and PPi hydrolysis. The Km value was around 0.6mM for pNPPase activity and ranged from 0.01 to 0.11mM for PPase activity, indicating that the enzyme has a higher affinity for this substrate. The study of pNPP and PPi hydrolysis by the enzyme revealed that the optimum pH of actuation for pNPP was 10.5, while for PPi, which is considered the true substrate of alkaline phosphatase, was 8.0, close to the physiological value. The results show that regardless of the ossification type that occurs, the same enzyme or isoenzymes act on the different bone regions and different life stages of anurans. The similarity of the results of studies with other vertebrates shows that anurans can be considered excellent animal models for the study of biological calcification.

Proposed three-phenylalanine motif involved in magnetoreception signalling of an Actinopterygii protein expressed in mammalian cells.

Studies at the cellular and molecular level of magnetoreception-sensing and responding to magnetic fields-are a relatively new research area. It appears that different mechanisms of magnetoreception in animals evolved from different origins, and, therefore, many questions about its mechanisms remain left open. Here we present new information regarding the Electromagnetic Perceptive Gene (EPG) from Kryptopterus vitreolus that may serve as part of the foundation to understanding and applying magnetoreception. Using HaloTag coupled with fluorescent ligands and phosphatidylinositol specific phospholipase C we show that EPG is associated with the membrane via glycosylphosphatidylinositol anchor. EPG's function of increasing intracellular calcium was also used to generate an assay using GCaMP6m to observe the function of EPG and to compare its function with that of homologous proteins. It was also revealed that EPG relies on a motif of three phenylalanine residues to function-stably swapping these residues using site directed mutagenesis resulted in a loss of function in EPG. This information not only expands upon our current understanding of magnetoreception but may provide a foundation and template to continue characterizing and discovering more within the emerging field.

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.

Systemic Review of Clot Retraction Modulators.

Through a process termed clot retraction, platelets cause thrombi to shrink and become more stable. After platelets are activated via inside-out signaling, glycoprotein αIIbβIII binds to fibrinogen and initiates a cascade of intracellular signaling that ends in actin remodeling, which causes the platelet to change its shape. Clot retraction is also important for wound healing. Although the detailed molecular biology of clot retraction is only partially understood, various substances and physiological conditions modulate clot retraction. In this review, we describe some of the current literature pertaining to clot retraction modulators. In addition, we discuss compounds from Cudrania trucuspidata, Arctium lappa, and Panax ginseng that diminish clot retraction and have numerous other health benefits. Caffeic acid and diindolylmethane, both common in plants and vegetables, likewise reduce clot retraction, as do all-trans retinoic acid (a vitamin A derivative), two MAP4K inhibitors, and the chemotherapeutic drug Dasatinib. Conversely, the endogenous anticoagulant Protein S (PS) and the matricellular protein secreted modular calcium-binding protein 1 (SMOC1) both enhance clot retraction. Most studies aiming to identify mechanisms of clot retraction modulators have focused on the increased phosphorylation of vasodilator-stimulated phosphoprotein and inositol 1,4,5-triphosphate receptor I and the decreased phosphorylation of various phospholipases (e.g., phospholipase A2 (PLA2) and phosphatidylinositol-specific phospholipase Cγ2 (PLCγ2), c-Jun N-terminal kinase, and (PI3Ks). One study focused on the decreased phosphorylation of Sarcoma Family Kinases (SFK), and others have focused on increased cAMP levels and the downregulation of inflammatory markers such as thromboxanes, including thromboxane A2 (TXA2) and thromboxane B2 (TXB2); prostaglandin A2 (PGE2); reactive oxygen species (ROS); and cyclooxygenase (COX) enzyme activity. Additionally, pregnancy, fibrinolysis, and the autoimmune condition systemic lupus erythematosus all seem to affect, or at least have some relation with, clot retraction. All the clot retraction modulators need in-depth study to explain these effects.

A live attenuated DIVA vaccine affords protection against Listeria monocytogenes challenge in sheep

Listeria monocytogenes (Lm) is a deadly foodborne pathogen that comprises 14 serotypes, among which, serotype 4b Lm is the primary cause of listeriosis outbreaks in humans and animals. Here, we evaluated the safety, immunogenicity, and protective efficacy of a serotype 4b vaccine candidate Lm NTSNΔactA/plcB/orfX in sheep. The infection dynamics, clinical features, and pathological observation verified that the triple genes deletion strain has adequate safety for sheep. Moreover, NTSNΔactA/plcB/orfX significantly stimulated humoral immune response and provided 78% immune protection to sheep against lethal wild-type strain challenge. Notably, the attenuated vaccine candidate could differentiate infected and vaccinated animals (DIVA) via serology determination of the antibody against listeriolysin O (LLO, encoded by hly) and phosphatidylinositol-specific phospholipase C (PI-PLC, encoded by plcB). These data suggest that the serotype 4b vaccine candidate has high efficacy, safety, and DIVA characteristics, and may be used to prevent Lm infection in sheep. Our study provides a theoretical basis for its future application in livestock and poultry breeding.

A lipid scramblase TMEM41B is involved in the processing and transport of GPI-anchored proteins.

Protein modification by glycosylphosphatidylinositol (GPI) takes place in the endoplasmic reticulum (ER). GPI-anchored proteins (GPI-APs) formed in the ER are transported to the cell surface through the Golgi apparatus. During transport, the GPI-anchor structure is processed. In most cells, an acyl chain modified to the inositol of GPI is removed by a GPI-inositol deacylase, PGAP1, in the ER. Inositol-deacylated GPI-APs become sensitive to bacterial phosphatidylinositol-specific phospholipase C (PI-PLC). We previously reported that GPI-APs are partially resistant to PI-PLC when PGAP1 activity is weakened by the deletion of selenoprotein T (SELT) or cleft lip and palate transmembrane protein 1 (CLPTM1). In this study, we found that the loss of TMEM41B, an ER-localized lipid scramblase, restored PI-PLC sensitivity of GPI-APs in SELT-knockout (KO) and CLPTM1-KO cells. In TMEM41B-KO cells, the transport of GPI-APs as well as transmembrane proteins from the ER to the Golgi was delayed. Furthermore, the turnover of PGAP1, which is mediated by ER-associated degradation, was slowed in TMEM41B-KO cells. Taken together, these findings indicate that inhibition of TMEM41B-dependent lipid scrambling promotes GPI-AP processing in the ER through PGAP1 stabilization and slowed protein trafficking.

Genome-wide investigation of the phospholipase C gene family in Solanum lycopersicum and abiotic stress analysis

Phospholipase C (PLC) can hydrolyze phospholipids to produce phospholipid signals and participate in plant stress response. In this study, we used the HMM method to identify PLC family members in the whole genome of tomato (Solanum lycopersicum L.). Based on the 7 known phosphatidylinositol-specific phospholipase C (SlPI-PLC) genes, 3 nonspecific phospholipase C (NPC) were newly identified. We found that SlPLCs contained a classical conserved domain and high homology with dicotyledonous Arabidopsis PLCs through the domain and evolutionary relationship of 10 genes. A variety of abiotic stress- and hormone-related elements were identified through cis-acting element analysis. Quantitative real-time polymerase chain reaction showed that most of the PLC members expressed differentially under abiotic stresses (low temperature, high temperature, drought, and high salt) and various developmental stages with condition/stage-specific expression and overlapping expression. The expression of SlPLC4 and SlPLC3 increased under low-temperature treatment and salt treatment. Under low temperature and salt stress, net photosynthetic rate and Fv/Fm decreased and electrical leakage increased in TRV-SlPLC4- or TRV-SlPLC3-silenced plants, indicating that PLC gene silencing can decrease abiotic stress resistance in tomato plants. Yeast Two-Hybrid experiment showed that 8 pairs of PLCs interact with each other, which may form dimer polymers within the family to perform specific functions. This study contributes to the understanding of the genome and function of SLPLCs and provides a foundation for further elucidation of the potential mechanism underlying the role of PLC in abiotic stress in tomatoes.

Development of a highly efficient oil degumming process using a novel phosphatidylinositol-specific phospholipase C enzyme from Bacillus thuringiensis PL14

The present work reports the heterologous expression and biochemical characterization of a novel phosphatidylinositol phospholipase C from Bacillus thuringiensis PL14 (PI-PLCBt) with potential for oil degumming. As degumming is a necessary refining step for all crude vegetable oils, enzymatic degumming tests were performed on crude soybean oil using rPI-PLCBt. Its efficiency was investigated by determining the phosphorus and diacylglycerol (DAG) contents after different types of treatment. Although the water and chemical degumming process can efficiently reduce the phosphorus content, a specific enzymatic process can still be selected giving its high degumming efficiency of 89.4%, with a noticeable reduction in phosphorus content to 13.7 mg kg−1, close to the values of the chemical approach, and a high increase in DAG. Accordingly, enzymatic degumming could be adopted as an alternative to traditional degumming making the process more efficient, eco-friendly and, at the same time, increasing the nutritional value of the final product.

Calcium Involved in the Enrichment of γ-Aminobutyric Acid (GABA) in Broccoli Sprouts under Fructose Treatment.

The effect of fructose on γ-aminobutyric acid (GABA) content and its metabolic pathway in broccoli sprouts was investigated. The results demonstrated that the fructose treatment not only significantly increased the fresh weight, GABA, and glutamate contents in sprouts, but also promoted the activity of glutamic acid decarboxylase (GAD) and the expressions of BoGAD1 and BoGAD2. Meanwhile, fructose treatment inhibited the stem length of broccoli sprouts and enhanced the abscisic acid (ABA) production in comparison with the control. Ca2+, CaM contents, and BoCaM2 expression in broccoli sprouts were also stimulated after fructose treatment. Exogenous fructose increased inositol trisphosphate (IP3) content and activated the activity of phosphatidylinositol-specific phospholipase C (PI-PLC) and the expression of BoPLC2, contributing to Ca2+ influx into the cells. These results suggested that Ca2+ played an essential role in GABA enrichment under fructose treatment, which may be associated with GAD and PI-PLC.

A 14-3-3 protein positively regulates rice salt tolerance by stabilizing phospholipase C1.

The phosphatidylinositol-specific phospholipase Cs (PI-PLCs) catalyze the hydrolysis of phosphatidylinositols, which play crucial roles in signaling transduction during plant development and stress response. However, the regulation of PI-PLC is still poorly understood. A previous study showed that a rice PI-PLC, OsPLC1, was essential to rice salt tolerance. Here, we identified a 14-3-3 protein, OsGF14b, as an interaction partner of OsPLC1. Similar to OsPLC1, OsGF14b also positively regulates rice salt tolerance, and their interaction can be promoted by NaCl stress. OsGF14b also positively regulated the hydrolysis activity of OsPLC1, and is essential to NaCl-induced activation of rice PI-PLCs. We further discovered that OsPLC1 was degraded via ubiquitin-proteasome pathway, and OsGF14b could inhibit the ubiquitination of OsPLC1 to protect OsPLC1 from degradation. Under salt stress, the OsPLC1 protein level in osgf14b was lower than the corresponding value of WT, whereas overexpression of OsGF14b results in a significant increase of OsPLC1 stability. Taken together, we propose that OsGF14b can interact with OsPLC1 and promote its activity and stability, thereby improving rice salt tolerance. This study provides novel insights into the important roles of 14-3-3 proteins in regulating protein stability and function in response to salt stress.

Molecular docking and dynamic simulation studies of terpenoid compounds against phosphatidylinositol-specific phospholipase C from Listeria monocytogenes

Listeriosis has emerged to be one of the life-threatening foodborne diseases worldwide, especially for immunocompromised individuals. The aim of this study was to develop a potential anti-bacterial drug with plant phytocompounds against phosphatidylinositol-specific phospholipase C from Listeria monocytogenes. A series of forty terpenoid compounds were selected based on Lipinski's rule of 5 and were subjected to molecular docking and dynamic simulation studies. From the results, Vouacapan was identified to be the promising compound out of the forty terpenoid compounds as it exhibited a good docking score of −7.7 kcal/mol and better stability with the protein in 100ns MD trajectory. Using MM/GBSA method, a total binding free energy value of −14.25 kcal/mol was observed for the Vouacapan-protein complex. The study offers a molecular level understanding of interactions of the selected compounds against the virulent protein.

Role of Integrins Involved in Mn2+-Dependent Adhesion to Fibronectin Peptide of Mastocytoma P-815 Cells and Peritoneal Mast Cells

Mn2+-dependent integrin-mediated adhesion to the extracellular matrix is extensively studied, however, its implication in mast cell biology remains unexplored. This study aims to investigate the role of Mn2+ in promoting adhesion in mouse mastocytoma P-815 cells (P-815) and peritoneal mast cells (PMC) to the Arg-Gly-Asp (RGD)-enriched fibronectin peptide (RGD matrix) within the culture medium. Our findings indicate that Mn2+ induces cell adhesion, with optimal results achieved when P-815 were exposed to 2 mM Mn2+ for 30 min at 37ºC, resulting in approximately 40% cell adhesion to the RGD matrix. The Mn2+-dependent P-815 adhesion was inhibited by anti-integrin α4, β1, and β3 subunit function-blocking antibodies, and by the integrin αIIbβ3 antagonist tirofiban, indicating the involvement of integrins α4β1 and αIIbβ3. Similarly, Mn2+-dependent PMC adhesion to the RGD matrix was inhibited by anti-integrin α4, α5, β1, β3, and β7 subunit function-blocking antibodies and tirofiban, demonstrating the involvement of integrins α4β1, α4β7, α5β1, and αIIbβ3. Integrins α4β1 and αIIbβ3 were consistently involved in Mn2+-induced adhesion reactions in both P-815 and PMC, while integrins α4β7 and α5β1 were specifically implicated in the response to PMC only. The addition of the actin inhibitor cytochalasin D, glycosylphosphatidylinositol-anchored protein (GPI-AP) cleaving enzyme phosphatidylinositol-specific phospholipase C, and the PKA inhibitor H-89 significantly reduced Mn2+-dependent P-815 adhesion to the RGD matrix. However, adding the myosin II inhibitor brebbistatin and the RhoA inhibitor Y27632 did not produce the same effect. Furthermore, cellular cholesterol removal with 6-O-α-maltosyl-β cyclodextrin significantly diminished Mn2+-dependent P-815 adhesion, concomitant with a decrease in the expression of integrin α4 and β1 subunits on the cell surface.

Phosphatidylcholine Cation-Tyrosine π Complexes: Motifs for Membrane Binding by a Bacterial Phospholipase C.

Phosphatidylinositol-specific phospholipase C (PI-PLC) enzymes are a virulence factor in many Gram-positive organisms. The specific activity of the Bacillus thuringiensis PI-PLC is significantly increased by adding phosphatidylcholine (PC) to vesicles composed of the substrate phosphatidylinositol, in part because the inclusion of PC reduces the apparent Kd for the vesicle binding by as much as 1000-fold when comparing PC-rich vesicles to PI vesicles. This review summarizes (i) the experimental work that localized a site on BtPI-PLC where PC is bound as a PC choline cation—Tyr-π complex and (ii) the computational work (including all-atom molecular dynamics simulations) that refined the original complex and found a second persistent PC cation—Tyr-π complex. Both complexes are critical for vesicle binding. These results have led to a model for PC functioning as an allosteric effector of the enzyme by altering the protein dynamics and stabilizing an ‘open’ active site conformation.

A subtractive proteomics and immunoinformatics approach towards designing a potential multi-epitope vaccine against pathogenic Listeriamonocytogenes

Listeria monocytogenes is the causative agent of listeriosis, which is dangerous for pregnant women, the elderly or individuals with a weakened immune system. Individuals with leukaemia, cancer, HIV/AIDS, kidney transplant and steroid therapy suffer from immunological damage are menaced. World Health Organization (WHO) reports that human listeriosis has a high mortality rate of 20–30% every year. To date, no vaccine is available to treat listeriosis. Thereby, it is high time to design novel vaccines against L. monocytogenes. Here, we present computational approaches to design an antigenic, stable and safe vaccine against the L. monocytogenes that could help to control the infections associated with the pathogen. Three vital pathogenic proteins of L. monocytogenes, such as Listeriolysin O (LLO), Phosphatidylinositol-specific phospholipase C (PI-PLC), and Actin polymerization protein (ActA), were selected using a subtractive proteomics approach to design the multi-epitope vaccine (MEV). A total of 5 Cytotoxic T-lymphocyte (CTL) and 9 Helper T-lymphocyte (HTL) epitopes were predicted from these selected proteins. To design the multi-epitope vaccine (MEV) from the selected proteins, CTL epitopes were joined with the AAY linker, and HTL epitopes were joined with the GPGPG linker. Additionally, a human β-defensin-3 (hBD-3) adjuvant was added to the N-terminal side of the final MEV construct to increase the immune response to the vaccine. The final MEV was predicted to be antigenic, non-allergen and non-toxic in nature. Physicochemical property analysis suggested that the MEV construct is stable and could be easily purified through the E. coli expression system. This in-silico study showed that MEV has a robust binding interaction with Toll-like receptor 2 (TLR2), a key player in the innate immune system. Current subtractive proteomics and immunoinformatics study provides a background for designing a suitable, safe and effective vaccine against pathogenic L. monocytogenes.

PLCγ2 impacts microglia-related effectors revealing variants and pathways important in Alzheimer’s disease

Alzheimer’s disease (AD) is an irreversible neurodegenerative disease mainly characterized by memory loss and cognitive decline. The etiology of AD is complex and remains incompletely understood. In recent years, genome-wide association studies (GWAS) have increasingly highlighted the central role of microglia in AD pathology. As a trans-membrane receptor specifically present on the microglia in the central nervous system, phosphatidylinositol-specific phospholipase C gamma 2 (PLCγ2) plays an important role in neuroinflammation. GWAS data and corresponding pathological research have explored the effects of PLCG2 variants on amyloid burden and tau pathologies that underline AD. The link between PLCγ2 and other AD-related effectors in human and mouse microglia has also been established, placing PLCγ2 downstream of the triggering receptor expressed on myeloid cells 2 (TREM2), toll-like receptor 4 (TLR4), Bruton’s tyrosine kinase (BTK), and colony-stimulating factor 1 receptor (CSF1R). Because the research on PLCγ2’s role in AD is still in its early stages, few articles have been published, therefore in this paper, we integrate the relevant research published to date, review the structural features, expression patterns, and related pathways of PLCγ2, and summarize the recent studies on important PLCG2 variants related to AD. Furthermore, the possibility and challenge of using PLCγ2 to develop therapeutic drugs for AD are also discussed.

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.

Soil Collected in the Great Smoky Mountains National Park Yielded a Novel Listeria sensu stricto Species, L. swaminathanii.

ABSTRACTSoil samples collected in the Great Smoky Mountains National Park yielded a Listeria isolate that could not be classified to the species level. Whole-genome sequence-based average nucleotide identity BLAST and in silico DNA-DNA Hybridization analyses confirmed this isolate to be a novel Listeria sensu stricto species with the highest similarity to L. marthii (ANI = 93.9%, isDDH = 55.9%). Additional whole-genome-based analysis using the Genome Taxonomy Database Toolkit further supported delineation as a novel Listeria sensu stricto species, as this tool failed to assign a species identification. Phenotypic and genotypic characterization results indicate that this species is nonpathogenic. Specifically, the novel Listeria species described here is phenotypically (i) nonhemolytic and (ii) negative for phosphatidylinositol-specific phospholipase C activity; the draft genome lacks all virulence genes found in the Listeria pathogenicity islands 1, 2, 3, and 4 as well as the internalin genes inlA and inlB. While the type strain contains an apparently intact catalase gene (kat), this strain is phenotypically catalase-negative (an unusual characteristic for Listeria sensu stricto species). Additional analyses identified a nonsynonymous mutation in a conserved codon of kat that is likely linked to the catalase-negative phenotype. Rapid species identification systems, including two biochemical and one matrix-assisted laser desorption/ionization, misidentified this novel species as either L. monocytogenes, L. innocua, or L. marthii. We propose the name L. swaminathanii, and the type strain is FSL L7-0020T (=ATCC TSD-239T).IMPORTANCE L. swaminathanii is a novel sensu stricto species that originated from a US National Park and it will be the first Listeria identified to date without official standing in the nomenclature. Validation was impeded by the National Park’s requirements for strain access, ultimately deemed too restrictive by the International Committee on Systematics of Prokaryotes. However, lack of valid status should not detract from the significance of adding a novel species to the Listeria sensu stricto clade. Notably, detection of non-monocytogenes sensu stricto species in a food processing environment indicate conditions that could facilitate the presence of the pathogen L. monocytogenes. If isolated, our data show a potential for L. swaminathanii to be misidentified as another sensu stricto, notably L. monocytogenes. Therefore, developers of Listeria spp. detection and identification methods, who historically only include validly published species in their validation studies, should include L. swaminathanii to ensure accurate results.

Identification and Characterisation of a Novel Phosphatidylinositol-Specific Phospholipase C Having Implications in the Pathogenesis of Leptospirosis

Identification and Characterisation of a Novel Phosphatidylinositol-Specific Phospholipase C Having Implications in the Pathogenesis of Leptospirosis