Recent approaches for the synthesis of pyridines and (iso)quinolines using propargylic Alcohols.

Propargylic alcohols are one of the readily available and highly explored building blocks in organic synthesis. They show distinct reactivities compared to simple alcohols and/or alkynes, and hence provide diverse possibilities to develop novel synthetic strategies for the construction of polycyclic systems, including heterocycles. The six-membered heterocycles, pyridines, quinolines, and isoquinolines, are very important privileged structures in medicinal chemistry and drug discovery due to their broad spectrum of biological activities. They are also part of vitamins, nucleic acids, pharmaceuticals, antibiotics, dyes, and agrochemicals. Many synthetic strategies have been developed for the rapid and efficient generation of these cyclic systems. One such strategy is employing the propargylic alcohols as reactants in the form of either a 3-carbon component or 2-carbon unit. Thus, in this review article, we aimed to summarize various approaches to pyridines, quinolines, and isoquinolines from propargylic alcohols. To the best of our knowledge, so far, no focused reviews have appeared on this topic in the literature. Due to the many reports available, we also restricted ourselves to the developments during the past 17 years, i.e., 2005-2021. We strongly believe that this review article provides comprehensive coverage of research articles on the title topic, and will be of great value for the organic synthetic community for further developments in this area of research.

Distribution and localization of 3ß-hydroxysteroid dehydrogenase (3ß-HSD) in the brain and its regions of the catfish Heteropneustes fossilis.

In vertebrates, steroids are synthesized de novo in the central and peripheral nervous system, independent of peripheral steroidogenic glands, such as the adrenal, gonads and placenta. 3ß-Hydroxysteroid dehydrogenase/Δ5-Δ4-isomerase (3ß-HSD) is a key steroidogenic enzyme in vertebrate gonads, placenta and adrenal. It mediates the oxidation and isomerization reactions of progesterone from pregnenolone, 17-hydroxyprogesterone from 17-hydroxypregnenolone and androstenedione from dehydroepiandrosterone. In the present study, we examined the expression of 3ß-HSD cDNA by real time-PCR and localization of the mRNA by in situ hybridization in the brain and its regions during the different phases of the reproductive cycle of the catfish Heteropneustes fossilis. Further, 3ß-HSD activity was assayed biochemically to show seasonal variations. We showed significant seasonal and sexual dimorphic changes in the levels of transcript abundance in the whole brain and its regions. In whole brain, level was the highest in post-spawning phase and lowest in spawning phase in males. In females, there was a progressive increase through resting phase to pre-spawning phase, a decline in the spawning phase and increase in the post-spawning phase. In the preparatory phase, the highest transcript level was seen in medulla oblongata and the lowest in pituitary in males. In females, the level was the highest in the hypothalamus and lowest in olfactory bulb and pituitary. However, in the pre-spawning phase, in males it was the highest in telencephalon and hypothalamus and lowest in pituitary. In females, the highest transcript level was in olfactory bulb and lowest in pituitary. 3ß-HSD enzyme activity showed significant seasonal variation in the brain, the highest in the resting phase and lowest in the preparatory and spawning phases. In situ hybridization showed the presence of 3ß-HSD transcript was especially high in the cerebellum region. The presence of 3ß-HSD in the brain may indicate steroidogenesis in the catfish brain.

An anaerobic bacterium, Bacteroides thetaiotaomicron, uses a consortium of enzymes to scavenge hydrogen peroxide.

Obligate anaerobes are periodically exposed to oxygen, and it has been conjectured that on such occasions their low-potential biochemistry will predispose them to rapid ROS formation. We sought to identify scavenging enzymes that might protect the anaerobe Bacteroides thetaiotaomicron from the H2 O2 that would be formed. Genetic analysis of eight candidate enzymes revealed that four of these scavenge H2 O2  in vivo: rubrerythrins 1 and 2, AhpCF, and catalase E. The rubrerythrins served as key peroxidases under anoxic conditions. However, they quickly lost activity upon aeration, and AhpCF and catalase were induced to compensate. The AhpCF is an NADH peroxidase that effectively degraded low micromolar levels of H2 O2 , while the catalytic cycle of catalase enabled it to quickly degrade higher concentrations that might arise from exogenous sources. Using a non-scavenging mutant we verified that endogenous H2 O2 formation was much higher in aerated B. thetaiotaomicron than in Escherichia coli. Indeed, the OxyR stress response to H2 O2 was induced when B. thetaiotaomicron was aerated, and in that circumstance this response was necessary to forestall cell death. Thus aeration is a serious threat for this obligate anaerobe, and to cope it employs a set of defences that includes a repertoire of complementary scavenging enzymes.

Drug-target prediction through self supervised learning with dual task ensemble approach.

Drug-Target interaction (DTI) prediction, a transformative approach in pharmaceutical research, seeks novel therapeutic applications for computational method based virtual screening, existing drugs to address untreated diseases and discovery of existing drugs side effects. The proposed model predict DTI through Heterogeneous biological network by combining drug, genes and disease related knowledge. For the purpose of embedding extraction Self-supervised learning (SSL) has been used which, trains models through pretext tasks, eliminating the need for manual annotations. The pretext tasks are related to either structural based information or similarity based information. To mitigate GNN vulnerability to non-robustness, ensemble learning can be incorporated into GNNs, harnessing multiple models to enhance robustness. This paper introduces a Graph neural network based architecture consisting of task based module and ensemble module for link prediction of DTI. The ensemble module of dual task combinations, both in cold start and warm start scenarios achieve very good performance as it provide 0.960 in cold start and 0.970 in warm start mean AUCROC score with less deviation.

Tissue specific tumor-gene link prediction through sampling based GNN using a heterogeneous network.

A tissue sample is a valuable resource for understanding a patient's symptoms and health status in relation to tumor growth. Recent research seeks to establish a connection between tissue-specific tumor samples and genetic markers (genes). This breakthrough has paved the way for personalized cancer therapies. With this motivation, the proposed model constructs a heterogeneous network based on tumor sample-gene relation data and gene-gene interaction data. This network also incorporates tissue-specific gene expression and primary site-based gene counts as features, enabling tissue-specific predictions. Graph neural networks (GNNs) have proven effective in modeling complex interactions and predicting links within this network. The proposed model has successfully predicted tumor-gene associations by leveraging sampling-based GNNs and link layer embedding. The model's performance metrics, such as AUC-ROC scores, reached approximately 94%, demonstrating the potential of this heterogeneous network in predicting tissue-specific tumor sample-gene links. This paper's findings highlight the importance of tissue-specific associations in cancer research.

Synthesis of Functionalized Pyrimidines from Propargylic alcohols and their Derivatives: Two Decades of Developments.

Pyrimidine is a six-membered diaza-heterocycle i. e., 1,3-diazine. It is found to be present in many biologically and pharmacologically active scaffolds like nucleotides, natural products, and drugs. The bioactivities of pyrimidine include anti-tubercular, anti-bacterial, anti-fungal, anti-viral, anti-inflammatory, anti-malarial, anti-cancer, anti-neoplastic and many more. In this review article we have summarized various synthetic approaches that involve the synthesis of these privileged building blocks by employing propargylic alcohols and their derivatives like propargylic esters and propargylic ynones as three carbon-components. Here, we have confined ourselves to the developments appeared during the period of 23 years i. e., 2000-2022.

Prevalence of Hyperglycemia in Pregnancy and Related Screening Practices in Rural Dehradun: The First Population-Based Study from Uttarakhand (PGDRD-1).

Aims: Phase I of the Prevalence of Gestational Diabetes Mellitus in Rural Dehradun (PGDRD) project estimates hyperglycemia in pregnancy (HIP) prevalence and identifies gaps in the utilization of community-related services in rural areas of the Dehradun district (western Uttarakhand); a state where notably no prior population-based study has ever been conducted despite being an Empowered Action Group state for more than two decades. Methods: Using a multistage random sampling technique, 1,223 pregnant women locally registered in the rural field practice area of a block were identified. Those requiring HIP screening were subjected to a 2-h 75 g oral glucose tolerance test during the house visit irrespective of their period-of-gestation and last meal timings, diagnosed using the Diabetes in Pregnancy Study Group India (DIPSI) criterion (when indicated). Data were collected by personal interviews using a pretested data collection tool. Statistical Package for Social Sciences version 20.0 was used for analysis. Results: The overall HIP prevalence recorded was 9.7% (95% CI: 8.1-11.5%); the majority (95.8%) were GDM followed by overt DIP (4.2%). Less than 1% of the subjects (0.7%) self-reported pre-GDM. Despite this burden, more than three-fourths were never screened for HIP in their pregnancy. Of those tested, the majority availed secondary healthcare facilities. Few even had to bear expenses in private with a very handful being tested free-of-cost by ANM in the community; findings that altogether sharply contrast to those recommended by national protocols. Conclusion: Despite the high HIP burden, beneficiaries are unable to utilize community-related universal screening protocols as desired.

Cardiolipin occupancy profiles of YidC paralogs reveal the significance of respective TM2 helix residues in determining paralog-specific phenotypes.

YidC belongs to an evolutionarily conserved family of insertases, YidC/Oxa1/Alb3, in bacteria, mitochondria, and chloroplasts, respectively. Unlike Gram-negative bacteria, Gram-positives including Streptococcus mutans harbor two paralogs of YidC. The mechanism for paralog-specific phenotypes of bacterial YidC1 versus YidC2 has been partially attributed to the differences in their cytoplasmic domains. However, we previously identified a W138R gain-of-function mutation in the YidC1 transmembrane helix 2. YidC1W138R mostly phenocopied YidC2, yet the mechanism remained unknown. Primary sequence comparison of streptococcal YidCs led us to identify and mutate the YidC1W138 analog, YidC2S152 to W/A, which resulted in a loss of YidC2- and acquisition of YidC1-like phenotype. The predicted lipid-facing side chains of YidC1W138/YidC2S152 led us to propose a role for membrane phospholipids in specific-residue dependent phenotypes of S. mutans YidC paralogs. Cardiolipin (CL), a prevalent phospholipid in the S. mutans cytoplasmic membrane during acid stress, is encoded by a single gene, cls. We show a concerted mechanism for cardiolipin and YidC2 under acid stress based on similarly increased promoter activities and similar elimination phenotypes. Using coarse grain molecular dynamics simulations with the Martini2.2 Forcefield, YidC1 and YidC2 wild-type and mutant interactions with CL were assessed in silico. We observed substantially increased CL interaction in dimeric versus monomeric proteins, and variable CL occupancy in YidC1 and YidC2 mutant constructs that mimicked characteristics of the other wild-type paralog. Hence, paralog-specific amino acid- CL interactions contribute to YidC1 and YidC2-associated phenotypes that can be exchanged by point mutation at positions 138 or 152, respectively.

Natural compounds as a potential modifier of stem cells renewal: Comparative analysis.

Cancer stem cells (CSCs) are indispensable for development, progression, drug resistance, and tumor metastasis. Current cancer-directed interventions target targeting rapidly dividing cancer cells and slow dividing CSCs, which are the root cause of cancer origin and recurrence. The most promising targets include several self-renewal pathways involved in the maintenance and renewal of CSCs, such as the Wnt/ß-Catenin, Sonic Hedgehog, Notch, Hippo, Autophagy, and Ferroptosis. In view of safety, natural compounds are coming to the front line of treatment modalities for modifying various signaling pathways simultaneously involved in maintaining CSCs. Therefore, targeting CSCs with natural compounds is a promising approach to treating various types of cancers. In view of this, here we provide a comprehensive update on the current status of natural compounds that effectively tune key self-renewal pathways of CSCs. In addition, we highlighted surface expression markers in several types of cancer. We also emphasize how natural compounds target these self-renewal pathways to reduce therapy resistance and cancer recurrence properties of CSCs, hence providing valuable cancer therapeutic strategies. The inclusion of nutraceuticals is believed to enhance the therapeutic efficacy of current cancer-directed interventions significantly.

Why do bacteria use so many enzymes to scavenge hydrogen peroxide?

Hydrogen peroxide (H(2)O(2)) is continuously formed by the autoxidation of redox enzymes in aerobic cells, and it also enters from the environment, where it can be generated both by chemical processes and by the deliberate actions of competing organisms. Because H(2)O(2) is acutely toxic, bacteria elaborate scavenging enzymes to keep its intracellular concentration at nanomolar levels. Mutants that lack such enzymes grow poorly, suffer from high rates of mutagenesis, or even die. In order to understand how bacteria cope with oxidative stress, it is important to identify the key enzymes involved in H(2)O(2) degradation. Catalases and NADH peroxidase (Ahp) are primary scavengers in many bacteria, and their activities and physiological impacts have been unambiguously demonstrated through phenotypic analysis and through direct measurements of H(2)O(2) clearance in vivo. Yet a wide variety of additional enzymes have been proposed to serve similar roles: thiol peroxidase, bacterioferritin comigratory protein, glutathione peroxidase, cytochrome c peroxidase, and rubrerythrins. Each of these enzymes can degrade H(2)O(2) in vitro, but their contributions in vivo remain unclear. In this review we examine the genetic, genomic, regulatory, and biochemical evidence that each of these is a bonafide scavenger of H(2)O(2) in the cell. We also consider possible reasons that bacteria might require multiple enzymes to catalyze this process, including differences in substrate specificity, compartmentalization, cofactor requirements, kinetic optima, and enzyme stability. It is hoped that the resolution of these issues will lead to an understanding of stress resistance that is more accurate and perceptive.

Clinical profile and in-hospital outcomes of COVID-19 patients: Findings from secondary data analysis.

Background: Uttarakhand, a hilly state in north India, reported the first coronavirus disease (COVID) case on 15 March 2020. Since then, the case numbers rose multiple folds. As Uttarakhand has been on a 'war-footing' amidst the recent second wave and is gearing up to fight against the third wave, the present study aims to uncover baseline clinical profile and in-hospital outcomes of COVID patients in Dehradun district (Uttarakhand) during the first wave. Methods: A record-based descriptive analysis was carried out for 671 COVID patients admitted to a private dedicated COVID hospital in Dehradun district between August 2020 and February 2021. Data was collected from medical records on a standardized abstraction form. Data was entered and analyzed using Statistical Package for Social Sciences (SPSS) version 20. Results: The present study showed most admitted COVID patients were males, aged 40 years and above, moderately ill, had co-morbidities with about one-fourth lately succumbed to death. The proportions of deaths, moderate-to-severe and severe category of illness were invariably high for those with co-morbidities irrespective of the gender. Females, age <60 years, and absence of co-morbidities had overall high mean survival estimates from COVID. Conclusion: Females, younger age group, and absence of co-morbidities are more likely to survive from COVID than males, older age groups, and those with co-morbidities.

The Cytoplasmic Domains of Streptococcus mutans Membrane Protein Insertases YidC1 and YidC2 Confer Unique Structural and Functional Attributes to Each Paralog.

Integral and membrane-anchored proteins are pivotal to survival and virulence of the dental pathogen, Streptococcus mutans. The bacterial chaperone/insertase, YidC, contributes to membrane protein translocation. Unlike Escherichia coli, most Gram-positive bacteria contain two YidC paralogs. Herein, we evaluated structural features that functionally delineate S. mutans YidC1 and YidC2. Bacterial YidCs contain five transmembrane domains (TMD), two cytoplasmic loops, and a cytoplasmic tail. Because S. mutans YidC1 (SmYidC1) and YidC2 (SmYidC2) cytoplasmic domains (CD) are less well conserved than are TMD, we engineered ectopic expression of the 14 possible YidC1-YidC2 CD domain swap combinations. Growth and stress tolerance of each was compared to control strains ectopically expressing unmodified yidC1 or yidC2. Acid and osmotic stress sensitivity are associated with yidC2 deletion. Sensitivity to excess zinc was further identified as a ΔyidC1 phenotype. Overall, YidC1 tolerated CD substitutions better than YidC2. Preferences toward particular CD combinations suggested potential intramolecular interactions. In silico analysis predicted salt-bridges between C1 and C2 loops of YidC1, and C1 loop and C-terminal tail of YidC2, respectively. Mutation of contributing residues recapitulated ΔyidC1- and ΔyidC2-associated phenotypes. Taken together, this work revealed the importance of cytoplasmic domains in distinct functional attributes of YidC1 and YidC2, and identified key residues involved in interdomain interactions.

Protein Interactomes of Streptococcus mutans YidC1 and YidC2 Membrane Protein Insertases Suggest SRP Pathway-Independent- and -Dependent Functions, Respectively.

Virulence properties of cariogenic Streptococcus mutans depend on integral membrane proteins. Bacterial cotranslational protein trafficking involves the signal recognition particle (SRP) pathway components Ffh and FtsY, the SecYEG translocon, and YidC chaperone/insertases. Unlike Escherichia coli, S. mutans survives loss of the SRP pathway and has two yidC paralogs. This study characterized YidC1 and YidC2 interactomes to clarify respective functions alone and in concert with the SRP and/or Sec translocon. Western blots of formaldehyde cross-linked or untreated S. mutans lysates were reacted with anti-Ffh, anti-FtsY, anti-YidC1, or anti-YidC2 antibodies followed by mass spectrometry (MS) analysis of gel-shifted bands. Cross-linked lysates of wild-type and ΔyidC2 strains were reacted with anti-YidC2-coupled Dynabeads, and cocaptured proteins were identified by MS. Last, YidC1 and YidC2 C-terminal tail-captured proteins were subjected to two-dimensional (2D) difference gel electrophoresis and MS analysis. Direct interactions of putative YidC1 and YidC2 binding partners were confirmed by bacterial two-hybrid assay. Our results suggest YidC2 works preferentially with the SRP pathway, while YidC1 is preferred for SRP-independent Sec translocon-mediated translocation. YidC1 and YidC2 autonomous pathways were also apparent. Two-hybrid assay identified interactions between holotranslocon components SecYEG/YajC and YidC1. Both YidC1 and YidC2 interacted with Ffh, FtsY, and chaperones DnaK and RopA. Putative membrane-localized substrates HlyX, LemA, and SMU_591c interacted with both YidC1 and YidC2. Identification of several Rgp proteins in the YidC1 interactome suggested its involvement in bacitracin resistance, which was decreased in ΔyidC1 and SRP-deficient mutants. Collectively, YidC1 and YidC2 interactome analyses has further distinguished these paralogs in the Gram-positive bacterium S. mutansIMPORTANCEStreptococcus mutans is a prevalent oral pathogen and major causative agent of tooth decay. Many proteins that enable this bacterium to thrive in its environmental niche and cause disease are embedded in its cytoplasmic membrane. The machinery that transports proteins into bacterial membranes differs between Gram-negative and Gram-positive organisms, an important difference being the presence of multiple YidC paralogs in Gram-positive bacteria. Characterization of a protein's interactome can help define its physiological role. Herein, we characterized the interactomes of S. mutans YidC1 and YidC2. Results demonstrated substantial overlap between their interactomes but also revealed several differences in their direct protein binding partners. Membrane transport machinery components were identified in the context of a large network of proteins involved in replication, transcription, translation, and cell division/cell shape. This information contributes to our understanding of protein transport in Gram-positive bacteria in general and informs our understanding of S. mutans pathogenesis.

Curcumin Oxidation Is Required for Inhibition of Helicobacter pylori Growth, Translocation and Phosphorylation of Cag A.

Curcumin is a potential natural remedy for preventing Helicobacter pylori-associated gastric inflammation and cancer. Here, we analyzed the effect of a phospholipid formulation of curcumin on H. pylori growth, translocation and phosphorylation of the virulence factor CagA and host protein kinase Src in vitro and in an in vivo mouse model of H. pylori infection. Growth of H. pylori was inhibited dose-dependently by curcumin in vitro. H. pylori was unable to metabolically reduce curcumin, whereas two enterobacteria, E. coli and Citrobacter rodentium, which efficiently reduced curcumin to the tetra- and hexahydro metabolites, evaded growth inhibition. Oxidative metabolism of curcumin was required for the growth inhibition of H. pylori and the translocation and phosphorylation of CagA and cSrc, since acetal- and diacetal-curcumin that do not undergo oxidative transformation were ineffective. Curcumin attenuated mRNA expression of the H. pylori virulence genes cagE and cagF in a dose-dependent manner and inhibited translocation and phosphorylation of CagA in gastric epithelial cells. H. pylori strains isolated from dietary curcumin-treated mice showed attenuated ability to induce cSrc phosphorylation and the mRNA expression of the gene encoding for IL-8, suggesting long-lasting effects of curcumin on the virulence of H. pylori. Our work provides mechanistic evidence that encourages testing of curcumin as a dietary approach to inhibit the virulence of CagA.

Asian management of hypertension: Current status, home blood pressure, and specific concerns in India.

Systemic hypertension and its related complications are the important contributing factors for major adverse cardiovascular events all over the world. Evidence from Asia and even from India reveals that both its incidence and prevalence are increasing even in young population both in urban and rural areas. The HOPE (Hypertension Cardiovascular Outcome Prevention and Evidence) Asia network data clearly say that most of these hypertensive patients are undiagnosed and undertreated. Even among the treated patients, the regular follow-up visits and compliance of antihypertensive drug intake are not effective. The blood pressure variability (BPV) and the exaggerated morning blood pressure surge (MBPS) leading to high cardiovascular mortality and morbidity have been demonstrated in many studies. The role of home blood pressure monitoring (HBPM) to detect BPV and MBPS to treat hypertensive patients more effectively has been published by the HOPE Asia Network. This article is to review the evidence and literature from the Indian perspective and the role of HBPM for the effective control of hypertension in general population.

Restricting rural-urban connect to combat infectious disease epidemic as India fights COVID-19.

With declaration of 2019 novel coronavirus disease (COVID-19) as a pandemic on 11 March 2020 by World Health Organization, India came to alert for its being at next potential risk. It reached alert Level 2, i.e. local transmission for virus spread in early March 2020 and soon thereafter alert Level 3, i.e. community transmission. With on-going rise in COVID-19 cases in country, Government of India (GoI) has been taking multiple intense measures in coordination with the state governments, such as urban lockdown, active airport screening, quarantining, aggressive calls for 'work from home', public awareness, and active case detection with contact tracing in most places. Feedback from other countries exhibits COVID-19 transmission levels to have shown within country variations. With two-third of Indian population living in rural areas, present editorial hypothesizes that if India enters Level 3, rural hinterland would also be at risk importation (at least Level 1). Hence, we have to call for stringent containment on rural-urban and inter-state fringes. This along with other on-going measures can result in flattening curve and also in staggering 'lockdowns', and thus, helping sustain national economy.

Effect of maternal perceived stress during pregnancy on gestational diabetes mellitus risk: A prospective case-control study.

BACKGROUND AND AIMS: A variety of risk factors have been reported for the development of gestational diabetes mellitus (GDM). But limited review on the role of antenatal perceived stress necessitated the design of the present study to evaluate the association between GDM and perceived stress during pregnancy. METHODS: A prospective case-control study was carried out among 100 GDM cases and 273 matched controls, attending regular antenatal clinic at two private hospitals of Karnataka. Data was collected by personal interviews using a standard questionnaire. Perceived stress was assessed using the Cohen 10-item Perceived Stress Scale. Score of ≥20 was identified as high stress. Statistical Package for the Social Sciences version 15 was used for analysis. RESULTS: Exposure rates for high maternal perceived stress among cases during pregnancy were noted. The odds of GDM were 13 folds higher among those with high antenatal stress (≥20) compared to those with low (<20) (p < 0.001) perceived stress. No correlation between maternal antenatal stress and blood glucose following OGTT was noted. CONCLUSIONS: The study identified high perceived stress during pregnancy as a potential risk factor for GDM.

A step towards real-time implementation of GDM guidelines in India: Review of Gaps in RCH Programme.

Gestational Diabetes Mellitus (GDM) risks the affected mother-child duos not only with respect to adverse perinatal outcomes but also for chronic diseases later in life. Therefore, in 2014, the Government of India (GoI) mandated universal GDM screening for all pregnant women as a part of essential obstetric care within the Reproductive and Child Health (RCH) programme. Later in 2018, the domain experts from GoI envisaged pan India implementation of GDM screening services within its RCH framework by 2023. As Uttarakhand-a hilly, EAG state of north India-would also be part of this nation-wide drive; prior identification of RCH services coverage in the State assumes paramount importance, as it reflects probable executability of GDM screening services within its delivery platform. Therefore, the present review aims to assess the readiness of Uttarakhand maternal health functionary system in view of GDM national guidelines implementation at both state and district levels. In this regard, freely accessible, full-text GoI documents pertaining to GDM implementation guidelines and maternal health program of India and Uttarakhand available in public domain in English language were reviewed. The present review favors the pilot implementation in district Dehradun prior to implementing in all districts of the state. It may, however, require overall improvement in maternal health programmatic services in all parts of the State for much efficient service delivery. Effective implementation of GDM guidelines requires urgent correction in the background performance of RCH program.

The Impacts of Sortase A and the 4'-Phosphopantetheinyl Transferase Homolog Sfp on Streptococcus mutans Extracellular Membrane Vesicle Biogenesis.

Extracellular membrane vesicles (EMVs) are produced by many Gram-positive organisms, but information regarding vesiculogenesis is incomplete. We used single gene deletions to evaluate the impacts on Streptococcus mutans EMV biogenesis of Sortase A (SrtA), which affects S. mutans EMV composition, and Sfp, a 4'-phosphopantetheinyl transferase that affects Bacillus subtilis EMV stability. ΔsrtA EMVs were notably larger than Δsfp and wild-type (WT) EMVs. EMV proteins identified from all three strains are known to be involved in cell wall biogenesis and cell architecture, bacterial adhesion, biofilm cell density and matrix development, and microbial competition. Notably, the AtlA autolysin was not processed to its mature active form in the ΔsrtA mutant. Proteomic and lipidomic analyses of all three strains revealed multiple dissimilarities between vesicular and corresponding cytoplasmic membranes (CMs). A higher proportion of EMV proteins are predicted substrates of the general secretion pathway (GSP). Accordingly, the GSP component SecA was identified as a prominent EMV-associated protein. In contrast, CMs contained more multi-pass transmembrane (TM) protein substrates of co-translational transport machineries than EMVs. EMVs from the WT, but not the mutant strains, were enriched in cardiolipin compared to CMs, and all EMVs were over-represented in polyketide flavonoids. EMVs and CMs were rich in long-chain saturated, monounsaturated, and polyunsaturated fatty acids, except for Δsfp EMVs that contained exclusively polyunsaturated fatty acids. Lipoproteins were less prevalent in EMVs of all three strains compared to their CMs. This study provides insight into biophysical characteristics of S. mutans EMVs and indicates discrete partitioning of protein and lipid components between EMVs and corresponding CMs of WT, ΔsrtA, and Δsfp strains.

Enhanced purification coupled with biophysical analyses shows cross-ß structure as a core building block for Streptococcus mutans functional amyloids.

Streptococcus mutans is an etiologic agent of human dental caries that forms dental plaque biofilms containing functional amyloids. Three amyloidogenic proteins, P1, WapA, and Smu_63c were previously identified. C123 and AgA are naturally occurring amyloid-forming fragments of P1 and WapA, respectively. We determined that four amyloidophilic dyes, ThT, CDy11, BD-oligo, and MK-H4, differentiate C123, AgA, and Smu_63c amyloid from monomers, but non-specific binding to bacterial cells in the absence of amyloid precludes their utility for identifying amyloid in biofilms. Congo red-induced birefringence is a more specific indicator of amyloid formation and differentiates biofilms formed by wild-type S. mutans from a triple ΔP1/WapA/Smu_63c mutant with reduced biofilm forming capabilities. Amyloid accumulation is a late event, appearing in older S. mutans biofilms after 60 hours of growth. Amyloid derived from pure preparations of all three proteins is visualized by electron microscopy as mat-like structures. Typical amyloid fibers become evident following protease digestion to eliminate non-specific aggregates and monomers. Amyloid mats, similar in appearance to those reported in S. mutans biofilm extracellular matrices, are reconstituted by co-incubation of monomers and amyloid fibers. X-ray fiber diffraction of amyloid mats and fibers from all three proteins demonstrate patterns reflective of a cross-ß amyloid structure.