Articoli di riviste sul tema "Gac/Rsm pathway"

In Pseudomonas fluorescens CHA0 and other fluorescent pseudomonads, the Gac/Rsm signal transduction pathway is instrumental for secondary metabolism and biocontrol of root pathogens via the expression of regulatory small RNAs (sRNAs). Furthermore, in strain CHA0, an imbalance in the Krebs cycle can affect the strain's ability to produce extracellular secondary metabolites, including biocontrol factors. Here, we report the metabolome of wild-type CHA0, a gacA-negative mutant, which has lost Gac/Rsm activities, and a retS-negative mutant, which shows strongly enhanced Gac/Rsm-dependent activities. Capillary electrophoresis-based metabolomic profiling revealed that the gacA and retS mutations had opposite effects on the intracellular levels of a number of central metabolites, suggesting that the Gac/Rsm pathway regulates not only secondary metabolism but also primary metabolism in strain CHA0. Among the regulated metabolites identified, the alarmone guanosine tetraphosphate (ppGpp) was characterized in detail by the construction of relA (for ppGpp synthase) and spoT (for ppGpp synthase/hydrolase) deletion mutants. In a relA spoT double mutant, ppGpp synthesis was completely abolished, the expression of Rsm sRNAs was attenuated, and physiological functions such as antibiotic production, root colonization, and plant protection were markedly diminished. Thus, ppGpp appears to be essential for sustaining epiphytic fitness and biocontrol activity of strain CHA0.

In Pseudomonas protegens CHA0 and other fluorescent pseudomonads, the Gac/Rsm signal transduction pathway is crucial for the expression of secondary metabolism and the biological control of fungi, nematodes, and insects. Based on the findings of a previous metabolomic study, the role of intracellular γ-aminobutyrate (GABA) as a potential signal in the Gac/Rsm pathway was investigated herein. The function and regulation of a gabDT (c01870-c01880) gene cluster in strain CHA0 were described. The gabT gene encoded GABA transaminase (GABAT) and enabled the growth of the bacterium on GABA, whereas the upstream gabD gene (annotated as a gene encoding succinic semialdehyde dehydrogenase) had an unknown function. A gacA mutant exhibited low GABAT activity, leading to the markedly greater intracellular accumulation of GABA than in the wild type. In the gacA mutant, the RsmA and RsmE proteins caused translational gabD repression, with concomitant gabT repression. Due to very low GABAT activity, the gabT mutant accumulated GABA to high levels. This trait promoted a planktonic lifestyle, reduced biofilm formation, and favored root colonization without exhibiting the highly pleiotropic gacA phenotypes. These results suggest an important role of GABA in the Gac/Rsm-regulated niche adaptation of strain CHA0 to plant roots.

Gall formation by Pantoea agglomerans pv. gypsophilae is dependent on the hypersensitive response and pathogenicity (hrp) system. Previous studies demonstrated that PagR and PagI, regulators of the quorum-sensing system, induce expression of the hrp regulatory cascade (i.e., hrpXY, hrpS, and hrpL) that activates the HrpL regulon. Here, we isolated the genes of the Gac/Rsm global regulatory pathway (i.e., gacS, gacA, rsmB, and csrD) and of the post-transcriptional regulator rsmA. Our results demonstrate that PagR and PagI also upregulate expression of the Gac/Rsm pathway. PagR acts as a transcriptional activator of each of the hrp regulatory genes and gacA in a N-butanoyl-L-homoserine lactone-dependent manner as shown by gel shift experiments. Mutants of the Gac/Rsm genes or overexpression of rsmA significantly reduced Pantoea agglomerans virulence and colonization of gypsophila. Overexpression of rsmB sRNA abolished gall formation, colonization, and hypersensitive reaction on nonhost plants and prevented transcription of the hrp regulatory cascade, indicating a lack of functional type III secretion system. Expression of rsmB sRNA in the background of the csrD null mutant suggests that CsrD may act as a safeguard for preventing excessive production of rsmB sRNA. Results presented indicate that the hrp regulatory cascade is controlled directly by PagR and indirectly by RsmA, whereas deficiency in RsmA activity is epistatic to PagR induction.

ABSTRACTInPseudomonasspp., the Gac-Rsm signal transduction system is required for the production of lipases. The current model assumes that the system induces lipase gene transcription mediated through the quorum-sensing (QS) system. However, there are no reports of a QS system based uponN-acyl homoserine lactones or the regulation of lipase gene expression inPseudomonas protegens. In this study, we investigated the regulatory mechanism acting onlipAexpression activated by the Gac-Rsm system inP. protegensPf-5 through deletion and overexpression ofgacA, overexpression ofrsmAorrsmE, expression of variouslacZfusions, reverse transcription-PCR analysis, and determination of whole-cell lipase activity. The results demonstrated that the GacS-GacA (GacS/A) system activateslipAexpression at both the transcriptional and the translational levels but that the translational level is the key regulatory pathway. Further results showed that the activation oflipAtranslation by the GacS/A system is mediated through RsmE, which inhibitslipAtranslation by binding to the ACAAGGAUGU sequence overlapping the Shine-Dalgarno (SD) sequence oflipAmRNA to hinder the access of the 30S ribosomal subunit to the SD sequence. Moreover, the GacS/A system promoteslipAtranscription through the mediation of RsmA inhibitinglipAtranscription via an unknown pathway. Besides the transcriptional repression, RsmA mainly activateslipAtranslation by negatively regulatingrsmEtranslation. In summary, inP. protegensPf-5, the Gac-RsmE system mainly and directly activateslipAtranslation and the Gac-RsmA system indirectly enhanceslipAtranscription.

Pseudomonas aeruginosa, a major inhabitant of numerous environmental reservoirs, is a momentous opportunistic human pathogen associated with severe infections even death in the patients suffering from immune deficiencies or metabolic diseases. Type III secretion system (T3SS) employed by P. aeruginosa to inject effector proteins into host cells is one of the pivotal virulence factors pertaining to acute infections caused by this pathogen. Previous studies showed that P. aeruginosa T3SS is activated by various environmental cues such as calcium concentration and the host signal spermidine. However, how T3SS is regulated and expressed particularly under the ever-changing environmental conditions remains largely elusive. In this study, we reported that a tRNA modification enzyme PA3980, designated as MiaB, positively regulated T3SS gene expression in P. aeruginosa and was essential for the induced cytotoxicity of human lung epithelial cells. Further genetic assays revealed that MiaB promoted T3SS gene expression by repressing the LadS-Gac/Rsm signaling pathway and through the T3SS master regulator ExsA. Interestingly, ladS, gacA, rsmY and rsmZ in the LadS-Gac/Rsm signaling pathway seemed potential targets under the independent regulation of MiaB. Moreover, expression of MiaB was found to be induced by the cAMP-dependent global regulator Vfr as well as the spermidine transporter-dependent signaling pathway and thereafter functioned to mediate their regulation on the T3SS gene expression. Together, these results revealed a novel regulatory mechanism for MiaB, with which it integrates different environmental cues to modulate T3SS gene expression in this important bacterial pathogen.

ABSTRACTPseudomonas fluorescens2P24 is a soilborne bacterium that synthesizes and excretes multiple antimicrobial metabolites. The polyketide compound 2,4-diacetylphloroglucinol (2,4-DAPG), synthesized by thephlACBDlocus, is its major biocontrol determinant. This study investigated two mutants defective in antagonistic activity againstRhizoctonia solani. Deletion of thegidA(PM701) ortrmE(PM702) gene from strain 2P24 completely inhibited the production of 2,4-DAPG and its precursors, monoacetylphloroglucinol (MAPG) and phloroglucinol (PG). The transcription of thephlAgene was not affected, but the translation of thephlAandphlDgenes was reduced significantly. Two components of the Gac/Rsm pathway, RsmA and RsmE, were found to be regulated bygidAandtrmE, whereas the other components, RsmX, RsmY, and RsmZ, were not. The regulation of 2,4-DAPG production bygidAandtrmE, however, was independent of the Gac/Rsm pathway. Both thegidAandtrmEmutants were unable to produce PG but could convert PG to MAPG and MAPG to 2,4-DAPG. Overexpression of PhlD in thegidAandtrmEmutants could restore the production of PG and 2,4-DAPG. Taken together, these findings suggest that GidA and TrmE are positive regulatory elements that influence the biosynthesis of 2,4-DAPG posttranscriptionally.

The GacS histidine kinase is the membrane sensor of the major upstream two-component system of the regulatory Gac/Rsm signal transduction pathway. This pathway governs the expression of a wide range of genes in pseudomonads and controls bacterial fitness and motility, tolerance to stress, biofilm formation, and virulence or plant protection. Despite the importance of these roles, the ligands binding to the sensor domain of GacS remain unknown, and their identification is an exciting challenge in this domain. At high population densities, the GacS signal triggers a switch from primary to secondary metabolism and a change in bacterial lifestyle. It has been suggested, based on these observations, that the GacS signal is a marker of the emergence of nutritional stress and competition. Biochemical investigations have yet to characterize the GacS signal fully. However, they portray this cue as a low-molecular weight, relatively simple and moderately apolar metabolite possibly resembling, but nevertheless different, from the aliphatic organic acids acting as quorum-sensing signaling molecules in other Proteobacteria. Significant progress in the development of metabolomic tools and new databases dedicated to Pseudomonas metabolism should help to unlock some of the last remaining secrets of GacS induction, making it possible to control the Gac/Rsm pathway.

ABSTRACTAzithromycin at clinically relevant doses does not inhibit planktonic growth of the opportunistic pathogenPseudomonas aeruginosabut causes markedly reduced formation of biofilms and quorum-sensing-regulated extracellular virulence factors. In the Gac/Rsm signal transduction pathway, which acts upstream of the quorum-sensing machinery inP. aeruginosa, the GacA-dependent untranslated small RNAs RsmY and RsmZ are key regulatory elements. As azithromycin treatment and mutational inactivation ofgacAhave strikingly similar phenotypic consequences, the effect of azithromycin onrsmYandrsmZexpression was investigated. In planktonically growing cells, the antibiotic strongly inhibited the expression of both small RNA genes but did not affect the expression of the housekeeping geneproC. The azithromycin treatment resulted in reduced expression ofgacAandrsmA, which are known positive regulators ofrsmYandrsmZ, and of the PA0588-PA0584 gene cluster, which was discovered as a novel positive regulatory element involved inrsmYandrsmZexpression. Deletion of this cluster resulted in diminished ability ofP. aeruginosato produce pyocyanin and to swarm. The results of this study indicate that azithromycin inhibitsrsmYandrsmZtranscription indirectly by lowering the expression of positive regulators of these small RNA genes.

The phytopathogenic bacterium Pseudomonas syringae pv. tomato DC3000 has a complex Gac-rsm global regulatory pathway that controls virulence, motility, production of secondary metabolites, carbon metabolism, and quorum sensing. However, despite the fact that components of this pathway are known, their physiological roles have not yet been established. Regarding the CsrA/RsmA type proteins, five paralogs, three of which are well conserved within the Pseudomonas genus (csrA1, csrA2, and csrA3), have been found in the DC3000 genome. To decipher their function, mutants lacking the three most conserved CsrA proteins have been constructed and their physiological outcomes examined. We show that they exert nonredundant functions and demonstrate that CsrA3 and, to a lesser extent, CsrA2 but not CsrA1 alter the expression of genes involved in a variety of pathways and systems important for motility, exopolysaccharide synthesis, growth, and virulence. Particularly, alginate synthesis, syringafactin production, and virulence are considerably de-repressed in a csrA3 mutant, whereas growth in planta is impaired. We propose that the linkage of growth and symptom development is under the control of CsrA3, which functions as a pivotal regulator of the DC3000 life cycle, repressing virulence traits and promoting cell division in response to environmental cues.

ABSTRACT In the biocontrol strain Pseudomonas fluorescens CHA0, the Gac/Rsm signal transduction pathway positively controls the synthesis of antifungal secondary metabolites and exoenzymes. In this way, the GacS/GacA two-component system determines the expression of three small regulatory RNAs (RsmX, RsmY, and RsmZ) in a process activated by the strain's own signal molecules, which are not related to N-acyl-homoserine lactones. Transposon Tn5 was used to isolate P. fluorescens CHA0 insertion mutants that expressed an rsmZ-gfp fusion at reduced levels. Five of these mutants were gacS negative, and in them the gacS mutation could be complemented for exoproduct and signal synthesis by the gacS wild-type allele. Furthermore, two thiamine-auxotrophic (thiC) mutants that exhibited decreased signal synthesis in the presence of 5 × 10−8 M thiamine were found. Under these conditions, a thiC mutant grew normally but showed reduced expression of the three small RNAs, the exoprotease AprA, and the antibiotic 2,4-diacetylphloroglucinol. In a gnotobiotic system, a thiC mutant was impaired for biological control of Pythium ultimum on cress. Addition of excess exogenous thiamine restored all deficiencies of the mutant. Thus, thiamine appears to be an important factor in the expression of biological control by P. fluorescens.

The GacS/GacA two-component system functions mechanistically in conjunction with global post-transcriptional regulators of the RsmA family to allow pseudomonads and other bacteria to adapt to changing environmental stimuli. Analysis of this Gac/Rsm signal transduction pathway in phytotoxin-producing pathovars of Pseudmonas syringae is incomplete, particularly with regard to rsmA. Our approach in studying it was to overexpress rsmA in P. syringae strains through introduction of pSK61, a plasmid constitutively expressing this gene. Disease and colonization of plant leaf tissue were consistently diminished in all P. syringae strains tested (pv. phaseolicola NPS3121, pv. syringae B728a, and BR2R) when harboring pSK61 relative to these isolates harboring the empty vector pME6031. Phaseolotoxin, syringomycin, and tabtoxin were not produced in any of these strains when transformed with pSK61. Production of protease and pyoverdin as well as swarming were also diminished in all of these strains when harboring pSK61. In contrast, alginate production, biofilm formation, and the hypersensitive response were diminished in some but not all of these isolates under the same growth conditions. These results indicate that rsmA is consistently important in the overarching phenotypes disease and endophtyic colonization but that its role varies with pathovar in certain underpinning phenotypes in the phytotoxin-producing strains of P. syringae.

ABSTRACT Production of the plant hormone indole-3-acetic acid (IAA) is widespread among plant-associated microorganisms. The non-gall-forming phytopathogen Erwinia chrysanthemi 3937 (strain Ech3937) possesses iaaM (ASAP16562) and iaaH (ASAP16563) gene homologues. In this work, the null knockout iaaM mutant strain Ech138 was constructed. The IAA production by Ech138 was reduced in M9 minimal medium supplemented with l-tryptophan. Compared with wild-type Ech3937, Ech138 exhibited reduced ability to produce local maceration, but its multiplication in Saintpaulia ionantha was unaffected. The pectate lyase production of Ech138 was diminished. Compared with wild-type Ech3937, the expression levels of an oligogalacturonate lyase gene, ogl, and three endopectate lyase genes, pelD, pelI, and pelL, were reduced in Ech138 as determined by a green fluorescent protein-based fluorescence-activated cell sorting promoter activity assay. In addition, the transcription of type III secretion system (T3SS) genes, dspE (a putative T3SS effector) and hrpN (T3SS harpin), was found to be diminished in the iaaM mutant Ech138. Compared with Ech3937, reduced expression of hrpL (a T3SS alternative sigma factor) and gacA but increased expression of rsmA in Ech138 was also observed, suggesting that the regulation of T3SS and pectate lyase genes by IAA biosynthesis might be partially due to the posttranscriptional regulation of the Gac-Rsm regulatory pathway.

ABSTRACT The Gac/Rsm signal transduction pathway positively regulates secondary metabolism, production of extracellular enzymes, and biocontrol properties of Pseudomonas fluorescens CHA0 via the expression of three noncoding small RNAs, termed RsmX, RsmY, and RsmZ. The architecture and function of the rsmY and rsmZ promoters were studied in vivo. A conserved palindromic upstream activating sequence (UAS) was found to be necessary but not sufficient for rsmY and rsmZ expression and for activation by the response regulator GacA. A poorly conserved linker region located between the UAS and the −10 promoter sequence was also essential for GacA-dependent rsmY and rsmZ expression, suggesting a need for auxiliary transcription factors. One such factor involved in the activation of the rsmZ promoter was identified as the PsrA protein, previously recognized as an activator of the rpoS gene and a repressor of fatty acid degradation. Furthermore, the integration host factor (IHF) protein was found to bind with high affinity to the rsmZ promoter region in vitro, suggesting that DNA bending contributes to the regulated expression of rsmZ. In an rsmXYZ triple mutant, the expression of rsmY and rsmZ was elevated above that found in the wild type. This negative feedback loop appears to involve the translational regulators RsmA and RsmE, whose activity is antagonized by RsmXYZ, and several hypothetical DNA-binding proteins. This highly complex network controls the expression of the three small RNAs in response to cell physiology and cell population densities.

Dickeya dadantii (Erwinia chrysanthemi 3937) secretes exoenzymes, including pectin-degrading enzymes, leading to the loss of structural integrity of plant cell walls. A type III secretion system (T3SS) is essential for full virulence of this bacterium within plant hosts. The GacS/GacA two-component signal transduction system participates in important biological roles in several gram-negative bacteria. In this study, a gacA deletion mutant (Ech137) of D. dadantii was constructed to investigate the effect of this mutation on pathogenesis and other phenotypes. Compared with wild-type D. dadantii, Ech137 had a delayed biofilm-pellicle formation. The production of pectate lyase (Pel), protease, and cellulase was diminished in Ech137 compared with the wild-type cells. Reduced transcription of two endo-Pel genes, pelD and pelL, was found in Ech137 using a green fluorescence protein-based fluorescence-activated cell sorter promoter activity assay. In addition, the transcription of T3SS genes dspE (an effector), hrpA (a structural protein of the T3SS pilus), and hrpN (a T3SS harpin) was reduced in Ech137. A lower amount of rsmB regulatory RNA was found in gacA mutant Ech137 compared with the wild-type bacterium by quantitative reverse-transcription polymerase chain reaction. Compared with wild-type D. dadantii, a lower amount of hrpL mRNA was observed in Ech137 at 12 h grown in medium. Although the role of RsmA, rsmB, and RsmC in D. dadantii is not clear, from the regulatory pathway revealed in E. carotovora, the lower expression of dspE, hrpA, and hrpN in Ech137 may be due to a posttranscriptional regulation of hrpL through the Gac-Rsm regulatory pathway. Consequently, the reduced exoenzyme production and Pel gene expression in the mutant may be partially due to the regulatory role of rsmB-RsmA on exoenzyme expression. Similar to in vitro results, a lower expression of T3SS and pectinase genes of Ech137 also was observed in bacterial cells inoculated into Saintpaulia ionantha leaves, perhaps accounting for the observed reduction in local maceration. Interestingly, compared with the wild-type D. dadantii, although a lower concentration of Ech137 was observed at day 3 and 4 postinoculation, there is no significant difference in bacterial concentration between the wild-type bacterium and Ech137 in the early stage of infection. Finally, the nearly abolished systemic invasion ability of Ech137 suggests that GacA of D. dadantii is essential for the pathogenicity and systemic movement of the bacterium in S. ionantha.

ABSTRACTPectobacterium wasabiae(previously known asErwinia carotovora) is an important plant pathogen that regulates the production of plant cell wall-degrading enzymes through anN-acyl homoserine lactone-based quorum sensing system and through the GacS/GacA two-component system (also known as ExpS/ExpA). At high cell density, activation of GacS/GacA induces the expression of RsmB, a noncoding RNA that is essential for the activation of virulence in this bacterium. A genetic screen to identify regulators of RsmB revealed that mutants defective in components of a putative Trk potassium transporter (trkHandtrkA) had decreasedrsmBexpression. Further analysis of these mutants showed that changes in potassium concentration influencedrsmBexpression and consequent tissue damage in potato tubers and that this regulation required an intact Trk system. Regulation ofrsmBexpression by potassium via the Trk system occurred even in the absence of the GacS/GacA system, demonstrating that these systems act independently and are both required for full activation of RsmB and for the downstream induction of virulence in potato infection assays. Overall, our results identified potassium as an essential environmental factor regulating the Rsm system, and the consequent induction of virulence, in the plant pathogenP. wasabiae.IMPORTANCECrop losses from bacterial diseases caused by pectolytic bacteria are a major problem in agriculture. By studying the regulatory pathways involved in controlling the expression of plant cell wall-degrading enzymes inPectobacterium wasabiae, we showed that the Trk potassium transport system plays an important role in the regulation of these pathways. The data presented further identify potassium as an important environmental factor in the regulation of virulence in this plant pathogen. We showed that a reduction in virulence can be achieved by increasing the extracellular concentration of potassium. Therefore, this work highlights how elucidation of the mechanisms involved in regulating virulence can lead to the identification of environmental factors that can influence the outcome of infection.

The opportunistic pathogen Pseudomonas aeruginosa is a significant cause of infection in immunocompromised individuals, cystic fibrosis patients, and burn victims. To benefit its survival, the bacterium adapt to either a motile or sessile lifestyle when infecting the host. The motile bacterium has an often activated type III secretion system (T3SS), which is virulent to the host, whereas the sessile bacterium harbors an active T6SS and lives in biofilms. Regulatory pathways involving Gac-Rsm or secondary messengers such as c-di-GMP determine which lifestyle is favorable for P. aeruginosa. Here, we introduce the RNA binding protein RtcB as a modulator of the switch between motile and sessile bacterial lifestyles. Using the wild-type P. aeruginosa PAO1, and a retS mutant PAO1(∆retS) in which T3SS is repressed and T6SS active, we show that deleting rtcB led to simultaneous expression of T3SS and T6SS in both PAO1(∆rtcB) and PAO1(∆rtcB∆retS). The deletion of rtcB also increased biofilm formation in PAO1(∆rtcB) and restored the motility of PAO1(∆rtcB∆retS). RNA-sequencing data suggested RtcB as a global modulator affecting multiple virulence factors, including bacterial secretion systems. Competitive killing and infection assays showed that the three T6SS systems (H1, H2, and H3) in PAO1(∆rtcB) were activated into a functional syringe, and could compete with Escherichia coli and effectively infect lettuce. Western blotting and RT-PCR results showed that RtcB probably exerted its function through RsmA in PAO1(∆rtcB∆retS). Quantification of c-di-GMP showed an elevated intracellular levels in PAO1(∆rtcB), which likely drove the switch between T6SS and T3SS, and contributed to the altered phenotypes and characteristics observed. Our data demonstrate a pivotal role of RtcB in the virulence of P. aeruginosa by controlling multiple virulence determinants, such as biofilm formation, motility, pyocyanin production, T3SS, and T6SS secretion systems towards eukaryotic and prokaryotic cells. These findings suggest RtcB as a potential target for controlling P. aeruginosa colonization, establishment, and pathogenicity.

Heme (iron-protoporphyrin IX) is an essential but potentially toxic cellular cofactor. While most organisms are heme prototrophs, many microorganisms can utilize environmental heme as iron source. The pathogenic yeast Candida albicans can utilize host heme in the iron-poor host environment, using an extracellular cascade of soluble and anchored hemophores, and plasma membrane ferric reductase-like proteins. To gain additional insight into the C. albicans heme uptake pathway, we performed an unbiased genetic selection for mutants resistant to the toxic heme analog Ga3+-protoporphyrin IX at neutral pH, and a secondary screen for inability to utilize heme as iron source. Among the mutants isolated were the genes of the pH-responsive RIM pathway, and a zinc finger transcription factor related to S. cerevisiae HAP1. In the presence of hemin in the medium, C. albicans HAP1 is induced, the Hap1 protein is stabilized and Hap1-GFP localizes to the nucleus. In the hap1 mutant, cytoplasmic heme levels are elevated, while influx of extracellular heme is lower. Gene expression analysis indicated that in the presence of extracellular hemin, Hap1 activates the heme oxygenase HMX1, which breaks down excess cytoplasmic heme, while at the same time it also activates all the known heme uptake genes. These results indicate that Hap1 is a heme-responsive transcription factor that plays a role both in cytoplasmic heme homeostasis and in utilization of extracellular heme. The induction of heme uptake genes by C. albicans Hap1 under iron satiety indicates that preferential utilization of host heme can be a dietary strategy in a heme prototroph.

B3 family transcription factors play an essential regulatory role in plant growth and development processes. This study performed a comprehensive analysis of the B3 family transcription factor in longan (Dimocarpus longan Lour.), and a total of 75 DlB3 genes were identified. DlB3 genes were unevenly distributed on the 15 chromosomes of longan. Based on the protein domain similarities and functional diversities, the DlB3 family was further clustered into four subgroups (ARF, RAV, LAV, and REM). Bioinformatics and comparative analyses of B3 superfamily expression were conducted in different light and with different temperatures and tissues, and early somatic embryogenesis (SE) revealed its specific expression profile and potential biological functions during longan early SE. The qRT-PCR results indicated that DlB3 family members played a crucial role in longan SE and zygotic embryo development. Exogenous treatments of 2,4-D (2,4-dichlorophenoxyacetic acid), NPA (N-1-naphthylphthalamic acid), and PP333 (paclobutrazol) could significantly inhibit the expression of the DlB3 family. Supplementary ABA (abscisic acid), IAA (indole-3-acetic acid), and GA3 (gibberellin) suppressed the expressions of DlLEC2, DlARF16, DlTEM1, DlVAL2, and DlREM40, but DlFUS3, DlARF5, and DlREM9 showed an opposite trend. Furthermore, subcellular localization indicated that DlLEC2 and DlFUS3 were located in the nucleus, suggesting that they played a role in the nucleus. Therefore, DlB3s might be involved in complex plant hormone signal transduction pathways during longan SE and zygotic embryo development.

ABSTRACT Swarming motility in pseudomonads typically requires both a functional flagellum and the production/secretion of a biosurfactant. Published work has shown that the wild-type Pseudomonas fluorescens Pf0-1 is swarming deficient due to a point mutation in the gacA gene, which until recently was thought to inactivate rather than attenuate the Gac/Rsm pathway. As a result, little is known about the underlying mechanisms that regulate swarming motility by P. fluorescens Pf0-1. Here, we demonstrate that a Δ rsmA Δ rsmE Δ rsmI mutant, which phenotypically mimics Gac/Rsm pathway overstimulation, is proficient at swarming motility. RsmA and RsmE appear to play a key role in this regulation. Transposon mutagenesis of the Δ rsmA Δ rsmE Δ rsmI mutant identified multiple factors that impact swarming motility, including pathways involved in flagellar synthesis and biosurfactant production/secretion. We find that loss of genes linked to biosurfactant Gacamide A biosynthesis or secretion impacts swarming motility, as does loss of the alternative sigma factor FliA, which results in a defect in flagellar function. Collectively, these findings provide evidence that P. fluorescens Pf0-1 can swarm if the Gac/Rsm pathway is activated, highlight the regulatory complexity of swarming motility in this strain, and demonstrate that the cyclic lipopeptide Gacamide A is utilized as a biosurfactant for swarming motility. IMPORTANCE Swarming motility is a coordinated process that allows communities of bacteria to collectively move across a surface. For P. fluorescens Pf0-1, this phenotype is notably absent in the parental strain, and to date, little is known about the regulation of swarming in this strain. Here, we identify RsmA and RsmE as key repressors of swarming motility via modulating the levels of biosurfactant production/secretion. Using transposon mutagenesis and subsequent genetic analyses, we further identify potential regulatory mechanisms of swarming motility and link Gacamide A biosynthesis and transport machinery to swarming motility.

In Pseudomonas aeruginosa, the second messenger cyclic-di-GMP and Gac/Rsm signaling pathways are associated with the transition from acute to chronic infection. Therefore, identifying the molecular mechanisms that govern lifestyle choice in bacteria is very important. Here, we identified a novel cyclic-di-GMP modulator, PrtR, which was shown to repress pyocin production by inhibition of PrtN and activate the Type III secretion system (T3SS) through PtrB. Compared to a wild-type strain or a prtN mutant, the prtR/prtN double mutant exhibited a wrinkly colony and hyperbiofilm phenotype as well as an increase in intracellular c-di-GMP levels. Interestingly, a diguanylate cyclase (DGC) gene, siaD, was repressed by PrtR. Further experiments revealed that PrtR directly interacts with SiaD, and facilitates the accumulation of c-di-GMP in cells. We also demonstrated that PrtR regulates the activity of Gac/Rsm system, thus affecting expression of the T3SS, type VI secretion system (T6SS) and the formation of biofilm. Taken together, the present findings indicate that PrtR, as a c-di-GMP modulator, plays key roles for the adaptation to the opportunistic infection of P. aeruginosa. Additionally, this study revealed a novel mechanism for PrtR-mediated regulation of the lifestyle transition via the Gac/Rsm and c-di-GMP signaling networks.

Pseudomonas fluorescens 2P24 is a plant root-associated bacterium that suppresses several soilborne plant diseases due to its production of the antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG). The biosynthesis of 2,4-DAPG is controlled by many regulatory elements, including the global regulator of the Gac/Rsm regulon and the pathway-specific repressor PhlF. In this work, a novel genetic element grxD, which encodes the monothiol glutaredoxin GrxD, was identified and characterized in the production of 2,4-DAPG in P. fluorescens 2P24. Our data showed that the mutation of grxD remarkably decreased 2,4-DAPG production. GrxD lost its ability to alter the production of 2,4-DAPG when the active-site CGFS motif of GrxD was mutated by site-directed mutagenesis. Further studies showed that the RsmA and RsmE proteins were essential for the GrxD-mediated regulation of 2,4-DAPG and exoprotease production. In addition, our data revealed that the deletion of grxD increased the expression of phlF, which negatively regulated the production of 2,4-DAPG. In addition, the grxD mutant was severely impaired in the biocontrol effect against the bacterial wilt of tomato. Overall, our results indicated that the monothiol glutaredoxin GrxD is involved in the production of 2,4-DAPG of P. fluorescens by influencing the Gac/Rsm global signaling pathway and transcriptional regulator PhlF and is essential for the biocontrol properties.

ABSTRACT Biofilm formation by the Gram-negative, Gammaproteobacteria Pseudomonas fluorescens relies on the repeats-in-toxin adhesins LapA and MapA in the cytoplasm, secretion of these adhesins through their respective type 1 secretion systems, and retention at the cell surface. Published work has shown that retention of the adhesins occurs via a post-translational mechanism involving the cyclic-di-GMP receptor LapD and the protease LapG. However, little is known about the underlying mechanisms that regulate the level of these adhesins. Here, we demonstrate that the master regulator FleQ modulates biofilm formation by both transcriptionally and post-transcriptionally regulating LapA and MapA. We find that a Δ fleQ mutant has a biofilm formation defect compared to the wild-type (WT) strain, which is attributed in part to a decrease in LapA and MapA abundance in the cell, despite the Δ fleQ mutant having increased levels of lapA and mapA transcripts compared to the WT strain. Through transposon mutagenesis and subsequent genetic analysis, we found that overstimulation of the Gac/Rsm pathway partially rescues biofilm formation in the Δ fleQ mutant background. Collectively, these findings provide evidence that FleQ regulates biofilm formation by both transcriptionally regulating the expression of the lapA and mapA genes and post-transcriptionally regulating the abundance of LapA and MapA, and that activation of the Gac/Rsm pathway can post-transcriptionally enhance biofilm formation by P. fluorescens . IMPORTANCE Biofilm formation is a highly coordinated process that bacteria undergo to colonize a variety of surfaces. For Pseudomonas fluorescens , biofilm formation requires the production and localization of repeats-in-toxin adhesins to the cell surface. To date, little is known about the underlying mechanisms that regulate biofilm formation by P. fluorescens . Here, we identify FleQ as a key regulator of biofilm formation that modulates both gene expression and abundance of LapA and MapA through both a transcriptional and post-transcriptional mechanism. We provide further evidence implicating activation of the Gac/Rsm system in FleQ-dependent regulation of biofilm formation. Together, our findings uncover evidence for a dual mechanism of transcriptional and post-transcriptional regulation of the LapA and MapA adhesins.

The perception and response to cellular death is an important aspect of multicellular eukaryotic life. For example, damage-associated molecular patterns activate an inflammatory cascade that leads to removal of cellular debris and promotion of healing. We demonstrate that lysis of Pseudomonas aeruginosa cells triggers a program in the remaining population that confers fitness in interspecies co-culture. We find that this program, termed P. aeruginosa response to antagonism (PARA), involves rapid deployment of antibacterial factors and is mediated by the Gac/Rsm global regulatory pathway. Type VI secretion, and, unexpectedly, conjugative type IV secretion within competing bacteria, induce P. aeruginosa lysis and activate PARA, thus providing a mechanism for the enhanced capacity of P. aeruginosa to target bacteria that elaborate these factors. Our finding that bacteria sense damaged kin and respond via a widely distributed pathway to mount a complex response raises the possibility that danger sensing is an evolutionarily conserved process.

Many root-colonizing Pseudomonas spp. exhibiting biocontrol activities produce a wide range of secondary metabolites that exert antibiotic effects against other microbes, nematodes, and insects in the rhizosphere. The expression of these secondary metabolites depends on the Gac/Rsm signal transduction pathway. Based on the findings of a previous genomic study on newly isolated biocontrol pseudomonad strains, we herein investigated the novel gene cluster OS3, which consists of four genes (Os1348–Os1351) that are located upstream of putative efflux transporter genes (Os1352–Os1355). Os1348 was predicted to encode an 85-aa small precursor protein, the expression of which was under the control of GacA, and an X-ray structural analysis suggested that the Os1348 protein formed a dimer. The mutational loss of the Os1348 gene decreased the antibiotic activity of Pseudomonas sp. Os17 without changing its growth rate. The Os1349–1351 genes were predicted to be involved in post-translational modifications. Intracellular levels of the Os1348 protein in the deficient mutant of each gene differed from that in wild-type cells. These results suggest that Os1348 is involved in antibiotic activity and that the structure or expression of this protein is under the control of downstream gene products.

Bacterial survival is fraught with antagonism, including that deriving from viruses and competing bacterial cells. It is now appreciated that bacteria mount complex antiviral responses; however, whether a coordinated defense against bacterial threats is undertaken is not well understood. Previously, we showed that Pseudomonas aeruginosa possess a danger-sensing pathway that is a critical fitness determinant during competition against other bacteria. Here, we conducted genome-wide screens in P. aeruginosa that reveal three conserved and widespread interbacterial antagonism resistance clusters (arc1-3). We find that although arc1-3 are coordinately activated by the Gac/Rsm danger-sensing system, they function independently and provide idiosyncratic defense capabilities, distinguishing them from general stress response pathways. Our findings demonstrate that Arc3 family proteins provide specific protection against phospholipase toxins by preventing the accumulation of lysophospholipids in a manner distinct from previously characterized membrane repair systems. These findings liken the response of P. aeruginosa to bacterial threats to that of eukaryotic innate immunity, wherein threat detection leads to the activation of specialized defense systems.

ABSTRACT Bacterial communities can sense their neighbors, regulating group behaviors in response to cell density and environmental changes. The diversity of signaling networks in a single species has been postulated to allow custom responses to different stimuli; however, little is known about how multiple signals are integrated and the implications of this integration in different ecological contexts. In the plant pathogen Pectobacterium wasabiae (formerly Erwinia carotovora), two signaling networks—the N-acyl homoserine lactone (AHL) quorum-sensing system and the Gac/Rsm signal transduction pathway—control the expression of secreted plant cell wall-degrading enzymes, its major virulence determinants. We show that the AHL system controls the Gac/Rsm system by affecting the expression of the regulatory RNA RsmB. This regulation is mediated by ExpR2, the quorum-sensing receptor that responds to the P. wasabiae cognate AHL but also to AHLs produced by other bacterial species. As a consequence, this level of regulation allows P. wasabiae to bypass the Gac-dependent regulation of RsmB in the presence of exogenous AHLs or AHL-producing bacteria. We provide in vivo evidence that this pivotal role of RsmB in signal transduction is important for the ability of P. wasabiae to induce virulence in response to other AHL-producing bacteria in multispecies plant lesions. Our results suggest that the signaling architecture in P. wasabiae was coopted to prime the bacteria to eavesdrop on other bacteria and quickly join the efforts of other species, which are already exploiting host resources. IMPORTANCE Quorum-sensing mechanisms enable bacteria to communicate through small signal molecules and coordinate group behaviors. Often, bacteria have various quorum-sensing receptors and integrate information with other signal transduction pathways, presumably allowing them to respond to different ecological contexts. The plant pathogen Pectobacterium wasabiae has two N-acyl homoserine lactone receptors with apparently the same regulatory functions. Our work revealed that the receptor with the broadest signal specificity is also responsible for establishing the link between the main signaling pathways regulating virulence in P. wasabiae. This link is essential to provide P. wasabiae with the ability to induce virulence earlier in response to higher densities of other bacterial species. We further present in vivo evidence that this novel regulatory link enables P. wasabiae to join related bacteria in the effort to degrade host tissue in multispecies plant lesions. Our work provides support for the hypothesis that interspecies interactions are among the major factors influencing the network architectures observed in bacterial quorum-sensing pathways. IMPORTANCE Quorum-sensing mechanisms enable bacteria to communicate through small signal molecules and coordinate group behaviors. Often, bacteria have various quorum-sensing receptors and integrate information with other signal transduction pathways, presumably allowing them to respond to different ecological contexts. The plant pathogen Pectobacterium wasabiae has two N-acyl homoserine lactone receptors with apparently the same regulatory functions. Our work revealed that the receptor with the broadest signal specificity is also responsible for establishing the link between the main signaling pathways regulating virulence in P. wasabiae. This link is essential to provide P. wasabiae with the ability to induce virulence earlier in response to higher densities of other bacterial species. We further present in vivo evidence that this novel regulatory link enables P. wasabiae to join related bacteria in the effort to degrade host tissue in multispecies plant lesions. Our work provides support for the hypothesis that interspecies interactions are among the major factors influencing the network architectures observed in bacterial quorum-sensing pathways.

ABSTRACT Pseudomonas fluorescens 2P24 is a rhizosphere bacterium that protects many crop plants against soilborne diseases caused by phytopathogens. The PcoI/PcoR quorum-sensing (QS) system and polyketide antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG) are particularly relevant to the strain’s biocontrol potential. In this study, we investigated the effects of c-di-GMP on the biocontrol activity of strain 2P24. The expression of the Escherichia coli diguanylate cyclase (YedQ) and phosphodiesterase (YhjH) in P. fluorescens 2P24 significantly increased and decreased the cellular concentration of c-di-GMP, respectively. The production of the QS signals N-acyl homoserine lactones (AHLs) and 2,4-DAPG was negatively regulated by c-di-GMP in 2P24. The regulatory proteins RsmA and RsmE were positively regulated by c-di-GMP. Genomic analysis revealed that 2P24 has 23 predicted proteins that contain c-di-GMP-synthesizing or -degrading domains. Among these proteins, C0J56_12915, C0J56_13325, and C0J56_27925 contributed to the production of c-di-GMP and were also involved in the regulation of the QS signal and antibiotic 2,4-DAPG production in P. fluorescens. Overexpression of C0J56_12915, C0J56_13325, and C0J56_27925 in 2P24 impaired its root colonization and biocontrol activities. Taken together, these results demonstrated that c-di-GMP played an important role in fine-tuning the biocontrol traits of P. fluorescens. IMPORTANCE In various bacteria, the bacterial second messenger c-di-GMP influences a wide range of cellular processes. However, the function of c-di-GMP on biocontrol traits in the plant-beneficial rhizobacteria remains largely unclear. The present work shows that the QS system and polyketide antibiotic 2,4-DAPG production are regulated by c-di-GMP through RsmA and RsmE proteins in P. fluorescens 2P24. The diguanylate cyclases (DGCs) C0J56_12915, C0J56_13325, and C0J56_27925 are especially involved in regulating the biocontrol traits of 2P24. Our work also demonstrated a connection between the Gac/Rsm cascade and the c-di-GMP signaling pathway in P. fluorescens.

IntroductionNF-YB transcription factor is an important regulatory factor in plant embryonic development.ResultsIn this study, 15 longan NF-YB (DlNF-YB) family genes were systematically identified in the whole genome of longan, and a comprehensive bioinformatics analysis of DlNF-YB family was performed. Comparative transcriptome analysis of DlNF-YBs expression in different tissues, early somatic embryogenesis (SE), and under different light and temperature treatments revealed its specific expression profiles and potential biological functions in longan SE. The qRT-PCR results implied that the expression patterns of DlNF-YBs were different during SE and the zygotic embryo development of longan. Supplementary 2,4-D, NPA, and PP333 in longan EC notably inhibited the expression of DlNF-YBs; ABA, IAA, and GA3 suppressed the expressions of DlNF-YB6 and DlNF-YB9, but IAA and GA3 induced the other DlNF-YBs. Subcellular localization indicated that DlNF-YB6 and DlNF-YB9 were located in the nucleus. Furthermore, verification by the modified 5'RNA Ligase Mediated Rapid Amplification of cDNA Ends (5' RLM-RACE) method demonstrated that DlNF-YB6 was targeted by dlo-miR2118e, and dlo-miR2118e regulated longan somatic embryogenesis (SE) by targeting DlNF-YB6. Compared with CaMV35S- actuated GUS expression, DlNF-YB6 and DlNF-YB9 promoters significantly drove GUS expression. Meanwhile, promoter activities were induced to the highest by GA3 but suppressed by IAA. ABA induced the activities of the promoter of DlNF-YB9, whereas it inhibited the promoter of DlNF-YB6.DiscussionHence, DlNF-YB might play a prominent role in longan somatic and zygotic embryo development, and it is involved in complex plant hormones signaling pathways.

Background:: Alzheimer's disease (AD) is a progressive neurodegenerative condition characterized by the gradual decline of cognitive abilities, primarily caused by impairments in the cholinergic system. AD is diagnosed based on the presence of specific pathological features, in-cluding senile plaques, neurofibrillary tangles, and the loss of neurons and synapses. Despite on-going efforts, the etiology of AD remains unclear, and there is a significant lack of effective treatments to meet the medical needs of affected individuals. The complex nature of AD, involv-ing multiple factors, presents challenges in the development of potential therapies. Numerous ob-stacles hinder the achievement of optimal pharmacological concentration of promising molecules for AD treatment. These obstacles include the presence of the blood-brain barrier (BBB), which restricts the entry of therapeutic agents into the brain, as well as issues related to poor bioavaila-bility and unfavorable pharmacokinetic profiles. Unfortunately, many therapeutically promising compounds have failed to overcome these hurdles and demonstrate efficacy in treating AD. background: Alzheimer’s disease (AD) is a progressive neurodegenerative disease that is manifested by depleted cognitive abilities resulted due to cholinergic impairments. AD is further diagnosed with pathological hallmarks including senile plaques, neurofibrillary tangles and neuronal and synaptic death. With constant efforts, few therapeutic targets and interventions have been identified but AD is still a disease with unclear etiopathology and unmet medical needs. The multifactorial nature of AD poses difficulties to develop a potential treatment. Unfortunately, large numbers of therapeutically efficient molecules for the treatment of AD failed to attain optimal pharmacological concentration due to numerous hurdles such as the presence of blood-brain barrier (BBB), poor bioavailability, or pharmacokinetic profile. Methods:: The PEGylated chitosan nanoconjugate was developed and evaluated for delivery of anti-Alzheimer natural extract of Salvia officinalis and Melissa officinalis to the brain. The nano-conjugates (S-PCN and M-PCN) were developed by ionic gelation technique. Result:: The nanoconjugates (S-PCN and M-PCN) were evaluated for various optical and in-vitro parameters. MTT assay on UCSD229i-SAD1-1 human astrocytoma cells indicated IC50 values of 0.42, 0.49, 0.67, and 0.75 μM for S-PCN, M-PCN formulations, and free Salvia officinalis and Melissa officinalis extracts, respectively. The In vitro assessments using cell lines have confirmed the improved uptake and distribution of nanoconjugates compared to free extracts. These findings were validated through confocal microscopy and apoptosis assays, revealing a substantial in-crease in the accumulation of nanoconjugates within the brain. The targeting potential OF M- PCN over S-PCN was found to be 2-fold significant. method: 1. Sample Preparation - Crude drug Salvia officinalis and Melissa officinalis, plants were collected from the botanical gardens of Warangal and Tirupathi and authenticated.The two plants, 1 g each, were crushed (using a lab mill) for 1 min, to obtain the corresponding powder. The extraction powder was performed as described in previous reports, via addition of 100 mL boiling water to 1 g of plant powder and after 5 min, the extract was filtered through a 0.45 mm filter. This procedure was optimized to obtain the highest potential activity of these plants. After the crude plant sedimentation, samples were filtered and maintained at 80 ͦ C, for freeze-drying procedures (Heto Holten A/S Drywinner, Allerød, Denmark). Then, solutions of 1% (w/v) of freeze-dried powder were dissolved in methanol for analytical evaluation other activity tests. Before injections, samples were filtered again through a 0.45 mm filter. 2. Preparation of PEGylated Chitosan Nanoparticles - Ionotropic gelation technique was employed for the synthesis of chitosan, encapsulating whole Salvia officinalis and Melissa officinalis extract separately. Accurately weighed 100 mg of Salvia officinalis and Melissa officinalis extract and 0.4% w/v of Chitosan were dissolved in 1% v/v aqueous glacial acetic acid (GAA) solution. Drop wise addition of 0.4% Sodium tripolyphosphate solution (TPP) was performed in drug polymer solution at the rate of 2ml/min (12 ml TPP in 20 ml drug polymer solution). The obtained particles dispersion were sonicated using a probe sonicator (S-4000; Misonix, Farmingdale, NY) at medium amplitude (50%) for 5 min to obtain nano sized particles. The dispersion was then filtered through a 0.2 um hydrophilic filter (Minsart, Sartorius) for isolation of smaller nanosize particles in order to achieve maximum transportation at targeted site. The nano sized particles, thus obtained were carefully purified by ultrafiltration (Amicon 8200 with a millipore PBMK membrane, MWCO 300000) against double distilled water at optimal temperature. The ultrafiltration facilitates elimination of residual of unreacted solvent and unbound drug. For the PEgylation process, accurately 50 mL of 0.3 % chitosan nanoparticles were added into polyethylene glycol (PEG) solution with a ratio of 3:1 and stirred at 500 rpm for 1 h. Further, dispersion was applied to the mixture for 60 seconds to produce homogeneous PEG-Chitosan nanoparticles. Result The formation of the PEGylated chitosan nanoparticles entrapping natural extract Salvia officinalis and Melissa officinalis ensued impulsively upon combination of the pawn anion TPP into the consistent Chitosan polymer solutions. Nanoparticle formation resulted from the ionic interactions between the negative charge ion TPP and the positively charged amino groups of Chitosan. The ratio of CS/TPP was optimized to attained stable dispersion and formation of nanosize particles. Preliminary experiments were performed in order to identify the optimal concentrations of CS and TPP for NP formation. The process parameters along with formulation parameters were thoroughly optimized for the achievement of physiochemical and thermal stable nanoparticles. The obtaining nano size particles were broadly characterized as either a clear solution, an opalescent suspension displaying a tyndall effect (NPs), or aggregate. 1 Particle size, Zeta Potential and Morphology The results achieved from the zeta sizer measurement displayed very distinct size of prepared S-PCN and M-PCN formulations ranging 150-250 nm (Figure 1- a & b). The nano size of the S-PCN, M-PCN formulations displayed decent encapsulation of extract in the polymer matrix due to the formulation and process optimization. The surface charge of both nanoformulation S-PCN, M-PCN was found to be -10.89 mV and -16.21 mV respectively (Figure 1- e & f) demonstrated negative charge nature of both formulation. The negative charge of formulation showed better stability and optimum candidature for enhance brain targeting. The pH of S-PCN, M-PCN formulations was measured as 6.9 ± 0.01 which play a vital role in nearly neutral microenvironment delivery for efficient brain targeting. The pH facilitate targeting mechanism act as the key element for the onsite degradation of the polymer matrix. This polymeric degradation activation mechanism enhanced the drug release at a controlled rate resulting into the desired therapeutic potential. 2 DLS Analysis The DLS outcomes again nanosize range dispersion of prepared S-PCN and M-PCN nanoformulation. The size distribution pattern of both nanoformulation is some identical to each other exhibiting size range of 160-240 nm for S-PCN and 150-230 nm for M-PCN formulation. The optimal nanosize range of both nanoformulation demonstrated the enhanced brain delivery and onsite targeting which efficiently comply the size of cells and its micro-environment. The DLS investigations showed diverse size distribution of and dispersion pattern. The PDI exhibited by S-PCN and M-PCN was found to be of 0.271 ± 0.08 and 0.259 ± 0.11. The DLS results showed enhance stability with even size distribution pattern of prepared nanoparticles between 100-500 nm (Figure 1-c & d). This nanosize stable pattern facilitates enhance diffusion of prepared nanoparticles across the blood brain barriers leading to optimal pharmacological potential during brain targeting. Therefore, it can be unswervingly state out that both the nanoformulations exhibited optimal and stable nano dispersal features for the operative brain targeting against Alzheimer management in clinical platform. 3 Transmission Electron Microscopy (TEM) The TEM analysis showed very discrete particles size exhibiting oval shape nanoparticles of both nanoformulation. The size revealed by TEM analysis for S-PCN and M-PCN was ranging 100-250 nm validating DLS measurement zeta sizer analysis (Figure 2- a & b). The formation of nanoparticles by entrapping natural extract showed better crosslinking between polymer and cross linker avoiding unwanted leakage. Also the aggregation of nanoparticles was found negligible showing better PEGylation process of chitosan boundaries. The TEM outcomes displayed suitable nano carrier system for the effective brain delivery, revealing decent BBB infiltration appearance of both nanoformulation. 4 Scanning Electron Microscopy (SEM) The SEM analysis significantly the results obtained by zeta sizer and TEM assay showing fine particles formation with spherical shape and smooth morphology. The SEM images noteworthy validates the sharp oval boundaries of both nanoformulation exhibiting better PEGylation process. The SEM images also clarifies no sign of clusters formation of agglomeration of particles showing significant PEG outer layer. The SEM analysis exhibiting size range of 150-250 nm again qualitatively validating the TEM, and zeta-sizer analysis and confirming the ideal brain targeting delivery characteristics of both S-PCN and M-PCN (Figure 2 – c & d). 5 In-vitro drug release studies In vitro drug release data of Salvia officinalis and Melissa officinalis extract associated with PEGylated nanoformulations is demonstrated in figure 3- a & b. The drug release pattern from both the nanoformulation S-PCN and M-PCN at different pH (4.0 & 7.4) exhibited a non-linear release profile characterized by a relatively faster initial drug release during the first 3-4 h, followed by slower release in later period. The two pH range was provided to deeply evaluate the effect of nanoformulations for better brain targeting and onsite delivery. The biphasic drug release pattern was observed by both nanoformulation with initial bursting of nanoparticles in early 1-8 h followed by slow release in 24 h. The in-vitro drug release studies suggested that initially both S-PCN and M-PCN provided burst release of drug extract at pH 4.0. The drug release was found to be 89.45 ± 3.67 % at 6h, 91.42 ± 2.11 at 8 h, 90.26 ± 1.84 % at 6 h and 95.67 ± 2.20 % at 8 h for S-PCN and M-PCN, respectively. On the contrary at pH 7 the drug release was significantly (P < 0> S-PCN. 6 In vitro cellular uptake The capacity of cellular targeting and intracellular transport of developed nanoformulation S-PCN and M-PCN evaluated and measured by using UCSD229i-SAD1-1 human astrocytoma cells line. The human astrocytoma cells line are imperative part of BBB and broadly engaged for the examination of brain delivery. The developed S-PCN and M-PCN showed noteworthy cellular acceptance and circulation compared to the free drug extract of Salvia officinalis and Melissa officinalis when evaluated by CLSM analysis. The CLSM signals for the developed S-PCN and M-PCN were resilient and sharp with enhance absorbance when treated with Rhodamine B isothiocyanate (RITC) compared to the free drug extract of Salvia officinalis and Melissa officinalis suspension on incubation for 12 h (Figure 4). In addition, the confocal laser scanning microscopic intense fluorescence signals displayed by nanoformulations showed the clear sign of vesicular localization of nanoparticles demonstrating enhance endocytic pathway progression. The CLSM signals showed by M-PCN samples treated UCSD229i-SAD1-1 human astrocytoma cells showed sharp red fluorescence signal around the cell nucleus when compared to the cells treated S-PCN incubated at 4 h and 12 h of time periods which is found enhanced and significant. The results of CLSM intensity examination showed 2 folds enhance cellular uptake and resilience in-vitro by M-PCN compared to S-PCN on the brain cell membranes. The S-PCN and M-PCN treated cells were also quantitatively observed inductively attached with the plasma optical emission (ICP-OE) spectrometry for 12 h of incubation. The results efficiently inveterate that the around ~45% of M-PCN and ~33% of S-PCN nanoformulation have pointedly traversed into the BBB layer, validated by the transwell assay at basolateral side. The free drug extracts showed scanty diffusion across BBB via UCSD229i-SAD1-1 human astrocytoma cells of ~16% signifying non-significant intracellular transport and penetrating efficiency due to early adsorption at cell membrane restricting direct diffusion to the cells (Figure 3c). Overall, at different incubation time interval, the cell uptake and transportation capability of M-PCN was remarkable compared to S-PCN with strong fluorescent adverts bereft of any morphological difference in cell lines, resulting in enhanced brain targeting efficiency. 7 In vitro cytotoxicity assay The MTT assay was employed for the investigation of developed M-PCN and S-PCN toward UCSD229i-SAD1-1 human astrocytoma cells. The MTT assay qualitatively showed significant anti-proliferation capability of nanoformulations in 24h of incubation. The investigations showed sharp cell viability of 100% and 10% by control Normal control (saline solution) and negative control group (Triton X 100 surfactant solution) respectively. The developed S-PCN and M-PCN showed notable cell viability of 96%, 89%, 76% & 65% and 98%, 90%, 80% & 71% at different concentration (0.1, 1, 10 and 20 μg/mL of individual concentration) on 24 h of incubation (Figure 3d). Whereas free drug extract of Salvia officinalis and Melissa officinalis showed cell viability of 96%, 88%, 68%, & 48% and 95%, 86%, 69% & 52% respectively on 24 h of incubation. The MTT investigation established non-significant cell cytotoxicity by different samples in 24h of incubation showing nonlinear relationship between incubation time and anti-proliferation efficiency. The MTT results clearly displayed significant cell viability of nanoformulation over free drug extract in 24 h of incubation expressing biologically safe brain targeting efficiency with negligible toxicity on human astrocytoma cells. The enhance cell viability showed by developed S-PCN and M-PCN is due to better physiochemical compatibility between nanocomposite resulting in efficient cellular transport and brain delivery. On inter-comparison of nanoformulation the cell viability of M-PCN is greater than S-PCN with less cell cytotoxicity at higher concentration. The inter-comparison results showed better endocytosis and resilience of M-PCN which is found statistically significant when analyzed by student’s T test. Overall the cell toxicity examinations clearly expounds that the developed nanocomposite may be used as novel drug carrier encapsulating natural extract for the treatment of brain diseases as targeted delivery system. 8 Apoptosis assay The Apoptosis investigation showed by free drug extract, S-PCN and M-PCN and verified striking apoptosis at all concentrations. The developed S-PCN and M-PCN showed inherent apoptosis compared to the free drug extract. It has been noted out that both S-PCN and M-PCN showed mitochondrial apoptosis phenomenon or death activator by provoking cell surface receptor. By activating cell surface receptor the activation of caspase cascade establishes optimum cell death which results in desired apoptosis process. The apoptosis index of free drug were found to be 0.39 and 0.42 for Salvia officinalis and Melissa officinalis respectively whereas the S-PCN and M-PCN showed apoptotic index of 0.66 and 0.79 respectively. The nanoformulation showed significant apoptosis action compared to plain free natural extract which is nearly two folds more and found significant (*P<0.01) (Figure 5). The chief cause for better apoptosis of nanoformulation over free drug extract is the nanosized particles, causing quick onsite drug transportation, sufficient distribution and better release. On inter-comparison of S-PCN and M-PCN the apoptosis potential is significant showed by m-PCN compared to S-PCN when analyzed by student T test. Overall PEgylation of chitosan nanoparticles facilitates better circulation of nanoparticles in brain microenvironment causing extended release and negligible drug toxicity resulting in better brain targeting against Alzheimer disease. Conclusion:: Based on the findings, it can be inferred that biodegradable PEGylated chitosan nanoconjugates hold promise as effective nano-targeting agents for delivering anti-Alzheimer drugs to the brain. The incorporation of PEGylated chitosan nanoparticles in this approach demonstrates enhanced delivery capabilities, ultimately leading to improved therapeutic out-comes. other: Characterization 1 Particle size, Zeta potential, pH and Morphology The developed S-PCN, M-PCN particle size and surface charge was measured by Malvern Zetasizer 3000 particle size and zeta potential analyzer (Malvern Instruments, Bedfordshire, UK). The Zeta potential of S-PCN, M-PCN was examined by smearing the principle of electrophoretic movement of particles in an applied electrical field. The concentration of both S-PCN, M-PCN formulation was attuned at 0.01% w/v by distilled water or in 0.01 M sodium chloride solution for potential assessment. The pH was calculated by using a digital pH meter (HI-TECH WATER TECH. New Delhi, India). The pH meter was first calibrated using buffer tablet, the pH meter was dipped in a beaker comprising S-PCN and M-PCN nanoformulations on post calibration. The nanoformulations, evaluation was triplicated and the measurement was repeated thrice with an average value along with SD was reported. 2 Dynamic Light Scattering (DLS) The S-PCN and M-PCN nanoformulations was examined for the Dynamic Light Scattering (DLS) investigating mean diameter and PDI by employing Brook-heaven BI 9000 AT instrument (Brookheaven Instrument Corporation, USA). The DLS examination was measured for the more distinct and significant evaluation of both S-PCN and M-PCN nanoformulations. The DLS evaluation were done at wavelength 417 and 215 nm for S-PCN and M-PCN nanoformulations receptively at temperature of 25°C. 3.3 Transmission Electron Microscopy (TEM) The TEM of both S-PCN and M-PCN nanoformulations was measured by using Hitachi H-7500 TEM analyzer. TEM metaphors were obtained to visualize the shape and structure of nanoformulaion. The S-PCN and M-PCN nanoformulations were coated with 2.5% w/v of phosphor-tungstic acid (PTA) solution and placed in a copper disc grid. The grid was then desiccated in 60 watt LED lamp (Philips, India Ltd) and was finally placed into the disc holder and scanned for TEM evaluation. 4 Scanning Electron Microscopy (SEM) The morphology and structure of prepared S-PCN and M-PCN nanoformulations were analyzed by SEM, Nova Nano SEM 450, Germany. Before the SEM assessment, the formulations were lyophilized by using freeze dry lyophilizer (REMI, New Delhi, India). The dried formulations were then placed on a SEM stub by using dual adhesive tape at 50mA 5-10 minutes via sputter (KYKY SBC-12, Beijing, China). A SEM aided with secondary electron detector was engaged to get the digital images of the developed S-PCN and M-PCN nanoformulations. 5 Entrapment Efficiency (EE): EE plays essential part in transporting the bioactive to the targeted site at detailed therapeutic dose in order to get the anticipated therapeutic value. To measure the EE, both the nanoformulations were centrifuged at 10000 rpm for 5 minutes to obtain the pellets. The collected supernatant was carefully diluted with PBS of pH 7 and the drug content was determined spectrophotometrically by using UV spectrophotometer (Schimadzu, Japan) at 317 nm and 215 nm for S-PCN and M-PCN nanoformulation respectively against a blank solvent. The EE can be measured by using the following formula: EE= weight of drug in nanoformulation / initial weight of drug taken x 100 6 In vitro Drug Release studies The release of from both S-PCN and M-PCN nanoformulations was tracked to predict the diffusion and kinetic behavior of the nanosystems for desired therapeutic efficiency. For release studies, both S-PCN and M-PCN obtained after centrifugation were suspended in 10 mL of a phosphate buffered saline (PBS) solution, pH 7.4. This nanoparticle suspension was transferred to clean Eppendorf’s tube and placed in a water bath at 37 °C under stirring. After 0.5, 1, 2, 4, 6, and 24 h, samples were collected from the bath and centrifuged at 14 000 rpm for 5 min (BOECO, Hamburg, Germany). Supernatants were analyzed by UV spectroscopy and used to calculate the amount of drug released from the nanoparticles over the specified time. Triplicate samples were analyzed at each time. 7 Cell Line studies 7.1 Cell Culture and Seeding The Human UCSD229i-SAD1-1 human astrocytoma cells line was obtained from NCCs Pune and was conserved in Dulbecco’s modified Eagles Medium. The cell line was then supplemented with 10