Search

Marine ecosystem associated organisms are an affluent source of bioactive compounds. Polysaccharides with unique structural and practical entities have gained special studies interest inside the current biomedical zone. Polysaccharides are the main components of marine algae, plants, animals, insects, and microorganisms. In recent times research on seaweed is more persistent for extraction of natural bioactive “Sulfated polysaccharides” (SP). The considerable amount of SP exists in the algae in the form of fucans, fucoidans, carrageenans, ulvan, etc. Major function of SP is to act as a defensive lattice towards the infective organism. All SP possess the high potential and possess a broad range of therapeutic applications as antitumor, immunomodulatory, vaccine adjuvant, anti-inflammatory, anticoagulant, antiviral, antiprotozoal, antimicrobial, antilipemic, therapy of regenerative medicine, also in drug delivery and tissue engineering application. This review aims to discuss the biomedicine applications of sulfated polysaccharides from marine seaweeds.

Sutures are widely used materials for closing the surgical wounds, and being an inert material, sutures are often colonized with drug-resistant polymicrobial biofilms. Surgical site infection (SSI) is a hospital-acquired infection caused by bacteria and fungi specifically in the sutured sites. Although most of the currently available sutures possess antibacterial property, their ability to prevent biofilm colonization by polymicrobial communities is underexplored. So, the present study shows that extracted chitosan (EC) from crab shells prevented the adherence of Staphylococcus epidermidis and Candida albicans, the predominant members that exist as mixed species at the site of SSI. In comparison with a commercial chitosan, EC showed profound inhibition of slime formation and mixed species biofilm inhibition. Intriguingly, EC-coated sutures could inhibit the growth of both bacterial and fungal pathogens when comparing with a commercial triclosan-coated suture which was active only against the bacterial pathogen. Scanning electron microscopy results revealed inhibition of C. albicans hyphal formation by the EC-coated sutures that is a crucial virulence factor responsible for tissue invasiveness. Collectively, the results of the present study showed that EC from crab shells (discarded material as a recalcitrant biowaste) could be used as an alternative to combat drug-resistant biofilms which are the prime cause for SSIs.

Genes encoding proteins with A20/AN1 zinc-finger domains, belonging to the stress associated protein (SAP) gene family, are present in all eukaryotes and play a decisive role in plant response to diverse physiological and molecular activities particularly on biotic and abiotic stresses (AbS). In this first and foremost study, global transcriptome analysis of members of the SAP gene family was carried out in C3 model-Oryza sativa (OsSAP) aiming at the identification of OsSAP genes activated in response to unique or Combined AbS (CAbS). Based on the available spatio-temporal and phytohormonal RNA-Seq expression profile datasets, nine OsSAP genes were filtered out and identified by a differential expression signature noted in various tissues as well as plant hormones. Comparative genome ideogram of OsSAP genes confirmed the orthologous collinearity with C4 panicoid genomes. Interactome of these genes, revealed the molecular cross-talks of OsSAP. Thus, the computational expression signature of OsSAP genes led to a better understanding of gene dynamism in diverse developmental tissues/organs. Transcriptional regulation analysis of key OsSAP genes in response to stress (drought and salinity) suggested the novel role of OsSAP1, OsSAP2, OsSAP5, OsSAP7, OsSAP8 and OsSAP11 in AbS. Altogether, the study provides deeper insights on molecular characteristics of OsSAP genes, which could be deployed further to decipher their precise functional roles in AbS responses.Communicated by Ramaswamy H. Sarma.

The SARS-CoV-2 virus has spread worldwide to cause a full blown pandemic since 2020. To date, several promising synthetic therapeutics are repurposed and vaccines through different stages of clinical trials were approved and being administered, but still the efficacy of the drugs and vaccines are yet to be decoded. This article highlights the importance of traditional medicinal plants and the phytomolecules derived from them, which possess in vitro antiviral and anti-CoV properties and further explores their potential as inhibitors to molecular targets of SARS-CoV-2 that were evaluated by in silico approaches. Botanicals in traditional medicinal systems have been investigated for anti-SARS-CoV-2 activity through in silico and in vitro studies. However, information linking structure of phytomolecules to their antiviral activity is limited. Most phytomolecules with anti-CoV activity were studied for inhibition of the human ACE2 receptor through which the virus enters host cells, and non-structural proteins 3CLpro and PLpro. Although the proteases are ideal anti-CoV targets, information on plant-based inhibitors for the CoV structural proteins, e.g., spike, envelope, membrane, nucleocapsid required further investigations. In absence of scientific evaluations through in vitro and biocompatibility studies, plant-based antivirals fall short as treatment options. Plant-based anti-SARS-CoV-2 therapeutics can be promising alternatives to their synthetic counterparts as they are economical and bear fewer chances of toxicity, side effects, and viral resistance. Our review could provide a systematic overview of the potential phytomolecules which can be repurposed and subjected to further modes of experimental evaluation to qualify for use in treatment and prophylaxis of SARS-CoV-2 infections.

Background. The direct or indirect effect of chemicals on the nervous system of humans or animals is referred to as neurotoxicity. Trimethyltin chloride (TMT) intoxication causes behavioral and cognitive deficiencies in humans and experimental animals. TMT has long been used as a model toxicant in the study of central nervous system (CNS) toxicity. Momordica charantia, which is used in traditional herbal medicine, has a variety of pharmacological functions. Mesoporous silica nanoparticles have a higher loading capacity, are less dense, and have a larger specific area. Objectives. To investigate a possible nanotherapy for Alzheimer’s disease caused by trimethyltin chloride in freshwater zebrafish. Methods. An aqueous extract of M.charantia was used to perform the primary and secondary screening. The DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging assay was used to determine the antioxidant capacity of crude aqueous extracts of M. charantia. Mesoporous silica nanoparticles are made using a CTAB surfactant chemical process and tetraethyl orthosilicate. UV-Vis spectroscopy, Fourier transform infrared spectroscopy, scanning electron microscope, and EDAX were used to characterize it. Danio rerio was used to test the trimethyltin chloride for Alzheimer’s disease. The M. charantia and mesoporous silica nanoparticles were then tested in the same method. Results. The extract has no adverse effects on zebrafish, indicating that M. charantia is safe for human consumption. The histopathological findings indicate that the tissues of the fish infected with the extract had no pathological modifications. Conclusion. The M. charantia showed higher antioxidant activity and anticholinesterase activity, and upon further characterization and assessment, this could be a safe and potential drug candidate for Neurotoxicity.

For the yeast population and diversity, marine habitats are the least explored niches and the lack of validated database is considered to be a drawback for yeast research. The aim of the present study is to create a comprehensive DNA barcode library for marine derived yeast species isolated from organic burial hotspots such as coastal sediment in mangrove and continental shelf ecosystems. As we enriched, isolated and ITS gene sequenced 1017 marine derived yeast isolates belonging to 157 marine species in 55 genera, 28 families, 14 orders, 8 classes of 2 Phyla (viz., Ascomycota and Basidiomycota) of which 13 yeast species were first time barcoded. We witnessed yeast species of both terrestrial and marine endemic origin in the barcode datasets. Due to the large volume of sequencing trace files, the variable length of extracted sequences, and the lack of reference sequences in public databases, difficulties were faced in taxonomic sequence validation. The length of the majority of the sequences (99.42%) were more than or equal to 600 base pairs. BLAST analysis revealed that 13 yeast species were barcoded for the first time. The genus, Candida was the speciose genera isolated in this study. K2P intra-species distance analysis performed for selective groups yielded an average of 0.33%, well below the previously proposed yeast barcode gap. ITS gene NJ-tree based identification conducted for selective species in Ascomycota and Basidomycota, precisely clustered the same species into one group, indicating the efficacy of ITS gene in yeast species delineation. Besides isolating some of the common marine yeast species such as Candida oceani, Bandonia marina and Yarrowia lipolytica, we found approximately 60% of the yeast species isolates were previously unrecorded from the marine environment (example; Cystobasidiopsis lactophilus, Slooffia cresolica, Udeniozyma ferulica, Colacogloea falcatus and Pichia guilliermondii), of which 16.5% were recognised as potential human pathogens (example; Candida orthopsilosis, C. rugosa, Debaryomyces fabryi and Yamadazyma triangularis). Apart from releasing the barcode data in GenBank, provisions were made to access the entire dataset along with meta-data in the Barcode of life database (http://dx.doi.org/10.5883/DS-MYIC). This research constitutes the largest dataset to date for collecting marine yeast isolates and their barcodes. As meta- and environmental barcoding analysis were expanding its scope including environmental assessment and monitoring, the datasets such as ours will be more useful.

Marine pollution is a significant issue in recent decades, with the increase in industries and their waste harming the environment and ecosystems. Notably, the rise in shellfish industries contributes to tons of shellfish waste composed of up to 58% chitin. Chitin, the second most ample polymer next to cellulose, is insoluble and resistant to degradation. It requires chemical-based treatment or enzymatic hydrolysis to cleave the chitin polymers. The chemical-based treatment can lead to environmental pollution, so to solve this problem, enzymatic hydrolysis is the best option. Moreover, the resulting biopolymer by-products can be used to boost the fish immune system and also as drug delivery agents. Many marine microbial strains have chitinase producing ability. Nevertheless, we still lack an economical and highly stable chitinase enzyme for use in the industrial sector. So we isolate a novel marine bacterial strain Achromobacter xylosoxidans from the shrimp waste disposal site using chitin minimal medium. Placket–Burman and central composite design statistical models for culture condition optimisation predicted a 464.2 U/ml of chitinase production. The culture conditions were optimised for maximum chitinase production recording up to 467 U/ml. This chitinase from the A. xylosoxidans was 100% active at an optimum temperature of 45 °C (withstand up to 55 °C) and pH 8 with 80% stability. The HPLC analysis of chitinase degraded shellfish waste reveals a major amino acid profile composition—arginine, lysine, aspartic acid, alanine, threonine and low levels of isoleucine and methionine. These chitinase degraded products and by-products can be used as supplements in the aquaculture industry.

Globally, the disposal of shellfishery waste is a major challenge and causes a risk to the coastal region. For potential development in aquaculture, the use of safe supplements to improve fish production and health is important. Chitosan (CS) used as feed additives for several fish species that enhanced production and immunity. The present study was intended to assess the effect of feed additives N-acetyl-d-glucosamine (NAG) loaded chitosan nanoparticles (CSNPs) on productivity, survival rate, and protein conversion efficiency of Oreochromis niloticus (L.). This is the first report on the effect of CSNPs and NAG loaded CSNPs as feed additives enhanced growth performance and non-specific immunity of O. niloticus. CSNPs and NAG loaded CSNPs were synthesized and characterized by scanning and transmission electron microscope, FT-IR, X-ray diffraction, particle size distribution, and zeta sizer. Fish (15.30 ± 0.23 g) administered diets fortified with 0.0, 0.25, 0.5, 1.0, and 2.0 g CSNPs/kg feed loaded with NAG for 45 d. The diets containing 1.0 g/kg NAG loaded CSNPs enhanced specific growth rate, weight gain, survival rate, respiratory burst, and lysozyme activities of tilapia compared control group. The data shows biologically active CSNPs and NAG loaded CSNPs are potent antimicrobial agents against selected bacterial pathogens. In conclusion, the findings suggested that the dietary supplement containing NAG loaded CSNPs significantly increased immune-modulatory properties, growth performance, and enhanced their disease resistance of Nile tilapia.

The effect of various concentrations of amyloid beta peptide (ABP) in different pH (pH 2, 6, 7, 8, 10) in aging at different time intervals was analysed using various techniques. The pH 7.4 and 8 were compared with other pH solution which indicated that the secondary structure content with high ?-sheet conformation is found at pH 7.4 and 8. Phosphate buffer solutions (pH) were detected using CD spectroscopy. The CD spectra of peptide in phosphate buffer (pH 2 and 10) on aging at time intervals were also analysed with the aging of sample (pH 2) on the 10th day. The results showed no major secondary structural changes in the peptide, which was indicative predominant random coil structure. The phosphate buffer pH 6, 7.4, 8 on the 10th day showed the occurrence of crossover point. This same sample smear on slide was mounded and the fibrils were visualized using the light microscope. The percent of viable cell in different pH was detected in MTT (3-(4,5-Dimethyazol-2-yl)-2, 5-diphenyl-tetrazolium bromide) assay using human keratinocyte cell line (HaCaT). Fluorescence spectroscopy was used to study the growth and development of fibrils. © 2019, Springer Nature B.V.

Antimicrobial resistance is a major public health concern in infection control. Hence, a multi-pronged approach is necessary to curb the severity of infections. The present study entails the identification of docosanol (fatty alcohol) from Streptomyces as a novel antibiofilm agent which can target the virulence factors of MRSA. Results showed that docosanol as a potent antibiofilm agent and found to inhibit several virulence factors of MRSA. The antibiofilm efficacy of docosanol analyzed through light and scanning electron microscopy showed a significant reduction in adherent cells. Moreover, analysis of three-dimensional structure of biofilm matrix by confocal laser scanning microscope demonstrated effective antibiofilm potential of docosanol. In addition, docosanol reduced the survival rate of MRSA in healthy human blood and enhanced the neutrophil-mediated killing by interfering with hemolysin production. RT-qPCR analysis revealed the down regulation of several virulence genes, possibly by affecting the expression of the accessory gene regulator (agr) system and transcriptional regulator sarA. These findings suggest that docosanol could effectively reduce the biofilm phenotype and virulence production, and thus becomes a promising candidate to treat MRSA infections.

A comparative study of the accumulation of Reactive Red 11 (RR11) and Acid Green 1 (AG1) dyes using live yeast culture Pichia pastoris (P. pastoris) was investigated. The changes in inoculum volume, pH, initial dye concentration and incubation temperature were studied. The maximum percentage of color removal efficiency, dye uptake capacity and bioaccumulation concentrations were found at pH 2. The maximum dried biomass concentration was found at pH 3. When the dye concentration increased, the percentage of color removal, specific growth rate and dried biomass concentrations decreased. However, the dye uptake capacity increased with an increase in the initial dye concentration. At an ambient temperature of 30 °C, the uptake efficiency for both the dyes was optimal The yeast biomass was characterized using scanning electron microscopic (SEM) image and fourier transform infrared (FTIR) spectral analysis. In this study, P. pastoris accumulated both dyes and was found to be more suitable for AG1 dye than RR11 dye.

Macro algae or Seaweeds contain many bio active compounds which can be used in various industries including agricultural, food, and pharmaceutical. In the present study we used Padina gymnospora, Gracilaria edulis and Ulva fasciata aqueous extracts used as a seaweed biofertilizers for the seed germination of Capsicum annuum. The seeds were soaked in different concentrations of seaweed extract (2, 4, 6, 8 and 10%) and water (as control) for 24 h. Percentage of seed germination (GP), shoot length (SL), germination index (GI), mean germination time (MGT), seedling vigor index (SVI), germination energy (GE), pH has been studied. The minimum concentration 8% of Padina gymnospora 6% of Gracilaria edulis and 8% of Ulva fasciata enhances the seed germination rate. The above mentioned three seaweed seed germination parameters on Capsicum annuum are as follows, the shoot length was (76,70 and 85 mm in dm), germination index (0.933, 0.866 and 0.7), germination percentage (70%,65% and 60%), mean germination time (10.5,5.25 and 9), seedling vigor index (53.2%,45.55% and 51%), germination energy (70% 65% and 60%), total protein content (2%,0.1% and 10.2%), total phenol content (15%,0.09% and 12%) and antioxidant inhibition percentage (43.1%,22.4% and 24.6) in Capsicum annuum. Between these three seaweeds Padina gymnospora aqueous extract 8% reveal superior retaliation. We conclude that Padina gymnospora 8% may improve the germination process and phytochemical presence in Capsicum annum.

Cyanobacteria are rich sources of structurally novel and biologically active metabolites with antibacterial, antiviral, antifungal, and anticancer activities. The present study aimed to evaluate the antimicrobial, antioxidant, and toxicity potential of freshwater filamentous cyanobacterial species isolated from a freshwater source in Kancheepuram district and Thiruverumbur, Tamilnadu. Among the tested two cyanobacterial species, methanol-chloroform extracts of Oscillatoria sp. SSCM01 exhibits higher antibacterial activity than Phormidium sp. SSCM02. Oscillatoria sp. SSCM01 exhibited higher inhibition activity of bacterial growth at 31.2 μg/mL and 7.8 μg/mL as MIC value against Staphylococcus aureus, Salmonella typhi, respectively, and for Candida albicans at 125 μg/mL. Antioxidant activity of Oscillatoria sp. SSCM01 extract assessed by DPPH assay exhibited potent radical scavenging activity of 48% inhibition at 60 μg/mL concentration. Two cyanobacterial extracts exhibited significant activity against brine shrimp larvae (Artemia salina). The active fraction was subsequently analyzed by gas chromatography-mass spectrometry (GC-MS), bioactive compounds like hexadecanoic acid (66.3%), 1, 2-benzene dicarboxylic acid, and dibutyl phthalate were detected in Oscillatoria sp. SSCM01. Thus, our findings indicate that freshwater cyanobacteria Oscillatoria sp. SSCM01 has an inherent capability that might make it beneficial as a promising source of antimicrobial and antioxidant therapeutic agents.

Introduction Vulvovaginal candidiasis(VVC) is an important health disorder among women which is caused due to drug resistance in Candida species. C.albicans is the most common pathogenic species among Candida. However, the Non albicans Candida (NAC) species is on the rise, so the appropriate identification of the Candida sp. will pave way for better management and minimising the emergence of drug resistance. The aim of this study is to analyse the species distribution and their antifungal susceptibility pattern of Candida sp. in patients with suspected vaginitis. Materials and methods A total of 153 women of age 15–40 years with complaints suggestive of vaginitis were clinically examined and high vaginal swabs were taken from them. Samples were inoculated on Sabouraud Dextrose agar and Hi-Crome Candida differential agar. Identification of Candida sp. was performed using conventional methods and with Vitek2 Compact (Biomerieux France) using vitek 2 cards for identification of yeast and yeast like organisms (ID-YST cards). Multidrug resistant Candida isolates were analysed for biofilm forming ability and their identification was confirmed with amplification and sequencing of internal transcribed space regions with specific PCR. Results In total, 63 Candida isolates were identified from the samples. Candida albicans (59%) was the most common species isolated in the study. The Non albicans Candida species (41%) isolated in the study were C. tropicalis, C. glabrata, C. krusei and C. auris. Discussion C. albicans is one of the common opportunistic fungal pathogen which causes mucocutaneous infections of genital and non-genital tracts of healthy individuals. It is a polymorphic yeast which exists in different morphologies which facilitates its survival under stress environments by forming biofilms. High prevalence of non albicans Candida sp. in this study indicates the importance of conducting epidemiological surveys to measure the occurrence of NAC. Multiplex PCR is a rapid and reliable method to identify the Candida sp. causing vulvovaginal candidiasis. Although fluconazole still appears to be effective against most of the Candida isolates, resistance rate of this drug to the Candida sp. emphasises the need for performing antifungal susceptibility testing and alternate drug therapy in the routine practice.

Human oral microbiome and dysbiotic infections have been recently evidently identified. One of the major reasons for such dysbiosis is impairment of the immune system. Periodontitis is a chronic inflammatory disease affecting the tissues that surround and support the teeth. In the United States., approximately 65 million people are affected by this condition. Its occurrence is also associated with many important systemic diseases such as cardiovascular disease, rheumatoid arthritis, and Alzheimer’s disease. Among the most important etiologies of periodontitis is Porphyromonas gingivalis, a keystone bacterial pathogen. Keystone pathogens can orchestrate inflammatory disease by remodeling a normally benign microbiota causing imbalance between normal and pathogenic microbiota (dysbiosis). The important characteristics of P. gingivalis causing dysbiosis are its virulence factors which cause effective subversion of host defenses to its advantage allowing other pathogens to grow. Some of the mechanisms involved in these processes are still not well-understood. However, various microbial strategies target host sialoglycoproteins for immune dysregulation. In addition, the enzymes that break down sialoglycoproteins and sialoglycans are the “sialoglycoproteases„, resulting in exposed terminal sialic acid. This process could lead to pathogen-toll like receptor (TLR) interactions mediated through sialic acid receptor ligand mechanisms. Assessing the function of P. gingivalis sialoglycoproteases, could pave the way to designing carbohydrate analogues and sialic acid mimetics to serve as drug targets.

The adaptation of Porphyromonas gingivalis to H 2 O 2 -induced stress while inducible is modulated by an unknown OxyR-independent mechanism. Previously, we reported that the PG_2212 gene was highly upregulated in P. gingivalis under conditions of prolonged oxidative stress. Because this gene may have regulatory properties, its function in response to H 2 O 2 was further characterized. PG2212, annotated as a hypothetical protein of unknown function, is a 10.3-kDa protein with a cysteine 2-histidine 2 (Cys 2 His 2 ) zinc finger domain. The isogenic mutant P. gingivalis FLL366 (ΔPG_2212) showed increased sensitivity to H 2 O 2 and decreased gingipain activity compared to the parent strain. Transcriptome analysis of P. gingivalis FLL366 revealed that approximately 11% of the genome displayed altered expression (130 downregulated genes and 120 upregulated genes) in response to prolonged H 2 O 2 -induced stress. The majority of the modulated genes were hypothetical or of unknown function, although some are known to participate in oxidative stress resistance. The promoter region of several of the most highly modulated genes contained conserved motifs. In electrophoretic mobility shift assays, the purified rPG2212 protein did not bind its own promoter region but bound a similar region in several of the genes modulated in the PG_2212-deficient mutant. A metabolome analysis revealed that PG2212 can regulate a number of genes coding for proteins involved in metabolic pathways critical for its survival under the conditions of oxidative stress. Collectively, our data suggest that PG2212 is a transcriptional regulator that plays an important role in oxidative stress resistance and virulence regulation in P. gingivalis .

Summary The adaptability and survival of Porphyromonas gingivalis in the oxidative microenvironment of the periodontal pocket are indispensable for survival and virulence, and are modulated by multiple systems. Among the various genes involved in P. gingivalis oxidative stress resistance, vimA gene is a part of the 6.15-kb locus. To elucidate the role of a P. gingivalis vimA-defective mutant in oxidative stress resistance, we used a global approach to assess the transcriptional profile, to study the unique metabolome variations affecting survival and virulence in an environment typical of the periodontal pocket. A multilayered protection strategy against oxidative stress was noted in P. gingivalis FLL92 with upregulation of detoxifying genes. The duration of oxidative stress was shown to differentially modulate transcription with 94 (87%) genes upregulated twofold during 10 min and 55 (83.3%) in 15 min. Most of the upregulated genes (55%), fell in the hypothetical/unknown/unassigned functional class. Metabolome variation showed reduction in fumarate and formaldehyde, hence resorting to alternative energy generation and maintenance of a reduced metabolic state. There was upregulation of transposases, genes encoding for the metal ion binding protein transport and secretion system.

Changes in periodontal status are associated with shifts in the composition of the bacterial community in the periodontal pocket. The relative abundances of several newly recognized microbial species, including Filifactor alocis , as-yet-unculturable organisms, and other fastidious organisms have raised questions on their impact on disease development. We have previously reported that the virulence attributes of F. alocis are enhanced in coculture with Porphyromonas gingivalis . We have evaluated the proteome of host cells and F. alocis during a polymicrobial infection. Coinfection of epithelial cells with F. alocis and P. gingivalis strains showed approximately 20% to 30% more proteins than a monoinfection. Unlike F. alocis ATCC 35896, the D-62D strain expressed more proteins during coculture with P. gingivalis W83 than with P. gingivalis 33277. Proteins designated microbial surface component-recognizing adhesion matrix molecules (MSCRAMMs) and cell wall anchor proteins were highly upregulated during the polymicrobial infection. Ultrastructural analysis of the epithelial cells showed formation of membrane microdomains only during coinfection. The proteome profile of epithelial cells showed proteins related to cytoskeletal organization and gene expression and epigenetic modification to be in high abundance. Modulation of proteins involved in apoptotic and cell signaling pathways was noted during coinfection. The enhanced virulence potential of F. alocis may be related to the differential expression levels of several putative virulence factors and their effects on specific host cell pathways.

Summary The VimA protein of Porphyromonas gingivalis is a multifunctional protein involved in cell surface biogenesis. To further determine if its acetyl coenzyme A (acetyl-CoA) transfer and putative sorting functions can affect the secretome, its role in peptidoglycan biogenesis and effects on the extracellular proteins of P. gingivalis FLL92, a vimA-defective mutant, were evaluated. There were structural and compositional differences in the peptidoglycan of P. gingivalis FLL92 compared with the wild-type strain. Sixty-eight proteins were present only in the extracellular fraction of FLL92. Fifteen proteins present in the extracellular fraction of the parent strain were missing in the vimA-defective mutant. These proteins had protein sorting characteristics that included a C-terminal motif with a common consensus Gly-Gly–CTERM pattern and a polar tail consisting of aromatic amino acid residues. These observations suggest that the VimA protein is likely involved in peptidoglycan synthesis, and corroborates our previous report, which suggests a role in protein sorting.

Porphyromonas gingivalis , the causative agent of adult periodontitis, must maintain nitric oxide (NO) homeostasis and surmount nitric oxide stress from host immune responses or other oral bacteria to survive in the periodontal pocket. To determine the involvement of a putative hydroxylamine reductase (PG0893) and a putative nitrite reductase-related protein (PG2213) in P. gingivalis W83 NO stress resistance, genes encoding those proteins were inactivated by allelic exchange mutagenesis. The isogenic mutants P. gingivalis FLL455 ( PG0893 ∷ ermF ) and FLL456 ( PG2213 ∷ ermF ) were black pigmented and showed growth rates and gingipain and hemolytic activities similar to those of the wild-type strain. P. gingivalis FLL455 was more sensitive to NO than the wild type. Complementation of P. gingivalis FLL455 with the wild-type gene restored the level of NO sensitivity to a level similar to that of the parent strain. P. gingivalis FLL455 and FLL456 showed sensitivity to oxidative stress similar to that of the wild-type strain. DNA microarray analysis showed that PG0893 and PG2213 were upregulated 1.4- and 2-fold, respectively, in cells exposed to NO. In addition, 178 genes were upregulated and 201 genes downregulated more than 2-fold. The majority of these modulated genes were hypothetical or of unknown function. PG1181 , predicted to encode a transcriptional regulator, was upregulated 76-fold. Transcriptome in silico analysis of the microarray data showed major metabolomic variations in key pathways. Collectively, these findings indicate that PG0893 and several other genes may play an important role in P. gingivalis NO stress resistance.

Filifactor alocis , a Gram-positive anaerobic rod, is one of the most abundant bacteria identified in the periodontal pockets of periodontitis patients. There is a gap in our understanding of its pathogenicity and ability to interact with other periodontal pathogens. To evaluate the virulence potential of F. alocis and its ability to interact with Porphyromonas gingivalis W83, several clinical isolates of F. alocis were characterized. F. alocis showed nongingipain protease and sialidase activities. In silico analysis revealed the molecular relatedness of several virulence factors from F. alocis and P. gingivalis . In contrast to P. gingivalis , F. alocis was relatively resistant to oxidative stress and its growth was stimulated under those conditions. Biofilm formation was significantly increased in coculture. There was an increase in adherence and invasion of epithelial cells in coculture compared with P. gingivalis or F. alocis monocultures. In those epithelial cells, endocytic vesicle-mediated internalization was observed only during coculture. The F. alocis clinical isolate had an increased invasive capacity in coculture with P. gingivalis compared to the ATCC 35896 strain. In addition, there was variation in the proteomes of the clinical isolates compared to the ATCC 35896 strain. Hypothetical proteins and those known to be important virulence factors in other bacteria were identified. These results indicate that F. alocis has virulence properties that may enhance its ability to survive and persist in the periodontal pocket and may play an important role in infection-induced periodontal disease.

The Porphyromonas gingivalis recombinant VimA can interact with the gingipains and several other proteins, including a sialidase. Sialylation can be involved in protein maturation; however, its role in virulence regulation in P. gingivalis is unknown. The three sialidase-related proteins in P. gingivalis showed the characteristic sialidase Asp signature motif (SXDXGXTW) and other unique domains. To evaluate the roles of the associated genes, randomly chosen P. gingivalis isogenic mutants created by allelic exchange and designated FLL401 ( PG0778 :: ermF ), FLL402 ( PG1724 :: ermF ), and FLL403 ( PG0352 :: ermF-ermAM ) were characterized. Similar to the wild-type strain, FLL402 and FLL403 displayed a black-pigmented phenotype in contrast to FLL401, which was not black pigmented. Sialidase activity in P. gingivalis FLL401 was reduced by approximately 70% in comparison to those in FLL402 and FLL403, which were reduced by approximately 42% and 5%, respectively. Although there were no changes in the expression of the gingipain genes, their activities were reduced by 60 to 90% in all the isogenic mutants compared to that for the wild type. Immunoreactive bands representing the catalytic domains for RgpA, RgpB, and Kgp were present in FLL402 and FLL403 but were missing in FLL401. While adhesion was decreased, the capacity for invasion of epithelial cells by the isogenic mutants was increased by 11 to 16% over that of the wild-type strain. Isogenic mutants defective in PG0778 and PG0352 were more sensitive to hydrogen peroxide than the wild type. Taken together, these results suggest that the P. gingivalis sialidase activity may be involved in regulating gingipain activity and other virulence factors and may be important in the pathogenesis of this organism.

The Porphyromonas gingivalis vimA gene has been previously shown to play a significant role in the biogenesis of gingipains. Further, in P. gingivalis FLL92, a vimA-defective mutant, there was increased auto-aggregation, suggesting alteration in membrane surface proteins. In order to determine the role of the VimA protein in cell surface biogenesis, the surface morphology of P. gingivalis FLL92 was further characterized. Transmission electron microscopy demonstrated abundant fimbrial appendages and a less well defined and irregular capsule in FLL92 compared with the wild-type. In addition, atomic force microscopy showed that the wild-type had a smoother surface compared with FLL92. Western blot analysis using anti-FimA antibodies showed a 41 kDa immunoreactive protein band in P. gingivalis FLL92 which was missing in the wild-type P. gingivalis W83 strain. There was increased sensitivity to globomycin and vancomycin in FLL92 compared with the wild-type. Outer membrane fractions from FLL92 had a modified lectin-binding profile. Furthermore, in contrast with the wild-type strain, nine proteins were missing from the outer membrane fraction of FLL92, while 20 proteins present in that fraction from FLL92 were missing in the wild-type strain. Taken together, these results suggest that the VimA protein affects capsular synthesis and fimbrial phenotypic expression, and plays a role in the glycosylation and anchorage of several surface proteins.

Background: While the oral cavity harbors more than 680 bacterial species, the interaction and association of selected bacterial species play a role in periodontal diseases. Bacterial species including Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia, a consortium previously designated as the ‘red complex’ is now being expanded to include other new emerging pathogens that are significantly associated with periodontal disease. Highlight: In addition to novel mechanisms for oxidative resistance of individual species, community dynamics may lead to an overall strategy for survival in the inflammatory environment of the periodontal pocket. Complex systems controlled by response regulators protect against oxidative and nitrosative stress. Conclusion: The combination of these multifaceted strategies would provide a comprehensive defense and support system against the repetitive host immune response to promote microbial persistence and disease.

Summary PG0162, annotated as an extracytoplasmic function (ECF) sigma factor in Porphyromonas gingivalis, is composed of 193 amino acids. As previously reported, the PG0162-deficient mutant, P. gingivalis FLL350 showed significant reduction in gingipain activity compared with the parental strain. Because this ECF sigma factor could be involved in the virulence regulation in P. gingivalis, its genetic properties were further characterized. A 5’-RACE analysis showed that the start of transcription of the PG0162 gene occurred from a guanine (G) residue 69 nucleotides upstream of the ATG translation initiation codon. The function of PG0162 as a sigma factor was confirmed in a run-off in vitro transcription assay using the purified rPG0162 and RNAP core enzyme from Escherichia coli with the PG0162 promoter as template. As an appropriate PG0162 inducing environmental signal is unknown, a strain overexpressing the PG0162 gene designated P. gingivalis FLL391 was created. Compared with the wild-type strain, transcriptome analysis of P. gingivalis FLL391 showed that approximately 24% of the genome displayed altered gene expression (260 upregulated genes; 286 downregulated genes). Two other ECF sigma factors (PG0985 and PG1660) were upregulated more than two-fold. The autoregulation of PG0162 was confirmed with the binding of the rPG0162 protein to the PG0162 promoter in electrophoretic mobility shift assay. In addition, the rPG0162 protein also showed the ability to bind to the promoter region of two genes (PG0521 and PG1167) that were most upregulated in P. gingivalis FLL391. Taken together, our data suggest that PG0162 is a sigma factor that may play an important role in the virulence regulatory network in P. gingivalis.

To survive in the periodontal pocket, Porphyromonas gingivalis, the main causative agent of periodontal disease, must overcome oxidative and nitric oxide (NO) stress. Previously, we reported that, in the presence of NO comparable to stress conditions, the transcriptome of P. gingivalis was differentially expressed, and genes belonging to the PG1178-81 cluster were significantly up regulated. To further evaluate their role(s) in NO stress resistance, these genes were inactivated by allelic exchange mutagenesis. Isogenic mutants P. gingivalis FLL460 (ΔPG1181::ermF) and FLL461 (ΔPG1178-81::ermF) were black-pigmented, with gingipain and hemolytic activities comparable to that of the wild-type strain. Whereas the recovery of these isogenic mutants from NO stress was comparable to the wild-type, there was increased sensitivity to hydrogen peroxide-induced stress. RNA-Seq analysis under conditions of NO stress showed that approximately 5 and 8 percent of the genome was modulated in P. gingivalis FLL460 and FLL461 respectively. The PG1178-81 gene cluster was shown to be part of the same transcriptional unit and is inducible in response to NO stress. In the presence of NO, PG1181, a putative transcriptional regulator, was shown to bind to its own promoter region and that of several other NO responsive genes including PG1181, PG0214 an extracytoplasmic function sigma factor, PG0893 and PG1236. Taken together, the data suggest that PG1181 is an NO responsive transcriptional regulator that may play an important role in the NO stress resistance regulatory network in P. gingivalis.

Filifactor alocis, a previously unrecognized Gram-positive anaerobic rod, is now considered a new emerging pathogen that may play a significant role in periodontal disease. F. alocis' unique characteristics and variations at the molecular level that may be responsible for the functional changes required to mediate the pathogenic process are discussed.

Oral Biofilms are one of the most complex and diverse ecosystem developed by successive colonization of more than 600 bacterial taxa. Development starts with the attachment of early colonizers such as Actinomyces species and oral streptococci on the acquired pellicle and tooth enamel. These bacteria not only adhere to tooth surface but also interact with each other and lay foundation for attachment of bridging colonizer such as Fusobacterium nucleatum followed by late colonizers including the red complex species: Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola-the founders of periodontal disease. As the biofilm progresses from supragingival sites to subgingival sites, the environment changes from aerobic to anaerobic thus favoring the growth of mainly Gram-negative obligate anaerobes while restricting the growth of the early Gram-positive facultative aerobes. Microbes present at supragingival level are mainly related to gingivitis and root-caries whereas subgingival species advance the destruction of teeth supporting tissues and thus causing periodontitis. This review summarizes our present understanding and recent developments on the characteristic features of supra- and subgingival biofilms, interaction between different genera and species of bacteria constituting these biofilms and draws our attention to the role of some of the recently discovered members of the oral community.

As an anaerobe, Porphyromonas gingivalis is significantly affected by the harsh inflammatory environment of the periodontal pocket during initial colonization and active periodontal disease. We reported previously that the repair of oxidative stress-induced DNA damage involving 8-oxo-7,8-dihydroguanine (8-oxoG) may occur by an undescribed mechanism in P. gingivalis. DNA affinity fractionation identified PG1037, a conserved hypothetical protein, among other proteins, that were bound to the 8-oxoG lesion. PG1037 is part of the uvrA-PG1037-pcrA operon in P. gingivalis which is known to be upregulated under H2O2 induced stress. A PCR-based linear transformation method was used to inactivate the uvrA and pcrA genes by allelic exchange mutagenesis. Several attempts to inactivate PG1037 were unsuccessful. Similar to the wild-type when plated on Brucella blood agar, the uvrA and pcrA-defective mutants were black-pigmented and beta-hemolytic. These isogenic mutants also had reduced gingipain activities and were more sensitive to H2O2 and UV irradiation compared to the parent strain. Additionally, glycosylase assays revealed that 8-oxoG repair activities were similar in both wild-type and mutant P. gingivalis strains. Several proteins, some of which are known to have oxidoreducatse activity, were shown to interact with PG1037. The purified recombinant PG1037 protein could protect DNA from H2O2-induced damage. Collectively, these findings suggest that the uvrA-PG1037-pcrA operon may play an important role in hydrogen peroxide stress-induced resistance in P. gingivalis.

Previously, we have reported that gingipain activity in Porphyromonas gingivalis, the major causative agent in adult periodontitis, is post-translationally regulated by the unique Vim proteins including VimF, a putative glycosyltransferase. To further characterize VimF, an isogenic mutant defective in this gene in a different P. gingivalis genetic background was evaluated. In addition, the recombinant VimF protein was used to further confirm its glycosyltransferase function. The vimF-defective mutant (FLL476) in the P. gingivalis ATCC 33277 genetic background showed a phenotype similar to that of the vimF-defective mutant (FLL95) in the P. gingivalis W83 genetic background. While hemagglutination was not detected and autoaggregation was reduced, biofilm formation was increased in FLL476. HeLa cells incubated with P. gingivalis FLL95 and FLL476 showed a 45% decrease in their invasive capacity. Antibodies raised against the recombinant VimF protein in E. coli immunoreacted only with the deglycosylated native VimF protein from P. gingivalis. In vitro glycosyltransferase activity for rVimF was observed using UDP-galactose and N-acetylglucosamine as donor and acceptor substrates, respectively. In the presence of rVimF and UDP-galactose, a 60 kDa protein from the extracellular fraction of FLL95 which was identified by mass spectrometry as Rgp gingipain, immunoreacted with the glycan specific mAb 1B5 antibody. Taken together, these results suggest the VimF glycoprotein is a galactosyltransferase that may be specific for gingipain glycosylation. Moreover, galatose is vital for the growing glycan chain.

The salivary glands of adults concentrate nitrate from the plasma into saliva where it is converted to nitrite by bacterial nitrate reductases. Nitrite can play a beneficial role in adult gastrointestinal and cardiovascular physiology. When nitrite is swallowed, some of it is converted to nitric oxide (NO) in the stomach and may then exert protective effects in the gastrointestinal tract and throughout the body. It has yet to be determined either when newborn infants acquire oral nitrate reducing bacteria or what the effects of antimicrobial therapy or premature birth may be on the bacterial processing of nitrate to nitrite. We measured nitrate and nitrite levels in the saliva of adults and both preterm and term human infants in the early weeks of life. We also measured oral bacterial reductase activity in the saliva of both infants and adults, and characterized the species of nitrate reducing bacteria present. Oral bacterial conversion of nitrate to nitrite in infants was either undetectable or markedly lower than the conversion rates of adults. No measurable reductase activity was found in infants within the first two weeks of life, despite the presence of oral nitrate reducing bacteria such as Actinomyces odontolyticus, and Veillonella atypica, and Rothia mucilaginosa. We conclude that relatively little nitrite reaches the infant gastrointestinal tract due to the lack of oral bacterial nitrate reductase activity. Given the importance of the nitrate-nitrite-NO axis in adults, the lack of oral nitrate-reducing bacteria in infants may be relevant to the vulnerability of newborns to hypoxic stress and gastrointestinal tract pathologies.

Filifactor alocis, a Gram-positive anaerobic rod, is now considered one of the marker organisms associated with periodontal disease. Although there was heterogeneity in its virulence potential, this bacterium was shown to have virulence properties that may enhance its ability to survive and persist in the periodontal pocket. To gain further insight into a possible mechanism(s) of pathogenesis, the proteome of F. alocis strains was evaluated. Proteins including several proteases, neutrophil-activating protein A and calcium-binding acid repeat protein, were identified in F. alocis. During the invasion of HeLa cells, there was increased expression of several of the genes encoding these proteins in the potentially more virulent F. alocis D-62D compared to F. alocis ATCC 35896, the type strain. A comparative protein in silico analysis of the proteome revealed more cell wall anchoring proteins in the F. alocis D-62D compared to F. alocis ATCC 35896. Their expression was enhanced by coinfection with Porphyromonas gingivalis. Taken together, the variation in the pathogenic potential of the F. alocis strains may be related to the differential expression of several putative virulence factors.

The Porphyromonas gingivalis VimA protein has multifunctional properties that can modulate several of its major virulence factors. To further characterize VimA, P. gingivalis FLL406 carrying an additional vimA gene and a vimA -defective mutant in a different P. gingivalis genetic background were evaluated. The vimA -defective mutant (FLL451) in the P. gingivalis ATCC 33277 genetic background showed a phenotype similar to that of the vimA -defective mutant (FLL92) in the P. gingivalis W83 genetic background. In contrast to the wild type, gingipain activity was increased in P. gingivalis FLL406, a vimA chimeric strain. P. gingivalis FLL451 had a five times higher biofilm-forming capacity than the parent strain. HeLa cells incubated with P. gingivalis FLL92 showed a decrease in invasion, in contrast to P. gingivalis FLL451 and FLL406, which showed increases of 30 and 40%, respectively. VimA mediated coenzyme A (CoA) transfer to isoleucine and reduced branched-chain amino acid metabolism. The lipid A content and associated proteins were altered in the vimA -defective mutants. The VimA chimera interacted with several proteins which were found to have an LXXTG motif, similar to the sorting motif of Gram-positive organisms. All the proteins had an N-terminal signal sequence with a putative sorting signal of L(P/T/S)X(T/N/D)G and two unique signatures of EXGXTX and HISXXGXG, in addition to a polar tail. Taken together, these observations further confirm the multifunctional role of VimA in modulating virulence possibly through its involvement in acetyl-CoA transfer and lipid A synthesis and possibly by protein sorting.

Recombinant VimA protein can interact with the gingipains and several other proteins that may play a role in its biogenesis in Porphyromonas gingivalis. In silico analysis of PG2096, a hypothetical protein that was shown to interact with VimA, suggests that it may have environmental stress resistance properties. To further evaluate the role(s) of PG2096, the predicted open reading frame was PCR amplified from P. gingivalis W83 and insertionally inactivated using the ermF-ermAM antibiotic-resistance cassette. One randomly chosen PG2096-defective mutant created by allelic exchange and designated FLL205 was further characterized. Under normal growth conditions at 37 °C, Arg-X and Lys-X gingipain activities in FLL205 were reduced by approximately 35 % and 21 %, respectively, compared to the wild-type strain. However, during prolonged growth at an elevated temperature of 42 °C, Arg-X activity was increased by more than 40 % in FLL205 in comparison to the wild-type strain. In addition, the PG2096-defective mutant was more resistant to oxidative stress when treated with 0.25 mM hydrogen peroxide. Taken together these results suggest that the PG2096 gene, designated regT (regulator of gingipain activity at elevated temperatures), may be involved in regulating gingipain activity at elevated temperatures and be important in oxidative stress resistance in P. gingivalis.

Tuberculosis (TB) is a communicable disease and remains one of the top 10 causes of death worldwide. One fourth of the world population is infected with TB at a risk of developing disease. The increase in the incidence of drug resistant TB around the world urges the need to develop a new candidate to fight against the disease. Plants were considered as the rich source of bioactive components to be used as potential drugs. Medicinal plants are used in pure as well as crude materials for their medicinal properties. Our research aims in identifying the phyto-molecules which have anti- tuberculosis property. Four medicinal plants namely, Acalyphaciliata (Kuppaimeni), Solanumtrilobatum (Thuthuvalai), Momordicacharantia (Bitter Gourd) and Sennaauriculata (Avaram) were chosen to evaluate their antimicrobial activity focusing on anti-tubercular activity. The methanol extracts of the medicinal plants showed significant inhibitory activity against bacterial and fungal pathogens. Sennaauriculata methanol extracts showed activity against S. aureus, E. coli, P. aeruginosa and C. albicans. In the screening of antimycobacterial activity done by LRP assay, among the plant extracts tested, the hexane crude extracts of Momordicacharantia (Bitter Gourd) showed 82.2% and 81.03% of inhibition against M. tuberculosis H37Rv at 500µg/ml and 250µg/ml concentration respectively. Similarly, the methanol crude extracts of Momordicacharantia showed 87.14% and 63.55% of inhibition at 500µg/ml and 250µg/ml concentration respectively.