Comparative proteomics and variations in extracellular matrix of Candida tropicalis biofilm in response to citral.

Candida tropicalis is an opportunistic human pathogen with an ability to cause superficial as well as systemic infections in immunocompromised patients. The formation of biofilm by C. tropicalis can cause dreadful and persistent infections which are difficult to treat due to acquired resistance. Presently, available anti-Candida drugs exhibit a high frequency of resistance, low specificity and toxicity at a higher dosage. In addition, the discovery of natural or synthetic anti-Candida drugs is slow paced and often does not pass clinical trials. Citral, a monoterpene aldehyde, has shown effective antimicrobial activities against various microorganisms. However, only few studies have elaborated the action of citral against the biofilm of C. tropicalis. In the present work, the aim was to study the fungicidal effect, differential expression of proteome and changes in extracellular matrix in response to the sub-lethal concentration (16 µg/mL) of citral. The administration of citral on C. tropicalis biofilm leads to a fungicidal effect. Furthermore, the differential expression of proteome has revealed twenty-five proteins in C. tropicalis biofilm, which were differentially expressed in the presence of citral. Among these, amino acid biosynthesis (Met6p, Gln1p, Pha2p); nucleotide biosynthesis (Xpt1p); carbohydrate metabolism (Eno1p, Fba1p, Gpm1p); sterol biosynthesis (Mvd1p/Erg19p, Hem13p); energy metabolism (Dnm1p, Coa1p, Ndk1p, Atp2p, Atp4p, Hts1p); oxidative stress (Hda2p, Gre22p, Tsa1p, Pst2p, Sod2p) and biofilm-specific (Adh1p, Ape1p, Gsp1p) proteins were identified. The overexpression of oxidative stress-related proteins indicates the response of biofilm cell to combating oxidative stress during citral treatment. Moreover, the upregulation of Adh1p is of particular interest because it subsidizes the biofilm inhibition through ethanol production as a cellular response. The augmented expression of Mvd1p/Erg19p signifies the effect of citral on ergosterol biosynthesis. The presence of citral has also shown an increment in hexosamine and ergosterol component in extracellular matrix of C. tropicalis biofilm. Hence, it is indicated that the cellular response towards citral acts through multifactorial processes. This study will further help in the interpretation of the effect of citral on C. tropicalis biofilm and development of novel antifungal agents against these potential protein targets.

Insights into COVID-19 Vaccine Development Based on Immunogenic Structural Proteins of SARS-CoV-2, Host Immune Responses, and Herd Immunity.

The first quarter of the 21st century has remarkably been characterized by a multitude of challenges confronting human society as a whole in terms of several outbreaks of infectious viral diseases, such as the 2003 severe acute respiratory syndrome (SARS), China; the 2009 influenza H1N1, Mexico; the 2012 Middle East respiratory syndrome (MERS), Saudi Arabia; and the ongoing coronavirus disease 19 (COVID-19), China. COVID-19, caused by SARS-CoV-2, reportedly broke out in December 2019, Wuhan, the capital of China's Hubei province, and continues unabated, leading to considerable devastation and death worldwide. The most common target organ of SARS-CoV-2 is the lungs, especially the bronchial and alveolar epithelial cells, culminating in acute respiratory distress syndrome (ARDS) in severe patients. Nevertheless, other tissues and organs are also known to be critically affected following infection, thereby complicating the overall aetiology and prognosis. Excluding H1N1, the SARS-CoV (also referred as SARS-CoV-1), MERS, and SARS-CoV-2 are collectively referred to as coronaviruses, and taxonomically placed under the realm Riboviria, order Nidovirales, suborder Cornidovirineae, family Coronaviridae, subfamily Orthocoronavirinae, genus Betacoronavirus, and subgenus Sarbecovirus. As of 23 September 2021, the ongoing SARS-CoV-2 pandemic has globally resulted in around 229 million and 4.7 million reported infections and deaths, respectively, apart from causing huge psychosomatic debilitation, academic loss, and deep economic recession. Such an unprecedented pandemic has compelled researchers, especially epidemiologists and immunologists, to search for SARS-CoV-2-associated potential immunogenic molecules to develop a vaccine as an immediate prophylactic measure. Amongst multiple structural and non-structural proteins, the homotrimeric spike (S) glycoprotein has been empirically found as the most suitable candidate for vaccine development owing to its immense immunogenic potential, which makes it capable of eliciting both humoral and cell-mediated immune responses. As a consequence, it has become possible to design appropriate, safe, and effective vaccines, apart from related therapeutic agents, to reduce both morbidity and mortality. As of 23 September 2021, four vaccines, namely, Comirnaty, COVID-19 vaccine Janssen, Spikevax, and Vaxzevria, have received the European Medicines Agency's (EMA) approval, and around thirty are under the phase three clinical trial with emergency authorization by the vaccine-developing country-specific National Regulatory Authority (NRA). In addition, 100-150 vaccines are under various phases of pre-clinical and clinical trials. The mainstay of global vaccination is to introduce herd immunity, which would protect the majority of the population, including immunocompromised individuals, from infection and disease. Here, we primarily discuss category-wise vaccine development, their respective advantages and disadvantages, associated efficiency and potential safety aspects, antigenicity of SARS-CoV-2 structural proteins and immune responses to them along with the emergence of SARS-CoV-2 VOC, and the urgent need of achieving herd immunity to contain the pandemic.

Characterisation of recombinant thermostable manganese-superoxide dismutase (NeMnSOD) from Nerium oleander.

Superoxide dismutase is one of the key antioxidant enzymes accountable for the eradication of free radicals generated during various metabolic processes. This is first study reporting a thermostable MnSOD obtained from a xerophytic plant, Nerium oleander. The full-length gene identified using Rapid amplification of cDNA ends revealed an open reading frame of 699 bp flanked by 5'UTR and 3'UTR of 134 bp and 198 bp respectively. The corresponding NeMnSOD protein was cloned and expressed in Escherichia coli. The purified protein yields a band of 25.4 kDa, which established a specific activity of 2617 units mg-1 of protein and under native condition yield bands of 52 kDa and 110 kDa, confirming the dimeric and tetrameric state of the protein. The Km and Vmax of 0.078 ± 0.008 mM and 1052.3 ± 33.59 units mg-1 of protein, respectively. The purified enzyme demonstrated thermostability by retaining more than 20% activity at a temperature 70 ℃. The enzyme functioned at pH range of 4-9.0 with maximum activity at pH 7.4. Sodium azide, effectively inhibited the activity of enzyme confirming it to be MnSOD. The enzyme activity was least affected on treatment with strong denaturants (Urea, guanidine HCl and SDS) and harsh chemicals (DTT, CHAPS and ß-mercapto-ethanol) These experimental data validated with Insilco analysis revealed that NeMnSOD possessed thermo as well as kinetically stable moiety which can be further exploited with its applications in the field of pharmaceutical, food and cosmetic industry, which urge for such thermostable enzyme.

Delineating the Biofilm Inhibition Mechanisms of Phenolic and Aldehydic Terpenes against Cryptococcus neoformans.

The recalcitrant biofilm formed by fungus Cryptococcus neoformans is a life-threatening pathogenic condition responsible for further intensifying cryptococcosis. Considering the enhanced biofilm resistance and toxicity of synthetic antifungal drugs, the search for efficient, nontoxic, and cost-effective natural therapeutics has received a major boost. Phenolic (thymol and carvacrol) and aldehydic (citral) terpenes are natural and safe alternatives capable of efficient microbial biofilm inhibition. However, the biofilm inhibition mechanism of these terpenes still remains unclear. In this study, we adopted an integrative biophysical and biochemical approach to elucidate the hierarchy of their action against C. neoformans biofilm cells. The microscopic analysis revealed disruption of the biofilm cell surface with elevation in surface roughness and reduction in cell height. Although all terpenes acted through ergosterol biosynthesis inhibition, the phenolic terpenes also selectively interacted via ergosterol binding. Further, the alterations in the fatty acid profile in response to terpenes attenuated the cell membrane fluidity with enhanced permeability, resulting in pore formation and efflux of the K+/intracellular content. Additionally, mitochondrial depolarization caused higher levels of reactive oxygen species, which led to increased lipid peroxidation and activation of the antioxidant defense system. Indeed, the oxidative stress caused a significant decline in the amount of extracellular polymeric matrix and capsule sugars (mannose, xylose, and glucuronic acid), leading to a reduced capsule size and an overall negative charge on the cell surface. This comprehensive data revealed the mechanistic insights into the mode of action of terpenes on biofilm inhibition, which could be exploited for formulating novel anti-biofilm agents.

In Vitro Anti-Biofilm Activities of Citral and Thymol Against Candida Tropicalis.

Candida tropicalis is an emerging non-albicans Candida species which is pathogenic to the immune-compromised humans, especially in tropical countries, including India. The acquired resistance of Candida species towards antifungal therapies is of major concern. Moreover, limited efficacy and dosage constraint of synthetic drugs have indicated the prerequisite of finding new and natural drugs for treatment. In the present study, we have compared the influence of citral and thymol on C. tropicalis and its biofilm along with expression levels of certain antifungal tolerance genes. The antifungal and anti-biofilm activities of the both were studied using 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide sodium salt (XTT) reduction assay, field emission scanning electron microscope (FE-SEM) and confocal laser scanning microscope (CLSM) and real-time reverse transcription polymerase chain reaction (RT-PCR) analysis. Citral and thymol have damaged the cells with distorted surface and less viability. Quantitative real-time PCR analysis showed augmented expression of the cell membrane biosynthesis genes including ERG11/CYT450 against citral and the cell wall related tolerance genes involving CNB1 against thymol thus, depicting their differential mode of actions.

In vitro metal catalyzed oxidative stress in DAH7PS: Methionine modification leads to structure destabilization and induce amorphous aggregation.

The first committed step of the shikimate pathway is catalyzed by a metalloenzyme 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (DAH7PS), which exhibits vulnerability to the oxidative stress. DAH7PS undergoes inactivation in multiple ways in the presence of redox metal, H2O2, and superoxide. The molecular mechanism and susceptibility of its inactivation might differ in different organisms and are presently unclear. In the present work, we have cloned, expressed and purified a DAH7PS from Providencia alcalifaciens (PaDAH7PS). The oligomeric state and effect of redox metal treatment on its stability were analyzed through the size exclusion chromatography. The FTIR, MALDI-TOF/TOF-MS studies revealed that methionine residues were modified to methionine sulfoxide in PaDAH7PS. During oxidation, PaDAH7PS is altered into partially folded protein and unfolded states as determined by CD and Fluorescence studies. A significant loss in enzymatic activity of PaDAH7PS was determined and the formation of amorphous aggregates was visualized using AFM imaging and also confirmed by ThT binding based assay. This is the first report where we have shown a hexameric DAH7PS and the methionine residues of PaDAH7PS get oxidize in the presence of oxidative stress. The partially folded and unfolded oligomeric states with high ß-content of PaDAH7PS might be the critical precursors for aggregation.

Antifungal and Anti-Biofilm Activity of Essential Oil Active Components against Cryptococcus neoformans and Cryptococcus laurentii.

Cryptococcosis is an emerging and recalcitrant systemic infection occurring in immunocompromised patients. This invasive fungal infection is difficult to treat due to the ability of Cryptococcus neoformans and Cryptococcus laurentii to form biofilms resistant to standard antifungal treatment. The toxicity concern of these drugs has stimulated the search for natural therapeutic alternatives. Essential oil and their active components (EO-ACs) have shown to possess the variety of biological and pharmacological properties. In the present investigation the effect of six (EO-ACs) sourced from Oregano oil (Carvacrol), Cinnamon oil (Cinnamaldehyde), Lemongrass oil (Citral), Clove oil (Eugenol), Peppermint oil (Menthol) and Thyme oil (thymol) against three infectious forms; planktonic cells, biofilm formation and preformed biofilm of C. neoformans and C. laurentii were evaluated as compared to standard drugs. Data showed that antibiofilm activity of the tested EO-ACs were in the order: thymol>carvacrol>citral>eugenol=cinnamaldehyde>menthol respectively. The three most potent EO-ACs, thymol, carvacrol, and citral showed excellent antibiofilm activity at a much lower concentration against C. laurentii in comparison to C. neoformans indicating the resistant nature of the latter. Effect of the potent EO-ACs on the biofilm morphology was visualized using scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM), which revealed the absence of extracellular polymeric matrix (EPM), reduction in cellular density and alteration in the surface morphology of biofilm cells. Further, to realize the efficacy of the EO-ACs in terms of human safety, cytotoxicity assays and co-culture model were evaluated. Thymol and carvacrol as compared to citral were the most efficient in terms of human safety in keratinocyte- Cryptococcus sp. co-culture infection model suggesting that these two can be further exploited as cost-effective and non-toxic anti-cryptococcal drugs.