Demonstrating the immunostimulatory and cytokine-augmentation effects of bacterial ghosts on natural killer cells and Caenorhabditis Elegans.

The potential of bacteria-based immunotherapy lies in its ability to inherently enhance immune responses. However, the "liveness" of bacteria poses risks of bacterial escape, nonspecific immuno-stimulation, and ethical concerns, limiting their acceptability in immunotherapy. In this scenario, nonliving empty bacterial-cell envelopes, named bacterial ghosts (BGs), have emerged as immuno-stimulants with the potential to side-step the limitations of live bacterial therapies. This study demonstrates the capability of BGs in modulating the functionality of NK-92 cells and Caenorhabditis elegans (C. elegans), as well as perform as cytokine-therapy adjuvants. BGs were obtained through a pH-driven culture method, and were validated for their structural and chemical integrity via electron microscopy and spectroscopy. In NK-92 cells, BGs have shown significant immuno-stimulation by boosting the gene-expression of perforin, granzyme-B, Fas-L, and interferon-gamma by factors of 3.5-, 1.5-, 12.5-, and 8.6-folds, respectively. Combined BG and IL-12 treatment yielded a notable 10.2-fold increase in interferon-gamma protein expression in 24 h. The BGs also significantly influenced the innate immune response in C. elegans through the upregulation of lysozyme genes viz., ilys-3 (8.8-fold) and lys-2 (3.1-fold). Our investigation into the impact of BGs on natural killer cells and C. elegans highlights its potential as a valid alternative approach for new-age immunotherapy and cytokine augmentation.

Investigating the pro-inflammatory differentiation of macrophages with bacterial ghosts in potential infection control.

In the complex realm of bacterial infections, particularly those caused by Staphylococcus aureus (S. aureus), macrophages play a pivotal role in orchestrating the immune response. During the initial stages of infection, the monocytes give rise to macrophages with a pro-inflammatory (M1 type) behaviour, engulfing and neutralizing the invading pathogens. However, under the sustained influence of S. aureus infection, monocytes can undergo a transition into an anti-inflammatory M2 state (pro-infection) rather than the M1 state (anti-infection), thereby compromising effective infection control. Therefore, it is necessary to develop a strategy that would preserve the pro-inflammatory functions of macrophages, in a safe and controlled manner. For this, we focused on harnessing the potential of S. aureus-derived ghost cells (GCs) which are non-live empty envelopes of bacterial cells, but with the antigenic determinants intact. Through a unique Lugol's-iodine treatment, we generated GCs and characterization of these GCs using gel electrophoresis, FTIR, flow cytometry, TEM, and SEM confirmed their structural integrity. Following this, we assessed the extend of cellular association of the GCs with RAW267.4 macrophages, and observed an immediate interaction between the two, as evident from the flowcytometry and microscopy studies. We then performed macrophage polarisation on a human monocyte-macrophage model cell line, THP-1. Our findings revealed that GCs effectively activated macrophages, and promoted a pro-inflammatory polarisation with the expression of M1 differentiation markers (CD86, TNFα, IL-1ß, IL-6, IL-12) evaluated through both qPCR and ELISA. Interestingly an intermediary expression of M2 markers viz., CD206 and IL-10 was also observed, but was overruled by the enhanced expression of M1 markers at a later time point. Overall, our study introduces a novel approach utilizing GCs to guide naïve macrophages towards M1 subtypes, thereby potentiating immune responses during microbial infections. This innovative strategy can modulate macrophage function, ultimately improving outcomes in S. aureus infections and beyond.

Appraisal of cytotoxicity and acrylamide mitigation potential of L-asparaginase SlpA from fish gut microbiome.

L-asparaginase catalyzes the hydrolysis of L-asparagine to L-aspartic acid and ammonia. It has application in the treatment of acute lymphoblastic leukemia in children, as well as in other malignancies, in addition to its role as a food processing aid for the mitigation of acrylamide formation in the baking industry. Its use in cancer chemotherapy is limited due to problems such as its intrinsic glutaminase activity and associated side effects, leading to an increased interest in the search for novel L-asparaginases without L-glutaminase activity. This study reports the cloning and expression of an L-asparaginase contig obtained from whole metagenome shotgun sequencing of Sardinella longiceps gut microbiota. Purified recombinant glutaminase-free L-asparaginase SlpA was a 74 kDa homodimer, with maximal activity at pH 8 and 30 °C. Km and Vmax of SlpA were determined to be 3.008 mM and 0.014 mM/min, respectively. SlpA displayed cytotoxic activity against K-562 (chronic myeloid leukemia) and MCF-7 (breast cancer) cell lines with IC50 values of 0.3443 and 2.692 U/mL, respectively. SlpA did not show any cytotoxic activity against normal lymphocytes and was proved to be hemocompatible. Pre-treatment of biscuit and bread dough with different concentrations of SlpA resulted in a clear, dose-dependent reduction of acrylamide formation during baking. KEY POINTS: • Cloned and expressed L-asparaginase (SlpA) from fish gut microbiota • Purified SlpA displayed good cytotoxicity against K-562 and MCF-7 cell lines • SlpA addition caused a significant reduction of acrylamide formation during baking.

A magainin-2 like bacteriocin BpSl14 with anticancer action from fish gut Bacillus safensis SDG14.

Bacteriocins are gaining utmost importance in antimicrobial and chemotherapy due to their diverse structure and activity. This study centres on magainin-2 like bacteriocin with anticancer action, produced by Bacillus safensis strain SDG14 isolated from gut of marine fish Sardinella longiceps. The purified bacteriocin designated as BpSl14 was thermostable and pH tolerant. The molecular weight of BpS114 was estimated to be 6061.2 Da using MALDI-ToF MS. The partial primary sequence was elucidated by peptide mass fingerprinting using MALDI MS/MS. The tertiary structure of the partial sequence was similar to that of two magainin-2 α-helices joined together by extended indolicidin. The BpSl14 protein inhibited the cells of lung carcinoma, one of the deadliest cancers. Docking studies conducted with DR5 and TGF-ß, two of the most prominent apoptotic receptors in adenocarcinoma, also proved the anti-apoptotic action of BpSl14.

Metagenomic landscape of taxonomy, metabolic potential and resistome of Sardinella longiceps gut microbiome.

Fish gut microbiota, encompassing a colossal reserve of microbes represents a dynamic ecosystem, influenced by a myriad of environmental and host factors. The current study presents a comprehensive insight into Sardinella longiceps gut microbiome using whole metagenome shotgun sequencing. Taxonomic profiling identified the predominance of phylum Proteobacteria, comprising of Photobacterium, Vibrio and Shewanella sp. Functional annotation revealed the dominance of Clustering based subsystems, Carbohydrate, and Amino acids and derivatives. Analysis of Virulence, disease and defense subsystem identified genes conferring resistance to antibiotics and toxic compounds, like multidrug resistance efflux pumps and resistance genes for fluoroquinolones and heavy metals like cobalt, zinc, cadmium and copper. The presence of overlapping genetic mechanisms of resistance to antibiotics and heavy metals, like the efflux pumps is a serious cause of concern as it is likely to aggravate co-selection pressure, leading to an increased dissemination of these resistance genes to fish and humans.

Insights into the response of mangrove sediment microbiomes to heavy metal pollution: Ecological risk assessment and metagenomics perspectives.

Rapid urbanisation and ensuing anthropogenic pollution lead to an escalated occurrence of heavy metals and metal-resistant bacteria in the soil ecosystem. Mangrove ecosystems are particularly vulnerable to heavy metal bioaccumulation and often act as metal sinks of the coastal areas. As a consequence, the microbial population in mangrove sediments develop multifarious metal tolerance mechanisms to combat metal toxicity. In this context, metagenomic investigation of two mangroves, viz. Mangalavanam and Puthuvypin from the heavily populated metropolitan city, Cochin (Central Kerala, India) was undertaken to discern the metal resistance functions and taxonomic diversity of the microbial consortia. Estimation of heavy metal content using Inductively Coupled Plasma Atomic Emission Spectrometer (ICP-MS) identified the abundance of zinc, chromium, nickel copper, lead, arsenic, and cadmium in the mangrove sediments. Ecological risk index values indicated high cadmium contamination of the two estuarine samples. Whole metagenome shotgun sequencing of the Central Kerala mangroves and comparative analysis with mangrove metal resistomes from other geographical regions revealed the prevalence of cobalt-zinc-cadmium resistance and preponderance of Proteobacteria in all the datasets. Cation efflux system protein CusA constituted the majority of the reads at the function level. Comparative analysis of taxonomy identified the dominance of Anaeromyxobacter, Geobacter, Pseudomonas, Candidatus Solibacter, and Pelobacter in the mangrove datasets. Non-metric multidimensional scaling analysis of the metal resistance genes depicted strong geographical clustering of the function and composition of metal resistant bacteria, suggesting a strong innate resilience of microbiome towards anthropogenic perturbations. More robust studies with intensive sampling will enhance our understanding of the occurrence, interactions, and functions of microbial heavy metal resistome in mangrove ecosystems.

Harnessing the innate immune system by revolutionizing macrophage-mediated cancer immunotherapy.

Immunotherapy is a promising and safer alternative to conventional cancer therapies. It involves adaptive T-cell therapy, cancer vaccines, monoclonal antibodies, immune checkpoint blockade (ICB), and chimeric antigen receptor (CAR) based therapies. However, most of these modalities encounter restrictions in solid tumours owing to a dense, highly hypoxic and immune-suppressive microenvironment as well as the heterogeneity of tumour antigens. The elevated intra-tumoural pressure and mutational rates within fastgrowing solid tumours present challenges in efficient drug targeting and delivery. The tumour microenvironment is a dynamic niche infiltrated by a variety of immune cells, most of which are macrophages. Since they form a part of the innate immune system, targeting macrophages has become a plausible immunotherapeutic approach. In this review, we discuss several versatile approaches (both at pre-clinical and clinical stages) such as the direct killing of tumour-associated macrophages, reprogramming pro-tumour macrophages to anti-tumour phenotypes, inhibition of macrophage recruitment into the tumour microenvironment, novel CAR macrophages, and genetically engineered macrophages that have been devised thus far. These strategies comprise a strong and adaptable macrophage-toolkit in the ongoing fight against cancer and by understanding their significance, we may unlock the full potential of these immune cells in cancer therapy.

Whole genome sequence analysis enabled affirmation of the probiotic potential of marine sporulater Bacillus amyloliquefaciens BTSS3 isolated from Centroscyllium fabricii.

Probiotics are microorganisms when administered in adequate amounts, confer health benefits on the host. Many probiotics find application in various industries however, probiotic bacteria linked to marine environments are less explored.Although Bifidobacteria, Lactobacilli, and Streptococcus thermophilus are the most frequently used probiotics, Bacillus spp. have acquired much acceptance in human functional foods due to their increased tolerance and enduring competence in harsh environments like the gastrointestinal (GI) tract. In this study, the 4 Mbp genome sequence of Bacillus amyloliquefaciens strain BTSS3, a marine spore former isolated from deep-sea shark Centroscyllium fabricii, with antimicrobial and probiotic properties was sequenced, assembled, and annotated. Analysis revealed the presence of numerous genes presenting probiotic traits like production of vitamins, secondary metabolites, amino acids, secretory proteins, enzymes and other proteins that allow survival in GI tract as well as adhesion to intestinal mucosa. Adhesion by colonization in the gut was studied in vivo in zebrafish (Danio rerio) using FITC labelled B.amyloliquefaciens BTSS3. Preliminary study revealed the ability of the marine Bacillus to attach to the intestinal mucosa of the fish gut. The genomic data and the in vivo experiment affirms that this marine spore former is a promising probiotic candidate with potential biotechnological applications.

Metabarcoding data of bacterial diversity of the deep sea shark, Centroscyllium fabricii.

This data article describes the bacterial diversity of the deep sea shark, Centroscyllium fabricii. The data was acquired by metabarcoding using 16S rDNA. Centroscyllium fabricii, a deep sea shark found at depths below 275 m was sampled during Sagar Sampada cruise no 305 in the Indian Ocean and metagenomic DNA was isolated from the gut contents using QIAamp DNA stool minikit. V3 region of 16S rDNA region was amplified and the amplicons were sequenced on Illumina MiSeq system using 151 bp × 2 paired end reads. The data of this metagenome is available in the BioSample Submission Portal as Bio-Project PRJNA431407and Sequence Read Archive (SRA) accession number SRR6507004.

PCR in Metagenomics.

Metagenomics approach involves direct genetic analysis of environmental samples, evading the tedious culturing process. Polymerase chain reaction is one invaluable tool used for such analyses. Here, we describe one protocol for metagenomic DNA isolation that gives inhibitor-free DNA suitable for PCR and other genetic manipulations. Subsequently, the chapter describes the use of PCR as an indicator of quality of DNA and to amplify a marker of phylogeny. Further, the application of PCR for detection of specific genes and screening of metagenomic libraries is outlined.