Insights into the genomic features and lifestyle of B1 subcluster mycobacteriophages.

Bacteriophages infecting Mycobacterium smegmatis mc2155 are numerous and, hence, are classified into clusters based on nucleotide sequence similarity. Analyzing phages belonging to clusters/subclusters can help gain deeper insights into their biological features and potential therapeutic applications. In this study, for genomic characterization of B1 subcluster mycobacteriophages, a framework of online tools was developed, which enabled functional annotation of about 55% of the previously deemed hypothetical proteins in B1 phages. We also studied the phenotype, lysogeny status, and antimycobacterial activity of 10 B1 phages against biofilm and an antibiotic-resistant M. smegmatis strain (4XR1). All 10 phages belonged to the Siphoviridae family, appeared temperate based on their spontaneous release from the putative lysogens and showed antibiofilm activity. The highest inhibitory and disruptive effects on biofilm were 64% and 46%, respectively. This systematic characterization using a combination of genomic and experimental tools is a promising approach to furthering our understanding of viral dark matter.

A cyclic lipopeptide produced by an antagonistic bacterium relies on its tail and transient receptor potential-type Ca2+ channels to immobilize a green alga.

The antagonistic bacterium Pseudomonas protegens secretes the cyclic lipopeptide (CLiP) orfamide A, which triggers a Ca2+ signal causing rapid deflagellation of the microalga Chlamydomonas reinhardtii. We performed chemical synthesis of orfamide A derivatives and used an aequorin reporter line to measure their Ca2+ responses. Immobilization of algae was studied using a modulator and mutants of transient receptor potential (TRP)-type channels. By investigating targeted synthetic orfamide A derivatives, we found that N-terminal amino acids of the linear part and the terminal fatty acid region are important for the specificity of the Ca2+ -signal causing deflagellation. Molecular editing indicates that at least two distinct Ca2+ -signaling pathways are triggered. One is involved in deflagellation (Thr3 change, fatty acid tail shortened by 4C), whereas the other still causes an increase in cytosolic Ca2+ in the algal cells, but does not cause substantial deflagellation (Leu1 change, fatty acid hydroxylation, fatty acid changes by 2C). Using mutants, we define four TRP-type channels that are involved in orfamide A signaling; only one (ADF1) responds additionally to low pH. These results suggest that the linear part of the CLiP plays one major role in Ca2+ signaling, and that orfamide A uses a network of algal TRP-type channels for deflagellation.

Antibody Polymer Conjugates (APCs) for Active Targeted Therapeutic Delivery.

Antibody drug conjugates (ADCs) are poised to have an enormous impact on targeted nanomedicine, especially in many cancer pathologies. The reach of the current format of ADCs is limited by their low drug-to-antibody ratio (DAR) because of the associated physiochemical instabilities. Here, we design antibody polymer conjugates (APCs) as a modular strategy to utilize polymers to address ADC's shortcomings. We show here that conjugation of polymer-based therapeutic molecules to antibodies helps increase the DAR, owing to the hydrophilic comonomer in the polymer that helps in masking the increased hydrophobicity caused by high drug loading. We show that the platform exhibits cell targetability and selective cell killing in multiple cell lines expressing disease-relevant antigens, viz., HER2 and EGFR. The ability to use different functionalities in the drug as the handle for polymer attachment further demonstrates the platform nature of APCs. The findings here could serve as an alternative design strategy for the next generation of active targeted nanomedicine.

What's Next after Lipid Nanoparticles? A Perspective on Enablers of Nucleic Acid Therapeutics.

Recent success of mRNA-based COVID-19 vaccines have bolstered the strength of nucleic acids as a therapeutic platform. The number of new clinical trial candidates is skyrocketing with the potential to address many unmet clinical needs. Despite advancements in other aspects, the systemic delivery of nucleic acids to target sites remains a major challenge. Thus, nucleic acid based therapy has yet to reach its full potential. In this review, we shed light on a select few prospective technologies that exhibit substantial potential over traditional nanocarrier designs for nucleic acid delivery. We critically analyze these systems with specific attention to the possibilities for clinical translation.

Disulfide Bridging Strategies in Viral and Nonviral Platforms for Nucleic Acid Delivery.

Self-assembled nanostructures that are sensitive to environmental stimuli are promising nanomaterials for drug delivery. In this class, disulfide-containing redox-sensitive strategies have gained enormous attention because of their wide applicability and simplicity of nanoparticle design. In the context of nucleic acid delivery, numerous disulfide-based materials have been designed by relying on covalent or noncovalent interactions. In this review, we highlight major advances in the design of disulfide-containing materials for nucleic acid encapsulation, including covalent nucleic acid conjugates, viral vectors or virus-like particles, dendrimers, peptides, polymers, lipids, hydrogels, inorganic nanoparticles, and nucleic acid nanostructures. Our discussion will focus on the context of the design of materials and their impact on addressing the current shortcomings in the intracellular delivery of nucleic acids.

Computational identification and characterization of antigenic properties of Rv3899c of Mycobacterium tuberculosis and its interaction with human leukocyte antigen (HLA).

A rise in drug-resistant tuberculosis (TB) cases demands continued efforts towards the discovery and development of drugs and vaccines. Secretory proteins of Mycobacterium tuberculosis (H37Rv) are frequently studied for their antigenicity and their scope as protein subunit vaccines requires further analysis. In this study, Rv3899c of H37Rv emerges as a potential vaccine candidate on its evaluation by several bioinformatics tools. It is a non-toxic, secretory protein with an 'immunoglobulin-like' fold which does not show similarity with a human protein. Through BlastP and MEME suite analysis, we found Rv3899c homologs in several mycobacterial species and its antigenic score (0.54) to compare well with the known immunogens such as ESAT-6 (0.56) and Rv1860 (0.52). Structural examination of Rv3899c predicted ten antigenic peptides, an accessibility profile of the antigenic determinants constituting B cell epitope-rich regions and a low abundance of antigenic regions (AAR) value. Significantly, STRING analysis showed ESX-2 secretion system proteins and antigenic PE/PPE proteins of H37Rv as the interacting partners of Rv3899c. Further, molecular docking predicted Rv3899c to interact with human leukocyte antigen HLA-DRB1*04:01 through its antigenically conserved motif (RAAEQQRLQRIVDAVARQEPRISWAAGLRDDGTT). Interestingly, the binding affinity was observed to increase on citrullination of its Arg1 residue. Taken together, the computational characterization and predictive information suggest Rv3899c to be a promising TB vaccine candidate, which should be validated experimentally.

Charge-Conversion Strategies for Nucleic Acid Delivery.

Nucleic acids are now considered as one of the most potent therapeutic modalities, as their roles go beyond storing genetic information and chemical energy or as signal transducer. Attenuation or expression of desired genes through nucleic acids have profound implications in gene therapy, gene editing and even in vaccine development for immunomodulation. Although nucleic acid therapeutics bring in overwhelming possibilities towards the development of molecular medicines, there are significant loopholes in designing and effective translation of these drugs into the clinic. One of the major pitfalls lies in the traditional design concepts for nucleic acid drug carriers, viz. cationic charge induced cytotoxicity in delivery pathway. Targeting this bottleneck, several pioneering research efforts have been devoted to design innovative carriers through charge-conversion approaches, whereby built-in functionalities convert from cationic to neutral or anionic, or even from anionic to cationic enabling the carrier to overcome several critical barriers for therapeutics delivery, such as serum deactivation, instability in circulation, low transfection and poor endosomal escape. This review will critically analyze various molecular designs of charge-converting nanocarriers in a classified approach for the successful delivery of nucleic acids. Accompanied by the narrative on recent clinical nucleic acid candidates, the review concludes with a discussion on the pitfalls and scope of these interesting approaches.

Synergistic Interplay of Covalent and Non-Covalent Interactions in Reactive Polymer Nanoassembly Facilitates Intracellular Delivery of Antibodies.

The primary impediments in developing large antibodies as drugs against intracellular targets involve their low transfection efficiency and suitable reversible encapsulation strategies for intracellular delivery with retention of biological activity. To address this, we outline an electrostatics-enhanced covalent self-assembly strategy to generate polymer-protein/antibody nanoassemblies. Through structure-activity studies, we down-select the best performing self-immolative pentafluorophenyl containing activated carbonate polymer for bioconjugation. With the help of an electrostatics-aided covalent self-assembly approach, we demonstrate efficient encapsulation of medium to large proteins (HRP, 44 kDa and ß-gal, 465 kDa) and antibodies (ca. 150 kDa). The designed polymeric nanoassemblies are shown to successfully traffic functional antibodies (anti-NPC and anti-pAkt) to cytosol to elicit their bioactivity towards binding intracellular protein epitopes and inducing apoptosis.

Unlocking prophage potential: In silico and experimental analysis of a novel Mycobacterium fortuitum LysinB containing a peptidoglycan-binding domain.

Endolysins are highly evolved bacteriophage-encoded lytic enzymes produced to damage the bacterial cell wall for phage progeny release. They offer promising potential as highly specific lytic proteins with a low chance of bacterial resistance. The diversity in lysin sequences and domain organization can be staggering. In silico analysis of bacteriophage and prophage genomes can help identify endolysins exhibiting unique features and high antibacterial activity, hence feeding the pipeline of narrow-spectrum protein antibiotics. Mycobacteriophage lysis cassettes mostly have two lytic enzymes, LysinA and LysinB. The enzyme LysinA targets peptidoglycan in the cell wall and possesses a modular architecture. LysinB typically contains a single domain and acts upon the mycolyl ester linkages in mycolyl-arabinogalactan-peptidoglycan (Payne et al., 2010). This study aimed to find novel LysinBs against Mycobacterium fortuitum. After a detailed in silico characterization of lysis cassettes from three M. fortuitum prophages, we chose to work on a LysinB (hereafter described as LysinB_MF) found in an incomplete prophage (phiE1336, 9.4 kb in strain E1336). LysinB_MF showed low sequence similarity with any other endolysins in the database and formed a separate clade on phylogenetic analysis. LysinB_MF's structure, extracted from the AlphaFold Protein Structure Database, demonstrated a modular architecture with two structurally distinct domains: a peptidoglycan-binding domain (PGBD) at the N-terminal and the characteristic alpha/beta hydrolase domain connected via a linker peptide. We found the alpha/beta hydrolase domain, which is the enzyme-active domain (EAD), contains the conserved Ser-Asp-His catalytic triad with a tunnel-like topology and forms intermolecular hydrogen bonds. The PGBD shows structural similarity to the cell-wall binding domain of an amidase from Clostridium acetobutylicum, hinting at its acquisition due to domain mobility. Our in silico electrostatic potential analysis suggested that PGBD might be essential to the enzyme activity. This was experimentally validated by generating a truncated version of the enzyme, which demonstrated about six-fold decreased activity compared to its native form. The antimycobacterial activity of this enzyme was also compromised in its absence. Based on our analysis, PGBD emerged as an integral constituent of enzymes with diverse functional properties and is predicted to be a conserved cross-kingdom. Overall, this study highlights the importance of mining mycobacterial prophages as a novel endolysin source. It also provides unique insights into the diverse architecture of mycobacteriophage-encoded endolysins and the importance of functional domains for their catalytic activities.

Functional characterization of a DNA-dependent AAA ATPase in a F-cluster mycobacteriophage.

Mycobacteriophages are viruses of Mycobacterium spp. with promising diagnostic and therapeutic potential. Phage genome exploration and characterization of their proteomes are essential to gaining a better understanding of their role in phage biology. So far, genomes of about 2113 mycobacteriophages have been defined and from among those, 1563 phage protein families (phamilies) are identified. However, the function of only a fraction (about 15%) is known since a majority of ORFs in phage genomes are hypothetical proteins. In this study, we have analyzed Gp65 (AQT25877.1), a putative AAA ATPase (Pham 9410) from a F1 cluster mycobacteriophage SimranZ1 (KY385384.1). Though homology analysis of Gp65-AAA ATPase showed the presence of this gene in 38 mycobacteriophages of the F1 cluster, however its further analysis has not been reported yet in any study. The sequence-based functional annotation predicted Gp65 to belong to the P-loop NTPase superfamily and to have AAA_24 and RecA/RadA domains, which are known to be involved in ATP-dependent DNA recombination/repair/maintenance mechanisms. Molecular docking of Gp65 with ATP identified Gly21 and Ser23 residues to be involved in the specific binding. The experimental validation of the DNA-dependent ATPase activity of Gp65 was done using a microtiter plate assay, where the ATPase activity was observed to increase in the presence of dsDNA. The structural characteristics of the protein are demonstrated by non-denaturing gel electrophoresis, showing Gp65 to exist in oligomeric states, which was confirmed by transmission electron microscopy (TEM). It was revealed to exist as a hexamer with a prominent central pore. In this study, based on the stated structural and functional characterization, we report the AAA ATPase to have a putative role in DNA recombination/repair/maintenance mechanism in mycobacteriophages.

Beyond antibiotics: phage-encoded lysins against Gram-negative pathogens.

Antibiotics remain the frontline agents for treating deadly bacterial pathogens. However, the indiscriminate use of these valuable agents has led to an alarming rise in AMR. The antibiotic pipeline is insufficient to tackle the AMR threat, especially with respect to the WHO critical category of priority Gram-negative pathogens, which have become a serious problem as nosocomial and community infections and pose a threat globally. The AMR pandemic requires solutions that provide novel antibacterial agents that are not only effective but against which bacteria are less likely to gain resistance. In this regard, natural or engineered phage-encoded lysins (enzybiotics) armed with numerous features represent an attractive alternative to the currently available antibiotics. Several lysins have exhibited promising efficacy and safety against Gram-positive pathogens, with some in late stages of clinical development and some commercially available. However, in the case of Gram-negative bacteria, the outer membrane acts as a formidable barrier; hence, lysins are often used in combination with OMPs or engineered to overcome the outer membrane barrier. In this review, we have briefly explained AMR and the initiatives taken by different organizations globally to tackle the AMR threat at different levels. We bring forth the promising potential and challenges of lysins, focusing on the WHO critical category of priority Gram-negative bacteria and lysins under investigation for these pathogens, along with the challenges associated with developing them as therapeutics within the existing regulatory framework.

Alternative Treatment Strategies for Secondary Bacterial and Fungal Infections Associated with COVID-19.

Antimicrobials are essential for combating infectious diseases. However, an increase in resistance to them is a major cause of concern. The empirical use of drugs in managing COVID-19 and the associated secondary infections have further exacerbated the problem of antimicrobial resistance. Hence, the situation mandates exploring and developing efficient alternatives for the treatment of bacterial and fungal infections in patients suffering from COVID-19 or other viral infections. In this review, we have described the alternatives to conventional antimicrobials that have shown promising results and are at various stages of development. An acceleration of efforts to investigate their potential as therapeutics can provide more treatment options for clinical management of drug-resistant secondary bacterial and fungal infections in the current pandemic and similar potential outbreaks in the future. The alternatives include bacteriophages and their lytic enzymes, anti-fungal enzymes, antimicrobial peptides, nanoparticles and small molecule inhibitors among others. What is required at this stage is to critically examine the challenges in developing the listed compounds and biomolecules as therapeutics and to establish guidelines for their safe and effective application within a suitable time frame. In this review, we have attempted to highlight the importance of rational use of antimicrobials in patients suffering from COVID-19 and boost the deployment of alternative therapeutics.

Core Hydrophobicity of Supramolecular Nanoparticles Induces NLRP3 Inflammasome Activation.

Designer nanomaterials capable of delivering immunomodulators to specific immune cells have been extensively studied. However, emerging evidence suggests that several of these nanomaterials can nonspecifically activate NLRP3 inflammasomes, an intracellular multiprotein complex controlling various immune cell functions, leading to undesirable effects. To understand what nanoparticle attributes activate inflammasomes, we designed a multiparametric polymer supramolecular nanoparticle system to modulate various surface and core nanoparticle-associated molecular patterns (NAMPs), one at a time. We also investigated several underlying signaling pathways, including lysosomal rupture-cathepsin B maturation and calcium flux-mitochondrial ROS production, to gain mechanistic insights into NAMPs-mediated inflammasome activation. Here, we report that out of the four NAMPs tested, core hydrophobicity strongly activates and positively correlates with the NLRP3 assembly compared to surface charge, core rigidity, and surface hydrophobicity. Moreover, we demonstrate different signaling inclinations and kinetics followed by differential core hydrophobicity patterns with the most hydrophobic ones exhibiting both lysosomal rupture and calcium influx early on. Altogether, this study will help design the next generation of polymeric nanomaterials for specific regulation of inflammasome activation, aiding efficient immunotherapy and vaccine delivery.

In Situ Forming Injectable Thermoresponsive Hydrogels for Controlled Delivery of Biomacromolecules.

Due to their relatively large molecular sizes and delicate nature, biologic drugs such as peptides, proteins, and antibodies often require high and repeated dosing, which can cause undesired side effects and physical discomfort in patients and render many therapies inordinately expensive. To enhance the efficacy of biologic drugs, they could be encapsulated into polymeric hydrogel formulations to preserve their stability and help tune their release in the body to their most favorable profile of action for a given therapy. In this study, a series of injectable, thermoresponsive hydrogel formulations were evaluated as controlled delivery systems for various peptides and proteins, including insulin, Merck proprietary peptides (glucagon-like peptide analogue and modified insulin analogue), bovine serum albumin, and immunoglobulin G. These hydrogels were prepared using concentrated solutions of poly(lactide-co-glycolide)-block-poly(ethylene glycol)-block-poly(lactide-co-glycolide) (PLGA-PEG-PLGA), which can undergo temperature-induced sol-gel transitions and spontaneously solidify into hydrogels near the body temperature, serving as an in situ depot for sustained drug release. The thermoresponsiveness and gelation properties of these triblock copolymers were characterized by dynamic light scattering (DLS) and oscillatory rheology, respectively. The impact of different hydrogel-forming polymers on release kinetics was systematically investigated based on their hydrophobicity (LA/GA ratios), polymer concentrations (20, 25, and 30%), and phase stability. These hydrogels were able to release active peptides and proteins in a controlled manner from 4 to 35 days, depending on the polymer concentration, solubility nature, and molecular sizes of the cargoes. Biophysical studies via size exclusion chromatography (SEC) and circular dichroism (CD) indicated that the encapsulation and release did not adversely affect the protein conformation and stability. Finally, a selected PLGA-PEG-PLGA hydrogel system was further investigated by the encapsulation of a therapeutic glucagon-like peptide analogue and a modified insulin peptide analogue in diabetic mouse and minipig models for studies of glucose-lowering efficacy and pharmacokinetics, where superior sustained peptide release profiles and long-lasting glucose-lowering effects were observed in vivo without any significant tolerability issues compared to peptide solution controls. These results suggest the promise of developing injectable thermoresponsive hydrogel formulations for the tunable release of protein therapeutics to improve patient's comfort, convenience, and compliance.