Structural mechanisms of Tad pilus assembly and its interaction with an RNA virus.

Caulobacter crescentus Tad (tight adherence) pili, part of the type IV pili family, are crucial for mechanosensing, surface adherence, bacteriophage (phage) adsorption, and cell-cycle regulation. Unlike other type IV pilins, Tad pilins lack the typical globular ß sheet domain responsible for pilus assembly and phage binding. The mechanisms of Tad pilus assembly and its interaction with phage ΦCb5 have been elusive. Using cryo-electron microscopy, we unveiled the Tad pilus assembly mechanism, featuring a unique network of hydrogen bonds at its core. We then identified the Tad pilus binding to the ΦCb5 maturation protein (Mat) through its ß region. Notably, the amino terminus of ΦCb5 Mat is exposed outside the capsid and phage/pilus interface, enabling the attachment of fluorescent and affinity tags. These engineered ΦCb5 virions can be efficiently assembled and purified in Escherichia coli, maintaining infectivity against C. crescentus, which presents promising applications, including RNA delivery and phage display.

Removal of Pseudomonas type IV pili by a small RNA virus.

The retractile type IV pilus (T4P) is important for virulence of the opportunistic human pathogen Pseudomonas aeruginosa. The single-stranded RNA (ssRNA) phage PP7 binds to T4P and is brought to the cell surface through pilus retraction. Using fluorescence microscopy, we discovered that PP7 detaches T4P, which impairs cell motility and restricts the pathogen's virulence. Using cryo-electron microscopy, mutagenesis, optical trapping, and Langevin dynamics simulation, we resolved the structure of PP7, T4P, and the PP7/T4P complex and showed that T4P detachment is driven by the affinity between the phage maturation protein and its bound pilin, plus the pilus retraction force and speed, and pilus bending. Pilus detachment may be widespread among other ssRNA phages and their retractile pilus systems and offers new prospects for antibacterial prophylaxis and therapeutics.

A Multi-epitope Vaccine Candidate Against Bolivian Hemorrhagic fever Caused by Machupo Virus.

Bolivian hemorrhagic fever (BHF) caused by Machupo virus (MACV) is a New World arenavirus having a reported mortality rate of 25-35%. The BHF starts with fever, followed by headache, and nausea which rapidly progresses to severe hemorrhagic phase within 7 days of disease onset. One of the key promoters for MACV viral entry into the cell followed by viral propagation is performed by the viral glycoprotein (GPC). GPC is post-transcriptionally cleaved into GP1, GP2 and a signal peptide. These proteins all take part in the viral infection in host body. Therefore, GPC protein is an ideal target for developing therapeutics against MACV infection. In this study, GPC protein was considered to design a multi-epitope, multivalent vaccine containing antigenic and immunogenic CTL and HTL epitopes. Different structural validations and physicochemical properties were analysed to validate the vaccine. Docking and molecular dynamics simulations were conducted to understand the interactions of the vaccine with various immune receptors. Finally, the vaccine was codon optimised in silico and along with which immune simulation studies was performed in order to evaluate the vaccine's effectiveness in triggering an efficacious immune response against MACV.

Computational design of candidate multi-epitope vaccine against SARS-CoV-2 targeting structural (S and N) and non-structural (NSP3 and NSP12) proteins.

The COVID-19 pandemic caused by SARS-CoV-2 virus has created a global damage and has exposed the vulnerable side of scientific research towards novel diseases. The intensity of the pandemic is huge, with mortality rates of more than 6 million people worldwide in a span of 2 years. Considering the gravity of the situation, scientists all across the world are continuously attempting to create successful therapeutic solutions to combat the virus. Various vaccination strategies are being devised to ensure effective immunization against SARS-CoV-2 infection. SARS-CoV-2 spreads very rapidly, and the infection rate is remarkably high than other respiratory tract viruses. The viral entry and recognition of the host cell is facilitated by S protein of the virus. N protein along with NSP3 is majorly responsible for viral genome assembly and NSP12 performs polymerase activity for RNA synthesis. In this study, we have designed a multi-epitope, chimeric vaccine considering the two structural (S and N protein) and two non-structural proteins (NSP3 and NSP12) of SARS-CoV-2 virus. The aim is to induce immune response by generating antibodies against these proteins to target the viral entry and viral replication in the host cell. In this study, computational tools were used, and the reliability of the vaccine was verified using molecular docking, molecular dynamics simulation and immune simulation studies in silico. These studies demonstrate that the vaccine designed shows steady interaction with Toll like receptors with good stability and will be effective in inducing a strong and specific immune response in the body.Communicated by Ramaswamy H. Sarma.

Immunoinformatics based designing a multi-epitope vaccine against pathogenic Chandipura vesiculovirus.

Chandipura vesiculovirus (CHPV) is a rapidly emerging pathogen responsible for causing acute encephalitis. Due to its widespread occurrence in Asian and African countries, this has become a global threat, and there is an urgent need to design an effective and nonallergenic vaccine against this pathogen. The present study aimed to develop a multi-epitope vaccine using an immunoinformatics approach. The conventional method of vaccine design involves large proteins or whole organism which leads to unnecessary antigenic load with increased chances of allergenic reactions. In addition, the process is also very time-consuming and labor-intensive. These limitations can be overcome by peptide-based vaccines comprising short immunogenic peptide fragments that can elicit highly targeted immune responses, avoiding the chances of allergenic reactions, in a relatively shorter time span. The multi-epitope vaccine constructed using CTL, HTL, and IFN-γ epitopes was able to elicit specific immune responses when exposed to the pathogen, in silico. Not only that, molecular docking and molecular dynamics simulation studies confirmed a stable interaction of the vaccine with the immune receptors. Several physicochemical analyses of the designed vaccine candidate confirmed it to be highly immunogenic and nonallergic. The computer-aided analysis performed in this study suggests that the designed multi-epitope vaccine can elicit specific immune responses and can be a potential candidate against CHPV.

Flavanones: A potential natural inhibitor of the ATP binding site of PknG of Mycobacterium tuberculosis.

Over the years, Mycobacterium tuberculosis has been one of the major causes of death worldwide. As several clinical isolates of the bacteria have developed drug resistance against the target sites of the current therapeutic agents, the development of a novel drug is the pressing priority. According to recent studies on Mycobacterium tuberculosis, ATP binding sites of Mycobacterium tuberculosis serine/threonine protein kinases (MTPKs) have been identified as the new promising drug target. Among the several other protein kinases (PKs), Protein kinase G (PknG) was selected for the study because of its crucial role in modulating bacterium's metabolism to survive in host macrophages. In this work, we have focused on the H37Rv strain of Mycobacterium tuberculosis. A list of 477 flavanones obtained from the PubChem database was docked one by one against the crystallized and refined structure of PknG by in-silico techniques. Initially, potential inhibitors were narrowed down by preliminary docking. Flavanones were then selected using binding energies ranging from -7.9 kcal.mol-1 to -10.8 kcal.mol-1. This was followed by drug-likeness prediction, redocking analysis, and molecular dynamics simulations. Here, we have used experimentally confirmed drug AX20017 as a reference to determine candidate compounds that can act as potential inhibitors for PknG. PubChem165506, PubChem242065, PubChem688859, PubChem101367767, PubChem3534982, and PubChem42607933 were identified as possible target site inhibitors for PknG with a desirable negative binding energy of -8.1, -8.3, -8.4, -8.8, -8.6 and -7.9 kcal.mol-1 respectively. Communicated by Ramaswamy H. Sarma.

In silico designing of vaccine candidate against Clostridium difficile.

Clostridium difficile is a spore-forming gram-positive bacterium, recognized as the primary cause of antibiotic-associated nosocomial diarrhoea. Clostridium difficile infection (CDI) has emerged as a major health-associated infection with increased incidence and hospitalization over the years with high mortality rates. Contamination and infection occur after ingestion of vegetative spores, which germinate in the gastro-intestinal tract. The surface layer protein and flagellar proteins are responsible for the bacterial colonization while the spore coat protein, is associated with spore colonization. Both these factors are the main concern of the recurrence of CDI in hospitalized patients. In this study, the CotE, SlpA and FliC proteins are chosen to form a multivalent, multi-epitopic, chimeric vaccine candidate using the immunoinformatics approach. The overall reliability of the candidate vaccine was validated in silico and the molecular dynamics simulation verified the stability of the vaccine designed. Docking studies showed stable vaccine interactions with Toll-Like Receptors of innate immune cells and MHC receptors. In silico codon optimization of the vaccine and its insertion in the cloning vector indicates a competent expression of the modelled vaccine in E. coli expression system. An in silico immune simulation system evaluated the effectiveness of the candidate vaccine to trigger a protective immune response.

A candidate multi-epitope vaccine against SARS-CoV-2.

In the past two decades, 7 coronaviruses have infected the human population, with two major outbreaks caused by SARS-CoV and MERS-CoV in the year 2002 and 2012, respectively. Currently, the entire world is facing a pandemic of another coronavirus, SARS-CoV-2, with a high fatality rate. The spike glycoprotein of SARS-CoV-2 mediates entry of virus into the host cell and is one of the most important antigenic determinants, making it a potential candidate for a vaccine. In this study, we have computationally designed a multi-epitope vaccine using spike glycoprotein of SARS-CoV-2. The overall quality of the candidate vaccine was validated in silico and Molecular Dynamics Simulation confirmed the stability of the designed vaccine. Docking studies revealed stable interactions of the vaccine with Toll-Like Receptors and MHC Receptors. The in silico cloning and codon optimization supported the proficient expression of the designed vaccine in E. coli expression system. The efficiency of the candidate vaccine to trigger an effective immune response was assessed by an in silico immune simulation. The computational analyses suggest that the designed multi-epitope vaccine is structurally stable which can induce specific immune responses and thus, can be a potential vaccine candidate against SARS-CoV-2.