Evolution of SARS-CoV-2: BA.4/BA.5 Variants Continues to Pose New Challenges.

The acquisition of a high number of mutations, notably, the gain of two mutations L452R and F486V in RBD, and the ability to evade vaccine/natural infection-induced immunity suggests that Omicron is continuing to use "immune-escape potential" as an evolutionary space to maintain a selection advantage within the population. Despite the low hospitalizations and lower death rate, the surges by these variants may offset public health measures and disrupt health care facilities as seen recently in Portugal and the USA. Interestingly these BA.4/BA.5 variants have been found to be more severe than the earlier-emerged Omicron variants. We believe that aggressive COVID-19 surveillance using affordable testing strategies might actually help understand the evolution and transmission pattern of new variants. The sudden dip in reporting of new cases in some of the low- and middle-income countries is an alarming situation and needs to be addressed as this could lead to undetected transmission of future variants of interest/concern of SARS-CoV-2 in large population settings, including advent of a 'super' virus. It would be interesting to examine the possible role/influence, if any, of the two different kinds of vaccines, the spike protein-based versus the inactivated whole virus, in the evolution of BA.4/BA.5.

Energetics of Spike Protein Opening of SARS-CoV-1 and SARS-CoV-2 and Its Variants of Concern: Implications in Host Receptor Scanning and Transmission.

The receptor binding domain(s) (RBD) of spike (S) proteins of SARS-CoV-1 and SARS-CoV-2 (severe acute respiratory syndrome coronavirus) undergoes closed to open transition to engage with host ACE2 receptors. In this study, using multi atomistic (equilibrium) and targeted (non-equilibrium) molecular dynamics simulations, we have compared energetics of RBD opening pathways in full-length (modeled from cryo-EM structures) S proteins of SARS-CoV-1 and SARS-CoV-2. Our data indicate that amino acid variations at the RBD interaction interface can culminate into distinct free energy landscapes of RBD opening in these S proteins. We further report that mutations in the S protein of SARS-CoV-2 variants of concern can reduce the protein-protein interaction affinity of RBD(s) with its neighboring domains and could favor its opening to access ACE2 receptors. The findings can also aid in predicting the impact of future mutations on the rate of S protein opening for rapid host receptor scanning.

ArgD of Mycobacterium tuberculosis is a functional N-acetylornithine aminotransferase with moonlighting function as an effective immune modulator.

Mycobacterium tuberculosis (M. tuberculosis) encodes an essential enzyme acetyl ornithine aminotransferase ArgD (Rv1655) of arginine biosynthetic pathway which plays crucial role in M. tuberculosis growth and survival. ArgD catalyzes the reversible conversion of N-acetylornithine and 2 oxoglutarate into glutamate-5-semialdehyde and L-glutamate. It also possesses succinyl diaminopimelate aminotransferase activity and can thus carry out the corresponding step in lysine biosynthesis. These essential roles played by ArgD in amino acid biosynthetic pathways highlight it as an important metabolic chokepoint thus an important drug target. We showed that M. tuberculosis ArgD rescues the growth of ΔargD E. coli grown in minimal media validating its functional importance. Phylogenetic analysis of M. tuberculosis ArgD showed homology with proteins in gram positive bacteria, pathogenic and non-pathogenic mycobacteria suggesting the essentiality of this protein. ArgD is a secretory protein that could be utilized by M. tuberculosis to modulate host innate immunity as its moonlighting function. In-silico analysis predicted it to be a highly antigenic protein. The recombinant ArgD protein when exposed to macrophage cells induced enhanced production of pro-inflammatory cytokines TNF, IL6 and IL12 in a dose dependent manner. ArgD also induced the increased production of innate immune effector molecule NOS2 and NO in macrophages. We also demonstrated ArgD mediated activation of the canonical NFkB pathway. Notably, we also show that ArgD is a specific TLR4 agonist involved in the activation of pro-inflammatory signaling for sustained production of effector cytokines. Intriguingly, ArgD protein treatment activated macrophages to acquire the M1 phenotype through the increased surface expression of MHCII and costimulatory molecules CD80 and CD86. ArgD induced robust B-cell response in immunized mice, validating its antigenicity potential as predicted by the in-silico analysis. These properties of M. tuberculosis ArgD signify its functional plasticity that could be exploited as a possible drug target to combat tuberculosis.

Mycobacterium tuberculosis Specific Protein Rv1509 Evokes Efficient Innate and Adaptive Immune Response Indicative of Protective Th1 Immune Signature.

Dissecting the function(s) of proteins present exclusively in Mycobacterium tuberculosis (M.tb) will provide important clues regarding the role of these proteins in mycobacterial pathogenesis. Using extensive computational approaches, we shortlisted ORFs/proteins unique to M.tb among 13 different species of mycobacteria and identified a hypothetical protein Rv1509 as a 'signature protein' of M.tb. This unique protein was found to be present only in M.tb and absent in all other mycobacterial species, including BCG. In silico analysis identified numerous putative T cell and B cell epitopes in Rv1509. Initial in vitro experiments using innate immune cells demonstrated Rv1509 to be immunogenic with potential to modulate innate immune responses. Macrophages treated with Rv1509 exhibited higher activation status along with substantial release of pro-inflammatory cytokines. Besides, Rv1509 protein boosts dendritic cell maturation by increasing the expression of activation markers such as CD80, HLA-DR and decreasing DC-SIGN expression and this interaction was mediated by innate immune receptor TLR2. Further, in vivo experiments in mice demonstrated that Rv1509 protein promotes the expansion of multifunctional CD4+ and CD8+T cells and induces effector memory response along with evoking a canonical Th1 type of immune response. Rv1509 also induces substantial B cell response as revealed by increased IgG reactivity in sera of immunized animals. This allowed us to demonstrate the diagnostic efficacy of this protein in sera of human TB patients compared to the healthy controls. Taken together, our results reveal that Rv1509 signature protein has immunomodulatory functions evoking immunological memory response with possible implications in serodiagnosis and TB vaccine development.

Structure-Function Analyses of New SARS-CoV-2 Variants B.1.1.7, B.1.351 and B.1.1.28.1: Clinical, Diagnostic, Therapeutic and Public Health Implications.

SARS-CoV-2 (Severe Acute Respiratory Syndrome-Coronavirus 2) has accumulated multiple mutations during its global circulation. Recently, three SARS-CoV-2 lineages, B.1.1.7 (501Y.V1), B.1.351 (501Y.V2) and B.1.1.28.1 (P.1), have emerged in the United Kingdom, South Africa and Brazil, respectively. Here, we have presented global viewpoint on implications of emerging SARS-CoV-2 variants based on structural-function impact of crucial mutations occurring in its spike (S), ORF8 and nucleocapsid (N) proteins. While the N501Y mutation was observed in all three lineages, the 501Y.V1 and P.1 accumulated a different set of mutations in the S protein. The missense mutational effects were predicted through a COVID-19 dedicated resource followed by atomistic molecular dynamics simulations. Current findings indicate that some mutations in the S protein might lead to higher affinity with host receptors and resistance against antibodies, but not all are due to different antibody binding (epitope) regions. Mutations may, however, result in diagnostic tests failures and possible interference with binding of newly identified anti-viral candidates against SARS-CoV-2, likely necessitating roll out of recurring "flu-like shots" annually for tackling COVID-19. The functional relevance of these mutations has been described in terms of modulation of host tropism, antibody resistance, diagnostic sensitivity and therapeutic candidates. Besides global economic losses, post-vaccine reinfections with emerging variants can have significant clinical, therapeutic and public health impacts.

Mycobacterium smegmatis Bacteria Expressing Mycobacterium tuberculosis-Specific Rv1954A Induce Macrophage Activation and Modulate the Immune Response.

Mycobacterium tuberculosis (M. tb), the intracellular pathogen causing tuberculosis, has developed mechanisms that endow infectivity and allow it to modulate host immune response for its survival. Genomic and proteomic analyses of non-pathogenic and pathogenic mycobacteria showed presence of genes and proteins that are specific to M. tb. In silico studies predicted that M.tb Rv1954A is a hypothetical secretory protein that exhibits intrinsically disordered regions and possess B cell/T cell epitopes. Treatment of macrophages with Rv1954A led to TLR4-mediated activation with concomitant increase in secretion of pro-inflammatory cytokines, IL-12 and TNF-α. In vitro studies showed that rRv1954A protein or Rv1954A knock-in M. smegmatis (Ms_Rv1954A) activates macrophages by enhancing the expression of CD80 and CD86. An upregulation in the expression of CD40 and MHC I/II was noted in the presence of Rv1954A, pointing to its role in enhancing the association of APCs with T cells and in the modulation of antigen presentation, respectively. Ms_Rv1954A showed increased infectivity, induction of ROS and RNS, and apoptosis in RAW264.7 macrophage cells. Rv1954A imparted protection against oxidative and nitrosative stress, thereby enhancing the survival of Ms_Rv1954A inside macrophages. Mice immunized with Ms_Rv1954A showed that splenomegaly and primed splenocytes restimulated with Rv1954A elicited a Th1 response. Infection of Ms_Rv1954A in mice through intratracheal instillation leads to enhanced infiltration of lymphocytes in the lungs without formation of granuloma. While Rv1954A is immunogenic, it did not cause adverse pathology. Purified Rv1954A or Rv1954A knock-in M. smegmatis (Ms_Rv1954A) elicited a nearly two-fold higher titer of IgG response in mice, and PTB patients possess a higher IgG titer against Rv1954A, also pointing to its utility as a diagnostic marker for TB. The observed modulation of innate and adaptive immunity renders Rv1954A a vital protein in the pathophysiology of this pathogen.

Immunodominant Mycobacterium tuberculosis Protein Rv1507A Elicits Th1 Response and Modulates Host Macrophage Effector Functions.

Mycobacterium tuberculosis (M. tb) persists as latent infection in nearly a quarter of the global population and remains the leading cause of death among infectious diseases. While BCG is the only vaccine for TB, its inability to provide complete protection makes it imperative to engineer BCG such that it expresses immunodominant antigens that can enhance its protective potential. In-silico comparative genomic analysis of Mycobacterium species identified M. tb Rv1507A as a "signature protein" found exclusively in M. tb. In-vitro (cell lines) and in-vivo experiments carried out in mice, using purified recombinant Rv1507A revealed it to be a pro-inflammatory molecule, eliciting significantly high levels of IL-6, TNF-α, and IL-12. There was increased expression of activation markers CD69, CD80, CD86, antigen presentation molecules (MHC I/MHCII), and associated Th1 type of immune response. Rv1507A knocked-in M. smegmatis also induced significantly higher pro-inflammatory Th1 response and higher survivability under stress conditions, both in-vitro (macrophage RAW264.7 cells) and in-vivo (mice). Sera derived from human TB patients showed significantly enhanced B-cell response against M. tb Rv1507A. The ability of M. tb Rv1507A to induce immuno-modulatory effect, B cell response, and significant memory response, renders it a putative vaccine candidate that demands further exploration.

Emerging genetic diversity among clinical isolates of SARS-CoV-2: Lessons for today.

Considering the current pandemic of COVID-19, it is imperative to gauge the role of molecular divergence in SARS-CoV-2 with time, due to clinical and epidemiological concerns. Our analyses involving molecular phylogenetics is a step toward understanding the transmission clusters that can be correlated to pathophysiology of the disease to gain insight into virulence mechanism. As the infections are increasing rapidly, more divergence is expected followed possibly by viral adaptation. We could identify mutational hotspots which appear to be major drivers of diversity among strains, with RBD of spike protein emerging as the key region involved in interaction with ACE2 and consequently a major determinant of infection outcome. We believe that such molecular analyses correlated with clinical characteristics and host predisposition need to be evaluated at the earliest to understand viral adaptability, disease prognosis, and transmission dynamics.

Dual direction CRISPR transcriptional regulation screening uncovers gene networks driving drug resistance.

Pooled CRISPR-Cas9 knock out screens provide a valuable addition to the methods available for novel drug target identification and validation. However, where gene editing is targeted to amplified loci, the resulting multiple DNA cleavage events can be a cause of false positive hit identification. The generation of nuclease deficient versions of Cas9 has enabled the development of two additional techniques - CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) - that enable the repression or overexpression, respectively, of target genes. Here we report the first direct combination of all three approaches (CRISPRko, CRISPRi and CRISPRa) in the context of genome-wide screens to identify components that influence resistance and sensitivity to the BRAF inhibitor, vemurafenib. The pairing of both loss- and gain-of-function datasets reveals complex gene networks which control drug response and illustrates how such data can add substantial confidence to target identification and validation analyses.

Reaction Decoder Tool (RDT): extracting features from chemical reactions.

UNLABELLED: Extracting chemical features like Atom-Atom Mapping (AAM), Bond Changes (BCs) and Reaction Centres from biochemical reactions helps us understand the chemical composition of enzymatic reactions. Reaction Decoder is a robust command line tool, which performs this task with high accuracy. It supports standard chemical input/output exchange formats i.e. RXN/SMILES, computes AAM, highlights BCs and creates images of the mapped reaction. This aids in the analysis of metabolic pathways and the ability to perform comparative studies of chemical reactions based on these features. AVAILABILITY AND IMPLEMENTATION: This software is implemented in Java, supported on Windows, Linux and Mac OSX, and freely available at https://github.com/asad/ReactionDecoder CONTACT: : asad@ebi.ac.uk or s9asad@gmail.com.

Exploring the chemistry and evolution of the isomerases.

Isomerization reactions are fundamental in biology, and isomers usually differ in their biological role and pharmacological effects. In this study, we have cataloged the isomerization reactions known to occur in biology using a combination of manual and computational approaches. This method provides a robust basis for comparison and clustering of the reactions into classes. Comparing our results with the Enzyme Commission (EC) classification, the standard approach to represent enzyme function on the basis of the overall chemistry of the catalyzed reaction, expands our understanding of the biochemistry of isomerization. The grouping of reactions involving stereoisomerism is straightforward with two distinct types (racemases/epimerases and cis-trans isomerases), but reactions entailing structural isomerism are diverse and challenging to classify using a hierarchical approach. This study provides an overview of which isomerases occur in nature, how we should describe and classify them, and their diversity.

Characterising Complex Enzyme Reaction Data.

The relationship between enzyme-catalysed reactions and the Enzyme Commission (EC) number, the widely accepted classification scheme used to characterise enzyme activity, is complex and with the rapid increase in our knowledge of the reactions catalysed by enzymes needs revisiting. We present a manual and computational analysis to investigate this complexity and found that almost one-third of all known EC numbers are linked to more than one reaction in the secondary reaction databases (e.g., KEGG). Although this complexity is often resolved by defining generic, alternative and partial reactions, we have also found individual EC numbers with more than one reaction catalysing different types of bond changes. This analysis adds a new dimension to our understanding of enzyme function and might be useful for the accurate annotation of the function of enzymes and to study the changes in enzyme function during evolution.

The Classification and Evolution of Enzyme Function.

Enzymes are the proteins responsible for the catalysis of life. Enzymes sharing a common ancestor as defined by sequence and structure similarity are grouped into families and superfamilies. The molecular function of enzymes is defined as their ability to catalyze biochemical reactions; it is manually classified by the Enzyme Commission and robust approaches to quantitatively compare catalytic reactions are just beginning to appear. Here, we present an overview of studies at the interface of the evolution and function of enzymes.

Comparative analyses of nonpathogenic, opportunistic, and totally pathogenic mycobacteria reveal genomic and biochemical variabilities and highlight the survival attributes of Mycobacterium tuberculosis.

UNLABELLED: Mycobacterial evolution involves various processes, such as genome reduction, gene cooption, and critical gene acquisition. Our comparative genome size analysis of 44 mycobacterial genomes revealed that the nonpathogenic (NP) genomes were bigger than those of opportunistic (OP) or totally pathogenic (TP) mycobacteria, with the TP genomes being smaller yet variable in size--their genomic plasticity reflected their ability to evolve and survive under various environmental conditions. From the 44 mycobacterial species, 13 species, representing TP, OP, and NP, were selected for genomic-relatedness analyses. Analysis of homologous protein-coding genes shared between Mycobacterium indicus pranii (NP), Mycobacterium intracellulare ATCC 13950 (OP), and Mycobacterium tuberculosis H37Rv (TP) revealed that 4,995 (i.e., ~95%) M. indicaus pranii proteins have homology with M. intracellulare, whereas the homologies among M. indicus pranii, M. intracellulare ATCC 13950, and M. tuberculosis H37Rv were significantly lower. A total of 4,153 (~79%) M. indicus pranii proteins and 4,093 (~79%) M. intracellulare ATCC 13950 proteins exhibited homology with the M. tuberculosis H37Rv proteome, while 3,301 (~82%) and 3,295 (~82%) M. tuberculosis H37Rv proteins showed homology with M. indicus pranii and M. intracellulare ATCC 13950 proteomes, respectively. Comparative metabolic pathway analyses of TP/OP/NP mycobacteria showed enzymatic plasticity between M. indicus pranii (NP) and M. intracellulare ATCC 13950 (OP), Mycobacterium avium 104 (OP), and M. tuberculosis H37Rv (TP). Mycobacterium tuberculosis seems to have acquired novel alternate pathways with possible roles in metabolism, host-pathogen interactions, virulence, and intracellular survival, and by implication some of these could be potential drug targets. IMPORTANCE: The complete sequence analysis of Mycobacterium indicus pranii, a novel species of Mycobacterium shown earlier to have strong immunomodulatory properties and currently in use for the treatment of leprosy, places it evolutionarily at the point of transition to pathogenicity. With the purpose of establishing the importance of M. indicus pranii in providing insight into the virulence mechanism of tuberculous and nontuberculous mycobacteria, we carried out comparative genomic and proteomic analyses of 44 mycobacterial species representing nonpathogenic (NP), opportunistic (OP), and totally pathogenic (TP) mycobacteria. Our results clearly placed M. indicus pranii as an ancestor of the M. avium complex. Analyses of comparative metabolic pathways between M. indicus pranii (NP), M. tuberculosis (TP), and M. intracellulare (OP) pointed to the presence of novel alternative pathways in M. tuberculosis with implications for pathogenesis and survival in the human host and identification of new drug targets.

The evolution of enzyme function in the isomerases.

The advent of computational approaches to measure functional similarity between enzymes adds a new dimension to existing evolutionary studies based on sequence and structure. This paper reviews research efforts aiming to understand the evolution of enzyme function in superfamilies, presenting a novel strategy to provide an overview of the evolution of enzymes belonging to an individual EC class, using the isomerases as an exemplar.

Gene cooption in mycobacteria and search for virulence attributes: comparative proteomic analyses of Mycobacterium tuberculosis, Mycobacterium indicus pranii and other mycobacteria.

Tuberculosis (TB), caused by Mycobacterium tuberculosis, is a leading infectious disease taking one human life every 15s globally. Mycobacterium undergoes reductive evolution; the ancestors have bigger genome size and rich in metabolic pathways. Mycobacterium indicus pranii (MIP) is placed much above Mycobacterium tuberculosis (M.tb) in evolutionary scale and is a non-pathogenic, saprophytic mycobacterium. Our in silico comparative proteomic analyses of virulence factors of M.tb and their homologs in 12 different Mycobacterial species, including MIP, point toward gene cooption as an important mechanism in evolution of mycobacteria. We propose that adaptive changes in niche factors of non-pathogenic mycobacterium, together with novel gene acquisitions, are key players in the evolution of pathogenicity. Antigenic analyses between M.tb and MIP highlighted the importance of PE/PPE family in host immunomodulation, further supporting the likely potential of MIP as an effective vaccine against TB.

Exploring the biological and chemical complexity of the ligases.

Using a novel method to map and cluster chemical reactions, we have re-examined the chemistry of the ligases [Enzyme Commission (EC) Class 6] and their associated protein families in detail. The type of bond formed by the ligase can be automatically extracted from the equation of the reaction, replicating the EC subclass division. However, this subclass division hides considerable complexities, especially for the C-N forming ligases, which fall into at least three distinct types. The lower levels of the EC classification for ligases are somewhat arbitrary in their definition and add little to understanding their chemistry or evolution. By comparing the multi-domain architecture of the enzymes and using sequence similarity networks, we examined the links between overall reaction and evolution of the ligases. These show that, whilst many enzymes that perform the same overall chemistry group together, both convergent (similar function, different ancestral lineage) and divergent (different function, common ancestor) evolution of function are observed. However, a common theme is that a single conserved domain (often the nucleoside triphosphate binding domain) is combined with ancillary domains that provide the variation in substrate binding and function.

EC-BLAST: a tool to automatically search and compare enzyme reactions.

We present EC-BLAST (http://www.ebi.ac.uk/thornton-srv/software/rbl/), an algorithm and Web tool for quantitative similarity searches between enzyme reactions at three levels: bond change, reaction center and reaction structure similarity. It uses bond changes and reaction patterns for all known biochemical reactions derived from atom-atom mapping across each reaction. EC-BLAST has the potential to improve enzyme classification, identify previously uncharacterized or new biochemical transformations, improve the assignment of enzyme function to sequences, and assist in enzyme engineering.