The multifaceted genus Acinetobacter: from infection to bioremediation.

Acinetobacter is a vast bacterial genus comprising of numerous species with variable characteristics. The enigma associated with clinical strains that have been implicated in many nosocomial outbreaks has prompted the need for continuous research on pathogens like Acinetobacter baumannii and members of the ACB complex. However, numerous species of Acinetobacter genus possess diverse metabolic capabilities and have the potential for a plethora of industrial and environment-based applications. Therefore, a comprehensive review on the entire genus, including many under-represented topics, would contribute extensive information to the scientific community indulged in Acinetobacter research. The current review is a unique compilation that attempts to provide the latest update on the genus covering its clinical as well as ecological aspects. Moreover, it is the first study of its kind that focuses on the entire genus and elaborates on the phylogenetic relationships, pathogenesis, and virulence mechanisms, followed by emerging biotechnological applications with future directions.

Codon usage behavior distinguishes pathogenic Clostridium species from the non-pathogenic species.

Genus Clostridium is of the largest genus in class Clostridia. It is comprised of spore-forming, anaerobic, gram-positive organisms. The members of this genus include human pathogens to free-living nitrogen fixing bacteria. In the present study, we have performed a comparison of the choice of preferred codons, codon usage patterns, dinucleotide and amino acid usage pattern of 76 species of Genus Clostridium. We found the pathogenic clostridium species to have smaller AT-rich genomes as compared to opportunistic and non-pathogenic clostridium species. The choice of preferred and optimal codons was also influenced by genomic GC/AT content of the respective clostridium species. The pathogenic clostridium species displayed a strict bias in the codon usage, employing 35 of the 61 codons encoding for 20 amino acids. Comparison of amino acid usage revealed an increased usage of amino acids with lower biosynthetic cost by pathogenic clostridium species as compared to opportunistic and non-pathogenic clostridium species. Smaller genome, strict codon usage bias and amino acid usage lead to lower protein energetic cost for the clostridial pathogens. Overall, we found the pathogenic members of genus Clostridium to prefer small, AT-rich codons to reduce biosynthetic costs and match the cellular environment of its AT-rich human host.

Evolution of codon and amino acid usage in bacterial protein toxins.

Toxin proteins are secreted by most pathogens as an integral part of pathogenic mechanism(s). The toxins act by either damaging the host cell membrane (for example, pore-forming toxins and RTX toxins) or by modulation of important cellular pathways (for example, inhibition of protein translation by ribosome-inactivating proteins). The mechanism of action of these toxins provides the pathogen with strategies for adaptation in the unfavorable host environment. Though, secreted by different pathogenic species, the protein toxins seem to share common features that allow the protein to bind to specific molecules and enter the host cell. Earlier studies have suggested role of several events like horizontal gene transfer and insertion-deletion mutations in evolution of protein toxins. The present study involving 125 bacterial protein toxins secreted by 49 pathogenic bacteria focuses on the role and constraints of the bacterial genome on evolution of codon and amino acid usage in respective bacterial protein toxins. We compare the nucleotide composition, codon and dinucleotide usage trends between different classes of bacterial protein toxins and between individual toxins and the parent bacterial genome expressing the toxin(s).

Expanding role of ribosome-inactivating proteins: From toxins to therapeutics.

Ribosome-inactivating proteins (RIPs) are toxic proteins with N-glycosidase activity. RIPs exert their action by removing a specific purine from 28S rRNA, thereby, irreversibly inhibiting the process of protein synthesis. RIPs can target both prokaryotic and eukaryotic cells. In bacteria, the production of RIPs aid in the process of pathogenesis whereas, in plants, the production of these toxins has been attributed to bolster defense against insects, viral, bacterial and fungal pathogens. In recent years, RIPs have been engineered to target a particular cell type, this has fueled various experiments testing the potential role of RIPs in many biomedical applications like anti-viral and anti-tumor therapies in animals as well as anti-pest agents in engineered plants. In this review, we present a comprehensive study of various RIPs, their mode of action, their significance in various fields involving plants and animals. Their potential as treatment options for plant infections and animal diseases is also discussed.

Base Composition and Host Adaptation of the SARS-CoV-2: Insight From the Codon Usage Perspective.

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been spreading rapidly all over the world and has raised grave concern globally. The present research aims to conduct a robust base compositional analysis of SARS-CoV-2 to reveal adaptive intricacies to the human host. Multivariate statistical analysis revealed a complex interplay of various factors including compositional constraint, natural selection, length of viral coding sequences, hydropathicity, and aromaticity of the viral gene products that are operational to codon usage patterns, with compositional bias being the most crucial determinant. UpG and CpA dinucleotides were found to be highly preferred whereas, CpG dinucleotide was mostly avoided in SARS-CoV-2, a pattern consistent with the human host. Strict avoidance of the CpG dinucleotide might be attributed to a strategy for evading a human immune response. A lower degree of adaptation of SARS-CoV-2 to the human host, compared to Middle East respiratory syndrome (MERS) coronavirus and SARS-CoV, might be indicative of its milder clinical severity and progression contrasted to SARS and MERS. Similar patterns of enhanced adaptation between viral isolates from intermediate and human hosts, contrasted with those isolated from the natural bat reservoir, signifies an indispensable role of the intermediate host in transmission dynamics and spillover events of the virus to human populations. The information regarding avoided codon pairs in SARS-CoV-2, as conferred by the present analysis, promises to be useful for the design of vaccines employing codon pair deoptimization based synthetic attenuated virus engineering.

Codon usage signatures in the genus Cryptococcus: A complex interplay of gene expression, translational selection and compositional bias.

The fungal genus Cryptococcus comprises of several diverse species. The pathogens forming Cryptococcus neoformans/ Cryptococcus gatti species complex are of immense clinical significance owing to the high frequency of infections and deaths globally. Three closely related non-pathogenic species namely, Cryptococcus amylolentus, Cryptococcus wingfieldii and Cryptococcus depauperatus are the non-pathogenic ancestral species from which pathogenic lineages have diverged. In the current study, a comprehensive analysis of factors influencing the codon and amino acid usage bias in six pathogenic and three non-pathogenic species was performed. Our results revealed that though compositional bias played a crucial role, translational selection and gene expression were the key determinants of codon usage variations. Analysis of relative dinucleotide abundance and codon context signatures revealed strict avoidance of TpA dinucleotide across genomes. Multivariate statistical analysis based on codon usage data resulted in discrete clustering of pathogens and non-pathogens which correlated with previous reports on their phylogenetic distribution.

Diverse species in the genus Cryptococcus: Pathogens and their non-pathogenic ancestors.

The genus Cryptococcus comprises of more than 30 species. It consists of clinically significant pathogenic Cryptococcus neoformans/Cryptococcus gattii species complex comprising of a minimum of seven species. These pathogens cost more than 200,000 lives annually by causing cryptococcal meningoencephalitis. The evolution of the pathogenic species from closely related non-pathogenic species of the Cryptococcus amylolentus complex is of particular importance and several advances have been made to understand their phylogenetic and genomic relationships. The current review briefly describes the sexual reproduction process followed by an individual description of the members focusing on their key attributes and virulence mechanisms of the pathogenic species. A special section on phylogenetic studies is aimed at understanding the evolutionary divergence of pathogens from non-pathogens. Recent findings from our group pertaining to parameters affecting codon usage bias in six pathogenic and three non-pathogenic ancestral species and their corroboration with existing phylogenetic reports are also included in the current review.

Production, Purification and Characterisation of a Potential Fibrinolytic Protease from Endophytic Xylaria curta by Solid Substrate Fermentation.

The present investigation highlights the optimal conditions for production of a non-toxic, bi-functional fibrinolytic enzyme xylarinase produced by endophytic fungus Xylaria curta by solid substrate fermentation using rice chaff medium. The purified enzyme is a monomeric protein with a molecular mass of ∼33 kDa. The enzyme exhibits cleavage of Aα and Bß chains of fibrin(ogen) and has no effect on γ chain. The optimal fibrinolytic activity of the enzyme was observed at 35 °C and pH 8. The fibrinolytic activity was enhanced in the presence of Ca2+, whereas it was completely inhibited in the presence of Fe2+ and Zn2+ ions and inhibitors like EDTA and EGTA suggesting it to be a metalloprotease. The K m and V max of the enzyme for azocasein were 326 µM and 0.13 µM min-1. The N-terminal sequence of the enzyme (SNGPLPGGVVWAG) was same when compared to xylarinase isolated from culture broth of X. curta. Thus, xylarinase could be exploited as a potent clot busting enzyme which could be produced on large scale using solid substrate fermentation.

Xylarinase: a novel clot busting enzyme from an endophytic fungus Xylaria curta.

Xylarinase is a bi-functional fibrinolytic metalloprotease isolated from the culture filtrate of endophytic fungus Xylaria curta which is monomeric with a molecular mass of ∼33.76 kDa. The enzyme displayed both plasmin and tissue plasminogen activator like activity under in vitro conditions. It hydrolyses Aα and Bß chains of the fibrinogen. Optimal fibrinolytic activity of xylarinase is observed at 35 °C, pH 8. Ca(2+) stimulated the fibrinolytic activity of xylarinase while Fe(2+) and Zn(2+) inhibited suggesting it to be a metalloprotease. The Km and Vmax values of xylarinase were 240.9 µM and 1.10 U/ml for fibrinogen and 246 µM and 1.22 U/ml for fibrin, respectively. Xylarinase was found to prolong the activated partial thromboplastin time and prothrombin time. The N-terminal sequence of xylarinase (SNGPLPGGVVWAG) did not show any homology with previously known fibrinolytic enzymes. Thus xylarinase is a novel fibrinolytic metalloprotease which could be possibly used as a new clot busting enzyme.

Functional characterization of Helicobacter pylori TlyA: pore-forming hemolytic activity and cytotoxic property of the protein.

Helicobacter pylori is a human specific gastric pathogen. H. pylori pathogenesis process involves a number of well-studied virulence factors that include the 'vacuolating cytotoxin' and the 'cytotoxin associated gene A'. Analysis of the H. pylori genome, however, indicates presence of additional virulence factors that are yet to be characterized in molecular detail. For example, H. pylori genome harbors a gene that has potential to encode a protein with sequence similarity to those of the TlyA-like proteins of several pathogenic bacteria. Earlier studies have indicated potential association of this H. pylori tlyA gene in the virulence mechanism of the organism. Despite such notions, however, the TlyA-like protein of H. pylori has not been studied previously in molecular detail. In particular, purified form of H. pylori TlyA has never been studied before toward exploring its functional properties. Here, we report characterization of the H. pylori TlyA protein purified from the recombinant over-expression system in Escherichia coli. Purified form of the recombinant TlyA exhibits prominent hemolytic activity against human erythrocytes, presumably via formation of pores of specific diameter in the cell membrane. Purified TlyA also triggers prominent cytotoxic responses in human gastric adenocarcinoma cells. Altogether, our study establishes H. pylori TlyA as a potential virulence factor of the organism.

Pre-pore oligomer formation by Vibrio cholerae cytolysin: insights from a truncated variant lacking the pore-forming pre-stem loop.

Vibrio cholerae cytolysin (VCC), a ß-barrel pore-forming toxin (ß-PFT), induces killing of the target eukaryotic cells by forming heptameric transmembrane ß-barrel pores. Consistent with the ß-PFT mode of action, binding of the VCC toxin monomers with the target cell membrane triggers formation of pre-pore oligomeric intermediates, followed by membrane insertion of the ß-strands contributed by the pre-stem motif within the central cytolysin domain of each protomer. It has been shown previously that blocking of membrane insertion of the VCC pre-stem motif arrests conversion of the pre-pore state to the functional transmembrane pore. Consistent with the generalized ß-PFT mechanism, it therefore appears that the VCC pre-stem motif plays a critical role toward forming the structural scaffold of the transmembrane ß-barrel pore. It is, however, still not known whether the pre-stem motif plays any role in the membrane interaction process, and subsequent pre-pore structure formation by VCC. In this direction, we have constructed a recombinant variant of VCC deleting the pre-stem region, and have characterized the effect(s) of physical absence of the pre-stem motif on the distinct steps of the membrane pore-formation process. Our results show that the deletion of the pre-stem segment does not affect membrane binding and pre-pore oligomer formation by the toxin, but it critically abrogates the functional pore-forming activity of VCC. Present study extends our insights regarding the structure-function mechanism associated with the membrane pore formation by VCC, in the context of the ß-PFT mode of action.

Functional mapping of the lectin activity site on the ß-prism domain of vibrio cholerae cytolysin: implications for the membrane pore-formation mechanism of the toxin.

Vibrio cholerae cytolysin (VCC) is a prominent member in the family of ß-barrel pore-forming toxins. It induces lysis of target eukaryotic cells by forming transmembrane oligomeric ß-barrel channels. VCC also exhibits prominent lectin-like activity in interacting with ß1-galactosyl-terminated glycoconjugates. Apart from the cytolysin domain, VCC harbors two lectin-like domains: the ß-Trefoil and the ß-Prism domains; however, precise contribution of these domains in the lectin property of VCC is not known. Also, role(s) of these lectin-like domains in the mode of action of VCC remain obscure. In the present study, we show that the ß-Prism domain of VCC acts as the structural scaffold to determine the lectin activity of the protein toward ß1-galactosyl-terminated glycoconjugates. Toward exploring the physiological implication of the ß-Prism domain, we demonstrate that the presence of the ß-Prism domain-mediated lectin activity is crucial for an efficient interaction of the toxin toward the target cells. Our results also suggest that such lectin activity may act to regulate the oligomerization ability of the membrane-bound VCC toxin. Based on the data presented here, and also consistent with the existing structural information, we propose a novel mechanism of regulation imposed by the ß-Prism domain's lectin activity, implicated in the process of membrane pore formation by VCC.

Single point mutation in Vibrio cholerae cytolysin compromises the membrane pore-formation mechanism of the toxin.

Vibrio cholerae cytolysin (VCC) belongs to the family of ß-barrel pore-forming protein toxins. VCC is secreted by the bacteria as water-soluble monomers, which upon binding to target eukaryotic cells form transmembrane heptameric ß-barrel channels. High-resolution 3D structures are described both for the water-soluble monomeric form and the transmembrane oligomeric pore; albeit that our understanding of the mechanistic details of the membrane pore-formation process remains incomplete. Here, we report the characterization of a nonfunctional VCC variant harboring a single point mutation of Ala425Val positioned within a potential membrane-interacting loop in the VCC structure. The mutation appears to affect interaction of the toxin with erythrocytes as well as cholesterol-containing liposome membrane, without affecting the oligomerization ability of the membrane-bound toxin molecules. The membrane-bound oligomers formed by this VCC mutant do not appear to represent the functional pore assembly of the toxin; rather, such assembly could be considered as being trapped in an abortive, nonfunctional oligomeric state. Our results suggest that the Ala425Val mutation in VCC critically compromises its cholesterol-dependent membrane-interaction mechanism and also abrogates the process of functional membrane pore formation by the toxin.

Cell signaling molecules as drug targets in lung cancer: an overview.

PURPOSE OF REVIEW: Lung being one of the vital and essential organs in the body, lung cancer is a major cause of mortality in the modern human society. Lung cancer can be broadly subdivided into nonsmall cell lung cancer (NSCLC) and small cell lung cancer (SCLC). Although NSCLC is sometimes treated with surgery, the advanced and metastatic NSCLC and SCLC usually respond better to chemotherapy and radiation. The most important targets of these chemotherapeutic agents are various intracellular signaling molecules. The primary focus of this review article is to summarize the description of various cell signaling molecules involved in lung cancer development and their regulation by chemotherapeutic agents. RECENT FINDINGS: Extensive research work in recent years has identified several cellular signaling molecules that may be intricately involved in the complexity of lung cancer. Some of these cell signaling molecules are epidermal growth factor receptors, vascular endothelial growth factor receptors, mammalian target of rapamycin, mitogen-activated protein kinase phosphatase-1, peroxisome proliferator-activated receptor-gamma, matrix metalloproteinases and receptor for advanced glycation end-products. SUMMARY: The present review will strengthen our current knowledge regarding the efficacy of the above-mentioned cell signaling molecules as potential beneficial drug targets against lung cancer.

Unfolding distinguishes the Vibrio cholerae cytolysin precursor from the mature form of the toxin.

Vibrio cholerae cytolysin (VCC) is a potent cytolytic toxin that induces colloid osmotic lysis of its target eukaryotic cells by forming transmembrane oligomeric ß-barrel channels. VCC is secreted by the bacteria as an inactive precursor (Pro-VCC) and is subsequently activated by proteolytic removal of an N-terminal "Pro-domain", thus generating the active form of the toxin (Mature-VCC). Earlier studies have indicated an intramolecular chaperone-like role of the Pro-domain favoring efficient secretion of the toxin from the periplasm into the extracellular space. However, the exact role of the Pro-domain in the VCC structure--function mechanism remains unclear. Here, we have compared the Pro-VCC and Mature-VCC molecules in terms of their structural and conformational properties. We have studied unfolding of the two variants of the VCC molecule in response to an array of denaturing conditions, including low-pH, chemical denaturant and heat-induced unfolding. Pro-VCC shows a more profound tendency to unfold in response to such denaturing conditions compared to Mature-VCC. Biophysical characterization of the isolated Pro-domain further suggests that the increased unfolding propensity of Pro-VCC does not arise because of an increased level of unfolding of the Pro-region itself. Altogether, our results imply that a natively folded architecture of the Pro-VCC molecule with sufficient structural and conformational plasticity presumably allows it to adopt a suitable configuration that is possibly required for its efficient secretion and subsequent proteolytic maturation under physiological conditions.