Microbial enzymes for deprivation of amino acid metabolism in malignant cells: biological strategy for cancer treatment.

Amino acid deprivation therapy (AADT) is emerging as a promising strategy for the development of novel therapeutics against cancer. This biological therapy relies upon the differences in the metabolism of cancer and normal cells. The rapid growth of tumors results in decreased expression of certain enzymes leading to auxotrophy for some specific amino acids. These auxotrophic tumors are targeted by amino acid-depleting enzymes. The depletion of amino acid selectively inhibits tumor growth as the normal cells can synthesize amino acids by their usual machinery. The enzymes used in AADT are mostly obtained from microbes for their easy availability. Microbial L-asparaginase is already approved by FDA for the treatment of acute lymphoblastic leukemia. Arginine deiminase and methionase are under clinical trials and the therapeutic potential of lysine oxidase, glutaminase and phenylalanine ammonia lyase is also being explored. The present review provides an overview of microbial amino acid depriving enzymes. Various attributes of these enzymes like structure, mode of action, production, formulations, and targeted cancers are discussed. The challenges faced and the combat strategies to establish AADT in standard cancer armamentarium are also reviewed.Key Points • Amino acid deprivation therapy is a potential therapy for auxotrophic tumors. • Microbial enzymes are used due to their ease of manipulation and high productivity. • Enzyme properties are improved by PEGylation, encapsulation, and genetic engineering. • AADT can be employed as combinational therapy for better containment of cancer.

Computational and mutational analysis of TatD DNase of Bacillus anthracis.

The role of TatD DNases as DNA repair enzymes or cell death (apoptotic) nucleases is well established in prokaryotes as well as eukaryotes. The current study aims to characterize the TatD nuclease from Bacillus anthracis (Ba TatD) and to explore its key histidine catalytic residues. Ba TatD was found to be a metal-dependent, nonspecific endonuclease which could efficiently cleave double-stranded DNA substrates. Moreover, Ba TatD nuclease was observed to be thermostable up to 55°C and act in a wide pH range indicating its industrial applicability. Diethyl pyrocarbonate-based histidine-selective alkylation of the Ba TatD resulted in a loss of its nuclease activity suggesting a crucial role of the histidine residues in its activity. The key residues of Ba TatD were predicted using sequence analysis and structure-based approaches, and then the predicted residues were further tested by mutational analysis. Upon mutational analysis, H128 and H153 have been found to be crucial for Ba TatD activity, though H153 seems to bear an important but a dispensable role for the Ba TatD nuclease. Ba TatD had a uniform expression in the cytosol of B. anthracis, which indicates a significant role of the protein in the pathogen's life cycle. This is the first study to identify and characterize the TatD DNase from B. anthracis and will be helpful in gaining more insights on the role of TatD proteins in Gram-positive bacteria where it remains unexplored.

Characterization of a two component system, Bas1213-1214, important for oxidative stress in Bacillus anthracis.

Microbial colonization is an outcome of appropriate sensing and regulation of its gene expression. Bacillus anthracis adapts and thrives in its environment through complex regulatory mechanisms, among them, the two component systems (TCS). Many bacteria respond to the oxygen fluctuations via TCS. In the present work, a previously uncharacterized TCS, Bas1213-1214, of B. anthracis with a probable role in oxygen sensing has been characterized as a functional TCS. A substantial increase in the expression of Bas1213 was observed during the stationary growth phase, in presence of bicarbonate ions, and under oxidative stress thereby speculating the role of Bas1213 in toxin production and adaptive responses. Electrophoretic mobility shift assay (EMSA) and ANS assay highlighted autoregulation of the system. Identification of Bas1213 regulon further suggested its regulatory function in metabolism and adaptive responses. A marked reduction in sporulation was observed on overexpression of Bas1213 in B. anthracis which can be correlated with the augmented expression of sporulation kinase D. Additionally, Bas1213 was shown to regulate catalase, and ABC transporter (mntH) further implicating its essential role during oxidative stress. Finally, crucial residues involved in the DNA binding activity of Bas1213 were also identified. This study reports that the role of Bas1213-1214 in the regulation of metabolism and adaptive responses during oxidative stress. Both sporulation and response to environmental oxygen are important for the maintenance of B. anthracis lifecycle, therefore, characterization of Bas1213-1214 provides a step closer toward understanding the regulatory network governing in B. anthracis.

Functional characterization of PhoPR two component system and its implication in regulating phosphate homeostasis in Bacillus anthracis.

BACKGROUND: Recent report on importance of phosphate starvation (PS) in Bacillus anthracis (BA) pathogenesis warrants further investigation of the underlying regulatory mechanism. Potential role of PhoPR two component system (TCS) in phosphate homeostasis and virulence of several pathogens necessitates the study of annotated PhoPR in BA. METHODS: Expression of phoP and phoR was analyzed using qRT-PCR. PhoPR was characterized biochemically. DNA-protein interaction was analyzed by EMSA. Docking was done to predict PhoPR interacting residues with further validation by mutational studies. pHCMC05 was used to overexpress PhoP in BA. RESULTS: In silico analysis revealed Bas4483-4484, as putative PhoR-PhoP. Their expression was decreased with increasing phosphate concentration reflecting some role in PS. Both PhoP (response regulator) and PhoR (histidine kinase) showed characteristic property of TCS i.e., autophosphorylation and phosphotransfer. PhoR showed both kinase and phosphatase activity. PhoP bound with promoter of PS marker genes. In silico and in vitro analysis revealed role of PhoRH370 and PhoPD10, PhoPD53, PhoPM55 in PhoPR interaction. Challenge studies showed decreased survival of mice infected with BApHCMC05-PhoP. CONCLUSION: This study affirms that PhoPR forms functional TCS which is upregulated under PS. PhoP binding with promoter of PS marker genes indicates its possible role in regulating PS response. Low survival of mice infected with BApHCMC05-PhoP suggests its role in BA virulence. GENERAL SIGNIFICANCE: Considering the significance of PS in BA infection, possible role of PhoPR in its regulation and exclusive presence of TCS in prokaryotes, PhoP can be proposed as potential drug target against anthrax.

In silico and in vitro analysis of arginine deiminase from Pseudomonas furukawaii as a potential anticancer enzyme.

Arginine deiminase (ADI), a promising anticancer enzyme from Mycoplasma hominis, is currently in phase III of clinical trials for the treatment of arginine auxotrophic tumors. However, it has been associated with several drawbacks in terms of low stability at human physiological conditions, high immunogenicity, hypersensitivity and systemic toxicity. In our previous work, Pseudomonas furukawaii 24 was identified as a potent producer of ADI with optimum activity under physiological conditions. In the present study, phylogenetic analysis of microbial ADIs indicated P. furukawaii ADI (PfADI) to be closely related to experimentally characterized ADIs of Pseudomonas sp. with proven anticancer activity. Immunoinformatics analysis was performed indicating lower immunogenicity of PfADI than MhADI (M. hominis ADI) both in terms of number of linear and conformational B-cell epitopes and T-cell epitope density. Overall antigenicity and allergenicity of PfADI was also lower as compared to MhADI, suggesting the applicability of PfADI as an alternative anticancer biotherapeutic. Hence, in vitro experiments were performed in which the ADI coding arcA gene of P. furukawaii was cloned and expressed in E. coli BL21. Recombinant ADI of P. furukawaii was purified, characterized and its anticancer activity was assessed. The enzyme was stable at human physiological conditions (pH 7 and 37 °C) with Km of 1.90 mM. PfADI was found to effectively inhibit the HepG2 cells with an IC50 value of 0.1950 IU/ml. Therefore, the current in silico and in vitro studies establish PfADI as a potential anticancer drug candidate with improved efficacy and low immunogenicity. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03292-2.

Novel application of trimethyl chitosan as an adjuvant in vaccine delivery.

The application of natural carbohydrate polysaccharides for antigen delivery and its adjuvanation potential has garnered interest in the scientific community in the recent years. These biomaterials are considered favorable candidates for adjuvant development due to their desirable properties like enormous bioavailability, non-toxicity, biodegradability, stability, affordability, and immunostimulating ability. Chitosan is the one such extensively studied natural polymer which has been appreciated for its excellent applications in pharmaceuticals. Trimethyl chitosan (TMC), a derivative of chitosan, possesses these properties. In addition it has the properties of high aqueous solubility, high charge density, mucoadhesive, permeation enhancing (ability to cross tight junction), and stability over a range of ionic conditions which makes the spectrum of its applicability much broader. It has also been seen to perform analogously to alum, complete Freund's adjuvant, incomplete Freund's adjuvant, and cyclic guanosine monophosphate adjuvanation, which justifies its role as a potent adjuvant. Although many review articles detailing the applications of chitosan in vaccine delivery are available, a comprehensive review of the applications of TMC as an adjuvant is not available to date. This article provides a comprehensive overview of structural and chemical properties of TMC which affect its adjuvant characteristics; the efficacy of various delivery routes for TMC antigen combination; and the recent advances in the elucidation of its mechanism of action.

Functional analysis of BAS2108-2109 two component system: Evidence for protease regulation in Bacillus anthracis.

BACKGROUND: Bacillus anthracis (BA) is a major bioterrorism concern which has evolved complex regulatory mechanisms for its virulence factors. Secreted proteases play an imperative role in the pathogenesis of BA, however their regulation remains elusive. Two component systems (TCS) are often employed by bacteria to sense and adapt to the environmental perturbations. In several pathogens, TCS are commonly associated with the regulation of virulence factors including proteases. The genome of BA encodes 41 TCS pairs, however, the role of any TCS in regulation of its proteases is not known. PRINCIPAL FINDINGS: The study established BAS2108-2109 as a prototypical TCS where BAS2108 functions as a histidine kinase and BAS2109 as the response regulator. The expression of BAS2109 was found to be elevated under host simulated conditions and in pellicle forming cells. Electrophoretic mobility shift assay (EMSA) and lacZ reporter assay revealed positive autoregulation of the BAS2108-2109 operon by BAS2109. Collective analysis of ANS assay and EMSA demonstrated Lys167, Thr179 and Thr182 residues are crucial for the DNA binding activity of BAS2109. EMSA analysis further highlighted BAS2109 as the transcriptional regulator for different genes of BA, particularly proteases. Upregulation of proteases in BA overexpressing BAS2109 further strengthen its role in protease regulation. SIGNIFICANCE: This is the first report to identify a TCS pair for its role in the regulation of proteases of BA. Importance of proteases in the pathogenesis of BA is well documented, therefore, studying the regulatory networks governing their expression will help in identification of new drug targets.

Detection, distribution and characterization of novel superoxide dismutases from Yersinia enterocolitica Biovar 1A.

BACKGROUND: Superoxide dismutases (SODs) cause dismutation of superoxide radicals to hydrogen peroxide and oxygen. Besides protecting the cells against oxidative damage by endogenously generated oxygen radicals, SODs play an important role in intraphagocytic survival of pathogenic bacteria. The complete genome sequences of Yersinia enterocolitica strains show presence of three different sod genes. However, not much is known about the types of SODs present in Y. enterocolitica, their characteristics and role in virulence and intraphagocytic survival of this organism. METHODOLOGY/PRINCIPAL FINDINGS: This study reports detection and distribution of the three superoxide dismutase (sodA, sodB and sodC) genes in 59 strains of Y. enterocolitica and related species. The majority (94%) of the strains carried all three genes and constitutive expression of sodA and sodB was detected in 88% of the strains. Expression of sodC was not observed in any of the strains. The sodA, sodB and sodC genes of Y. enterocolitica were cloned in pET28a (+) vector. Recombinant SodA (82 kDa) and SodB (21 kDa) were expressed as homotetramer and monomer respectively, and showed activity over a broad range of pH (3.0-8.0) and temperature (4-70°C). SodA and SodB showed optimal activity at 4°C under acidic pH of 6.0 and 4.0 respectively. The secondary structures of recombinant SodA and SodB were studied using circular dichroism. Production of YeSodC was not observed even after cloning and expression in E. coli BL21(DE3) cells. A SodA(-) SodB(-) Escherichia coli strain which was unable to grow in medium supplemented with paraquat showed normal growth after complementation with Y. enterocolitica SodA or SodB. CONCLUSIONS/SIGNIFICANCE: This is the first report on the distribution and characterization of superoxide dismutases from Y. enterocolitica. The low pH optima of both SodA and SodB encoded by Y. enterocolitica seem to implicate their role in acidic environments such as the intraphagocytic vesicles.