Computational exploration of the genomic assignments, molecular structure, and dynamics of the ccdABXn2 toxin-antitoxin homolog with its bacterial target, the DNA gyrase, in the entomopathogen Xenorhabdus nematophila.

Toxin-antitoxin (TA) modules, initially discovered on bacterial plasmids and subsequently identified within chromosomal contexts, hold a pivotal role in the realm of bacterial physiology. Among these, the pioneering TA system, ccd (Control of Cell Death), primarily localized on the F-plasmid, is known for its orchestration of plasmid replication with cellular division. Nonetheless, the precise functions of such systems within bacterial chromosomal settings remain a compelling subject that demands deeper investigation. To bridge this knowledge gap, our study focuses on exploring ccdABXn2, a chromosomally encoded TA module originating from the entomopathogenic bacterium Xenorhabdus nematophila. We meticulously delved into the system's genomic assignments, structural attributes, and functional interplay. Our findings uncovered intriguing patterns-CcdB toxin homologs exhibited higher conservation levels compared to their CcdA antitoxin counterparts. Moreover, we constructed secondary as well as tertiary models for both the CcdB toxin and CcdA antitoxin using threading techniques and subsequently validated their structural integrity. Our exploration extended to the identification of key interactions, including the peptide interaction with gyrase for the CcdB homolog and CcdB toxin interactions for the CcdA homolog, highlighting the intricate TA interaction network. Through docking and simulation analyses, we unequivocally demonstrated the inhibition of replication via binding the CcdB toxin to its target, DNA gyrase. These insights provide valuable knowledge about the metabolic and physiological roles of the chromosomally encoded ccdABXn2 TA module within the context of X. nematophila, significantly enhancing our comprehension of its functional significance within the intricate ecosystem of the bacterial host.Communicated by Ramaswamy H. Sarma.

Genomic assortment and interactive insights of the chromosomal encoded control of cell death (ccd) toxin-antitoxin (TA) module in Xenorhabdus nematophila.

In the present circumstances, toxin-antitoxin (TA) modules have a great consideration due to their elusive role in bacterial physiology. TA modules consist of a toxic part and a counteracting antitoxin part and these are abundant genetic loci harbored on bacterial plasmids and chromosomes. The control of cell death (ccd) TA locus was the first identified TA module and its unitary function (such as plasmid maintenance) has been described, however, the function of its chromosomal counterparts is still ambiguous. Here, we are exploring the genomic assortment, structural and functional association of chromosomally encoded ccdAB TA homolog (ccdABXn1) in the genome of an entomopathogenic bacterium Xenorhabdus nematophila. This bacterium is a symbiotic model with the nematode Steinernema carpocapsae that infects and kills the host insect. By genomic assortment analysis, our observations suggested that CcdA antitoxin homologs are not more closely related than CcdB toxin homologs. Further results suggest that the ccdABXn1 TA homolog has sulphonamide (such as 4C6, for CcdA homolog) and peptide (such as gyrase, for CcdB homolog) ligand partners with a typical TA interaction network that may affect essential cellular metabolism of the X. nematophila. Collectively, our results improve the knowledge and conception of the metabolic interactive role of ccdAB TA homologs in X. nematophila physiology.Communicated by Ramaswamy H. Sarma.

Functional and transcriptional analysis of chromosomal encoded hipBAXn2 type II toxin-antitoxin (TA) module from Xenorhabdus nematophila.

Xenorhabdus nematophila is an entomopathogenic bacterium that synthesizes numerous toxins and kills its larval insect host. Apart from such toxins, its genome also has a plethora of toxin-antitoxin (TA) systems. The role of TA systems in bacterial physiology is debatable; however, they are associated with maintaining bacterial genomic stability and their survival under adverse environmental conditions. Here, we explored the functionality and transcriptional regulation of the type II hipBAXn2 TA system. This TA system was identified in the genome of X. nematophila ATCC 19061, which consists of the hipAXn2 toxin gene encoding 278 amino acid residues and hipBXn2 encoding antitoxin of 135 amino acid residues. We showed that overexpression of HipAXn2 toxin reduced the growth of Escherichia coli cells in a bacteriostatic manner, and amino-acids G8, H164, N167, and S169 were key residues for this growth reduction. Promoter activity and expression profiling of the hipBAXn2 TA system was showed that transcription was induced in both E. coli as well as X. nematophila upon exposure to different stress conditions. Further, we have exhibited the binding features of HipAXn2 toxin and HipBXn2 antitoxin to their promoter. This study provides evidence for the presence of a functional and well-regulated hipBAXn2 TA system in X. nematophila.

Bacterial toxin-antitoxin modules: classification, functions, and association with persistence.

Toxin-antitoxin (TA) modules are ubiquitous gene loci among bacteria and are comprised of a toxin part and its cognate antitoxin part. Under normal physiological conditions, antitoxin counteracts the toxicity of the toxin whereas, during stress conditions, TA modules play a crucial role in bacterial physiology through involvement in the post-segregational killing, abortive infection, biofilms, and persister cell formation. Most of the toxins are proteinaceous that affect translation or DNA replication, although some other intracellular molecular targets have also been described. While antitoxins may be a protein or RNA, that generally neutralizes its cognate toxin by direct interaction or with the help of other signaling elements and thus helps in the TA module regulation. In this review, we have discussed the current state of the multifaceted TA (type I-VIII) modules by highlighting their classification and specific targets. We have also discussed the presence of TA modules in the various pathogens and their role in antibiotic persistence development as well as biofilm formation, by influencing the different cellular processes. In the end, assembling knowledge about ubiquitous TA systems from pathogenic bacteria facilitated us to propose multiple novel antibacterial strategies involving artificial activation of TA modules.

Novel Toxin-antitoxin System Xn-mazEF from Xenorhabdus nematophila: Identification, Characterization and Functional Exploration.

BACKGROUND: Xenorhabdus nematophila maintains species-specific mutual interaction with nematodes of Steinernema genus. Type II Toxin Antitoxin (TA) systems, the mazEF TA system controls stress and programmed cell death in bacteria. OBJECTIVE: This study elucidates the functional characterization of Xn-mazEF, a mazEF homolog in X. nematophila by computational and in vitro approaches. METHODS: 3D- structural models for Xn-MazE toxin and Xn-MazF antitoxin were generated, validated and characterized for protein - RNA interaction analysis. Further biological and cellular functions of Xn-MazF toxin were also predicted. Molecular dynamics simulations of 50ns for Xn- MazF toxin complexed with nucleic acid units (DU, RU, RC, and RU) were performed. The MazF toxin and complete MazEF operon were endogenously expressed and monitored for the killing of Escherichia coli host cells under arabinose induced tightly regulated system. RESULTS: Upon induction, E. coli expressing toxin showed rapid killing within four hours and attained up to 65% growth inhibition, while the expression of the entire operon did not show significant killing. The observation suggests that the Xn-mazEF TA system control transcriptional regulation in X. nematophila and helps to manage stress or cause toxicity leading to programmed death of cells. CONCLUSION: The study provides insights into structural and functional features of novel toxin, Xn- MazF and provides an initial inference on control of X. nematophila growth regulated by TA systems.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), a newly emerged pathogen: an overview.

Coronavirus disease 2019 (COVID-19) is a viral pneumonia, responsible for the recent pandemic, and originated from Wuhan, China, in December 2019. The causative agent of the outbreak was identified as coronavirus and designated as severe acute respiratory syndrome coronavirus 2 (SARS- CoV-2). Few years back, the severe acute respiratory syndrome coronavirus (SARS- CoV) and the Middle East respiratory syndrome coronavirus (MERS-CoV) were reported to be highly pathogenic and caused severe infections in humans. In the current situation SARS-CoV-2 has become the third highly pathogenic coronavirus that is responsible for the present outbreak in human population. At the time of this review, there were more than 14 007 791 confirmed COVID-19 patients which associated with over 597 105 deaths in more then 216 countries across the globe (as reported by World Health Organization). In this review we have discussed about SARS-CoV, MERS-CoV and SARC-CoV-2, their reservoirs, role of spike proteins and immunogenicity. We have also covered the diagnosis, therapeutics and vaccine status of SARS-CoV-2.

The hipBAXn operon from Xenorhabdus nematophila functions as a bonafide toxin-antitoxin module.

Here, for the first time, we have investigated the hipBAXn toxin-antitoxin (TA) module from entomopathogenic bacterium Xenorhabdus nematophila. It is a type II TA module that consists of HipAXn toxin and HipBXn antitoxin protein and located in the complementary strand of chromosome under XNC1_operon 0810 locus tag. For functional analysis, hipAXn toxin, hipBXn antitoxin, and an operon having both genes were cloned in pBAD/His C vector and transformed in Escherichia coli cells. The expression profiles and endogenous toxicity assay were performed in these cells. To determine the active amino acid residues responsible for the toxicity of HipAXn toxin, site-directed mutagenesis (SDM) was performed. SDM results showed that amino acid residues S149, D306, and D329 in HipAXn toxin protein were significantly essential for its toxicity. For transcriptional analysis, the 157 bp upstream region of the hipBAXn TA module was identified as a promoter with bioinformatics tools. Further, the LacZ reporter construct with promoter region was prepared and LacZ assays as well as reverse transcriptase-polymerase chain reaction (RT-PCR) analysis was performed under different stress conditions. Electrophoretic mobility shift assay (EMSA) was also performed with recombinant HipAXn toxin, HipBXn antitoxin protein, and 157 bp promoter region. Results showed that the hipBAXn TA module is a well-regulated system in which the upregulation of gene expression was also found compulsive in different SOS conditions. KEY POINTS: •Functional characterization of hipBA Xn TA module from Xenorhabdus nematophila. •hipBA Xn TA module is a functional type II TA module. •Transcriptional characterization of hipBA Xn TA module. •hipBA Xn TA module is a well regulated TA module. Graphical abstract.

TAome analysis of type-II toxin-antitoxin system from Xenorhabdus nematophila.

Here we report the first essentially complete TAome analysis for type II toxin-antitoxin (TA) system, a major class of TA modules found in bacterial system, from entomopathogenic bacterium Xenorhabdus nematophila ATCC 19061 (NCBI RefSeq NC_014228). We summarize this analysis in terms of TA locus, accession identifier, hits in conserved domain database, toxin superfamily, antitoxin superfamily and chromosomal/mobile genome/plasmid occurrences. Moreover, for TA context analyses we use six different specifications namely virulence factors, mobile genetic elements (MGE), antibiotic resistance genes, secretion systems, prophage and a classification of mobile genetic elements (ACLAME); among these hits are found for only MGE, ACLAME and prophage. A total 39 sets of TA have been discovered in which numbers of TA encoded for MGE, ACLAME and prophage are 15, 15 and 5 respectively while the remaining four have no context hit. In addition, a comparative analysis of TAome was also done with closely related bacterium Photorhabdus luminescens subsp. laumondii TTO1 (NCBI RefSeq NC_005126) and results shows that a total 8 sets of TA are conserved. Further, a bootstrap Neighbor-Joining phylogenetic tree was also constructed for major toxin protein superfamily found namely RelE, HigB, GNAT, CcdB and MazF explored in the TAome of X. nematophila. We also characterized, the most abundantly found TA module (relBE) in this TAome, functionally and transcriptionally. This first TAome analysis of type II TA modules provides new insights in multi-drug tolerance in bacterial populations.

Functional annotation of a novel toxin-antitoxin system Xn-RelT of Xenorhabdus nematophila; a combined in silico and in vitro approach.

Toxin-antitoxin (TA) complexes play an important role in stress responses and programmed cell death in bacteria. The RelB-RelE toxin antitoxin system is well studied in Escherichia coli. In this study, we used combined in silico and in vitro approaches to study a novel Xn-RelT toxin from Xenorhabdus nematophila bearing its own antitoxin Xn-RelAT-a RelB homolog of E. coli. The structure for this toxin-antitoxin pair is yet unknown. We generated homology-based models of X. nematophila RelT toxin and antitoxin. The deduced models were further characterized for protein-nucleic acid, protein-protein interactions and gene ontology. A detrimental effect of recombinant Xn-RelT on host E. coli was determined through endogenous toxicity assay. When expressed from a isopropyl ß-D-1-thiogalactopyranoside-regulated LacZ promoter, Xn-RelT toxin showed a toxic effect on E. coli cells. These observations imply that the conditional cooperativity governing the Xn-RelT TA operon in X. nematophila plays an important role in stress management and programmed cell death.

Protective role of Th17 cells in pulmonary infection.

Th17 cells are characterized as preferential producer of interleukins including IL-17A, IL-17F, IL-21 and IL-22. Corresponding receptors of these cytokines are expressed on number of cell types found in the mucosa, including epithelial cells and fibroblasts which constitute the prime targets of the Th17-associated cytokines. Binding of IL-17 family members to their corresponding receptors lead to modulation of antimicrobial functions of target cells including alveolar epithelial cells. Stimulated alveolar epithelial cells produce antimicrobial peptides and are involved in granulepoesis, neutrophil recruitment and tissue repair. Mucosal immunity mediated by Th17 cells is protective against numerous pulmonary pathogens including extracellular bacterial and fungal pathogens. This review focuses on the protective role of Th17 cells during pulmonary infection, highlighting subset differentiation, effector cytokines production, followed by study of the binding of these cytokines to their corresponding receptors, the subsequent signaling pathway they engender and their effector role in host defense.

Expression, purification, and functional analysis of novel RelE operon from X. nematophila.

Bacterial toxin-antitoxin (TA) complexes induce programmed cell death and also function to relieve cell from stress by various response mechanisms. Escherichia coli RelB-RelE TA complex consists of a RelE toxin functionally counteracted by RelB antitoxin. In the present study, a novel homolog of RelE toxin designated as Xn-relE toxin from Xenorhabdus nematophila possessing its own antitoxin designated as Xn-relEAT has been identified. Expression and purification of recombinant proteins under native conditions with GST and Ni-NTA chromatography prove the existence of novel TA module. The expression of recombinant Xn-relE under tightly regulated ara promoter in E. coli Top 10 cells confirms its toxic nature in endogenous toxicity assay. The neutralization activity in endogenous toxicity assay by Xn-relEAT antitoxin confirms its antidote nature when studying the whole TA operon under ara regulated promoter. This study promotes newly discovered TA module to be regarded as important as other proteins of type II toxin-antitoxin system.

Expression, Purification, and Functional Characterization of Atypical Xenocin, Its Immunity Protein, and Their Domains from Xenorhabdus nematophila.

Xenorhabdus nematophila, a gram-negative bacterium belonging to the family Enterobacteriaceae is a natural symbiont of a soil nematode from the family Steinernematidae. In this study cloning, expression, and purification of broad range iron regulated multidomain bacteriocin called xenocin from X. nematophila (66 kDa, encoded by xcinA gene) and its multidomain immunity protein (42 kDa, encoded by ximB gene) have been done. xcinA-ximB (N' terminal 270 bp), translocation, and translocation-receptor domain of xcinA, ximB, and its hemolysin domain were cloned, expressed, and purified by single step Ni-NTA chromatography under native conditions. In the functional characterization, neutralization of xcinA toxicity by immunity domain of ximB gene was determined by endogenous assay. Exogenous toxic assays results showed that only the purified recombinant xenocin-immunity domain (10 kDa) protein complex had toxic activity. Atypical cognate immunity protein (42 kDa) of xenocin was fusion of immunity domain (10 kDa) and hemolysin domain (32 kDa). In silico analysis of immunity protein revealed its similarity with hemolysin and purine NTPase like proteins. Hemolytic activity was not observed in immunity protein or in its various domains; however, full-length immunity protein lacking Walker motif showed ATPase activity. Finally, using circular dichroism performed secondary structural analyses of all the recombinant proteins/protein complexes.

Induction of somatic embryogenesis in gum arabic tree [Acacia senegal (L.) Willd].

Factors affecting somatic embryogenesis from immature cotyledon of gum arabic tree [Acacia senegal (L.) Willd.] were investigated. Induction of somatic embryogenesis was influenced by plant growth regulator concentrations and addition of amino acids in medium. Best induction of somatic embryogenesis was obtained on MS medium supplemented with 0.45 µM 2, 4-D, 2.32 µM Kin and 15 mM L-glutamine. L-glutamine plays a significant role in the maturation of somatic embryos and most of embryos attained maturity only on L-glutamine (15 mM) containing medium. Maximum percent (75.0 ± 2.5) germination of somatic embryos was recorded on medium containing 0.22 µM BAP.