Yogendra Singh

AAA+ proteins function as molecular machines that utilize ATP to perform diverse cellular functions, including protein homeostasis, stress regulation, and cell cycle/developmental processes. In this study, we identified a novel AAA+ ATPase-related Bacillus anthracis protein BAS0518 (BA PrkA) which has 88% amino acid homology to Bacillus subtilis PrkA. Conserved domain analysis showed that BA PrkA has an N-terminal AAA+ ATPase domain with characteristic Walker A and Walker B motifs and a conserved secondary region of homology (SRH) domain, along with a C-terminal cAMP-dependent protein kinase domain. Based on AlphaFold 3 predicted structures, we classified BA PrkA as part of Clade III of the AAA+ superfamily. Our results suggest that BA PrkA has negligible protease and kinase activities under in vitro conditions. Nonetheless, BA PrkA was found to play a significant role in sporulation. It is temporally expressed during sporulation Stages II to VI. A null mutant lacking BA PrkA exhibits severe sporulation defects, with reduced spore viability and downregulation of genes related to spore integuments formation. These defects were reversed in a complementation strain expressing BA PrkA ectopically. Additionally, the null mutant strain showed compromised growth under ionic-osmotic stress conditions and showed a 13-fold induction of BA prkA in wild-type B. anthracis when exposed to 1 M NaCl. Analysis of the BA PrkA interactome revealed enrichment of two proteins, ProA and EzrA, which are implicated in osmotic stress response and the sporulation process, respectively. These findings show that BA PrkA is essential for sporulation and resistance to osmotic stress. IMPORTANCE: Sporulation and stress adaptation are vital for the survival of Bacillus anthracis in harsh environments. We characterize a novel AAA+ ATPase, B. anthracis BAS0518 (BA PrkA), which shares 88% similarity with Bacillus subtilis PrkA. Analysis of BA PrkA null mutant shows that BA PrkA is essential for proper spore development and stress resilience. BA PrkA is expressed during sporulation Stages II–VI, and its deletion causes defective, less viable spores, reduced osmotic stress tolerance, and downregulation of key sporulation genes. Interaction analyses identified ProA and EzrA, proteins linked to stress response and sporulation, as potential partners. These findings establish BA PrkA as a regulatory link between stress adaptation and sporulation in B. anthracis .

For more than a century, tuberculosis (TB) has been known as a lung disease that is caused by a species of pathogenic bacteria called Mycobacterium tuberculosis. Most infections show no symptoms, but a small fraction of infections become active and, if untreated, can be fatal. Indeed, TB is responsible for over one million deaths every year (Bagcchi, 2023).

Introduction Tuberculosis remains one of the world’s deadliest infectious diseases. The pathophysiology of the two manifestations of Mycobacterium tuberculosis infection, pulmonary TB (PTB) and extrapulmonary TB (EPTB) is still not fully understood. Understanding the metabolic profile of both disease manifestations in patients is important for developing therapeutic approaches and molecular diagnosis. Objective The current study aimed to elucidate differences in the gut metabolic profile of PTB and EPTB patients compared to healthy controls (HCs). Method We used an untargeted approach through ¹H Nuclear Magnetic Resonance (NMR) spectroscopy to perform metabolomic profiling of stool samples from 77 TB patients [pulmonary TB (PTB, n = 33), cervical lymph node TB (CrLNTB, n = 30), abdominal TB (ATB, n = 14)], and 30 HCs. Multivariate and univariate analyses were performed to identify the differential gut metabolites associated with TB patients. Results PTB patients showed greater metabolic perturbation than either EPTB group, with 24 metabolites significantly altered compared to 13 in ATB and 12 in CrLNTB, relative to HCs (adjusted p < 0.05). Each TB subtype displayed distinct metabolic profiles, yet several metabolites were commonly altered across all TB groups, including valine, N-formyl-L-methionine, choline, dimethylsulfone, tryptophan, valerate, N-acetylglutamate, creatine, and malonate. In the combined TB cohort versus controls, the most discriminatory metabolites were valine and N-formyl-L-methionine (AUC ≥ 0.80). Overall, these findings offer insights into the gut metabolome of TB patients in India and characterize for the first time metabolic perturbations in EPTB patients. Conclusion The study highlights the metabolic disruptions associated with PTB and EPTB patients.

Protein toxins are biologically active polypeptides produced by a variety of organisms, including bacteria, plants, fungi, and animals. These molecules exert potent and specific toxic effects on target cells and are primarily associated with pathogenicity and defense mechanisms of the organisms. In the past few decades, significant progress has been made in understanding their structure, mechanisms of action, and regulation. Among these, bacterial protein toxins have emerged as valuable tools particularly in the development of targeted therapies. A notable example is Botulinum toxin, originally known for its neurotoxic effects, which was approved as a therapeutic agent in 1989 for strabismus treatment, paving way for repurposing bacterial toxins for clinical use. This review provides an overview of the different classes of bacterial toxin-based therapeutics, with a particular focus on Pseudomonas exotoxin A (PE) from Pseudomonas aeruginosa and anthrax toxin from Bacillus anthracis. The modular architecture and potent cytotoxicity of these A-B type toxins have enabled their successful adaptation into targeted cancer therapies. The clinical approval of the PE-based immunotoxin, moxetumomab pasudotox, for the treatment of hairy cell leukemia, underscores the potential of this strategy. This review also discusses current challenges and outlines future directions for the advancement of bacterial toxin-based therapeutics.

AAA+ proteins function as molecular machines that utilize ATP to perform diverse cellular functions, including protein homeostasis, stress regulation, and cell cycle/developmental processes. In this study, we identified a novel AAA+ ATPase BAS PrkA in B. anthracis Sterne 34F2 which has 88 % protein homology to Bacillus subtilis PrkA. Conserved domain analysis confirms BAS PrkA has an N-terminal AAA+ ATPase domain with characteristic Walker A and Walker B motifs and a conserved secondary region of homology (SRH) domain, along with a C-terminal cAMP-dependent protein kinase domain. Based on Alpha Fold3 predicted structure, we classified BAS PrkA as part of Clade III of the AAA+ superfamily. Contrary to the reported enzymatic activity in B. subtilis PrkA, we observed that BAS PrkA has negligible protease and kinase activity under in-vitro conditions. Nonetheless, BAS PrkA plays a significant role in regulating sporulation. It is temporally expressed during Stages II to VI during sporulation. A null mutant of BAS PrkA exhibits severe sporulation defects, with reduced spore viability, and down regulation of genes related to spore-coat formation. These phenotypes were restored in a complementation strain expressing BAS PrkA ectopically. Additionally, the null mutant strain showed compromised growth under ionic-osmotic stress conditions. Analysis of the BAS PrkA interactome revealed enrichment of two proteins, ProA and EzrA, that are implicated in osmotic stress response and the sporulation process, respectively. These findings show that BAS PrkA plays a critical role in sporulation and osmotic stress response in B. anthracis .

Bacillus anthracis is a deadly pathogen that under unfavourable conditions forms highly resistant spores which enable them to survive for a long period of time. Spores of B. anthracis are transmitted through the contaminated soil or animal products and enter to the host through the skin, lungs or oral route and can cause cutaneous, injection, inhalation and gastrointestinal anthrax, respectively. The disease is caused by the toxin which is produced by them once they germinate within the host cell. Anthrax toxin is the major virulence factor which has the ability to kill the host cell. The role of protein kinases and phosphatases of B. anthracis in toxin production and other virulence related properties have also been reported. There are two vaccines, BioThrax and CYFENDUS^TM, which are approved by the FDA-USA to prevent anthrax disease. Recently, anthrax toxin has also been shown to be a potential candidate for cancer therapeutics. Through present review, we aim to provide insights into sporulation, transmission and pathogenesis of B. anthracis as well as the current state of its prevention, treatment, vaccines and possible therapeutic uses in cancer.

Dickeya dadantii is the causal agent of bacterial stalk rot and one of the most destructive and widespread diseases of the sorghum in the world. Here, we explored microbe-based approaches for managing this destructive pathogen, intending to provide alternatives for integrated disease management. The objective of the research was to decipher the effect of antagonistic microbes on systemic defense enzymes, histochemical changes, plant growth attributes, reduction in disease severity, and interaction of these antagonistic microbes with host. Trichoderma, Pseudomonas, and Bacillus isolates were collected from rhizospheric soil and characterized using morphological and molecular tools. ITS and 16S rRNA sequences were analyzed to determine the molecular characterization of all antagonist microbes, and they were identified as T. asperellum, T. viride, T. harzianum, B. subtilis, and P. flourescens. These isolates were evaluated for antibacterial properties against D. dadantii under in vitro conditions and showed the higher inhibition in a dual culture method. Further, the effects of seed bio-priming and soil application of these isolates were tested under glasshouse and field conditions. T. viride outperformed the other isolates, significantly enhancing the plant growth parameters and induced resistance to Dickeya dadantii (BSR). T. viride showed a significantly higher accumulation of defensive enzymes, viz. PAL (1.02), PO (1.70), PPO (1.25), CAT (1.11), and TPC (0.91) at 48 h after pathogen challenge, as compared to the control. Histochemical tests confirmed lignification and callose deposition in the cell walls of the treated plants. Antagonist microbes were further evaluated under field conditions against D. dadantii infection. Compared to the control, there is a significant enhancement of plant growth parameters and yield with a simultaneous decrease in disease severity in T. viride treated plants. Results showed that the potential benefits of T. viride could not only effectively induce resistance in plants, enhance plant growth, increase yield, and suppress pathogen infection but also reduce the use of hazardous pesticides. As a result of correlation, PCA and heat map analyses indicated that T. viride is interconnected to determine the crop ability to sustain its growth under pathogen stress.

Protein phosphorylation is a universal mechanism regulating a wide range of cellular responses across all domains of life. The antagonistic activities of kinases and phosphatases can orchestrate the life cycle of an organism. The availability of bacterial genome sequences, particularly Bacillus species, followed by proteomics and functional studies have aided in the identification of putative protein kinases and protein phosphatases, and their downstream substrates. Several studies have established the role of phosphorylation in different physiological states of Bacillus species as they pass through various life stages such as sporulation, germination, and biofilm formation. The most common phosphorylation sites in Bacillus proteins are histidine, aspartate, tyrosine, serine, threonine, and arginine residues. Protein phosphorylation can alter protein activity, structural conformational and protein-protein interactions, ultimately affecting the downstream pathways. In this review, we summarize the knowledge available in the field of Bacillus signaling, with a focus on the role of protein phosphorylation in its physiological processes.