Sergii Krysenko

Long COVID, also termed post-acute sequelae of SARS-CoV-2 infection (PASC), affects a substantial minority of people after acute COVID-19 and is now recognized as a distinct clinical entity rather than slow convalescence. This review has two aims: to critically appraise the evidence for the principal proposed mechanisms of long COVID and to evaluate whether heat shock protein 90 (Hsp90) represents a candidate molecular node that intersects several of these mechanisms and may provide a therapeutically tractable target, without implying primacy over other pathways involved in disease pathogenesis. We first summarise the clinical presentation and the diagnostic difficulty created by symptom heterogeneity and by overlap with myalgic encephalomyelitis/chronic fatigue syndrome and other post-infectious syndromes. Then the leading candidate mechanisms, including immune dysregulation, viral antigen persistence, reactivation of latent herpesviruses, autoimmunity, endothelial and microvascular dysfunction, and skeletal muscle and mitochondrial abnormality, grading the strength of the supporting human evidence and stating the principal uncertainties for each, are evaluated. Further, we discuss the roles of Hsp90 on SARS-CoV-2 replication, immune evasion, endothelial barrier dysfunction, and chaperone-directed autoimmunity. Evidence on Hsp90 inhibition is deliberately presented in both directions: several preclinical studies report reduced viral load and inflammation, whereas one inhibitor failed to reduce viral shedding and increased mortality in infected mice, indicating that Hsp90 is a potential therapeutic target because of its involvement across multiple biological processes relevant to long COVID. Management is discussed by symptom domain, with clinical evidence distinguished throughout from in vitro, in silico and animal data, and candidate repurposed agents are summarised with the highest level of evidence currently available for each. In summary, no pharmacological therapy is yet supported by adequate randomised evidence in long COVID, and the value of Hsp90 as a target will depend on isoform selectivity, treatment timing and biomarker-defined patient selection.

Polyamine metabolism has emerged as an important factor in pathogen survival, proliferation, virulence and persistence, highlighting polyamine-associated proteins and metabolic pathways as promising therapeutic targets. This is particularly relevant to major infectious diseases, including tuberculosis, malaria, leishmaniasis, and trypanosomiasis, in which pathogens exploit and remodel host cellular processes to evade immune responses and establish intracellular survival. Increasing evidence indicates that alterations in host–pathogen polyamine metabolism contribute to pathogen adaptation and persistence, providing opportunities for therapeutic intervention. Over the past decade, several components of polyamine biosynthesis, transport, and utilization have been identified and validated as potential drug targets in clinically relevant pathogens. Concurrently, advances in medicinal chemistry have led to the development of polyamine-derived compounds with enhanced specificity toward pathogen-associated polyamine proteins and metabolic pathways. These advancements have improved approaches to target-based drug discovery, which can be a novel complementary strategy to conventional antimicrobial development. This is particularly relevant in the context of the increasing prevalence of multidrug-resistant (MDR) pathogens. In this review, recent advances in the identification and validation of polyamine-related drug targets in pathogens causing tuberculosis, malaria, leishmaniasis, and trypanosomiasis are summarized. Furthermore, emerging strategies for the design and optimization of polyamine-derived therapeutic compounds, with a focus on their molecular targets, mechanisms of action, and potential for combating antimicrobial resistance, are discussed. Current challenges and future opportunities for targeting polyamine metabolism in the development of next-generation therapeutics are highlighted.:

Polyamines such as putrescine, spermidine, and spermine are small, positively charged organic cations that interact with negatively charged molecules, notably nucleic acids. These molecules play a vital role in cell proliferation, differentiation, and growth. Their involvement in wound healing has attracted considerable research interest, as they are key regulators of keratinocyte and fibroblast proliferation—two cell types essential for re-epithelialization and tissue regeneration. Polyamines also influence gene expression and protein synthesis, facilitating the transition of cells from a quiescent to an active state during the healing process. Enzymes such as Adenosylmethionine Decarboxylase 1 (AMD1), which are involved in polyamine biosynthesis, are upregulated during wound healing and contribute to gene regulation at multiple levels. In parasitic protozoa—including Plasmodium falciparum (malaria), Trypanosoma spp. (sleeping sickness), and Leishmania spp. (leishmaniasis)—polyamines are crucial for DNA stabilization, protein synthesis, and cellular proliferation. These parasites depend on both endogenous polyamine synthesis and host-derived polyamines to sustain their metabolic functions. Notably, Plasmodium falciparum, the causative agent of malaria, possesses a bifunctional enzyme complex linking S-Adenosylmethionine decarboxylase (AdoMetDC) and ornithine decarboxylase (ODC), unlike the human form, where these enzymes are separate. This structural distinction makes the parasite’s AdoMetDC/ODC complex a promising target for drug development. In Mycobacterium tuberculosis (Mtb), polyamine metabolites have been shown to influence DNA methylation patterns, thereby affecting gene expression and bacterial adaptation. A 2025 study revealed that altered polyamine levels in the blood of patients with multidrug-resistant TB were associated with changes in Mtb DNA methylation, suggesting a potential role in resistance mechanisms. This chapter explores the multifaceted roles of polyamines in wound healing, parasitic infections, and tuberculosis-related bacteria. It also considers emerging technologies for wound treatment and highlights parasite-specific targets that may inform novel therapeutic strategies against TB.

Microorganisms naturally produce many pharmaceutically and industrially relevant secondary metabolites. For this process they usually use biosynthetic units. For example, microbes from the genus Streptomyces possess great ability to produce a variety of natural products in such manner, which is possible due to complicated crosstalk between primary and secondary metabolism. These microbial cell factories produce more than 2/3 of antibiotics used in medicine, and a large variety of other bioactive compounds. Although bacterial producer hosts, including Bacillus spp. and Streptomyces spp., have been studied for decades, the engineering of these bacteria remains challenging, and the genetic potential has not been fully utilized. This is due to limited genetic toolbox, restriction activity and occurrence of silent biosynthetic gene clusters. Recent advancements in genetic manipulation of microorganisms allowed to improve the turnaround time of strain engineering, but still has strain-specific limitations. However, a new perspective offered by synthetic biology to exploit the potential of existing and novel pathways in primary and secondary metabolism allows combining of different biosynthetic steps originating from diverse bacteria using a limited toolbox. Synthetic biology has emerged as a robust strategy to understand, investigate, design, and engineer the biosynthetic capability of bacterial antibiotics machinery, including such in Streptomyces. Innovative synthetic biology and metabolic engineering tools have rapidly accelerated the discovery of new natural products as well as engineering of Streptomyces, e.g. enzymatic modules for secondary metabolite production can be combined in synthetic cells to produce new derivatives of natural products. Furthermore, with the recent advances in molecular biology and genome editing, Synthetic biology has focused at generation of controlled phenotypes from a given input and at other sophisticated approaches. In this review, developments of novel approaches of Synthetic biology for microbial engineering with focus on antibiotics producers like Streptomyces spp. are discussed.

Nitrogen is an essential element required for bacterial homeostasis. It serves as a building block for the biosynthesis of macromolecules and provides precursors for secondary metabolites. Actinomycetes have developed the ability to use various nitrogen sources to ensure their survival in ecological niches with fluctuating nutrient availability. A complex nitrogen metabolism of Actinobacteria allows the utilization of various compounds as N sources, including ammonium, nitrate, urea, amino acids, amino sugars, and amines. One such adaptation is the ability to acquire nitrogen from alternative amine sources like monoamines or polyamines putrescine, cadaverine, spermidine, and spermine, ensuring both nutrient availability (C and N sources) and resistance against high polyamine concentrations. Actinobacterial nitrogen degradation, including the catabolism of amines, is not only important under low nitrogen availability, but also required to survive under high concentrations of these compounds. The purpose of this review is to summarize the knowledge on nitrogen degradation and, more specifically, catabolism of amines in Actinobacterial survival and its role in nitrogen metabolism. Applying critical analysis of the recent available literature and sequencing data, this work aims to explore strategies of pathogenic and non-pathogenic Actinobacteria to survive in the presence of different nitrogen sources, and their impact on primary and secondary metabolism. The knowledge about nitrogen degradation pathways in Actinobacteria including mono- and polyamine catabolism collected in the scope of this review paper is brought in connection with possibilities to combat pathogens by using their capability to metabolize polyamines as an antibiotic drug target. This might offer new directions for target-based drug design to combat Actinobacterial infections.

Nitrogen is an essential element required for bacterial homeostsis. It serves as a building block for the biosynthesis of macromolecules and provides precursors for secondary metabolites. Actinomycetes have developed the ability to use various nitrogen sources and possess two central enzyme systems for nitrogen assimilation involving glutamine synthetase / glutamate synthase and glutamate dehydrogenase. Microorganisms living in habitats with changeable availability of nutrients have to cope with limited nitrogen availability in their ecological niches and developed strategies to survive under nitrogen limitation. A complex nitrogen metabolism of Actinobacteria allows the utilization of various compounds as N-sources. One such adaptation is the ability to acquire nitrogen from alternative sources like monoamines or polyamines putrescine, cadaverine, spermidine and spermine, ensuring both nutrients availability (C- and N-source) and resistance against high polyamine concentrations. Bacterial polyamine catabolism is not only important under low nitrogen availability, but it is also required to survive under high concentrations of these compounds. Such conditions can occur in diverse habitats like soil, plant tissues and human cells. Strategies of pathogenic and non-pathogenic Actinobacteria to survive in the presence of mono- and polyamines offer the possibility to combat pathogens by using their capability to metabolize polyamines as an antibiotic drug target. This work aims to summarize the knowledge on nitrogen utilization and, more specifically, catabolism of amines in actinobacterial survival and its role in nitrogen metabolism.

Bacterial natural ecological niches are characterized by variations in the availability of nutrients, resulting in a complex metabolism. Their impressive ability to adapt to changeable nutrient conditions is possible through the utilization of large amounts of substrates. Recent discoveries in bacterial metabolism have suggested the importance of polyamine metabolism in bacteria, particularly in those of the order Actinomycetales, in enabling them to survive in their natural habitats. This makes such enzymes promising targets to inhibit their growth. Since the polyamine metabolisms of soil bacteria of the genus Streptomyces and the human pathogenic Mycobacteria are surprisingly similar, target-based drug development in Streptomyces and Mycobacterium spp. is an alternative approach to the classical search for antibiotics. The recent development of drugs to treat epidemic diseases like tuberculosis (TB) has gained attention due to the occurrence of multidrug-resistant strains. In addition, drug repurposing plays a crucial role in the treatment of various complex diseases, such as malaria. With that notion, the treatment of TB could also benefit from this approach. For example, molecular chaperones, proteins that help other proteins to fold properly, are found in almost all living organisms, including the causative agents of TB. Therefore, targeting these molecules could help in the treatment of TB. We aim to summarize our knowledge of the nitrogen and carbon metabolism of the two closely related actinobacterial genera, Streptomyces and Mycobacterium, and of the identification of new potential drug targets.

Actinobacterial natural ecological niches are characterized by variations in the availability of nutrients, resulting in a complex metabolism. Their remarkable ability to adapt to fluctuating nutrient conditions is possible through the utilization of large amounts of substrates. Recent discoveries in bacterial metabolism suggested the importance of polyamine metabolism in bacteria, particularly in those of the order Actinomycetales, to survive in their natural habitats. This makes such enzymes promising targets to inhibit their growth. Since the polyamine metabolism of soil bacteria of the genus Streptomyces and the human pathogenic Mycobacteria is surprisingly similar, a target-based drug development in Streptomyces and Mycobacterium spp. is an alternative approach to the classical search of antibiotics. The recent development of drugs to treat epidemic diseases like tuberculosis (TB) has gained attention due to the occurrence of multi-drug-resistant strains. In addition, drug repurposing plays a crucial role in the treatment of various complex diseases, such as malaria. With that notion, the treatment of TB could also benefit from this approach. For example, molecular chaperones, proteins that help other proteins to fold properly are found in almost all living organisms including the causative agents of TB. Therefore, targeting these molecules could help in the treatment of TB. We aim to summarize the knowledge of nitrogen and carbon metabolism of the two closely related actinobacterial genera, Streptomyces and Mycobacterium, and of the identification of new potential drug targets.

Mycobacterium tuberculosis is well adapted to survive and persist in the infected host, escaping the host’s immune response. Since polyamines such as spermine, which are synthesized by infected macrophages, are able to inhibit the growth of M. tuberculosis , the pathogen needs strategies to cope with these toxic metabolites. The actinomycete Streptomyces coelicolor , a close relative of M. tuberculosis, makes use of a gamma-glutamylation pathway to functionally neutralize spermine. We therefore considered whether a similar pathway would be functional in M. tuberculosis . In the current study, we demonstrated that M. tuberculosis growth was inhibited by the polyamine spermine. Using in vitro enzymatic assays we determined that GlnA3 _Mt (Rv1878) possesses genuine gamma-glutamylspermine synthetase catalytic activity. We further showed that purified His-Strep-GlnA3 _Mt , as well as native GlnA3 _Mt , prefer spermine as a substrate over putrescine, cadaverine, spermidine, or other monoamines and amino acids, suggesting that GlnA3 _Mt may play a specific role in the detoxification of the polyamine spermine. However, the deletion of the glnA3 gene in M. tuberculosis did not result in growth inhibition or enhanced sensitivity of M. tuberculosis in the presence of high spermine concentrations. Gene expression analysis of spermine-treated M. tuberculosis revealed no difference in the level of glnA3 _Mt expression relative to untreated cells, whereas a gene encoding a previously characterized efflux pump (Mmr; rv3065 ) was significantly upregulated. This suggests that bacterial survival under elevated spermine concentrations can not only be achieved by detoxification of spermine itself but also by mechanisms resulting in decreased spermine levels in the bacteria. IMPORTANCE: Upon Mycobacterium tuberculosis infection macrophages synthesize the polyamine spermine, which at elevated concentrations is toxic for M. tuberculosis . Based on our investigations of spermine resistance in the closely related actinomycete Streptomyces coelicolor , we hypothesized that the glutamylspermine synthetase GlnA3 may be responsible for the resistance of M. tuberculosis against toxic spermine. Here we show that GlnA3 _Mt can indeed covalently modify spermine via glutamylation. However, GlnA3 _Mt is probably not the only resistance mechanism since a glnA3 null mutant of M. tuberculosis can survive under spermine stress. Gene expression studies suggest that an efflux pump may participate in resistance. Thus a combination of GlnA3 _Mt and specific efflux pumps acting as putative spermine transporters may constitute an active spermine-detoxification system in M. tuberculosis .

Organisms from the genus Streptomyces feature actinobacteria with complex developmental cycles and a great ability to produce a variety of natural products. These soil bacteria produce more than 2/3 of antibiotics used in medicine, and a large variety of bioactive compounds for industrial, medical and agricultural use. Although Streptomyces spp. have been studied for decades, the engineering of these bacteria remains challenging, and the available genetic tools are rather limited. Furthermore, most biosynthetic gene clusters in these bacteria are silent and require strategies to activate them and exploit their production potential. In order to explore, understand and manipulate the capabilities of Streptomyces spp. as a key bacterial for biotechnology, synthetic biology strategies emerged as a valuable component of Streptomyces research. Recent advancements in strategies for genetic manipulation of Streptomyces involving proposals of a large variety of synthetic components for the genetic toolbox, as well as new approaches for genome mining, assembly of genetic constructs and their delivery into the cell, allowed facilitation of the turnaround time of strain engineering and efficient production of new natural products at an industrial scale, but still have strain- and design-dependent limitations. A new perspective offered recently by technical advances in DNA sequencing, analysis and editing proposed strategies to overcome strain- and construct-specific difficulties in the engineering of Streptomyces. In this review, challenges and recent developments of approaches for Streptomyces engineering are discussed, an overview of novel synthetic biology strategies is provided and examples of successful application of new technologies in molecular genetic engineering of Streptomyces are highlighted.