Resolving the polycistronic aftermath: Essential role of topoisomerase IA in preventing R-loops in Leishmania.

Kinetoplastid parasites are "living bridges" in the evolution from prokaryotes to higher eukaryotes. The near-intronless genome of the kinetoplastid Leishmania exhibits polycistronic transcription which can facilitate R-loop formation. Therefore, to prevent such DNA-RNA hybrids, Leishmania has retained prokaryotic-like DNA Topoisomerase IA (LdTOPIA) in the course of evolution. LdTOPIA is an essential enzyme that is expressed ubiquitously and is adapted for the compartmentalized eukaryotic form in harboring functional bipartite nuclear localization signals. Although exhibiting greater homology to mycobacterial TOPIA, LdTOPIA could functionally complement the growth lethality of Escherichia coli TOPIA null GyrB ts strain at non-permissive temperatures. Purified LdTOPIA exhibits Mg2+-dependent relaxation of only negatively supercoiled DNA and preference towards single-stranded DNA substrates. LdTOPIA prevents nuclear R-loops as conditional LdTOPIA downregulated parasites exhibit R-loop formation and thereby parasite killing. The clinically used tricyclic antidepressant, norclomipramine could specifically inhibit LdTOPIA and lead to R-loop formation and parasite elimination. This comprehensive study therefore paves an avenue for drug repurposing against Leishmania.

Impact of the Hydrocarbon Chain Length of Biodegradable Ester-Bonded Cationic Gemini Surfactants on Self-Assembly, In Vitro Gene Transfection, Cytotoxicity, and Antimicrobial Activity.

Gemini surfactants, due to their unique structural features and enhanced properties compared to conventional surfactants, are becoming more popular in the domain of colloid and interface science, drug delivery, and gene delivery science. This distinct class of surfactants forms a wide range of self-assembled aggregates depending on their chemical structure and environmental conditions. The present work aims to develop Gemini with three distinct chain lengths linked through the ester group and quaternary nitrogen head groups that can bind DNA molecules and ultimately serve as vectors for DNA transfection. Thus, we synthesized three distinct cationic Gemini with 12, 14, and 16 carbons in their tails and studied the effect of the hydrocarbon chain length on their physicochemical properties and biological applications. The self-assembly of these Geminis in aqueous solution was investigated by a number of techniques, including surface tension, electrical conductivity, fluorescence probe, calorimetry, dynamic light scattering, and atomic force microscopy. All three Gemini were extremely surface active and self-assembled above a very low critical micelle concentration. Calorimetric studies suggested the formation of thermodynamically favorable aggregates in an aqueous medium. Chain length dependence was observed in the size as well as the morphology of the aggregates. These Gemini ions were found to bind DNA strongly, as indicated by the high binding constant values. In vitro gene transfection studies using the RAW 264.7 cell line suggested that all three cationic Gemini had transfection efficiencies comparable to that of commercial standard turbofectamine. MTT assay was also performed for concentration selection while using these Gemini as transfection vectors. Overall, it was observed that Gemini had very little cytotoxicity within the investigated concentration range, highlighting the significance of the ester link within the structure. When compared with known antimicrobials such as kanamycin and ampicillin, all three Gemini furnished excellent antimicrobial activity in both Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) microorganisms.

Self-Assembly, In Vitro Gene Transfection, and Antimicrobial Activity of Biodegradable Cationic Bolaamphiphiles.

Bolaamphiphiles or bolaforms have drawn particular interest in drug and gene delivery, and studies of bolaforms have been growing continuously. Bolaforms, due to their unique structure, exhibit specific self-assembly behavior in water. The present work aims to develop biodegradable cationic bolaforms with a better gene transfection ability. In this work, a novel cationic bolaform (Bola-1) with head groups bearing hydroxyl (OH) functionality was designed and synthesized to investigate self-assembly and gene transfection efficiency. The self-assembly behavior of Bola-1 in water was compared with that of the hydrochloride salt (Bola-2) of its precursor molecule to investigate the effect of the -OH functionality on their solution properties. Several techniques, including surface tension, electrical conductivity, fluorescence probe, calorimetry, dynamic light scattering, and atomic force microscopy, were employed for the physicochemical characterization of Bola-1 and Bola-2. Despite the presence of polar urea groups in the spacer chain, both bolaforms were found to form spherical or elongated micelles above a relatively low critical aggregation concentration (CAC). The presence of the OH group was found to significantly affect the CAC value. The results of calorimetric measurements suggested a thermodynamically favorable aggregate formation in salt-free water. Despite stronger binding efficiency with calf thymus DNA, in vitro gene transfection studies performed using adherent cell Hek 293 suggested that both Bola-1 and Bola-2 have gene transfection efficiency comparable to that of turbofectamine standard. Both bolaforms were found to exhibit significant in vitro cytotoxicity at higher concentrations. Also, the bolaforms showed beneficial antibacterial activity at higher concentrations.

Impact of Linker Groups on Self-Assembly, Gene Transfection, Antibacterial Activity, and In Vitro Cytotoxicity of Cationic Bolaamphiphiles.

Cationic bolaamphiphiles have gained significant attention in various research fields, including materials science, drug delivery, and gene therapy, due to their unique properties and potential applications. The objective of the current research is to develop more effective cationic bolaamphiphiles. Thus, we have designed and synthesized two cationic bolaamphiphiles (-(CH2)12(2,3-dihydroxy-N,N-dimethyl-N-(3-ureidopropyl)propan-1-aminium chloride))2 (C12(DDUPPAC)2)) and (-(CH2)12(N-(3-(carbamoyloxy)propyl)-2,3-dihydroxy-N,N-dimethylpropan-1-aminium chloride)2 (C12(CPDDPAC)2) containing urea and urethane linkages, respectively. We have investigated their self-assembly properties in water using several techniques, including surface tension, electrical conductivity, fluorescence probe, calorimetry, dynamic light scattering, and atomic force microscopy. Their biological applications, e.g., in vitro gene transfection, antibacterial activity, and cytotoxicity, were studied. Both bolaamphiphiles were observed to produce aggregates larger than spherical micelles above a relatively low critical aggregation concentration (cac). The calorimetric experiments suggested the thermodynamically favorable spontaneous aggregation of both bolaforms in water. The results of interaction studies led to the conclusion that C12(CPDDPAC)2 binds DNA with a greater affinity than C12(DDUPPAC)2. Also, C12(CPDDPAC)2 is found to act as a more efficient gene transfection vector than C12(DDUPPAC)2 in 264.7 cell lines. The in vitro cytotoxicity assay using MTT, however, revealed that neither of the bolaamphiphiles was toxic, even at higher quantities. Additionally, both bolaforms show beneficial antibacterial activity.

A Bumpy Ride of Mycobacterial Phagosome Maturation: Roleplay of Coronin1 Through Cofilin1 and cAMP.

Phagosome-lysosome fusion in innate immune cells like macrophages and neutrophils marshal an essential role in eliminating intracellular microorganisms. In microbe-challenged macrophages, phagosome-lysosome fusion occurs 4 to 6 h after the phagocytic uptake of the microbe. However, live pathogenic mycobacteria hinder the transfer of phagosomes to lysosomes, up to 20 h post-phagocytic uptake. This period is required to evade pro-inflammatory response and upregulate the acid-stress tolerant proteins. The exact sequence of events through which mycobacteria retards phagolysosome formation remains an enigma. The macrophage coat protein Coronin1(Cor1) is recruited and retained by mycobacteria on the phagosome membrane to retard its maturation by hindering the access of phagosome maturation factors. Mycobacteria-infected macrophages exhibit an increased cAMP level, and based on receptor stimulus, Cor1 expressing cells show a higher level of cAMP than non-Cor1 expressing cells. Here we have shown that infection of bone marrow-derived macrophages with H37Rv causes a Cor1 dependent rise of intracellular cAMP levels at the vicinity of the phagosomes. This increased cAMP fuels cytoskeletal protein Cofilin1 to depolymerize F-actin around the mycobacteria-containing phagosome. Owing to reduced F-actin levels, the movement of the phagosome toward the lysosomes is hindered, thus contributing to the retarded phagosome maturation process. Additionally, Cor1 mediated upregulation of Cofilin1 also contributes to the prevention of phagosomal acidification, which further aids in the retardation of phagosome maturation. Overall, our study provides first-hand information on Cor1 mediated retardation of phagosome maturation, which can be utilized in developing novel peptidomimetics as part of host-directed therapeutics against tuberculosis.

ESX secretion system: The gatekeepers of mycobacterial survivability and pathogenesis.

Mycobacterium tuberculosis, the causative agent of Tuberculosis has plagued humankind for ages and has surfaced stronger than ever with the advent of drug resistance. Mycobacteria are adept at evading the host immune system and establishing infection by engaging host factors and secreting several virulence factors. Hence these secretion systems play a key role in mycobacterial pathogenesis. The type VII secretion system or ESX (early secretory antigenic target (ESAT6) secretion) system is one such crucial system that comprises five different pathways having distinct roles in mycobacterial proliferation, pathogenesis, cytosolic escape within macrophages, regulation of macrophage apoptosis, metal ion homeostasis, etc. ESX 1-5 systems are implicated in the secretion of a plethora of proteins, of which only a few are functionally characterized. Here we summarize the current knowledge of ESX secretion systems of mycobacteria with a special focus on ESX-1 and ESX-5 systems that subvert macrophage defenses and help mycobacteria to establish their niche within the macrophage.