Legionella uses host Rab GTPases and BAP31 to create a unique ER niche.

Upon entry into host cells, the facultative intracellular bacterium Legionella pneumophila ( L.p .) uses its type IV secretion system, Dot/Icm, to secrete ~330 bacterial effector proteins into the host cell. Some of these effectors hijack endoplasmic reticulum (ER)-derived vesicles to form the Legionella -containing vacuole (LCV). Despite extensive investigation over decades, the fundamental question persists: Is the LCV membrane distinct from or contiguous with the host ER network? Here, we employ advanced photobleaching techniques, revealing a temporal acquisition of both smooth and rough ER (sER and rER) markers on the LCV. In the early stages of infection, the sER intimately associates with the LCV. Remarkably, as the infection progresses, the LCV evolves into a distinct niche comprising an rER membrane that is independent of the host ER network. We discover that the L.p. effector LidA binds to and recruits two host proteins of the Rab superfamily, Rab10, and Rab4, that play significant roles in acquiring sER and rER membranes, respectively. Additionally, we identify the pivotal role of a host ER-resident protein, BAP31, in orchestrating the transition from sER to rER. While previously recognized for shuttling between sER and rER, we demonstrate BAP31's role as a Rab effector, mediating communication between these ER sub-compartments. Furthermore, using genomic deletion strains, we uncover a novel L.p. effector, Lpg1152, essential for recruiting BAP31 to the LCV and facilitating its transition from sER to rER. Depletion of BAP31 or infection with an isogenic L.p. strain lacking Lpg1152 results in a growth defect. Collectively, our findings illuminate the intricate interplay between molecular players from both host and pathogen, elucidating how L.p. orchestrates the transformation of its residing vacuole membrane from a host-associated sER to a distinct rER membrane that is not contiguous with the host ER network.

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.

Gelatin-decorated Graphene oxide: A nanocarrier for delivering pH-responsive drug for improving therapeutic efficacy against atherosclerotic plaque.

The progressive inflammatory disease atherosclerosis promotes myocardial infarction, stroke, and heart attack. Anti-inflammatory drugs treat severe atherosclerosis. They are inadequate bioavailability and cause adverse effects at higher doses. A new nanomaterial coupled pH-apperceptive drug delivery system for atherosclerotic plaque is outlined here. We have synthesized a Graphene Oxide-Gelatin-Atorvastatin (GO-Gel-ATR) nanodrug characterized by spectroscopic and imaging techniques. The encapsulation efficiency of GO-Gel-ATR (79.2%) in the loading process is observed to be better than GO-ATR (66.8%). The internal milieu of the plaque cells has a pH of 6.8. The GO-Gel-ATR displays sustained and cumulative release profile at pH 6.8 compared to ATR and GO-ATR. Our proposed nanocomposite demonstrated high cytocompatibility up to 100µg/mL in foam cells induced by Oxidized-Low Density Lipoprotein (Ox-LDL) and Lipopolysaccharides (LPS) compared to normal macrophages for 24 and 48 h. The uptake efficacy of the nanodrugs is shown to be enhanced in foam cells compared to normal macrophage. Oil red O staining of foam cells with and without drugs confirmed therapeutic efficacy. Foam cells treated with nanocomposite had more lipids efflux than ATR. The finding of the in-vitro study reveals that the GO-Gel-ATR nanocomposite carriers have the potential to deliver anti-atherosclerotic drugs effectively and inhibit atherosclerotic plaque progression.

Putative staphylococcal enterotoxin possesses two common structural motifs for MHC-II binding.

Staphylococcus aureus has become a significant cause of health risks in humankind. Staphylococcal superantigens (SAgs) or enterotoxins are the key virulent factors that can exhibit acute diseases to severe life-threatening conditions. Recent literature reports S. aureus has steadily gained new enterotoxin genes over the past few decades. In spite of current knowledge of the established SAgs, several questions on putative enterotoxins are still remaining unanswered. Keeping that in mind, this study sheds light on a putative enterotoxin SEl26 to characterize its structural and functional properties. In-silico analyses indicate its close relation with the conventional SAgs, especially the zinc-binding SAgs. Additionally, important residues that are vital for the T-cell receptor (TcR) and major histocompatibility complex class II (MHC-II) interaction were predicted and compared with established SAgs. Besides, our biochemical analyses exhibited the binding of this putative enterotoxin with MHC-II, followed by regulating pro-inflammatory and anti-inflammatory cytokines.

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.

The ultimate fate determinants of drug induced cell-death mechanisms in Trypanosomatids.

Chemotherapy constitutes a major part of modern-day therapy for infectious and chronic diseases. A drug is said to be effective if it can inhibit its target, induce stress, and thereby trigger an array of cell death pathways in the form of programmed cell death, autophagy, necrosis, etc. Chemotherapy is the only treatment choice against trypanosomatid diseases like Leishmaniasis, Chagas disease, and sleeping sickness. Anti-trypanosomatid drugs can induce various cell death phenotypes depending upon the drug dose and growth stage of the parasites. The mechanisms and pathways triggering cell death in Trypanosomatids serve to help identify potential targets for the development of effective anti-trypanosomatids. Studies show that the key proteins involved in cell death of trypanosomatids are metacaspases, Endonuclease G, Apoptosis-Inducing Factor, cysteine proteases, serine proteases, antioxidant systems, etc. Unlike higher eukaryotes, these organisms either lack the complete set of effectors involved in cell death pathways, or are yet to be deciphered. A detailed summary of the existing knowledge of different drug-induced cell death pathways would help identify the lacuna in each of these pathways and therefore open new avenues for research and thereby new therapeutic targets to explore. The cell death pathway associated complexities in metazoans are absent in trypanosomatids; hence this summary can also help understand the trigger points as well as cross-talk between these pathways. Here we provide an in-depth overview of the existing knowledge of these drug-induced trypanosomatid cell death pathways, describe their associated physiological changes, and suggest potential interconnections amongst them.

"It Takes Two to Tango": Role of Neglected Macrophage Manipulators Coronin 1 and Protein Kinase G in Mycobacterial Pathogenesis.

Macrophages being the connecting link between innate and adaptive immune system plays a crucial role in microbial antigen presentation and orchestrates the subsequent clearance of microorganisms. Microbial invasion of macrophages trigger a plethora of signaling cascades, which interact among them to generate a dynamically altered hostile environment, that ultimately leads to disruption of microbial pathogenesis. Paradoxically, Mycobacterium sp. exploits macrophage proteins such as Coronin 1, Calcineurin, LRG47, SOCS1, CISH, Gbp5 etc. and secretes virulence proteins such as PknG, PtpA, SapM, Eis etc. to hijack these intra-macrophage, signaling cascades and thereby develop its own niche. Coronin 1, being a cortical protein is transiently recruited to all mycobacteria containing phagosomes, but only pathogenic mycobacteria can retain it on the phagosome, to hinder its maturation. Additionally, mycobacterial infection linked secretion of virulence factor Protein Kinase G through its phosphorylation, manipulates several macrophage signaling pathways and thus promotes pathogenesis at various stages, form early infection to latency to granuloma formation. Here we discuss the present status of mycobacteria engaged Coronin 1-dependent signaling cascades and secreted PknG related sequence of events promoting mycobacterial pathogenesis. Current knowledge about these two proteins in context of macrophage signaling manipulation encompassing diverse mechanisms like calcium-calcineurin signaling, reduced proinflamtory cytokine secretion, cytoskeletal changes, and adaptation in acidic environment, which ultimately converge toward mycobacterial survival inside the macrophages has been discussed.