Host Organelles Research Articles

Apicomplexan Espionage: Orchestrated Miscommunication at the Host-Parasite Interface.

Intracellular parasites, including Toxoplasma and Plasmodium, are entirely reliant on the active scavenging of host-derived nutrients to fuel their replicative cycle, as they are confined within a specialized membrane-bound compartment, the parasitophorous vacuole (PV). Initial observations, based on the proximity of host vesicles to the parasitophorous vacuole membrane (PVM), suggested that parasites utilize host vesicles to obtain essential nutrients. However, mounting evidence has now unequivocally demonstrated that intracellular pathogens establish membrane contacts with host organelles, establishing control over host cellular machinery. These intimate interactions enable the parasites to gain unimpeded access to cytosolic resources critical for development while evading host immune responses. This review consolidates the latest advancements in understanding the molecular machinery driving these transkingdom contacts and their functional roles. Further investigation into these processes promises to revolutionize our understanding of organelle communication, with profound implications for identifying new therapeutic targets and strategies.

Double infection of Nicotiana benthamiana with AMV and WCMV increases both virus concentrations and synergistically changes both host organelle ultrastructure and chlorophyll content

To clarify the synergistic pathogenic mechanism of Nicotiana benthamiana double infection with alfalfa mosaic virus (AMV) and white clover mosaic virus (WCMV), AMV and WCMV co-inoculation of N. benthamiana as treatment and single inoculation of AMV or WCMV and phosphate buffer solution (pH 7.0, PBS) as control, respectively. The concentrations and the relative expression of AMV and WCMV coat proteins were determined by a double antibody sandwich enzyme-linked immune sorbent assay (DAS-ELISA) and real-time fluorescence quantitative PCR (RT-qPCR) in a double infection of N. benthamiana with AMV and WCMV. Meanwhile, virion morphology, ultrastructure morphology, and chlorophyll content were observed and determined by electron microscopy. The results showed that the diseased symptoms were more serious, and virus concentration and relative expression of AMV and WCMV coat proteins were also higher in N. benthamiana double infection with AMV and WCMV than in AMV or WCMV single infection. The main symptoms manifested as severe mottle mosaic, shrinkage, and chlorosis. The concentrations of AMV and WCMV were 182.23pg/mL and 148.77pg/mL of double infection with AMV and WCMV, which were 1.75-fold and 1.62-fold than AMV and WCMV single infection, respectively. The relative expression of AMV and WCMV coat proteins was 4.25-fold and 2.50-fold than the single virus infection, respectively. Electron microscopy also observed that chloroplast malformation, cell membrane deformation, contents dissolution, grana lamella disorder, fat granules increased and enlarged, starch granules enlarged, and mitochondria were seriously malformed in a double infection of N. benthamiana with AMV and WCMV. The chlorophyll content was significantly lower for double infection with AMV and WCMV than for AMV or WCMV single-infected and CK, reduced by 31.52%, 22.83%, and 76.09%, respectively. This is the first report of a double infection of N. benthamiana with AMV and WCMV that increases both virus concentrations and synergistically changes both host organelle ultrastructure and chlorophyll content.

Positive-strand RNA virus replication organelles at a glance.

Membrane-bound replication organelles (ROs) are a unifying feature among diverse positive-strand RNA viruses. These compartments, formed as alterations of various host organelles, provide a protective niche for viral genome replication. Some ROs are characterised by a membrane-spanning pore formed by viral proteins. The RO membrane separates the interior from immune sensors in the cytoplasm. Recent advances in imaging techniques have revealed striking diversity in RO morphology and origin across virus families. Nevertheless, ROs share core features such as interactions with host proteins for their biogenesis and for lipid and energy transfer. The restructuring of host membranes for RO biogenesis and maintenance requires coordinated action of viral and host factors, including membrane-bending proteins, lipid-modifying enzymes and tethers for interorganellar contacts. In this Cell Science at a Glance article and the accompanying poster, we highlight ROs as a universal feature of positive-strand RNA viruses reliant on virus-host interplay, and we discuss ROs in the context of extensive research focusing on their potential as promising targets for antiviral therapies and their role as models for understanding fundamental principles of cell biology.

Transcriptional and ultrastructural changes of macrophages after african swine fever virus infection

African swine fever (ASF) is a highly impactful infectious disease in the swine industry, leading to substantial economic losses globally. The causative agent, African swine fever virus (ASFV), possesses intricate pathogenesis, warranting further exploration. In this study, we investigated the impact of ASFV infection on host gene transcription and organelle changes through macrophage transcriptome sequencing and ultrastructural transmission electron microscopy observation. According to the results of the transcriptome sequencing, ASFV infection led to significant alterations in the gene expression pattern of porcine bone marrow derived macrophages (BMDMs), with 2404 genes showing upregulation and 1579 genes downregulation. Cytokines, and chemokines were significant changes in the expression of BMDMs; there was significant activation of pattern recognition receptors such as Toll-like receptors and Nod-like receptors. According to the observation of the ultrastructure, mitochondrial damage and mitochondrial autophagy were widely present in ASFV-infected cells. The reduced number of macrophage pseudopodia suggested that virus-induced structural changes may compromise pathogen recognition, phagocytosis, and signal communication in macrophages. Additionally, the decreased size and inhibited acidification of secondary lysosomes in macrophages implied suppressed phagocytosis. Overall, ASFV infection resulted in significant changes in the expression of cytokines and chemokines, accompanied by the activation of NLR and TLR signaling pathways. We reported for the first time that ASFV infection led to a reduction in pseudopodia numbers and a decrease in the size and acidification of secondary lysosomes.

Correlative light-electron microscopy methods to characterize the ultrastructural features of the replicative and dormant liver stages of Plasmodium parasites

BackgroundThe infection of the liver by Plasmodium parasites is an obligatory step leading to malaria disease. Following hepatocyte invasion, parasites differentiate into replicative liver stage schizonts and, in the case of Plasmodium species causing relapsing malaria, into hypnozoites that can lie dormant for extended periods of time before activating. The liver stages of Plasmodium remain elusive because of technical challenges, including low infection rate. This has been hindering experimentations with well-established technologies, such as electron microscopy. A deeper understanding of hypnozoite biology could prove essential in the development of radical cure therapeutics against malaria.ResultsThe liver stages of the rodent parasite Plasmodium berghei, causing non-relapsing malaria, and the simian parasite Plasmodium cynomolgi, causing relapsing malaria, were characterized in human Huh7 cells or primary non-human primate hepatocytes using Correlative Light-Electron Microscopy (CLEM). Specifically, CLEM approaches that rely on GFP-expressing parasites (GFP-CLEM) or on an immunofluorescence assay (IFA-CLEM) were used for imaging liver stages. The results from P. berghei showed that host and parasite organelles can be identified and imaged at high resolution using both CLEM approaches. While IFA-CLEM was associated with more pronounced extraction of cellular content, samples’ features were generally well preserved. Using IFA-CLEM, a collection of micrographs was acquired for P. cynomolgi liver stage schizonts and hypnozoites, demonstrating the potential of this approach for characterizing the liver stages of Plasmodium species causing relapsing malaria.ConclusionsA CLEM approach that does not rely on parasites expressing genetically encoded tags was developed, therefore suitable for imaging the liver stages of Plasmodium species that lack established protocols to perform genetic engineering. This study also provides a dataset that characterizes the ultrastructural features of liver stage schizonts and hypnozoites from the simian parasite species P. cynomolgi.Graphical

Diurnal transcriptional variation is reduced in a nitrogen-fixing diatom endosymbiont.

Many organisms have formed symbiotic relationships with nitrogen (N)-fixing bacteria to overcome N limitation. Diatoms in the family Rhopalodiaceae host unicellular, N-fixing cyanobacterial endosymbionts called spheroid bodies (SBs). Although this relationship is relatively young, SBs share many key features with older endosymbionts, including coordinated cell division and genome reduction. Unlike free-living relatives that fix N exclusively at night, SBs fix N largely during the day; however, how SB metabolism is regulated and coordinated with the host is not yet understood. We compared four SB genomes, including those from two new host species (Rhopalodia gibba and Epithemia adnata), to build a genome-wide phylogeny which provides a better understanding of SB evolutionary origins. Contrary to models of endosymbiotic genome reduction, the SB chromosome is unusually stable for an endosymbiont genome, likely due to the early loss of all mobile elements. Transcriptomic data for the R. gibba SB and host organelles addressed whether and how the allocation of transcriptional resources depends on light and nitrogen availability. Although allocation to the SB was high under all conditions, relative expression of chloroplast photosynthesis genes increased in the absence of nitrate, but this pattern was suppressed by nitrate addition. SB expression of catabolism genes was generally greater during daytime rather than at night, although the magnitude of diurnal changes in expression was modest compared to free-living Cyanobacteria. We conclude that SB daytime catabolism likely supports N-fixation by linking the process to host photosynthetic carbon fixation.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) culture and sample preparation for correlative light electron microscopy.

Correlative Light Electron Microscopy (CLEM) is a powerful technique to investigate the ultrastructure of specific cells and organelles at sub-cellular resolution. Transmission Electron Microscopy (TEM) is particularly useful to the field of virology, given the small size of the virion, which is below the limit of detection by light microscopy. Furthermore, viral infection results in the rearrangement of host organelles to form spatially defined compartments that facilitate the replication of viruses. With the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), there has been great interest to study the viral replication complex using CLEM. In this chapter we provide an exemplary workflow describing the safe preparation and processing of cells grown on coverslips and infected with SARS-CoV-2.

Picornavirus 3C Proteins Intervene in Host Cell Processes through Proteolysis and Interactions with RNA.

The Picornaviridae family comprises a large group of non-enveloped viruses with enormous impact on human and animal health. The picornaviral genome contains one open reading frame encoding a single polyprotein that can be processed by viral proteases. The picornaviral 3C proteases share similar three-dimensional structures and play a significant role in the viral life cycle and virus-host interactions. Picornaviral 3C proteins also have conserved RNA-binding activities that contribute to the assembly of the viral RNA replication complex. The 3C protease is important for regulating the host cell response through the cleavage of critical host cell proteins, acting to selectively 'hijack' host factors involved in gene expression, promoting picornavirus replication, and inactivating key factors in innate immunity signaling pathways. The protease and RNA-binding activities of 3C are involved in viral polyprotein processing and the initiation of viral RNA synthesis. Most importantly, 3C modifies critical molecules in host organelles and maintains virus infection by subtly subverting host cell death through the blocking of transcription, translation, and nucleocytoplasmic trafficking to modulate cell physiology for viral replication. Here, we discuss the molecular mechanisms through which 3C mediates physiological processes involved in promoting virus infection, replication, and release.

Ultrastructure of the bacteriome and bacterial symbionts in the Asian citrus psyllid, Diaphorina citri.

Omics analyses suggested a mutually indispensable tripartite association among the host D. citri and organelle-like bacteriome associates, Carsonella and Profftella, which are vertically transmitted through host generations. This relationship is based on the metabolic complementarity among these organisms, which is partly enabled by horizontal gene transfer between partners. However, little was known about the fine morphology of the symbionts and the bacteriome, the interface among these organisms. As a first step to address this issue, the present study performed transmission electron microscopy, which revealed previously unrecognized ultrastructures, including aggregations of ribosomes in Carsonella, numerous tubes and occasional protrusions of Profftella, apparently degrading Profftella, and host organelles with different abundance and morphology in distinct cell types. These findings provide insights into the behaviors of the symbionts and host cells to maintain the symbiotic relationship in D. citri.

Quantifying spatial dynamics of Mycobacterium tuberculosis infection of human macrophages using microfabricated patterns

Macrophages provide a first line of defense against invading pathogens, including the leading cause of bacterial mortality, Mycobacterium tuberculosis (Mtb). A challenge for quantitative characterization of host-pathogen processes in differentially polarized primary human monocyte-derived macrophages (MDMs) is their heterogeneous morphology. Here, we describe the use of microfabricated patterns that constrain the size and shape of cells, mimicking the physiological spatial confinement cells experience in tissues, to quantitatively characterize interactions during and after phagocytosis at the single-cell level at high resolution. Comparing pro-inflammatory (M1) and anti-inflammatory (M2) MDMs, we find interferon-γ stimulation increases the phagocytic contraction, while contraction and bacterial uptake decrease following silencing of phagocytosis regulator NHLRC2 or bacterial surface lipid removal. We identify host organelle position alterations within infected MDMs and differences in Mtb subcellular localization in line with M1 and M2 cellular polarity. Our approach can be adapted to study other host-pathogen interactions and coupled with downstream automated analytical approaches.

Contact sites between host organelles and pathogens: boon or bane?

A microbe and its host are in constant communication. An emerging platform for direct communication is the membrane contact sites that form between several pathogens and host organelles. Here, we review our progress on the molecular mechanisms underlying contact sites between host mitochondria and the human parasite Toxoplasma gondii. We discuss open questions regarding their function during infection as well as those formed between the host endoplasmic reticulum and Toxoplasma.

Gold nanoparticles combat enveloped RNA virus by affecting organelle dynamics

Enveloped RNA viruses are a group of viruses with an outer membrane derived from a host cell and a genome consisting of ribonucleic acid (RNA). These viruses rely on host cell machinery and organelles to replicate and assemble new virus particles. However, the interaction between viruses and host organelles may be disrupted by nanomaterials, such as gold nanoparticles (AuNPs) with unique physical and chemical properties. In this study, we investigated the effects of AuNPs with different surface charge properties on the subcellular structure and function of mammalian cells, and their effects on two representative enveloped RNA viruses: lentivirus and human coronavirus OC43 (HCoV- OC43) antiviral potential. By comparing the subcellular effects of AuNPs with different surface charge properties, we found that treatment with AuNPs with positive surface charges induced more significant disruption of subcellular structures than neutrally charged AuNPs and negatively charged AuNPs, mainly manifested in lysosomes and Cytoskeletal disorders. The antiviral effect of the surface positively charged AuNPs was further evaluated using lentivirus and HCoV-OC43. The results showed that AuNPs had a significant inhibitory effect on both lentivirus and HCoV-OC43 without obvious side effects. In conclusion, our study provides insights into the mechanism of action and biocompatibility of AuNP in biological systems, while supporting the potential of targeting organelle dynamics against enveloped RNA viruses.

A Golgi-resident GPR108 cooperates with E3 ubiquitin ligase Smurf1 to suppress antiviral innate immunity

The regulation of antiviral immunity is crucial in maintaining host immune homeostasis, a process that involves dynamic modulations of host organelles. The Golgi apparatus is increasingly perceived as a host organelle functioning as a critical platform for innate immunity, but the detailed mechanism by which it regulates antiviral immunity remains elusive. Here, we identify the Golgi-localized G protein-coupled receptor 108 (GPR108) as a regulator of type Ι interferon responses by targeting interferon regulatory factor 3 (IRF3). Mechanistically, GPR108 enhances the ubiquitin ligase Smad ubiquitylation regulatory factor 1 (Smurf1)-mediated K63-linked polyubiquitination of phosphorylated IRF3 for nuclear dot 10 protein 52 (NDP52)-dependent autophagic degradation, leading to suppression of antiviral immune responses against DNA or RNA viruses. Taken together, our study provides insight into the crosstalk between the Golgi apparatus and antiviral immunity via a dynamic and spatiotemporal regulation of GPR108-Smurf1 axis, thereby indicating a potential target for treating viral infection.

Metatranscriptomic analysis uncovers prevalent viral ORFs compatible with mitochondrial translation.

RNA viruses are ubiquitous components of the global virosphere, yet relatively little is known about their genetic diversity or the cellular mechanisms by which they exploit the biology of their diverse eukaryotic hosts. A hallmark of (+)ssRNA (positive single-stranded RNA) viruses is the ability to remodel host endomembranes for their own replication. However, the subcellular interplay between RNA viruses and host organelles that harbor gene expression systems, such as mitochondria, is complex and poorly understood. Here we report the discovery of 763 new virus sequences belonging to the family Mitoviridae by metatranscriptomic analysis, the identification of previously uncharacterized mitovirus clades, and a putative new viral class. With this expanded understanding of the diversity of mitovirus and encoded RNA-dependent RNA polymerases (RdRps), we annotate mitovirus-specific protein motifs and identify hallmarks of mitochondrial translation, including mitochondrion-specific codons. This study expands the known diversity of mitochondrial viruses and provides additional evidence that they co-opt mitochondrial biology for their survival. IMPORTANCE Metatranscriptomic studies have rapidly expanded the cadre of known RNA viruses, yet our understanding of how these viruses navigate the cytoplasmic milieu of their hosts to survive remains poorly characterized. In this study, we identify and assemble 763 new viral sequences belonging to the Mitoviridae, a family of (+)ssRNA viruses thought to interact with and remodel host mitochondria. We exploit this genetic diversity to identify new clades of Mitoviridae, annotate clade-specific sequence motifs that distinguish the mitoviral RdRp, and reveal patterns of RdRp codon usage consistent with translation on host cell mitoribosomes. These results serve as a foundation for understanding how mitoviruses co-opt mitochondrial biology for their proliferation.

Phagocytosis underpins the biotrophic lifestyle of intracellular parasites in the class Phytomyxea (Rhizaria).

Phytomyxea are intracellular biotrophic parasites infecting plants and stramenopiles, including the agriculturally impactful Plasmodiophora brassicae and the brown seaweed pathogen Maullinia ectocarpii. They belong to the clade Rhizaria, where phagotrophy is the main mode of nutrition. Phagocytosis is a complex trait of eukaryotes, well documented for free-living unicellular eukaryotes and specific cellular types of animals. Data on phagocytosis in intracellular, biotrophic parasites are scant. Phagocytosis, where parts of the host cell are consumed at once, is seemingly at odds with intracellular biotrophy. Here we provide evidence that phagotrophy is part of the nutritional strategy of Phytomyxea, using morphological and genetic data (including a novel transcriptome of M. ectocarpii). We document intracellular phagocytosis in P. brassicae and M. ectocarpii by transmission electron microscopy and fluorescent insitu hybridization. Our investigations confirm molecular signatures of phagocytosis in Phytomyxea and hint at a small specialized subset of genes used for intracellular phagocytosis. Microscopic evidence confirms the existence of intracellular phagocytosis, which in Phytomyxea targets primarily host organelles. Phagocytosis seems to coexist with the manipulation of host physiology typical of biotrophic interactions. Our findings resolve long debated questions on the feeding behaviour of Phytomyxea, suggesting an unrecognized role for phagocytosis in biotrophic interactions.

Toxoplasma gondii scavenges mammalian host organelles through the usurpation of host ESCRT-III and Vps4A.

Intracellular pathogens exploit cellular resources through host cell manipulation. Within its nonfusogenic parasitophorous vacuole (PV), Toxoplasma gondii targets host nutrient-filled organelles and sequesters them into the PV through deep invaginations of the PV membrane (PVM) that ultimately detach from this membrane. Some of these invaginations are generated by an intravacuolar network (IVN) of parasite-derived tubules attached to the PVM. Here, we examined the usurpation of host ESCRT-III and Vps4A by the parasite to create PVM buds and vesicles. CHMP4B associated with the PVM/IVN, and dominant-negative (DN) CHMP4B formed many long PVM invaginations containing CHMP4B filaments. These invaginations were shorter in IVN-deficient parasites, suggesting cooperation between the IVN and ESCRT. In infected cells expressing Vps4A-DN, enlarged intra-PV structures containing host endolysosomes accumulated, reflecting defects in PVM scission. Parasite mutants lacking T. gondii (Tg)GRA14 or TgGRA64, which interact with ESCRT, reduced CHMP4B-DN-induced PVM invaginations and intra-PV host organelles, with greater defects in a double knockout, revealing the exploitation of ESCRT to scavenge host organelles by Toxoplasma.

First person – Julia Romano

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping researchers promote themselves alongside their papers. Julia Romano is first author on ‘ Toxoplasma gondii scavenges mammalian host organelles through the usurpation of host ESCRT-III and Vps4A’, published in JCS. Julia is a Senior Research Associate in the lab of Isabelle Coppens at Johns Hopkins University Bloomberg School of Public Health, Baltimore, USA, investigating how intracellular parasites interact with host cells to scavenge host materials such as lipids and vesicles.

Effectors from a Bacterial Vector-Borne Pathogen Exhibit Diverse Subcellular Localization, Expression Profiles, and Manipulation of Plant Defense.

Climate change is predicted to increase the prevalence of vector-borne disease due to expansion of insect populations. 'Candidatus Liberibacter solanacearum' is a phloem-limited pathogen associated with multiple economically important diseases in solanaceous crops. Little is known about the strategies and pathogenicity factors 'Ca. L. solanacearum' uses to colonize its vector and host. We determined the 'Ca. L. solanacearum' effector repertoire by predicting proteins secreted by the general secretory pathway across four different 'Ca. L. solanacearum' haplotypes, investigated effector localization in planta, and profiled effector expression in the vector and host. The localization of 'Ca. L. solanacearum' effectors in Nicotiana spp. revealed diverse eukaryotic subcellular targets. The majority of tested effectors were unable to suppress plant immune responses, indicating they possess unique activities. Expression profiling in tomato and the psyllid Bactericera cockerelli indicated 'Ca. L. solanacearum' differentially interacts with its host and vector and can switch effector expression in response to these environments. This study reveals 'Ca. L. solanacearum' effectors possess complex expression patterns, target diverse host organelles and the majority are unable to suppress host immune responses. A mechanistic understanding of 'Ca. L. solanacearum' effector function will reveal novel targets and provide insight into phloem biology. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Pseudomonas quinolone signal induces organelle stress and dysregulates inflammation in human macrophages

Pseudomonas quinolone signal (PQS) is a quorum-sensing molecule associated with Pseudomonas aeruginosa that regulates quorum sensing, extracellular vesicle biogenesis, iron acquisition, and the secretion of virulence factors. PQS has been shown to have immunomodulatory effects on the host. It induces oxidative stress, modulates cytokine levels, and activates regulated cell death in the host. In this study, we investigated the effects of PQS (10 μM) on host organelle dynamics and dysfunction in human macrophages at the interphase of endoplasmic reticulum (ER), mitochondria, and lysosome. This study showed that PQS increases cytosolic Ca+2 levels and elevates ER stress, as evidenced by increased expression of BiP and activation of the PERK-CHOP axis of unfolded protein response (UPR). Moreover, PQS also negatively affects mitochondria by disrupting mitochondrial membrane potential and increasing mitochondrial ROS generation (mROS). Additionally, PQS stimulation decreased the number of acridine orange-positive lysosomes, indicating lysosomal destabilization. Furthermore, PQS-induced lysosomal destabilization also induces overexpression of the lysosomal stress-responsive gene TFEB. Besides organelle dysfunction, PQS dysregulates inflammation-related genes by upregulating NLRC4, TMS1, and Caspase 1 while downregulating NLRP3 and IL-1β. Also, PQS increases gene expression of pro-inflammatory cytokines (IL-6, TNF-α, and IFN-γ). In conclusion, our findings suggest that PQS negatively affects human macrophages by interfering with organelle function and dysregulating inflammatory response. Consequently, this study provides crucial insight into PQS-driven macrophage dysfunction and may contribute to a better understanding of Pseudomonas aeruginosa-associated infections.

Role of C-terminal domain of Mycobacterium tuberculosis PE6 (Rv0335c) protein in host mitochondrial stress and macrophage apoptosis.

PE/PPE proteins of Mycobacterium tuberculosis (Mtb) target the host organelles to dictate the outcome of infection. This study investigated the significance of PE6/Rv0335c protein's unique C-terminal in causing host mitochondrial perturbations and apoptosis. In-silico analysis revealed that similar to eukaryotic apoptotic Bcl2 proteins, Rv0335c had disordered, hydrophobic C-terminal and two BH3-like motifs in which one was located at C-terminal. Also, Rv0335c’s N terminal had mitochondrial targeting sequence. Since, C-terminal of Bcl2 proteins are crucial for mitochondria targeting and apoptosis; it became relevant to evaluate the role of Rv0335c’s C-terminal domain in modulating host mitochondrial functions and apoptosis. To confirm this, in-vitro experiments were conducted with Rv0335c whole protein and Rv0335c∆Cterm (C-terminal domain deleted Rv0335c) protein. Rv0335c∆Cterm caused significant reduction in mitochondrial perturbations and Caspase-mediated apoptosis of THP1 macrophages in comparison to Rv0335c. However, the deletion of C-terminal domain didn’t affect Rv0335c’s ability to localize to mitochondria. Nine Ca2+ binding residues were predicted within Rv0335c and four of them were at the C-terminal. In-vitro studies confirmed that Rv0335c caused significant increase in intracellular calcium influx whereas Rv0335c∆Cterm had insignificant effect on Ca2+ influx. Rv0335c has been reported to be a TLR4 agonist and, we observed a significant reduction in the expression of TLR4-HLA-DR-TNF-α in response to Rv0335c∆Cterm protein also suggesting the role of Rv0335c’s C-terminal domain in host–pathogen interaction. These findings indicate the possibility of Rv0335c as a molecular mimic of eukaryotic Bcl2 proteins which equips it to cause host mitochondrial perturbations and apoptosis that may facilitate pathogen persistence.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10495-022-01778-1.