SARS-CoV-2 virulence factor ORF3a blocks lysosome function by modulating TBC1D5-dependent Rab7 GTPase cycle.

SARS-CoV-2, the causative agent of COVID-19, uses the host endolysosomal system for entry, replication, and egress. Previous studies have shown that the SARS-CoV-2 virulence factor ORF3a interacts with the lysosomal tethering factor HOPS complex and blocks HOPS-mediated late endosome and autophagosome fusion with lysosomes. Here, we report that SARS-CoV-2 infection leads to hyperactivation of the late endosomal and lysosomal small GTP-binding protein Rab7, which is dependent on ORF3a expression. We also observed Rab7 hyperactivation in naturally occurring ORF3a variants encoded by distinct SARS-CoV-2 variants. We found that ORF3a, in complex with Vps39, sequesters the Rab7 GAP TBC1D5 and displaces Rab7 from this complex. Thus, ORF3a disrupts the GTP hydrolysis cycle of Rab7, which is beneficial for viral production, whereas the Rab7 GDP-locked mutant strongly reduces viral replication. Hyperactivation of Rab7 in ORF3a-expressing cells impaired CI-M6PR retrieval from late endosomes to the trans-Golgi network, disrupting the biosynthetic transport of newly synthesized hydrolases to lysosomes. Furthermore, the tethering of the Rab7- and Arl8b-positive compartments was strikingly reduced upon ORF3a expression. As SARS-CoV-2 egress requires Arl8b, these findings suggest that ORF3a-mediated hyperactivation of Rab7 serves a multitude of functions, including blocking endolysosome formation, interrupting the transport of lysosomal hydrolases, and promoting viral egress.

SifA SUMOylation governs Salmonella Typhimurium intracellular survival via modulation of lysosomal function.

One of the mechanisms shaping the pathophysiology during the infection of enteric pathogen Salmonella Typhimurium is host PTM machinery utilization by the pathogen encoded effectors. Salmonella Typhimurium (S. Tm) during infection in host cells thrives in a vacuolated compartment, Salmonella containing vacuole (SCV), which sequentially acquires host endosomal and lysosomal markers. Long tubular structures, called as Salmonella induced filaments (SIFs), are further generated by S. Tm, which are known to be required for SCV's nutrient acquisition, membrane maintenance and stability. A tightly coordinated interaction involving prominent effector SifA and various host adapters PLEKHM1, PLEKHM2 and Rab GTPases govern SCV integrity and SIF formation. Here, we report for the first time that the functional regulation of SifA is modulated by PTM SUMOylation at its 11th lysine. S. Tm expressing SUMOylation deficient lysine 11 mutants of SifA (SifAK11R) is defective in intracellular proliferation due to compromised SIF formation and enhanced lysosomal acidification. Furthermore, murine competitive index experiments reveal defective in vivo proliferation and weakened virulence of SifAK11R mutant. Concisely, our data reveal that SifAK11R mutant nearly behaves like a SifA knockout strain which impacts Rab9-MPR mediated lysosomal acidification pathway, the outcome of which culminates in reduced bacterial load in in vitro and in vivo infection model systems. Our results bring forth a novel pathogen-host crosstalk mechanism where the SUMOylation of effector SifA regulated S. Tm intracellular survival.

RUFY1 binds Arl8b and mediates endosome-to-TGN CI-M6PR retrieval for cargo sorting to lysosomes.

Arl8b, an Arf-like GTP-binding protein, regulates cargo trafficking and positioning of lysosomes. However, it is unknown whether Arl8b regulates lysosomal cargo sorting. Here, we report that Arl8b binds to the Rab4 and Rab14 interaction partner, RUN and FYVE domain-containing protein (RUFY) 1, a known regulator of cargo sorting from recycling endosomes. Arl8b determines RUFY1 endosomal localization through regulating its interaction with Rab14. RUFY1 depletion led to a delay in CI-M6PR retrieval from endosomes to the TGN, resulting in impaired delivery of newly synthesized hydrolases to lysosomes. We identified the dynein-dynactin complex as an RUFY1 interaction partner, and similar to a subset of activating dynein adaptors, the coiled-coil region of RUFY1 was required for interaction with dynein and the ability to mediate dynein-dependent organelle clustering. Our findings suggest that Arl8b and RUFY1 play a novel role on recycling endosomes, from where this machinery regulates endosomes to TGN retrieval of CI-M6PR and, consequently, lysosomal cargo sorting.

Detection of SARS-CoV-2 in the air in Indian hospitals and houses of COVID-19 patients.

To understand the transmission characteristics of severe acute respiratory syndrome corona virus-2 (SARS-CoV-2) through air, samples from different locations occupied by coronavirus disease (COVID-19) patients were analyzed. Three sampling strategies were used to understand the presence of virus in the air in different environmental conditions. In the first strategy, which involved hospital settings, air samples were collected from several areas of hospitals like COVID-intensive-care units (ICUs), nurse-stations, COVID-wards, corridors, non-COVID-wards, personal protective equipment (PPE) doffing areas, COVID rooms, out-patient (OP) corridors, mortuary, COVID casualty areas, non-COVID ICUs and doctors' rooms. Out of the 80 air samples collected from 6 hospitals from two Indian cities- Hyderabad and Mohali, 30 samples showed the presence of SARS-CoV-2 nucleic acids. In the second sampling strategy, that involved indoor settings, one or more COVID-19 patients were asked to spend a short duration of time in a closed room. Out of 17 samples, 5 samples, including 4 samples collected after the departure of three symptomatic patients from the room, showed the presence of SARS-CoV-2 nucleic acids. In the third strategy, involving indoor settings, air samples were collected from rooms of houses of home-quarantined COVID-19 patients and it was observed that SARS-CoV-2 RNA could be detected in the air in the rooms occupied by COVID-19 patients but not in the other rooms of the houses. Taken together, we observed that the air around COVID-19 patients frequently showed the presence of SARS-CoV-2 RNA in both hospital and indoor residential settings and the positivity rate was higher when 2 or more COVID-19 patients occupied the room. In hospitals, SARS-CoV-2 RNA could be detected in ICUs as well as in non-ICUs, suggesting that the viral shedding happened irrespective of the severity of the infection. This study provides evidence for the viability of SARS-CoV-2 and its long-range transport through the air. Thus, airborne transmission could be a major mode of transmission for SARS-CoV-2 and appropriate precautions need to be followed to prevent the spread of infection through the air.

RUFY3 links Arl8b and JIP4-Dynein complex to regulate lysosome size and positioning.

The bidirectional movement of lysosomes on microtubule tracks regulates their whole-cell spatial arrangement. Arl8b, a small GTP-binding (G) protein, promotes lysosome anterograde trafficking mediated by kinesin-1. Herein, we report an Arl8b effector, RUFY3, which regulates the retrograde transport of lysosomes. We show that RUFY3 interacts with the JIP4-dynein-dynactin complex and facilitates Arl8b association with the retrograde motor complex. Accordingly, RUFY3 knockdown disrupts the positioning of Arl8b-positive endosomes and reduces Arl8b colocalization with Rab7-marked late endosomal compartments. Moreover, we find that RUFY3 regulates nutrient-dependent lysosome distribution, although autophagosome-lysosome fusion and autophagic cargo degradation are not impaired upon RUFY3 depletion. Interestingly, lysosome size is significantly reduced in RUFY3 depleted cells, which could be rescued by inhibition of the lysosome reformation regulatory factor PIKFYVE. These findings suggest a model in which the perinuclear cloud arrangement of lysosomes regulates both the positioning and size of these proteolytic compartments.

Methods for binding analysis of small GTP-binding proteins with their effectors.

Proteins often do not function as a single biomolecular entity; instead, they frequently interact with other proteins and biomolecules forming complexes. There is increasing evidence depicting the essentiality of protein-protein interactions (PPIs) governing a wide array of cellular processes. Thus, it is crucial to understand PPIs. Commonly used approaches like genetic (e.g., Yeast Two-Hybrid, Y2H), optical (e.g., Surface Plasmon Resonance, SPR; Fluorescence Resonance Energy Transfer, FRET), and biochemical have rendered ease in developing interactive protein maps as freely available information in protein databases on the web. The underlying basis of traditional protein interaction analysis is the core of biochemical methodologies providing direct evidence of interactions. Co-Immunoprecipitation (Co-IP) is a powerful biochemical technique that facilitates identifying novel interacting partners of a protein of interest in vivo, allowing specific capture of their complexes on an immunoglobulin. Here, using Arf-like (Arl) GTPase-8b (Arl8b) and Pleckstrin Homology Domain-Containing Family M Member 1 (PLEKHM1) as an example of small GTPase-effector pair, we provide a detailed protocol for performing Y2H and Co-IP assays to confirm the interaction between a small GTPase and its effector protein.

How to do business with lysosomes: Salmonella leads the way.

Pathogens have devised various strategies to alter the host endomembrane system towards building their replicative niche. This is aptly illustrated by Salmonella Typhimurium, whereby it remodels the host endolysosomal system to form a unique niche, also known as Salmonella-containing vacuole (SCV). Decades of research using in vitro cell-based infection studies have revealed intricate details of how Salmonella effectors target endocytic trafficking machinery of the host cell to acquire membrane and nutrients for bacterial replication. Unexpectedly, Salmonella requires host factors involved in endosome-lysosome fusion for its intravacuolar replication. Understanding how Salmonella obtains selective content from lysosomes, that is nutrients, but not active hydrolases, needs further exploration. Recent studies have described heterogeneity in the composition and pH of lysosomes, which will be highly relevant to explore, not only in the context of Salmonella infection, but also for other intracellular pathogens that interact with the endolysosomal pathway.

ARL11 regulates lipopolysaccharide-stimulated macrophage activation by promoting mitogen-activated protein kinase (MAPK) signaling.

ADP-ribosylation factor-like GTPase 11 (ARL11) is a cancer-predisposing gene that has remained functionally uncharacterized to date. In this study, we report that ARL11 is endogenously expressed in mouse and human macrophages and regulates their activation in response to lipopolysaccharide (LPS) stimulation. Accordingly, depletion of ARL11 impaired both LPS-stimulated pro-inflammatory cytokine production by macrophages and their ability to control intracellular replication of Salmonella. LPS-stimulated activation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) was substantially compromised in Arl11-silenced macrophages. In contrast, increased expression of ARL11 led to constitutive ERK1/2 phosphorylation, resulting in macrophage exhaustion. Finally, we found that ARL11 forms a complex with phospho-ERK in macrophages within minutes of LPS stimulation. Taken together, our findings establish ARL11 as a novel regulator of ERK signaling in macrophages, required for macrophage activation and immune function.

Lysosome-Mediated Plasma Membrane Repair Is Dependent on the Small GTPase Arl8b and Determines Cell Death Type in Mycobacterium tuberculosis Infection.

Mycobacterium tuberculosis is an extremely successful pathogen, and its success is widely attributed to its ability to manipulate the intracellular environment of macrophages. A central phenomenon of tuberculosis pathology enabling immune evasion is the capacity of virulent M. tuberculosis (H37Rv) to induce macrophage necrosis, which facilitates the escape of the mycobacteria from the macrophage and spread of infection. In contrast, avirulent M. tuberculosis (H37Ra) induces macrophage apoptosis, which permits Ag presentation and activation of adaptive immunity. Previously, we found that H37Rv induces plasma membrane microdisruptions, leading to necrosis in the absence of plasma membrane repair. In contrast, H37Ra permits plasma membrane repair, which changes the host cell death modality to apoptosis, suggesting that membrane repair is critical for sequestering the pathogen in apoptotic vesicles. However, mechanisms of plasma membrane repair induced in response to M. tuberculosis infection remain unknown. Plasma membrane repair is known to induce a Ca2+-mediated signaling, which recruits lysosomes to the area of damaged plasma membrane sites for its resealing. In this study, we found that the small GTPase Arl8b is required for plasma membrane repair by controlling the exocytosis of lysosomes in cell lines and in human primary macrophages. Importantly, we found that the Arl8b secretion pathway is crucial to control the type of cell death of the M. tuberculosis-infected macrophages. Indeed, Arl8b-depleted macrophages infected with avirulent H37Ra undergo necrotic instead of apoptotic cell death. These findings suggest that membrane repair mediated by Arl8b may be an important mechanism distinguishing avirulent from virulent M. tuberculosis-induced necrotic cell death.

Method for Studying the Effect of Gene Silencing on Bacterial Infection-induced ERK1/2 Signaling in Bone-marrow Derived Macrophages.

Macrophages are highly phagocytic cells that utilize various pathogen recognition receptors (PRRs) to recognize pathogen-associated molecular patterns (PAMPs). These PAMPs can be present within the microbe, such as bacterial CpG DNA, and are recognized by Toll-like receptor 9 (TLR9), a PRR present on the endosomal membrane of macrophages. PAMPs can also be present on the surface of microbes, such as Lipopolysaccharide (LPS), which decorates the outer membrane of gram-negative bacteria like Salmonella typhimurium and Escherichia coli. LPS is recognized by TLR4 present on the plasma membrane of macrophages, and LPS-TLR4 association leads to activation of signaling cascades including MAPK phosphorylation, which in turn promotes macrophage activation and microbial killing. This protocol describes the method for studying the role of a gene of interest in Extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) signaling, induced by bacterial infection in primary bone-marrow derived macrophages (BMDMs).

Salmonella exploits the host endolysosomal tethering factor HOPS complex to promote its intravacuolar replication.

Salmonella enterica serovar typhimurium extensively remodels the host late endocytic compartments to establish its vacuolar niche within the host cells conducive for its replication, also known as the Salmonella-containing vacuole (SCV). By maintaining a prolonged interaction with late endosomes and lysosomes of the host cells in the form of interconnected network of tubules (Salmonella-induced filaments or SIFs), Salmonella gains access to both membrane and fluid-phase cargo from these compartments. This is essential for maintaining SCV membrane integrity and for bacterial intravacuolar nutrition. Here, we have identified the multisubunit lysosomal tethering factor-HOPS (HOmotypic fusion and Protein Sorting) complex as a crucial host factor facilitating delivery of late endosomal and lysosomal content to SCVs, providing membrane for SIF formation, and nutrients for intravacuolar bacterial replication. Accordingly, depletion of HOPS subunits significantly reduced the bacterial load in non-phagocytic and phagocytic cells as well as in a mouse model of Salmonella infection. We found that Salmonella effector SifA in complex with its binding partner; SKIP, interacts with HOPS subunit Vps39 and mediates recruitment of this tethering factor to SCV compartments. The lysosomal small GTPase Arl8b that binds to, and promotes membrane localization of Vps41 (and other HOPS subunits) was also required for HOPS recruitment to SCVs and SIFs. Our findings suggest that Salmonella recruits the host late endosomal and lysosomal membrane fusion machinery to its vacuolar niche for access to host membrane and nutrients, ensuring its intracellular survival and replication.

The Rab7 effector PLEKHM1 binds Arl8b to promote cargo traffic to lysosomes.

Endocytic, autophagic, and phagocytic vesicles move on microtubule tracks to fuse with lysosomes. Small GTPases, such as Rab7 and Arl8b, recruit their downstream effectors to mediate this transport and fusion. However, the potential cross talk between these two GTPases is unclear. Here, we show that the Rab7 effector PLEKHM1 simultaneously binds Rab7 and Arl8b, bringing about clustering and fusion of late endosomes and lysosomes. We show that the N-terminal RUN domain of PLEKHM1 is necessary and sufficient for interaction with Arl8b and its subsequent localization to lysosomes. Notably, we also demonstrate that Arl8b mediates recruitment of HOPS complex to PLEKHM1-positive vesicle contact sites. Consequently, Arl8b binding to PLEKHM1 is required for its function in delivery and, therefore, degradation of endocytic and autophagic cargo in lysosomes. Finally, we also show that PLEKHM1 competes with SKIP for Arl8b binding, which dictates lysosome positioning. These findings suggest that Arl8b, along with its effectors, orchestrates lysosomal transport and fusion.

Amyloid precursor protein and amyloid precursor-like protein 2 in cancer.

Amyloid precursor protein (APP) and its family members amyloid precursor-like protein 1 (APLP1) and amyloid precursor-like protein 2 (APLP2) are type 1 transmembrane glycoproteins that are highly conserved across species. The transcriptional regulation of APP and APLP2 is similar but not identical, and the cleavage of both proteins is regulated by phosphorylation. APP has been implicated in Alzheimer's disease causation, and in addition to its importance in neurology, APP is deregulated in cancer cells. APLP2 is likewise overexpressed in cancer cells, and APLP2 and APP are linked to increased tumor cell proliferation, migration, and invasion. In this present review, we discuss the unfolding account of these APP family members' roles in cancer progression and metastasis.

MHC class II presentation is controlled by the lysosomal small GTPase, Arl8b.

Dendritic cells (DCs) are specialized APCs with the ability to prime naive T cells. DCs first sample Ags from the environment and then orchestrate their processing and loading onto MHC class II (MHC II) Ag-presenting molecules in lysosomes. Once MHC II molecules have bound a peptide, the MHC II-peptide complex is delivered to the cell surface for presentation to CD4(+) T cells. Regulation of Ag uptake via macropinocytosis and phagocytosis has been extensively studied, as well as trafficking in early endocytic vesicles notably regulated by the small GTPase Rab5 and its effectors. However, little is known about the regulators of Ag delivery from early endosomes to lysosomal compartments where the proper pH, proteases, MHC II, invariant chain, and HLA-DM reside, awaiting exogenous Ags for loading. In this article, we report the crucial role of the small GTPase ADP-ribosylation factor-like 8b (Arl8b) in MHC II presentation in DCs. We show for the first time, to our knowledge, that Arl8b localizes to MHC II compartments in DCs and regulates formation of MHC II-peptide complexes. Arl8b-silenced DCs display a defect in MHC II-Ag complex formation and its delivery to the cell surface during infection resulting in a defect in T cell recognition. Our results highlight the role of Arl8b as a trafficking regulator of the late stage of complex formation and MHC II presentation in DCs.

Arf-like GTPase Arl8b regulates lytic granule polarization and natural killer cell-mediated cytotoxicity.

Natural killer (NK) lymphocytes contain lysosome-related organelles (LROs), known as lytic granules, which upon formation of immune synapse with the target cell, polarize toward the immune synapse to deliver their contents to the target cell membrane. Here, we identify a small GTP-binding protein, ADP-ribosylation factor-like 8b (Arl8b), as a critical factor required for NK cell-mediated cytotoxicity. Our findings indicate that Arl8b drives the polarization of lytic granules and microtubule-organizing centers (MTOCs) toward the immune synapse between effector NK lymphocytes and target cells. Using a glutathione S-transferase pull-down approach, we identify kinesin family member 5B (KIF5B; the heavy chain of kinesin-1) as an interaction partner of Arl8b from NK cell lysates. Previous studies showed that interaction between kinesin-1 and Arl8b is mediated by SifA and kinesin-interacting protein (SKIP) and the tripartite complex drives the anterograde movement of lysosomes. Silencing of both KIF5B and SKIP in NK cells, similar to Arl8b, led to failure of MTOC-lytic granule polarization to the immune synapse, suggesting that Arl8b and kinesin-1 together control this critical step in NK cell cytotoxicity.

Relevance of amyloid precursor-like protein 2 C-terminal fragments in pancreatic cancer cells.

In some cellular systems, particularly neurons, amyloid precursor-like protein 2 (APLP2), and its highly homologous family member amyloid precursor protein (APP), have been linked to cellular growth. APLP2 and APP undergo regulated intramembrane proteolysis to produce C-terminal fragments. In this study, we found comprehensive expression of APLP2 C-terminal fragments in a panel of pancreatic cancer cell lines; however, APP C-terminal fragments were notably limited to the BxPC3 cell line. Extensive glycosaminoglycan modification on APLP2 was also found in the majority of pancreatic cancer cell lines. Glycosaminoglycan-modified and -unmodified APLP2, and particularly APLP2 C-terminal fragments, also demonstrated increased expression in oncogene-transformed pancreatic ductal cells. Additionally, elevated APLP2 levels were confirmed in human pancreatic cancer tissue. Downregulation of APLP2 and APP expression, alone or in combination, caused a decrease in the growth of a pancreatic cancer cell line with representatively low APP C-terminal fragment expression, the S2-013 cell line. Furthermore, we found that treatment with ß-secretase inhibitors to block formation of APLP2 C-terminal fragments decreased the growth and viability of S2-013 cells, without affecting the survival of a non-transformed pancreatic ductal cell line. In conclusion, our studies demonstrate that abundant APLP2, but not APP, C-terminal fragment expression is conserved in pancreatic cancer cell lines; however, APP and APLP2 equally regulated the growth of S2-013 pancreatic cancer cells. Chiefly, our discoveries establish a role for APLP2 in the growth of pancreatic cancer cells and show that inhibitors preventing APLP2 cleavage reduce the viability of pancreatic cancer cells.

Invariant natural killer T cells recognize lipid self antigen induced by microbial danger signals.

Invariant natural killer T cells (iNKT cells) have a prominent role during infection and other inflammatory processes, and these cells can be activated through their T cell antigen receptors by microbial lipid antigens. However, increasing evidence shows that they are also activated in situations in which foreign lipid antigens would not be present, which suggests a role for lipid self antigen. We found that an abundant endogenous lipid, ß-D-glucopyranosylceramide (ß-GlcCer), was a potent iNKT cell self antigen in mouse and human and that its activity depended on the composition of the N-acyl chain. Furthermore, ß-GlcCer accumulated during infection and in response to Toll-like receptor agonists, contributing to iNKT cell activation. Thus, we propose that recognition of ß-GlcCer by the invariant T cell antigen receptor translates innate danger signals into iNKT cell activation.

Regulation of major histocompatibility complex class I molecule expression on cancer cells by amyloid precursor-like protein 2.

The three members of the amyloid precursor protein family in mammals [amyloid precursor protein, amyloid precursor-like protein 1, and amyloid precursor-like protein 2 (APLP2)] have been implicated in a large array of intracellular processes, which include development, transcription, apoptosis, metabolism, and the cell cycle. A series of studies by our laboratories has demonstrated that APLP2 is highly expressed by many cancer cell lines (with the highest expression in pancreatic cancer cell lines) and that it facilitates major histocompatibility complex (MHC) class I molecule endocytosis. This review focuses on this recently revealed function of APLP2 relevant to tumor immunology: that it acts as a novel regulator of MHC class I molecule surface expression.

Lysosomal trafficking, antigen presentation, and microbial killing are controlled by the Arf-like GTPase Arl8b.

Antigen presentation and microbial killing are critical arms of host defense that depend upon cargo trafficking into lysosomes. Yet, the molecular regulators of traffic into lysosomes are only partly understood. Here, using a lysosome-dependent immunological screen of a trafficking shRNA library, we identified the Arf-like GTPase Arl8b as a critical regulator of cargo delivery to lysosomes. Homotypic fusion and vacuole protein sorting (HOPS) complex members were identified as effectors of Arl8b and were dependent on Arl8b for recruitment to lysosomes, suggesting that Arl8b-HOPS plays a general role in directing traffic to lysosomes. Moreover, the formation of CD1 antigen-presenting complexes in lysosomes, their delivery to the plasma membrane, and phagosome-lysosome fusion were all markedly impaired in Arl8b silenced cells resulting in corresponding defects in T cell activation and microbial killing. Together, these results define Arl8b as a key regulator of lysosomal cellular and immunological functions.

Effect of a tapasin mutant on the assembly of the mouse MHC class I molecule H2-K(d).

Major histocompatibility complex (MHC) class I heavy chain/beta(2)m heterodimers assemble with antigenic peptides through interactions with peptide-loading complex proteins, including tapasin and ERp57. In human cells, a cysteine residue within tapasin (C95) has been shown to form a covalent bond with ERp57. In this study, we focused on the effect of this tapasin amino-acid residue in mouse cells expressing the MHC class I molecule H2-K(d). We showed that a large disulfide-bonded complex was present in the mouse cells that included ERp57, tapasin, and K(d). Furthermore, in mouse cells, unlike human cells, we found that tapasin mutated at C95 can participate in a non-covalent complex with ERp57. Comparison of our findings to earlier findings with a human molecule (HLA-B(*)4402) also revealed that a tapasin C95 mutation has a stronger effect on the maturation and stability of K(d) than HLA-B(*)4402. Overall, our results characterize the influence of this tapasin cysteine residue on the stable surface expression of a mouse MHC class I molecule and reveal differences in tapasin C95 interactions and effects between mouse and human systems.