John Brumell

• PhD
• Senior Researcher at SickKids

Salmonella utilizes type 3 secreted effector proteins to induce plasma membrane (PM) perturbations during invasion of host cells¹. The effectors drive mobilization of host membranes to generate cell surface ruffles, followed by invagination and scission of the PM to generate Salmonella-containing vacuoles (SCVs)². Here, we show that LRRK2 kinase ge...

Peroxisomes are essential for mitochondrial health, as the absence of peroxisomes leads to altered mitochondria. However, it is unclear whether the changes in mitochondria are a function of preserving cellular function or a response to cellular damage caused by the absence of peroxisomes. To address this, we developed conditional hepatocyte-specifi...

SLIT/ROBO signaling impacts many aspects of tissue development and homeostasis, in part, through the regulation of cell growth and proliferation. Recent studies have also linked SLIT/ROBO signaling to the regulation of diverse phagocyte functions. However, the mechanisms by which SLIT/ROBO signaling acts at the nexus of cellular growth control and...

[This corrects the article DOI: 10.1016/j.isci.2022.105188.].

Cell proliferation is dependent on growth factors insulin and IGF1. We sought to identify interactors of IRS1, the most proximal mediator of insulin/IGF1 signaling, that regulate cell proliferation. Using proximity-dependent biotin identification (BioID) we detected 40 proteins displaying proximal interactions with IRS1, including DCAF7 and its int...

Endothelia determine blood-to-tissue solute delivery, yet glucose transit is poorly understood. To illuminate mechanisms, we tracked [3H]-2-deoxyglucose (2-DG) in human adipose-tissue microvascular endothelial cells. 2-DG uptake was largely facilitated by glucose transporters GLUT1 and GLUT3. Once in the cytosol, >80% of 2-DG became phosphorylated...

Non-canonical autophagy is a key cellular pathway in immunity, cancer, and neurodegeneration, characterized by conjugation of ATG8 to endolysosomal single membranes (CASM). CASM is activated by engulfment (endocytosis, phagocytosis), agonists (STING, TRPML1), and infection (influenza), dependent on K490 in the ATG16L1 WD40-domain. However, factors...

Despite their low abundance, phosphoinositides play a central role in membrane traffic and signalling. PtdIns(3,4,5)P3 and PtdIns(3,4)P2 are uniquely important, as they promote cell growth, survival and migration. Pathogenic organisms have developed means to subvert phosphoinositide metabolism to promote successful infection and their survival in h...

Adaptive changes in lysosomal capacity are driven by the transcription factors TFEB and TFE3 in response to increased autophagic flux and endolysosomal stress, yet the molecular details of their activation are unclear. LC3 and GABARAP members of the ATG8 protein family are required for selective autophagy and sensing perturbation within the endolys...

Macroautophagy is a highly conserved eukaryotic cellular pathway involving the engulfment of macromolecules, organelles, and invading microbes by a double-membrane compartment and subsequent lysosomal degradation. The mechanisms that regulate macroautophagy, and the interaction of its components with other cellular pathways, have remained unclear....

Depolarized mitochondria can be degraded via mitophagy, a selective form of autophagy. The RAB GTPase RAB7A was recently shown to play a key role in this process. RAB7A regulates late endocytic trafficking under normal growth conditions but is translocated to the mitochondrial surface following depolarization. However, how RAB7A activity is regulat...

The type I interferon (IFN) signaling pathway has important functions in resistance to viral infection, with the downstream induction of interferon stimulated genes (ISG) protecting the host from virus entry, replication and spread. Listeria monocytogenes ( Lm ), a facultative intracellular foodborne pathogen, can exploit the type I IFN response as...

Salmonella utilizes translocated virulence proteins (termed effectors) to promote host cell invasion. The effector SopD contributes to invasion by promoting scission of the plasma membrane, generating Salmonella-containing vacuoles. SopD is expressed in all Salmonella lineages and plays important roles in animal models of infection, but its host ce...

Insulin is a paramount anabolic hormone that promotes energy-storage in adipose tissue, skeletal muscle and liver, and these responses are significantly attenuated in insulin resistance leading to type 2 diabetes. Contrasting with insulin’s function, macroautophagy/autophagy is a physiological mechanism geared to the degradation of intracellular co...

Despite their comparatively low abundance, phosphoinositides play a central role in membrane traffic and signalling. PtdIns(3,4,5)P3 and PtdIns(3,4)P2 are uniquely important, as they promote cell growth, survival, and migration. Pathogenic organisms have developed means to subvert phosphoinositide metabolism to promote successful infection and thei...

Despite their comparatively low abundance, phosphoinositides play a central role in membrane traffic and signalling. PtdIns(3,4,5)P3 and PtdIns(3,4)P2 are uniquely important, as they promote cell growth, survival, and migration. Pathogenic organisms have developed means to subvert phosphoinositide metabolism to promote successful infection and thei...

Non-canonical autophagy is a key cellular pathway in immunity, cancer and neurodegeneration, characterised by Conjugation of ATG8 to endolysosomal Single-Membranes (CASM). CASM is activated by engulfment (endocytosis, phagocytosis), agonists (STING, TRPML1) and infection (influenza), dependent on the ATG16L1 WD40-domain, and specifically K490. Howe...

Plasma membrane integrity is essential for cellular homeostasis. In vivo, cells experience plasma membrane damage from a multitude of stressors in the extra- and intra-cellular environment. To avoid lethal consequences, cells are equipped with repair pathways to restore membrane integrity. Here, we assess plasma membrane damage and repair from a wh...

Rab5 is required for macropinosome formation, but its site and mode of action remain unknown. We report that Rab5 acts at the plasma membrane, downstream of ruffling, to promote macropinosome sealing and scission. Dominant-negative Rab5, which obliterates macropinocytosis, had no effect on the development of membrane ruffles. However, Rab5-containi...

Increased autophagic flux and endolysosomal stress coordinates lysosomal capacity through the TFEB/TFE3 transcription factors, yet the molecular details of their activation are unclear. LC3 and GABARAP members of the ATG8 protein family are required for selective autophagy and sensing perturbation within the endolysosomal system. Here we show that...

The NOX2 NADPH oxidase (NOX2) produces reactive oxygen species to kill phagosome-confined bacteria. However, we previously showed that Listeria monocytogenes is able to avoid the NOX2 activity in phagosomes and escape to the cytosol. Thus, despite the established role of NOX2 limiting L. monocytogenes infection in mice, the underlying mechanisms of...

A bstract Rab5 is required for macropinosome formation, but its site and mode of action remain unknown. We report that Rab5 acts at the plasma membrane, downstream of ruffling, to promote macropinosome sealing and scission. Dominant-negative Rab5, which obliterates macropinocytosis, had no effect on the development of membrane ruffles. However, Rab...

Many bacterial pathogens express virulence proteins that are translocated into host cells (herein referred to as effectors), where they can interact with target proteins to manipulate host cell processes. These effector–host protein interactions are often dynamic and transient in nature, making them difficult to identify using traditional interacti...

NODs require S-palmitoylation to signal The compartmentalization of proteins within the cell is essential for their function. The addition of lipid molecules redistributes proteins to the cell surface or to membrane-bound organelles. Working in transgenic mice and in tissue cultured cells, Lu et al. found that nucleotide oligomerization domain–like...

Insulin resistance is a defining feature of type 2 diabetes, yet our understanding of the progression and development of insulin resistance is incomplete. Recently, deficient autophagy, a bulk degradation pathway, was associated with the induction of insulin resistance, although the causative mechanism remains unknown. We sought to investigate the...

There is growing evidence in the literature for unconventional roles of autophagy-related (ATG) proteins, outside of their function in canonical autophagy. Here we discuss our recent study that revealed a novel ATG16L1-dependent pathway that promotes plasma membrane repair upon bacterial pore-forming toxin damage. Disruption of the ATG16L1-dependen...

Supplemental Figure 2. Knockdown of Septin expression with siRNA in HeLa and Henle 407 cells. HeLa and Henle 407 cells were transfected with SEPTIN7 siRNA pools. Each siRNA pool is made of two independent siRNA's targeting SEPTIN7. (A) 48 h post‐siRNA transfection, cells were infected with S. Typhimurium for 30 min. Differential antibody staining w...

Supplemental Figure 1. Knockdown of SEPTIN2, 7 and 9 expression with siRNA. HeLa cells were transfected with the indicated siRNA. 48 h post siRNA transfection, cells were lysed and lysates were prepared as described in Methods and Experimental Procedures. (A) HeLa cells were transfected with SEPTIN2 or SEPTIN9 siRNA. Lysates were probed with antibo...

Supplemental Figure 3. Recruitment of endogenous SEPTIN2 and 9 to Salmonella invasion ruffles in SEPTIN7 depleted cells. HeLa cells were transfected with the indicated siRNA and 48 h post siRNA transfection septin recruitment to the invasion ruffle was assessed in HeLa cells. HeLa cells were infected with S. Typhimurium and fixed 10 min post‐invasi...

Supplemental Movie 1. Dynamics of S. Typhimurium invasion sites in control siRNA treated cells. HeLa cells were transfected with control siRNA for 24 h and then transfected with RFP‐LifeAct for 24 h. Cells were then infected with S. Typhimurium. Invasion was recorded in live cells using spinning‐disk confocal microscopy

Supplemental Movie 2. Dynamics of S. Typhimurium invasion sites in SEPT7 siRNA treated cells. HeLa cells were transfected with SEPTIN7 siRNA for 24 h and then transfected with RFP‐LifeAct for 24 h. Cells were then infected with S. Typhimurium. Invasion was recorded in live cells using spinning‐disk confocal microscopy

Salmonella uses type 3 secretion systems (T3SS) to deliver virulence factors, called effectors, into host cells during infection. The T3SS effectors promote invasion into host cells and the generation of a replicative niche. SopB is a T3SS effector that plays an important role in Salmonella pathogenesis through its lipid phosphatase activity. Here...

Autophagy is a degradation pathway persisting in all cells at basal levels and further activated by various stimuli, such as starvation. This cellular process is mediated by autophagy‐related (Atg) proteins, which govern cargo recognition and formation of a nascent organelle called the autophagosome. Several Atg proteins have been identified, inclu...

Listeria monocytogenes is a facultative intracellular bacterial pathogen that is frequently associated with food-borne infection. Of particular concern is the ability of L. monocytogenes to breach the blood-brain barrier, leading to life-threatening meningitis and encephalitis. The mechanisms used by bacterial pathogens to infect the brain are not...

Listeria monocytogenes (Lm) is a Gram-positive facultative intracellular pathogen. Infections in humans can lead to listeriosis, a systemic disease with a high mortality rate. One important mechanism ofLmdissemination involves cell-to-cell spread after bacteria have entered the cytosol of host cells. Listeriolysin O (LLO; encoded by thehlygene) is...

Human actin-related protein 2/3 complex (Arp2/3), required for actin filament branching, has two ARPC1 component isoforms, with ARPC1B prominently expressed in blood cells. Here we show in a child with microthrombocytopenia, eosinophilia and inflammatory disease, a homozygous frameshift mutation in ARPC1B (p.Val91Trpfs*30). Platelet lysates reveal...

Supplementary Figures, Supplementary Tables, Supplementary Notes and Supplementary Reference.

Whole exome sequencing (WES) data for patients.

Type I interferons (IFNs) play a critical role in anti-viral immune responses, but can be deleterious to the host during some bacterial infections. Listeria monocytogenes (Lm) induces a type I IFN response by activating cytosolic anti-viral surveillance pathways. This is beneficial to the bacteria as mice lacking the type I IFN receptor (IFNAR1-/-)...

Phagocytosis plays a central role in immunity and tissue homeostasis. After internalization of cargo into single-membrane phagosomes, these compartments undergo a maturation sequences that terminates in lysosome fusion and cargo degradation. Components of the autophagy pathway have recently been linked to phagosome maturation in a process called LC...

Background & aims: Severe forms of inflammatory bowel disease (IBD) that develop in very young children can be caused by variants in a single gene. We performed whole-exome sequence (WES) analysis to identify genetic factors that might cause granulomatous colitis and severe perianal disease, with recurrent bacterial and viral infections, in an inf...

Intracellular bacterial pathogens of a diverse nature share the ability to evade host immunity by impairing trafficking of endocytic cargo to lysosomes for degradation, a process that is poorly understood. Here, we show that the Salmonella enterica type 3 secreted effector SopD2 mediates this process by binding the host regulatory GTPase Rab7 and i...

Efficient acquisition of extracellular nutrients is essential for bacterial pathogenesis, however the identities and mechanisms for transport of many of these substrates remain unclear. Here, we investigate the predicted iron-binding transporter AfuABC and its role in bacterial pathogenesis in vivo. By crystallographic, biophysical and in vivo appr...

Defects in intestinal innate defense systems predispose patients to inflammatory bowel disease (IBD). Reactive oxygen species (ROS) generated by nicotinamide-adenine dinucleotide phosphate (NADPH) oxidases in the mucosal barrier maintain gut homeostasis and defend against pathogenic attack. We hypothesized that molecular genetic defects in intestin...

Listeria monocytogenes is an intracellular bacterial pathogen that can replicate in the cytosol of host cells. These bacteria undergo actin-based motility in the cytosol via expression of ActA, which recruits host actin-regulatory proteins to the bacterial surface. L. monocytogenes is thought to evade killing by autophagy using ActA-dependent mecha...

The Gram-positive bacterium Listeria monocytogenes is a facultative intracellular pathogen whose virulence depends on its ability to spread from cell to cell within an infected host. Although the actin-related protein 2/3 (Arp2/3) complex is necessary and sufficient for Listeria actin tail assembly, previous studies suggest that other actin polymer...

The host actin nucleation machinery is subverted by many bacterial pathogens to facilitate their entry, motility, replication, and survival. The majority of research conducted in the past primarily focused on exploitation of a host actin nucleator, the Arp2/3 complex, by bacterial pathogens. Recently, new studies have begun to explore the role of f...

Background & aims: The colitis observed in patients with very early onset inflammatory bowel disease (VEOIBD; defined as onset of disease at younger than 6 years of age) often resembles that of chronic granulomatous disease (CGD) in extent and features of colonic inflammation observed by endoscopy and histology. CGD is a severe immunodeficiency ca...

Efferocytosis, the process by which dying or dead cells are removed by phagocytosis, has an important role in development, tissue homeostasis and innate immunity. Efferocytosis is mediated, in part, by receptors that bind to exofacial phosphatidylserine (PS) on cells or cellular debris after loss of plasma membrane asymmetry. Here we show that a ba...

The NOS2 gene encodes for the inducible nitric oxide synthase (iNOS), responsible for nitric oxide (NO) production, which contributes to antimicrobial and antipathogenic activities. Higher levels of both iNOS and NO-induced damage have been observed in inflammatory bowel disease (IBD) patients. NOS2 may have a role in a specific subset of IBD patie...

Very early onset inflammatory bowel diseases (VEOIBD), including infant disorders, are a diverse group of diseases found in children less than 6 years of age. They have been associated with several gene variants. We aimed to identify genes that cause VEOIBD. We performed whole-exome sequencing of DNA from 1 infants with severe enterocolitis and her...

Autophagy is a cellular process that targets proteins, lipids and organelles to lysosomes for degradation, but it has also been shown to combat infection with various pathogenic bacteria. In turn, bacteria have developed diverse strategies to avoid autophagy by interfering with autophagy signalling or the autophagy machinery and, in some cases, the...

Research in autophagy continues to accelerate,(1) and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.(2,3) There are many useful an...

SopB is a type 3 secreted effector with phosphatase activity that Salmonella employs to manipulate host cellular processes, allowing the bacteria to establish their intracellular niche. One important function of SopB is activation of the pro-survival kinase Akt/protein kinase B in the infected host cell. Here, we examine the mechanism of Akt activa...

SopB expression is sufficient to activate Akt. HeLa cells were transiently transfected with plasmids encoding untagged SopB, GFP-SopB or GFP. As a control, cells were stimulated with 100 ng/mL EGF for 5 min. Akt activation of cells was assessed 8 h after transfection by immunoblotting the cell lysates with a phospho-specific anti-Ser473 Akt antibod...

SopB is not sufficient to produce formation of PI(3–5) P3in vitro. Reaction mixtures of FL-PIP2 or FL-PIP3 and His-tagged recombinant SopB or catalytically inactive SopB (C460S) were separated on TLC. As a control, pure lipids were directly separated on TLC and liposomes without recombinant SopB were also examined. (TIF)

SopB-mediated Akt activation in HeLa cells is only partially sensitive to PI3-Kinase inhibitor Wortmannin. HeLa cells were treated with different concentrations of Wortmannin (10 nM to 1000 nM) for 30 min. Cells were then infected with wild type S. Typhimurium for 30 min or incubated with 100 ng/mL EGF for 5 min. As controls, cells were either unin...

IPMK does not contribute to SopB-mediated Akt activation in mouse embryonic fibroblasts (MEF). (A) Wild type or IPMK knockout mouse embryonic fibroblasts were infected with wild type or ΔsopB mutant S. Typhimurium for 30 min. As controls, cells were uninfected or treated with 100 ng/mL EGF for 5 min. Where indicated, cells were treated with 100 µM...

Salmonella invade host cells using Type 3 secreted effectors, which modulate host cellular targets to promote actin rearrangements at the cell surface that drive bacterial uptake. The Arp2/3 complex contributes to Salmonella invasion but is not essential, indicating other actin regulatory factors are involved. Here, we show a novel role for FHOD1,...

Neutrophils recruited to the postischemic kidney contribute to the pathogenesis of ischemia-reperfusion injury (IRI), which is the most common cause of renal failure among hospitalized patients. The Slit family of secreted proteins inhibits chemotaxis of leukocytes by preventing activation of Rho-family GTPases, suggesting that members of this fami...

To date, the utility of single genetic markers to improve disease risk assessment still explains only a small proportion of genetic variance for many complex diseases. This missing heritability may be explained by additional variants with weak effects. To discover and incorporate these additional genetic factors, statistical and computational metho...

Recent genetic-based studies have implicated a number of immune-related genes in the pathogenesis of inflammatory bowel disease (IBD). Our recent genetic studies showed that RAC2 is associated with human IBD; however, its role in disease pathogenesis is unclear. Given Rac2's importance in various fundamental immune cell processes, we investigated w...

Histological Score. (DOC)

Clinical Symptoms Score. (DOC)

Listeria monocytogenes is a bacterial pathogen that can escape the phagosome and replicate in the cytosol of host cells during infection. We previously observed that a population (up to 35%) of L. monocytogenes strain 10403S colocalize with the macroautophagy marker LC3 at 1 h postinfection. This is thought to give rise to spacious Listeria-contain...

An enzyme protects mice from bacterial infection by inducing pyroptosis, an inflammatory form of cell death.

Macrophages are immune cells that participate in the host defense against bacterial pathogens. These cells mediate bacterial clearance by internalizing bacteria into a phagosome, which ultimately fuses with lysosomes to kill bacteria. One bacterial strategy to evade killing in the phagosome is to escape from this compartment prior to lysosomal fusi...

Phosphoinositides (PI) play an important role in many different cellular processes. Their generation and functions, however, are very dynamic, and the detection of localized events usually requires very precise imaging techniques. Recent advances in lipid research raised the possibility of designing molecular probes to specifically detect lipids in...

In autophagic processes a variety of cargos is delivered to the degradative compartment of cells. Recent progress in autophagy research has provided support for the notion that when autophagic processes are operating in selective mode, a receptor protein complex will process the cargo. Here we present a concept of receptor protein complexes as comp...

Lysosomes provide a niche for molecular digestion and are a convergence point for endocytic trafficking, phagosome maturation and autophagy. Typically, lysosomes are small, globular organelles that appear punctate under the fluorescence microscope. However, activating agents like phorbol esters transform macrophage lysosomes into tubular lysosomes...

Background Inflammatory complications after ileal pouch-anal anastomosis (IPAA) for ulcerative colitis (UC) are common.Objective To investigate whether genetic factors are associated with adverse pouch outcomes such as chronic pouchitis (CP) and a Crohn's disease-like (CDL) phenotype.Design866 patients were recruited from three centres in North Ame...

Autophagy is a conserved cellular degradative pathway that is now established to be a vital part of the host immune response to microbial infection. Autophagy can directly eliminate intracellular pathogens by mediating their delivery to lysosomes. Canonical autophagy is characterized by the formation of a double-membrane autophagosome and the invol...

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring au...

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring au...

Cells can destroy invading bacteria through a digestive process called autophagy. A study finds that sugar molecules, exposed by bacterial damage to the cell's membrane, can trigger this process. See Letter p.414

Some bacteria invade host cells by triggering a process akin to phagocytosis. We analyzed the mechanisms underlying invasion vacuole formation by the bacterium Yersinia pseudotuberculosis. After engaging integrins on host cells, Yersinia resided in PI(4,5)P₂-rich, membrane-bound compartments called "prevacuoles" that were inaccessible to extracellu...

Autophagy involves lysosomal-mediated degradation of cellular components and contributes to host immunity. Some pathogens avoid autophagy-mediated killing, while others exploit it to acquire host cell nutrients. Starr et al. reveal that the intracellular bacterial pathogen Brucella abortus can "hitch a ride" with autophagy, subverting autophagy mac...

The intracellular bacterial pathogen Listeria monocytogenes produces phospholipases C (PI-PLC and PC-PLC) and the pore-forming cytolysin listeriolysin O (LLO) to escape the phagosome and replicate within the host cytosol. We found that PLCs can also activate the phagocyte NADPH oxidase during L. monocytogenes infection, a response that would advers...

The study of autophagy is rapidly expanding, and our knowledge of the molecular mechanism and its connections to a wide range of physiological processes has increased substantially in the past decade. The vocabulary associated with autophagy has grown concomitantly. In fact, it is difficult for readers--even those who work in the field--to keep up...

The NOX2 NADPH oxidase complex produces reactive oxygen species and plays a critical role in the killing of microbes by phagocytes. Genetic mutations in genes encoding components of the complex result in both X-linked and autosomal recessive forms of chronic granulomatous disease (CGD). Patients with CGD often develop intestinal inflammation that i...

Autophagy plays a significant role in innate and adaptive immune responses to microbial infection. Some pathogenic bacteria have developed strategies to evade killing by host autophagy. These include the use of 'camouflage' proteins to block targeting to the autophagy pathway and the use of pore-forming toxins to block autophagosome maturation. How...