Robin C. May

Involvement of the Arp2/3 complex in phagocytosis mediated by FcgammaR or CR3.

hagocytosis, the process by which cells internalize particles larger than 0.5 μm in diameter, is central to immunity. Fcγ receptors (FcγRs) and complement receptors (CRs) represent two well-studied classes of phagocytic receptor that are involved in the clearance of invading pathogens. Particles coated (‘opsonized’) with IgG or with the complement product C3bi can be internalized by cells expressing FcγRs or the complement receptor CR3, respectively. Localized actin polymerization provides the driving force for engulfment in both cases, although the morphology of internalization and the molecular pathways involved differ between the two receptors. Here we investigate whether the Arp2/3 complex, a multifunctional actin organizer, is involved in actin remodelling during phagocytosis. We show that this complex is required for both FcγRand CR3-mediated phagocytosis, although the upstream signals that recruit the Arp2/3 complex to phagosomes differ for the two receptors. We studied phagocytosis in the mouse macrophage cell line J774.A1, and in COS cells transiently transfected with complementary DNA constructs encoding phagocytic receptors. Expression of FcγRIIA or of the CD18 and CD11b chains of CR3 enables COS cells to phagocytose particles coated with IgG or C3bi, respectively. This system allows single receptor classes to be expressed in the absence of other phagocytic receptors, while producing a response that is morphologically similar to that of professional phagocytes. Following transfection with FcγRIIA, IgG-opsonized beads were bound efficiently by receptor-expressing COS cells, but not by neighbouring cells that did not express FcγRIIA. Actin filaments (Factin) appeared rapidly beneath bound particles (Fig. 1a) and colocalized there with the Arp2/3 complex (Fig. 1b) in an intense and complete ring of varying intensity (Fig. 1c, d). Recruitment of Factin and the Arp2/3 complex peaked 10 min after the cells were warmed to 37 °C (with about 75% of particles showing staining for both proteins), and subsequently declined (by 60 min, about 25% of particles stained positively for both proteins; data not shown). An identical pattern was observed when IgG-opsonized beads were fed to J774.A1 macrophages, with both F-actin and the Arp2/3 complex localizing rapidly beneath bound particles (Fig. 1e, f). The Arp2/3 complex was also recruited to phagosomes during complement-mediated phagocytosis. Twenty-four hours after transfection with plasmids encoding the CD11b (αM) and CD18 (β2) chains of CR3, COS cells were able to bind and internalize C3bi-opsonized beads. As described previously, actin filaments localized to CR3-mediated phagosomes but showed lower-intensity staining than at FcγR-mediated phagosomes; a faint ‘ring’ of actin, punctuated by bright F-actin foci, was often seen (Fig. 1g). Arp2/3 was also recruited to CR3-mediated phagosomes (Fig. 1h), showed lower-intensity staining than at FcγR-mediated phagosomes, and frequently appeared as punctate foci (Fig. 1i, j), as reported previously for other cytoskeletal proteins. Similarly, in J774.A1 macrophages, the Arp2/3 complex and F-actin co-localized below bound C3bi-opsonized latex beads (Fig. 1k, l). ScarWA, a carboxy-terminal fragment of the Arp2/3-binding protein Scar1, delocalizes the Arp2/3 complex by preventing its recruitment by endogenous proteins of the Wiskott–Aldrich syndrome protein (WASP) family. In vitro, ScarWA can bypass normal signalling processes to stimulate actin nucleation by the Arp2/3 complex. In cells expressing ScarWA, a mass of diffuse actin filaments is apparent throughout the cytoplasm, presumably reflecting the dual effect of ScarWA in delocalizing and activating the Arp2/3 complex. We coexpressed ScarWA and FcγRIIA in COS cells. Although binding of these cells to IgG-opsonized beads was normal, recruitment of the Arp2/3 complex and formation of an actin ring beneath the particles was completely abrogated, at both 20 min and 60 min, resulting in a failure of the cells to internalize particles (Table 1). This inhibition was specific, as expression of another truncated Scar1 protein, ScarW (which does not bind the Arp2/3 complex), had no effect on internalization. In contrast, we were unable to block FcγR-mediated phagocytosis in J774.A1 cells following microinjection of the ScarWA plasmid into the nucleus. J774.A1 cells expressing detectable levels of ScarWA showed a loss of filopodia and a flattened phenotype, as shown previously, but remained able to recruit Arp2/3 and F-actin to nascent FcγR-mediated phagoP

Emergence and pathogenicity of highly virulent Cryptococcus gattii genotypes in the northwest United States.

Cryptococcus gattii causes life-threatening disease in otherwise healthy hosts and to a lesser extent in immunocompromised hosts. The highest incidence for this disease is on Vancouver Island, Canada, where an outbreak is expanding into neighboring regions including mainland British Columbia and the United States. This outbreak is caused predominantly by C. gattii molecular type VGII, specifically VGIIa/major. In addition, a novel genotype, VGIIc, has emerged in Oregon and is now a major source of illness in the region. Through molecular epidemiology and population analysis of MLST and VNTR markers, we show that the VGIIc group is clonal and hypothesize it arose recently. The VGIIa/IIc outbreak lineages are sexually fertile and studies support ongoing recombination in the global VGII population. This illustrates two hallmarks of emerging outbreaks: high clonality and the emergence of novel genotypes via recombination. In macrophage and murine infections, the novel VGIIc genotype and VGIIa/major isolates from the United States are highly virulent compared to similar non-outbreak VGIIa/major-related isolates. Combined MLST-VNTR analysis distinguishes clonal expansion of the VGIIa/major outbreak genotype from related but distinguishable less-virulent genotypes isolated from other geographic regions. Our evidence documents emerging hypervirulent genotypes in the United States that may expand further and provides insight into the possible molecular and geographic origins of the outbreak.

Expulsion of Live Pathogenic Yeast by Macrophages

Phagocytic cells, such as neutrophils and macrophages, perform a critical role in protecting organisms from infection by engulfing and destroying invading microbes . Although some bacteria and fungi have evolved strategies to survive within a phagocyte after uptake, most of these pathogens must eventually kill the host cell if they are to escape and infect other tissues . However, we now demonstrate that the human fungal pathogen Cryptococcus neoformans is able to escape from within macrophages without killing the host cell by a novel expulsive mechanism. This process occurs in both murine J774 cells and primary human macrophages. It is extremely rapid and yet can occur many hours after phagocytosis of the pathogen. Expulsion occurs independently of the initial route of phagocytic uptake and does not require phagosome maturation . After the expulsive event, both the host macrophage and the expelled C. neoformans appear morphologically normal and continue to proliferate, suggesting that this process may represent an important mechanism by which pathogens are able to escape from phagocytic cells without triggering host cell death and thus inflammation .

Rho-Kinase and Myosin-II Control Phagocytic Cup Formation during CR, but Not FcγR, Phagocytosis

Phagocytosis through Fcgamma receptor (FcgammaR) or complement receptor 3 (CR) requires Arp2/3 complex-mediated actin polymerization, although each receptor uses a distinct signaling pathway. Rac and Cdc42 are required for actin and Arp2/3 complex recruitment during FcgammaR phagocytosis, while Rho controls actin assembly at CR phagosomes. To better understand the role of Rho in CR phagocytosis, we tested the idea that a known target of Rho, Rho-kinase (ROK), might control phagocytic cup formation and/or engulfment of particles. Inhibitors of ROK (dominant-negative ROK and Y-27632) and of the downstream target of ROK, myosin-II (ML7, BDM, and dominant-negative myosin-II), were used to test this idea. We found that inhibition of the Rho --> ROK --> myosin-II pathway caused a decreased accumulation of Arp2/3 complex and F-actin around bound particles, which led to a reduction in CR-mediated phagocytic engulfment. FcgammaR-mediated phagocytosis, in contrast, was independent of Rho or ROK activity and was only dependent on myosin-II for particle internalization, not for actin cup formation. While myosins have been previously implicated in FcgammaR phagocytosis, to our knowledge, this is the first demonstration of a role for myosin-II in CR phagocytosis.

The fatal fungal outbreak on Vancouver Island is characterized by enhanced intracellular parasitism driven by mitochondrial regulation

In 1999, the population of Vancouver Island, Canada, began to experience an outbreak of a fatal fungal disease caused by a highly virulent lineage of Cryptococcus gattii. This organism has recently spread to the Canadian mainland and Pacific Northwest, but the molecular cause of the outbreak remains unknown. Here we show that the Vancouver Island outbreak (VIO) isolates have dramatically increased their ability to replicate within macrophages of the mammalian immune system in comparison with other C. gattii strains. We further demonstrate that such enhanced intracellular parasitism is directly linked to virulence in a murine model of cryptococcosis, suggesting that this phenotype may be the cause of the outbreak. Finally, microarray studies on 24 C. gattii strains reveals that the hypervirulence of the VIO isolates is characterized by the up-regulation of a large group of genes, many of which are encoded by mitochondrial genome or associated with mitochondrial activities. This expression profile correlates with an unusual mitochondrial morphology exhibited by the VIO strains after phagocytosis. Our data thus demonstrate that the intracellular parasitism of macrophages is a key driver of a human disease outbreak, a finding that has significant implications for a wide range of other human pathogens.

Genes required for systemic RNA interference in Caenorhabditis elegans

RNA interference (RNAi) in the nematode worm, Caenorhabditis elegans, occurs systemically. Double-stranded RNA (dsRNA) provided in the diet can be absorbed from the gut lumen and distributed throughout the body, triggering RNAi in tissues that are not exposed to the initial dsRNA trigger. This is in marked contrast to other animals, in which RNAi does not spread from targeted tissues to neighboring cells. Here, we report the characterization of mutants defective in the systemic aspect of RNAi, but not in the core RNAi process itself. Analysis of these mutants suggests that dsRNA uptake is a specific process involving several unique proteins.

Ancient Dispersal of the Human Fungal Pathogen Cryptococcus gattii from the Amazon Rainforest

Over the past two decades, several fungal outbreaks have occurred, including the high-profile ‘Vancouver Island’ and ‘Pacific Northwest’ outbreaks, caused by Cryptococcus gattii, which has affected hundreds of otherwise healthy humans and animals. Over the same time period, C. gattii was the cause of several additional case clusters at localities outside of the tropical and subtropical climate zones where the species normally occurs. In every case, the causative agent belongs to a previously rare genotype of C. gattii called AFLP6/VGII, but the origin of the outbreak clades remains enigmatic. Here we used phylogenetic and recombination analyses, based on AFLP and multiple MLST datasets, and coalescence gene genealogy to demonstrate that these outbreaks have arisen from a highly-recombining C. gattii population in the native rainforest of Northern Brazil. Thus the modern virulent C. gattii AFLP6/VGII outbreak lineages derived from mating events in South America and then dispersed to temperate regions where they cause serious infections in humans and animals.

Cytokine Signaling Regulates the Outcome of Intracellular Macrophage Parasitism by Cryptococcus neoformans

ABSTRACT The pathogenic yeast Cryptococcus neoformans and C. gattii commonly cause severe infections of the central nervous system in patients with impaired immunity but also increasingly in immunocompetent individuals. Cryptococcus is phagocytosed by macrophages but can then survive and proliferate within the phagosomes of these infected host cells. Moreover, Cryptococcus is able to escape into the extracellular environment via a recently discovered nonlytic mechanism (termed expulsion or extrusion). Although it is well established that the hosts cytokine profile dramatically affects the outcome of cryptococcal disease, the molecular basis for this effect is unclear. Here, we report a systematic analysis of the influence of Th1, Th2, and Th17 cytokines on the outcome of the interaction between macrophages and cryptococci. We show that Th1 and Th17 cytokines activate, whereas Th2 cytokines inhibit, anticryptococcal functions. Intracellular yeast proliferation and cryptococcal expulsion rates were significantly lower after treatment with the Th1 cytokines gamma interferon and tumor necrosis factor alpha and the Th17 cytokine interleukin-17 (IL-17). Interestingly, however, the Th2 cytokines IL-4 and IL-13 significantly increased intracellular yeast proliferation while reducing the occurrence of pathogen expulsion. These results help explain the observed poor prognosis associated with the Th2 cytokine profile (e.g., in human immunodeficiency virus-infected patients).

Cryptococcal Interactions with the Host Immune System

ABSTRACT Opportunistic pathogens have become of increasing medical importance over the last decade due to the AIDS pandemic. Not only is cryptococcosis the fourth-most-common fatal infectious disease in sub-Saharan Africa, but also Cryptococcus is an emerging pathogen of immunocompetent individuals. The interaction between Cryptococcus and the hosts immune system is a major determinant for the outcome of disease. Despite initial infection in early childhood with Cryptococcus neoformans and frequent exposure to C. neoformans within the environment, immunocompetent individuals are generally able to contain the fungus or maintain the yeast in a latent state. However, immune deficiencies lead to disseminating infections that are uniformly fatal without rapid clinical intervention. This review will discuss the innate and adaptive immune responses to Cryptococcus and cryptococcal strategies to evade the hosts defense mechanisms. It will also address the importance of these strategies in pathogenesis and the potential of immunotherapy in cryptococcosis treatment.

The Arp2/3 complex is essential for the actin-based motility of Listeria monocytogenes

Actin polymerisation is thought to drive the movement of eukaryotic cells and some intracellular pathogens such as Listeria monocytogenes. The Listeria surface protein ActA synergises with recruited host proteins to induce actin polymerisation, propelling the bacterium through the host cytoplasm [1]. The Arp2/3 complex is one recruited host factor [2] [3]; it is also believed to regulate actin dynamics in lamellipodia [4] [5]. The Arp2/3 complex promotes actin filament nucleation in vitro, which is further enhanced by ActA [6] [7]. The Arp2/3 complex also interacts with members of the Wiskott-Aldrich syndrome protein (WASP) [8] family - Scar1 [9] [10] and WASP itself [11]. We interfered with the targeting of the Arp2/3 complex to Listeria by using carboxy-terminal fragments of Scar1 that bind the Arp2/3 complex [11]. These fragments completely blocked actin tail formation and motility of Listeria, both in mouse brain extract and in Ptk2 cells overexpressing Scar1 constructs. In both systems, Listeria could initiate actin cloud formation, but tail formation was blocked. Full motility in vitro was restored by adding purified Arp2/3 complex. We conclude that the Arp2/3 complex is a host-cell factor essential for the actin-based motility of L. monocytogenes, suggesting that it plays a pivotal role in regulating the actin cytoskeleton.