New roles for perforins and proteases in apicomplexan egress

Egress is a pivotal step in the life cycle of intracellular pathogens initiating the transition from an expiring host cell to a fresh target cell. While much attention has been focused on understanding cell invasion by intracellular pathogens, recent work is providing a new appreciation of mechanisms and therapeutic potential of microbial egress. This review highlights recent insight into cell egress by apicomplexan parasites and emerging contributions of membranolytic and proteolytic secretory products, along with host proteases. New findings suggest that Toxoplasma gondii secretes a pore-forming protein, TgPLP1, during egress that facilitates parasite escape from the cell by perforating the parasitophorous membrane. Also, in a cascade of proteolytic events, Plasmodium falciparum late-stage schizonts activate and secrete a subtilisin, PfSUB1, which processes enigmatic putative proteases called serine-repeat antigens that contribute to merozoite egress. A new report also suggests that calcium-activated host proteases called calpains aid parasite exit, possibly by acting upon the host cytoskeleton. Together these discoveries reveal important new molecular players involved in the principal steps of egress by apicomplexans.     

How does Legionella pneumophila exit the host cell?

In recent years, tremendous progress has been made in unraveling the elegant mechanisms by which intracellular pathogens invade host cells and establish intracellular infections. By contrast, our knowledge of the mechanisms of host cell cytolysis and the egress of intracellular pathogens is still in its infancy. Temporal pore-formation-mediated lysis of the host and exit by Legionella pneumophila and Leishmania could provide a new model of egress for other intracellular pathogens, many of which exhibit pore-forming or cytolysin activity     

Sweet host revenge: Galectins and GBPs join forces at broken membranes

Most bacterial pathogens enter and exit eukaryotic cells during their journey through the vertebrate host. In order to endure inside a eukaryotic cell, bacterial invaders commonly employ bacterial secretion systems to inject host cells with virulence factors that co‐opt the host's membrane trafficking systems and thereby establish specialised pathogen‐containing vacuoles (PVs) as intracellular niches permissive for microbial growth and survival. To defend against these microbial adversaries hiding inside PVs, host organisms including humans evolved an elaborate cell‐intrinsic armoury of antimicrobial weapons that include noxious gases, antimicrobial peptides, degradative enzymes, and pore‐forming proteins. This impressive defence machinery needs to be accurately delivered to PVs, in order to fight off vacuole‐dwelling pathogens. Here, I discuss recent evidence that the presence of bacterial secretion systems at PVs and the associated destabilisation of PV membranes attract such antimicrobial delivery systems consisting of sugar‐binding galectins as well as dynamin‐like guanylate‐binding proteins (GBPs). I will review recent advances in our understanding of intracellular immune recognition of PVs by galectins and GBPs, discuss how galectins and GBPs control host defence, and highlight important avenues of future research in this exciting area of cell‐autonomous immunity.     

Manipulation of rab GTPase function by intracellular bacterial pathogens.

Intracellular bacterial pathogens have evolved highly specialized mechanisms to enter and survive within their eukaryotic hosts. In order to do this, bacterial pathogens need to avoid host cell degradation and obtain nutrients and biosynthetic precursors, as well as evade detection by the host immune system. To create an intracellular niche that is favorable for replication, some intracellular pathogens inhibit the maturation of the phagosome or exit the endocytic pathway by modifying the identity of their phagosome through the exploitation of host cell trafficking pathways. In eukaryotic cells, organelle identity is determined, in part, by the composition of active Rab GTPases on the membranes of each organelle. This review describes our current understanding of how selected bacterial pathogens regulate host trafficking pathways by the selective inclusion or retention of Rab GTPases on membranes of the vacuoles that they occupy in host cells during infection.     

Prison Break: Pathogens' Strategies To Egress from Host Cells

Summary: A wide spectrum of pathogenic bacteria and protozoa has adapted to an intracellular life-style, which presents several advantages, including accessibility to host cell metabolites and protection from the host immune system. Intracellular pathogens have developed strategies to enter and exit their host cells while optimizing survival and replication, progression through the life cycle, and transmission. Over the last decades, research has focused primarily on entry, while the exit process has suffered from neglect. However, pathogen exit is of fundamental importance because of its intimate association with dissemination, transmission, and inflammation. Hence, to fully understand virulence mechanisms of intracellular pathogens at cellular and systemic levels, it is essential to consider exit mechanisms to be a key step in infection. Exit from the host cell was initially viewed as a passive process, driven mainly by physical stress as a consequence of the explosive replication of the pathogen. It is now recognized as a complex, strategic process termed “egress,” which is just as well orchestrated and temporally defined as entry into the host and relies on a dynamic interplay between host and pathogen factors. This review compares egress strategies of bacteria, pathogenic yeast, and kinetoplastid and apicomplexan parasites. Emphasis is given to recent advances in the biology of egress in mycobacteria and apicomplexans.     

The outcome of phagocytic cell division with infectious cargo depends on single phagosome formation

Given that macrophages can proliferate and that certain microbes survive inside phagocytic cells, the question arises as to the post-mitotic distribution of microbial cargo. Using macrophage-like cells we evaluated the post-mitotic distribution of intracellular Cryptococcus yeasts and polystyrene beads by comparing experimental data to a stochastic model. For beads, the post-mitotic distribution was that expected from chance alone. However, for yeast cells the post-mitotic distribution was unequal, implying preferential sorting to one daughter cell. This mechanism for unequal distribution was phagosomal fusion, which effectively reduced the intracellular particle number. Hence, post-mitotic intracellular particle distribution is stochastic, unless microbial and/or host factors promote unequal distribution into daughter cells. In our system unequal cargo distribution appeared to benefit the microbe by promoting host cell exocytosis. Post-mitotic infectious cargo distribution is a new parameter to consider in the study of intracellular pathogens since it could potentially define the outcome of phagocytic-microbial interactions.     

The P2X7 receptor and intracellular pathogens: a continuing struggle

The purinergic receptor, P2X₇, has recently emerged as an important component of the innate immune response against microbial infections. Ligation of P2X₇ by ATP can stimulate inflammasome activation and secretion of proinflammatory cytokines, but it can also lead directly to killing of intracellular pathogens in infected macrophages and epithelial cells. Thus, while some intracellular pathogens evade host defense responses by modulating with membrane trafficking or cell signaling in the infected cells, the host cells have also developed mechanisms for inhibiting infection. This review will focus on the effects of P2X₇ on control of infection by intracellular pathogens, microbial virulence factors that interfere with P2X₇ activity, and recent evidence linking polymorphisms in human P2X₇ with susceptibility to infection.     

Current status of immune mechanisms of killing of intracellular microorganisms

The interaction between intracellular pathogens and the mammalian host follows different pathways that reflect evolved survival mechanisms of both the pathogen and the host to assure each one's own survival. From the host's perspective, different immune mechanisms predominate at different stages of infection. Both phagocytic and non-phagocytic target cells participate in microbial uptake and, in some cases, intracellular destruction. In addition, the development of specific immunity ensures sustained activation of intracellular microbicidal mechanisms in the target cells, and induction of apoptotic or lytic target cell death by cytotoxic T lymphocytes. From the pathogen's perspective, different evasion strategies are employed to counteract host defenses. Understanding microbial survival strategies and the immune mechanisms that result in killing of intracellular pathogens will deepen our insight into the pathogenesis of infection that could be applied towards the development of effective vaccination and immunotherapy.     

Non-lytic, actin-based exit of intracellular parasites from C. elegans intestinal cells

The intestine is a common site for invasion by intracellular pathogens, but little is known about how pathogens restructure and exit intestinal cells in vivo. The natural microsporidian parasite N. parisii invades intestinal cells of the nematode C. elegans, progresses through its life cycle, and then exits cells in a transmissible spore form. Here we show that N. parisii causes rearrangements of host actin inside intestinal cells as part of a novel parasite exit strategy. First, we show that N. parisii infection causes ectopic localization of the normally apical-restricted actin to the basolateral side of intestinal cells, where it often forms network-like structures. Soon after this actin relocalization, we find that gaps appear in the terminal web, a conserved cytoskeletal structure that could present a barrier to exit. Reducing actin expression creates terminal web gaps in the absence of infection, suggesting that infection-induced actin relocalization triggers gap formation. We show that terminal web gaps form at a distinct stage of infection, precisely timed to precede spore exit, and that all contagious animals exhibit gaps. Interestingly, we find that while perturbations in actin can create these gaps, actin is not required for infection progression or spore formation, but actin is required for spore exit. Finally, we show that despite large numbers of spores exiting intestinal cells, this exit does not cause cell lysis. These results provide insight into parasite manipulation of the host cytoskeleton and non-lytic escape from intestinal cells in vivo.     

Manipulation of the host cell death pathway by Shigella

Host cells deploy multiple defences against microbial infection. One prominent host defence mechanism, the death of infected cells, plays a pivotal role in clearing damaged cells, eliminating pathogens, removing replicative niches, exposing intracellular bacterial pathogens to extracellular immune surveillance and presenting bacteria‐derived antigens to the adaptive immune system. Although cell death can occur under either physiological or pathophysiological conditions, it acts as an innate defence mechanism against bacterial pathogens by limiting their persistent colonization. However, many bacterial pathogens, including Shigella, have evolved mechanisms that manipulate host cell death for their own benefit.     

Extrusions are phagocytosed and promote Chlamydia survival within macrophages

The precise strategies that intracellular pathogens use to exit host cells have a direct impact on their ability to disseminate within a host, transmit to new hosts, and engage or avoid immune responses. The obligate intracellular bacterium Chlamydia trachomatis exits the host cell by two distinct exit strategies, lysis and extrusion. The defining characteristics of extrusions, and advantages gained by Chlamydia within this unique double‐membrane structure, are not well understood. Here, we define extrusions as being largely devoid of host organelles, comprised mostly of Chlamydia elementary bodies, and containing phosphatidylserine on the outer surface of the extrusion membrane. Extrusions also served as transient, intracellular‐like niches for enhanced Chlamydia survival outside the host cell. In addition to enhanced extracellular survival, we report the key discovery that chlamydial extrusions are phagocytosed by primary bone marrow‐derived macrophages, after which they provide a protective microenvironment for Chlamydia. Extrusion‐derived Chlamydia staved off macrophage‐based killing and culminated in the release of infectious elementary bodies from the macrophage. Based on these findings, we propose a model in which C. trachomatis extrusions serve as “trojan horses” for bacteria, by exploiting macrophages as vehicles for dissemination, immune evasion, and potentially transmission.     

Apoptosis paves the detour path for CD8 T cell activation against intracellular bacteria

Intracellular bacteria such as Mycobacterium tuberculosis primarily infect macrophages. Within these host cells, the pathogens are confined to phagosomes and their antigens are secluded from the classical MHC I presentation pathway. Moreover, macrophages fail to express certain antigen presenting molecules like CD1 proteins. As a result of this intracellular lifestyle, the pathways for the induction of MHC I- and CD1-restricted CD8 T cells by such microorganisms remain elusive. Based on recent findings in tuberculosis and salmonellosis, we propose a new detour pathway for CD8 T cell activation against intracellular bacteria through apoptotic blebs from infected macrophages. Pathogen-derived antigens including proteins and lipids are delivered from infected cells to non-infected dendritic cells. Subsequently, these professional antigen presenting cells display microbial antigens through MHC I and CD1 to T cells. Thus, cross-priming mediated by apoptotic vesicles is not just a matter of antigen distribution, but an intrinsic immunological function due to the nature of phagosomally located intracellular bacteria. We consider infection-induced apoptosis the conditio sine qua non for antigen-specific CD8 T cell activation by phagosome-enclosed pathogens. This important new function of cell death in antibacterial immunity requires consideration for rational vaccine design.     

Reprogramming the phagocytic pathway—intracellular pathogens and their vacuoles (Review)

Phagocytic immune cells (particularly macrophages and neutrophils) take up and digest particles that have invaded our bodies. In doing so, they represent a very early line of defence against a microbial attack. During uptake, the particles are wrapped by a portion of the phagocyte's plasma membrane, and a new endocytic compartment, the phagosome, is formed. The typical fate of a phagosome is its fusion with lysosomes to yield a phagolysosome in which the particle is digested. Recent data show that some ‘intracellular microorganisms’ that can cause severe illnesses (tuberculosis, leprosy, legionaire's disease and others) manage to reprogramme the host phagocytes not to deliver them to the lysosomal compartment. This probably results in increased survival of the pathogens. The analysis of the composition of such ‘novel’ compartments and research on the molecular mechanisms underlying the microbial interference with host cell functions are likely to yield important insights into: (1) which endocytic/phagocytic compartments phagocytes employ to handle ingested material in general; (2) how some pathogenic microorganisms can reprogramme the phagocytic pathway; and possibly (3) how infections caused by these microorganisms can be treated more effectively. Here, some studies are presented analysing which compartments intracellular pathogens inhabit and how microbes might be able to reprogramme their host cells.     

Actin Recruitment to the Chlamydia Inclusion Is Spatiotemporally Regulated by a Mechanism That Requires Host and Bacterial Factors

The ability to exit host cells at the end of their developmental growth is a critical step for the intracellular bacterium Chlamydia. One exit strategy, extrusion, is mediated by host signaling pathways involved with actin polymerization. Here, we show that actin is recruited to the chlamydial inclusion as a late event, occurring after 20 hours post-infection (hpi) and only within a subpopulation of cells. This event increases significantly in prevalence and extent from 20 to 68 hpi, and actin coats strongly correlated with extrusions. In contrast to what has been reported for other intracellular pathogens, actin nucleation on Chlamydia inclusions did not ‘flash’, but rather exhibited moderate depolymerization dynamics. By using small molecule agents to selectively disrupt host signaling pathways involved with actin nucleation, modulate actin polymerization dynamics and also to disable the synthesis and secretion of chlamydial proteins, we further show that host and bacterial proteins are required for actin coat formation. Transient disruption of either host or bacterial signaling pathways resulted in rapid loss of coats in all infected cells and a reduction in extrusion formation. Inhibition of Chlamydia type III secretion also resulted in rapid loss of actin association on inclusions, thus implicating chlamydial effector proteins(s) as being central factors for engaging with host actin nucleating factors, such as formins. In conclusion, our data illuminate the host and bacterial driven process by which a dense actin matrix is dynamically nucleated and maintained on the Chlamydia inclusion. This late stage event is not ubiquitous for all infected cells in a population, and escalates in prevalence and extent throughout the developmental cycle of Chlamydia, culminating with their exit from the host cell by extrusion. The initiation of actin recruitment by Chlamydia appears to be novel, and may serve as an upstream determinant of the extrusion mechanism.     

Chlamydia--host cell interactions: recent advances on bacterial entry and intracellular development

Bacteria of the Chlamydiales order are very successful intracellular organisms that grow in human and animal cells, and even in amoebae. They fulfill several essential functions to enter their host cells, establish an intracellular environment favorable for their multiplication and exit the host cell. They multiply in a unique organelle called the inclusion, which is isolated from the endocytic but not the exocytic pathway. A combination of host cell factors and of proteins secreted by the bacteria, from within the inclusion, contribute to the establishment and development of this inclusion. Here we review recent data on the entry mechanisms and maturation of the inclusion.     

Intracellular innate resistance to bacterial pathogens

Mammalian innate immunity stimulates antigen-specific immune responses and acts to control infection prior to the onset of adaptive immunity. Some bacterial pathogens replicate within the host cell and are therefore sheltered from some protective aspects of innate immunity such as complement. Here we focus on mechanisms of innate intracellular resistance encountered by bacterial pathogens and how some bacteria can evade destruction by the innate immune system. Major strategies of intracellular antibacterial defence include pathogen compartmentalization and iron limitation. Compartmentalization of pathogens within the host endocytic pathway is critical for generating high local concentrations of antimicrobial molecules, such as reactive oxygen species, and regulating concentrations of divalent cations that are essential for microbial growth. Cytosolic sensing, autophagy, sequestration of essential nutrients and membrane attack by antimicrobial peptides are also discussed.     

Role of autophagy in disease resistance and hypersensitive response-associated cell death

Ancient autophagy pathways are emerging as key defense modules in host eukaryotic cells against microbial pathogens. Apart from actively eliminating intracellular intruders, autophagy is also responsible for cell survival, for example by reducing the deleterious effects of endoplasmic reticulum stress. At the same time, autophagy can contribute to cellular suicide. The concurrent engagement of autophagy in these processes during infection may sometimes mask its contribution to differing pro-survival and pro-death decisions. The importance of autophagy in innate immunity in mammals is well documented, but how autophagy contributes to plant innate immunity and cell death is not that clear. A few research reports have appeared recently to shed light on the roles of autophagy in plant-pathogen interactions and in disease-associated host cell death. We present a first attempt to reconcile the results of this research.     

Caenorhabditis elegans as a model for intracellular pathogen infection

The genetically tractable nematode Caenorhabditis elegans is a convenient host for studies of pathogen infection. With the recent identification of two types of natural intracellular pathogens of C. elegans, this host now provides the opportunity to examine interactions and defence against intracellular pathogens in a whole‐animal model for infection. C. elegans is the natural host for a genus of microsporidia, which comprise a phylum of fungal‐related pathogens of widespread importance for agriculture and medicine. More recently, C. elegans has been shown to be a natural host for viruses related to the Nodaviridae family. Both microsporidian and viral pathogens infect the C. elegans intestine, which is composed of cells that share striking similarities to human intestinal epithelial cells. Because C. elegans nematodes are transparent, these infections provide a unique opportunity to visualize differentiated intestinal cells in vivo during the course of intracellular infection. Together, these two natural pathogens of C. elegans provide powerful systems in which to study microbial pathogenesis and host responses to intracellular infection.     

Exploitation of the endoplasmic reticulum by bacterial pathogens

The endoplasmic reticulum (ER) has unique properties that are exploited by microbial pathogens. Exotoxins secreted by bacteria take advantage of the host transport pathways that deliver proteins from the Golgi to the ER. Transport to the ER is necessary for the unfolding and translocation of these toxins into the cytosol where their host targets reside. Intracellular pathogens subvert host vesicle transport to create ER-like vacuoles that support their intracellular replication. Investigations on how bacterial pathogens can use the ER during host infection are providing important details on transport pathways involving this specialized organelle.