Respiration supports intraphagosomal filamentation and escape of Candida albicans from macrophages.

IMPORTANCE: Candida albicans is a leading human fungal pathogen that often causes life-threatening infections in immunocompromised individuals. The ability of C. albicans to transition between yeast and filamentous forms is key to its virulence, and this occurs in response to many host-relevant cues, including engulfment by host macrophages. While previous efforts identified C. albicans genes required for filamentation in other conditions, the genes important for this morphological transition upon internalization by macrophages remained largely enigmatic. Here, we employed a functional genomic approach to identify genes that enable C. albicans filamentation within macrophages and uncovered a role for the mitochondrial ribosome, respiration, and the SNF1 AMP-activated kinase complex. Additionally, we showed that glucose uptake and glycolysis by macrophages support C. albicans filamentation. This work provides insights into the metabolic dueling that occurs during the interaction of C. albicans with macrophages and identifies vulnerabilities in C. albicans that could serve as promising therapeutic targets.

The chemorepellent, SLIT2, bolsters innate immunity against Staphylococcus aureus.

Neutrophils are essential for host defense against Staphylococcus aureus (S. aureus). The neuro-repellent, SLIT2, potently inhibits neutrophil chemotaxis, and might, therefore, be expected to impair antibacterial responses. We report here that, unexpectedly, neutrophils exposed to the N-terminal SLIT2 (N-SLIT2) fragment kill extracellular S. aureus more efficiently. N-SLIT2 amplifies reactive oxygen species production in response to the bacteria by activating p38 mitogen-activated protein kinase that in turn phosphorylates NCF1, an essential subunit of the NADPH oxidase complex. N-SLIT2 also enhances the exocytosis of neutrophil secondary granules. In a murine model of S. aureus skin and soft tissue infection (SSTI), local SLIT2 levels fall initially but increase subsequently, peaking at 3 days after infection. Of note, the neutralization of endogenous SLIT2 worsens SSTI. Temporal fluctuations in local SLIT2 levels may promote neutrophil recruitment and retention at the infection site and hasten bacterial clearance by augmenting neutrophil oxidative burst and degranulation. Collectively, these actions of SLIT2 coordinate innate immune responses to limit susceptibility to S. aureus.

Lysosomal swelling and lysis mediate delivery of C3 toxins to their cytoplasmic targets.

Unlike other cholera-like toxins that contain separate binding/translocation and catalytic subunits, C3-like mono-ADP-ribosyltransferases consist of a single subunit that serves both functions. The manner whereby C3 toxins reach the host cell cytoplasm is poorly understood and was addressed in this study by monitoring the fate of fluorescently labeled C3larvinA. Following binding to the macrophage membrane in a discontinuous punctate pattern, the toxin was internalized, traversing the endocytic pathway to reach lysosomes. Strikingly, the lysosomes of C3larvinA-treated cells underwent massive swelling over the course of 1-4 h. Lysosomal swelling preceded the extensive rearrangement of the cellular F-actin caused by ADP-ribosylation of cytosolic Rho-GTPases. This suggested that lysosome swelling might be required for the escape of the toxin into the cytoplasm where the GTPases reside. Accordingly, preventing swelling by osmotic manipulation or by arresting macropinocytosis precluded the F-actin rearrangement. Toxin-induced swelling was associated with leakage of sulforhodamine B and dextran from the lysosomes, implying membrane rupture or activation of mechano-sensitive pores, enabling the toxin itself to reach the cytosol. Finally, comparison of the cellular traffic and actin remodeling activities of C3larvinA with that of two related toxins, C3larvintrunc and Plx2A, highlighted the importance of the N-terminal α1 -helix for lysosomal swelling and successful intoxication.

Calcium-dependent ESCRT recruitment and lysosome exocytosis maintain epithelial integrity during Candida albicans invasion.

Candida albicans is both a commensal and an opportunistic fungal pathogen. Invading hyphae of C. albicans secrete candidalysin, a pore-forming peptide toxin. To prevent cell death, epithelial cells must protect themselves from direct damage induced by candidalysin and by the mechanical forces exerted by expanding hyphae. We identify two key Ca2+-dependent repair mechanisms employed by epithelial cells to withstand candidalysin-producing hyphae. Using camelid nanobodies, we demonstrate candidalysin secretion directly into the invasion pockets induced by elongating C. albicans hyphae. The toxin induces oscillatory increases in cytosolic [Ca2+], which cause hydrolysis of PtdIns(4,5)P2 and loss of cortical actin. Epithelial cells dispose of damaged membrane regions containing candidalysin by an Alg-2/Alix/ESCRT-III-dependent blebbing process. At later stages, plasmalemmal tears induced mechanically by invading hyphae are repaired by exocytic insertion of lysosomal membranes. These two repair mechanisms maintain epithelial integrity and prevent mucosal damage during both commensal growth and infection by C. albicans.

Maintaining phagosome integrity during fungal infection: do or die?

Professional phagocytes represent a critical node in innate immunity and tissue homeostasis through their specialized ability to eat, drink, and digest material from the extracellular milieu. The degradative and microbicidal functions of phagocytes rely on the fusion of lysosomes with endosomal compartments such as phagosomes, resulting in the digestion and recycling of internalized prey and debris. Despite these efforts, several particularly dangerous infections result from a class of tenacious pathogens that resist digestion, often surviving and even proliferating within the confines of the phagosomal membrane. One such example, Candida albicans, is a commensal polymorphic fungus that colonizes ~50% of the population and can cause life-threatening infections in immunocompromised patients. Not only can C. albicans survive within phagosomes, but its ingestion by macropahges triggers a yeast-to-hyphal transition promoting rapid intraphagosomal growth (several microns per hour) while imposing a substantial mechanical burden on the phagosomal membrane surrounding the fungus. Preservation of membrane integrity is essential to maintain the hostile internal environment of the phagosome, a functionality of degradative enzymes and oxidative stress. Yet, biological membranes such as phagosomes have a limited capacity to stretch. Using C. albicans as a model intracellular pathogen, our recent work reveals a mechanism by which phagosomes respond to intraphagosomal growth of pathogens by expanding their surface area, and as a result, maintain the integrity of the phagosomal membrane. We hypothesized that this expansion would be facilitated by the delivery and fusion of membrane from extraneous sources with the phagosome. Consistently, macrophages respond to the yeast-to-hyphal transition through a stretch-induced release of phagosomal calcium, leading to recruitment and insertion of lysosomes that accommodate the expansion of the phagolysosome and preserve its integrity. Below, we discuss this calcium-dependent mechanism of lysosome insertion as a means of avoiding phagosomal rupture. Further, we examine the implications of membrane integrity on the delicate balance between the host and pathogen by focusing on fungal stress responses, nutrient acquisition, inflammasome activation, and cell death.

Lysosome Fusion Maintains Phagosome Integrity during Fungal Infection.

Phagosomes must maintain membrane integrity to exert their microbicidal function. Some microorganisms, however, survive and grow within phagosomes. In such instances, phagosomes must expand to avoid rupture and microbial escape. We studied whether phagosomes regulate their size to preserve integrity during infection with the fungal pathogen Candida albicans. Phagosomes release calcium as C. albicans hyphae elongate, inducing lysosome recruitment and insertion, thereby increasing the phagosomal surface area. As hyphae grow, the expanding phagosome consumes the majority of free lysosomes. Simultaneously, lysosome biosynthesis is stimulated by activation of TFEB, a transcriptional regulator of lysosomal biogenesis. Preventing lysosomal insertion causes phagosomal rupture, NLRP3 inflammasome activation, IL-1ß secretion and host-cell death. Whole-genome transcriptomic analysis demonstrate that stress responses elicited in C. albicans upon engulfment are reversed if phagosome expansion is prevented. Our findings reveal a mechanism whereby phagosomes maintain integrity while expanding, ensuring that growing pathogens remain entrapped within this microbicidal compartment.

Unconventional role of lysosomes in phagocytosis.

Lysosomes are generally thought to be required only for the late stages of phagosome maturation, providing the proton pumps (V-ATPases) and hydrolases needed to acidify and degrade the ingested prey. A recent paper by Davis et al. (EMBO J. [2020], doi:10.15252/embj.2019104058) reports the involvement of lysosomes at a much earlier stage, namely in scission of phagosomes from the plasma membrane. Here we analyze these findings, highlighting a number of unexpected observations and unresolved questions.

I want to break free - macrophage strategies to recognize and kill Candida albicans, and fungal counter-strategies to escape.

Candida albicans is a major cause of fungal nosocomial infections. Host defense against disseminated infections caused by this yeast strongly relies on myeloid cells of the innate immune system. Recently, several breakthroughs have been made that significantly improved our understanding of the role of macrophages during candidiasis and how C. albicans and macrophages interact. Resident tissue macrophages and macrophages derived from monocytes that infiltrate infected tissues are essential for the initiation of the antifungal immune response, as well as elimination of C. albicans from the bloodstream and infected organs. These cells engulf and try to eliminate the invading fungi through specialized mechanisms. Concurrently, C. albicans tries to survive the stresses imposed by the macrophage, acquires nutrients, and can break free from their captive environment. This review focuses on the most recent insights into the strategies of macrophages to eliminate C. albicans and the fungal counterstrategies to overcome these threats.

Determinants of Phagosomal pH During Host-Pathogen Interactions.

The ability of phagosomes to halt microbial growth is intimately linked to their ability to acidify their luminal pH. Establishment and maintenance of an acidic lumen requires precise co-ordination of H+ pumping and counter-ion permeation to offset the countervailing H+ leakage. Despite the best efforts of professional phagocytes, however, a number of specialized pathogens survive and even replicate inside phagosomes. In such instances, pathogens target the pH-regulatory machinery of the host cell in an effort to survive inside or escape from phagosomes. This review aims to describe how phagosomal pH is regulated during phagocytosis, why it varies in different types of professional phagocytes and the strategies developed by prototypical intracellular pathogens to manipulate phagosomal pH to survive, replicate, and eventually escape from the phagocyte.

A human antithrombin isoform dampens inflammatory responses and protects from organ damage during bacterial infection.

Severe infectious diseases are often characterized by an overwhelming and unbalanced systemic immune response to microbial infections. Human antithrombin (hAT) is a crucial coagulation inhibitor with anti-inflammatory activities. Here we identify three hAT-binding proteins (CD13, CD300f and LRP-1) on human monocytes that are involved in blocking the activity of nuclear factor-κB. We found that the modulating effect is primarily restricted to the less abundant ß-isoform (hßAT) of hAT that lacks N-glycosylation at position 135. Individuals with a mutation at this position have increased production of hßAT and analysis of their blood, which was stimulated ex vivo with lipopolysaccharide, showed a decreased inflammatory response. Similar findings were recorded when heterozygotic mice expressing hAT or hßAT were challenged with lipopolysaccharide or infected with Escherichia coli bacteria. Our results finally demonstrate that in a lethal E. coli infection model, survival rates increased when mice were treated with hßAT one hour and five hours after infection. The treatment also resulted in a reduction of the inflammatory response and less severe organ damage.

Revisiting the role of calcium in phagosome formation and maturation.

Like other membrane receptor-mediated responses, execution of phagocytosis requires the transduction of signals to cytoplasmic effectors. Signaling in this case is particularly complex as the process involves not only the formation of phagosomes but also their subsequent maturation and resolution. Transient increases in cytosolic calcium, which mediate a variety of other transduction pathways, also feature prominently in phagocytosis. However, despite intensive study over the course of nearly 30 years, the occurrence, source, and functional relevance of such calcium bursts remain the subject of debate. Here, we have attempted to consolidate the information that was reviewed in the past with more recent studies in an effort to shed some light on the existing controversies.

Integrity under stress: Host membrane remodelling and damage by fungal pathogens.

Membrane bilayers of eukaryotic cells are an amalgam of lipids and proteins that distinguish organelles and compartmentalise cellular functions. The mammalian cell has evolved mechanisms to sense membrane tension or damage and respond as needed. In the case of the plasma membrane and phagosomal membrane, these bilayers act as a barrier to microorganisms and are a conduit by which the host interacts with pathogens, including fungi such as Candida, Cryptococcus, Aspergillus, or Histoplasma species. Due to their size, morphological flexibility, ability to produce long filaments, secrete pathogenicity factors, and their potential to replicate within the phagosome, fungi can assault host membranes in a variety of physical and biochemical ways. In addition, the recent discovery of a fungal pore-forming peptide toxin further highlights the importance of membrane biology in the outcomes between host and fungal cells. In this review, we discuss the apparent "stretching" of membranes as a sophisticated biological response and the role of vesicular transport in combating membrane stress and damage. We also review the known pathogenicity factors and physical properties of fungal pathogens in the context of host membranes and discuss how this may contribute to pathogenic interactions between fungal and host cells.

Phagocytosis of Necrotic Debris at Sites of Injury and Inflammation.

Clearance of cellular debris is required to maintain the homeostasis of multicellular organisms. It is intrinsic to processes such as tissue growth and remodeling, regeneration and resolution of injury and inflammation. Most of the removal of effete and damaged cells is performed by macrophages and neutrophils through phagocytosis, a complex phenomenon involving ingestion and degradation of the disposable particles. The study of the clearance of cellular debris has been strongly biased toward the removal of apoptotic bodies; as a result, the mechanisms underlying the removal of necrotic cells have remained relatively unexplored. Here, we will review the incipient but growing knowledge of the phagocytosis of necrotic debris, from their recognition and engagement to their internalization and disposal. Critical insights into these events were gained recently through the development of new in vitro and in vivo models, along with advances in live-cell and intravital microscopy. This review addresses the classes of "find-me" and "eat-me" signals presented by necrotic cells and their cognate receptors in phagocytes, which in most cases differ from the extensively characterized counterparts in apoptotic cell engulfment. The roles of damage-associated molecular patterns, chemokines, lipid mediators, and complement components in recruiting and activating phagocytes are reviewed. Lastly, the physiological importance of necrotic cell removal is emphasized, highlighting the key role of impaired debris clearance in autoimmunity.

The fungal peptide toxin Candidalysin activates the NLRP3 inflammasome and causes cytolysis in mononuclear phagocytes.

Clearance of invading microbes requires phagocytes of the innate immune system. However, successful pathogens have evolved sophisticated strategies to evade immune killing. The opportunistic human fungal pathogen Candida albicans is efficiently phagocytosed by macrophages, but causes inflammasome activation, host cytolysis, and escapes after hypha formation. Previous studies suggest that macrophage lysis by C. albicans results from early inflammasome-dependent cell death (pyroptosis), late damage due to glucose depletion and membrane piercing by growing hyphae. Here we show that Candidalysin, a cytolytic peptide toxin encoded by the hypha-associated gene ECE1, is both a central trigger for NLRP3 inflammasome-dependent caspase-1 activation via potassium efflux and a key driver of inflammasome-independent cytolysis of macrophages and dendritic cells upon infection with C. albicans. This suggests that Candidalysin-induced cell damage is a third mechanism of C. albicans-mediated mononuclear phagocyte cell death in addition to damage caused by pyroptosis and the growth of glucose-consuming hyphae.

Candida albicans Hyphal Expansion Causes Phagosomal Membrane Damage and Luminal Alkalinization.

Macrophages rely on phagosomal acidity to destroy engulfed microorganisms. To survive this hostile response, opportunistic fungi such as Candida albicans developed strategies to evade the acidic environment. C. albicans is polymorphic and able to convert from yeast to hyphae, and this transition is required to subvert the microbicidal activity of the phagosome. However, the phagosomal lumen, which is acidic and nutrient deprived, is believed to inhibit the yeast-to-hypha transition. To account for this apparent paradox, it was recently proposed that C. albicans produces ammonia that alkalinizes the phagosome, thus facilitating yeast-to-hypha transition. We reexamined the mechanism underlying phagosomal alkalinization by applying dual-wavelength ratiometric pH measurements. The phagosomal membrane was found to be highly permeable to ammonia, which is therefore unlikely to account for the pH elevation. Instead, we find that yeast-to-hypha transition begins within acidic phagosomes and that alkalinization is a consequence of proton leakage induced by excessive membrane distension caused by the expanding hypha.IMPORTANCEC. albicans is the most common cause of nosocomial fungal infection, and over 3 million people acquire life-threatening invasive fungal infections every year. Even if antifungal drugs exist, almost half of these patients will die. Despite this, fungi remain underestimated as pathogens. Our study uses quantitative biophysical approaches to demonstrate that yeast-to-hypha transition occurs within the nutrient-deprived, acidic phagosome and that alkalinization is a consequence, as opposed to the cause, of hyphal growth.

Neutrophil extracellular trap-microparticle complexes enhance thrombin generation via the intrinsic pathway of coagulation in mice.

Abdominal sepsis is associated with dysfunctional hemostasis. Thrombin generation (TG) is a rate-limiting step in systemic coagulation. Neutrophils can expell neutrophil extracellular traps (NETs) and/or microparticles (MPs) although their role in pathological coagulation remains elusive. Cecal ligation and puncture (CLP)-induced TG in vivo was reflected by a reduced capacity of plasma from septic animals to generate thrombin. Depletion of neutrophils increased TG in plasma from CLP mice. Sepsis was associated with increased histone 3 citrullination in neutrophils and plasma levels of cell-free DNA and DNA-histone complexes and administration of DNAse not only eliminated NET formation but also elevated TG in sepsis. Isolated NETs increased TG and co-incubation with DNAse abolished NET-induced formation of thrombin. TG triggered by NETs was inhibited by blocking factor XII and abolished in factor XII-deficient plasma but intact in factor VII-deficient plasma. Activation of neutrophils simultaneously generated large amount of neutrophil-derived MPs, which were found to bind to NETs via histone-phosphatidylserine interactions. These findings show for the first time that NETs and MPs physically interact, and that NETs might constitute a functional assembly platform for MPs. We conclude that NET-MP complexes induce TG via the intrinsic pathway of coagulation and that neutrophil-derived MPs play a key role in NET-dependent coagulation.

The role of lipids in host-pathogen interactions.

Innate immunity relies on the effective recognition and elimination of pathogenic microorganisms. This entails sequestration of pathogens into phagosomes that promptly acquire microbicidal and degradative properties. This complex series of events, which involve cytoskeletal reorganization, membrane remodeling and the activation of multiple enzymes, is orchestrated by lipid signaling. To overcome this immune response, intracellular pathogens acquired mechanisms to subvert phosphoinositide-mediated signaling and use host lipids, notably cholesterol, as nutrients. We present brief overviews of the role of phosphoinositides in phagosome formation and maturation as well as of cholesterol handling by host cells, and selected Salmonella, Shigella, Chlamydia and Mycobacterium tuberculosis to exemplify the mechanisms whereby intracellular pathogens co-opt lipid metabolism in host cells. © 2018 IUBMB Life, 70(5):384-392, 2018.

Protein SIC Secreted from Streptococcus pyogenes Forms Complexes with Extracellular Histones That Boost Cytokine Production.

Innate immunity relies on an effective recognition of the pathogenic microorganism as well as on endogenous danger signals. While bacteria in concert with their secreted virulence factors can cause a number of inflammatory reactions, danger signals released at the site of infection may in addition determine the amplitude of such responses and influence the outcome of the disease. Here, we report that protein SIC, Streptococcal Inhibitor of Complement, an abundant secreted protein from Streptococcus pyogenes, binds to extracellular histones, a group of danger signals released during necrotizing tissue damage. This interaction leads to the formation of large aggregates in vitro. Extracellular histones and SIC are abundantly expressed and seen colocalized in biopsies from patients with necrotizing soft-tissue infections caused by S. pyogenes. In addition, binding of SIC to histones neutralized their antimicrobial activity. Likewise, the ability of histones to induce hemolysis was inhibited in the presence of SIC. However, when added to whole blood, SIC was not able to block the pro-inflammatory effect of histones. Instead SIC boosted the histone-triggered release of a broad range of cytokines and chemokines, including IL-6, TNF-α, IL-8, IL-1ß, IL-1ra, G-CSF, and IFN-γ. These results demonstrate that the interaction between SIC and histones has multiple effects on the host response to S. pyogenes infection.

Globular C1q receptor (p33) binds and stabilizes pro-inflammatory MCP-1: a novel mechanism for regulation of MCP-1 production and function.

The protein gC1qR (globular C1q receptor), also named p33, was originally identified as a binding partner of the globular heads of C1q in the complement system. gC1qR/p33 is abundantly expressed in many cell types, but the functional importance of this protein is not completely understood. Here, we investigate the impact of gC1qR/p33 on the production and function of the pathophysiologically important chemokine monocyte chemoattractant protein-1 (MCP-1) and the underlying molecular mechanisms. Knockdown of gC1qR/p33 negatively regulated the production of MCP-1, but had no effect on the expression of transcript for MCP-1 in human periodontal ligament cells, suggesting a translational/post-translational mechanism of action. Laser scanning confocal microscopy showed considerable cytosolic co-localization of gC1qR/p33 and MCP-1, and co-immunoprecipitation disclosed direct physical interaction between gC1qR/p33 and MCP-1. Surface plasmon resonance analysis revealed a high-affinity binding (KD = 10.9 nM) between gC1qR/p33 and MCP-1. Using a transwell migration assay, we found that recombinant gC1qR/p33 enhances MCP-1-induced migration of human THP-1 monocytes, pointing to a functional importance of the interaction between gC1qR/p33 and MCP-1. An in vitro assay revealed a rapid turnover of the MCP-1 protein and that gC1qR/p33 stabilizes MCP-1, hence preventing its degradation. We propose that endogenous gC1qR/p33 physically interacts with MCP-1 causing stabilization of the MCP-1 protein and stimulation of its activity in human periodontal ligament cells, suggesting a novel gC1qR/p33-mediated pro-inflammatory mechanism of action.

Immunoregulation of Neutrophil Extracellular Trap Formation by Endothelial-Derived p33 (gC1q Receptor).

The formation of neutrophil extracellular traps (NETs) is a host defence mechanism, known to facilitate the entrapment and growth inhibition of many bacterial pathogens. It has been implicated that the translocation of myeloperoxidase (MPO) from neutrophilic granules to the nucleus is crucial to this process. Under disease conditions, however, excessive NET formation can trigger self-destructive complications by releasing pathologic levels of danger-associated molecular pattern molecules (DAMPs). To counteract such devastating immune reactions, the host has to rely on precautions that help circumvent these deleterious effects. Though the induction of DAMP responses has been intensively studied, the mechanisms that are used by the host to down-regulate them are still not understood. In this study, we show that p33 is an endothelial-derived protein that has the ability to annul NET formation. We found that the expression of human p33 is up-regulated in endothelial cells upon infections with Streptococcus pyogenes bacteria. Using tissue biopsies from a patient with streptococcal necrotising fasciitis, we monitored co-localisation of p33 with MPO. Further in vitro studies revealed that p33 is able to block the formation of DAMP-induced NET formation by inhibiting the enzymatic activity of MPO. Additionally, mice challenged with S. pyogenes bacteria demonstrated diminished MPO activity when treated with p33. Together, our results demonstrate that host-derived p33 has an important immunomodulating function that helps to counterbalance an overwhelming DAMP response.