Candida albicansis a major life-threatening human fungal pathogen in the immunocompromised host. Host defense against systemicCandidainfection relies heavily on the capacity of professional phagocytes of the innate immune system to ingest and destroy fungal cells. A number of pathogens, includingC. albicans, have evolved mechanisms that attenuate the efficiency of phagosome-mediated inactivation, promoting their survival and replication within the host. Here we visualize host-pathogen interactions using live-cell imaging and show that viable, but not heat- or UV-killedC. albicanscells profoundly delay phagosome maturation in macrophage cell lines and primary macrophages. The ability ofC. albicansto delay phagosome maturation is dependent on cell wall composition and fungal morphology. Loss of cell wallO-mannan is associated with enhanced acquisition of phagosome maturation markers, distinct changes in Rab GTPase acquisition by the maturing phagosome, impaired hyphal growth within macrophage phagosomes, profound changes in macrophage actin dynamics, and ultimately a reduced ability of fungal cells to escape from macrophage phagosomes. The loss of cell wallO-mannan leads to exposure of β-glucan in the inner cell wall, facilitating recognition by Dectin-1, which is associated with enhanced phagosome maturation.IMPORTANCEInnate cells engulf and destroy invading organisms by phagocytosis, which is essential for the elimination of fungal cells to protect against systemic life-threatening infections. Yet comparatively little is known about what controls the maturation of phagosomes following ingestion of fungal cells. We used live-cell microscopy and fluorescent protein reporter macrophages to understand howC. albicansviability, filamentous growth, and cell wall composition affect phagosome maturation and the survival of the pathogen within host macrophages. We have demonstrated that cell wall glycosylation and yeast-hypha morphogenesis are required for disruption of host processes that function to inactivate pathogens, leading to survival and escape of this fungal pathogen from within host phagocytes. The methods employed here are applicable to study interactions of other pathogens with phagocytic cells to dissect how specific microbial features impact different stages of phagosome maturation and the survival of the pathogen or host.

Significance: Macrophages protect against microbial infection, in part by engulfing and killing invading microbes. Fungal pathogens such as Candida albicans are known to evade phagocytic killing by forming hyphae that are physically challenging to engulf because of their length. We now find that macrophages can respond by folding the hyphae of C. albicans (and other fungal species). Hyphal folding implies that immune cells can continue to apply mechanical force after their cargo has been internalized. The involvement of Dectin-1, β2-integrin, and actin–myosin polymerization provides initial mechanistic insight. Folding damages hyphae, inhibits their growth, and facilitates their complete engulfment. Therefore, hyphal folding represents an additional weapon in the immune cell armory that presumably contributes to fungal clearance.

Avoidance of innate immune defense is an important mechanism contributing to the pathogenicity of microorganisms. The fungal pathogenCandida albicansundergoes morphogenetic switching from the yeast to the filamentous hyphal form following phagocytosis by macrophages, facilitating its escape from the phagosome, which can result in host cell lysis. We show that the intracellular host trafficking GTPase Rab14 plays an important role in protecting macrophages from lysis mediated byC. albicanshyphae. Live-cell imaging of macrophages expressing green fluorescent protein (GFP)-tagged Rab14 or dominant negative Rab14, or with small interfering RNA (siRNA)-mediated knockdown of Rab14, revealed the temporal dynamics of this protein and its influence on the maturation of macrophage phagosomes following the engulfment ofC. albicanscells. Phagosomes containing liveC. albicanscells became transiently Rab14 positive within 2 min following engulfment. The duration of Rab14 retention on phagosomes was prolonged for hyphal cargo and was directly proportional to hyphal length. Interference with endogenous Rab14 did not affect the migration of macrophages towardC. albicanscells, the rate of engulfment, the overall uptake of fungal cells, or early phagosome processing. However, Rab14 depletion delayed the acquisition of the late phagosome maturation markers LAMP1 and lysosomal cathepsin, indicating delayed formation of a fully bioactive lysosome. This was associated with a significant increase in the level of macrophage killing byC. albicans. Therefore, Rab14 activity promotes phagosome maturation duringC. albicansinfection but is dysregulated on the phagosome in the presence of the invasive hyphal form, which favors fungal survival and escape.

The theoretical physicist and Nobel laureate Richard Feynman outlined in his 1959 lecture, “There’s plenty of room at the bottom”, the enormous possibility of producing and visualising things at smaller scales. The advent of advanced scanning and transmission electron microscopy and high-resolution microscopy has begun to open the door to visualise host-pathogen interactions at smaller scales, and spinning disc confocal and two-photon microscopy has improved our ability to study these events in real time in three dimensions. The aim of this review is to illustrate some of the advances in understanding host-fungal interactions that have been made in recent years in particular those relating to the interactions of live fungal pathogens with phagocytes. Dynamic imaging of host-pathogen interactions has recently revealed novel detail and unsuspected mechanistic insights, facilitating the dissection of the phagocytic process into its component parts. Here, we will highlight advances in our knowledge of host-fungal pathogen interactions, including the specific effects of fungal cell viability, cell wall composition and morphogenesis on the phagocytic process and try to define the relative contributions of neutrophils and macrophages to the clearance of fungal pathogens in vitro and the infected host.

Candida albicans is an opportunistic fungal pathogen that infects immunocompromised patients. Infection control requires phagocytosis by innate immune cells, including macrophages. Migration towards, and subsequent recognition of, C. albicans fungal cell wall components by macrophages is critical for phagocytosis. Using live-cell imaging of phagocytosis, the macrophage cell line J774.1 showed enhanced movement in response to C. albicans cell wall mutants, particularly during the first 30 min, irrespective of the infection ratio. However, phagocyte migration was reduced up to 2-fold within a C. albicans biofilm compared to planktonic fungal cells. Biofilms formed from C. albicans glycosylation mutant cells also inhibited macrophage migration to a similar extent as wildtype Candida biofilms, suggesting that the physical structure of the biofilm, rather than polysaccharide matrix composition, may hamper phagocyte migration. These data illustrate differential macrophage migratory capacities, dependent upon the form of C. albicans encountered. Impaired migration of macrophages within a C. albicans biofilm may contribute to the recalcitrant nature of clinical infections in which biofilm formation occurs.

• The rate of uptake of fungal cells by macrophages varies by up to 26-fold between different pathogenic fungal species. • Phagosome acidification times for macrophages varies by as much as 29-fold for different pathogenic fungi. • Heat-killing affects the kinetics of the interaction with macrophages in a species-dependent manner.