Stage Specific Assessment of Candida albicans Phagocytosis by Macrophages Identifies Cell Wall Composition and Morphogenesis as Key Determinants

Abstract

Candida albicans is a major life-threatening human fungal pathogen. Host defence against systemic Candida infection relies mainly on phagocytosis of fungal cells by cells of the innate immune system. In this study, we have employed video microscopy, coupled with sophisticated image analysis tools, to assess the contribution of distinct C. albicans cell wall components and yeast-hypha morphogenesis to specific stages of phagocytosis by macrophages. We show that macrophage migration towards C. albicans was dependent on the glycosylation status of the fungal cell wall, but not cell viability or morphogenic switching from yeast to hyphal forms. This was not a consequence of differences in maximal macrophage track velocity, but stems from a greater percentage of macrophages pursuing glycosylation deficient C. albicans during the first hour of the phagocytosis assay. The rate of engulfment of C. albicans attached to the macrophage surface was significantly delayed for glycosylation and yeast-locked morphogenetic mutant strains, but enhanced for non-viable cells. Hyphal cells were engulfed at a slower rate than yeast cells, especially those with hyphae in excess of 20 µm, but there was no correlation between hyphal length and the rate of engulfment below this threshold. We show that spatial orientation of the hypha and whether hyphal C. albicans attached to the macrophage via the yeast or hyphal end were also important determinants of the rate of engulfment. Breaking down the overall phagocytic process into its individual components revealed novel insights into what determines the speed and effectiveness of C. albicans phagocytosis by macrophages.

Live-cell Video Microscopy of Fungal Pathogen Phagocytosis

“…More importantly, the method described enables differential analysis of the individual stages of phagocytosis. We have, for example, shown that changes in overall uptake of C. albicans glycosylation and morphogenesis mutants by macrophage cell lines and primary macrophages can be either a consequence of changes in macrophage migration towards target cells or rate of engulfment once cell-cell contact is established 7 .…”

“…However, target size is an important determinant for feasibility, as image analysis becomes increasingly difficult with a reduction in target cell size. Sophisticated image analysis software is essential for rapid video microscopy analysis, and this should be coupled with appropriate bioinformatics support to facilitate the generation of algorithms to study migration of individual cells and entire cell populations 7 . Live-cell video microscopy in combination with sophisticated image analysis software for the minute-by-minute analysis of migration and individual macrophage-C.albicans interactions, provides unique insight into the complexity of C. albicans phagocytosis by macrophages.…”

“…These studies have revealed novel aspects of phagocytosis that could only be observed using systematic temporal analysis of the molecular and cellular interactions between human phagocytes and fungal pathogens and infectious microorganisms more generally. For example, we have begun to define the following: (a) the components of the cell surface required for each stage of the process of recognition, engulfment and killing of fungal cells 1,6,7,8 ; (b) how surface geometry influences the efficiency of macrophage uptake and killing of yeast and hyphal cells 7 ; and (c) how engulfment leads to alteration of the cell cycle and behavior of macrophages 9,10 . In contrast to single time point snapshots, live-cell video microscopy enables a wide variety of host cells and pathogens to be studied as continuous sequences over lengthy time periods, providing spatial and temporal information on a broad range of dynamic processes, including cell migration, replication and vesicular trafficking.…”

Evidence, illness, and causation: An epidemiological perspective on the Russo–Williamson Thesis

“…Its prevalence may be growing, however due to the use of technologies that enable continuous monitoring of biological processes. One recent example of activity-based mechanistic evidence comes from the article, "Stage Specific (Lewis, Bain et al 2012). Supporting video S3 from this article uses video microscopy to show a macrophage moving toward and subsequently engulfing a C. albicans yeast cell.…”

Caracterização das bases moleculares da suscetibilidade imunológica à infecção por Candida albicans

“…As taxas de fagocitose encontram-se dentro do descrito para este modelo, sendo que com trinta minutos de interação, já era possível observar taxas de fagocitose próximas à máxima decorrente de maiores tempos de cocultivo, conforme descrito na literatura (Lewis et al, 2012). A fagocitose é um fenômeno importante pois várias vias de transdução de sinal dependem da internalização da célula fúngica para serem ativadas (Bourgeois et al, 2011;Kagan et al, 2008 Choi et al, 2014;Longo et al, 2014;Lowe et al, 2014;Tierney et al, 2012).…”