The Human Fungal Pathogen Cryptococcus neoformans Escapes Macrophages by a Phagosome Emptying Mechanism That Is Inhibited by Arp2/3 Complex-Mediated Actin Polymerisation

The lysis of infected cells by disease-causing microorganisms is an efficient but risky strategy for disseminated infection, as it exposes the pathogen to the full repertoire of the host's immune system. Cryptococcus neoformans is a widespread fungal pathogen that causes a fatal meningitis in HIV and other immunocompromised patients. Following intracellular growth, cryptococci are able to escape their host cells by a non-lytic expulsive mechanism that may contribute to the invasion of the central nervous system. Non-lytic escape is also exhibited by some bacterial pathogens and is likely to facilitate long-term avoidance of the host immune system during latency. Here we show that phagosomes containing intracellular cryptococci undergo repeated cycles of actin polymerisation. These actin ‘flashes’ occur in both murine and human macrophages and are dependent on classical WASP-Arp2/3 complex mediated actin filament nucleation. Three dimensional confocal imaging time lapse revealed that such flashes are highly dynamic actin cages that form around the phagosome. Using fluorescent dextran as a phagosome membrane integrity probe, we find that the non-lytic expulsion of Cryptococcus occurs through fusion of the phagosome and plasma membranes and that, prior to expulsion, 95% of phagosomes become permeabilised, an event that is immediately followed by an actin flash. By using pharmacological agents to modulate both actin dynamics and upstream signalling events, we show that flash occurrence is inversely related to cryptococcal expulsion, suggesting that flashes may act to temporarily inhibit expulsion from infected phagocytes. In conclusion, our data reveal the existence of a novel actin-dependent process on phagosomes containing cryptococci that acts as a potential block to expulsion of Cryptococcus and may have significant implications for the dissemination of, and CNS invasion by, this organism.\ud \u

FRET Detection of Lymphocyte Function-Associated Antigen-1 Conformational Extension

Lymphocyte function-associated antigen 1 (LFA-1, CD11a/CD18, αLβ2-integrin) and its ligands are essential for adhesion between T-cells and antigen-presenting cells, formation of the immunological synapse, and other immune cell interactions. LFA-1 function is regulated through conformational changes that include the modulation of ligand binding affinity and molecular extension. However, the relationship between molecular conformation and function is unclear. Here fluorescence resonance energy transfer (FRET) with new LFA-1-specific fluorescent probes showed that triggering of the pathway used for T-cell activation induced rapid unquenching of the FRET signal consistent with extension of the molecule. Analysis of the FRET quenching at rest revealed an unexpected result that can be interpreted as a previously unknown LFA-1 conformation

Tyrosine kinase activity and remodelling of the actin cytoskeleton are co-temporally required for degranulation by cytotoxic T lymphocytes

In this study, we examined the contribution of the actin cytoskeleton to T-cell receptor (TCR)-initiated signalling in cytotoxic T lymphocytes (CTLs). We demonstrate that cytoskeletal remodelling is required for sustaining TCR-stimulated signals that lead to degranulation by CTLs. Disruption of the actin cytoskeleton in CTLs already undergoing signalling responses results in an almost immediate loss of essentially all protein tyrosine phosphorylation. This signal reversal is not restricted to tyrosine phosphorylation, as disruption of the actin cytoskeleton also reverses the phosphorylation of the more downstream serine/threonine kinase extracellular signal regulated kinase (Erk). An intact cytoskeleton and cell spreading are not sufficient for maintaining signals, as stabilization of actin filaments, at a point when peak tyrosine phosphorylation is occurring, also leads to the rapid loss of protein tyrosine phosphorylation. Disruption of tyrosine kinase activity after TCR signals are maximally induced causes the immediate reversal of tyrosine phosphorylation as well as cytoskeletal disruption, as indicated by loss of cell spreading, adhesion and CTL degranulation. Taken together, our results indicate that actin remodelling occurs co-temporally with ongoing tyrosine kinase activity, leading to CTL degranulation. We hypothesize that continuous actin remodelling is important for sustaining productive signals, even after downstream signalling molecules such as Erk have been activated, and that the actin cytoskeleton is not solely required for initiating and maintaining the T cell in contact with its stimulus

Adaptor protein 3-dependent microtubule-mediated movement of lytic granules to the immunological synapse

Hermansky-Pudlak syndrome (HPS) is a rare autosomal recessive disease characterized by platelet defects and oculocutaneous albinism. Individuals with HPS type 2 (HPS2) lack the cytosolic adaptor protein 3 (AP-3) involved in lysosomal sorting, and are also immunodeficient. Here we characterize an HPS2 mutation and demonstrate that AP-3 deficiency leads to a loss of cytotoxic T lymphocyte (CTL)-mediated cytotoxicity. Although the lysosomal protein CD63 was mislocalized to the plasma membrane, perforin and granzymes were correctly localized to the lytic granules in AP-3-deficient CTLs. However, the lytic granules of AP-3-deficient CTLs were enlarged and were unable to move along microtubules and dock within the secretory domain of the immunological synapse. These data show that AP-3 is essential for polarized secretion from CTLs