Transcription-dependent spatial arrangements of CFTR and adjacent genes in human cell nuclei.

We investigated in different human cell types nuclear positioning and transcriptional regulation of the functionally unrelated genes GASZ, CFTR, and CORTBP2, mapping to adjacent loci on human chromosome 7q31. When inactive, GASZ, CFTR, and CORTBP2 preferentially associated with the nuclear periphery and with perinuclear heterochromatin, whereas in their actively transcribed states the gene loci preferentially associated with euchromatin in the nuclear interior. Adjacent genes associated simultaneously with these distinct chromatin fractions localizing at different nuclear regions, in accordance with their individual transcriptional regulation. Although the nuclear localization of CFTR changed after altering its transcription levels, the transcriptional status of CFTR was not changed by driving this gene into a different nuclear environment. This implied that the transcriptional activity affected the nuclear positioning, and not vice versa. Together, the results show that small chromosomal subregions can display highly flexible nuclear organizations that are regulated at the level of individual genes in a transcription-dependent manner.

Characterisation of the phagocytic uptake of Aspergillus fumigatus conidia by macrophages.

Aspergillus fumigatus is an opportunistic fungal pathogen responsible for severe, life-threatening infections in immunocompromised patients. Airborne conidia are the infectious agent and can reach the lower parts of the respiratory system. In the lung, phagocytes represent the first line of defence. Resident macrophages are able to track down, engulf and kill the invading spores. Phagocytosis of the conidia is therefore a prerequisite for their efficient elimination. Using human and murine macrophages we analysed the phagocytic uptake of A. fumigatus conidia. We found that conidial phagocytosis is an actin-depending process that additionally requires myosin motor, phosphoinositide-3-phosphate kinase and tyrosine kinase activity. Both broad range tyrosine kinase inhibitors and inhibitors that specifically block src kinases had a strong impact on the conidial uptake. Immunofluorescence data demonstrate the recruitment of tyrosine-phosphorylated proteins to the vicinity of engulfed conidia. Uptake of the conidia was accompanied by a strong and local reorganisation of the actin cytoskeleton, whereas no prominent reorganisation was apparent for the microtubules. Both confocal immunofluorescence and electron microscopic data revealed the presence of large ruffle-like structures engaged in the uptake of conidia. This suggests that the internalisation of A. fumigatus spores can be mediated by a process resembling macropinocytosis, which is furthermore supported by the detection of intracellular conidia within spacious vacuoles. Taken together, our data provide new insights into the internalisation of A. fumigatus spores by macrophages, a key process in the early immune defence against an Aspergillus infection.

Toll-like receptors: Recent advances, open questions and implications for aspergillosis control.

Aspergillus fumigatus is a pathogenic mould that can cause severe and life-threatening infections in immunocompromised patients. Apart from novel and improved antifungals, additional strategies are required to protect patients at risk from developing invasive aspergillosis. Given the problems in diagnosis of this disease, important perspectives lie in attempts to elicit and strengthen a protective immunity. The innate immune system is the first line of defence against A. fumigatus. Phagocytes engulf and kill inhaled conidia, but also closely communicate with the adaptive immune system. Recognition of invading microbes is mediated by pattern recognition receptors (PRRs), and Toll-like receptors (TLR) 2 and TLR4 have been implicated in the immune response to A. fumigatus. The analysis of this process is hampered by the fact that A. fumigatus infections are inevitably coupled to germination resulting in the appearance of different fungal morphotypes, like conidia and hyphae. While conflicting data still exist on the relative importance of TLR2 and 4 in recognition of distinct A. fumigatus morphotypes, recent evidence suggests that certain TLR agonists can be used to divert the immune response towards an optimal fungicidal activity in the absence of detrimental inflammatory consequences.

Quantification of phagocytosis of Aspergillus conidia by macrophages using a novel antibody-independent assay.

The pathogenic mould Aspergillus fumigatus can cause severe infections in immunocompromised patients. Phagocytosis of inhaled conidia is an early and crucial event in the defense of A. fumigatus infections. Here we describe a novel antibody-independent assay for quantification of phagocytosis, that in this study has been applied to different Aspergillus species, but that is in principle suitable for many fungi.

Phagocytosis of Aspergillus fumigatus conidia by murine macrophages involves recognition by the dectin-1 beta-glucan receptor and Toll-like receptor 2.

Aspergillus fumigatus is a fungal pathogen causing severe infections in immunocompromised patients. For clearance of inhaled conidia, an efficient response of the innate immune system is required. Macrophages represent the first line of defence and ingest and kill conidia. C-type lectins represent a family of receptors, which recognize pathogen-specific carbohydrates. One of them is beta1-3 glucan, a major component of the fungal cell wall. Here we provide evidence that beta1-3 glucan plays an important role for the elimination of A. fumigatus conidia. Laminarin, a soluble beta1-3 glucan and antibodies to dectin-1, a well known beta1-3 glucan receptor, significantly inhibited conidial phagocytosis. On resting conidia low amounts of surface accessible beta1-3 glucan were detected, whereas high amounts were found on small spores that appear early during germination and infection as well as on resting conidia of a pksP mutant strain. Swollen conidia also display larger quantities of beta1-3 glucan, although in an irregular spotted pattern. Resting pksP mutant conidia and swollen wild-type conidia are phagocytosed with high efficiency thereby confirming the relevance of beta1-3 glucans for conidial phagocytosis. Additionally we found that TLR2 and the adaptor protein MyD88 are required for efficient conidial phagocytosis, suggesting a link between the TLR2-mediated recognition of A. fumigatus and the phagocytic response.