Sequestration of human cytomegalovirus by human renal and mammary epithelial cells

AbstractUrine and breast milk represent the main routes of human cytomegalovirus (HCMV) transmission but the contribution of renal and mammary epithelial cells to viral excretion remains unclear. We observed that kidney and mammary epithelial cells were permissive to HCMV infection and expressed immediate early, early and late antigens within 72h of infection. During the first 24h after infection, high titers of infectious virus were measured associated to the cells and in culture supernatants, independently of de novo synthesis of virus progeny. This phenomenon was not observed in HCMV-infected fibroblasts and suggested the sequestration and the release of HCMV by epithelial cells. This hypothesis was supported by confocal and electron microscopy analyses. The sequestration and progressive release of HCMV by kidney and mammary epithelial cells may play an important role in the excretion of the virus in urine and breast milk and may thereby contribute to HCMV transmission

Human high density lipoproteins are platforms for the assembly of multi-component innate immune complexes

Author Posting. © American Society for Biochemistry and Molecular Biology, 2005. This article is posted here by permission of American Society for Biochemistry and Molecular Biology for personal use, not for redistribution. The definitive version was published in Journal of Biological Chemistry 280 (2005): 32578-3258, doi:10.1074/jbc.M503510200.Human innate immunity to non-pathogenic species of African trypanosomes is provided by human high density lipoprotein (HDL) particles. Here we show that native human HDLs containing haptoglobin-related protein (Hpr), apolipoprotein L-I (apoL-I) and apolipoprotein A-I (apoA-I) are the principle antimicrobial molecules providing protection from trypanosome infection. Other HDL subclasses containing either apoA-I and apoL-I or apoA-I and Hpr have reduced trypanolytic activity, whereas HDL subclasses lacking apoL-I and Hpr are non-toxic to trypanosomes. Highly purified, lipid-free Hpr and apoL-I were both toxic to Trypanosoma brucei brucei but with specific activities at least 500-fold less than those of native HDLs, suggesting that association of these apolipoproteins within the HDL particle was necessary for optimal cytotoxicity. These studies show that HDLs can serve as platforms for the assembly of multiple synergistic proteins and that these assemblies may play a critical role in the evolution of primate-specific innate immunity to trypanosome infection.These studies were supported by National Institutes of Health Grants AI39033 and AI054496 and a grant from the Ellison Medical Foundation. Mass spectrometry was carried out at the University of Alabama at Birmingham Mass Spectrometry Shared Facility and was supported in part by NCI, National Institutes of Health Core Research Support Grant P30 CA 1314 to the University of Alabama at Birmingham Comprehensive Cancer Center

Structural asymmetry and discrete nucleic acid subdomains in the Trypanosoma brucei kinetoplast

The mitochondrial genome of Trypanosoma brucei is contained in a specialized structure termed the kinetoplast. Kinetoplast DNA (kDNA) is organized into a concatenated network of mini and maxicircles, positioned at the base of the flagellum, to which it is physically attached. Here we have used electron microscope cytochemistry to determine structural and functional domains involved in replication and segregation of the kinetoplast. We identified two distinct subdomains within the kinetoflagellar zone (KFZ) and show that the unilateral filaments are composed of distinct inner and outer filaments. Ethanolic phosphotungstic acid (E-PTA) and EDTA regressive staining indicate that basic proteins and DNA are major constituents of the inner unilateral filaments adjoining the kDNA disc. This evidence for an intimate connection of the unilateral filaments in the KFZ with DNA provides support for models of minicircle replication involving vectorial export of free minicircles into the KFZ. Unexpectedly however, detection of DNA in the KFZ throughout the cell cycle suggests that other processes involving kDNA occur in this domain. We also describe a hitherto unrecognized, intramitochondrial, filamentous structure rich in basic proteins that links the kDNA discs during their segregation and is maintained between them for an extended period of the cell cycle

Coupling of lysosomal and mitochondrial membrane permeabilization in trypanolysis by APOL1

Humans resist infection by the African parasite Trypanosoma brucei owing to the trypanolytic activity of the serum apolipoprotein L1 (APOL1). Following uptake by endocytosis in the parasite, APOL1 forms pores in endolysosomal membranes and triggers lysosome swelling. Here we show that APOL1 induces both lysosomal and mitochondrial membrane permeabilization (LMP and MMP). Trypanolysis coincides with MMP and consecutive release of the mitochondrial TbEndoG endonuclease to the nucleus. APOL1 is associated with the kinesin TbKIFC1, of which both the motor and vesicular trafficking VHS domains are required for MMP, but not for LMP. The presence of APOL1 in the mitochondrion is accompanied by mitochondrial membrane fenestration, which can be mimicked by knockdown of a mitochondrial mitofusin-like protein (TbMFNL). The BH3-like peptide of APOL1 is required for LMP, MMP and trypanolysis. Thus, trypanolysis by APOL1 is linked to apoptosis-like MMP occurring together with TbKIFC1-mediated transport of APOL1 from endolysosomal membranes to the mitochondrion

Persistent DNA Damage Foci and DNA Replication with a Broken Chromosome in the African Trypanosome

Damaged DNA typically imposes stringent controls on eukaryotic cell cycle progression, ensuring faithful transmission of genetic material. Some DNA breaks, and the resulting rearrangements, are advantageous, however. For example, antigenic variation in the parasitic African trypanosome, Trypanosoma brucei, relies upon homologous recombination-based rearrangements of telomeric variant surface glycoprotein (VSG) genes, triggered by breaks. Surprisingly, trypanosomes with a severed telomere continued to grow while progressively losing subtelomeric DNA, suggesting a nominal telomeric DNA damage checkpoint response. Here, we monitor the single-stranded DNA-binding protein replication protein A (RPA) in response to induced, locus-specific DNA breaks in T. brucei. RPA foci accumulated at nucleolar sites following a break within ribosomal DNA and at extranucleolar sites following a break elsewhere, including adjacent to transcribed or silent telomeric VSG genes. As in other eukaryotes, RPA foci were formed in S phase and H2A and RAD51 damage foci were disassembled prior to mitosis. Unlike in other eukaryotes, however, and regardless of the damaged locus, RPA foci persisted through the cell cycle, and these cells continued to replicate their DNA. We conclude that a DNA break, regardless of the damaged locus, fails to trigger a stringent cell cycle checkpoint in T. brucei. This DNA damage tolerance may facilitate the generation of virulence-enhancing genetic diversity, within subtelomeric domains in particular. Stringent checkpoints may be similarly lacking in some other eukaryotic cells. Importance: Chromosome damage must be repaired to prevent the proliferation of defective cells. Alternatively, cells with damage must be eliminated. This is true of human and several other cell types but may not be the case for single-celled parasites, such as trypanosomes. African trypanosomes, which cause lethal diseases in both humans and livestock, can actually exploit chromosomal damage to activate new surface coat proteins and to evade host immune responses, for example. We monitored responses to single chromosomal breaks in trypanosomes using a DNAbinding protein that, in response to DNA damage, forms nuclear foci visible using a microscope. Surprisingly, and unlike what is seen in mammalian cells, these foci persist while cells continue to divide. We also demonstrate chromosome replication even when one chromosome is broken. These results reveal a remarkable degree of damage tolerance in trypanosomes, which may suit the lifestyle of a single-celled parasite, potentially facilitating adaptation and enhancing virulence

Fat1 deletion promotes hybrid EMT state, tumour stemness and metastasis

FAT1, which encodes a protocadherin, is one of the most frequently mutated genes in human cancers1–5. However, the role and the molecular mechanisms by which FAT1 mutations control tumour initiation and progression are poorly understood. Here, using mouse models of skin squamous cell carcinoma and lung tumours, we found that deletion of Fat1 accelerates tumour initiation and malignant progression and promotes a hybrid epithelial-to-mesenchymal transition (EMT) phenotype. We also found this hybrid EMT state in FAT1-mutated human squamous cell carcinomas. Skin squamous cell carcinomas in which Fat1 was deleted presented increased tumour stemness and spontaneous metastasis. We performed transcriptional and chromatin profiling combined with proteomic analyses and mechanistic studies, which revealed that loss of function of FAT1 activates a CAMK2–CD44–SRC axis that promotes YAP1 nuclear translocation and ZEB1 expression that stimulates the mesenchymal state. This loss of function also inactivates EZH2, promoting SOX2 expression, which sustains the epithelial state. Our comprehensive analysis identified drug resistance and vulnerabilities in FAT1-deficient tumours, which have important implications for cancer therapy. Our studies reveal that, in mouse and human squamous cell carcinoma, loss of function of FAT1 promotes tumour initiation, progression, invasiveness, stemness and metastasis through the induction of a hybrid EMT state

Automated Nuclear Analysis of Leishmania major Telomeric Clusters Reveals Changes in Their Organization during the Parasite's Life Cycle

Parasite virulence genes are usually associated with telomeres. The clustering of the telomeres, together with their particular spatial distribution in the nucleus of human parasites such as Plasmodium falciparum and Trypanosoma brucei, has been suggested to play a role in facilitating ectopic recombination and in the emergence of new antigenic variants. Leishmania parasites, as well as other trypanosomes, have unusual gene expression characteristics, such as polycistronic and constitutive transcription of protein-coding genes. Leishmania subtelomeric regions are even more unique because unlike these regions in other trypanosomes they are devoid of virulence genes. Given these peculiarities of Leishmania, we sought to investigate how telomeres are organized in the nucleus of Leishmania major parasites at both the human and insect stages of their life cycle. We developed a new automated and precise method for identifying telomere position in the three-dimensional space of the nucleus, and we found that the telomeres are organized in clusters present in similar numbers in both the human and insect stages. While the number of clusters remained the same, their distribution differed between the two stages. The telomeric clusters were found more concentrated near the center of the nucleus in the human stage than in the insect stage suggesting reorganization during the parasite's differentiation process between the two hosts. These data provide the first 3D analysis of Leishmania telomere organization. The possible biological implications of these findings are discussed