Proteomic analysis of Legionella-containing phagosomes isolated from Dictyostelium.

Legionella pneumophila, the agent of Legionnaires' disease, replicates intracellularly within specialized phagosomes of human macrophages and amoebae. In this study, we have developed a protocol for the isolation of Legionella-containing phagosomes from Dictyostelium discoideum. Cell fractionation, two-dimensional gel electrophoresis and MALDI-TOF MS combined with genomic data identified 157 phagosome host proteins. In addition to proteins with an evident role in phagosome maturation, we identified proteins for which a function remains to be elucidated. Possible interactions of coronin with cytosolic NADPH oxidase components and protein kinase C inhibitors which together may lead to an inhibition of phagosomal superoxide generation are discussed. Comparative proteomics of phagosomes containing highly virulent L. pneumophila Corby versus less virulent L. hackeliae revealed distinctive protein expression patterns, e.g., an abundance of RhoGDI in L. hackeliae degrading phagosomes versus little RhoGDI in L. pneumophila Corby replicative phagosomes. We present a kinetic dissection of phagosome maturation including the complex alterations of the phagosome protein composition. A reference flow chart suggests so far unrecognized consequences of infection for host cell physiology, actin degradation on phagosomes, and a putative cysteine proteinase inhibitor interference with lysosomal enzyme sorting and activation processes.

Cytoplasmic polyadenylation-element-binding protein (CPEB)1 and 2 bind to the HIF-1alpha mRNA 3'-UTR and modulate HIF-1alpha protein expression.

The heterodimeric HIF (hypoxia-inducible factor)-1 is a transcriptional master regulator of several genes involved in mammalian oxygen homoeostasis. Besides the well described regulation of the HIF-1alpha subunit via hydroxylation-mediated protein stability in hypoxia, there are several indications of an additional translational control of the HIF-1alpha mRNA, especially after growth factor stimulation. We identified an interaction of CPEB (cytoplasmic polyadenylation-element-binding protein) 1 and CPEB2 with the 3'-UTR (untranslated region) of HIF-1alpha mRNA. Overexpression of CPEB1 and CPEB2 affected HIF-1alpha protein levels mediated by the 3'-UTR of HIF-1alpha mRNA. Stimulation of neuroblastoma SK-N-MC cells with insulin and thus activation of endogenous CPEBs increased the expression of a luciferase reporter gene fused to the 3'-UTR of HIF-1alpha as well as endogenous HIF-1alpha protein levels. This could be abrogated by treating the cells with CPEB1 or CPEB2 siRNAs (short interfering RNAs). Injection of HIF-1alpha cRNA into Xenopus oocytes verified the elongation of the poly(A)+ (polyadenylated) tail by cytoplasmic polyadenylation. Thus CPEB1 and CPEB2 are involved in the regulation of HIF-1alpha following insulin stimulation.

Enhanced hypoxia susceptibility in hippocampal slices from a mouse model of rett syndrome.

Rett syndrome is a neurodevelopmental disorder caused by mutations in the X-chromosomal MECP2 gene encoding for the transcriptional regulator methyl CpG binding protein 2 (MeCP2). Rett patients suffer from episodic respiratory irregularities and reduced arterial oxygen levels. To elucidate whether such intermittent hypoxic episodes induce adaptation/preconditioning of the hypoxia-vulnerable hippocampal network, we analyzed its responses to severe hypoxia in adult Rett mice. The occurrence of hypoxia-induced spreading depression (HSD)--an experimental model for ischemic stroke--was hastened in Mecp2-/y males. The extracellular K+ rise during HSD was attenuated in Mecp2-/y males and the input resistance of CA1 pyramidal neurons decreased less before HSD onset. CA1 pyramidal neurons were smaller and more densely packed, but the cell swelling during HSD was unaffected. The intrinsic optical signal and the propagation of HSD were similar among the different genotypes. Basal synaptic function was intact, but Mecp2-/y males showed reduced paired-pulse facilitation and higher field potential/fiber volley ratios, but no increased seizure susceptibility. Synaptic failure during hypoxia was complete in all genotypes and the final degree of posthypoxic synaptic recovery indistinguishable. Cellular ATP content was normal in Mecp2-/y males, but their hematocrit was increased as was HIF-1alpha expression throughout the brain. This is the first study showing that in Rett syndrome, the susceptibility of telencephalic neuronal networks to hypoxia is increased; the underlying molecular mechanisms apparently involve disturbed K+ channel function. Such an increase in hypoxia susceptibility may potentially contribute to the vulnerability of male Rett patients who are either not viable or severely disabled.

TSGA10 prevents nuclear localization of the hypoxia-inducible factor (HIF)-1alpha.

The hypoxia-inducible factor (HIF)-1 is a transcriptional regulator of genes involved in oxygen homeostasis. We previously described testis-specific isoforms of HIF-1alpha (mHIF-1alphaI.1 and hHIF-1alphaTe). Using mHIF-1alpha exon I.1 knock-out mice we confirmed the specific expression of mHIF-1alphaI.1 in the sperm tail. A protein-protein interaction between HIF-1alpha and the testis specific gene antigen 10 (TSGA10) was identified by yeast two-hybrid screening. TSGA10 is expressed in testis but also in other organs and malignant tissues. Immunofluorescence analysis indicated that the C-terminal part of TSGA10 accumulates in the midpiece of spermatozoa, where it co-localizes with HIF-1alpha. HIF-1alpha nuclear localization and HIF-1 transcriptional activity were significantly affected by overexpressed TSGA10.

A dominant-negative isoform of hypoxia-inducible factor-1 alpha specifically expressed in human testis.

Spermatogenesis in the seminiferous tubuli of the testis occurs under a high proliferation rate, suggesting considerable oxygen consumption. Because of the lack of blood vessels, the oxygen partial pressure in the lumen of these tubuli is very low. We previously identified a testis isoform of the hypoxia-inducible factor (HIF)-1alpha in the mouse, termed mHIF-1alphaI.1. Here, we demonstrate that expression of mHIF-1alphaI.1 increases during puberty, further demonstrating its gene induction in postmeiotic germ cells. Using 5'-rapid amplification of cDNA ends, we identified a novel HIF-1alpha isoform in the human testis, called hHIF-1alphaTe. Like mHIF-1alphaI.1, hHIF-1alphaTe mRNA is derived from an alternative promoter-first exon combination, but with a different genomic organization and a different nucleotide sequence. Reverse transcription-polymerase chain reaction analysis confirmed that hHIF-1alphaTe is exclusively expressed in the testis. As determined by immunofluorescence of ejaculated sperm cells, HIF-1alpha protein is mainly localized in the postacrosomal head and in the midpiece of spermatozoa. Though overlapping with mitochondrial localization in human and mouse spermatozoa, neither hHIF-1alphaTe nor hHIF-1alpha associated with mitochondria. In contrast with the ubiquitously expressed HIF-1alpha protein and the mouse testis-specific mHIF-1alphaI.1 isoform, the hHIF-1alphaTe mRNA sequence predicts a protein with an N-terminal truncation of the DNA-binding domain. As shown by yeast two-hybrid assays, hHIF-1alphaTe still formed heterodimeric complexes with HIF-1beta. However, hHIF-1alphaTe was incapable of forming a DNA-binding HIF-1 complex. Overexpression of exogenous hHIF-1alphaTe resulted in the inhibition of the endogenous HIF-1 transcriptional activity, demonstrating that the testis-specific hHIF-1alphaTe isoform is a dominant-negative regulator of normal HIF-1 activity.

Various bacterial pathogens and symbionts infect the amoeba Dictyostelium discoideum.

The haploid soil amoeba Dictyostelium discoideum is a suitable model organism to study host-pathogen interactions with Legionella pneumophila. In this study we show that D. discoideum AX2 is also susceptible to infection with other important human pathogens and obligate intracellular symbionts. Infection assays demonstrated that Legionella-like amoebal pathogens (LLAP K62), Mycobacterium avium and the obligate intracellular endosymbionts of Acanthamoeba sp. strains TUME1, UWE25 and UWC6 were able to multiply within Dictyostelium. Salmonella typhimurium and Pseudomonas aeruginosa also invaded Dictyostelium, however were degraded shortly after uptake. Comitin-minus host cells were more permissive to infections with L. pneumophila and LLAP K62. Furthermore, this mutation significantly delayed the degradation of S. typhimurium. Accompanying electron and fluorescence microscopy of infected AX2 cells revealed that L. pneumophila and M. avium replicate within vacuoles, while LLAP K62, TUME1 and UWE25 were tightly enclosed by membranous structures within the cytoplasm. The beta-proteobacterium UWC6 was found to persist in the cytoplasm. The observed subcellular locations which correspond to the locations within the respective natural hosts suggest that D. discoideum is a representative model system for these pathogens and symbionts.