Improved split-ubiquitin screening technique to identify surface membrane protein-protein interactions.

Yeast-based methods are still the workhorse for the detection of protein-protein interactions (PPIs) in vivo. Yeast two-hybrid (Y2H) systems, however, are limited to screening for a specific group of molecules that interact in a particular cell compartment. For this reason, the split-ubiquitin system (SUS) was developed to allow screening of cDNA libraries of full-length membrane proteins for protein-protein interactions in Saccharomyces cerevisiae. Here we demonstrate that a modification of the widely used membrane SUS involving the transmembrane (TM) domain of the yeast receptor Wsc1 increases the stringency of screening and improves the selectivity for proteins localized in the plasma membrane (PM).

Resistance to intranasal infection with Balamuthia mandrillaris amebae is T-cell dependent.

T and B cell-deficient BALB/c SCID mice become severely ill and die of amebic encephalitis after intranasal infection with Balamuthia mandrillaris, while adult immunocompetent BALB/c wild-type (WT) mice are resistant. To further investigate the role of lymphocytes in protection from Balamuthia amebic encephalitis (BAE), SCID mice were reconstituted with and WT mice selectively depleted of lymphocytes before infection. Reconstitution of SCID mice with whole spleen cells from WT mice rendered the recipients as resistant to BAE as WT mice. SCID mice that had received spleen cells depleted of CD4(+) T cells remained susceptible. When adult WT mice were depleted of both CD4(+) and CD8(+) T cells or of CD4(+) T cells alone, these mice also became susceptible to BAE. Depletion of CD8(+) T cells alone increased susceptibility only marginally. All morbidity and mortality data were underpinned by histological analysis of the brain.

Oral infection of immunocompetent and immunodeficient mice with Balamuthia mandrillaris amebae.

Balamuthia mandrillaris is an opportunistic agent of lethal granulomatous amebic encephalitis (GAE). In mice, we have shown that intranasally instilled B. mandrillaris amebae infect the brain via the olfactory nerve pathway. In this study, we raised the question whether this ameba might also reach the brain after an oral/gastrointestinal infection. Immunocompetent (WT) and immunodeficient (RAG) mice received B. mandrillaris amebae by gavage into the esophagus. Mice of both groups became ill and some died (WT 20%, RAG 40%) within 42 days. All orally infected mice revealed B. mandrillaris amebae in the central nervous system. Outwardly intact amebae and/or specific antigen were found widely distributed in various organs and the stool. The data indicate that oral infection with B. mandrillaris leading to GAE is possible. Exit from the gastrointestinal tract and dissemination remains unresolved. Though stool cultures were negative, transmission of this highly pathogenic ameba via stool cannot be ruled out.

Cytopathogenicity of Balamuthia mandrillaris, an opportunistic causative agent of granulomatous amebic encephalitis.

Balamuthia mandrillaris is a free-living ameba and an opportunistic agent of lethal granulomatous amebic encephalitis in humans and other mammals. Balamuthia mandrillaris is highly cytopathic but, in contrast to the related Acanthamoeba, does not feed on bacteria and seems to feed only on eukaryotic cells instead. Most likely, the cytopathogenicity of B. mandrillaris is inseparable from its infectivity and pathogenicity. To better understand the mechanisms of B. mandrillaris cytopathogenicity, an assay for measuring amebic cytolytic activity was adapted that is based on the release of a reporter enzyme by damaged target cells. The ameba is shown to lyse murine mastocytoma cells very efficiently in a time- and dose-related manner. Furthermore, experiments involving semipermeable membranes and phagocytosis inhibitors indicate that the cytolytic activity of B. mandrillaris is essentially cell contact-dependent. Standard and fluorescence light microscopy, as well as scanning and transmission electron microscopy support and extend these findings at the ultrastructural level.

Balamuthia mandrillaris, free-living ameba and opportunistic agent of encephalitis, is a potential host for Legionella pneumophila bacteria.

Balamuthia mandrillaris is a free-living ameba and an opportunistic agent of granulomatous encephalitis in humans and other mammalian species. Other free-living amebas, such as Acanthamoeba and Hartmannella, can provide a niche for intracellular survival of bacteria, including the causative agent of Legionnaires' disease, Legionella pneumophila. Infection of amebas by L. pneumophila enhances the bacterial infectivity for mammalian cells and lung tissues. Likewise, the pathogenicity of amebas may be enhanced when they host bacteria. So far, the colonization of B. mandrillaris by bacteria has not been convincingly shown. In this study, we investigated whether this ameba could host L. pneumophila bacteria. Our experiments showed that L. pneumophila could initiate uptake by B. mandrillaris and could replicate within the ameba about 4 to 5 log cycles from 24 to 72 h after infection. On the other hand, a dotA mutant, known to be unable to propagate in Acanthamoeba castellanii, also did not replicate within B. mandrillaris. Approaching completion of the intracellular cycle, L. pneumophila wild-type bacteria were able to destroy their ameboid hosts. Observations by light microscopy paralleled our quantitative data and revealed the rounding, collapse, clumping, and complete destruction of the infected amebas. Electron microscopic studies unveiled the replication of the bacteria in a compartment surrounded by a structure resembling rough endoplasmic reticulum. The course of intracellular infection, the degree of bacterial multiplication, and the ultrastructural features of a L. pneumophila-infected B. mandrillaris ameba resembled those described for other amebas hosting Legionella bacteria. We hence speculate that B. mandrillaris might serve as a host for bacteria in its natural environment.

Balamuthia mandrillaris, an opportunistic agent of granulomatous amebic encephalitis, infects the brain via the olfactory nerve pathway.

Balamuthia mandrillaris is a free-living ameba and an opportunistic agent of lethal granulomatous amebic encephalitis (GAE) in humans and other mammals. Its supposed routes of infection have been largely assumed from what is known about Acanthamoeba spp. and Naegleria fowleri, other free-living amebae and opportunistic encephalitis agents. However, formal proof for any migratory pathway, from GAE patients or from animal models, has been lacking. Here, immunodeficient mice were infected with B. mandrillaris amebae by intranasal instillation, the most likely natural portal of entry. By means of classical and immunohistology, the amebae are shown to adhere to the nasal epithelium, progress along the olfactory nerves, traverse the cribriform plate of the ethmoid bone, and finally infect the brain. A similar invasion pathway has been described for N. fowleri. The data suggest that the olfactory nerve pathway is a likely route for natural infection of the brain by B. mandrillaris amebae.

Characterisation of the gptA gene, encoding UDP N-acetylglucosamine: dolichol phosphate N-acetylglucosaminylphosphoryl transferase, from the filamentous fungus, Aspergillus niger.

The production of asparagine (N)-linked oligosaccharides is of vital importance in the formation of glycosylated proteins in eukaryotes and is mediated by the dolichol pathway. As part of studies to allow manipulation of this pathway, the gene coding for the production of the enzyme UDP N-acetylglucosamine: dolichol phosphate N-acetylglucosaminylphosphoryl transferase (GPT), catalysing the first step in the assembly of dolichol-linked oligosaccharides, was cloned from the filamentous fungus Aspergillus niger. Degenerate-PCR was used to amplify a 470-bp fragment of the gene, which was labelled as a probe to obtain a full-length clone from a genomic library of A. niger. This contained a 1557-bp open reading frame encoding a highly hydrophobic protein of 468 amino acids with a predicted molecular weight of 51.4 kDa. The gene contained two intron sequences and putative dolichol recognition sites (PDRSs) were present in the deduced amino acid sequence. Comparison with other eukaryotic GPTs revealed the A. niger GPT to share 45-47% identity with yeasts (Saccharomyces cerevisiae and Schizosaccharomyces pombe) and 41-42% identity with mammals (mouse, hamster, human). Nested-PCR of a cDNA library was used to confirm the position of an intron. A complete cDNA clone of A. niger gpt was obtained by employing a recombinant PCR approach. This was used to rescue a conditional lethal mutant of S. cerevisiae carrying a dysfunctional gpt gene by heterologous expression, confirming that the gpt genes from A. niger and S. cerevisiae are functionally equivalent.