A Gß protein and the TupA Co-Regulator Bind to Protein Kinase A Tpk2 to Act as Antagonistic Molecular Switches of Fungal Morphological Changes.

The human pathogenic fungus Paracoccidioides brasiliensis (Pb) undergoes a morphological transition from a saprobic mycelium to pathogenic yeast that is controlled by the cAMP-signaling pathway. There is a change in the expression of the Gß-protein PbGpb1, which interacts with adenylate cyclase, during this morphological transition. We exploited the fact that the cAMP-signaling pathway of Saccharomyces cerevisiae does not include a Gß-protein to probe the functional role of PbGpb1. We present data that indicates that PbGpb1 and the transcriptional regulator PbTupA both bind to the PKA protein PbTpk2. PbTPK2 was able to complement a TPK2Δ strain of S. cerevisiae, XPY5a/α, which was defective in pseudohyphal growth. Whilst PbGPB1 had no effect on the parent S. cerevisiae strain, MLY61a/α, it repressed the filamentous growth of XPY5a/α transformed with PbTPK2, behaviour that correlated with a reduced expression of the floculin FLO11. In vitro, PbGpb1 reduced the kinase activity of PbTpk2, suggesting that inhibition of PbTpk2 by PbGpb1 reduces the level of expression of Flo11, antagonizing the filamentous growth of the cells. In contrast, expressing the co-regulator PbTUPA in XPY5a/α cells transformed with PbTPK2, but not untransformed cells, induced hyperfilamentous growth, which could be antagonized by co-transforming the cells with PbGPB1. PbTUPA was unable to induce the hyperfilamentous growth of a FLO8Δ strain, suggesting that PbTupA functions in conjunction with the transcription factor Flo8 to control Flo11 expression. Our data indicates that P. brasiliensis PbGpb1 and PbTupA, both of which have WD/ß-propeller structures, bind to PbTpk2 to act as antagonistic molecular switches of cell morphology, with PbTupA and PbGpb1 inducing and repressing filamentous growth, respectively. Our findings define a potential mechanism for controlling the morphological switch that underpins the virulence of dimorphic fungi.

Analgesia via blockade of NGF/TrkA signaling does not influence fracture healing in mice.

Abatement of fracture-related pain is important in patient welfare. However, the frequently used non-steroidal anti-inflammatory drugs are considered to impair fracture healing through blockade of cyclooxygenase-2. An alternative for fracture-related pain treatment may be blockade of nerve growth factor (NGF)/neurotrophic tyrosine kinase receptor type 1 (TrkA) signaling. Because the effect of blocking this signal-pathway on bone healing has not been extensively investigated, we addressed this issue by applying neutralizing antibodies that target NGF and TrkA, respectively, in a mouse fracture model. Mice with a knock-in for human TrkA underwent femur osteotomy and were randomly allocated to phosphate-buffered-saline, anti-NGF-antibody, or anti-TrkA-antibody treatment. The analgesic effect of the antibodies was determined from the activity and the ground reaction force of the operated limb. The effect of antibody administration on fracture healing was assessed by histomorphometry, micro-computed tomography, and biomechanics. NGF/TrkA-signaling blockade had no negative effect on fracture healing as callus formation and maturation were not altered. Mice treated with anti-TrkA antibody displayed significantly greater activity on post-operative day 2 compared to PBS treatment indicating effective analgesia. Our data indicate, that blockade of NGF/TrkA signaling via specific neutralizing antibodies for pain reduction during fracture healing does not influence fracture healing.

Evidence for the assembly of a bacterial tripartite multidrug pump with a stoichiometry of 3:6:3.

The multiple transferable resistance (mTR) pump from Neisseria gonorrhoeae MtrCDE multidrug pump is assembled from the inner and outer membrane proteins MtrD and MtrE and the periplasmic membrane fusion protein MtrC. Previously we established that while there is a weak interaction of MtrD and MtrE, MtrC binds with relatively high affinity to both MtrD and MtrE. MtrD conferred antibiotic resistance only when it was expressed with MtrE and MtrC, suggesting that these proteins form a functional tripartite complex in which MtrC bridges MtrD and MtrE. Furthermore, we demonstrated that MtrC interacts with an intraprotomer groove on the surface of MtrE, inducing channel opening. However, a second groove is apparent at the interface of the MtrE subunits, which might also be capable of engaging MtrC. We have now established that MtrC can be cross-linked to cysteines placed in this interprotomer groove and that mutation of residues in the groove impair the ability of the pump to confer antibiotic resistance by locking MtrE in the closed channel conformation. Moreover, MtrE K390C forms an intermolecular disulfide bond with MtrC E149C locking MtrE in the open channel conformation, suggesting that a functional salt bridge forms between these residues during the transition from closed to open channel conformations. MtrC forms dimers that assemble into hexamers, and electron microscopy studies of single particles revealed that these hexamers are arranged into ring-like structures with an internal aperture sufficiently large to accommodate the MtrE trimer. Cross-linking of single cysteine mutants of MtrC to stabilize the dimer interface in the presence of MtrE, trapped an MtrC-MtrE complex with a molecular mass consistent with a stoichiometry of 3:6 (MtrE(3)MtrC(6)), suggesting that dimers of MtrC interact with MtrE, presumably by binding to the two grooves. As both MtrE and MtrD are trimeric, our studies suggest that the functional pump is assembled with a stoichiometry of 3:6:3.

MacB ABC transporter is a dimer whose ATPase activity and macrolide-binding capacity are regulated by the membrane fusion protein MacA.

Gram-negative bacteria utilize specialized machinery to translocate drugs and protein toxins across the inner and outer membranes, consisting of a tripartite complex composed of an inner membrane secondary or primary active transporter (IMP), a periplasmic membrane fusion protein, and an outer membrane channel. We have investigated the assembly and function of the MacAB/TolC system that confers resistance to macrolides in Escherichia coli. The membrane fusion protein MacA not only stabilizes the tripartite assembly by interacting with both the inner membrane protein MacB and the outer membrane protein TolC, but also has a role in regulating the function of MacB, apparently increasing its affinity for both erythromycin and ATP. Analysis of the kinetic behavior of ATP hydrolysis indicated that MacA promotes and stabilizes the ATP-binding form of the MacB transporter. For the first time, we have established unambiguously the dimeric nature of a noncanonic ABC transporter, MacB that has an N-terminal nucleotide binding domain, by means of nondissociating mass spectrometry, analytical ultracentrifugation, and atomic force microscopy. Structural studies of ABC transporters indicate that ATP is bound between a pair of nucleotide binding domains to stabilize a conformation in which the substrate-binding site is outward-facing. Consequently, our data suggest that in the presence of ATP the same conformation of MacB is promoted and stabilized by MacA. Thus, MacA would facilitate the delivery of drugs by MacB to TolC by enhancing the binding of drugs to it and inducing a conformation of MacB that is primed and competent for binding TolC. Our structural studies are an important first step in understanding how the tripartite complex is assembled.

alpha-cleavage of the prion protein occurs in a late compartment of the secretory pathway and is independent of lipid rafts.

Endoproteolysis of the cellular prion protein (PrP(C)) modulates both the normal function of the protein and the pathogenesis of the neurodegenerative prion diseases. PrP(C) undergoes alpha-cleavage to generate the N-terminally truncated fragment C1. Utilizing various constructs of PrP(C) expressed in human neuroblastoma cells we investigated the subcellular compartment where alpha-cleavage occurs. C1 was detected at the cell surface and the generation of C1 occurred in mutants of PrP(C) incapable of Cu2+-mediated endocytosis. A transmembrane-anchored form that is not lipid raft-localised, as well as a secreted construct lacking the glycosyl-phosphatidylinositol membrane anchor, were also subject to alpha-cleavage. However, when this transmembrane-anchored form was modified with an endoplasmic reticulum retention motif, C1 was not formed. Inhibition of protein export from the Golgi by temperature block increased the amount of C1. Our data thus demonstrate that the alpha-cleavage of PrP(C) occurs predominantly in a raft-independent manner in a late compartment of the secretory pathway.

The cAMP pathway is important for controlling the morphological switch to the pathogenic yeast form of Paracoccidioides brasiliensis.

Paracoccidioides brasiliensis is a human pathogenic fungus that switches from a saprobic mycelium to a pathogenic yeast. Consistent with the morphological transition being regulated by the cAMP-signalling pathway, there is an increase in cellular cAMP levels both transiently at the onset (< 24 h) and progressively in the later stages (> 120 h) of the transition to the yeast form, and this transition can be modulated by exogenous cAMP. We have cloned the cyr1 gene encoding adenylate cyclase (AC) and established that its transcript levels correlate with cAMP levels. In addition, we have cloned the genes encoding three Galpha (Gpa1-3), Gbeta (Gpb1) and Ggamma (Gpg1) G proteins. Gpa1 and Gpb1 interact with one another and the N-terminus of AC, but neither Gpa2 nor Gpa3 interacted with Gpb1 or AC. The interaction of Gpa1 with Gpb1 was blocked by GTP, but its interaction with AC was independent of bound nucleotide. The transcript levels for gpa1, gpb1 and gpg1 were similar in mycelium, but there was a transient excess of gpb1 during the transition, and an excess of gpa1 in yeast. We have interpreted our findings in terms of a novel signalling mechanism in which the activity of AC is differentially modulated by Gpa1 and Gpb1 to maintain the signal over the 10 days needed for the morphological switch.

Elucidation of the hrp clusters of Xanthomonas oryzae pv. oryzicola that control the hypersensitive response in nonhost tobacco and pathogenicity in susceptible host rice.

Xanthomonas oryzae pv. oryzicola, the cause of bacterial leaf streak in rice, possesses clusters of hrp genes that determine its ability to elicit a hypersensitive response (HR) in nonhost tobacco and pathogenicity in host rice. A 27-kb region of the genome of X. oryzae pv. oryzicola (RS105) was identified and sequenced, revealing 10 hrp, 9 hrc (hrp conserved), and 8 hpa (hrp-associated) genes and 7 regulatory plant-inducible promoter boxes. While the region from hpa2 to hpaB and the hrpF operon resembled the corresponding genes of other xanthomonads, the hpaB-hrpF region incorporated an hrpE3 gene that was not present in X. oryzae pv. oryzae. We found that an hrpF mutant had lost the ability to elicit the HR in tobacco and pathogenicity in adult rice plants but still caused water-soaking symptoms in rice seedlings and that Hpa1 is an HR elicitor in nonhost tobacco whose expression is controlled by an hrp regulator, HrpX. Using an Hrp phenotype complementation test, we identified a small hrp cluster containing the hrpG and hrpX regulatory genes, which is separated from the core hrp cluster. In addition, we identified a gene, prhA (plant-regulated hrp), that played a key role in the Hrp phenotype of X. oryzae pv. oryzicola but was neither in the core hrp cluster nor in the hrp regulatory cluster. A prhA mutant failed to reduce the HR in tobacco and pathogenicity in rice but caused water-soaking symptoms in rice. This is the first report that X. oryzae pv. oryzicola possesses three separate DNA regions for HR induction in nonhost tobacco and pathogenicity in host rice, which will provide a fundamental base to understand pathogenicity determinants of X. oryzae pv. oryzicola compared with those of X. oryzae pv. oryzae.

Ectodomain shedding of human Nogo-66 receptor homologue-1 by zinc metalloproteinases.

The Nogo-66 receptor (NgR) plays a pivotal role in the inhibition of neuroregeneration as the receptor for multiple neurite outgrowth inhibitors such as Nogo-A. We have previously shown that NgR undergoes zinc metalloproteinase-mediated ectodomain shedding in neuroblastoma cells. Here, we demonstrate that the NgR-related protein NgR homologue-1 is released from neuroblastoma cells as a full-length ectodomain (NgRH1-ecto) and an N-terminal fragment (NTF-NgRH1) containing the leucine-rich repeat region of the protein. Inhibitors of the major protease classes failed to block the release of NgRH1-ecto, suggesting that this occurs via a protease-independent mechanism, presumably by a phospholipase-like enzyme. The release of NTF-NgRH1 was blocked by a hydroxamate-based zinc metalloproteinase inhibitor and tissue inhibitor of metalloproteinases-2 and -3, but not -1, implicating the involvement of membrane-type matrix metalloproteinases in this process. Our findings thus highlight the parallels between the ectodomain shedding of NgRH1 and that previously described for NgR.

Structural understanding of efflux-mediated drug resistance: potential routes to efflux inhibition.

The active efflux of cytotoxic drugs mediated by multidrug transporters is the basis of multidrug resistance in prokaryotic and eukaryotic cells. Individual multidrug transporters can be extremely versatile, often exhibiting a staggering range of substrate specificity that can negate the effects of clinically relevant therapies. The effective treatment of bacterial, fungal and protozoan infections, along with certain cancer treatments, has been compromised by the presence of multidrug transporters. Traditionally, advances in the understanding of multidrug transporters have been made through biochemical analyses; more recently, however, fundamental advances have been made with the elucidation of several three dimensional structures of representative multidrug pumps. Biochemical and structural analysis of multidrug pumps could lead to the development of novel 'anti-efflux' therapies.

Zinc metalloproteinase-mediated cleavage of the human Nogo-66 receptor.

The central nervous system myelin components oligodendrocyte-myelin glycoprotein, myelin-associated glycoprotein and the Nogo-66 domain of Nogo-A inhibit neurite outgrowth by binding the neuronal glycosyl-phosphatidylinositol-anchored Nogo-66 receptor (NgR) that transduces the inhibitory signal to the cell interior via a transmembrane co-receptor, p75NTR. Here, we demonstrate that human NgR expressed in human neuroblastoma cells is constitutively cleaved in a post-ER compartment to generate a lipid-raft associated C-terminal fragment that is present on the cell surface and a soluble N-terminal fragment that is released into the medium. Mass spectrometric analysis demonstrated that the N-terminal fragment terminated just after the C-terminus of the ligand-binding domain of NgR. In common with other shedding mechanisms, the release of this fragment was blocked by a hydroxamate-based inhibitor of zinc metalloproteinases, but not by inhibitors of other protease classes and up-regulated by treatment with the cellular cholesterol depleting agent methyl-beta-cyclodextrin. The N-terminal fragment bound Nogo-66 and blocked Nogo-66 binding to cell surface NgR but failed to associate with p75NTR, indicative of a role as a Nogo-66 antagonist. Furthermore, the N- and C-terminal fragments of NgR were detectable in human brain cortex and the N-terminal fragment was also present in human cerebrospinal fluid, demonstrating that NgR proteolysis occurs within the human nervous system. Our findings thus identify a potential cellular mechanism for the regulation of NgR function at the level of the receptor.

Structure and function of efflux pumps that confer resistance to drugs.

Resistance to therapeutic drugs encompasses a diverse range of biological systems, which all have a human impact. From the relative simplicity of bacterial cells, fungi and protozoa to the complexity of human cancer cells, resistance has become problematic. Stated in its simplest terms, drug resistance decreases the chance of providing successful treatment against a plethora of diseases. Worryingly, it is a problem that is increasing, and consequently there is a pressing need to develop new and effective classes of drugs. This has provided a powerful stimulus in promoting research on drug resistance and, ultimately, it is hoped that this research will provide novel approaches that will allow the deliberate circumvention of well understood resistance mechanisms. A major mechanism of resistance in both microbes and cancer cells is the membrane protein-catalysed extrusion of drugs from the cell. Resistant cells exploit proton-driven antiporters and/or ATP-driven ABC (ATP-binding cassette) transporters to extrude cytotoxic drugs that usually enter the cell by passive diffusion. Although some of these drug efflux pumps transport specific substrates, many are transporters of multiple substrates. These multidrug pumps can often transport a variety of structurally unrelated hydrophobic compounds, ranging from dyes to lipids. If we are to nullify the effects of efflux-mediated drug resistance, we must first of all understand how these efflux pumps can accommodate a diverse range of compounds and, secondly, how conformational changes in these proteins are coupled to substrate translocation. These are key questions that must be addressed. In this review we report on the advances that have been made in understanding the structure and function of drug efflux pumps.

The pfr1 gene from the human pathogenic fungus Paracoccidioides brasiliensis encodes a half-ABC transporter that is transcribed in response to treatment with fluconazole.

We have isolated a gene that encodes a half-ABC-transporter, designated Pfr1, from the dimorphic human pathogenic fungus Paracoccidioides brasiliensis, which has high identity with members of the ABC-superfamily involved in multidrug resistance. The pfr1 gene is predicted to encode a 827 amino acid protein that, in common with mammalian Mdr1, has a TM-NBD topology. The transcription of the pfr1 gene is induced by the triazole drug fluconazole but not by amphotericin B, suggesting a role in transport-mediated azole resistance. However, Pfr1 has greatest identity to the mitochondrial ABC transporters Mdl1 and Mdl2 from Saccharomyces cerevisiae and mammalian ABC-me, with identities of 47.2%, 40.6% and 39.5%, respectively, over the length of these proteins. Furthermore, the N-terminus of Pfr1 is rich in positively charged residues, a feature of mitochondrial targeting sequences. Considering these features, it seems likely that Pfr1 is a mitochondrial protein. Previous studies have revealed that the acquisition of azole resistance in S. cerevisiae is linked to mitochondrial loss and, conversely, that mitochondrial dysfunction can lead to the upregulation of PDR transporters mediated by the transcription factor Pdr3. Our studies suggest that a mitochondrial ABC transporter is induced as part of the cellular response to drug treatment. The promoter region of pfr1 contains a PDRE-like consensus sequence to which Pdr3 binds, which may be the element responsible for the upregulation of Pfr1 in response to fluconazole. The nucleotide binding domain of Pfr1 was expressed and purified from Escherichia coli and shown to retain ATPase activity, consistent with Pfr1 functioning as a homodimeric transport ATPase.

The N-terminal region of the prion protein ectodomain contains a lipid raft targeting determinant.

The association of the prion protein (PrP) with sphingolipid- and cholesterol-rich lipid rafts is instrumental in the pathogenesis of the neurodegenerative prion diseases. Although the glycosylphosphatidylinositol (GPI) anchor is an exoplasmic determinant of raft association, PrP remained raft-associated in human neuronal cells even when the GPI anchor was deleted or substituted for a transmembrane anchor indicating that the ectodomain contains a raft localization signal. The raft association of transmembrane-anchored PrP occurred independently of Cu(II) binding as it failed to be abolished by either deletion of the octapeptide repeat region (residues 51-90) or treatment of cells with a Cu(II) chelator. Raft association of transmembrane-anchored PrP was only abolished by the deletion of the N-terminal region (residues 23-90) of the ectodomain. This region was sufficient to confer raft localization when fused to the N terminus of a non-raft transmembrane-anchored protein and suppressed the clathrin-coated pit localization signal in the cytoplasmic domain of the amyloid precursor protein. These data indicate that the N-terminal region of PrP acts as a cellular raft targeting determinant and that residues 23-90 of PrP represent the first proteinaceous raft targeting signal within the ectodomain of a GPI-anchored protein.

Glycosylation efficiency of Asn-Xaa-Thr sequons is independent of distance from the C-terminus in membrane dipeptidase.

In vitro transcription/translation studies with model proteins have shown that glycosylation of Asn-Xaa-Thr sequons is reduced when the sequon is within 60 residues of the C-terminus of the protein. We have previously shown that in living cells N-glycosylation of the prion protein (PrP) is also abolished when its Asn-Ile-Thr and Asn-Phe-Thr sequons are less than 60 residues from the C-terminus (Walmsley and Hooper [2003] Biochemical Journal, 370, 351-355). To investigate whether sequon distance to the C-terminus is a general determinant of N-glycosylation in living cells, Asn-Ile/Phe-Thr sequons were introduced into another glycosylphosphatidylinositol (GPI) anchored protein, membrane dipeptidase (MDP), at similar distances from the C-terminus as those in PrP. When expressed in the human neuroblastoma SH-SY5Y cell line, the introduced sequons were fully N-glycosylated even when they were less than 60 residues from the C-terminus in both GPI-anchored and secreted forms of MDP. These data demonstrate that the utilization of sequons in some proteins is independent of their distance from the C-terminus.

Tethering the N-terminus of the prion protein compromises the cellular response to oxidative stress.

The role of the N-terminal half of the prion protein (PrPC) in normal cellular function and pathology remains enigmatic. To investigate the biological role of the N-terminus of PrP, we examined the cellular properties of a construct of murine PrP, PrP-DA, in which the N-terminus is tethered to the membrane by an uncleaved signal peptide and which retains the glycosyl-phosphatidylinositol anchor. Human neuroblastoma SH-SY5Y cells expressing PrP-DA were more susceptible to hydrogen peroxide and copper induced toxicity than wtPrP expressing cells. The PrP-DA expressing cells had an increased level of intracellular free radicals and reduced levels of superoxide dismutase and glutathione peroxidase as compared to the wtPrP expressing cells. The membrane topology, cell surface location, lipid raft localisation, intracellular trafficking and copper-mediated endocytosis of PrP-DA were not significantly different from wtPrP. However, cells expressing PrP-DA accumulated an N-terminal fragment that was resistant to proteinase K. The data presented here are consistent with the N-terminal region of PrPC having a role in the cellular response to oxidative stress, and that tethering this region of the protein to the membrane compromises this function through the accumulation of a protease-resistant N-terminal fragment, similar to that seen in some forms of human prion disease.

Distance of sequons to the C-terminus influences the cellular N-glycosylation of the prion protein.

Cell-specific differences in the utilization of the two N-glycosylation sequons (Asn180-Ile-Thr and Asn196-Phe-Thr) of the prion protein (PrP) have been proposed to influence the aetiology of the neurodegenerative prion diseases. As the N-glycosylation of PrP is ablated by deletion of the C-terminal glycosyl-phosphatidylinositol (GPI) anchor signal sequence, we have investigated the determinants for PrP sequon utilization in human neuronal cells using the novel approach of restoring N-glycosylation to secreted forms of PrP lacking a GPI anchor. N-glycosylation was restored to an efficiency comparable with that of GPI anchored PrP when the distance of the sequon to the C-terminus was increased so that it was sufficient to reach the active site of oligosaccharyltransferase before chain termination. Our findings indicate that sequon utilization in PrP is a co-translational process that precedes GPI anchor addition and, as such, will be greatly influenced by the dynamics of the translocon-oligosaccharyltransferase complex.

Evidence for cooperativity between the four binding sites of dimeric ArsD, an As(III)-responsive transcriptional regulator.

ArsD is a trans-acting repressor of the arsRDABC operon that confers resistance to arsenicals and antimonials in Escherichia coli. It possesses two-pairs of vicinal cysteine residues, Cys(12)-Cys(13) and Cys(112)-Cys(113), that potentially form separate binding sites for the metalloids that trigger dissociation of ArsD from the operon. However, as a homodimer it has four vicinal cysteine pairs. Titration of the steady-state fluorescence of ArsD with metalloids revealed positive cooperativity, with a Hill coefficient of 2, between these sites. Disruption of the Cys(112)-Cys(113) site by mutagenesis of arsD, but not the Cys(12)-Cys(13) site, largely abolished this cooperativity, indicative of interactions between adjacent Cys(112)-Cys(113) sites within the dimer. The kinetics of metalloid binding were determined by stopped flow spectroscopy; the rate increased in a sigmoidal manner, with a Hill coefficient of 4, indicating that the pre-steady-state measurements reported cooperativity between all four sites of the dimer rather than just the intermolecular interactions reported by the steady-state measurements. The kinetics of Sb(III) displacement by As(III) revealed that the metalloid-binding sites behave differentially, with the rapid exchange of As(III) for Sb(III) at one site retarding the release of Sb(III) from the other sites. We propose a model involving the sequential binding and release of metalloids by the four binding sites of dimeric ArsD, with only one site releasing free metalloids.

TAPAS-1, a novel microdomain within the unique N-terminal region of the PDE4A1 cAMP-specific phosphodiesterase that allows rapid, Ca2+-triggered membrane association with selectivity for interaction with phosphatidic acid.

Here we identify an 11-residue helical module in the unique N-terminal region of the cyclic AMP-specific phosphodiesterase PDE4A1 that determines association with phospholipid bilayers and shows a profound selectivity for interaction with phosphatidic acid (PA). This module contains a core bilayer insertion unit that is formed by two tryptophan residues, Trp(19) and Trp(20), whose orientation is optimized for bilayer insertion by the Leu(16):Val(17) pairing. Ca(2+), at submicromolar levels, interacts with Asp(21) in this module and serves to gate bilayer insertion, which is completed within 10 ms. Selectivity for interaction with PA is suggested to be achieved primarily through the formation of a charge network of the form (Asp(21-):Ca(2+):PA(2-):Lys(24+)) with overall neutrality at the bilayer surface. This novel phospholipid-binding domain, which we call TAPAS-1 (tryptophan anchoring phosphatidic acid selective-binding domain 1), is here identified as being responsible for membrane association of the PDE4A1 cAMP-specific phosphodiesterase. TAPAS-1 may not only serve as a paradigm for other PA-binding domains but also aid in detecting related phospholipid-binding domains and in generating simple chimeras for conferring membrane association and intracellular targeting on defined proteins.

The pathobiology of Paracoccidioides brasiliensis.

Paracoccidioides brasiliensis causes one of the most prevalent systemic mycoses in Latin America--paracoccidioidomycosis. It is a dimorphic fungus that undergoes a complex transformation in vivo, with mycelia in the environment producing conidia, which probably act as infectious propagules upon inhalation into the lungs, where they transform to the pathogenic yeast form. This transition is readily induced in vitro by temperature changes, resulting in modulation of the composition of the cell wall. Notably, the polymer linkages change from beta-glucan to alpha-glucan, possibly to avoid beta-glucan triggering the inflammatory response. Mammalian oestrogens inhibit this transition, giving rise to a higher incidence of disease in males. Furthermore, the susceptibility of individuals to paracoccidioidomycosis has a genetic basis, which results in a depressed cellular immune response in susceptible patients; resistance is conferred by cytokine-stimulated granuloma formation and nitric oxide production. The latency period and persistence of the disease and the apparent lack of efficacy of humoral immunity are consistent with P. brasiliensis existing as a facultative intracellular pathogen.