A role for the unfolded protein response (UPR) in virulence and antifungal susceptibility in Aspergillus fumigatus.

Filamentous fungi rely heavily on the secretory pathway, both for the delivery of cell wall components to the hyphal tip and the production and secretion of extracellular hydrolytic enzymes needed to support growth on polymeric substrates. Increased demand on the secretory system exerts stress on the endoplasmic reticulum (ER), which is countered by the activation of a coordinated stress response pathway termed the unfolded protein response (UPR). To determine the contribution of the UPR to the growth and virulence of the filamentous fungal pathogen Aspergillus fumigatus, we disrupted the hacA gene, encoding the major transcriptional regulator of the UPR. The DeltahacA mutant was unable to activate the UPR in response to ER stress and was hypersensitive to agents that disrupt ER homeostasis or the cell wall. Failure to induce the UPR did not affect radial growth on rich medium at 37 degrees C, but cell wall integrity was disrupted at 45 degrees C, resulting in a dramatic loss in viability. The DeltahacA mutant displayed a reduced capacity for protease secretion and was growth-impaired when challenged to assimilate nutrients from complex substrates. In addition, the DeltahacA mutant exhibited increased susceptibility to current antifungal agents that disrupt the membrane or cell wall and had attenuated virulence in multiple mouse models of invasive aspergillosis. These results demonstrate the importance of ER homeostasis to the growth and virulence of A. fumigatus and suggest that targeting the UPR, either alone or in combination with other antifungal drugs, would be an effective antifungal strategy.

Metallomic analysis of macrophages infected with Histoplasma capsulatum reveals a fundamental role for zinc in host defenses.

The fungal pathogen Histoplasma capsulatum evades the innate and adaptive immune responses and thrives within resting macrophages. Cytokines that induce antimicrobial activity, such as granulocyte macrophage colony-stimulating factor (GM-CSF), inhibit H. capsulatum growth in macrophages. Conversely, interleukin 4 inhibits the killing of intracellular pathogens. Using inductively coupled plasma mass spectrometry, we examined alterations in the metal homeostasis of murine H. capsulatum-infected macrophages that were exposed to activating cytokines. Decreases in the levels of iron (Fe(2+) and Fe(3+)) and zinc (Zn(2+)) were observed in infected, GM-CSF-treated macrophages compared with those in infected controls. Interleukin 4 reversed the antifungal activity of GM-CSF-activated macrophages and was associated with increased intracellular Zn(2+) levels. Chelation of Zn(2+) inhibited yeast replication in both the absence of macrophages and the presence of macrophages. Treatment of cells with GM-CSF altered the host Zn(2+) binding species profile. These results establish that Zn(2+) deprivation may be a host defense mechanism utilized by macrophages.

Paracoccidioides brasiliensis enolase is a surface protein that binds plasminogen and mediates interaction of yeast forms with host cells.

Paracoccidioidomycosis (PCM), caused by the dimorphic fungus Paracoccidioides brasiliensis, is a disseminated, systemic disorder that involves the lungs and other organs. The ability of the pathogen to interact with host components, including extracellular matrix (ECM) proteins, is essential to further colonization, invasion, and growth. Previously, enolase (EC 4.2.1.11) was characterized as a fibronectin binding protein in P. brasiliensis. Interaction of surface-bound enolase with plasminogen has been incriminated in tissue invasion for pathogenesis in several pathogens. In this paper, enolase was expressed in Escherichia coli as a recombinant glutathione S-transferase (GST) fusion protein (recombinant P. brasiliensis enolase [rPbEno]). The P. brasiliensis native enolase (PbEno) was detected at the fungus surface and cytoplasm by immunofluorescence with an anti-rPbEno antibody. Immobilized purified rPbEno bound plasminogen in a specific, concentration-dependent fashion. Both native enolase and rPbEno activated conversion of plasminogen to plasmin through tissue plasminogen activator. The association between PbEno and plasminogen was lysine dependent. In competition experiments, purified rPbEno, in its soluble form, inhibited plasminogen binding to fixed P. brasiliensis, suggesting that this interaction required surface-localized PbEno. Plasminogen-coated P. brasiliensis yeast cells were capable of degrading purified fibronectin, providing in vitro evidence for the generation of active plasmin on the fungus surface. Exposure of epithelial cells and phagocytes to enolase was associated with an increased expression of surface sites of adhesion. In fact, the association of P. brasiliensis with epithelial cells and phagocytes was increased in the presence of rPbEno. The expression of PbEno was upregulated in yeast cells derived from mouse-infected tissues. These data indicate that surface-associated PbEno may contribute to the pathogenesis of P. brasiliensis.

Dynamic structural changes during complement C3 activation analyzed by hydrogen/deuterium exchange mass spectrometry.

Proteolytic cleavage of component C3 to C3b is a central step in the activation of complement. Whereas C3 is largely biologically inactive, C3b is directly involved in various complement activities. While the recently described crystal structures of C3 and C3b provide a molecular basis of complement activation, they do not reflect the dynamic changes that occur in solution. In addition, the available C3b structures diverge in some important aspects. Here we have utilized hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) to investigate relative changes in the solution-phase structures of C3 and C3b. By combining two forms of mass spectrometry we could maximize the primary sequence coverage of C3b and demonstrate the feasibility of this method for large plasma proteins. While the majority of the 82 peptides that could be followed over time showed only minor alterations in HDX, we observed clear changes in solvent accessibility for 16 peptides, primarily in the alpha-chain (alpha'NT, MG6-8, CUB, TED, C345C domains). Most of these peptides could be directly linked to the structural transitions visible in the crystal structures and revealed additional information about the probability of the structural variants of C3b. In addition, a discontinuous cluster of seven peptides in the MG3, MG6, LNK and alpha'NT domains showed a decreased accessibility after activation to C3b. Although no gross conformational changes are detected in the crystal structure, this area may reflect a structurally flexible region in solution that contributes to C3 activation and function.

The catalases of Paracoccidioides brasiliensis are differentially regulated: protein activity and transcript analysis.

Paracoccidioides brasiliensis is a fungal pathogen of humans. The P. brasiliensis response to oxidative stress is largely unexplored. We report the analysis of three catalases, PbCatA, PbCatP and PbCatC. The former are monofunctional catalases and the latter is a catalase-peroxidase. Differential expression of catalases as measured by activity and by quantitative analysis of transcripts was observed in the morphological conversion and in response to different stress conditions. PbCatA manifested higher activity in the mycelial phase, showed increased activity during transition from mycelium to yeast and during conditions of endogenous oxidative stress. Consistent with our previous studies, PbCatP manifested higher activity in yeast cells since it is putatively involved in the control of exogenous reactive oxygen species. P. brasiliensis displays an oxidative stress response following phagocytosis by macrophages, inducing the expression of catalase A and P transcripts. PbCatC displayed a relatively constant pattern of expression, being modestly induced in cells exposed to osmotic and heat stress.

Histoplasma capsulatum proteome response to decreased iron availability.

BACKGROUND: A fundamental pathogenic feature of the fungus Histoplasma capsulatum is its ability to evade innate and adaptive immune defenses. Once ingested by macrophages the organism is faced with several hostile environmental conditions including iron limitation. H. capsulatum can establish a persistent state within the macrophage. A gap in knowledge exists because the identities and number of proteins regulated by the organism under host conditions has yet to be defined. Lack of such knowledge is an important problem because until these proteins are identified it is unlikely that they can be targeted as new and innovative treatment for histoplasmosis. RESULTS: To investigate the proteomic response by H. capsulatum to decreasing iron availability we have created H. capsulatum protein/genomic databases compatible with current mass spectrometric (MS) search engines. Databases were assembled from the H. capsulatum G217B strain genome using gene prediction programs and expressed sequence tag (EST) libraries. Searching these databases with MS data generated from two dimensional (2D) in-gel digestions of proteins resulted in over 50% more proteins identified compared to searching the publicly available fungal databases alone. Using 2D gel electrophoresis combined with statistical analysis we discovered 42 H. capsulatum proteins whose abundance was significantly modulated when iron concentrations were lowered. Altered proteins were identified by mass spectrometry and database searching to be involved in glycolysis, the tricarboxylic acid cycle, lysine metabolism, protein synthesis, and one protein sequence whose function was unknown. CONCLUSION: We have created a bioinformatics platform for H. capsulatum and demonstrated the utility of a proteomic approach by identifying a shift in metabolism the organism utilizes to cope with the hostile conditions provided by the host. We have shown that enzyme transcripts regulated by other fungal pathogens in response to lowering iron availability are also regulated in H. capsulatum at the protein level. We also identified H. capsulatum proteins sensitive to iron level reductions which have yet to be connected to iron availability in other pathogens. These data also indicate the complexity of the response by H. capsulatum to nutritional deprivation. Finally, we demonstrate the importance of a strain specific gene/protein database for H. capsulatum proteomic analysis.

Mechanisms of action of differentiation inducers: detection of inducer binding protein(s) in murine erythroleukemia cells.

We have shown previously that murine erythroleukemia (MEL) and human neuroectodermal RD/TE-671 cells are induced to differentiate by ureido derivatives of pyridine (UDPs) and may contain inducer binding protein(s). In the present study, we prepared radiolabeled [3H]UDP [2-(3-ethylureido)-6-[3H]-acetylaminopyridine] as ligand and investigated whether it interacts selectively with novel binding proteins. MEL and RD/TE-671 cells, incubated with the inducer [3H]UDP and subsequently fractionated, yielded a radiolabeled postmitochondrial soluble fraction containing the [3H]UDP-protein complex. We purified the UDP binding protein by using UDP-sepharose affinity chromatography, gel filtration, and SDS-PAGE electrophoresis and analyzed its structure. The data presented here indicate for the first time that the inducer UDP interacts with a 38,333 +/- 30 Da binding protein(s) (p38), of unknown function, in both cell lines. Microsequencing and sequence alignment search revealed that the p38 protein(s) contains at least two homologous domains, one being part of ABC-type transporters and another found in the Wingless-type (Wnt) proteins. Kinetic analysis revealed that the p38 forms a relatively stable protein complex with [3H]UDP that accumulates within the cytosol and nucleus of MEL cells during the precommitment period. This complex, however, decays later on after commitment to erythroid maturation has been initiated. De novo protein and mRNA synthesis is needed for the UDP-p38 complex to form, as shown by the use of metabolic inhibitors. Purified p38 was used to develop an anti-p38 polyclonal serum, and Western blot analysis revealed that the level of p38 was quite similar in both UDP-inducible and -resistant MEL subclones that we developed. Although only a portion of the primary structure of the p38 is known from microsequencing, the mechanism by which the UDP-p38 complex contributes to induction of differentiation in both UDP-responsive mouse MEL and human RD/TE-671 cells is discussed.

Purification of Paracoccidioides brasiliensis catalase P: subsequent kinetic and stability studies.

Catalases are essential components of the cellular equipment to cope with oxidative stress. Here we have purified a highly abundant catalase P of Paracoccidioides brasiliensis (PbCatP) that is preferentially expressed in the parasitic yeast phase. This oxidative stress-induced protein was isolated from yeast cells grown in the presence of 15 mM of hydrogen peroxide (H(2)O(2)). We have used consecutive steps of protein precipitation and gel filtration chromatography to achieve the purified protein. Protein purification was validated using matrix-assisted laser desorption ionization time-of-flight mass spectrometry and bioinformatics analysis. The purified enzyme showed strong similarity to small-subunit catalases. Like most monofunctional catalases, PbCatP is a homotetramer, resistant to inactivation by acidic conditions, temperature and denaturants. Furthermore, the kinetic behaviour of catalase P was observed to be different at low compared to high H(2)O(2) concentrations. The results demonstrated that a purified PbCatP is a homotetrameric enzyme, classified as a small subunit catalase.

Solvent accessibility of native and hydrolyzed human complement protein 3 analyzed by hydrogen/deuterium exchange and mass spectrometry.

Complement protein C3 is a 187-kDa (1641-aa) protein that plays a key role in complement activation and immune responses. Its hydrolyzed form, C3(H2O), is responsible for the initiation of the activation of alternative complement pathway. Previous analyses using mAbs, anilinonaphthalenesulfonate dyes, and functional studies have suggested that C3 is conformationally different from C3(H2O). We have used amide hydrogen/deuterium exchange and MALDI-TOF mass spectrometry to identify and localize structural differences between native C3 and C3(H2O). Both proteins were incubated in D2O for varying amounts of time, digested with pepsin, and then subjected to mass-spectrometric analysis. Of 111 C3 peptides identified in the MALDI-TOF analysis, 31 had well-resolved isotopic mass envelopes in both C3 and C3(H2O) spectra. Following the conversion of native C3 to C3(H2O), 17 of these 31 peptides exhibited a change in deuterium incorporation, suggesting a conformational change in these regions. Among the identified peptides, hydrogen/deuterium exchange data were obtained for peptides 944-967, 1211-1228, 1211-1231, 1259-1270, 1259-1273, 1295-1318, and 1319-1330, which span the factor H binding site on C3d and factor I cleavage sites, and peptides 1034-1048, 1049-1058, 1069-1080, 1130-1143, 1130-1145, 1211-1228, 1211-1231, 1259-1270, and 1259-1273, spanning 30% of the C3d region of C3. Our results suggest that hydrolysis may produce a looser (more open) structure in the C3d region, in which some of the changes affect the conversion of helical segments into coil segments facilitating interactions with factors I and H. This study represents the first detailed study mapping the regions of C3 involved in conformational transition when hydrolyzed to C3(H2O).

Detecting protein-protein interactions in the intact cell of Bacillus subtilis (ATCC 6633).

The salt bridge, paired group-specific reagent cyanogen (ethanedinitrile; C(2)N(2)) converts naturally occurring pairs of functional groups into covalently linked products. Cyanogen readily permeates cell walls and membranes. When the paired groups are shared between associated proteins, isolation of the covalently linked proteins allows their identity to be assigned. Examination of organisms of known genome sequence permits identification of the linked proteins by mass spectrometric techniques applied to peptides derived from them. The cyanogen-linked proteins were isolated by polyacrylamide gel electrophoresis. Digestion of the isolated proteins with proteases of known specificity afforded sets of peptides that could be analyzed by mass spectrometry. These data were compared with those derived theoretically from the Swiss Protein Database by computer-based comparisons (Protein Prospector; http://prospector.ucsf.edu). Identification of associated proteins in the ribosome of Bacillus subtilis strain ATCC 6633 showed that there is an association homology with the association patterns of the ribosomal proteins of Haloarcula marismortui and Thermus thermophilus. In addition, other proteins involved in protein biosynthesis were shown to be associated with ribosomal proteins.

Identification of amino acid residues participating in intermolecular salt bridges between self-associating proteins.

Salt bridges between self-associating hen egg white (HEW) lysozyme and bovine insulin molecules were converted to covalent links by ethanedinitrile (cyanogen) and identified using mass spectrometry. Peptides resulting from cyanogen-mediated intermolecular cross-linking of HEW lysozyme were detected using in-gel digestion and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Sequence data from electrospray ionization-quadrupole-time of flight mass spectrometry (ESI-Q-TOF MS) revealed that one of the peptides has a covalent bond between Asp 66 and Arg 14. The self-assembly of bovine insulin was also investigated using cyanogen. Using high-performance liquid chromatography (HPLC) coupled with ESI-Q-TOF MS, an intermolecular salt bridge association was identified by covalently linking the B chain C-terminal carboxyl group of Ala 30 and the charged imidazole of His 5 (B chain). A method was developed incorporating cyanogen, enzymatic digestion, one-dimensional gel electrophoresis, MALDI-TOF MS, and HPLC ESI-Q-TOF MS to identify amino acid residues participating in salt bridge formations at protein-protein interfaces. The novelty of this approach is the ease with which cyanogen can be administered to a protein sample and the apparent lack of nonspecific cross-linking side reactions interfering with the analysis.