Defects in DNA Lesion Bypass Lead to Spontaneous Chromosomal Rearrangements and Increased Cell Death

Rev3 polymerase and Mph1 DNA helicase participate in error-prone and error-free pathways, respectively, for the bypassing of template lesions during DNA replication. Here we have investigated the role of these pathways and their genetic interaction with recombination factors, other nonreplicative DNA helicases, and DNA damage checkpoint components in the maintenance of genome stability, viability, and sensitivity to the DNA-damaging agent methyl methanesulfonate (MMS). We find that cells lacking Rev3 and Mph1 exhibit a synergistic, Srs2-dependent increase in the rate of accumulating spontaneous, gross chromosomal rearrangements, suggesting that the suppression of point mutations by deletion of REV3 may lead to chromosomal rearrangements. While mph1Δ is epistatic to homologous recombination (HR) genes, both Rad51 and Rad52, but not Rad59, are required for normal growth of the rev3Δ mutant and are essential for survival of rev3Δ cells during exposure to MMS, indicating that Mph1 acts in a Rad51-dependent, Rad59-independent subpathway of HR-mediated lesion bypass. Deletion of MPH1 helicase leads to synergistic DNA damage sensitivity increases in cells with chl1Δ or rrm3Δ helicase mutations, whereas mph1Δ is hypostatic to sgs1Δ. Previously reported slow growth of mph1Δ srs2Δ cells is accompanied by G₂/M arrest and fully suppressed by disruption of the Mec3-dependent DNA damage checkpoint. We propose a model for replication fork rescue mediated by translesion DNA synthesis and homologous recombination that integrates the role of Mph1 in unwinding D loops and its genetic interaction with Rev3 and Srs2-regulated pathways in the suppression of spontaneous genome rearrangements and in mutation avoidance.Nonreplicative DNA helicases play an important role in the maintenance of genome stability from bacteria to humans, most likely by affecting the formation and/or resolution of recombination intermediates and by facilitating replication fork progression through chromosomal regions with a propensity to adopt unusual DNA structures or those bound by proteins. In Saccharomyces cerevisiae, this group of DNA helicases includes the 3′-to-5′ helicases Sgs1 and Srs2 and the 5′-to-3′ DNA helicase Rrm3. In the absence of any two of these three helicases, unresolved recombination intermediates accumulate and lead to extremely slow growth that is fully suppressed by deletion of genes encoding early homologous recombination (HR) factors (4, 6, 17, 20, 37, 46). In the absence of Sgs1, cells exhibit increased rates of mitotic recombination, frequent chromosome missegregation, accumulation of extrachromosomal ribosomal DNA (rDNA) circles, and increased rates of gross chromosomal rearrangements (GCRs) involving nonhomologous chromosomes (5, 24, 25, 38, 40, 43, 49, 50). Based on the increased crossover frequency during HO endonuclease-induced double-strand breaks (DSBs) in cells lacking Sgs1, it has also been proposed that Sgs1 may function in decatenation of Holliday junctions (HJs) to yield noncrossovers (12, 22). Like Sgs1, Srs2 acts to favor noncrossover outcomes during DSB repair but appears to act earlier than Sgs1 in regulating recombination outcomes through its ability to dislodge Rad51 from recombinogenic 3′ overhangs, thereby promoting a noncrossover synthesis-dependent single-strand annealing (SDSA) pathway (12, 33, 35). In contrast, Rrm3 has not been implicated in DNA repair but is thought to be important for avoidance of recombination substrate formation by removal of DNA protein complexes in certain chromosomal locations, such as chromosome ends and replication fork barriers at the rDNA locus, thus facilitating replication fork progression (13, 14).In addition to Sgs1, Rrm3, and Srs2, the yeast genome encodes two other nonreplicative DNA helicases with proposed functions in DNA repair, Mph1 and Chl1. Mph1 possesses 3′-to-5′ helicase activity, and its ATPase activity requires a relatively long fragment of single-stranded DNA (ssDNA) (≥40 nucleotides [nt]) for full activity in vitro (32). Mph1 is also necessary for resistance to the DNA damaging agents methyl methanesulfonate (MMS) and 4-nitroquinoline-1-oxide (4-NQO) and suppresses spontaneous mutations toward canavanine resistance (3, 41). The modest mutator phenotype of the mph1Δ mutant is enhanced by additional mutations in base excision repair (apn1Δ and apn2Δ) and is suppressed by mutations in translesion DNA synthesis (TLS) (rev3Δ) (36, 41). These findings, in combination with the observation of an epistatic relationship between mph1Δ and homologous recombination mutations, have led to the proposal that Mph1 may act in Rad52-dependent, error-free bypassing of DNA lesions (41). Like the 3′-to-5′ DNA helicases Sgs1 and Srs2, Mph1 was recently shown to affect crossover frequency during repair of an HO endonuclease-induced DNA DSB, favoring noncrossovers as the outcome (33). The authors showed that Mph1 can unwind intermediates of homologous recombination in vitro, specifically D loops that are thought to form early during homologous recombination when a homoduplex is invaded by a Rad51 filament. While Srs2 has been shown to be able to disassemble Rad51 filaments in vitro, it does not appear to possess Mph1''s ability to dissociate D loops once they have formed (19, 47).Although Chl1 has been shown to be required for the establishment of sister chromatid cohesion, a possible role in DNA repair by homologous recombination has also been proposed (11, 28, 30, 42). While Chl1 possesses a conserved helicase domain, helicase activity has so far been shown only for its putative human homolog, hCHLR1 (10).To further elucidate the functional interaction between nonreplicative DNA helicases and DNA repair pathways, we generated a series of mutants with combinations of mph1Δ, chl1Δ, rrm3Δ, srs2Δ, and sgs1Δ mutations and mutations in translesion DNA synthesis (TLS), base excision repair (BER), homologous recombination (HR), and DNA damage checkpoints. In addition to synthetic fitness defects due to aberrant HR and checkpoint activation, we identified epistatic and synergistic relationships with regard to fitness, the accumulation of gross chromosomal rearrangements (GCRs), and sensitivity to DNA damage. We propose that Mph1 functions in a Rad51-dependent, Rad59-independent pathway of HR for DNA lesion bypass and interacts genetically with REV3 in the suppression of gross chromosomal rearrangements.     

F-Actin Dynamics in Neurospora crassa

This study demonstrates the utility of Lifeact for the investigation of actin dynamics in Neurospora crassa and also represents the first report of simultaneous live-cell imaging of the actin and microtubule cytoskeletons in filamentous fungi. Lifeact is a 17-amino-acid peptide derived from the nonessential Saccharomyces cerevisiae actin-binding protein Abp140p. Fused to green fluorescent protein (GFP) or red fluorescent protein (TagRFP), Lifeact allowed live-cell imaging of actin patches, cables, and rings in N. crassa without interfering with cellular functions. Actin cables and patches localized to sites of active growth during the establishment and maintenance of cell polarity in germ tubes and conidial anastomosis tubes (CATs). Recurrent phases of formation and retrograde movement of complex arrays of actin cables were observed at growing tips of germ tubes and CATs. Two populations of actin patches exhibiting slow and fast movement were distinguished, and rapid (1.2 μm/s) saltatory transport of patches along cables was observed. Actin cables accumulated and subsequently condensed into actin rings associated with septum formation. F-actin organization was markedly different in the tip regions of mature hyphae and in germ tubes. Only mature hyphae displayed a subapical collar of actin patches and a concentration of F-actin within the core of the Spitzenkörper. Coexpression of Lifeact-TagRFP and β-tubulin–GFP revealed distinct but interrelated localization patterns of F-actin and microtubules during the initiation and maintenance of tip growth.Actins are highly conserved proteins found in all eukaryotes and have an enormous variety of cellular roles. The monomeric form (globular actin, or G-actin) can self-assemble, with the aid of numerous actin-binding proteins (ABPs), into microfilaments (filamentous actin, or F-actin), which, together with microtubules, form the two major components of the fungal cytoskeleton. Numerous pharmacological and genetic studies of fungi have demonstrated crucial roles for F-actin in cell polarity, exocytosis, endocytosis, cytokinesis, and organelle movement (6, 7, 20, 34, 35, 51, 52, 59). Phalloidin staining, immunofluorescent labeling, and fluorescent-protein (FP)-based live-cell imaging have revealed three distinct subpopulations of F-actin-containing structures in fungi: patches, cables, and rings (1, 14, 28, 34, 60, 63, 64). Actin patches are associated with the plasma membrane and represent an accumulation of F-actin around endocytic vesicles (3, 26, 57). Actin cables are bundles of actin filaments stabilized with cross-linking proteins, such as tropomyosins and fimbrin, and are assembled by formins at sites of active growth, where they form tracks for myosin V-dependent polarized secretion and organelle transport (10, 16, 17, 27, 38, 47, 48). Cables, unlike patches, are absolutely required for polarized growth in the budding yeast Saccharomyces cerevisiae (34, 38). Contractile actomyosin rings are essential for cytokinesis in budding yeast, whereas in filamentous fungi, actin rings are less well studied but are known to be involved in septum formation (20, 28, 34, 39, 40).Actin cables and patches have been particularly well studied in budding yeast. However, there are likely to be important differences between F-actin architecture and dynamics in budding yeast and those in filamentous fungi, as budding yeasts display only a short period of polarized growth during bud formation, which is followed by isotropic growth over the bud surface (10). Sustained polarized growth during hyphal morphogenesis is a defining feature of filamentous fungi (21), making them attractive models for studying the roles of the actin cytoskeleton in cell polarization, tip growth, and organelle transport.In Neurospora crassa and other filamentous fungi, disruption of the actin cytoskeleton leads to rapid tip swelling, which indicates perturbation of polarized tip growth, demonstrating a critical role for F-actin in targeted secretion to particular sites on the plasma membrane (7, 22, 29, 56). Immunofluorescence studies of N. crassa have shown that F-actin localizes to hyphal tips as “clouds” and “plaques” (7, 54, 59). However, immunolabeling has failed to reveal actin cables in N. crassa and offers limited insights into F-actin dynamics. Live-cell imaging of F-actin architecture and dynamics has not been accomplished in N. crassa, yet it is expected to yield key insights into cell polarization, tip growth, and intracellular transport.We took advantage of a recently developed live-cell imaging probe for F-actin called Lifeact (43). Lifeact is a 17-amino-acid peptide derived from the N terminus of the budding yeast actin-binding protein Abp140 (5, 63) and has recently been demonstrated to be a universal live-cell imaging marker for F-actin in eukaryotes (43). Here, we report the successful application of fluorescent Lifeact fusion constructs for live-cell imaging of F-actin in N. crassa. We constructed two synthetic genes consisting of Lifeact fused to “synthetic” green fluorescent protein (sGFP) (S65T) (henceforth termed GFP) (12) or red fluorescent protein (TagRFP) (33) and expressed these constructs in various N. crassa strains. In all strain backgrounds, fluorescent Lifeact constructs clearly labeled actin patches, cables, and rings and revealed a direct association of F-actin structures with sites of cell polarization and active tip growth. Our results demonstrate the efficacy of Lifeact as a nontoxic live-cell imaging probe in N. crassa.     

Structure and Function of Glycosylated Tandem Repeats from Candida albicans Als Adhesins

Tandem repeat (TR) regions are common in yeast adhesins, but their structures are unknown, and their activities are poorly understood. TR regions in Candida albicans Als proteins are conserved glycosylated 36-residue sequences with cell-cell aggregation activity (J. M. Rauceo, R. De Armond, H. Otoo, P. C. Kahn, S. A. Klotz, N. K. Gaur, and P. N. Lipke, Eukaryot. Cell 5:1664–1673, 2006). Ab initio modeling with either Rosetta or LINUS generated consistent structures of three-stranded antiparallel β-sheet domains, whereas randomly shuffled sequences with the same composition generated various structures with consistently higher energies. O- and N-glycosylation patterns showed that each TR domain had exposed hydrophobic surfaces surrounded by glycosylation sites. These structures are consistent with domain dimensions and stability measurements by atomic force microscopy (D. Alsteen, V. Dupres, S. A. Klotz, N. K. Gaur, P. N. Lipke, and Y. F. Dufrene, ACS Nano 3:1677–1682, 2009) and with circular dichroism determination of secondary structure and thermal stability. Functional assays showed that the hydrophobic surfaces of TR domains supported binding to polystyrene surfaces and other TR domains, leading to nonsaturable homophilic binding. The domain structures are like “classic” subunit interaction surfaces and can explain previously observed patterns of promiscuous interactions between TR domains in any Als proteins or between TR domains and surfaces of other proteins. Together, the modeling techniques and the supporting data lead to an approach that relates structure and function in many kinds of repeat domains in fungal adhesins.Yeast adhesins are a diverse set of cell adhesion proteins that mediate adhesion to host cells, environmental substrates, other fungi, and coinfecting bacteria (6, 8, 20, 21, 23, 29). The adhesins share common features, including compact N-terminal domains similar to Ig or lectin domains, Thr-rich midpieces, often in tandem repeats, and long highly glycosylated Ser/Thr-rich C-terminal regions that extend the functional domains out from the cell surface. No structures for the Thr-rich midpieces are known, but they can mediate aggregation of fungal cells (33, 35, 47). The prevalence and conservation of such repeats argue that they are functionally important, despite limited data on their structure and function.In Candida albicans, the Als adhesins are homologous proteins, products of 8 loci that encode numerous alleles of cell surface adhesins (16). In each mature Als protein, there are, from the N terminus, three tandem Ig-like domains, a β-sheet-rich conserved 127-residue amyloid-forming T region, a variable number of 36-residue tandem repeats (TRs), and a highly glycosylated stalk region that extends the N-terminal domains away from the cell surface (Fig. 1) (16, 33, 41). The C termini of these and other wall-associated adhesins are covalently cross-linked into the cell wall through transglycosylation of a modified glycosylphosphatidylinositol (GPI) anchor (18, 25). This modular design, including tandem repeats, is typical of fungal adhesins (8).Open in a separate windowFig. 1.Schematic diagram of the sequence of Als5p. The regions are named above, and the number of amino acid residues in each region is shown below. The modeled sequences are in the TR region.The Als protein Ig-like region, T region, and TR region all have protein-protein interaction activities (26, 33, 35). The Ig-like regions can interact with diverse mammalian proteins, presumably in a way analogous to antibody-antigen binding, as has been shown in the homologous protein α-agglutinin from Saccharomyces cerevisiae (8, 24, 26, 35). The T regions interact through formation of amyloid-like structures both in vivo and in vitro (33, 34a, 36). An insight into the function of the tandem repeats followed from observations that Als proteins initiate and maintain cell-to-cell aggregations, either spontaneously (“autoaggregation”) or following adhesion to a bead-bound defined ligand (10, 11, 36). Aggregation is more extensive for Als proteins with more tandem repeats (26, 35). This result suggested that the tandem repeats are uniquely structured to facilitate or mediate the aggregative function. Circular dichroism spectroscopy of the TR region of Als5p shows a β-sheet-rich structure in the soluble protein (35).In support of their direct involvement in aggregation, the repeat region of the C. albicans adhesin Als5p mediates cell-cell aggregation in the absence of the Ig-like and T domains (35). Moreover, the repeats can also potentiate binding of Als5p to fibronectin (35). Thus, the TR domains mediate cellular aggregation and increased binding to fibronectin. In addition, TR domains and their amino acid sequences are highly conserved across several Candida species (3). These properties need to be explained by their three-dimensional structure.Because there are no homologous structures known, we modeled by two independent ab initio methods. Rosetta assembles structures by combining short peptide structures extracted from the protein structural database PDB (38), then combines structures in a Monte Carlo approach, and assesses energetics of assembled structures. Rosetta has recently been shown to generate accurate models for protein-sized domains (40). We also predicted structures with LINUS, which generates randomized structures and rapidly estimates energetics to choose low-energy models (45). The models were supported by structural analyses with atomic force microscopy and circular dichroism spectroscopy. Functional assays showed that the TR domains can mediate binding activities predicted from the calculated structures.     

Diversity Within the O-linked Protein Glycosylation Systems of Acinetobacter Species

The opportunistic human pathogen Acinetobacter baumannii is a concern to health care systems worldwide because of its persistence in clinical settings and the growing frequency of multiple drug resistant infections. To combat this threat, it is necessary to understand factors associated with disease and environmental persistence of A. baumannii. Recently, it was shown that a single biosynthetic pathway was responsible for the generation of capsule polysaccharide and O-linked protein glycosylation. Because of the requirement of these carbohydrates for virulence and the non-template driven nature of glycan biogenesis we investigated the composition, diversity, and properties of the Acinetobacter glycoproteome. Utilizing global and targeted mass spectrometry methods, we examined 15 strains and found extensive glycan diversity in the O-linked glycoproteome of Acinetobacter. Comparison of the 26 glycoproteins identified revealed that different A. baumannii strains target similar protein substrates, both in characteristics of the sites of O-glycosylation and protein identity. Surprisingly, glycan micro-heterogeneity was also observed within nearly all isolates examined demonstrating glycan heterogeneity is a widespread phenomena in Acinetobacter O-linked glycosylation. By comparing the 11 main glycoforms and over 20 alternative glycoforms characterized within the 15 strains, trends within the glycan utilized for O-linked glycosylation could be observed. These trends reveal Acinetobacter O-linked glycosylation favors short (three to five residue) glycans with limited branching containing negatively charged sugars such as GlcNAc3NAcA4OAc or legionaminic/pseudaminic acid derivatives. These observations suggest that although highly diverse, the capsule/O-linked glycan biosynthetic pathways generate glycans with similar characteristics across all A. baumannii.Acinetobacter baumannii is an emerging opportunistic pathogen of increasing significance to health care institutions worldwide (1–3). The growing number of identified multiple drug resistant (MDR)¹ strains (2–4), the ability of isolates to rapidly acquire resistance (3, 4), and the propensity of this agent to survive harsh environmental conditions (5) account for the increasing number of outbreaks in intensive care, burn, or high dependence health care units since the 1970s (2–5). The burden on the global health care system of MDR A. baumannii is further exacerbated by standard infection control measures often being insufficient to quell the spread of A. baumannii to high risk individuals and generally failing to remove A. baumannii from health care institutions (5). Because of these concerns, there is an urgent need to identify strategies to control A. baumannii as well as understand the mechanisms that enable its persistence in health care environments.Surface glycans have been identified as key virulence factors related to persistence and virulence within the clinical setting (6–8). Acinetobacter surface carbohydrates were first identified and studied in A. venetianus strain RAG-1, leading to the identification of a gene locus required for synthesis and export of the surface carbohydrates (9, 10). These carbohydrate synthesis loci are variable yet ubiquitous in A. baumannii (11, 12). Comparison of 12 known capsule structures from A. baumannii with the sequences of their carbohydrate synthesis loci has provided strong evidence that these loci are responsible for capsule synthesis with as many as 77 distinct serotypes identified by molecular serotyping (11). Because of the non-template driven nature of glycan synthesis, the identification and characterization of the glycans themselves are required to confirm the true diversity. This diversity has widespread implications for Acinetobacter biology as the resulting carbohydrate structures are not solely used for capsule biosynthesis but can be incorporated and utilized by other ubiquitous systems, such as O-linked protein glycosylation (13, 14).Although originally thought to be restricted to species such as Campylobacter jejuni (15, 16) and Neisseria meningitidis (17), bacterial protein glycosylation is now recognized as a common phenomenon within numerous pathogens and commensal bacteria (18, 19). Unlike eukaryotic glycosylation where robust and high-throughput technologies now exist to enrich (20–22) and characterize both the glycan and peptide component of glycopeptides (23–25), the diversity (glycan composition and linkage) within bacterial glycosylation systems makes few technologies broadly applicable to all bacterial glycoproteins. Because of this challenge a deeper understanding of the glycan diversity and substrates of glycosylation has been largely unachievable for the majority of known bacterial glycosylation systems. The recent implementation of selective glycopeptide enrichment methods (26, 27) and the use of multiple fragmentation approaches (28, 29) has facilitated identification of an increasing number of glycosylation substrates independent of prior knowledge of the glycan structure (30–33). These developments have facilitated the undertaking of comparative glycosylation studies, revealing glycosylation is widespread in diverse genera and far more diverse then initially thought. For example, Nothaft et al. were able to show N-linked glycosylation was widespread in the Campylobacter genus and that two broad groupings of the N-glycans existed (34).During the initial characterization of A. baumannii O-linked glycosylation the use of selective enrichment of glycopeptides followed by mass spectrometry analysis with multiple fragmentation technologies was found to be an effective means to identify multiple glycosylated substrates in the strain ATCC 17978 (14). Interestingly in this strain, the glycan utilized for protein modification was identical to a single subunit of the capsule (13) and the loss of either protein glycosylation or glycan synthesis lead to decreases in biofilm formation and virulence (13, 14). Because of the diversity in the capsule carbohydrate synthesis loci and the ubiquitous distribution of the PglL O-oligosaccharyltransferase required for protein glycosylation, we hypothesized that the glycan variability might be also extended to O-linked glycosylation. This diversity, although common in surface carbohydrates such as the lipopolysaccharide of numerous Gram-negative pathogens (35), has only recently been observed within bacterial proteins glycosylation system that are typically conserved within species (36) and loosely across genus (34, 37).In this study, we explored the diversity within the O-linked protein glycosylation systems of Acinetobacter species. Our analysis complements the recent in silico studies of A. baumannii showing extensive glycan diversity exists in the carbohydrate synthesis loci (11, 12). Employing global strategies for the analysis of glycosylation, we experimentally demonstrate that the variation in O-glycan structure extends beyond the genetic diversity predicted by the carbohydrate loci alone and targets proteins of similar properties and identity. Using this knowledge, we developed a targeted approach for the detection of protein glycosylation, enabling streamlined analysis of glycosylation within a range of genetic backgrounds. We determined that; O-linked glycosylation is widespread in clinically relevant Acinetobacter species; inter- and intra-strain heterogeneity exist within glycan structures; glycan diversity, although extensive results in the generation of glycans with similar properties and that the utilization of a single glycan for capsule and O-linked glycosylation is a general feature of A. baumannii but may not be a general characteristic of all Acinetobacter species such as A. baylyi.     

Yeast Cell Adhesion Molecules Have Functional Amyloid-Forming Sequences

The occurrence of highly conserved amyloid-forming sequences in Candida albicans Als proteins (H. N. Otoo et al., Eukaryot. Cell 7:776–782, 2008) led us to search for similar sequences in other adhesins from C. albicans and Saccharomyces cerevisiae. The β-aggregation predictor TANGO found highly β-aggregation-prone sequences in almost all yeast adhesins. These sequences had an unusual amino acid composition: 77% of their residues were β-branched aliphatic amino acids Ile, Thr, and Val, which is more than 4-fold greater than their prevalence in the S. cerevisiae proteome. High β-aggregation potential peptides from S. cerevisiae Flo1p and C. albicans Eap1p rapidly formed insoluble amyloids, as determined by Congo red absorbance, thioflavin T fluorescence, and fiber morphology. As examples of the amyloid-forming ability of the native proteins, soluble glycosylphosphatidylinositol (GPI)-less fragments of C. albicans Als5p and S. cerevisiae Muc1p also formed amyloids within a few days under native conditions at nM concentrations. There was also evidence of amyloid formation in vivo: the surfaces of cells expressing wall-bound Als1p, Als5p, Muc1p, or Flo1p were birefringent and bound the fluorescent amyloid-reporting dye thioflavin T. Both of these properties increased upon aggregation of the cells. In addition, amyloid binding dyes strongly inhibited aggregation and flocculation. The results imply that amyloid formation is an intrinsic property of yeast cell adhesion proteins from many gene families and that amyloid formation is an important component of cellular aggregation mediated by these proteins.Protein amyloids are characteristic of pathological conditions, including neurodegenerative diseases (4, 11, 17, 38). These protein aggregates can also occur naturally in adhesive bacterial curli (3), melanosomes (14), condensed peptide hormone arrays (24), as regulatory prions in yeast (2, 5), and fungal hydrophobins, which are nonantigenic coats to some fungi (1, 33, 39). Nevertheless, such natural occurrences are relatively few, considering the negative free energy for amyloid formation (28).We have recently discovered that there are amyloid-forming sequences in the cell surface Als adhesins of Candida albicans. Cells that express these adhesins aggregate readily, and the aggregation has amyloid-like properties, including protein conformational shifting, surface birefringence, and ability to bind the amyloid-active dyes Congo red and amino-naphthalene sulfonic acid (ANS) (29). A five- to seven-residue sequence in Als1p, Als3p, and Als5p has extremely high potential for formation of β-aggregates, according to the protein state prediction program TANGO (13, 27, 31). Such β-aggregates include amyloids, which are ordered structures with paracrystalline regions of stacked parallel β-strands that are perpendicular to the long axis of micrometer-long fibrils. The strands are stabilized by interaction of identical sequences from many protein molecules (31, 32). Where TANGO analyses have shown that specific sequences have β-aggregate potentials greater than 20%, an insoluble β-aggregate state is likely to form. These β-aggregates nucleate formation of amyloids if the proteins can associate to form fibers (13, 27, 31). Sequences in the conserved 127-residue T region of Als1p, Als3p, and Als5p have β-aggregation potentials of >90% (27). An oligopeptide with this sequence, as well as 412- and 645-residue fragments of Als5p formed authentic amyloids, as determined by characteristic dye binding and fiber morphology. The amyloid-forming sequences were rich in the β-branched amino acids Thr, Val, and Ile. This amino acid composition is unusual among proteins in general, but is common in the Thr-rich mid-piece domains of yeast adhesins.Yeasts display many cell-wall-bound adhesins that mediate colonial and biofilm interactions as well as host-pathogen binding (9, 21, 41). Such adhesins have a common mosaic structure. In general, the adhesins have N-terminal globular binding domains (often immunoglobulin-like or lectin-like), Thr-rich mid-piece sequences including tandem repeats, and 300- to 800-residue heavily glycosylated Ser and Thr-rich “stalk” domains near the C-terminal domain that extend the active regions from the surface of the wall. The adhesins are covalently cross-linked to wall polysaccharides through modified glycosylphosphatidylinositol (GPI) anchors and/or glycosyl esters of glutamic acid (9, 18).Because the yeast adhesins share this common modular domain structure, we searched among known and putative yeast adhesins for sequences with high β-aggregation potential. We have found that many of these proteins share amyloid-forming sequences and amyloid-like behavior on activation.     

Nuclear Dynamics during Germination,Conidiation, and Hyphal Fusion of Fusarium oxysporum

In many fungal pathogens, infection is initiated by conidial germination. Subsequent stages involve germ tube elongation, conidiation, and vegetative hyphal fusion (anastomosis). Here, we used live-cell fluorescence to study the dynamics of green fluorescent protein (GFP)- and cherry fluorescent protein (ChFP)-labeled nuclei in the plant pathogen Fusarium oxysporum. Hyphae of F. oxysporum have uninucleated cells and exhibit an acropetal nuclear pedigree, where only the nucleus in the apical compartment is mitotically active. In contrast, conidiation follows a basopetal pattern, whereby mononucleated microconidia are generated by repeated mitotic cycles of the subapical nucleus in the phialide, followed by septation and cell abscission. Vegetative hyphal fusion is preceded by directed growth of the fusion hypha toward the receptor hypha and followed by a series of postfusion nuclear events, including mitosis of the apical nucleus of the fusion hypha, migration of a daughter nucleus into the receptor hypha, and degradation of the resident nucleus. These previously unreported patterns of nuclear dynamics in F. oxysporum could be intimately related to its pathogenic lifestyle.Fusarium oxysporum is a soilborne pathogen that causes substantial losses in a wide variety of crops (12) and has been reported as an emerging human pathogen (36, 38). Similar to other fungal pathogens (18), the early stages of interaction between F. oxysporum and the host are crucial for the outcome of infection (11). Key processes occurring during these initial stages include spore germination, adhesion to the host surface, establishment of hyphal networks through vegetative hyphal fusion, differentiation of infection hyphae, and penetration of the host (53). Surprisingly, very little is known about the cytology of basic processes, such as spore germination and hyphal development, which play key roles during infection by F. oxysporum.F. oxysporum produces three types of asexual spores: microconidia, macroconidia, and chlamydospores (9, 26). Germination usually represents the first step in the colonization of a new environment, including the host. Once dormancy is broken, spores undergo a defined set of morphogenetic changes that lead to the establishment of a polarized growth axis and the emergence of one or multiple germ tubes (reviewed by d''Enfert and Hardham [10, 19]). In certain fungi, such as Aspergillus nidulans, germ tube emergence and septum formation are subject to precise spatial controls and are tightly coordinated with nuclear division (20, 22, 34, 42, 54). In contrast, in spores from other filamentous fungi, such as macroconidia of Fusarium graminearum, nuclear division is not required for the emergence of germ tubes (21, 48). During hyphal growth, multinucleate fungi display distinct mitotic patterns, such as asynchronous nuclear division in Neurospora crassa and Ashbya gossypii (15, 16, 29, 30, 33, 49), parasynchronous in A. nidulans (7, 15, 23, 46), and synchronous in Ceratocystis fagacearum (1, 15).Vegetative hyphal fusion, or anastomosis, is a common developmental process during the life cycle of filamentous fungi that is thought to serve important functions in intrahyphal communication, nutrient transport, and colony homeostasis (41). F. oxysporum undergoes anastomosis (8, 25, 32, 40), and although this process is not strictly required for plant infection, it appears to contribute to efficient colonization of the root surface (39).The aim of this study was to explore nuclear dynamics during different developmental stages of F. oxysporum that are of key relevance during the establishment of infection. They include germination of microconidia, vegetative hyphal development, and conidiation, as well as vegetative hyphal fusion during colony establishment. Fusion PCR-mediated gene targeting (55) was used to C-terminally label histone H1 in F. oxysporum (FoH1) with either green fluorescent protein (GFP) or the cherry variant (ChFP), allowing us to perform, for the first time, live-cell analysis of nuclear dynamics in this species. Our study revealed distinct patterns of nuclear divisions in F. oxysporum. Moreover, we report, for the first time in an ascomycete, that hyphal fusion initiates a series of nuclear events, including mitosis in the fusing hypha and nuclear migration into the receptor hypha, followed by degradation of the resident nucleus.     

Downregulation of an Entamoeba histolytica Rhomboid Protease Reveals Roles in Regulating Parasite Adhesion and Phagocytosis

Entamoeba histolytica is a deep-branching eukaryotic pathogen. Rhomboid proteases are intramembrane serine proteases, which cleave transmembrane proteins in, or in close proximity to, their transmembrane domain. We have previously shown that E. histolytica contains a single functional rhomboid protease (EhROM1) and has unique substrate specificity. EhROM1 is present on the trophozoite surface and relocalizes to internal vesicles during erythrophagocytosis and to the base of the cap during surface receptor capping. In order to further examine the biological function of EhROM1 we downregulated EhROM1 expression by >95% by utilizing the epigenetic silencing mechanism of the G3 parasite strain. Despite the observation that EhROM1 relocalized to the cap during surface receptor capping, EhROM1 knockdown [ROM(KD)] parasites had no gross changes in cap formation or complement resistance. However, ROM(KD) parasites demonstrated decreased host cell adhesion, a result recapitulated by treatment of wild-type parasites with DCI, a serine protease inhibitor with activity against rhomboid proteases. The reduced adhesion phenotype of ROM(KD) parasites was noted exclusively with healthy cells, and not with apoptotic cells. Additionally, ROM(KD) parasites had decreased phagocytic ability with reduced ingestion of healthy cells, apoptotic cells, and rice starch. Decreased phagocytic ability is thus independent of the reduced adhesion phenotype, since phagocytosis of apoptotic cells was reduced despite normal adhesion levels. The defect in host cell adhesion was not explained by altered expression or localization of the heavy subunit of the Gal/GalNAc surface lectin. These results suggest no significant role of EhROM1 in complement resistance but unexpected roles in parasite adhesion and phagocytosis.Entamoeba histolytica is an extracellular protozoan parasite and is a leading parasitic cause of death worldwide (48). The factors, which determine the outcome of amebic infection, are currently unknown, although it is likely that a combination of host and parasite determinants influence clinical outcome. A number of parasite factors required for amebic pathogenesis have been identified, including the Gal/GalNAc surface lectin, pore-forming proteins, and cysteine proteases (36,–38, 41).Recently, we identified several members of a class of intramembrane rhomboid proteases in the E. histolytica genome (4). Rhomboid proteases are seven-pass transmembrane proteases first identified in Drosophila melanogaster whose active site lies within the lipid bilayer, allowing them to cleave transmembrane proteins (6, 32). Substrates of rhomboid proteases are largely single-pass transmembrane proteins whose transmembrane domain contains helix-breaking residues (52). Recent work has revealed that there are multiple classes of rhomboid proteases that recognize different types of sequences within the transmembrane domains of their substrates (3). Despite low sequence similarity between individual rhomboid proteases of each class, these enzymes share a remarkable ability to functionally replace one another (16, 28, 52).Rhomboid proteases have been studied in flies, bacteria, mammals, and parasites, and roles ranging from quorum sensing to host cell entry have been identified (3, 11, 25, 33, 35, 46, 47, 49, 54, 59). In apicomplexan parasites, such as Plasmodium falciparum and Toxoplasma gondii, it has been suggested that rhomboid proteases mediate cleavage of surface adhesin proteins to facilitate host cell entry (3, 11, 46, 47). The E. histolytica genome encodes four rhomboid-like genes, with only a single gene containing the necessary catalytic residues for proteolytic activity (4). This gene, EhROM1, is a functional protease with substrate specificity similar to the P. falciparum ROM4 (PfROM4) (3, 4). In trophozoites EhROM1 is localized to the parasite surface and relocalizes to internal vesicles during erythrophagocytosis and to the base of the cap during surface receptor capping. We have shown that the heavy subunit of the amebic surface Gal/GalNAc lectin (Hgl) is a substrate of EhROM1 in vitro. Mutational analyses using a COS cell cleavage assay demonstrated that the cleavage of Hgl requires the catalytic serine in EhROM1 as well as a helix-breaking glycine residue in the transmembrane domain of Hgl (4). These data indicate that EhROM1 is a functional rhomboid protease whose physiological substrate may be Hgl.In order to further elucidate the biological function of EhROM1 we have utilized the epigenetic silencing mechanism of the E. histolytica G3 strain (8, 9). The mechanism of gene silencing in G3 ameba is not well understood. However, it is known that the silencing mechanism is epigenetically maintained, and epigenetic changes in the chromatin state of the silenced genes have been noted (22). G3 parasites transfected with a plasmid containing an upstream region of the 5′ end of EhROM showed almost complete downregulation of expression; we have named these parasites ROM(KD) for ROM knockdown. Phenotypes examined in ROM(KD) parasites included cap formation, complement resistance, adhesion, phagocytosis, hemolysis, and motility. We observed defects in both adhesion and phagocytosis in the ROM(KD) parasites compared to the parent G3 strain but no changes in cap formation or complement resistance. Importantly, the reduced phagocytosis phenotype appears independent of the reduced adhesion phenotype, implying that EhROM1 has distinct roles in both pathways.     

Using in Vivo Biotinylated Ubiquitin to Describe a Mitotic Exit Ubiquitome from Human Cells

Mitotic division requires highly regulated morphological and biochemical changes to the cell. Upon commitment to exit mitosis, cells begin to remove mitotic regulators in a temporally and spatially controlled manner to bring about the changes that reestablish interphase. Ubiquitin-dependent pathways target these regulators to generate polyubiquitin-tagged substrates for degradation by the 26S proteasome. However, the lack of cell-based assays to investigate in vivo ubiquitination limits our knowledge of the identity of substrates of ubiquitin-mediated regulation in mitosis. Here we report an in vivo ubiquitin tagging system used in human cells that allows efficient purification of ubiquitin conjugates from synchronized cell populations. Coupling purification with mass spectrometry, we have identified a series of mitotic regulators targeted for polyubiquitination in mitotic exit. We show that some are new substrates of the anaphase-promoting complex/cyclosome and validate KIFC1 and RacGAP1/Cyk4 as two such targets involved respectively in timely mitotic spindle disassembly and cell spreading. We conclude that in vivo biotin tagging of ubiquitin can provide valuable information about the role of ubiquitin-mediated regulation in processes required for rebuilding interphase cells.Ubiquitination has emerged as a major post-translational modification determining the fate of cellular proteins. One of these fates is proteolysis, whereby the assembly of polyubiquitin chains creates signatures on target proteins that specify delivery to the 26S proteasome for proteolytic destruction. Targeted proteolysis is critical to the control of cell division. For example, the universally conserved mechanism of mitotic exit depends upon rapid proteolysis of mitotic cyclins and securins to drive the transition from mitosis to interphase. This transition is under surveillance by the spindle assembly checkpoint (SAC),¹ which controls the activity of a multi-subunit ubiquitin ligase, the anaphase-promoting complex/cyclosome (APC/C) (1, 2).Much of the known specificity in the ubiquitin-proteasome system (UPS) is mediated at the level of substrate targeting by ubiquitin ligase (E3) enzymes, of which there are more than 600 in human cells. Given these facts, it is perhaps surprising that the APC/C is almost the only known engineer of the protein landscape after anaphase onset, targeting mitotic regulators for destruction with high temporal specificity (2–4). Some roles for nondegradative ubiquitination in regulating the localization of mitotic kinases Aurora B and Plk1 have been described (5–9), and a growing list of reported ubiquitin interactors can modulate ubiquitin-dependent events during mitosis (10). However, the majority of ubiquitination events that have so far been described as occurring at the transition from mitosis to interphase are APC/C-dependent.Two co-activator subunits, Cdc20 and Cdh1, play vital roles in APC/C-dependent substrate recognition (11) by recognizing two widely characterized degrons, the D-box and the KEN motif (12, 13). Computational approaches that have been used to calculate the total number of APC/C substrates from the prevalence of degrons in the human proteome estimate that there are between 100 and 200 substrates (14), and experiments using in vitro ubiquitination of protein arrays have given rise to estimates in the same range (15). Most of the mitotic regulators targeted by the APC/C during mitotic exit in human cells have been identified via in vitro degradation assays or ubiquitination assays on in vitro–expressed pools of substrates (15–18). These approaches have identified several important substrates, but in the absence of in vivo parameters they may not identify substrates whose targeting depends on post-translational modifications or substrates that are only recognized in vivo as components of higher-order complexes. Not all substrates identified in this way have been validated as polyubiquitinated proteins in vivo. Multiple recent proteomic studies have identified large numbers of in vivo ubiquitin-modified sites from yeast (19–21) and human cells (22–29). None of these studies have used synchronized cell populations to provide information on the timing or regulation of substrate ubiquitination.We reasoned that a better view of ubiquitin-mediated processes that regulate mitotic exit would come from identifying proteins that are ubiquitinated in vivo during mitotic exit. With this goal in mind we adopted a system for in vivo tagging of ubiquitin chains with biotin, previously used to identify ubiquitin-conjugated proteins from the Drosophila neural system (30), and applied it to a human cell line (U2OS) that can be tightly synchronized at mitosis. In contrast to several recent studies that employed antibodies specific to the diGly-Lys remnant that marks ubiquitination sites following trypsin digestion (19, 25), an in vivo ubiquitin tagging strategy allows direct validation of candidate ubiquitinated proteins (whether mono- or polyubiquitinated) through immunoblotting of samples. Moreover, in contrast to other methods for affinity tagging of ubiquitin, or affinity purification via ubiquitin-binding domains, the use of the biotin tag enables purification under highly denaturing conditions for stringent isolation of ubiquitin-conjugated material from higher eukaryotes. His₆-tagged ubiquitin is also available for use under denaturing conditions, but it is not generally useful in higher eukaryotic cells, where a high frequency of proteins containing multiple histidine residues confounds the specificity of nickel-affinity pulldowns (as discussed in detail in Ref. 30). Therefore, in this paper we describe the reproducible identification and validation of mitoticphase-specific polyubiquitinated proteins via the in vivo biotinylation of ubiquitin. A large number of polyubiquitinated proteins that we identified are specific to mitotic exit, when the APC/C is active, and we expect that many of them are substrates for the APC/C. We formally identified KIFC1/HSET and Cyk4/RACGAP1 as targets of APC/C-dependent ubiquitin-mediated proteolysis after anaphase onset and investigated the role of their ubiquitination in the regulation of mitotic exit. Cell cycle phase-specific information on protein ubiquitination and the generation of ubiquitinated protein networks provides a framework for further investigation of ubiquitin-controlled processes occurring during the rebuilding of interphase cells.     

Melanin Externalization in Candida albicans Depends on Cell Wall Chitin Structures

The fungal pathogen Candida albicans produces dark-pigmented melanin after 3 to 4 days of incubation in medium containing l-3,4-dihydroxyphenylalanine (l-DOPA) as a substrate. Expression profiling of C. albicans revealed very few genes significantly up- or downregulated by growth in l-DOPA. We were unable to determine a possible role for melanin in the virulence of C. albicans. However, we showed that melanin was externalized from the fungal cells in the form of electron-dense melanosomes that were free or often loosely bound to the cell wall exterior. Melanin production was boosted by the addition of N-acetylglucosamine to the medium, indicating a possible association between melanin production and chitin synthesis. Melanin externalization was blocked in a mutant specifically disrupted in the chitin synthase-encoding gene CHS2. Melanosomes remained within the outermost cell wall layers in chs3Δ and chs2Δ chs3Δ mutants but were fully externalized in chs8Δ and chs2Δ chs8Δ mutants. All the CHS mutants synthesized dark pigment at equivalent rates from mixed membrane fractions in vitro, suggesting it was the form of chitin structure produced by the enzymes, not the enzymes themselves, that was involved in the melanin externalization process. Mutants with single and double disruptions of the chitinase genes CHT2 and CHT3 and the chitin pathway regulator ECM33 also showed impaired melanin externalization. We hypothesize that the chitin product of Chs3 forms a scaffold essential for normal externalization of melanosomes, while the Chs8 chitin product, probably produced in cell walls in greater quantity in the absence of CHS2, impedes externalization.Candida albicans is a major opportunistic fungal human pathogen that causes a wide variety of infections (9, 68). In healthy individuals C. albicans resides as a commensal within the oral cavity and gastrointestinal and urogenital tracts. However, in immunocompromised hosts, C. albicans causes infections ranging in severity from mucocutaneous infections to life-threatening disseminated diseases (9, 68). Research into the pathogenicity of C. albicans has revealed a complex mix of putative virulence factors (7, 60), perhaps reflecting the fine balance this species strikes between commensal colonization and opportunistic invasion of the human host.Melanins are biological pigments, typically dark brown or black, formed by the oxidative polymerization of phenolic compounds. They are negatively charged hydrophobic molecules with high molecular weights and are insoluble in both aqueous and organic solvents. Their insolubility makes melanins difficult to study, and no definitive structure has yet been found for them; they probably represent an amorphous mixture of polymers (35). There are various types of melanin in nature, including eumelanin and phaeomelanin (76). Two principal types of melanin are found in the fungal kingdom. The majority are 1.8-dihydroxynapthalene (DNH) melanins synthesized from acetyl-coenzyme A (CoA) via the polyketide pathway (5). DNH melanins have been found in a wide range of opportunistic fungal pathogens of humans, including dark (dematiaceous) molds, such as Cladosporium, Fonsecaea, Phialophora, and Wangiella species, and as conidial pigments in Aspergillus fumigatus and Aspergillus niger (41, 80, 87, 88). However, several other fungal pathogens, including Blastomyces dermatitidis, Coccidioides posadasii, Cryptococcus neoformans, Histoplasma capsulatum, Paracoccidioides brasiliensis, and Sporothrix schenckii, produce eumelanin (3,4-dihydroxyphenylalanine [DOPA]-melanin) through the activity of a polyphenol oxidase (laccase) and require an exogenous o-diphenolic or p-diphenolic substrate, such as l-DOPA (16, 30, 63,–65, 67, 79).The production of melanin in humans and other mammals is a function of specialized cells called melanocytes. Particles of melanin polymers, sometimes, including more than one melanin type, are built up within membrane-bound organelles called melanosomes (76), and these are actively transported along microtubules to the tips of dendritic outgrowths of melanocytes, from where they are transferred to neighboring cells (32, 81). The mechanism of intercellular transfer of melanosomes has not yet been established, but the export process probably involves the fusion of cell and vesicular membranes rather than secretion of naked melanin (82). In pathogenic fungi, melanins are often reported to be associated with or “in” the cell wall (35, 36, 50, 72, 79). However, there is variation between species: the melanin may be located external to the wall, e.g., in P. brasiliensis (79); within the wall itself (reviewed in reference 42); or as a layer internal to the wall and external to the cell membrane, e.g., in C. neoformans (22, 45, 85). However, mutants of C. neoformans bearing disruptions of three CDA genes involved in the biosynthesis of cell wall chitosan, or of CHS3, encoding a chitin synthase, or of CSR2, which probably regulates Chs3, all released melanin into the culture supernatant, suggesting a role for chitin or chitosan in retaining the pigment polymer in its normal intracellular location (3, 4). However, vesicles externalized from C. neoformans cells also show laccase activity (21), so the effect of chitin may be on vesicle externalization rather than on melanin itself. Internal structures compatible with mammalian melanosomes have been observed in Cladosporium carrionii (73) and in Fonsecaea pedrosoi (2, 26). Remarkably, F. pedrosoi also secretes melanin and locates the polymer within the cell wall (1, 2, 25, 27, 74).Melanization has been found to play an important role in the virulence of several human fungal pathogens, such as C. neoformans, A. fumigatus, P. brasiliensis, S. schenckii, H. capsulatum, B. dermatitidis, and C. posadasii (among recent reviews are references 29, 42, 62, 74, and 79). From these and earlier reviews of the extensive literature, melanin has been postulated to be involved in a range of virulence-associated properties, including interactions with host cells; protection against oxidative stresses, UV light, and hydrolytic enzymes; resistance to antifungal agents; iron-binding activities; and even the harnessing of ionizing radiation in contaminated soils (15). The most extensively studied fungal pathogen for the role of melanization is C. neoformans, which possesses two genes, LAC1 and LAC2, encoding melanin-synthesizing laccases (52, 69, 90). It has been known since early studies with naturally occurring albino variants of C. neoformans (39) that melanin-deficient strains are attenuated in mouse models of cryptococcosis. Deletion of both the LAC1 and LAC2 genes reduced survival of C. neoformans in macrophages (52), and a study based on otherwise isogenic LAC1⁺ and LAC1⁻ strains confirmed the importance of LAC1 in experimental virulence (66). Other genes in the regulatory pathway for LAC1 are similarly known to be essential to virulence (12, 84).C. albicans has been shown to produce melanin with DOPA as a substrate for production of the polymer (53). The cells could be treated with hot acids to produce typical melanin “ghosts,” and antibodies specific for melanin reacted with the fungal cells by immunohistochemistry with tissues from experimentally infected mice, demonstrating that C. albicans produces melanin in vivo (53). However, no candidate genes encoding laccases have yet been identified in the C. albicans genome (http://www.candidagenome.org/). In this study, we investigated the production of melanin by C. albicans and showed that its normal externalization from wild-type cells, including formation of melanosomes, can be altered to an intracellular and intrawall location by mutation of genes involved in chitin synthesis. C. albicans has four genes encoding chitin synthase enzymes. CHS1 is an essential gene under normal conditions (59), and its product is the main enzyme involved in septum formation (83). Chs3 forms the bulk of the chitin in the cell wall and the chitinous ring at sites of bud emergence (8, 51, 57), while Chs2 contributes to differential chitin levels found between yeast and hyphal forms of the fungus, and Chs8 influences the architecture of chitin microfibrils (43, 51, 55, 57, 58). We found that melanin externalization was unaffected in a chs8Δ mutant but was reduced or abrogated in chs2Δ and chs3Δ mutants. Expression profiles of melanin-producing cells grown in the presence of l-DOPA did not identify any potential laccase-synthesizing genes.