The Aspergillus fumigatus cspA Gene Encoding a Repeat-Rich Cell Wall Protein Is Important for Normal Conidial Cell Wall Architecture and Interaction with Host Cells

cspA (for cell surface protein A) encodes a repeat-rich glycophosphatidylinositol (GPI)-anchored cell wall protein (CWP) in the pathogenic fungus Aspergillus fumigatus. The number of repeats in cspA varies among isolates, and this trait is used for typing closely related strains of A. fumigatus. We have previously shown that deletion of cspA is associated with rapid conidial germination and reduced adhesion of dormant conidia. Here we show that cspA can be extracted with hydrofluoric acid (HF) from the cell wall, suggesting that it is a GPI-anchored CWP. The cspA-encoded CWP is unmasked during conidial germination and is surface expressed during hyphal growth. Deletion of cspA results in weakening of the conidial cell wall, whereas its overexpression increases conidial resistance to cell wall-degrading enzymes and inhibits conidial germination. Double mutant analysis indicates that cspA functionally interacts with the cell wall protein-encoding genes ECM33 and GEL2. Deletion of cspA together with ECM33 or GEL2 results in strongly reduced conidial adhesion, increased disorganization of the conidial cell wall, and exposure of the underlying layers of chitin and β-glucan. This is correlated with increasing susceptibility of the ΔcspA, ΔECM33, and ΔcspA ΔECM33 mutants to conidial phagocytosis and killing by human macrophages and hyphal damage induced by neutrophils. However, these strains did not exhibit altered virulence in mice with infected lungs. Collectively, these results suggest a role for cspA in maintaining the strength and integrity of the cell wall.The saprophytic mold Aspergillus fumigatus is an emerging pathogen and the major causative agent of invasive aspergillosis, a life-threatening disease primarily affecting immunocompromised patients (12, 16, 38).Molecular analyses have revealed numerous virulence attributes that enable A. fumigatus to infect the human host, including the production of toxins, the ability to acquire nutrients and iron under limiting conditions, and the presence of protective mechanisms that degrade oxygen radicals released by the host immune cells (7).The fungal cell wall plays a crucial role in infection. In A. fumigatus, as in other pathogenic fungi, the cell wall protects the fungus and interacts directly with the host immune system. It is an elastic, dynamic, and highly regulated structure and is essential for growth, viability, and infection. The fungal cell wall is a unique structure and therefore a specific target for antifungal drugs. The cell wall of A. fumigatus is composed of a polysaccharide skeleton interlaced and coated with cell wall proteins (CWPs). The main building blocks of the polysaccharide skeleton are an interconnected network of glucan, chitin, and galactomannan polymers (26). The major class of fungal CWPs is the glycophosphatidylinositol (GPI)-modified proteins (8,–11, 14).We recently identified and characterized A. fumigatus CWPs containing tandem repeats (27). Repeats are hot spots of genetic change: because of replication slippage and recombination, repeats can undergo rapid changes in copy number, leading to natural variability among different isolates and allowing faster adaptation to new environments (23). In Saccharomyces cerevisiae, for example, an increase in the number of coding repeats in the FLO1 adhesin-encoding gene correlates with an increase in adhesion to the plastics used in medical devices (44,–46). Similarly, repeat variation in the Candida albicans ALS3 adhesin changes its cellular binding specificity (34). Moreover, clinical C. albicans isolates show variability in the number of repeats in various cell surface genes, suggesting that this recombination process could play a role during infection, allowing cells to adapt rapidly to a fluctuating environment and/or evade the host immune system (34, 49, 50).We identified four genes encoding putative A. fumigatus GPI-anchored CWPs (AFUA_3G08990 [termed cspA for cell-surface protein A [4], AFUA_2G05150 [MP-2], AFUA_4G09600, and AFUA_6G14090) containing variable numbers of repeats among patient isolates (27). In A. fumigatus WT strain AF 293, cspA encodes a 433-amino-acid-long protein containing a putative leader sequence and GPI modification site. cspA lacks recognizable catalytic domains, and homologous genes are found only in species of Aspergillus. Most interesting is that the gene encodes a 188-amino-acid-long serine-threonine-proline-rich N-terminal region followed by a large size-variable six-amino-acid serine-proline [P-G-Q-P-S-(A/V)]-rich tandem repeat region showing significant homology to the repeat domains found in mammalian type XXI collagen. The number of repeats varies between 18 and 47 (24 to 65% of the length of the protein) in different isolates of A. fumigatus. The strains used in this study, AF 293 and CBS 144.89, contain 32 and 28 repeats, respectively.Deletion of cspA resulted in a phenotype characterized by rapid conidial germination and reduced adhesion to extracellular matrix (ECM), which suggests that cspA participates in defining cell surface properties. Highlighting the importance of this gene, Balajee et al. (4) showed that variations in the cspA nucleotide repeat sequence can be used to type closely related pathogenic isolates of A. fumigatus and identify outbreak clusters occurring in hospitals (3, 4).In this work, we undertook a detailed study of cspA. We analyzed the expression pattern of the protein encoded by cspA and its attachment to the cell wall. We prepared and analyzed A. fumigatus mutant strains in which cspA was overexpressed or deleted in combination with additional cell wall-associated genes. Results indicate that the protein encoded by cspA is GPI anchored to the cell wall and is unmasked during conidial germination. cspA deletion weakens the cell wall and results in rapid conidial germination, whereas cspA overexpression increases conidial resistance to protoplasting and inhibits conidial germination. cspA functionally interacts with the genes ECM33 and GEL2, which encode cell wall-associated proteins, resulting primarily in profound defects in conidial cell wall organization. The cspA ECM33 double mutant exhibited greater susceptibility to killing by human macrophages and hyphal damage induced by neutrophils. The implications of our findings are discussed.     

Splitting the BLOSUM Score into Numbers of Biological Significance

Mathematical tools developed in the context of Shannon information theory were used to analyze the meaning of the BLOSUM score, which was split into three components termed as the BLOSUM spectrum (or BLOSpectrum). These relate respectively to the sequence convergence (the stochastic similarity of the two protein sequences), to the background frequency divergence (typicality of the amino acid probability distribution in each sequence), and to the target frequency divergence (compliance of the amino acid variations between the two sequences to the protein model implicit in the BLOCKS database). This treatment sharpens the protein sequence comparison, providing a rationale for the biological significance of the obtained score, and helps to identify weakly related sequences. Moreover, the BLOSpectrum can guide the choice of the most appropriate scoring matrix, tailoring it to the evolutionary divergence associated with the two sequences, or indicate if a compositionally adjusted matrix could perform better.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29]     

Analysis of Free Energy Signals Arising from Nucleotide Hybridization Between rRNA and mRNA Sequences during Translation in Eubacteria

A decoding algorithm is tested that mechanistically models the progressive alignments that arise as the mRNA moves past the rRNA tail during translation elongation. Each of these alignments provides an opportunity for hybridization between the single-stranded, -terminal nucleotides of the 16S rRNA and the spatially accessible window of mRNA sequence, from which a free energy value can be calculated. Using this algorithm we show that a periodic, energetic pattern of frequency 1/3 is revealed. This periodic signal exists in the majority of coding regions of eubacterial genes, but not in the non-coding regions encoding the 16S and 23S rRNAs. Signal analysis reveals that the population of coding regions of each bacterial species has a mean phase that is correlated in a statistically significant way with species () content. These results suggest that the periodic signal could function as a synchronization signal for the maintenance of reading frame and that codon usage provides a mechanism for manipulation of signal phase.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Synergy between Ficolin-2 and Pentraxin 3 Boosts Innate Immune Recognition and Complement Deposition

The long pentraxin 3 (PTX3) is a multifunctional soluble pattern recognition molecule that is crucial in innate immune protection against opportunistic fungal pathogens such as Aspergillus fumigatus. The mechanisms that mediate downstream effects of PTX3 are largely unknown. However, PTX3 interacts with C1q from the classical pathway of the complement. The ficolins are recognition molecules of the lectin complement pathway sharing structural and functional characteristics with C1q. Thus, we investigated whether the ficolins (Ficolin-1, -2, and -3) interact with PTX3 and whether the complexes are able to modulate complement activation on A. fumigatus. Ficolin-2 could be affinity-isolated from human plasma on immobilized PTX3. In binding studies, Ficolin-1 and particularly Ficolin-2 interacted with PTX3 in a calcium-independent manner. Ficolin-2, but not Ficolin-1 and Ficolin-3, bound A. fumigatus directly, but this binding was enhanced by PTX3 and vice versa. Ficolin-2-dependent complement deposition on the surface of A. fumigatus was enhanced by PTX3. A polymorphism in the FCN2 gene causing a T236M amino acid change in the fibrinogen-like binding domain of Ficolin-2, which affects the binding to GlcNAc, reduced Ficolin-2 binding to PTX3 and A. fumigatus significantly. These results demonstrate that PTX3 and Ficolin-2 may recruit each other on pathogens. The effect was alleviated by a common amino acid change in the fibrinogen-like domain of Ficolin-2. Thus, components of the humoral innate immune system, which activate different complement pathways, cooperate and amplify microbial recognition and effector functions.The ficolins are multimeric collagen-like proteins consisting of an N-terminal domain, a collagen-like domain (CD),² and a C-terminal fibrinogen-like (FBG) domain involved in innate immune defense (1, 2). In humans, three types of ficolins have been identified as follows: Ficolin-1 (M-ficolin), Ficolin-2 (L-ficolin), and Ficolin-3 (H-ficolin/Hakata antigen). They function as recognition molecules in the lectin complement pathway along with mannose-binding lectin but with differentiated complement activating capacity (3). Ficolin-2 and Ficolin-3 circulate in the blood with a median concentration of 5 and 25 μg/ml, respectively (4, 5). Ficolin-2 is mainly produced in the liver, whereas Ficolin-3 is synthesized in both the liver and lungs, with the highest expression in the lungs (3). Ficolin-1 is primarily expressed by bone marrow-derived cells and lung epithelial cells (3, 6–8) and has recently been shown to be present in the blood with a median plasma concentration of 60 ng/ml (9). The ficolin genes (FCN1, -2, and -3) are polymorphic, and particularly polymorphisms in FCN2 regulate both the level and function of Ficolin-2 (4, 10, 11). In this respect, a base substitution in exon 8 at position 6359 (C→T) causing a threonine to be replaced by a methionine (T236M) in the FBG domain of Ficolin-2 has been shown to cause decreased binding activity toward GlcNAc (10).Ficolin-1 has been reported to bind to GlcNAc, GalNAc, and sialic acid (8, 12). It may opsonize Staphylococcus aureus via GlcNAc and interact with a smooth-type strain of Salmonella typhimurium through an unknown ligand, the binding of which is not diminished by GlcNAc (8). Ficolin-2 has been shown to recognize specific pathogen-associated molecular patterns, which are typically located in pathogen cell membranes, such as lipoteichoic acid and peptidoglycan in Gram-positive bacteria cell walls, lipopolysaccharide in Gram-negative bacteria cell walls, and 1,3-β-d-glucan in yeast and fungal cell walls (13, 14). The ligand specificity of Ficolin-2 has also been defined as acetyl groups, including those of N-acetylmannosamine, GlcNAc, GalNAc as well as acetyl groups on cysteine, glycine, and choline (15). Ficolin-2 recognizes clinically important pathogens, like S. typhimurium, S. aureus, and Streptococcus pneumoniae (13, 16, 17). Ficolin-3 shows affinity for GlcNAc, GalNAc, and d-fucose and may interact with S. typhimurium, Salmonella minnesota, and Aerococcus viridans (17, 18).The long pentraxin 3 (PTX3) is a soluble pattern recognition molecule mediating innate immune recognition (19). PTX3 is a glycoprotein of 45 kDa, which assembles into an octameric structure through protomer linkage by disulfide bonds (20). PTX3 shares C-terminal structural similarity with the classic short pentraxins, C-reactive protein (CRP), and serum amyloid P component, whereas the N-terminal sequence differs from the other proteins (21). Myeloid cells are a major source of PTX3, but PTX3 has also been shown in vitro to be produced by a variety of cells in response to inflammatory signals (21). During inflammation PTX3 is rapidly up-regulated and released into the surrounding tissue and into the bloodstream. PTX3 interacts with C1q and participates in activation of the classical complement pathway (22, 23). Moreover, it has also been shown that PTX3 binds the complement regulatory factor H and that this interaction regulates the alternative pathway of complement (24).PTX3 can interact with a number of different pathogens, bacteria as well as fungi and viruses. A specific binding has been observed for selected Gram-positive and Gram-negative bacteria, including S. aureus, Pseudomonas aeruginosa, S. typhimurium, Klebsiella pneumoniae, S. pneumoniae, and Neisseria meningitidis (21). PTX3 also binds zymosan and conidia from Aspergillus fumigatus) (25). Furthermore, it has been shown that ptx3 knock-out mice are extremely susceptible to invasive pulmonary aspergillosis. The phenotypic defect can be completely reversed by treatment with recombinant PTX3 (25, 26). These data indicate that PTX3 is important in protection against A. fumigatus, which has become a major cause of morbidity in medical institutions because of the increasing number of immunosuppressed patients (27).Based on the knowledge of the structural and functional similarities between C1q and the ficolins, this study was designed to characterize a possible interaction between the ficolins and PTX3 using A. fumigatus as a model. Based on our data, we propose an important role for previously unlinked collaboration of PTX3 and Ficolin-2, but not Ficolin-1 and Ficolin-3, in the recognition of A. fumigatus and amplification of complement activation. Moreover, our results demonstrate functional consequences of the Ficolin-2 T236M substitution in the interaction between PTX3 and A. fumigatus.     

Algorithms for Finding Small Attractors in Boolean Networks

A Boolean network is a model used to study the interactions between different genes in genetic regulatory networks. In this paper, we present several algorithms using gene ordering and feedback vertex sets to identify singleton attractors and small attractors in Boolean networks. We analyze the average case time complexities of some of the proposed algorithms. For instance, it is shown that the outdegree-based ordering algorithm for finding singleton attractors works in time for , which is much faster than the naive time algorithm, where is the number of genes and is the maximum indegree. We performed extensive computational experiments on these algorithms, which resulted in good agreement with theoretical results. In contrast, we give a simple and complete proof for showing that finding an attractor with the shortest period is NP-hard.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]