Amandine Gastebois

Aspergillus fumigatus: cell wall polysaccharides, their biosynthesis and organization

Aspergillus fumigatus is the most prevalent thermophilic inhabitants of decaying vegetation and one of the most important human opportunistic fungal pathogens. Like other fungi, A. fumigatus cells are covered by a cell wall, which is both a protective, rigid exoskeleton and a dynamic structure, undergoing constant modification depending on its environment. The cell wall, in the majority of fungi, is composed of polysaccharides, and understanding the biochemical organization and biogenesis of an A. fumigatus cell wall is essential as this envelop is continuously in contact with the environment/host cell and acts as a sieve and reservoir for molecules, such as enzymes and toxins that play an active role during infection. This article is intended to give an overview of the biosynthesis of constituent cell wall polysaccharides and their postsynthetic modification in A. fumigatus, it also discusses the antifungal drugs that affect cell wall polysaccharide biosynthesis.

Characterization of a New β(1–3)-Glucan Branching Activity of Aspergillus fumigatus

A new HPLC method was developed to separate linear from β(1–6)-branched β(1–3)-glucooligosaccharides. This methodology has permitted the isolation of the first fungal β(1–6)/β(1–3)-glucan branching transglycosidase using a cell wall autolysate of Aspergillus fumigatus (Af). The encoding gene, AfBGT2 is an ortholog of AfBGT1, another transglycosidase of A. fumigatus previously analyzed (Mouyna, I., Hartland, R. P., Fontaine, T., Diaquin, M., Simenel, C., Delepierre, M., Henrissat, B., and Latgé, J. P. (1998) Microbiology 144, 3171–3180). Both enzymes release laminaribiose from the reducing end of a β(1–3)-linked oligosaccharide and transfer the remaining chain to another molecule of the original substrate. The AfBgt1p transfer occurs at C-6 of the non-reducing end group of the acceptor, creating a kinked β(1–3;1–6) linear molecule. The AfBgt2p transfer takes place at the C-6 of an internal group of the acceptor, resulting in a β(1–3)-linked product with a β(1–6)-linked side branch. The single Afbgt2 mutant and the double Afbgt1/Afbgt2 mutant in A. fumigatus did not display any cell wall phenotype showing that these activities were not responsible for the construction of the branched β(1–3)-glucans of the cell wall.

Microbial antioxidant defense enzymes

Free radicals are often described as chemical compounds characterized by unpaired electrons in their outer orbital rendering them highly reactive species. In mammalians, studies on free radicals were focused on reactive oxygen species (ROS) or reactive nitrogen species (RNS) due to their relative importance in physiological as well as in pathological processes. These cellular compounds are produced by different physiological systems such as the aerobic metabolism and play a major role in cell signaling pathways but also in the host immune defenses against pathogenic microorganisms. ROS and RNS are highly reactive species with potentially harmful effects on any cellular components (lipids, proteins and nucleic acids) when produced with a high level. To maintain ROS and RNS at a non-toxic concentration, enzymatic and non-enzymatic cellular antioxidants coordinate the balance between their production and their degradation. Superoxide dismutases, catalases, glutathione system, thioredoxin system, peroxidase systems, flavohemoglobins and nitrate or nitrite reductases represent the prominent enzymatic antioxidants used to scavenge excess of internal as well as external ROS and RNS. Bacteria, fungi and parasites also display similar enzymatic activities to escape the host oxidative defenses during the immune response against infectious processes. Here we summarize current knowledge on the enzymatic systems that allow microorganisms to fight against ROS and RNS, and shed light on the role that take some of them in microbial infections. Such microbial protective systems are considered as virulence factors, and therefore represent key targets for diagnosis of the infections or development of anti-infectious drugs.

A Multifaceted Study of Scedosporium boydii Cell Wall Changes during Germination and Identification of GPI-Anchored Proteins

Scedosporium boydii is a pathogenic filamentous fungus that causes a wide range of human infections, notably respiratory infections in patients with cystic fibrosis. The development of new therapeutic strategies targeting S. boydii necessitates a better understanding of the physiology of this fungus and the identification of new molecular targets. In this work, we studied the conidium-to-germ tube transition using a variety of techniques including scanning and transmission electron microscopy, atomic force microscopy, two-phase partitioning, microelectrophoresis and cationized ferritin labeling, chemical force spectroscopy, lectin labeling, and nanoLC-MS/MS for cell wall GPI-anchored protein analysis. We demonstrated that the cell wall undergoes structural changes with germination accompanied with a lower hydrophobicity, electrostatic charge and binding capacity to cationized ferritin. Changes during germination also included a higher accessibility of some cell wall polysaccharides to lectins and less CH3/CH3 interactions (hydrophobic adhesion forces mainly due to glycoproteins). We also extracted and identified 20 GPI-anchored proteins from the cell wall of S. boydii, among which one was detected only in the conidial wall extract and 12 only in the mycelial wall extract. The identified sequences belonged to protein families involved in virulence in other fungi like Gelp/Gasp, Crhp, Bglp/Bgtp families and a superoxide dismutase. These results highlighted the cell wall remodeling during germination in S. boydii with the identification of a substantial number of cell wall GPI-anchored conidial or hyphal specific proteins, which provides a basis to investigate the role of these molecules in the host-pathogen interaction and fungal virulence.

Major Sensing Proteins in Pathogenic Fungi: The Hybrid Histidine Kinase Family.

The pioneering discovery of histidine kinases (HKs) from Escherichia coli was made in the early 1980s with the identification of the envZ gene [1] (Fig 1A). Further biochemical characterization of the corresponding protein revealed a new type of protein kinase activity, namely HK, to add to the well-known serine/threonine and tyrosine kinases. For a decade, HKs were considered to be restricted to bacteria, but in the 1990s, HKs were identified in plants [2], fungi [3], archaea [4], cyanobacteria [5], and amoebae (Fig 1A) [6]. Soon after, evidence suggested that HKs regulate essential processes in pathogenic bacteria and fungi [7]. Although some HKs appear to be present in humans, typical bacterial or fungal HK-like sensor proteins have not been reported yet in mammals [8], promoting these proteins as ideal targets for future therapeutics [9]. It is now accepted that HKs are involved in cell signaling systems referred to as His-to-Asp phosphorelays and several canonical schemes depicting transduction pathways involving HKs in bacteria, amoebae, plants, and fungi have emerged (Fig 1B). To date, HKs act as primary sensors for various environmental stimuli, and, upon activation, initiate phosphate transfer events between various proteins, leading to an adaptive response. Although these mechanistic models are largely described in bacteria and plants, limited evidence is available for amoebae and fungi.

The Identification of Phytohormone Receptor Homologs in Early Diverging Fungi Suggests a Role for Plant Sensing in Land Colonization by Fungi

ABSTRACT Histidine kinases (HKs) are among the most prominent sensing proteins studied in the kingdom Fungi. Their distribution and biological functions in early diverging fungi (EDF), however, remain elusive. We have taken advantage of recent genomic resources to elucidate whether relationships between the occurrence of specific HKs in some EDF and their respective habitat/lifestyle could be established. This led to the unexpected discovery of fungal HKs that share a high degree of similarity with receptors for plant hormones (ethylene and cytokinin). Importantly, these phytohormone receptor homologs are found not only in EDF that behave as plant root symbionts or endophytes but also in EDF species that colonize decaying plant material. We hypothesize that these particular sensing proteins promoted the interaction of EDF with plants, leading to the conquest of land by these ancestral fungi.

Developing collaborative works for faster progress on fungal respiratory infections in cystic fibrosis

Cystic fibrosis (CF) is the major genetic inherited disease in Caucasian populations. The respiratory tract of CF patients displays a sticky viscous mucus, which allows for the entrapment of airborne bacteria and fungal spores and provides a suitable environment for growth of microorganisms, including numerous yeast and filamentous fungal species. As a consequence, respiratory infections are the major cause of morbidity and mortality in this clinical context. Although bacteria remain the most common agents of these infections, fungal respiratory infections have emerged as an important cause of disease. Therefore, the International Society for Human and Animal Mycology (ISHAM) has launched a working group on Fungal respiratory infections in Cystic Fibrosis (Fri-CF) in October 2006, which was subsequently approved by the European Confederation of Medical Mycology (ECMM). Meetings of this working group, comprising both clinicians and mycologists involved in the follow-up of CF patients, as well as basic scientists interested in the fungal species involved, provided the opportunity to initiate collaborative works aimed to improve our knowledge on these infections to assist clinicians in patient management. The current review highlights the outcomes of some of these collaborative works in clinical surveillance, pathogenesis and treatment, giving special emphasis to standardization of culture procedures, improvement of species identification methods including the development of nonculture-based diagnostic methods, microbiome studies and identification of new biological markers, and the description of genotyping studies aiming to differentiate transient carriage and chronic colonization of the airways. The review also reports on the breakthrough in sequencing the genomes of the main Scedosporium species as basis for a better understanding of the pathogenic mechanisms of these fungi, and discusses treatment options of infections caused by multidrug resistant microorganisms, such as Scedosporium and Lomentospora species and members of the Rasamsonia argillacea species complex.

Diversity and Evolution of Sensor Histidine Kinases in Eukaryotes

Abstract Histidine kinases (HKs) are primary sensor proteins that act in cell signaling pathways generically referred to as “two-component systems” (TCSs). TCSs are among the most widely distributed transduction systems used by both prokaryotic and eukaryotic organisms to detect and respond to a broad range of environmental cues. The structure and distribution of HK proteins are now well documented in prokaryotes, but information is still fragmentary for eukaryotes. Here, we have taken advantage of recent genomic resources to explore the structural diversity and the phylogenetic distribution of HKs in the prominent eukaryotic supergroups. Searches of the genomes of 67 eukaryotic species spread evenly throughout the phylogenetic tree of life identified 748 predicted HK proteins. Independent phylogenetic analyses of predicted HK proteins were carried out for each of the major eukaryotic supergroups. This allowed most of the compiled sequences to be categorized into previously described HK groups. Beyond the phylogenetic analysis of eukaryotic HKs, this study revealed some interesting findings: 1) characterization of some previously undescribed eukaryotic HK groups with predicted functions putatively related to physiological traits; 2) discovery of HK groups that were previously believed to be restricted to a single kingdom in additional supergroups, and 3) indications that some evolutionary paths have led to the appearance, transfer, duplication, and loss of HK genes in some phylogenetic lineages. This study provides an unprecedented overview of the structure and distribution of HKs in the Eukaryota and represents a first step toward deciphering the evolution of TCS signaling in living organisms.

Progressive loss of hybrid histidine kinase genes during the evolution of budding yeasts (Saccharomycotina)

Two-component systems (TCSs) are widely distributed cell signaling pathways used by both prokaryotic and eukaryotic organisms to cope with a wide range of environmental cues. In fungi, TCS signaling routes, that mediate perception of stimuli, correspond to a multi-step phosphorelay between three protein families including hybrid histidine kinases (HHK), histidine phosphotransfer proteins (HPt) and response regulators (RR). The best known of these fungal transduction pathways remains the Sln1(HHK)–Ypd1(HPt)–Ssk1(RR) system that governs the high-osmolarity glycerol (HOG) mitogen-activated protein kinase (MAPK) pathway for osmo-adaptation in Saccharomyces cerevisiae. Although recent advances have provided a preliminary overview of the distribution of TCS proteins in the kingdom Fungi, underlying mechanisms that drive the remarkable diversity among HHKs and other TCS proteins in different fungal lineages remain unclear. More precisely, evolutionary paths that led to the appearance, transfer, duplication, and loss of the corresponding TCS genes in fungi have never been hitherto addressed. In the present study, we were particularly interested in studying the distribution of TCS modules across the so-called “budding yeasts clade” (Saccharomycotina) by interrogating the genome of 82 species. With the exception of the emergence of an additional RR (named Srr1) in the fungal CTG clade, TCS proteins Ypd1 (HPt), Ssk1 (RR), Skn7 (RR), and Rim15 (RR) are well conserved within the Saccharomycotina. Surprisingly, some species from the basal lineages, especially Lipomyces starkeyi, harbor several filamentous-type HHKs that appear as relict genes that have been likely retained from a common ancestor of Saccharomycotina. Overall, this analysis revealed a progressive diminution of the initial pool of HHK-encoding genes during Saccharomycotina yeast evolution.