Blanc – Accords bilatéraux 2013 - SVSE 3 - Blanc – Accords bilatéraux 2013 - SVSE 3 - Microbiologie, immunologie, infectiologie

Soluble mediators of the immune system against Aspergillus fumigatus – COMASPIN

COMSAPSIN: SOLUBLE MEDIATORS OF THE HOST INNATE IMMUNE SYSTEM AGAINST ASPERGILLUS FUMIGATUS

A. fumigatus is the most ubiquitous human fungal pathogen. Host innate immune system provides first line of defense when A. fumigatus tries to establish its pathogenicity. The role played by the soluble mediators of the innate immune system (complement system, collectins and antimicrobial peptides) is not well studied. Deciphering the role of these soluble mediators in defending against A. fumigatus pathogenicity is the main goal of our project.

Interaction between A. fumigatus and host soluble mediators of the innate immune system

1. To decipher the influence of Aspergillus fumigatus morphotypes and their cell wall components on the activation of the soluble mediators of the immune system: A. fumigatus cells are covered by a thick cell wall, which is mainly composed of different polysaccharides, pigments and proteins that are unique. Being on the surface, the cell wall is the first fungal component to come in contact with the host immune system. However, the composition of this cell wall varies during fungal morphogenesis. The influence of different morphotypes and their respective cell wall components on the complement activation and interaction with collectins and antimicrobial peptides will be studied. <br />2. To analyse the role of A. fumigatus secreted proteins on the complement system, collectins and antimicrobial peptides: Secreted proteins are other major A. fumigatus cell-surface associated components, contributing to the fungal defensive mechanism and host immune invasion. A recent study showed that 64 proteins are released into the culture medium in which A. fumigatus is grown. One such secreted protein, Alp1, an alkaline protease, has been reported to inactivate the complement components. Studying the effect of other secreted proteins on the complement system and PRPs is another objective of this project proposal. <br />3. To study the antifungal effect of host defence (antimicrobial) peptides – alone or in combination with other drugs: The characteristic of fungi is the presence of a cell wall and for the antimicrobial to be active this cell wall has to be permeable. Thus either modifying the cell wall permeability through the deletion of cell wall genes or using the cell wall directed antifungal drugs in combination will lead to the better characterization of the antifungal activity of these peptides.

We obtained A. fumigatus secreted proteins of interest using yeast Pichia pastoris/bacterial Escherichia coli expression system. Thus obtained recombinant proteins were tested for different complement component activation/degradation activity. The protein(s) showing activity were further chosen to be added during opsonization of A. fumigatus conidia with the normal human serum (NHS) and thus opsonized conidia were checked for phagocytosis by human monocyte derived macrophages.

The binding efficiency of different A. fumigatus morphotypes with C3b, the major component of the complement system, one of the soluble mediators of the innate immune system, was checked by FACS analysis, as well as chemical methods to check for the chemical nature of the linkage. C3b binding component on the A. fumigatus conidia was checked by opsonization of conidia with NHS followed by removal of the surface components by chemical method and analyzing the component removed.

Binding efficiency of SP-D, a collectin that is a another component of the soluble mediator of the innate immune system, with different cell wall components of the A. fumigatus was checked by ELISA method. Further, to check binding efficiency of different domains of SP-D with A. fumigatus cell wall components, truncated forms of recombinant SP-D was used.

We started initially with second objective we proposed, the role of A. fumigatus secreted proteins on the complement system. However, we also screened the complement C3 binding efficiency with different A. fumigatus morphotypes and the binding efficiency of collectin SP-D with different cell wall components of A. fumigatus.

1. We could show that, in addition to Alp1, there is a secreted A. fumigatus Mep1, a metalloprotease, which could also degrade complement components
2. Unlike Alp1, which is getting secreted after around 15-h of growth, wherein A. fumigatus is in mycelial form, Mep1 was found in the conidial cell wall upon contact with the collagen medium. Conidia are the A. fumigatus morphotype which enter human lungs and lungs contain collagen
3. In the presence of Mep1, there was less opsonization of conidia and thus lower phagocytosis by human macrophages
4. Collectin SP-D contains collagen and carbohydrate biding domains; collagen domain was found to bind to A. fumigatus conidial melanin and carbohydrate binding domain with the mycelial cell wall galactosaminogalactan
5. Complement C3 binding with A. fumigatus conidia was mainly by ester-linkage
6. There was interaction with A. fumigatus conidial surface RodA and complement C3

The present project led us to collaborate with Frank Veerdonk/Katharina Becker, Radboud University Medical Center, Nijmegen, The Netherlands, wherein we showed differential pro- and anti-inflammatory properties of A. fumigatus cell wall chitin in the presence and absence of serum immunoglobulins, one of the soluble mediators of the innate immune system. Based on this work, a manuscript is been prepared and is ready to submit. While working with collectins, addition to obtain them commercially, we started collaborating with Dr. Uday Kishor, Brunel University, London UK, who had the repertoire of full length and truncated/mutated forms of SP-A and SP-D, who has agreed and sent them to us (IP, Pasteur and NIRRH, Pune) to work on.

Opsonization of A. fumigatus conidia is considered to be important for conidial phagocytosis by the host phagocytes, but the mechanism of opsonization and fungal surface component involved in the opsonization were not known. Here we show that there is the formation of adduct between A. fumigatus conidial surface RodA, which is known to mask the conidial recognition by the host immune cells upon forming a surface rodlet layer, and the host complement component C3b. Presently we are looking at the amino acid residues involved in the binding between C3b and RodA.

On the other hand, A. fumigatus secretome is known plays an important role in evading host defense system, and we identified secreted Mep1 as the complement degrading factor in addition to Alp1, an alkaline protease, which has been identified earlier as another complement degrading factor. However, Alp1 is secreted by the mycelial form of A. fumigatus whereas Mep1 is found in the conidial form. This identifies secreted proteases as A. fumigatus virulence factor and hence these proteases could be worth checking to be antifungal targets.

In addition, collectins, other soluble mediators of the innate immune system, is supposed to play a role against A. fumigatus pathogenicity; however their antifungal mechanism is not known. We identified A. fumigatus cell wall components binding with one of the collectins, SP-D. Further we check for the importance of such binding between SP-D and A. fumigatus.

1. Manuscript on ‘The role of Mep1, an A. fumigatus secreted metalloprotease, on the complement system’ is under preparation based on the results obtained while working on the present project - COMASPIN.

2. The present project led us to colla

The threat posed to hospitalized patients due to fungal infection is attributable to: (i) Lack of a sensitive, rapid and accurate diagnostic assays for invasive fungal infection, (ii) antifungal drugs have only modest success in reducing the high mortality rates of invasive mycoses and are inactive against newly emerging antifungal-resistant strains; the cost of treating Aspergillus (the most common human fungal pathogens) infections in Europe is approximately €80,000 per patient and the annual cost of administering antifungal agents often overrides that of antibacterial agents, (iii) The pathobiology of fungal infections remains poorly understood and (iv) The population of patients at risk is steadily increasing. Even in India, Aspergillus is becoming medically important as they cause most prevalent invasive fungal infections.

Accordingly, this project proposal is centered on Aspergillus fumigatus, though a saprophyte growing on decaying vegetation is the most ubiquitous opportunistic human fungal pathogen world-wide. It causes a number of diseases such as lung/sinus aspergilloma and allergic bronchopulmonary aspergillosis in the predisposed immunocompetent human population. However, the most fatal one is the invasive aspergillosis (IA), a systemic infection in the immunocompromised individuals; the frequency of IA has risen more than 10-folds worldwide in the last two decades.

A. fumigatus conidia entering the human lung alveoli are confronted with the innate immune system, both cellular barriers and soluble mediators. The role played by the cellular barriers in evading A. fumigatus is quite well studied but that of the soluble mediators (the complement system, collectins and antimicrobial peptides) is mostly ignored. The cellular barriers are mainly resident alveolar macrophages, polymorphonuclear neutrophils (PMNs) and respiratory epithelial cells. The soluble mediators are playing a central role as innate immune effector elements against invading microbes; but their mechanisms of action against A. fumigatus are unknown.

On the other hand, A. fumigatus exploit several strategies to evade host defense: in the conidial stage, the surface rodlet layer masks their immune recognition and during vegetative growth proteins secreted are reported to evade host defense thus contributing for the pathogenicity.

Our proposed project is focussed on the interaction between A. fumigatus and the soluble mediators of the innate immune system. Accordingly, the main goals are:

Host - fungus: To understand the exact role played by the soluble mediators as immune effector elements against A. fumigatus.
Fungus - host: To understand the role of A. fumigatus secreted proteins (secretome) in establishing pathogenicity by acting against defense mechanisms played by the soluble mediators of the immune system.

Our objectives are innovative and of medical importance, combining the three themes of Blanc SVSE3 microbiology-immunology-infection program. Interest in this International collaboration is that, combating invasive fungal infection is the aim of both the countries participating in the proposed project and the partners complement one another in achieving proposed goals. A face-to-face discussion was made among the partners during the Franco-Indian Symposium (HOPE IN RED) sponsored by CEFIPRA [Bangalore, India, 2010] to initiate this collaboration. We (Partner n°1) have experience in the field of fungal biology. Dr. Taruna Madan’s (Partner n°3; India) theme of work is the role of collectins in the A. fumigatus infection, while Dr. Arvind Sahu (Partner n°2; India) has got expertise in the complement system field (particularly, viral complement invasion) and enthusiastic to enter the fungal-field. Due to the development of resistance, search for the new antifungal drugs is of global thrust and outcome of our proposed project might bring out future collaborations in developing new such antifungal drugs.

Project coordinator

Monsieur Vishukumar Aimanianda (Aspergillus Unit) – vkumar@pasteur.fr

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

NIRRH National Institute for Research In Reproductive Health, Mumbai, India
IP Aspergillus Unit
NCCS National Centre for Cell Science, Pune, India

Help of the ANR 270,000 euros
Beginning and duration of the scientific project: December 2013 - 36 Months

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