Presented by

Lionel Williamson

Aspergillus species

Kingdom: Fungi Phylum: AscomycotaOrder: EurotialesFamily: TrichocomaceaeGenus gi: Aspergillus

Aspergillus is a filamentous, ubiquitous fungus found in nature. It is commonly isolated from soil, plant debris, and indoor air environments.

Types of Aspergillus

The genus Aspergillus includes over 185 species. Around 20 species have so far been reported as causative agents of opportunistic infections in man

A. flavus produces aflotoxins A. nidulans produces cutaneous

aspergillosisA. oryzae used to make soy sauce, and sake

A. fumigatus

Is the second most common opportunistic fungal infection (Candida)

This species is the most common agent of aspergillosis in both man and animals. It is a thermophilic species (growth at 40°C and beyond) that may be extremely angioinvasive, particularly in the compromised patient

Key words

T helper 1- TH1 Cytokine response that deals with pro inflammatory response

T helper 2- TH2 Cytokine response that deals with anti-inflammatory response

PBMC are macrophages and are involved with the innate immune response

Innate immunity refers to antigen-nonspecific defense mechanisms that a host uses immediately or within several hours after exposure to almost any antigen. This is the immunity one is born with and is the initial response by the body to eliminate microbes and prevent infection.

Cytokines

Proteins produced by white blood cells that act as chemical messengers between cells. They can stimulate or inhibit the growth and activity of various immune cells

Pro-inflammatory response ( TNF, IL-1)Anti-inflammatory response (IL-10)

Structure of Aspergillus

Conidia are asexual spores that grown on elaborate structures called conidiophores. These are usually stalked, lifting the conidia off the substrate for better dispersal and to avoid microscopic grazing animals. They often produce hundreds or thousands of conidia at a time

Hyphae A hypha (plural hyphae) is a long, branching filament that, with other hyphae, forms the feeding thallus of a fungus called the mycelium. A typical hypha consists of a tubular wall, usually made of chitin, which surrounds, supports, and protects the cells that compose a hypha. For most fungi, a cell within a hyphal filament is separated from other cells by internal cross-walls called septa

Hyphae vs. conidia

  The University of Adelaide

Life cycle of Aspergillus

A fungus begins its existence as an independent biont as some sort of propagule, most often a spore. From the spore, the fungus grows in a thread-like, branching formation known as a hypha.

The hypha grow and intertwine and form mycelia which become the body of the fungus.

Germinating conidiaRequirement of spermidine for developmental transitions in Aspergillus nidulans Yuan Jin 1 , Jin Woo Bok 2 , Doralinda Guzman-de-Peña 3 and Nancy P. Keller

Toll like receptors

Are pattern-recognition receptors believed to play a role in innate immunity

The receptor was first isolated from Drosophila as a gene required for ontogenesis and antifungal resistance

There have been several TLR’s isolated from humans

Types of TLR’s

TLR 2 recognizes peptidoglycan that are associated with gram positive bacteria

TLR 4 recognizes LPS associated with gram negative bacteria

TLR5 recognizes flagellaTLR9 recognizes bacterial DNA

Experiment 1

Challenged mouse macrophages with conidia or hyphae from A. fumigatus

They found that maximal cytokine release induced by stimulation with 10^7 cfu

Assessed TLR4’s role by using a TLR4-deficient mutant compared to a control

Assessed TLR2’s role by using a TLR2-deficient mutant compared to a control

Exp 1 cont

Experiment 2

To prove that TLR4 was involved in conidia and not hyphae recognition

Using a NF-kB reporter plasmid that drives CD 25 expression

Used a cell line (3E10) that expressed TLR4 but not TLR2

Stimulated the cells with LPS (TLR4 antagonist) Pam3cys (TLR2 antagonist)

Then they transfected cDNA for TLR2

Exp 2

Experiment 3

They wanted to see what roles TLR4 and TLR2 played on the Pro-inflammatory response

Used PBMC’s with antibodies blocking the TLR4 then stimulating with 10^7 cfu

The same experiment was repeated with TLR2

Exp 3 cont.

Experiment 4

Which TLR stimulated IL-10 Recent data suggests that TLR2 stimulates TH-2

response and TLR4 stimulates a TH-1 response Role of IL-10 in invasive aspergillosis:

increased resistance of IL-10 gene knockout mice to lethal systemic aspergillosis

K. V. Clemons, G. Grunig* , R. A. Sobel , L. F. Mirels, D. M. Rennick* & D. A. Stevens

Exp 4 cont.

My conclusion

I think that what happens is that Aspergillus conidia gets into the body and meets the macrophages that expresses TLR4. This causes an increase of expression of IL-1 and TNF, this kills the most of the fungus.

The fungus counteracts this by germinating from the conidia to the hyphae forms. TLR4 does not recognize the new form and the hyphae grows. In a normal immune system TLR2 which has 2 functions (pro-inflammatory and anti-inflammatory) with the help of the cell mediated response the body is able to fight off the fungal infection

Immunocompromised

The same things occur but there is a diminished cell mediated response due to the disease process

TLR2 is releasing both pro-inflammatory and an Anti-inflammatory response

This decreases the destruction of the hyphae and they continue to grow.

Where they form mycelia and cause Aspergillosis.

Aspergillosis

- Pulmonary aspergillosis 1- CNS aspergillosis- Sinonasal aspergillosis- Osteomyelitis - Endophthalmitis- Endocarditis- Renal abscesses- Cutaneous

Why do you think pulmonary aspergillosis is the number one form?

This is the air sacs of a hen during an outbreak of aspergillosis

  The University of Adelaide

Fungal balls made up of hyphae isolated from the lungs

  The University of Adelaide

Aspergillus fumigatus in lung tissue, note conidial heads forming in an alveolus

  The University of Adelaide

Aspergilloma found at post-mortem in the lung of a child with leukemia.  Note fungus ball occupying cavity

  The University of Adelaide

I’m so glad we had this time together