Lionel Williamson

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• Presented by
• Lionel Williamson

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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.

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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 aspergillosis
• A. oryzae used to make soy sauce, and sake

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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 40C and beyond)
  that may be extremely angioinvasive, particularly
  in the compromised patient

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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.

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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)

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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

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Hyphae vs. conidia
   The University of Adelaide
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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.

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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
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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 TLRs isolated from humans

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Types of TLRs

• TLR 2 recognizes peptidoglycan that are
  associated with gram positive bacteria
• TLR 4 recognizes LPS associated with gram
  negative bacteria
• TLR5 recognizes flagella
• TLR9 recognizes bacterial DNA

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Experiment 1

• Challenged mouse macrophages with conidia or
  hyphae from A. fumigatus
• They found that maximal cytokine release induced
  by stimulation with 107 cfu
• Assessed TLR4s role by using a TLR4-deficient
  mutant compared to a control
• Assessed TLR2s role by using a TLR2-deficient
  mutant compared to a control

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Exp 1 cont
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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

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Exp 2
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Experiment 3

• They wanted to see what roles TLR4 and TLR2
  played on the Pro-inflammatory response
• Used PBMCs with antibodies blocking the TLR4
  then stimulating with 107 cfu
• The same experiment was repeated with TLR2

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Exp 3 cont.
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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

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Exp 4 cont.
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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

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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.

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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?

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This is the air sacs of a hen during an outbreak
of aspergillosis
   The University of Adelaide
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Fungal balls made up of hyphae isolated from the
lungs
   The University of Adelaide
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Aspergillus fumigatus in lung tissue, note
conidial heads forming in an alveolus
   The University of Adelaide
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Aspergilloma found at post-mortem in the lung of
a child with leukemia.  Note fungus ball
occupying cavity
  The University of Adelaide
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Im so glad we had this time together