Conidia charge and basal lamina binding

Wasylnka JA, Moore MM. Adhesion of Aspergillus species to extracellular matrix proteins: evidence for involvement of negatively charged carbohydrates on the conidial surface. Infect Immun. 2000 Jun;68(6):3377-84. PMC free article

This study came out of Simon Fraser University in Burnaby, BC, Canada. 

Those of us in the desert southwestern U.S. are very familiar with fungal lung infections.  Wasylnka and Moore mention in their introduction that cytotoxic chemotherapy patients and those with AIDS are immunocompromised and more susceptible to fungal infections.  Here in the United States where advertising drugs on prime time t.v. is legal, immunosuppressive therapies for autoimmune diseases are advised not to be taken by patients who visit areas where “certain fungal infections are prevalent.”  Indeed, most countries require testing for Aspergillis species on marijuana used for both medical and recreational purposes.  Wasylnka and Moore followed the literature suggesting that conidia of Aspergillis species may adhere to components of the extracellular matrix of our airways.  They tested the hypothesis that the conidia adhere to our airways via electrostatic interactions of negatively charged glycosaminoglycans to positively charged regions of these matrix proteins

Panels A and B came from the CDC. Panel C is from an extensive blog on medical mycology and shows conidia of A fumagatis.

MATERIALS AND METHODS

Aspergillus strains and growth conditions.

The Aspergillus strains were purchased from the American Type Culture Collection, ATTC, or isolated from patients in B.C.  The fungi were grown on a standard fungal agar until the spores/conidia were ready to harvest and enumerate.  

Preparation of basal lamina from cultured lung cells.

The type II pneumocyte cell line A549 was also obtained from ATCC.  These cells were gown in culture dishes in a  a standard medium.  The cells were removed with 0.1 M NH4OH (ammonium hydroxide) and then rinsed.  The protein remaining consisted of what is called the basal lamina. 

Peroxidase labeling of A. fumigatus conidia

Biotin is a small molecule that can be attached to large molecules like proteins while still exposing the Vidin site.  Avidin can be attached to other molecules like peroxidases that make the whole assemble easy to detect.  Peroxidase labeling of conidia was determined by previous work to not interfere with adhesion of the conidia to fibronectin, laminin, or type IV collagen….proteins of the basal lamina.

Adherence assays on glass slides.

Non specific sites on the fibronectin or authentic cell culture basal lamina were blocked with albumin, a major blood protein which was subsequently rinsed off.  Conidia were allowed to adhere to the protein in PBS, phosphate buffered saline, a solution that buffers the mix to the same pH as blood using the same major buffering salts.  After giving the conidia a time to bind, the slides were rinsed with a mild detergent to remove nonspecific, weakly bound conidia.

The strategy in images

The basic experimental strategy. A. Domains of a fibronectin dimer. Image can be found at this link to a slideshare presentation of the extracellular matrix..

Summary of results

The bar graphs of Figures 1-4 illustrate These findings also available PMC free article in full form.

  • This site has discussed the D614G mutation in the Covid spike protein giving it the ability to bind to integrins on the surface of many cells, that recognize the RGD peptide sequence.  A slightly longer version of this sequence, GRGDS, failed to result in statistically significant less binding of the conidia to fibronectin compared to a sequence SGGDR.    In simple lay terms, the conidia are not pretending to be endothelial cells binding to RGD motifs on the fibronectin.
  • The ability of three fragments of fibronectin were tested for their ability to bind spores.  Only the glycosamino glycan binding domain was able to bind the spores, albeit at a lower affinity than full length fibonectin.   Neither the 45-kDa gelatin-binding domain nor the 120-kDa cell binding domain were able to  bind conidia.
  •  Removal of glycosylation of fibronectin did not affect conidia binding.  Denaturization (unfolding) of the fibronectin did affect binding.
  • Removal of sialic acid glycosylation had no effect on conidia binding.
  • We must remember that the conidia also have glycosylation.  Three different polysaccharides were used to complete with conidia for sites of fibronectin and the cell culture grown basal lamina:  dextran sulfate, chondroitin sulfate,  keratan sulfate, and dextran.  All four inhibited conidia from binding to  fibronectin and cell culture basal lamina.  High concentrations of NaCl also inhibited binding further implicating ion-ion interactions.

While electrostatic interactions were claimed to be part of the interaction, charges of strong binders versus weak binders were not measured.

Discussion highlights

The Wasylnka and Moore study presents evidence that negatively charged carbohydrates on the surface of A. fumigatus conidia may mediate adherence to fibronectin and intact basal lamina.  These are some highlights of the discussion and some comments on possible copper binding:

  • Alveolar basal lamina is a specialized ECM composed of laminin, type IV and V collagen, entactin, chondroitin sulfate proteoglycan, heparan sulfate proteoglycan, and fibronectin that might originate from the circulation and therefore be absent from the cell culture produced substrate.
  • Extracellular matrix damage is thought to predispose a patient to aspirgilliosis.  CopperOne and lysyl oxidase
  • Diseased lungs were thought to have more fibronectin than healthy lungs.
  • In the damaged lung, inhaled conidia may have increased access to fibronectin and other ECM proteins as a result of detached epithelial cells.
  • The finding that A. fumigatus binds to fibronectin correlates with the observation that  A. fumigatus infections account for over 90% of all cases of invasive aspirgiliosis

Published by BL

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