Molecular imaging of fungal infection using a Trojan horse

Invasive fungal diseases are key causes of morbidity and death in immunocompromised patients and represent major drivers of increased healthcare costs. For example, up to 10% of patients with hematological malignancies suffer from these infections after their immune systems are weakened by high-dose chemotherapy. Unable to mount an effective immune response to the infection, up to 50% of these patients die as a consequence.

Severe fungal pneumonia (aspergillous) due to immunocompromised state. The fungal hyphae are present as radiating filaments stained light blue to gray.

© vetpathologist / Shutterstock.com

Early diagnosis and management of invasive fungal infections are key to improving survival among immunocompromised patients, but current diagnostic procedures are limited, particularly in terms of sensitivity and specificity. A major challenge faced by researchers is the development of highly specific and sensitive imaging techniques that enable localization of the infection site and early diagnosis.

PET and radiolabeled probes

Positron-Emission Tomography (PET) has become established as a major clinical imaging technique over the last ten years, particularly in the field of oncology. This is mainly due to the highly effective radiotracers used to carry out “molecular trapping” and generate intense radioactive signals in target cells. Researchers have also been trying to develop radiotracers for the imaging of fungal infections, but many of those have lacked specificity for the pathogens being targeted. Aside from the ability to accumulate specifically in target sites, radiotracers also need to demonstrate favorable pharmacokinetics such as rapid transport, low retention in non-target sites and efficient elimination from the body.

Aspergillus fumigatus

One of the most common types of fungus to cause infection in humans is Aspergillus. Usually, people are not affected when they breathe in Aspergillus spores, but immunocompromised individuals are at a significantly greater risk of developing health problems. Of approximately 200 Aspergilli so far identified, the most prevalent is Aspergillus fumigatus, which is mainly responsible for the increased incidence of the devastating condition invasive aspergillosis in patients with weakened immune systems.

Fungi Aspergillus, Aspergillus fumigatus, Aspergillus niger on colorful background, black mold, which produce aflatoxins, cause pulmonary infection aspergillosis

© Kateryna Kon / Shutterstock.com

Scientists have been trying to develop radiotracers that will target and localize A. fumigatus infection. Although these radiotracers have demonstrated the potential to specifically target A.fumigatus, a lack of active uptake by the pathogen causes signal intensification at sites of infection. This has led Austrian researchers Hubertus Haas (Innsbruck Medical University) and colleagues to investigate an iron-uptake mechanism used by A.fumigatus, as a potential target for a radiotracer.

Iron starvation and siderophores

Iron plays a vital role as a nutrient for A. fumigatus and other pathogens during infection. However, under infection conditions, host proteins tightly sequester available iron, creating what is essentially an iron-free environment for pathogens. As a result, pathogens have evolved highly efficient mechanisms for “stealing” this iron from the host and one of the mechanisms used by A. fumigatus, is siderophore-mediated iron acquisition. A siderophore is an iron chelating compound produced and secreted by A. fumigatus in order to scavenge for ferric iron (Fe[III]).

A. fumigatus produces two hydroxamate-type siderophores; fusarinine C (FSC) and its derivative tria-cetylfusarinine C (TAFC). Following iron chelation, the pathogen’s uptake of the ferri-siderophores is mediated by specific transporter proteins.

Radiolabeling siderophores

Siderophores can be radiolabeled by replacing the FE (III) inside them with an iron-mimicking radionuclide substitute. No iron isotope with a suitable half-life and photon emission has yet been identified for this, but it is known that a radiotracer called Ga (III) is a Fe (III) substitute that has previously been used to investigate siderophores and their uptake.

With the establishment of PET as a major clinical imaging modality, interest has grown significantly in 68Ga, which has a physical half-life of 68 minutes and a very low radiation burden to patients. In a proof of concept study, Haas and team showed that radiolabeling of desferri-siderophores, and in particular TAFC, was easily achievable with 68GA. Under iron starvation conditions in A. fumigatus infection, uptake of the 68Ga–TAFC complex was upregulated and could be blocked with NaN3 or an excess of siderophore, demonstrating specific, energy-dependent uptake (Figure 1A). Furthermore, in a rat model of aspergillosis, intact 68Ga–TAFC was eliminated rapidly, exclusively via renal excretion, suggesting excellent metabolic stability.

PET imaging of A. fumigatus

In another rat model, high contrast imaging of A. fumigatus pulmonary infection showed pronounced accumulation of 68GA–TAFC at affected sites (Figure 1B). This was shown early on after the onset of infection and 68GA–TAFC accumulation increased with the severity of infection. The researchers obtained the images using Micro-PET/computed tomography. Specifically, they used the Albira PET/SPECT/CT small animal imaging system developed by Bruker BioSpin. Equipped with a unique, patented PET detector, this system generates extremely high resolution, accurate images.

Copyright: © 2015 Haas et al. PLOS Pathogens - DOI:10.1371/journal.ppat.1004568 January 29, 2015

Figure 1. In vitro and in vivo uptake of 68Ga-TAFC by A. fumigatus.

(A) In-vitro uptake of 68Ga-TAFC in A. fumigatus cultures, showing induction of uptake during iron starvation, energy-dependence, and saturation by excess of ferric TAFC. (B) Micro-PET/CT (Albira PET/SPECT/CT small animal imaging system, Bruker Biospin Corporation, Woodbridge, CT, USA) imaging of A. fumigatus (coronal slices) in a rat infection model 45 minutes post intravenous injection of 68Ga-TAFC showing clear accumulation (blue arrow) in infected lung tissue (M. Petrik, unpublished). Accumulation of 68Ga-TAFC in kidney (yellow arrow) and bladder (red arrow) is caused by rapid renal excretion of the tracer. The colors reflect the signal intensity increasing from blue to green, yellow and red.

Haas and team believe 68Ga-labeled siderophores and in particular 68Ga–TAFC, have the potential to be used as radiotracers for the specific targeting and localization of fungal infections. The 68Ga–TAFC complex rapidly reaches infection sites, where this “Trojan horse” is mistaken for a source of iron and taken up by the pathogen.

References

  1. Haas H, et al. An Iron-Mimicking, Trojan Horse-Entering Fungi —Has the Time Come for Molecular
  2. Imaging of Fungal Infections? PloS Pathog 11(1): e1004568
  3. Petrik M, et al. Ga-Siderophores for PET Imaging of Invasive Pulmonary Aspergillosis: Proof of Principle. The Journal of Nuclear Medicine 2010;5:639–645
  4. Haas H. Fungal siderophore metabolism with a focus on Aspergillus fumigatus. Natural Product Reports 2014; 31:1266–1276
  5. Schrettl M and Haas H. Iron homeostasis—Achilles’ heel of Aspergillus fumigatus? Current opinion in Microbiology 2011;14:400–405
  6. Centers for Disease Control and Prevention. Aspergillosis. Available at: http://www.cdc.gov/fungal/diseases/aspergillosis/index.html?s_cid=cs_748
  7. Tutt B. Diagnosis and Treatment of Invasive Fungal Infections in Patients with Hematological Malignancies. Oncolog 2013;58 No.5:4–5.
  8. Dagenais T and Keller N. Pathogenesis of Aspergillus fumigatus in Invasive Aspergillosis. Clinical Microbiology Reviews 2008 22(3):447–465.

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Last updated: Dec 3, 2015 at 11:47 AM

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