Objectives: 

Mucormycosis, infections caused by fungi of the order Mucorales, are a group of emerging infectious diseases characterized by rapid progression and high aggressiveness. The genera Mucor is one of the most common etiological agents, being Mucor circinelloides the most frequently isolated species of the genera. Moreover, their virulence mechanisms are still unknown and there is no effective diagnostic method for this disease apart from classical methods. 
In this context, the aim of this study was to identify the antigens of M. circinelloides recognized specifically by immunosuppressed infected mice sera. 

Methods:

M. circinelloides was grown into potato dextrose broth at 37ºC for 24 hours with 120 rpm agitation. After that, the fungal material was separated from the medium by filtration and incubated for 20 hours in PBS 2% glucose. Then, the supernatant was filtered, sterilized and dialyzed, and finally, proteins were precipitated and resuspended in 2-DE buffer.
Protein separation was carried out by two-dimensional electrophoresis and antigen detection was performed by Western Blot against a pool of sera obtained from 5 mice infected intravenously with 1x105conidia of M. circinelloides NRRL 3631 and 5 sera from non-infected mice. These mice had been previously immunosuppressed by giving them 100 mg/kg cyclophosphamide intraperitoneally at -4 days and, then, every three days. Mice were sacrificed at day 20. 
Finally, the most immunoreactive proteins detected specifically by infected mice were identified by LC-MS/MS.

Results:


Eight immunoreactive proteins were specifically detected by sera from infected immunosupresed mice. Four of them were detected at isoelectric point (pI) of 5,4-5,7 and a molecular weight (Mr) of 50 kDa and the other four at 4,8 -5 pI and Mr 25 kDa. These four antigens were the most immunoreactive and were identified as triosephosphate isomerase by LC-MS/MS. The reactivity of these antigens was also observed when sera were tested individually.

Conclusions:

This study identifies Triosephosphate isomerase as a major antigen of M. circinelloides, which is recognized by sera from infected mice even in an immunosuppressant state. The study of the reactivity of this antigen might be very useful to develop a sensitive and specific serological diagnostic test, taking into account that infections caused by these fungi occur mainly in immunosuppressed patients.

Triosephosphate isomerase of Mucorcircinelloides is recognized as a main antigen by sera from infected immunosuppressed mice