Mixed infections and In Vivo evolution in the human fungal pathogen Cryptococcus neoformans

Abstract

Koch's postulates are criteria establishing a causal relationship between a microbe and a disease that lead to the assumption that diseases are caused by a single strain or its evolved forms. Cryptococcus neoformans is a life-threatening human fungal pathogen responsible for an estimated 1 million cases of cryptococcosis/year, predominantly meningoencephalitis. To assess the molecular diversity of clinical isolates and gain knowledge of C. neoformans biology in the host, we analyzed clinical cultures collected during the prospective CryptoA/D study. Using molecular analysis of unpurified isolates, we demonstrated that mixed infections in humans are more common than previously thought, occurring in almost 20% of patients diagnosed with cryptococcosis. These mixed infections are composed of different mating types, serotypes, and/or genotypes. We also identified genetically related haploid and diploid strains in the same patients. Experimental infections and quantitative PCR show that these ploidy changes can result from endoreplication (duplication of DNA content) and that shuttling between haploid and diploid states can occur, suggesting in vivo evolution. Thus, the concept of one strain/one infection does not hold true for C. neoformans and may apply to other environmentally acquired fungal pathogens. Furthermore, the possibility of mixed and/or evolving infections should be taken into account when developing therapeutic strategies against these pathogens.

FIG 1

Comparison of DNA contents and virulence of haploid and diploid Aα C. neoformans isolates recovered during human or murine infections. (A) Cells were stained with propidium iodide to measure DNA content in comparison with that of reference haploid strain H99. Ploidy was determined for single-colony isolates (AD 7-99 and AD 8-18) purified from clinical sample AD 1-77, as well as a single colony (AD 10-30) recovered from the brain of a mouse inoculated with AD 7-99. (B) Three groups of seven outbred male mice were inoculated intravenously with 10⁵ cells of haploid (AD 7-99) or diploid (AD 8-18) strains purified from the clinical sample or the diploid strain (AD 10-30) recovered from the mouse brain. Differences in survival were analyzed using Stata (SE) version 10 (Stata Corporation, College Station, TX). The strains of clinical origin showed no statistically significant differences in virulence (P = 0.056). Mice infected with the diploid strain of murine origin (AD 10-30) survived significantly longer than did mice infected with the strains of clinical origin (P < 0.001, log-rank test).

FIG 2

Colocalization of different strains following intravenous inoculation. C. neoformans strains KN99a and KN99α were stained with Alexa Fluor 594 (red) and 350 (blue), respectively. Mice were inoculated via the tail vein with 5 × 10⁶ KN99a cells and then 5 × 10⁶ KN99α cells. At 24 h postinoculation, the animals were sacrificed and their brains were harvested for immunofluorescence. Frozen sections were immunostained with anti-collagen IV primary antibody with an FITC (green)-labeled secondary antibody to identify capillary endothelial cells. Bar, 20 µm.