Figure 1.
Correlation between spore size and virulence in M. c. f. lusitanicus.
(A) Several strains of Mcl display differences in spore size. Two (−) strains (R7B and ATCC1216a) produce larger spores (12.32±2.71 µm long for R7B), and two (+) strains (NRRL3631 and ATCC1216b) produce smaller spores (4.70±0.91 µm long for NRRL3631). One (−) isolate, NRRL1443, produces intermediate sized spores. Note that R7B is an auxotrophic mutant (leucine−) of CBS277.49 and the mutation in the leuA gene did not impact virulence. (B) Nuclei were stained with DAPI and cell walls were stained with calcofluor. Combined Z-stack images show a difference in the number of nuclei in the (−) and (+) mating type spores. The left panel shows multinucleated spores; however, smaller spores of the (−) mating type (small rectangular area) are uni- or bi-nucleate. The (+) spores in the right panel are uninucleate. Scale = 10 µm. N.A. = 1.4 with oil. (C) Virulence in the wax moth host is correlated with spore size. Three (−) strains (R7B, CBS277.49, and ATCC1216a) are significantly more virulent compared to the (+) strains with a smaller spore size (see the text for statistics). (D) In the survival curves of wax moth larvae infected with different numbers of (+) spores (NRRL3631), only 50,000 (+) spores are similarly virulent to 500 (−) spores (R7B) (p = 0.8826). See Figure 1S for the analysis of additional large (−) and small (+) sporangiospore producing strains further substantiating the link of spore size to virulence.
Table 1.
Mucor circinelloides strains used in this study.
Table 2.
M. circinelloides f. lusitanicus (−) isolates produce larger spores than (+) isolates and other subspecies.
Figure 2.
Delay in isotropic to polarized growth transition during germination of small vs. large spores.
(A) Large spores (R7B) have a very short isotropic growth stage or bypass it entirely to send germ tubes. (B) Small spores (NRRL3631) grow isotropically to reach the size comparable to that of large spores, and then germ tubes emerge. Time-lapse (every ∼15 min) images of each strain are presented. Note that in panel B, there are 3 rows of images to show the delay (∼225 mins) prior to germ tube emergence in the smaller (+) spores. See Videos S1 and S2 for corresponding videos. Scale = 40 µm. (C) When isotropically grown, the enlarged (+) spores became as virulent as larger spores in the wax moth larva host (p = 0.9878).
Figure 3.
Time-lapse analysis of response of the murine macrophage to large spores (LS), small spores (SS), and isotropically grown spores (IS).
The macrophage (J774) engulfs each of the different-sized spores successfully. Both LS and IS germinate inside macrophages indicating that their invasive hyphal growth is not inhibited by the macrophage. In contrast, SS are trapped inside the macrophage and either do not undergo isotropic growth or exhibit very limited growth when compared to the spores outside of the macrophage that grow isotropically. Time progresses across each row in the images from left to right.
Figure 4.
SEM and TEM images of large and small spores.
(A) Large spores are decorated with ‘bumps’ on their surface. (B) The surface of small spores is smooth. (C) The subpopulation of small spores in the larger spore producing isolates (CBS277.49) has a smooth surface (black arrowhead) compared to larger spores (white arrowhead). (D) Trafficking to the cell wall is observed in the large spores (left) but not in the small spore (right) indicating that the larger spores may be metabolically active for further development, such as, invasive hyphal growth. Scales are 1 µm for A, B, and C, and 100 nm for D.
Figure 5.
Disruption of the sexM gene and mating and virulence tests of the sexM mutants.
(A) Southern blot analysis shows that the sexM gene was replaced with the pyrG gene resulting in disruption of the sexM gene in two transformants. The sexM probe corresponds to a 1.3-kb EcoRI fragment of the 3′ region of sexM. (B) Two sexM mutants [MU423 (sexMΔ1) and MU424 (sexMΔ2)] are sterile in mating assays with (+) or (−) strains (see also Figure 3S). The parental MU402 strain is a pyrG leuA double mutant. (C) The sexM mutants do not display differences in spore size or virulence in the wax moth host. See Figure 3S for additional PCR validation of the sexM mutant strains.
Figure 6.
Phylogenetic relationships of the M. circinelloides subspecies.
(A) Mucor circinelloides spp. compared with R. oryzae are shown using concatenated RBP1, rDNA2, and ITS loci. (B) Phylogenetic relationships constructed with the RBP1, rDNA2, and ITS loci in Mcg, Mcc, and Mcl. Both phylogenetic analyses provide evidence that there are three subspecies within the M. circinelloides complex. Mcc: M. circinelloides f. circinelloides, Mcl: M. circinelloides f. lusitanicus, and Mcg: M. circinelloides f. griseocyanus.
Figure 7.
Virulence tests of the three M. circinelloides subspecies in the wax moth and murine hosts.
(A) Larvae of the wax moth Galleria mellonella were used as the host. Mcl was found to be the most virulent, causing 100% mortality in 3 days. All other strains were less virulent. Injections were repeated three times with similar results. PBS injection served as a negative control. (B) Virulence of M. circinelloides subspecies in the murine host. Mice infected with NRRL3615 (Mcc) show 100% mortality in four days, and those with NRRL3614 (Mcc) show 40% mortality in four days. Both NRRL3615 and NRRL3614 are Mcc isolates that are commonly found in clinical isolates tested in this study. (C) Mcc (NRRL3614 and NRRL3615) exhibits more robust grows at 37°C compared to Mcl (CBS277.49), which may result in Mcc being highly virulent in the diabetic murine host compared to Mcl, unlike our observations in the wax moth host.
Figure 8.
Sexual development of Mucor circinelloides.
Light and scanning electron microscopy images were obtained with mounted samples of zygospores and other mating structures. (A) Images show a zygospore (enlarged insert) as viewed by light microscopy and the dark zygospore line that forms during mating. (+) and (−) designations indicate mating types of strains (ATCC1216a (−) and ATCC1216b (+) Mcl strains). A distinct dark zygospore line was found in (+)/(−) co-cultures but not in same-sex mating pairs. All matings were performed for 7 days in the dark with the exception of the bottom right plate that was incubated in the light at room temperature. Mating occurred in the dark and not in the light. (B) The sporangium (upper panels), the asexual spore harboring structure, and zygospores (lower panels), the sexual spores, are shown at higher magnification by SEM. (C) The formation of the zygospore structure is depicted by SEM. Zygospore formation is initiated by the production of coiled hyphae that entangle to form the mature zygospore. Coiled hyphae in a mating between ATCC1207a and ATCC1207b are presented in the small rectangular area (light microscopic image). Scale = 10 µm.
Table 3.
Sexual reproduction of the M. circinelloides isolates.
Figure 9.
Defining the (+) and (−) sex locus alleles in M. circinelloides f. griseocyanus and Mucor circinelloides f. circinelloides.
(A) Dot plot comparison of the sex locus alleles of the (+) and (−) strains of Mcg and Mcc show the sex specific regions with conserved flanking gene regions. The tptA and rnhA genes encoding the TPT and RNA helicase, respectively, are highly conserved between the (+) and (−) alleles; however, the sex genes encoding HMG domain proteins are divergent in sequence. Interestingly, the promoters for the TPT and RNA helicase genes are part of the sex locus in Mcg (A) but only the TPT promoter is part of the sex locus in Mcc (B). The regions of the tptA and rnhA genes sequenced are depicted (5′ regions of the genes). sex loci of ATCC1207a (+) and ATCC1207b were sequenced for Mcg and those of NRRL3614(−) and NRRL3615 (+) were sequenced for Mcc. Dotted lines indicate the start of the rnhA gene. Asterisk indicates the start of the RNA helicase gene. (C) The promoters for the TPT and RNA helicase genes are part of the sex locus in Mcg but only the TPT promoter is part of the sex locus in Mcc.
Table 4.
DNA and deduced protein sequence comparison between three subspecies.
Table 5.
Comparison of SexP (left) and SexM (right) in the three subspecies.