An Adult Zebrafish Model Reveals that Mucormycosis Induces Apoptosis of Infected Macrophages

Abstract

Mucormycosis is a life-threatening fungal infection caused by various ubiquitous filamentous fungi of the Mucorales order, although Rhizopus spp. and Mucor spp. are the most prevalent causal agents. The limited therapeutic options available together with a rapid progression of the infection and a difficult early diagnosis produce high mortality. Here, we developed an adult zebrafish model of Mucor circinelloides infection which allowed us to confirm the link between sporangiospore size and virulence. Transcriptomic studies revealed a local, strong inflammatory response of the host elicited after sporangiospore germination and mycelial tissue invasion, while avirulent and UV-killed sporangiospores failed to induce inflammation and were rapidly cleared. Of the 857 genes modulated by the infection, those encoding cytokines, complement factors, peptidoglycan recognition proteins, and iron acquisition are particularly interesting. Furthermore, neutrophils and macrophages were similarly recruited independently of sporangiospore virulence and viability, which results in a robust depletion of both cell types in the hematopoietic compartment. Strikingly, our model also reveals for the first time the ability of mucormycosis to induce the apoptosis of recruited macrophages but not neutrophils. The induction of macrophage apoptosis, therefore, might represent a key virulence mechanism of these fungal pathogens, providing novel targets for therapeutic intervention in this lethal infection.

Figure 1

M. circinelloides R7B is highly pathogenic for zebrafish. Zebrafish were i.p. injected with 2.5 × 10⁵ or 10⁷ (high dose: HD) live or UV-killed sporangiospores of the R7B or NRRL strains. (A) The survival rates of infected zebrafish are shown. Each experimental group contained 20 zebrafish, and each experiment was performed in duplicate. ^*P < 0.05; ^**P < 0.01; ^***P < 0.001. (B) Gross morphological examinations of adult zebrafish infected with M. circinelloides R7B and NRRL were imaged at 16 hpi. (C) Histological analysis of M circinelloides-infected zebrafish. Transverse sections were obtained from zebrafish injected with 2.5 × 10⁵ R7B spores/fish or 10⁷ NRRL spores/fish at 16 hpi. Note that the mycelium (black arrows) can invade several organs and promotes a strong leukocyte infiltration (red arrows). L, liver; G, gut; M, muscle; O, ovary; T, testis. Scale bars 10 µm

Figure 2

Differential expression analysis. (A) Heat-map shows the 857 differentially expressed genes clustered by their expression levels in response to M. circinelloides R7B strain infection. Roman numbers and left column colors indicate independent clusters. Color key indicates the log2 fold change values. (B) The graph shows the most specific biological process enriched. (C) The graph shows the most specific molecular function enriched. In (B,C), X-axis indicates the enrichment ratio obtained by dividing the sequences percentage of the test set versus the sequences percentage of the reference set (see methods section). Positive and negative values, respectively represent biological processes and molecular function enriched in up-regulated, and down-regulated genes. AMC, aminoglycan catabolic process; ATA, ATPase activity; BCU, blood coagulation; CBI, calcium ion binding; CRE, cellular response to estrogen stimulus; CTT, carbohydrate transmembrane transport; DRB, defense response to bacterium; EIA, endopeptidase inhibitor activity; HEB, heme binding; HOG, hydrolase activity, hydrolyzing O-glycosyl compounds; INB, iron ion binding; IRE, immune response; LPT, lipid transport; MOM, monocarboxylic acid metabolic process; ORP, oxidation-reduction process; OXI, oxidoreductase activity; PAC, protein activation cascade; PEC, peptidoglycan catabolic process; PRO, proteolysis; RIR, regulation of immune system process; ROC, response to organic cyclic compound; SAR, sarcomere organization; SEP, serine-type endopeptidase activity; SMC, small molecule catabolic process; STT, sodium ion transmembrane transporter activity; TAC, transporter activity; TTA, active transmembrane transporter activity; UTT, urea transmembrane transport; VCM, ventricular cardiac myofibril assembly. Asterisks indicate the statistical significance level, FDR ≤ 0.001 (***), FDR ≤ 0.01 (**) and FDR ≤ 0.05 (*).

Figure 3

R7B is more potent than NRRL inducing immune-related gene expression in zebrafish. Zebrafish were i.p injected with 2.5 × 10⁵ or 10⁷ (high dose: HD) live or UV-inactivated sporangiospores of the R7B or NRRL strains. At 16 hpi, the expression of the indicated immune-related genes was analyzed by RT-qPCR in the infection site and compared to those of control fish injected with PBS (n = 4). ^*P < 0.05; ^***P < 0.001. ns, nonsignificant.

Figure 4

M circinelloides can activate mouse macrophages. IL-1β mRNA levels assayed by RT-qPCR in the macrophage cell line J774 at 3 hpi with live or UV-killed R7B and NRRL strains (MOI = 1) alone or in combination with 10 ng/ml LPS. ^**p < 0.01, ^***P < 0.001. ns, nonsignificant.

Figure 5

M. circinelloides inhibits the expression of immune-related genes in the head kidney of zebrafish. The mRNA levels of the genes coding for the indicated pro-inflammatory and neutrophil (mpx) and macrophage (mpeg1) markers were determined by RT-qPCR at 16 hpi in the head kidney from zebrafish challenged with 2.5 × 10⁵ R7B spores/fish or 10⁷ NRRL spores/fish (n = 8). ^*P < 0.05; ^**P < 0.01; ^***P < 0.001. ns, nonsignificant.

Figure 6

M. circinelloides promotes neutrophils and macrophages depletion in the head kidney. (A) Representative sections of zebrafish head kidneys infected with 2.5 × 10⁵ R7B spores/fish at 16 hpi and immunostained with anti-Lcp1 (pan-leukocyte marker) and anti-Mpx (neutrophil marker) antibodies. Note the robust depletion of myeloid cells. Scale bars 20 µm. (B) Quantification of Lcp1⁺ and Mpx⁺ cells is shown as the mean ± SEM of the immunostained area/total area of 4 randomly distributed optical areas from 4 fish at ×200 magnification. ^*P<0.05; ^**P<0.01; ^***P<0.001.

Figure 7

M. circinelloides altered the expression profile of gene encoding neutrophil and macrophages markers in the infection site. The mRNA levels of the genes coding for neutrophil (mpx) (A) and macrophages (mpeg1) (B) markers were determined by RT-qPCR at 16 hpi in the head kidney of zebrafish challenged with live or UV-killed 2.5×10⁵ R7B spores/fish and 2.5×10⁵ or 10⁷ (high dose, HD) NRRL spores/fish (n=4). ^*P<0.05; ^***P<0.001. ns, nonsignificant.

Figure 8

M. circinelloides promotes the recruitment and apoptosis of macrophages in the infection site. (A) Representative serial sections of zebrafish head kidneys challenged with PBS, 2.5×10⁵ R7B spores/fish and 10⁷ (high dose, HD) NRRL spores/fish at 16 hpi and immunostained with anti-Lcp1, anti-Mpx, and anti-active Casp3 antibodies. Infiltrated leukocytes (black arrows) at 16 hpi were PAS⁺, Lcp1⁺ and most of them Casp3⁺ in the fish infected with the virulent strain (R7B) and the HD of the avirulent NRRL strain. Scale bars 20 µm. (B) Quantification of Lcp1⁺, Mpx^+, and Casp3⁺ cells is shown as the mean ± SEM of the immunostained area/total area of 4 randomly distributed optical regions from 4 fish at ×200 magnification. ^*P<0.05.