Deciphering the molecular and cellular dynamics of early phagocytosis and granuloma formation in response to Mucorales

Résumé

Invasive fungal infections have emerged in the last decades, primarily due to the increasing population of immunocompromised individuals, which is attributed to the use of immunosuppressive treatments, and to the prevalence of chronic diseases such as HIV. The use of these treatments impairs their immune system, making these patientsmore susceptible to opportunistic fungal pathogens, such as the group of Mucorales. Mucorales members, classified by the Word Health Organization as a high-priority group, cause the life-threatening infection known as mucormycosis, which is characterized by high mortality rates and limited antifungal treatments. The immune response to Mucorales and overall, to fungal pathogens is complex and requires multiple cellular and molecular events. On the other hand, fungal pathogens have evolved several mechanisms to evade the immune response. Here, using asmodel organismsMucorlusitanicus and Rhizopusmicrosporus, which are two causal agents of mucormycosis, we have elucidated the molecular events that operate in the early phagocytosisof fungal sporangiospores and in the incipient granulomasformed when the fungal pathogen evades the first mechanism of defense (phagocytosis) and germinates constituting hyphal structures. Additionally, the gene regulation mediated by RNA interference and predicting the fungal lncRNAs and their involvement in the host-pathogen interface were conducted. To characterize the incipient granulomas formed in response to germinated fungal spores, a protocol was developed to isolate these cell aggregates in a mouse model within 3h post-infection. The integration of the molecular characterization of the cell clusters together by transcriptomic analysis with their cellular description by electron and optic microscopy analysis elucidated that neutrophils are the main components of the incipient granulomas. On the other hand, the fungal gene networks that are activated in the host-pathogen interactions, either in the internalization of fungal spores by macrophages or in the incipient granulomas, were elucidated determining that the transcription factors BRCA1 and HMG-BOX, the chromatin related proteins Histone 1 and the histone deacetylase 1 (HDA1), and a glutamate synthase are necessary for the full virulence of R. microsporus. Interestingly, the BRCA1 and Histone1 also seem to participate in M. lusitanicus virulence, constituting common virulence factors. Furthermore, the gene regulation mediated by therelated-virulence RNA degradation mechanism knownas Non-canonical RNA interference pathway (NCRIP) because it uses the atypical ribonuclease III R3B2inM. lusitanicuswas also assessed.The simultaneous sequencingofsmall RNAs and messenger RNAs from a wild-type and ther3b2-mutant strains in saprophytic conditions and with macrophages, and analysing their transcriptomic profileenabled the discovery and description ofthe direct targets of the NCRIP. Most of the identified target genes are transposable elements. Interestingly, these reporters include LTR transposons, which are newly described in the M. lusitanicus genome within this work. Interestingly, using the NCRIP markers, thus the HNH endonuclease and an LTR-transposon, reporter bioluminescent strains containing the fusion of these markersto the luciferasegene were generated to quickly analyse the regulation of the NCRIP. Finally, to have a more comprehensive understanding of gene regulation in the fungal pathogen during phagocytosis and in the incipient granuloma formation, a deep screening of lncRNAs in R.microsporusand M.lusitanicuswas performed. Thisanalysis identified hundreds of intergenic lncRNAs. The differential expression analysis shows that some of the predicted lncRNAs may be involved in the interaction with immune cellsin vitro and in vivo during mice infection. Mouse RNA-seq data analysis revealedthat host lncRNAs are also involved in the response to fungal infection. The disruption of four lncRNAs that are involved in mice in vivo infection by R. microsporus and where incipient granulomas are formed enabled the discovery of two essential lncRNAs: lncRNA2 and lncRNA4. Finally, both protein-coding genes described as virulence factors and lncRNAs 2 and 4 constitute new direct targets to design novel antifungal treatments and block fungal growth and dissemination, and molecular markers of the mucormycosis infection.