SUMMARY Aspergilli produce conidia for reproduction or to survive hostile conditions, and they are highly effective in the distribution of conidia through the environment. In immunocompromised individuals, inhaled conidia can germinate inside the respiratory tract, which may result in invasive pulmonary aspergillosis. The management of invasive aspergillosis has become more complex, with new risk groups being identified and the emergence of antifungal resistance. Patient survival is threatened by these developments, stressing the need for alternative therapeutic strategies. As germination is crucial for infection, prevention of this process might be a feasible approach. A broader understanding of conidial germination is important to identify novel antigermination targets. In this review, we describe conidial resistance against various stresses, transition from dormant conidia to hyphal growth, the underlying molecular mechanisms involved in germination of the most common Aspergillus species, and promising antigermination targets. Germination of Aspergillus is characterized by three morphotypes: dormancy, isotropic growth, and polarized growth. Intra- and extracellular proteins play an important role in the protection against unfavorable environmental conditions. Isotropically expanding conidia remodel the cell wall, and biosynthetic machineries are needed for cellular growth. These biosynthetic machineries are also important during polarized growth, together with tip formation and the cell cycle machinery. Genes involved in isotropic and polarized growth could be effective antigermination targets. Transcriptomic and proteomic studies on specific Aspergillus morphotypes will improve our understanding of the germination process and allow discovery of novel antigermination targets and biomarkers for early diagnosis and therapy.

Aspergillus fumigatus is a saprophytic fungus that extensively produces conidia. These microscopic asexually reproductive structures are small enough to reach the lungs. Germination of conidia followed by hyphal growth inside human lungs is a key step in the establishment of infection in immunocompromised patients. RNA-Seq was used to analyze the transcriptome of dormant and germinating A. fumigatus conidia. Construction of a gene co-expression network revealed four gene clusters (modules) correlated with a growth phase (dormant, isotropic growth, polarized growth). Transcripts levels of genes encoding for secondary metabolites were high in dormant conidia. During isotropic growth, transcript levels of genes involved in cell wall modifications increased. Two modules encoding for growth and cell cycle/DNA processing were associated with polarized growth. In addition, the co-expression network was used to identify highly connected intermodular hub genes. These genes may have a pivotal role in the respective module and could therefore be compelling therapeutic targets. Generally, cell wall remodeling is an important process during isotropic and polarized growth, characterized by an increase of transcripts coding for hyphal growth and cell cycle/DNA processing when polarized growth is initiated.

Aspergillus fumigatus is one of the most common airborne fungal pathogens for humans. Low-molecular-mass iron chelators, termed siderophores, have been shown to play a central role in iron homeostasis and, consequently, virulence of this mold.

The opportunistic pathogen Aspergillus fumigatus is found on all continents and thrives in soil and agricultural environments. Its ability to readily adapt to novel environments and to produce billions of spores led to the spread of azole-resistant A. fumigatus across the globe, posing a threat to many immunocompromised patients, including critically ill patients with severe influenza or COVID-19. In our study, we sought to compare the adaptational response to azoles from A. fumigatus isolates that differ in azole susceptibility and genetic background. To gain more insight into how short-term adaptation to stressful azole compounds is managed through gene expression, we conducted an RNA-sequencing study on the response of A. fumigatus to itraconazole and the newest clinically approved azole, isavuconazole. We observed many similarities in ergosterol biosynthesis up-regulation across isolates, with the exception of the pan-azole-resistant isolate, which showed very little differential regulation in comparison to other isolates. Additionally, we found differential regulation of membrane efflux transporters, secondary metabolites, iron metabolism, and various stress response and cell signaling mechanisms.

Germination of inhaled Aspergillus fumigatus conidia is a necessary sequitur for infection. Germination of conidia starts with the breaking of dormancy, which is initiated by an increase of the cellular perimeter in a process termed isotropic growth. This swelling phase is followed by polarized growth, resulting in the formation of a germ tube. The multinucleate tubular cells exhibit tip growth from the hyphae, after which lateral branches emerge to form the mycelial network. The regulatory mechanisms governing conidial germination are not well defined. In this study, we identified a novel role for the transcription factor SltA in the orchestration of germination and hyphal development. Conidia lacking sltA fail to appropriately regulate isotropic growth and begin to swell earlier and subsequently switch to polarized growth faster. Additionally, hyphal development is distorted in a ∆ sltA isolate as hyphae are hyper-branching and wider, and show branching at the apical tip. ∆ sltA conidia are more tolerant to cell wall stressors on minimal medium compared to the wild-type (WT) strain. A transcriptome analysis of different stages of early growth was carried out to assess the regulatory role of SltA. Null mutants generated for three of the most dysregulated genes showed rapid germ tube emergence. Distinct from the phenotype observed for ∆ sltA , conidia from these strains lacked defects in isotropic growth, but switched to polarized growth faster. Here, we characterize and describe several genes in the regulon of SltA, highlighting the complex nature of germination. IMPORTANCE: Aspergillus fumigatus is the main human fungal pathogen causing aspergillosis. For this fungus, azoles are the most commonly used antifungal drugs for treatment of aspergillosis. However, the prevalence of azole resistance is alarmingly increasing and linked with elevated mortality. Germination of conidia is crucial within its asexual life cycle and plays a critical role during the infection in the human host. Precluding germination could be a promising strategy considering the role of germination in Aspergillus spp. pathogenicity. Here, we identify a novel role for SltA in appropriate maintenance of dormancy, germination, and hyphal development. Three genes in the regulon of SltA were also essential for appropriate germination of conidia. With an expanding knowledge of germination and its different morphotypes, more advances can be made toward potential anti-germination targets for therapy.

Phase-variable type I restriction modification (RM) systems are epigenetic regulatory systems that have been identified in numerous human bacterial pathogens. We previously showed that an emerging zoonotic lineage of Streptococcus suis acquired a phase-variable type I RM system, named SsuCC20p. The SsuCC20p locus was identified in the genome of multiple streptococcal species, indicating that it is not restricted to S. suis and can be acquired through horizontal gene transfer. We demonstrate that SsuCC20p phase variability relies on a recombinase present within the locus. SsuCC20p is the single RM system responsible for the genome methylation profiles that were detected in the representative zoonotic S. suis isolate 861160 in vitro . In addition, we show that, contrary to previous observations, hsdS genes located downstream of hsdM and the recombinase gene contribute to the SsuCC20p genome methylation profile. SsuCC20p locked mutants expressing a single hsdS each show a unique genome methylation profile and, when grown in human serum, have distinct transcriptomes. In a zebrafish larvae infection model, we observed significant differences in virulence between the locked mutants and a corresponding shift in hsdS allele distribution in the wild type. These data indicate that the streptococcal phase-variable type I RM system SsuCC20p can impact bacterial fitness via epigenetic regulation of gene expression, which impacts the virulence of S. suis in the zebrafish larvae infection model. IMPORTANCE: Phase variation allows a single strain to produce phenotypic diverse subpopulations. Phase-variable restriction modification (RM) systems are systems that allow for such phase variation via epigenetic regulation of gene expression levels. The phase-variable RM system SsuCC20p was found in multiple streptococcal species and was acquired by an emerging zoonotic lineage of Streptococcus suis . We show that the phase variability of SsuCC20p is dependent on a recombinase encoded within the SsuCC20p locus. We characterized the genome methylation profiles of the different phases of SsuCC20p and demonstrated the consequential impact on the transcriptome and virulence in a zebrafish infection model. Acquiring mobile genetic elements containing epigenetic regulatory systems, like phase-variable RM systems, enables bacterial pathogens to produce diverse phenotypic subpopulations that are better adapted to specific (host) environments encountered during infection.