Divergent Targets of Aspergillus fumigatus AcuK and AcuM Transcription Factors during Growth In Vitro versus Invasive Disease

Abstract

eIn Aspergillus nidulans, the AcuK and AcuM transcription factors form a complex that regulates gluconeogenesis. In Aspergillus fumigatus, AcuM governs gluconeogenesis and iron acquisition in vitro and virulence in immunosuppressed mice. However, the function of AcuK was previously unknown. Through in vitro studies, we found that A. fumigatus ⌬acuK single and ⌬acuK ⌬acuM double mutants had impaired gluconeogenesis and iron acquisition, similar to the ⌬acuM mutant. Also, the ⌬acuK, ⌬acuM, and ⌬acuK ⌬acuM mutants had similar virulence defects in mice. However, the ⌬acuK mutant had a milder defect in extracellular siderophore activity and induction of epithelial cell damage in vitro than did the ⌬acuM mutant. Moreover, overexpression of acuM in the ⌬acuK mutant altered expression of 3 genes and partially restored growth under iron-limited conditions, suggesting that AcuM can govern some genes independently of AcuK. Although the ⌬acuK and ⌬acuM mutants had very similar transcriptional profiles in vitro, their transcriptional profiles during murine pulmonary infection differed both from their in vitro profiles and from each other. While AcuK and AcuM governed the expression of only a few iron-responsive genes in vivo, they influenced the expression of other virulence-related genes, such as hexA and dvrA. Therefore, in A. fumigatus, while AcuK and AcuM likely function as part of the same complex, they can also function independently of each other. Furthermore, AcuK and AcuM have different target genes in vivo than in vitro, suggesting that in vivo infection stimulates unique transcriptional regulatory pathways in A. fumigatus. Aspergillus fumigatus causes a variety of diseases, including allergic bronchopulmonary aspergillosis, aspergilloma, invasive pulmonary aspergillosis, and hematogenously disseminated aspergillosis (1, 2). The incidence of invasive aspergillosis is rising due to the increasing number of immunocompromised patients, who are at risk for this disease (3, 4). Furthermore, because invasive aspergillosis remains difficult to diagnose and treat, this infection is still associated with significant mortality (5). One approach to developing new strategies to treat this frequently deadly infection is to target the factors that enable A. fumigatus to survive and proliferate within the host.Iron is an essential trace element for most living organisms, including pathogenic microorganisms. Consequently, sequestration of free iron by the host is a key factor for inhibiting the virulence of microbial pathogens (6-8). Furthermore, most successful pathogens have evolved mechanisms to obtain iron while inside the host. For example, A. fumigatus acquires iron from the host by both reductive iron assimilation and secretion of siderophores (9-11). Siderophore secretion is more important for growth within the host, because A. fumigatus mutants with defects in siderophore synthesis have dramatically attenuated virulence, whereas those with defects in reductive iron assimilation do not (11-13).Previously, we determined tha...

Functional convergence of gliP and aspf1 in Aspergillus fumigatus pathogenicity

“…To isolate fungal RNA from the infected mouse lungs, the RNeasy minikit (Qiagen) was used with modifications [8]. Approximately 2.4 ml of buffer RLT with 1% β-mercaptoethanol was added to the lungs from each mouse and the tissue was homogenized in an M tube (Miltenyi Biotec) using a gentleMACS dissociator (Miltenyi Biotec) on setting RNA_02.01.…”

“…The capacity of the various strains to damage the cells was determined using a minor modification of our previously described method [8,50]. RAW cells were grown to confluency in 24-well tissue culture pates and then incubated with 51 Cr overnight.…”

“…Distinct features of the infection environment have been revealed by diverse approaches that include histological analysis [23,24], imaging mass spectrometry [25], and transcriptional profiling [26]. We have previously used transcriptional profiling to understand the genetic control of pathogenicity in infected tissue [8,27,28]. We have found that many mutants that are defective in proliferation during infection present alterations of gene expression that can reveal the role of the mutated gene in pathogenicity.…”

Aspergillosis and stem cell transplantation: An overview of experimental pathogenesis studies

“…73 Attenuated virulence has also been reported in other strains of A. fumigatus with mutations in the siderophore synthesis pathway and with other mutations affecting iron metabolism, including HapX, AcuM, and AcuK. 72,[74][75][76] Taken together, these studies strongly suggest that the ability of A. fumigatus to acquire and utilize iron from the host is essential for pathogenicity. This hypothesis is supported by the observation that iron overload within HSCT patients is a risk factor for the development of IA.…”

In vivo veritas: Aspergillus fumigatusproliferation and pathogenesis – conditionally speaking

“…Recent in vivo gene expression studies in Candida albicans and A. fumigatus highlight how different fungal genetic programs are in vivo vs. favorite in vitro culture conditions. 35,36 As filamentous fungal RNA is often a limiting factor in in vivo fungal gene expression profiling, the DOX system presented here also presents an opportunity to regulate expression of key virulence associated genes in vivo allowing fungal growth to levels needed to acquire fungal RNA. Additionally, it is often under appreciated that the murine models utilized to probe virulence place pressure on the genes under study from the very initiation of infection.…”