Disparate Proteome Responses of Pathogenic and Nonpathogenic Aspergilli to Human Serum Measured by Activity-Based Protein Profiling (ABPP)

Abstract

Aspergillus fumigatus is the primary pathogen causing the devastating pulmonary disease Invasive Aspergillosis in immunocompromised individuals. There is high genomic synteny between A. fumigatus and closely related rarely pathogenic Neosartorya fischeri and Aspergillus clavatus genomes. We applied activity-based protein profiling to compare unique or overexpressed activitybased probe-reactive proteins of all three fungi over time in minimal media growth and in response to human serum. We found 360 probe-reactive proteins exclusive to A. fumigatus, including known virulence associated proteins, and 13 proteins associated with stress response exclusive to A. fumigatus culture in serum. Though the fungi are highly orthologous, A. fumigatus has a significantly greater number of ABP-reactive proteins across varied biological process. Only 50% of expected orthologs of measured A. fumigatus reactive proteins were observed in N. fischeri and A. clavatus. Activity-based protein profiling identified a number of processes that were induced by human serum in A. fumigatus relative to N. fischeri and A. clavatus. These included actin organization and assembly, transport, and fatty acid, cell membrane, and cell wall synthesis. Additionally, signaling proteins regulating vegetative growth, conidiation, and cell wall integrity, required for appropriate cellular response to external stimuli, had higher activity-based probe-protein reaction over time in A. fumigatus and N. fisheri, but not in A. clavatus. Together, we show that measured proteins and physiological processes identified solely or significantly over-represented in A. fumigatus reveal a unique adaptive response to human protein not found in closely related, but rarely pathogenic aspergilli. These unique activity-based probe-protein responses to culture condition may reveal how A. fumigatus initiates pulmonary invasion leading to Invasive Aspergillosis. Invasive aspergillosis (IA) 1 is a devastating infection caused by the ubiquitous saprophytic filamentous fungus Aspergillus fumigatus (Af) (1). Af is an opportunistic pathogen with no true virulence factors. Its pathogenicity is often attributed to its thermotolerance, response to oxidative stress, ability to grow in hypoxic or iron limiting environments, and its ability to use a variety of carbon and nitrogen sources as nutrients, such as proteins derived from the human host (2). A thorough understanding of biological processes or factors that facilitate pathogenic Af infection compared with other microbial infections is needed to assist treatment and diagnosis of IA.Af protein activity regulation and function, attenuated by environmental response and adaptation, is critical for opportunistic infection and development of IA (3). Activity-based protein profiling (ABPP), coupled to mass spectrometry (MS), is a powerful chemical biology approach for directly identifying a subset of proteins (4). ABPP employs activity-based probes (ABPs) to covalently label and enrich functional families of proteins, thereby reducing th...

Activity-based protein profiling of microbes

“…Using a FP-probe multiplexed with a probe that targets enzymes with reactive cysteines in the fungus Aspergillus fumigatus, a human pathogen that is difficult to detect and for which no known virulence proteins are known, our group found that in the presence of host serum the organism alters physiological functions throughout numerous metabolic processes [12]. A follow-up study comparing A. fumigatus to two highly related but non-pathogenic fungi further confirmed the metabolic attenuation of A. fumigatus in the presence of the host, and the disparate metabolism in the presence of host was not identified in the other fungi [13]. Thereby, ABPP facilitated an improved understanding of the fungi"s pathogenesis mechanism, and an array of proteins was identified as potential disease candidate biomarkers.…”

“…Studies to identify virulence mechanisms in A. fumigatus [12,13]. Characterized Glpg in E. coli [14].…”

Fungal sensing of host environment

“…While the mechanisms exploited by fungi to evade the immune system have been studied in detail, the subsequent fungal intracellular response after binding and sensing of host serum proteins is less clear. Proteomic analysis of Aspergillus species after incubation with human serum revealed a divergent response in pathogenic A. fumigatus compared with less pathogenic species (Wiedner et al, 2013). Thus, a direct connection between sensing of host factors and adaptation to a host environment with virulence and resistance seems to exist.…”

“…The authors would like to apologize to all the scientists with relevant contributions in the field, whose work could not be cited as a result of space limitations. Moreover, the authors would like to thank the organizers of the fungal cell wall meeting, held in Paris 26-28 October (Wiedner et al, 2013) Fungal sensing 1195 2015, for enabling fruitful scientific exchange between different fields of fungal research and initiating the writing of this review. We declare that there is no conflict of interest.…”

Activity-based proteomic and metabolomic approaches for understanding metabolism

“…The same group used a non-directed phenyl sulfonate ester probe to target a broad range of enzyme families and showed that HCV infection led to dysregulation of several protein activities that may be relevant to HCV replication (15). Wright and colleagues recently used a cysteine-reactive sulfonate ester probe and the serine hydrolase probe to identify several dysregulated enzyme activities in Aspergillus fumigatus, the primary pathogen causing the devastating pulmonary disease Invasive Aspergillosis (16). The same group also developed activity-based probes for cellulose degrading enzymes in Clostridium thermocellum which may have applications in biofuel development (17).…”