A protein complex network of Drosophila melanogaster.

Determining the composition of protein complexes is an essential step toward understanding the cell as an integrated system. Using coaffinity purification coupled to mass spectrometry analysis, we examined protein associations involving nearly 5,000 individual, FLAG-HA epitope-tagged Drosophila proteins. Stringent analysis of these data, based on a statistical framework designed to define individual protein-protein interactions, led to the generation of a Drosophila protein interaction map (DPiM) encompassing 556 protein complexes. The high quality of the DPiM and its usefulness as a paradigm for metazoan proteomes are apparent from the recovery of many known complexes, significant enrichment for shared functional attributes, and validation in human cells. The DPiM defines potential novel members for several important protein complexes and assigns functional links to 586 protein-coding genes lacking previous experimental annotation. The DPiM represents, to our knowledge, the largest metazoan protein complex map and provides a valuable resource for analysis of protein complex evolution.

Characterization of PdCP1, a serine carboxypeptidase from Pseudogymnoascus destructans, the causal agent of White-nose Syndrome.

Pseudogymnoascus destructans is a pathogenic fungus responsible for White-nose Syndrome (WNS), a disease afflicting multiple species of North American bats. Pseudogymnoascus destructans infects susceptible bats during hibernation, invading dermal tissue and causing extensive tissue damage. In contrast, other Pseudogymnoascus species are non-pathogenic and cross-species comparisons may therefore reveal factors that contribute to virulence. In this study, we compared the secretome of P. destructans with that from several closely related Pseudogymnoascus species. A diverse set of hydrolytic enzymes were identified, including a putative serine peptidase, PdCP1, that was unique to the P. destructans secretome. A recombinant form of PdCP1 was purified and substrate preference determined using a multiplexed-substrate profiling method based on enzymatic degradation of a synthetic peptide library and analysis by mass spectrometry. Most peptide substrates were sequentially truncated from the carboxyl-terminus revealing that this enzyme is a bona fide carboxypeptidase. Peptides with arginine located close to the carboxyl-terminus were rapidly cleaved, and a fluorescent substrate containing arginine was therefore used to characterize PdCP1 activity and to screen a selection of peptidase inhibitors. Antipain and leupeptin were found to be the most potent inhibitors of PdCP1 activity.

Destructin-1 is a collagen-degrading endopeptidase secreted by Pseudogymnoascus destructans, the causative agent of white-nose syndrome.

Pseudogymnoascus destructans is the causative agent of white-nose syndrome, a disease that has caused the deaths of millions of bats in North America. This psychrophilic fungus proliferates at low temperatures and targets hibernating bats, resulting in their premature arousal from stupor with catastrophic consequences. Despite the impact of white-nose syndrome, little is known about the fungus itself or how it infects its mammalian host. P. destructans is not amenable to genetic manipulation, and therefore understanding the proteins involved in infection requires alternative approaches. Here, we identify hydrolytic enzymes secreted by P. destructans, and use a novel and unbiased substrate profiling technique to define active peptidases. These experiments revealed that endopeptidases are the major proteolytic activities secreted by P. destructans, and that collagen, the major structural protein in mammals, is actively degraded by the secretome. A serine endopeptidase, hereby-named Destructin-1, was subsequently identified, and a recombinant form overexpressed and purified. Biochemical analysis of Destructin-1 showed that it mediated collagen degradation, and a potent inhibitor of peptidase activity was identified. Treatment of P. destructans-conditioned media with this antagonist blocked collagen degradation and facilitated the detection of additional secreted proteolytic activities, including aminopeptidases and carboxypeptidases. These results provide molecular insights into the secretome of P. destructans, and identify serine endopeptidases that have the clear potential to facilitate tissue invasion and pathogenesis in the mammalian host.

Gut Biogeography Accentuates Sex-Related Differences in the Murine Microbiome.

Recent studies have highlighted the influence of factors such as sex and sex-linked hormones on microbiome composition, raising concerns about the generalizability of findings. Here, we explore whether gut geography, specifically the upper and lower gastrointestinal tract (GI), contributes to sex-linked microbiome differences in mice. We collected microbial samples throughout the length of the GI from male and female C57B6/J mice at 6- and 8-weeks old, and conducted 16S rRNA sequencing. Our findings revealed significant sex-related differences, with Clostridium_sensu_stricto_1 more abundant in the male colon, while females exhibited higher levels of Dubosiella newyorkensis across all organs at 6 weeks. We also observed decreased Shannon alpha diversity in the small intestine compared to the lower GI, and this diversity decreased further at 8 weeks. Interestingly, our results suggest that age mitigates sex-related, but not gut geography-related differences in beta diversity, with implications for experimental outcomes and treatment strategies. This study underscores the dynamic nature of microbial diversity, influenced by sex, age, and GI localization, emphasizing the need for a more comprehensive understanding of microbiome dynamics in experimental research and clinical interventions.

Short-Term Dietary Intervention with Whole Oats Protects from Antibiotic-Induced Dysbiosis.

Antibiotic-induced gut microbiome dysbiosis (AID) is known to be influenced by host dietary composition. However, how and when diet modulates gut dysbiosis remains poorly characterized. Thus, here, we utilize a multi-omics approach to characterize how a diet supplemented with oats, a rich source of microbiota-accessible carbohydrates, or dextrose impacts amoxicillin-induced changes to gut microbiome structure and transcriptional activity. We demonstrate that oat administration during amoxicillin challenge provides greater protection from AID than the always oats or recovery oats diet groups. In particular, the group in which oats were provided at the time of antibiotic exposure induced the greatest protection against AID while the other oat diets saw greater effects after amoxicillin challenge. The oat diets likewise reduced amoxicillin-driven elimination of Firmicutes compared to the dextrose diet. Functionally, gut communities fed dextrose were carbohydrate starved and favored respiratory metabolism and consequent metabolic stress management while oat-fed communities shifted their transcriptomic profile and emphasized antibiotic stress management. The metabolic trends were exemplified when assessing transcriptional activity of the following two common gut commensal bacteria: Akkermansia muciniphila and Bacteroides thetaiotaomicron. These findings demonstrate that while host diet is important in shaping how antibiotics effect the gut microbiome composition and function, diet timing may play an even greater role in dietary intervention-based therapeutics. IMPORTANCE We utilize a multi-omics approach to demonstrate that diets supplemented with oats, a rich source of microbiota-accessible carbohydrates, are able to confer protection against antibiotic-induced dysbiosis (AID). Our findings affirm that not only is host diet important in shaping antibiotics effects on gut microbiome composition and function but also that the timing of these diets may play an even greater role in managing AID. This work provides a nuanced perspective on dietary intervention against AID and may be informative on preventing AID during routine antibiotic treatment.

Comparative genomics of white and opaque cell states supports an epigenetic mechanism of phenotypic switching in Candida albicans.

Several Candida species can undergo a heritable and reversible transition from a 'white' state to a mating proficient 'opaque' state. This ability relies on highly interconnected transcriptional networks that control cell-type-specific gene expression programs over multiple generations. Candida albicans, the most prominent pathogenic Candida species, provides a well-studied paradigm for the white-opaque transition. In this species, a network of at least eight transcriptional regulators controls the balance between white and opaque states that have distinct morphologies, transcriptional profiles, and physiological properties. Given the reversible nature and the high frequency of white-opaque transitions, it is widely assumed that this switch is governed by epigenetic mechanisms that occur independently of any changes in DNA sequence. However, a direct genomic comparison between white and opaque cells has yet to be performed. Here, we present a whole-genome comparative analysis of C. albicans white and opaque cells. This analysis revealed rare genetic changes between cell states, none of which are linked to white-opaque switching. This result is consistent with epigenetic mechanisms controlling cell state differentiation in C. albicans and provides direct evidence against a role for genetic variation in mediating the switch.

Galleria mellonella as an insect model for P. destructans, the cause of White-nose Syndrome in bats.

Pseudogymnoascus destructans is the fungal pathogen responsible for White-nose Syndrome (WNS), a disease that has killed millions of bats in North America over the last decade. A major obstacle to research on P. destructans has been the lack of a tractable infection model for monitoring virulence. Here, we establish a high-throughput model of infection using larvae of Galleria mellonella, an invertebrate used to study host-pathogen interactions for a wide range of microbial species. We demonstrate that P. destructans can kill G. mellonella larvae in an inoculum-dependent manner when infected larvae are housed at 13°C or 18°C. Larval killing is an active process, as heat-killed P. destructans spores caused significantly decreased levels of larval death compared to live spores. We also show that fungal spores that were germinated prior to inoculation were able to kill larvae 3-4 times faster than non-germinated spores. Lastly, we identified chemical inhibitors of P. destructans and used G. mellonella to evaluate these inhibitors for their ability to reduce virulence. We demonstrate that amphotericin B can effectively block larval killing by P. destructans and thereby establish that this infection model can be used to screen biocontrol agents against this fungal pathogen.