Trained innate immunity (TII) is induced following immunization with live attenuated microbes and represents a clinically important strategy to enhance innate defenses. TII was initially demonstrated following intravenous inoculation with low-virulence Candida albicans , with protection against a subsequent lethal C. albicans intravenous bloodstream infection (BSI) mediated by monocytes with enhanced cytokine responses.

Many successful pathogens cause latent infections, remaining dormant within the host for years but retaining the ability to reactivate to cause symptomatic disease. The human opportunistic fungal pathogen Cryptococcus neoformans establishes latent pulmonary infections in immunocompetent individuals upon inhalation from the environment.

Fungal-bacterial intra-abdominal infections (IAI) can lead to sepsis with significant morbidity and mortality. We have established a murine model of Candida albicans (Ca) and Staphylococcus aureus (Sa) IAI that results in acute lethal sepsis. Prior intraperitoneal or intravenous inoculation with low virulence Candida dubliniensis (Cd) confers high level protection against lethal Ca/Sa IAI and sepsis. Protection via Cd immunization is associated with decreased pro-inflammatory cytokines and mediated by Gr-1+ putative myeloid-derived suppressor cells (MDSCs) representing a novel form of trained innate immunity (TII). The objective of these studies was to determine the extent of Cd-mediated TII against sepsis of broad origin and explore the potential of fungal cell wall components as abiotic immunogen alternatives to induce TII, including zymosan depleted of TLR2 activity (d-zymosan), or purified preparations of β-glucan. Immunized mice were challenged 14 days post-immunization with a lethal array of live or abiotic inducers of sepsis, including Ca/Sa, Ca/Escherichia coli (Ca/Ec), LPS or untreated zymosan. Results showed that live Cd immunization was protective against sepsis induced by Ca/Ec and zymosan, but not LPS. Similar to protection against Ca/Sa, survival was dependent on Gr-1+ cells with no role for macrophages. Among the fungal cell wall compounds as immunogens, immunization with d-zymosan and an alkali-treated form of β-glucan also resulted in significant protection against sepsis induced by Ca/Sa or Ca/Ec, but not LPS sepsis. Again, there was a strong dependence on Gr-1+ cells for protection with one exception, an added role for macrophages in the case of protection induced by alkali-treated β-glucan. Overall, these results demonstrate that immunization with Cd as well as abiotic fungal cell components are capable of Gr-1+ cell-mediated trained innate immune protection against sepsis of broad microbial origin. In addition, abiotic β-glucans represent potential alternatives to live Cd for protection against lethal polymicrobial sepsis.

RNA sequencing (RNA-Seq) experiments focused on gene expression involve removal of ribosomal RNA (rRNA) because it is the major RNA constituent of cells. This process, called RNA enrichment, is done primarily to reduce cost: without rRNA removal, deeper sequencing must be performed to compensate for the sequencing reads wasted on rRNA. The ideal RNA enrichment method removes all rRNA without affecting other RNA in the sample. We tested the performance of three RNA enrichment methods on RNA isolated from Cryptococcus neoformans, a fungal pathogen of humans. We find that the RNase H depletion method is more efficient in depleting rRNA and more specific in recapitulating non-rRNA levels present in unenriched controls than the commonly-used Poly(A) isolation method. The RNase H depletion method is also more effective than the Ribo-Zero depletion method as measured by rRNA depletion efficiency and recapitulation of protein-coding RNA levels present in unenriched controls, while the Ribo-Zero depletion method more closely recapitulates annotated non-coding RNA (ncRNA) levels. Finally, we leverage these data to accurately map the C. neoformans mitochondrial rRNA genes, and also demonstrate that RNA-Seq data generated with the RNase H and Ribo-Zero depletion methods can be used to explore novel C. neoformans long non-coding RNA genes.

Introduction: Microbial pathogens undergo significant physiological changes during interactions with the infected host, including alterations in metabolism and cell architecture. The Cryptococcus neoformans Mar1 protein is required for the proper ordering of the fungal cell wall in response to host-relevant stresses. However, the precise mechanism by which this Cryptococcus-specific protein regulates cell wall homeostasis was not defined.Methods: Here, we use comparative transcriptomics, protein localization, and phenotypic analysis of a mar1D loss-of-function mutant strain to further define the role of C. neoformans Mar1 in stress response and antifungal resistance.Results: We demonstrate that C. neoformans Mar1 is highly enriched in mitochondria. Furthermore, a mar1Δ mutant strain is impaired in growth in the presence of select electron transport chain inhibitors, has altered ATP homeostasis, and promotes proper mitochondrial morphogenesis. Pharmacological inhibition of complex IV of the electron transport chain in wild-type cells promotes similar cell wall changes as the mar1Δ mutant strain, supporting prior associations between mitochondrial function and cell wall homeostasis. Although Mar1 is not required for general susceptibility to the azole antifungals, the mar1Δ mutant strain displays increased tolerance to fluconazole that correlates with repressed mitochondrial metabolic activity.Discussion: Together, these studies support an emerging model in which the metabolic activity of microbial cells directs cell physiological changes to allow persistence in the face of antimicrobial and host stress.