Th17 cells provide protection at barrier tissues but may also contribute to immune pathology. The relevance and induction mechanisms of pathologic Th17 responses in humans are poorly understood. Here, we identify the mucocutaneous pathobiont Candida albicans as the major direct inducer of human anti-fungal Th17 cells. Th17 cells directed against other fungi are induced by cross-reactivity to C. albicans. Intestinal inflammation expands total C. albicans and cross-reactive Th17 cells. Strikingly, Th17 cells cross-reactive to the airborne fungus Aspergillus fumigatus are selectively activated and expanded in patients with airway inflammation, especially during acute allergic bronchopulmonary aspergillosis. This indicates a direct link between protective intestinal Th17 responses against C. albicans and lung inflammation caused by airborne fungi. We identify heterologous immunity to a single, ubiquitous member of the microbiota as a central mechanism for systemic induction of human antifungal Th17 responses and as a potential risk factor for pulmonary inflammatory diseases.

Numerous antibiotics have proven to be effective at ameliorating the clinical symptoms of urinary tract infections (UTIs), but recurrent and chronic infections continue to plague many individuals. Most UTIs are caused by strains of uropathogenic Escherichia coli (UPEC), which can form both extra-and intracellular biofilm-like communities within the bladder. UPEC also persist inside host urothelial cells in a more quiescent state, sequestered within late endosomal compartments. Here, we tested a panel of 17 different antibiotics, representing seven distinct functional classes, for their effects on the survival of the reference UPEC isolate UTI89 within both biofilms and host bladder urothelial cells. All but one of the tested antibiotics prevented UTI89 growth in broth culture, and most were at least modestly effective against bacteria present within in vitro-grown biofilms. In contrast, only a few of the antibiotics, including nitrofurantoin and the fluoroquinolones ciprofloxacin and sparfloxacin, were able to eliminate intracellular bacteria in bladder cell culture-based assays. However, in a mouse UTI model system in which these antibiotics reached concentrations in the urine specimens that far exceeded minimal inhibitory doses, UPEC reservoirs in bladder tissues were not effectively eradicated. We conclude that the persistence of UPEC within the bladder, regardless of antibiotic treatments, is likely facilitated by a combination of biofilm formation, entry of UPEC into a quiescent or semiquiescent state within host cells, and the stalwart permeability barrier function associated with the bladder urothelium.

The mammalian immune system relies on recognition of pathogen surface antigens for targeting and clearance. In the absence of immune evasion strategies, pathogen clearance is rapid. In the case of Aspergillus fumigatus, the successful fungus must avoid phagocytosis in the lung to establish invasive infection. In healthy individuals, fungal spores are cleared by immune cells; however, in immunocompromised patients, clearance mechanisms are impaired. Here, using proteome analyses, we identified CcpA as an important fungal spore protein involved in pathogenesis. A. fumigatus lacking CcpA was more susceptible to immune recognition and prompt eradication and, consequently, exhibited drastically attenuated virulence. In infection studies, CcpA was required for virulence in infected immunocompromised mice, suggesting that it could be used as a possible immunotherapeutic or diagnostic target in the future. In summary, our report adds a protein to the list of those known to be critical to the complex fungal spore surface environment and, more importantly, identifies a protein important for conidial immunogenicity during infection.

Polymorphonuclear granulocytes (PMNs) are indispensable for controlling life-threatening fungal infections. In addition to various effector mechanisms, PMNs also produce extracellular vesicles (EVs). Their contribution to antifungal defense has remained unexplored. We reveal that the clinically important human-pathogenic fungus Aspergillus fumigatus triggers PMNs to release a distinct set of antifungal EVs (afEVs). Proteome analyses indicated that afEVs are enriched in antimicrobial proteins. The cargo and the release kinetics of EVs are modulated by the fungal strain confronted. Tracking of afEVs indicated that they associated with fungal cells and even entered fungal hyphae, resulting in alterations in the morphology of the fungal cell wall and dose-dependent antifungal effects. To assess as a proof of concept whether the antimicrobial proteins found in afEVs might contribute to growth inhibition of hyphae when present in the fungal cytoplasm, two human proteins enriched in afEVs, cathepsin G and azurocidin, were heterologously expressed in fungal hyphae. This led to reduced fungal growth relative to that of a control strain producing the human retinol binding protein 7. In conclusion, extracellular vesicles produced by neutrophils in response to A. fumigatus infection are able to associate with the fungus, limit growth, and elicit cell damage by delivering antifungal cargo. This finding offers an intriguing, previously overlooked mechanism of antifungal defense against A. fumigatus. IMPORTANCE Invasive fungal infections caused by the mold Aspergillus fumigatus are a growing concern in the clinic due to the increasing use of immunosuppressive therapies and increasing antifungal drug resistance. These infections result in high rates of mortality, as treatment and diagnostic options remain limited. In healthy individuals, neutrophilic granulocytes are critical for elimination of A. fumigatus from the host; however, the exact extracellular mechanism of neutrophil-mediated antifungal activity remains unresolved. Here, we present a mode of antifungal defense employed by human neutrophils against A. fumigatus not previously described. We found that extracellular vesicles produced by neutrophils in response to A. fumigatus infection are able to associate with the fungus, limit growth, and elicit cell damage by delivering antifungal cargo. In the end, antifungal extracellular vesicle biology provides a significant step forward in our understanding of A. fumigatus host pathogenesis and opens up novel diagnostic and therapeutic possibilities.

Intracellular quiescent reservoirs of uropathogenic Escherichia coli (UPEC), which can seed the bladder mucosa during the acute phase of a urinary tract infection (UTI), are protected from antibiotic treatments and are extremely difficult to eliminate. These reservoirs are a potential source for recurrent UTIs that affect millions annually. Here, using murine infection models and the bladder cell exfoliant chitosan, we demonstrate that intracellular UPEC populations shift within the stratified layers of the urothelium during the course of a UTI. Following invasion of the terminally differentiated superficial layer of epithelial cells that line the bladder lumen, UPEC can multiply and disseminate, eventually establishing reservoirs within underlying immature host cells. If given access, UPEC can invade the superficial and immature bladder cells equally well. As infected immature host cells differentiate and migrate towards the apical surface of the bladder, UPEC can reinitiate growth and discharge into the bladder lumen. By inducing the exfoliation of the superficial layers of the urothelium, chitosan stimulates rapid regenerative processes and the reactivation and efflux of quiescent intracellular UPEC reservoirs. When combined with antibiotics, chitosan treatment significantly reduces bacterial loads within the bladder and may therefore be of therapeutic value to individuals with chronic, recurrent UTIs.

Endogenous double-stranded RNA (dsRNA) must be intricately regulated in mammals to prevent aberrant activation of host inflammatory pathways by cytosolic dsRNA binding proteins. Here, we define the long, endogenous dsRNA repertoire in mammalian macrophages and monocytes during the inflammatory response to bacterial lipopolysaccharide. Hyperediting by adenosine deaminases that act on RNA (ADAR) enzymes was quantified over time using RNA-seq data from activated mouse macrophages to identify 342 Editing Enriched Regions (EERs), indicative of highly structured dsRNA. Analysis of publicly available data sets for samples of human peripheral blood monocytes resulted in discovery of 3438 EERs in the human transcriptome. Human EERs had predicted secondary structures that were significantly more stable than those of mouse EERs and were located primarily in introns, whereas nearly all mouse EERs were in 3′ UTRs. Seventy-four mouse EER-associated genes contained an EER in the orthologous human gene, although nucleotide sequence and position were only rarely conserved. Among these conserved EER-associated genes were several TNF alpha-signaling genes, including Sppl2a and Tnfrsf1b, important for processing and recognition of TNF alpha, respectively. Using publicly available data and experimental validation, we found that a significant proportion of EERs accumulated in the nucleus, a strategy that may prevent aberrant activation of proinflammatory cascades in the cytoplasm. The observation of many ADAR-edited dsRNAs in mammalian immune cells, a subset of which are in orthologous genes of mouse and human, suggests a conserved role for these structured regions.

Summary Bacterial populations utilize a variety of signaling strategies to exchange information, including the secretion of quorum-sensing molecules and contact-dependent signaling cascades. Although quorum sensing has received the bulk of attention for many years, contact-dependent signaling is forging a niche in the research world with the identification of novel systems and the emergence of more mechanistic data. Contact-dependent signaling is likely a common strategy by which bacteria in close contact, such as within biofilms, can modulate the growth and behavior of both siblings and competitors. Ongoing work with diverse bacterial systems, including Myxococcus xanthus, pathogenic Escherichia coli strains, Bacillus subtilis, and dissimilatory metal-reducing soil bacteria, is providing increasingly detailed insight into the dynamic mechanisms and potential of contact-dependent signaling processes.

Fungal spores and hyphal fragments play an important role as allergens in respiratory diseases. In this study, we performed trypsin shaving and secretome analyses to identify the surface-exposed proteins and secreted/shed proteins of Aspergillus fumigatus conidia, respectively. We investigated the surface proteome under different conditions, including temperature variation and germination. We found that the surface proteome of resting A. fumigatus conidia is not static, but instead unexpectedly dynamic, as evidenced by drastically different surface proteomes under different growth conditions. Knockouts of two abundant A. fumigatus surface proteins, ScwA and CweA, were found to function only in fine-tuning the cell wall stress response, implying that the conidial surface is very robust against perturbations. We then compared the surface proteome of A. fumigatus to other allergy-inducing molds, including Alternaria alternata, Penicillium rubens, and Cladosporium herbarum, and performed comparative proteomics on resting and swollen conidia, as well as secreted proteins from germinating conidia. We detected 125 protein ortholog groups, including 80 with putative catalytic activity, in the extracellular region of all four molds, and 42 nonorthologous proteins produced solely by A. fumigatus.Ultimately, this study highlights the dynamic nature of the A. fumigatus conidial surface and provides targets for future diagnostics and immunotherapy.