Pseudomonas Synergizes with Fluconazole against Candida during Treatment of Polymicrobial Infection.

Polymicrobial infections are challenging to treat because we don't fully understand how pathogens interact during infection and how these interactions affect drug efficacy. Candida albicans and Pseudomonas aeruginosa are opportunistic pathogens that can be found in similar sites of infection such as in burn wounds and most importantly in the lungs of CF and mechanically ventilated patients. C. albicans is particularly difficult to treat because of the paucity of antifungal agents, some of which lack fungicidal activity. In this study, we investigated the efficacy of anti-fungal treatment during C. albicans-P. aeruginosa coculture in vitro and co-infection in the mucosal zebrafish infection model analogous to the lung. We find that P. aeruginosa enhances the activity of fluconazole (FLC), an anti-fungal drug that is fungistatic in vitro, to promote both clearance of C. albicans during co-infection in vivo and fungal killing in vitro. This synergy between FLC treatment and bacterial antagonism is partly due to iron piracy, as it is reduced upon iron supplementation and knockout of bacterial siderophores. Our work demonstrates that FLC has enhanced activity in clinically relevant contexts and highlights the need to understand antimicrobial effectiveness in the complex environment of the host with its associated microbial communities.

Redundant Trojan horse and endothelial-circulatory mechanisms for host-mediated spread of Candida albicans yeast.

The host innate immune system has developed elegant processes for the detection and clearance of invasive fungal pathogens. These strategies may also aid in the spread of pathogens in vivo, although technical limitations have previously hindered our ability to view the host innate immune and endothelial cells to probe their roles in spreading disease. Here, we have leveraged zebrafish larvae as a model to view the interactions of these host processes with the fungal pathogen Candida albicans in vivo. We examined three potential host-mediated mechanisms of fungal spread: movement inside phagocytes in a "Trojan Horse" mechanism, inflammation-assisted spread, and endothelial barrier passage. Utilizing both chemical and genetic tools, we systematically tested the loss of neutrophils and macrophages and the loss of blood flow on yeast cell spread. Both neutrophils and macrophages respond to yeast-locked and wild type C. albicans in our model and time-lapse imaging revealed that macrophages can support yeast spread in a "Trojan Horse" mechanism. Surprisingly, loss of immune cells or inflammation does not alter dissemination dynamics. On the other hand, when blood flow is blocked, yeast can cross into blood vessels but they are limited in how far they travel. Blockade of both phagocytes and circulation reduces rates of dissemination and significantly limits the distance of fungal spread from the infection site. Together, this data suggests a redundant two-step process whereby (1) yeast cross the endothelium inside phagocytes or via direct uptake, and then (2) they utilize blood flow or phagocytes to travel to distant sites.

Candidalysin is a fungal peptide toxin critical for mucosal infection.

Cytolytic proteins and peptide toxins are classical virulence factors of several bacterial pathogens which disrupt epithelial barrier function, damage cells and activate or modulate host immune responses. Such toxins have not been identified previously in human pathogenic fungi. Here we identify the first, to our knowledge, fungal cytolytic peptide toxin in the opportunistic pathogen Candida albicans. This secreted toxin directly damages epithelial membranes, triggers a danger response signalling pathway and activates epithelial immunity. Membrane permeabilization is enhanced by a positive charge at the carboxy terminus of the peptide, which triggers an inward current concomitant with calcium influx. C. albicans strains lacking this toxin do not activate or damage epithelial cells and are avirulent in animal models of mucosal infection. We propose the name 'Candidalysin' for this cytolytic peptide toxin; a newly identified, critical molecular determinant of epithelial damage and host recognition of the clinically important fungus, C. albicans.

Yeast and Filaments Have Specialized, Independent Activities in a Zebrafish Model of Candida albicans Infection.

Candida albicans dimorphism is a crucial virulence factor during invasive candidiasis infections, which claim the lives of nearly one-half of those afflicted. It has long been believed that filaments drive tissue invasion and yeast mediates bloodstream dissemination, but observation of these activities during infection has been prevented by technical limitations. We used a transparent zebrafish infection model to analyze more comprehensively how C. albicans utilizes shape to disseminate and invade. This model facilitated the use of diverse, complementary strategies to manipulate shape, allowing us to monitor dissemination, invasion, and pathogenesis via intravital imaging of individual fungal cells throughout the host. To control fungal cell shape, we employed three different strategies: gene deletion (efg1Δ/Δ cph1Δ/Δ, eed1Δ/Δ), overexpression of master regulators (NRG1 or UME6), and modulation of the infection temperature (21°C, 28°C, or 33°C). The effects of these orthogonal manipulations were consistent, support the proposed specialized roles of yeast in dissemination and filaments in tissue invasion and pathogenesis, and indicate conserved mechanisms in zebrafish. To test if either morphotype changes the effectiveness of the other, we infected fish with a known mixture of shape-locked strains. Surprisingly, mixed-strain infections were associated with additive, but not synergistic, filament invasion and yeast dissemination. These findings provide the most complete view of morphotype-function relationships for C. albicans to date, revealing independent roles of yeast and filaments during disseminated candidiasis.

Neutrophil Attack Triggers Extracellular Trap-Dependent Candida Cell Wall Remodeling and Altered Immune Recognition.

Pathogens hide immunogenic epitopes from the host to evade immunity, persist and cause infection. The opportunistic human fungal pathogen Candida albicans, which can cause fatal disease in immunocompromised patient populations, offers a good example as it masks the inflammatory epitope ß-glucan in its cell wall from host recognition. It has been demonstrated previously that ß-glucan becomes exposed during infection in vivo but the mechanism behind this exposure was unknown. Here, we show that this unmasking involves neutrophil extracellular trap (NET) mediated attack, which triggers changes in fungal cell wall architecture that enhance immune recognition by the Dectin-1 ß-glucan receptor in vitro. Furthermore, using a mouse model of disseminated candidiasis, we demonstrate the requirement for neutrophils in triggering these fungal cell wall changes in vivo. Importantly, we found that fungal epitope unmasking requires an active fungal response in addition to the stimulus provided by neutrophil attack. NET-mediated damage initiates fungal MAP kinase-driven responses, particularly by Hog1, that dynamically relocalize cell wall remodeling machinery including Chs3, Phr1 and Sur7. Neutrophil-initiated cell wall disruptions augment some macrophage cytokine responses to attacked fungi. This work provides insight into host-pathogen interactions during disseminated candidiasis, including valuable information about how the C. albicans cell wall responds to the biotic stress of immune attack. Our results highlight the important but underappreciated concept that pattern recognition during infection is dynamic and depends on the host-pathogen dialog.

Control of Mucosal Candidiasis in the Zebrafish Swim Bladder Depends on Neutrophils That Block Filament Invasion and Drive Extracellular-Trap Production.

Candida albicans is a ubiquitous mucosal commensal that is normally prevented from causing acute or chronic invasive disease. Neutrophils contribute to protection in oral infection but exacerbate vulvovaginal candidiasis. To dissect the role of neutrophils during mucosal candidiasis, we took advantage of a new, transparent zebrafish swim bladder infection model. Intravital microscopic tracking of individual animals revealed that the blocking of neutrophil recruitment leads to rapid mortality in this model through faster disease progression. Conversely, artificial recruitment of neutrophils during early infection reduces disease pressure. Noninvasive longitudinal tracking showed that mortality is a consequence of C. albicans breaching the epithelial barrier and invading surrounding tissues. Accordingly, we found that a hyperfilamentous C. albicans strain breaches the epithelial barrier more frequently and causes mortality in immunocompetent zebrafish. A lack of neutrophils at the infection site is associated with less fungus-associated extracellular DNA and less damage to fungal filaments, suggesting that neutrophil extracellular traps help to protect the epithelial barrier from C. albicans breach. We propose a homeostatic model where C. albicans disease pressure is balanced by neutrophil-mediated damage of fungi, maintaining this organism as a commensal while minimizing the risk of damage to host tissue. The unequaled ability to dissect infection dynamics at a high spatiotemporal resolution makes this zebrafish model a unique tool for understanding mucosal host-pathogen interactions.

Candida albicans and Pseudomonas aeruginosa Interact To Enhance Virulence of Mucosal Infection in Transparent Zebrafish.

Polymicrobial infections often include both fungi and bacteria and can complicate patient treatment and resolution of infection. Cross-kingdom interactions among bacteria, fungi, and/or the immune system during infection can enhance or block virulence mechanisms and influence disease progression. The fungus Candida albicans and the bacterium Pseudomonas aeruginosa are coisolated in the context of polymicrobial infection at a variety of sites throughout the body, including mucosal tissues such as the lung. In vitro, C. albicans and P. aeruginosa have a bidirectional and largely antagonistic relationship. Their interactions in vivo remain poorly understood, specifically regarding host responses in mediating infection. In this study, we examine trikingdom interactions using a transparent juvenile zebrafish to model mucosal lung infection and show that C. albicans and P. aeruginosa are synergistically virulent. We find that high C. albicans burden, fungal epithelial invasion, swimbladder edema, and epithelial extrusion events serve as predictive factors for mortality in our infection model. Longitudinal analyses of fungal, bacterial, and immune dynamics during coinfection suggest that enhanced morbidity is associated with exacerbated C. albicans pathogenesis and elevated inflammation. The P. aeruginosa quorum-sensing-deficient ΔlasR mutant also enhances C. albicans pathogenicity in coinfection and induces extrusion of the swimbladder. Together, these observations suggest that C. albicans-P. aeruginosa cross talk in vivo can benefit both organisms to the detriment of the host.

ß-(1,3)-Glucan Unmasking in Some Candida albicans Mutants Correlates with Increases in Cell Wall Surface Roughness and Decreases in Cell Wall Elasticity.

Candida albicans is among the most common human fungal pathogens, causing a broad range of infections, including life-threatening systemic infections. The cell wall of C. albicans is the interface between the fungus and the innate immune system. The cell wall is composed of an outer layer enriched in mannosylated glycoproteins (mannan) and an inner layer enriched in ß-(1,3)-glucan and chitin. Detection of C. albicans by Dectin-1, a C-type signaling lectin specific for ß-(1,3)-glucan, is important for the innate immune system to recognize systemic fungal infections. Increased exposure of ß-(1,3)-glucan to the immune system occurs when the mannan layer is altered or removed in a process called unmasking. Nanoscale changes to the cell wall during unmasking were explored in live cells with atomic force microscopy (AFM). Two mutants, the cho1Δ/Δ and kre5Δ/Δ mutants, were selected as representatives that exhibit modest and strong unmasking, respectively. Comparisons of the cho1Δ/Δ and kre5Δ/Δ mutants to the wild type reveal morphological changes in their cell walls that correlate with decreases in cell wall elasticity. In addition, AFM tips functionalized with Dectin-1 revealed that the forces of binding of Dectin-1 to all of the strains were similar, but the frequency of binding was highest for the kre5Δ/Δ mutant, decreased for the cho1Δ/Δ mutant, and rare for the wild type. These data show that nanoscale changes in surface topology are correlated with increased Dectin-1 adhesion and decreased cell wall elasticity. AFM, using tips functionalized with immunologically relevant molecules, can map epitopes of the cell wall and increase our understanding of pathogen recognition by the immune system.

NADPH oxidase-driven phagocyte recruitment controls Candida albicans filamentous growth and prevents mortality.

Candida albicans is a human commensal and clinically important fungal pathogen that grows as both yeast and hyphal forms during human, mouse and zebrafish infection. Reactive oxygen species (ROS) produced by NADPH oxidases play diverse roles in immunity, including their long-appreciated function as microbicidal oxidants. Here we demonstrate a non-traditional mechanistic role of NADPH oxidase in promoting phagocyte chemotaxis and intracellular containment of fungi to limit filamentous growth. We exploit the transparent zebrafish model to show that failed NADPH oxidase-dependent phagocyte recruitment to C. albicans in the first four hours post-infection permits fungi to germinate extracellularly and kill the host. We combine chemical and genetic tools with high-resolution time-lapse microscopy to implicate both phagocyte oxidase and dual-specific oxidase in recruitment, suggesting that both myeloid and non-myeloid cells promote chemotaxis. We show that early non-invasive imaging provides a robust tool for prognosis, strongly connecting effective early immune response with survival. Finally, we demonstrate a new role of a key regulator of the yeast-to-hyphal switching program in phagocyte-mediated containment, suggesting that there are species-specific methods for modulation of NADPH oxidase-independent immune responses. These novel links between ROS-driven chemotaxis and fungal dimorphism expand our view of a key host defense mechanism and have important implications for pathogenesis.

The complex roles of NADPH oxidases in fungal infection.

NADPH oxidases play key roles in immunity and inflammation that go beyond the production of microbicidal reactive oxygen species (ROS). The past decade has brought a new appreciation for the diversity of roles played by ROS in signalling associated with inflammation and immunity. NADPH oxidase activity affects disease outcome during infections by human pathogenic fungi, an important group of emerging and opportunistic pathogens that includes Candida, Aspergillus and Cryptococcus species. Here we review how alternative roles of NADPH oxidase activity impact fungal infection and how ROS signalling affects fungal physiology. Particular attention is paid to roles for NADPH oxidase in immune migration, immunoregulation in pulmonary infection, neutrophil extracellular trap formation, autophagy and inflammasome activity. These recent advances highlight the power and versatility of spatiotemporally controlled redox regulation in the context of infection, and point to a need to understand the molecular consequences of NADPH oxidase activity in the cell.

Masking of ß(1-3)-glucan in the cell wall of Candida albicans from detection by innate immune cells depends on phosphatidylserine.

The virulence of Candida albicans in a mouse model of invasive candidiasis is dependent on the phospholipids phosphatidylserine (PS) and phosphatidylethanolamine (PE). Disruption of the PS synthase gene CHO1 (i.e., cho1Δ/Δ) eliminates PS and blocks the de novo pathway for PE biosynthesis. In addition, the cho1Δ/Δ mutant's ability to cause invasive disease is severely compromised. The cho1Δ/Δ mutant also exhibits cell wall defects, and in this study, it was determined that loss of PS results in decreased masking of cell wall ß(1-3)-glucan from the immune system. In wild-type C. albicans, the outer mannan layer of the wall masks the inner layer of ß(1-3)-glucan from exposure and detection by innate immune effector molecules like the C-type signaling lectin Dectin-1, which is found on macrophages, neutrophils, and dendritic cells. The cho1Δ/Δ mutant exhibits increases in exposure of ß(1-3)-glucan, which leads to greater binding by Dectin-1 in both yeast and hyphal forms. The unmasking of ß(1-3)-glucan also results in increased elicitation of TNF-α from macrophages in a Dectin-1-dependent manner. The role of phospholipids in fungal pathogenesis is an emerging field, and this is the first study showing that loss of PS in C. albicans results in decreased masking of ß(1-3)-glucan, which may contribute to our understanding of fungus-host interactions.

Regulation of progenitor cell proliferation and granulocyte function by microRNA-223.

MicroRNAs are abundant in animal genomes and have been predicted to have important roles in a broad range of gene expression programmes. Despite this prominence, there is a dearth of functional knowledge regarding individual mammalian microRNAs. Using a loss-of-function allele in mice, we report here that the myeloid-specific microRNA-223 (miR-223) negatively regulates progenitor proliferation and granulocyte differentiation and activation. miR-223 (also called Mirn223) mutant mice have an expanded granulocytic compartment resulting from a cell-autonomous increase in the number of granulocyte progenitors. We show that Mef2c, a transcription factor that promotes myeloid progenitor proliferation, is a target of miR-223, and that genetic ablation of Mef2c suppresses progenitor expansion and corrects the neutrophilic phenotype in miR-223 null mice. In addition, granulocytes lacking miR-223 are hypermature, hypersensitive to activating stimuli and display increased fungicidal activity. As a consequence of this neutrophil hyperactivity, miR-223 mutant mice spontaneously develop inflammatory lung pathology and exhibit exaggerated tissue destruction after endotoxin challenge. Our data support a model in which miR-223 acts as a fine-tuner of granulocyte production and the inflammatory response.

Candida albicans induces arginine biosynthetic genes in response to host-derived reactive oxygen species.

The interaction of Candida albicans with phagocytes of the host's innate immune system is highly dynamic, and its outcome directly impacts the progression of infection. While the switch to hyphal growth within the macrophage is the most obvious physiological response, much of the genetic response reflects nutrient starvation: translational repression and induction of alternative carbon metabolism. Changes in amino acid metabolism are not seen, with the striking exception of arginine biosynthesis, which is upregulated in its entirety during coculture with macrophages. Using single-cell reporters, we showed here that arginine biosynthetic genes are induced specifically in phagocytosed cells. This induction is lower in magnitude than during arginine starvation in vitro and is driven not by an arginine deficiency within the phagocyte but instead by exposure to reactive oxygen species (ROS). Curiously, these genes are induced in a narrow window of sublethal ROS concentrations. C. albicans cells phagocytosed by primary macrophages deficient in the gp91(phox) subunit of the phagocyte oxidase do not express the ARG pathway, indicating that the induction is dependent on the phagocyte oxidative burst. C. albicans arg pathway mutants are retarded in germ tube and hypha formation within macrophages but are not notably more sensitive to ROS. We also find that the ARG pathway is regulated not by the general amino acid control response but by transcriptional regulators similar to the Saccharomyces cerevisiae ArgR complex. In summary, phagocytosis induces this single amino acid biosynthetic pathway in an ROS-dependent manner.

Ex-chitin-g news on drug-induced fungal epitope unmasking.

The microbial cell wall is an essential cellular organelle commonly targeted by antimicrobials. It is also a battleground of innate immune recognition where microbes can evade immune recognition by masking essential cell wall components. A recent study (A. S. Wagner, S. W. Lumsdaine, M. M. Mangrum, and T. B. Reynolds, mBio https://doi.org/10.1128/mbio.00074-23, 2023) provides insight into how echinocandin antifungals cause exposure of proinflammatory ß(1,3)-glucan by driving excess chitin production in the weakened cell wall. Although many environmental and biological activities perturb cell wall integrity and regulate ß(1,3)-glucan exposure, we still know little about which intracellular signaling components regulate the cell wall changes that result in disrupted cell wall architecture. Wagner et al. showed that calcineurin and the Mkc1p kinase regulate chitin deposition and ß(1,3)-glucan unmasking. They further identified chitin synthesis as a key driving force in cell wall structure disruption leading to epitope exposure. Their findings highlight how fungal cell wall dynamics have important implications for antifungal immunity and future drug development.

A New Phenotype in Candida-Epithelial Cell Interaction Distinguishes Colonization- versus Vulvovaginal Candidiasis-Associated Strains.

Vulvovaginal candidiasis (VVC) affects nearly 3/4 of women during their lifetime, and its symptoms seriously reduce quality of life. Although Candida albicans is a common commensal, it is unknown if VVC results from a switch from a commensal to pathogenic state, if only some strains can cause VVC, and/or if there is displacement of commensal strains with more pathogenic strains. We studied a set of VVC and colonizing C. albicans strains to identify consistent in vitro phenotypes associated with one group or the other. We find that the strains do not differ in overall genetic profile or behavior in culture media (i.e., multilocus sequence type [MLST] profile, rate of growth, and filamentation), but they show strikingly different behaviors during their interactions with vaginal epithelial cells. Epithelial infections with VVC-derived strains yielded stronger fungal proliferation and shedding of fungi and epithelial cells. Transcriptome sequencing (RNA-seq) analysis of representative epithelial cell infections with selected pathogenic or commensal isolates identified several differentially activated epithelial signaling pathways, including the integrin, ferroptosis, and type I interferon pathways; the latter has been implicated in damage protection. Strikingly, inhibition of type I interferon signaling selectively increases fungal shedding of strains in the colonizing cohort, suggesting that increased shedding correlates with lower interferon pathway activation. These data suggest that VVC strains may intrinsically have enhanced pathogenic potential via differential elicitation of epithelial responses, including the type I interferon pathway. Therefore, it may eventually be possible to evaluate pathogenic potential in vitro to refine VVC diagnosis. IMPORTANCE Despite a high incidence of VVC, we still have a poor understanding of this female-specific disease whose negative impact on women's quality of life has become a public health issue. It is not yet possible to determine by genotype or laboratory phenotype if a given Candida albicans strain is more or less likely to cause VVC. Here, we show that Candida strains causing VVC induce more fungal shedding from epithelial cells than strains from healthy women. This effect is also accompanied by increased epithelial cell detachment and differential activation of the type I interferon pathway. These distinguishing phenotypes suggest it may be possible to evaluate the VVC pathogenic potential of fungal isolates. This would permit more targeted antifungal treatments to spare commensals and could allow for displacement of pathogenic strains with nonpathogenic colonizers. We expect these new assays to provide a more targeted tool for identifying fungal virulence factors and epithelial responses that control fungal vaginitis.

Live imaging of disseminated candidiasis in zebrafish reveals role of phagocyte oxidase in limiting filamentous growth.

Candida albicans is a human commensal and a clinically important fungal pathogen that grows in both yeast and hyphal forms during human infection. Although Candida can cause cutaneous and mucosal disease, systemic infections cause the greatest mortality in hospitals. Candidemia occurs primarily in immunocompromised patients, for whom the innate immune system plays a paramount role in immunity. We have developed a novel transparent vertebrate model of candidemia to probe the molecular nature of Candida-innate immune system interactions in an intact host. Our zebrafish infection model results in a lethal disseminated disease that shares important traits with disseminated candidiasis in mammals, including dimorphic fungal growth, dependence on hyphal growth for virulence, and dependence on the phagocyte NADPH oxidase for immunity. Dual imaging of fluorescently marked immune cells and fungi revealed that phagocytosed yeast cells can remain viable and even divide within macrophages without germinating. Similarly, although we observed apparently killed yeast cells within neutrophils, most yeast cells within these innate immune cells were viable. Exploiting this model, we combined intravital imaging with gene knockdown to show for the first time that NADPH oxidase is required for regulation of C. albicans filamentation in vivo. The transparent and easily manipulated larval zebrafish model promises to provide a unique tool for dissecting the molecular basis of phagocyte NADPH oxidase-mediated limitation of filamentous growth in vivo.

Linking high-resolution metabolic flux phenotypes and transcriptional regulation in yeast modulated by the global regulator Gcn4p.

Genome sequencing dramatically increased our ability to understand cellular response to perturbation. Integrating system-wide measurements such as gene expression with networks of protein-protein interactions and transcription factor binding revealed critical insights into cellular behavior. However, the potential of systems biology approaches is limited by difficulties in integrating metabolic measurements across the functional levels of the cell despite their being most closely linked to cellular phenotype. To address this limitation, we developed a model-based approach to correlate mRNA and metabolic flux data that combines information from both interaction network models and flux determination models. We started by quantifying 5,764 mRNAs, 54 metabolites, and 83 experimental (13)C-based reaction fluxes in continuous cultures of yeast under stress in the absence or presence of global regulator Gcn4p. Although mRNA expression alone did not directly predict metabolic response, this correlation improved through incorporating a network-based model of amino acid biosynthesis (from r = 0.07 to 0.80 for mRNA-flux agreement). The model provides evidence of general biological principles: rewiring of metabolic flux (i.e., use of different reaction pathways) by transcriptional regulation and metabolite interaction density (i.e., level of pairwise metabolite-protein interactions) as a key biosynthetic control determinant. Furthermore, this model predicted flux rewiring in studies of follow-on transcriptional regulators that were experimentally validated with additional (13)C-based flux measurements. As a first step in linking metabolic control and genetic regulatory networks, this model underscores the importance of integrating diverse data types in large-scale cellular models. We anticipate that an integrated approach focusing on metabolic measurements will facilitate construction of more realistic models of cellular regulation for understanding diseases and constructing strains for industrial applications.

The dominoes fall after long-term antibiotic exposure.

Broad-spectrum antibiotics should prevent disease, right? In this issue of Cell Host & Microbe, Drummond et al. turn logic on its head and show they actually drive more deadly invasive fungal-bacterial systemic co-infection. Prophylactic antibiotics increase susceptibility to these infections by targeting the commensal microbes required for gut-derived IL-17-mediated immunity.

IL-23 signaling prevents ferroptosis-driven renal immunopathology during candidiasis.

During infection the host relies on pattern-recognition receptors to sense invading fungal pathogens to launch immune defense mechanisms. While fungal recognition and immune effector responses are organ and cell type specific, during disseminated candidiasis myeloid cells exacerbate collateral tissue damage. The ß-glucan receptor ephrin type-A 2 receptor (EphA2) is required to initiate mucosal inflammatory responses during oral Candida infection. Here we report that EphA2 promotes renal immunopathology during disseminated candidiasis. EphA2 deficiency leads to reduced renal inflammation and injury. Comprehensive analyses reveal that EphA2 restrains IL-23 secretion from and migration of dendritic cells. IL-23 signaling prevents ferroptotic host cell death during infection to limit inflammation and immunopathology. Further, host cell ferroptosis limits antifungal effector functions via releasing the lipid peroxidation product 4-hydroxynonenal to induce various forms of cell death. Thus, we identify ferroptotic cell death as a critical pathway of Candida-mediated renal immunopathology that opens a new avenue to tackle Candida infection and inflammation.

Passive sampling to scale wastewater surveillance of infectious disease: Lessons learned from COVID-19.

Much of what is known and theorized concerning passive sampling techniques has been developed considering chemical analytes. Yet, historically, biological analytes, such as Salmonella typhi, have been collected from wastewater via passive sampling with Moore swabs. In response to the COVID-19 pandemic, passive sampling is re-emerging as a promising technique to monitor SARS-CoV-2 RNA in wastewater. Method comparisons and disease surveillance using composite, grab, and passive sampling for SARS-CoV-2 RNA detection have found passive sampling with a variety of materials routinely produced qualitative results superior to grab samples and useful for sub-sewershed surveillance of COVID-19. Among individual studies, SARS-CoV-2 RNA concentrations derived from passive samplers demonstrated heterogeneous correlation with concentrations from paired composite samples ranging from weak (R2 = 0.27, 0.31) to moderate (R2 = 0.59) to strong (R2 = 0.76). Among passive sampler materials, electronegative membranes have shown great promise with linear uptake of SARS-CoV-2 RNA observed for exposure durations of 24 to 48 h and in several cases RNA positivity on par with composite samples. Continuing development of passive sampling methods for the surveillance of infectious diseases via diverse forms of fecal waste should focus on optimizing sampler materials for the efficient uptake and recovery of biological analytes, kit-free extraction, and resource-efficient testing methods capable of rapidly producing qualitative or quantitative data. With such refinements passive sampling could prove to be a fundamental tool for scaling wastewater surveillance of infectious disease, especially among the 1.8 billion persons living in low-resource settings served by non-traditional wastewater collection infrastructure.