ABSTRACT Extracellular vesicles (EVs) are nano-sized lipid bilayer structures released by diverse cell types. For pathogenic fungi, EVs play critical roles by modulating immune responses and influencing disease progression. Previously, we demonstrated that vaccination with EVs from Candida albicans promoted protection in immunosuppressed mice subsequently infected with lethal doses of this organism. However, the impact of EVs in the presence of fungi is poorly investigated in vivo . Here, we demonstrated that co-injection of C. albicans yeasts and their EVs into Galleria mellonella larvae led to a rapid larval death compared to yeast cells alone. This effect seems to be associated with thermosensitive compounds, such as secreted aspartic proteases (Saps), virulence factors carried by C. albicans EVs. EVs from SAP knockout (KO) strains ( sap1 / 2 / 3 Δ and sap9 Δ) impaired this harmful effect, confirming the crucial role of Saps. Deletion of SAP1–3 prevented the EV-associated decrease in hemocyte density, whereas SAP9 deletion impaired the increase in oxidative response and melanin production. In a murine model of disseminated candidiasis with intraperitoneal infection, the deleterious effect of C. albicans EVs was observed only when co-injected intraperitoneally with yeasts, but not when EVs were administered intramuscularly. Therefore, both fungal cells and EVs are required for the pathogenic effects. Our findings confirm the aggressive role of fungal EVs in the presence of C. albicans and reinforce their contribution to candidiasis, elucidating EV–pathogen interactions. Although these results do not preclude the use of C. albicans EVs in vaccine formulations, they emphasize the need to consider delivery route and presence of virulence factors. IMPORTANCE Fungal infections caused by Candida albicans represent a significant threat, particularly to immunocompromised individuals. Extracellular vesicles (EVs) from fungi have been explored as promising vaccine formulations, with demonstrated protective effects. However, previous studies have also suggested that EVs may contribute to disease progression. Here, we demonstrate that EVs enhance fungal pathogenicity in vivo and identify key factors driving this effect. We show that EV-associated aspartyl proteases (secreted aspartic proteases [Saps]), along with other virulence components, directly contribute to accelerated host mortality when EVs are present alongside fungal cells, and that this outcome depends on both their composition and route of delivery. By demonstrating the involvement of EV-associated Saps in disease progression, this work advances our understanding of host–pathogen interactions and reveals critical considerations for the safe development of EV-based therapeutic and vaccine strategies.
ABSTRACT Pediatric infections with highly resistant Klebsiella pneumoniae (HR-Kp) disproportionately affect low-income countries where childhood malnutrition is prevalent. Malnutrition is an identified risk factor for infection with HR-Kp and other antimicrobial-resistant Enterobacterales, but the mechanistic underpinnings of this link and independence from other common exposures, like antibiotics, in children are unclear. Using a murine model of pediatric malnutrition, we found that mice fed a protein-deficient diet (PD) were more susceptible to colonization with HR-Kp at a 4-log lower challenge inoculum and developed a 3-log higher intestinal burden in comparison to adequately nourished mice. HR-Kp colonization in PD-fed mice persisted beyond 5 weeks, and colonization was transmitted to diet-matched cage mates. Antibiotic treatment resulted in similar HR-Kp colonization burdens regardless of diet, suggesting that nutrition-dependent colonization risk is driven by altered intestinal microbiota. Despite reduced dietary protein intake, intestinal amino acid content was maintained in PD-fed mice, supporting optimal HR-Kp growth. Furthermore, secondary bile acids, prototypic intestinal microbiota-derived molecules, were reduced in PD-fed mice, demonstrated an inverse correlation with HR-Kp burden in vivo , and partially inhibited the growth of HR-Kp in vitro . Collectively, these findings reveal a role for dietary protein intake in determining nutrition-dependent colonization resistance to HR-Kp. IMPORTANCE Antimicrobial-resistant Klebsiella pneumoniae are responsible for a growing mortality burden worldwide, with disproportionate rates of infection in low-income countries where childhood malnutrition is prevalent. Several publications have demonstrated high rates of intestinal colonization with antimicrobial-resistant K. pneumoniae in children admitted with severe malnutrition in sub-Saharan Africa. However, numerous confounding risk factors in malnourished children (antibiotic use, healthcare exposure, and immune dysfunction) make it difficult to assess the direct impact of nutrition on K. pneumoniae colonization. In this study, we utilized a novel mouse model of protein malnutrition to demonstrate that dietary protein deficiency renders mice susceptible to a higher burden of intestinal colonization at a lower inoculating dose of K. pneumoniae . Furthermore, reduced microbiota-derived bile acid metabolites correlate with increased burden of colonization in protein-deficient mice. These results are the first to establish a direct mechanistic link associating dietary protein deficiency, bile acid metabolism, and intestinal colonization with antimicrobial-resistant K. pneumoniae .
ABSTRACT Peromyscus leucopus ( P. leucopus ) is an important reservoir host for several zoonoses, including Borrelia burgdorferi ( B. burgdorferi ), the causative agent of Lyme disease. However, the immunoglobulin germline repertoire underlying antibody responses in this reservoir host remains poorly defined. In this study, we characterized the immunoglobulin variable genes of P. leucopus and identified 12 IGHV, 12 IGKV, and 3 IGLV gene families comprising 129, 87, and 12 genes, respectively. These genes exhibited similar usage patterns to those of Mus musculus ( M. musculus ). We also identified a high-divergence region (HDR) within the germline IGHV loci of three Borrelia sp.-susceptible Cricetidae species, P. leucopus , Peromyscus maniculatus ( P. maniculatus ), and Arvicola amphibius ( A. amphibius ). Compared with the corresponding regions in Muridae species, the Cricetidae HDRs are expanded and contain a large number of IGHV genes. To investigate the antigen-specific features of the repertoire, we analyzed the antibody responses of P. leucopus to B. burgdorferi outer surface protein A (OspA), a highly immunogenic tick midgut adhesin involved in midgut colonization. Comparative analysis of more than 140 IGHV genes from immunized P. leucopus showed that germline IGHV genes encoding anti-OspA antibodies exhibited higher sequence similarity to corresponding germline IGHV genes in P. maniculatus and A. amphibius than did P. leucopus germline IGHV genes not associated with anti-OspA responses. IMPORTANCE This study characterized the immunoglobulin gene repertoire of Peromyscus leucopus and revealed extensive diversity across its variable-region loci. Analysis of antibody response to outer surface protein A (OspA) showed that germline IGHV genes encoding anti-OspA antibodies showed higher sequence similarity to their corresponding germline IGHV genes in other Borrelia -susceptible cricetid rodents, suggesting that conserved germline IGHV features are associated with OspA recognition. These findings lay the foundation for further understanding of antibody responses in this important reservoir host.
ABSTRACT Agrobacterium fabrum infects a wide range of plants, resulting in significant economic losses. In the rhizosphere, the bacterium is exposed to several stressors, including compounds secreted from other bacteria, fungi, or plants. A. fabrum encodes the efflux pump PecM for which the substrate is 4-hydroxybenzaldehyde, a compound that is both present in plant exudates and generated by bacteria during degradation of aromatic compounds. As an aromatic aldehyde, 4-hydroxybenzaldehyde is a chemically reactive electrophile that can react with cellular nucleophiles such as thiol groups on proteins and glutathione, leading to disruption of redox homeostasis. To assess the physiological consequences of pecM disruption, we compared the transcriptome and proteome profiles of wild-type and pecM disruption strains. While 30 genes were seen to be differentially expressed in the pecM disruption strain, 118 proteins differentially accumulated. Accumulating proteins included glutathione S-transferase and glutathione-dependent peroxiredoxin, both of which protect from oxidative stress. Consistent with this observation, the pecM disrupted strain was more resistant to oxidative stress. Across the genes and proteins detected in both data sets, there was no overall linear relationship, as reflected in a Pearson correlation of ~0. A marked depletion of bS21C, which is a component of the small ribosomal subunit, was observed; as one of three paralogs, its differential expression suggests the possibility that ribosome heterogeneity is part of adaptation to stress. Taken together, our data suggest that absence of PecM imposes a 4-hydroxybenzaldehyde-induced oxidative or electrophile stress and that PecM serves as a critical metabolic relief valve to maintain cellular homeostasis. IMPORTANCE Efflux pumps often receive attention in the context of protecting bacteria from environmental toxins such as antibiotics. However, they are also important for exporting metabolic intermediates that may otherwise disrupt cellular processes. Bacterial metabolism of aromatic compounds can generate 4-hydroxybenzaldehyde, which the bacteria may also encounter in the rhizosphere, and its intracellular accumulation would be expected to disrupt redox homeostasis. Here, we show that disruption of the Agrobacterium fabrum efflux pump PecM, which exports 4-hydroxybenzaldehyde, results in widespread proteomic remodeling, with marked accumulation of proteins associated with oxidative and electrophile stress. These findings emphasize the importance of efflux systems in preventing the accumulation of reactive intermediates, and they expand our understanding of how such efflux systems contribute to metabolic balance and global cellular adaptation in complex environmental niches such as the rhizosphere.
