Fungal pathogens are a growing threat to public health. As human immunodeficiency becomes increasingly common, fungal infections are becoming more prevalent. The use of antifungal agents for prophylaxis and treatment of fungal infections has favored the emergence of previously rare or unidentified species of drug-resistant fungal pathogens, including several Candida and Cryptococcus species, as well as mold pathogens. As these new and increasingly drug-resistant fungal pathogens continue to emerge, novel strategies for rapid identification and treatment are necessary to combat these life-threatening infections.

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Fungal infections are a global concern and the evolution of intrinsic resistance to current antifungals presents an alarming problem. For Cryptococcus neoformans, a human fungal pathogen of primarily immunocompromised individuals, resistance toward treatment strategies demands alternative approaches. Given the prevalence of virulence factor production during cryptococcal infection, an emerging and important field of research encompasses the development of novel antivirulence therapies proposed to improve host immune responses and promote fungal clearance. To accomplish this task, information regarding the presence and role of virulence factors, the mechanisms of action within the host, and the ability to influence fungal susceptibility to antifungals is pertinent. Research into mechanisms of antifungal resistance for C. neoformans is limited but extrapolation from successful studies in other fungal species can improve our understanding of mechanisms employed by C. neoformans and suggest targeted strategies to enhance our ability to combat the pathogen. In this Review, we highlight antifungal therapy options against Cryptococcus, explore current knowledge of underlying mechanisms promoting resistance, and present new opportunities for novel and effective strategies to overcome fungal infections and reduce, or possibly even reverse, the effects of resistance evolution.

PurposeThe purpose of this study was to evaluate mechanisms controlling secretory IgA (SIgA) production, thereby ensuring maintenance of ocular surface health.MethodsTo determine whether the presence of specific gut commensal species regulates SIgA levels and IgA transcripts in the eye-associated lymphoid tissues (EALT), specific-pathogen-free (SPF) Swiss Webster (SW) mice were treated with antibiotic cocktails, germ-free (GF) SW mice were reconstituted with diverse commensal gut microbiota, or monocolonized with gut-specific commensals. Proteomic profiling and quantitative real-time polymerase chain reaction (qRT-PCR) were used to quantify SIgA and IgA levels. 16S rDNA sequencing was carried out to characterize commensal microbiota.ResultsCommensal presence regulated ocular surface SIgA levels and mRNA IgA transcripts in EALT. Oral antibiotic cocktail intake significantly reduced gut commensal presence, while maintaining ocular surface commensal levels reduced SIgA and IgA transcripts in EALT. Analysis of gut microbial communities revealed that SPF SW mice carried abundant Bacteroides organisms when compared to SPF C57BL6/N mice, with B. acidifaciens being the most prominent species in SPF SW mice. Monocolonization of GF SW mice with B. acidifaciens, a strict gut anaerobe, resulted in significant increase of IgA transcripts in the EALT, implying generation of B-cell memory.ConclusionsThese data illustrated a “gut-eye” axis of immune regulation. Exposure of the host to gut commensal species may serve as a priming signal to generate B-cell repertoires at sites different from the gut, such as EALT, thereby ensuring broad protection.

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Peptidoglycan (PG) is a critical component of the bacterial cell wall and is composed of a repeating β-1,4–linked disaccharide of N-acetylglucosamine and N-acetylmuramic acid appended with a highly conserved stem peptide. In Gram-negative bacteria, PG is assembled in the cytoplasm and exported into the periplasm where it undergoes considerable maturation, modification, or degradation depending on the growth phase or presence of environmental stressors. These modifications serve important functions in diverse processes, including PG turnover, cell elongation/division, and antibiotic resistance. Conventional methods for analyzing PG composition are complex and time-consuming. We present here a streamlined MS-based method that combines differential analysis with statistical 1D annotation approaches to quantitatively compare PGs produced in planktonic- and biofilm-cultured Pseudomonas aeruginosa. We identified a core assembly of PG that is present in high abundance and that does not significantly differ between the two growth states. We also identified an adaptive PG assembly that is present in smaller amounts and fluctuates considerably between growth states in response to physiological changes. Biofilm-derived adaptive PG exhibited significant changes compared with planktonic-derived PG, including amino acid substitutions of the stem peptide and modifications that indicate changes in the activity of amidases, deacetylases, and lytic transglycosylases. The results of this work also provide first evidence of de-N-acetylated muropeptides from P. aeruginosa. The method developed here offers a robust and reproducible workflow for accurately determining PG composition in samples that can be used to assess global PG fluctuations in response to changing growth conditions or external stimuli.

Pseudomonas aeruginosa ‐induced corneal keratitis is a sight‐threatening disease. The rise of antibiotic resistance among P. aeruginosa keratitis isolates makes treatment of this disease challenging, emphasizing the need for alternative therapeutic modalities. By comparing the responses to P. aeruginosa infection between an outbred mouse strain (Swiss Webster, SW) and a susceptible mouse strain (C57BL6/N), we found that the inherent neutrophil‐killing abilities of these strains correlated with their susceptibility to infection. Namely, SW‐derived neutrophils were significantly more efficient at killing P. aeruginosa in vitro than C57BL6/N‐derived neutrophils. To interrogate whether the distinct neutrophil killing capacities were dependent on endogenous or exogenous factors, neutrophil progenitor cell lines were generated. The in vitro differentiated neutrophils from either SW or C57BL6/N progenitors retained the differential killing abilities, illustrating that endogenous factors conferred resistance. Consistently, quantitative LC‐MS/MS analysis revealed strain‐specific and infection‐induced alterations of neutrophil proteomes. Among the distinctly elevated proteins in the SW‐derived proteomes were α‐mannosidases, potentially associated with protection. Inhibition of α‐mannosidases reduced neutrophil bactericidal functions in vitro. Conversely, topical application of α‐mannosidases reduced bacterial biofilms and burden of infected corneas. Cumulatively, these data suggest novel therapeutic approaches to control bacterial biofilm assembly and improve bacterial clearance via enzymatic treatments.