James Muse Davis

SUMMARY: Meningoencephalitis is the most severe form of cryptococcal infection. Pathogenic cryptococcal species enter the body primarily via the respiratory system. The central nervous system (CNS) is by far the most common site of cryptococcal dissemination, despite its reputation as a privileged anatomical space. Results from both in vitro and in vivo experiments have suggested multiple cellular and molecular mechanisms for entering the CNS, but no single one has been proven responsible for this remarkable neurotropism. While experimental approaches to the problem have centered around a uniform conception of the blood-brain barrier, a review of the histopathological forms of CNS disease shows marked variety in the locations and forms of lesions and their relevant anatomical barriers. Based on the microanatomy, it is likely that the pathway from blood into the CNS differs from lesion type to lesion type. In considering the genesis of cryptococcal CNS infection, we will first summarize cryptococcal virulence determinants of relevance to CNS infection and the conceptualization of the blood-brain barrier, its history, and functions. Next, we will summarize modes of cryptococcal entry through the blood-brain barrier and the interplay between fungal virulence determinants and host factors. We will outline the common histological findings of cryptococcal meningoencephalitis and examine the relevant vascular structures, discussing their implications for mechanisms of dissemination in the context of the vasculature, the host cellular and metabolic environment, and cryptococcal virulence factors in different parts of the CNS. Finally, we will discuss the value of different animal and in vitro models of cryptococcal infection and the endothelial glycocalyx, a ubiquitous feature of endothelial surfaces seldom considered in microbial pathogenesis.

Cryptococcus is the leading cause of fungal meningitis in humans and a major source of mortality in people living with HIV. Infection starts in the lungs and results in meningoencephalitis after dissemination via the bloodstream. Alveolar macrophages are the first immune cells to encounter the infection and are usually capable of clearing it. In immunocompromised hosts, Cryptococcus survives and grows inside macrophages, establishing pulmonary and ultimately central nervous system infection. The initial interaction with macrophages is a key event in pathogenesis, and we have demonstrated that macrophage exposure induces an enhanced virulence phenotype in Cryptococcus . This process can be replicated in vitro : yeast cells exposed to macrophages in culture demonstrate enhanced virulence in both zebrafish and mouse models. To understand this induction process, we investigated its effect on the cryptococcal transcriptome to determine what cryptococcal factors are required. Ada2 is a subunit of the transcription modulating Spt-Ada-Gcn5-acetyltransferase complex and is required for cryptococcal virulence. We find that a cryptococcal strain lacking Ada2 is not capable of enhanced virulence after macrophage exposure. We further show that Pdr802, a transcription factor with a significant but poorly understood role in cryptococcal pathogenesis, acts downstream of Ada2 during macrophage exposure, and loss of Pdr802 function is sufficient to block macrophage-enhanced virulence. These findings shed light on the earliest stages of cryptococcal pathogenesis and offer new clues to the functions of Pdr802. IMPORTANCE: Cryptococcus is a fungal pathogen affecting mostly immunocompromised people, especially those with HIV. Infection starts in the lungs but ultimately disseminates to cause meningitis, leading to over 50% mortality in patients with AIDS. Cryptococcal meningitis is difficult to treat, and understanding the early stages of infection will be the first step toward preventing it. Lung macrophages are the first immune cells to encounter Cryptococcus , and while they clear the infection in some cases, in immunocompromised patients, they instead serve as a niche for cryptococcal growth. How can macrophage interaction promote infection? We have shown that in a susceptible host, cryptococcal virulence is increased after initial contact with macrophages. This effect can be replicated in vitro , as cryptococcal cells pre-exposed to cultured macrophages are more virulent in animal models. We have identified two cryptococcal genes that regulate this change in virulence, setting the stage for a better understanding of cryptococcal pathogenesis.

Dissemination from one organ system to another is common to many pathogens and often the key process separating simple illness from fatal infection. The pathogenic Cryptococcus species offer a prime example. Cryptococcal infection is thought to begin in the lungs, as a mild or asymptomatic pneumonia. However, bloodborne dissemination from the lungs to the brain is responsible for the most devastating forms of infection. As with other disseminating infections, the transition likely depends on rare but crucial events, such as the crossing of a tissue barrier. By their nature, these events are difficult to study. Francis et al. (mBio 15:e03078-23, 2024, https://doi.org/10.1128/mbio.03078-23 ) have addressed this difficulty by developing a powerful imaging pipeline to scan through unprecedented volumes of tissue from mice infected with Cryptococcus at multiple stages of infection. Their observations challenge some of our basic assumptions about cryptococcal pathogenesis, including when and how the organism reaches the bloodstream and the central nervous system.

Cryptococcus is a ubiquitous environmental fungus and frequent colonizer of human lungs. Colonization can lead to diverse outcomes, from clearance to long-term colonization to life-threatening meningoencephalitis. Regardless of the outcome, the process starts with an encounter with phagocytes. Using the zebrafish model of this infection, we have noted that cryptococcal cells first spend time inside macrophages before they become capable of pathogenic replication and dissemination. What “licensing” process takes place during this initial encounter, and how are licensed cryptococcal cells different? To address this, we isolated cryptococcal cells after phagocytosis by cultured macrophages and found these macrophage-experienced cells to be markedly more virulent in both zebrafish and mouse models. Despite producing a thick polysaccharide capsule, they were still subject to phagocytosis by macrophages in the zebrafish. Analysis of antigenic cell wall components in these licensed cells demonstrated that components of mannose and chitin are more available for staining than they are in culture-grown cells or cells with capsule production induced in vitro . Cryptococcus is capable of exiting or transferring between macrophages in vitro , raising the likelihood that this fungus alternates between intracellular and extracellular life during growth in the lungs. Our results raise the possibility that intracellular life has its advantages over time, and phagocytosis-induced alteration in mannose and chitin exposure is one way that makes subsequent rounds of phagocytosis more beneficial to the fungus. IMPORTANCE: Cryptococcosis begins in the lungs and can ultimately travel through the bloodstream to cause devastating infection in the central nervous system. In the zebrafish model, small amounts of cryptococcus inoculated into the bloodstream are initially phagocytosed and become far more capable of dissemination after they exit macrophages. Similarly, survival in the mouse lung produces cryptococcal cell types with enhanced dissemination. In this study, we have evaluated how phagocytosis changes the properties of Cryptococcus during pathogenesis. Macrophage-experienced cells (MECs) become “licensed” for enhanced virulence. They out-disseminate culture-grown cells in the fish and out-compete non-MECs in the mouse lung. Analysis of their cell surface demonstrates that MECs have increased availability of cell wall components mannose and chitin substances involved in provoking phagocytosis. These findings suggest how Cryptococcus might tune its cell surface to induce but survive repeated phagocytosis during early pathogenesis in the lung.

Cryptococcal meningitis is a fungal infection of the brain and a major cause of death in people with uncontrolled HIV. Infection begins in the lungs but can enter the bloodstream and disseminate to the brain.

Although we have recognized cryptococcosis as a disease entity for well over 100 years, there are many details about its pathogenesis which remain unknown. A major barrier to better understanding is the very broad range of clinical and pathological forms cryptococcal infections can take. One such form has been historically called the cryptococcal granuloma, or the cryptococcoma. These words have been used to describe essentially any mass lesion associated with infection, due to their presumed similarity to the quintessential granuloma, the tubercle in tuberculosis. Although clear distinctions between tuberculosis and cryptococcal disease have been discovered, cellular and molecular studies still confirm some important parallels between these 2 diseases and what we now call granulomatous inflammation. In this review, we shall sketch out some of the history behind the term “granuloma” as it pertains to cryptococcal disease, explore our current understanding of the biology of granuloma formation, and try to place that understanding in the context of the myriad pathological presentations of this infection. Finally, we shall summarize the role of the granuloma in cryptococcal latency and present opportunities for future investigations.

The zebrafish has become a widely accepted model host for studies of infectious disease, including fungal infections. The species is genetically tractable, and the larvae are transparent and amenable to prolonged in vivo imaging and small molecule screening. The aim of this review is to provide a thorough introduction into the published studies of fungal infection in the zebrafish and the specific ways in which this model has benefited the field. In doing so, we hope to provide potential new zebrafish researchers with a snapshot of the current toolbox and prior results, while illustrating how the model has been used well and where the unfulfilled potential of this model can be found.

Bacterial serine/threonine kinases are increasingly sought after as drug targets for new antibiotics. PknB, an essential kinase in Mycobacteria tuberculosis, is intensely targeted, and many inhibitors are in the developmental pipeline. These inhibitors typically are derived from screens of known kinase inhibitors and most share similar chemical properties as their parent compounds were all designed for optimal pharmacokinetic properties in the human body. Here, we investigate the reactivity and toxicity of a proposed PknB inhibitor, YH-8, which does not follow traditional drug design rules. We found that the compound is highly reactive with thiolating agents and has appreciable toxicity in a zebrafish animal model. Furthermore, we find minimal anti-mycobacterial activity with non-tubercular mycobacteria strains. These data suggest that further investigation is needed into its efficacy and physiochemical properties if it is to be further developed as an effective antibiotic.

Candida auris has recently emerged as the first fungal pathogen to cause a global public health threat. The reason this species is causing hospital-associated outbreaks of invasive candidiasis with high mortality is unknown. In this study, we examine the interaction of C. auris with neutrophils, leukocytes critical for control of invasive fungal infections. We show that human neutrophils do not effectively kill C. auris . Compared to Candida albicans , neutrophils poorly recruited to C. auris and failed to form neutrophil extracellular traps (NETs), which are structures of DNA, histones, and proteins with antimicrobial activity. In mixed cultures, neutrophils preferentially engaged and killed C. albicans over C. auris . Imaging of neutrophils in a zebrafish larval model of invasive candidiasis revealed the recruitment of approximately 50% fewer neutrophils in response to C. auris compared to C. albicans . Upon encounter with C. albicans in the zebrafish hindbrain, neutrophils produced clouds of histones, suggesting the formation of NETs. These structures were not observed in C. auris infection. Evasion of neutrophil attack and innate immunity offers an explanation for the virulence of this pathogen. IMPORTANCE The emerging fungal pathogen Candida auris has produced numerous outbreaks of invasive disease in hospitals worldwide. Why this species causes deadly disease is unknown. Our findings reveal a failure of neutrophils to kill C. auris compared to the most commonly encountered Candida species, C. albicans . While neutrophils produce neutrophil extracellular traps (NETs) upon encounter with C. albicans , these antimicrobial structures are not formed in response to C. auris . Using human neutrophils and a zebrafish model of invasive candidiasis, we show that C. auris poorly recruits neutrophils and evades immune attack. Identification of this impaired innate immune response to C. auris sheds light on the dismal outcomes for patients with invasive disease.

Kingella kingae musculoskeletal infections continue to be under-diagnosed and there remains a paucity of literature on its imaging features. The purpose of this manuscript is to review the imaging, clinical, and laboratory findings of microbiology-proven K. kingae infections.