A deep cut into early 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.

Survival in macrophages induces enhanced virulence in Cryptococcus.

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.IMPORTANCECryptococcosis 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.

Roles for Microglia in Cryptococcal Brain Dissemination in the Zebrafish Larva.

Cryptococcal infection begins in the lungs, but yeast cells subsequently access the bloodstream, from which they can reach the central nervous system (CNS). The resulting meningoencephalitis is the most common presentation and is very difficult to treat. How this fungus interacts with the blood-brain barrier (BBB) and establishes growth in the brain parenchyma remains a central question in fungal pathogenesis. We and others have developed the zebrafish larva as a model host for cryptococcosis and demonstrated that hematogenous CNS infection is replicated in this model. Here, we have used this model to examine the details of BBB crossing and the events immediately before and after. We have observed multiple mechanisms of BBB crossing and found that microglia, the resident phagocytes of the brain, likely have multiple roles. First, microglia either actively transfer yeast cells across the BBB or take up a significant proportion of them immediately after crossing. Second, microglia are capable of clearing individual cryptococcal cells at a developmental stage before adaptive immune cells have emerged. Third, microglia serve to maintain endothelial integrity, preventing other, phagocyte-independent forms of crossing. These proposed microglial functions during infection in the zebrafish larva generate new hypotheses concerning the establishment and control of cryptococcal meningoencephalitis. IMPORTANCE 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. A structure called the blood-brain barrier must be crossed for the fungus to enter and cause meningitis. Learning how Cryptococcus crosses the blood-brain barrier will be crucial to understanding and possibly preventing brain infection. Using the zebrafish larva as a model host, we show that microglia, the resident phagocytes of the brain, potentially play multiple previously unappreciated roles in cryptococcal infection of the brain. These roles include reinforcing the integrity of the blood-brain barrier, clearing cryptococcal cells after they have crossed, and possibly participating directly in crossing via a previously unknown mechanism.

The granuloma in cryptococcal disease.

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 as a Model Host for Invasive Fungal Infections.

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.

Emerging Fungal Pathogen Candida auris Evades Neutrophil Attack.

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.

Imaging of Kingella kingae musculoskeletal infections in children: a series of 5 cases.

PURPOSE: 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. MATERIALS AND METHODS: A retrospective review of musculoskeletal infections between January 1, 2013 and Dec 31, 2016 yielded 134 patients from whom 5 patients had confirmed K. kingae infections (3 boys and 2 girls, mean age of 16 months, range 9-38 months). Picture archiving and communication system and electronic medical records were reviewed. RESULTS: At presentation, none of the patients had a fever and not all patients had abnormal inflammatory markers. Three patients had septic arthritis (2 knee and 1 sternomanubrial joints), one had epiphyseal osteomyelitis, and one had lumbar spondylodiscitis. The case of epiphyseal osteomyelitis of the distal humerus also had elbow joint involvement. A combination of radiography (n = 4), ultrasound (n = 2), and magnetic resonance (MR) imaging (n = 5) were performed. Prominent synovial thickening was observed for both knee and elbow joints and extensive regional myositis for all except for the patient with sternomanubrial joint infection. The diagnosis of K. kingae infection resulted in a change in the antibiotic regimen in 80% of the patients. CONCLUSION: Disproportionate synovial thickening, prominent peri-articular myositis, and/or characteristic sites of involvement demonstrating imaging features of infection or inflammation in a young child with mild infectious symptoms and elevated inflammatory markers should invoke the possibility of an underlying K. kingae infection.

A Zebrafish Model of Cryptococcal Infection Reveals Roles for Macrophages, Endothelial Cells, and Neutrophils in the Establishment and Control of Sustained Fungemia.

Cryptococcal meningoencephalitis is a fungal infection that predominantly affects immunocompromised patients and is uniformly fatal if left untreated. Timely diagnosis is difficult, and screening or prophylactic measures have generally not been successful. Thus, we need a better understanding of early, asymptomatic pathogenesis. Inhaled cryptococci must survive the host immune response, escape the lung, and persist within the bloodstream in order to reach and invade the brain. Here we took advantage of the zebrafish larval infection model to assess the process of cryptococcal infection and disease development sequentially in a single host. Using yeast or spores as infecting particles, we discovered that both cell types survived and replicated intracellularly and that both ultimately established a sustained, low-level fungemia. We propose that the establishment and maintenance of this sustained fungemia is an important stage of disease progression that has been difficult to study in other model systems. Our data suggest that sustained fungemia resulted from a pattern of repeated escape from, and reuptake by, macrophages, but endothelial cells were also seen to play a role as a niche for cryptococcal survival. Circulating yeast collected preferentially in the brain vasculature and eventually invaded the central nervous system (CNS). As suggested previously in a mouse model, we show here that neutrophils can play a valuable role in limiting the sustained fungemia, which can lead to meningoencephalitis. This early stage of pathogenesis-a balanced interaction between cryptococcal cells, macrophages, endothelial cells, and neutrophils-could represent a window for timely detection and intervention strategies for cryptococcal meningoencephalitis.

Evaluation of the pathogenesis and treatment of Mycobacterium marinum infection in zebrafish.

Mycobacterium marinum-infected zebrafish are used to study tuberculosis pathogenesis, as well as for antitubercular drug discovery. The small size of zebrafish larvae coupled with their optical transparency allows for rapid analysis of bacterial burdens and host survival in response to genetic and pharmacological manipulations of both mycobacteria and host. Automated fluorescence microscopy and automated plate fluorimetry (APF) are coupled with facile husbandry to facilitate large-scale, repeated analysis of individual infected fish. Both methods allow for in vivo screening of chemical libraries, requiring only 0.1 µmol of drug per fish to assess efficacy; they also permit a more detailed evaluation of the individual stages of tuberculosis pathogenesis. Here we describe a 16-h protocol spanning 22 d, in which zebrafish larvae are infected via the two primary injection sites, the hindbrain ventricle and caudal vein; this is followed by the high-throughput evaluation of pathogenesis and antimicrobial efficacy.

Neutrophils exert protection in the early tuberculous granuloma by oxidative killing of mycobacteria phagocytosed from infected macrophages.

Neutrophils are typically the first responders in host defense against invading pathogens, which they destroy by both oxidative and nonoxidative mechanisms. However, despite a longstanding recognition of neutrophil presence at disease sites in tuberculosis, their role in defense against mycobacteria is unclear. Here we exploit the genetic tractability and optical transparency of zebrafish to monitor neutrophil behavior and its consequences during infection with Mycobacterium marinum, a natural fish pathogen. In contrast to macrophages, neutrophils do not interact with mycobacteria at initial infection sites. Neutrophils are subsequently recruited to the nascent granuloma in response to signals from dying infected macrophages within the granuloma, which they phagocytose. Some neutrophils then rapidly kill the internalized mycobacteria through NADPH oxidase-dependent mechanisms. Our results provide a mechanistic link to the observed patterns of neutrophils in human tuberculous granulomas and the susceptibility of humans with chronic granulomatous disease to mycobacterial infection.

Tuberculous granuloma induction via interaction of a bacterial secreted protein with host epithelium.

Granulomas, organized aggregates of immune cells, are a hallmark of tuberculosis and have traditionally been thought to restrict mycobacterial growth. However, analysis of Mycobacterium marinum in zebrafish has shown that the early granuloma facilitates mycobacterial growth; uninfected macrophages are recruited to the granuloma where they are productively infected by M. marinum. Here, we identified the molecular mechanism by which mycobacteria induce granulomas: The bacterial secreted protein 6-kD early secreted antigenic target (ESAT-6), which has long been implicated in virulence, induced matrix metalloproteinase-9 (MMP9) in epithelial cells neighboring infected macrophages. MMP9 enhanced recruitment of macrophages, which contributed to nascent granuloma maturation and bacterial growth. Disruption of MMP9 function attenuated granuloma formation and bacterial growth. Thus, interception of epithelial MMP9 production could hold promise as a host-targeting tuberculosis therapy.

Leptospira interrogans stably infects zebrafish embryos, altering phagocyte behavior and homing to specific tissues.

Leptospirosis is an extremely widespread zoonotic infection with outcomes ranging from subclinical infection to fatal Weil's syndrome. Despite the global impact of the disease, key aspects of its pathogenesis remain unclear. To examine in detail the earliest steps in the host response to leptospires, we used fluorescently labelled Leptospira interrogans serovar Copenhageni to infect 30 hour post fertilization zebrafish embryos by either the caudal vein or hindbrain ventricle. These embryos have functional innate immunity but have not yet developed an adaptive immune system. Furthermore, they are optically transparent, allowing direct visualization of host-pathogen interactions from the moment of infection. We observed rapid uptake of leptospires by phagocytes, followed by persistent, intracellular infection over the first 48 hours. Phagocytosis of leptospires occasionally resulted in formation of large cellular vesicles consistent with apoptotic bodies. By 24 hours, clusters of infected phagocytes were accumulating lateral to the dorsal artery, presumably in early hematopoietic tissue. Our observations suggest that phagocytosis may be a key defense mechanism in the early stages of leptospirosis, and that phagocytic cells play roles in immunopathogenesis and likely in the dissemination of leptospires to specific target tissues.

Pseudomonas aeruginosa Type III secretion system interacts with phagocytes to modulate systemic infection of zebrafish embryos.

Pseudomonas aeruginosa is an opportunistic human pathogen that can cause serious infection in those with deficient or impaired phagocytes. We have developed the optically transparent and genetically tractable zebrafish embryo as a model for systemic P. aeruginosa infection. Despite lacking adaptive immunity at this developmental stage, zebrafish embryos were highly resistant to P. aeruginosa infection, but as in humans, phagocyte depletion dramatically increased their susceptibility. The virulence of an attenuated P. aeruginosa strain lacking a functional Type III secretion system was restored upon phagocyte depletion, suggesting that this system influences virulence through its effects on phagocytes. Intravital imaging revealed bacterial interactions with multiple blood cell types. Neutrophils and macrophages rapidly phagocytosed and killed P. aeruginosa, suggesting that both cell types play a role in protection against infection. Intravascular aggregation of erythrocytes and other blood cells with resultant circulatory blockage was observed immediately upon infection, which may be relevant to the pathogenesis of thrombotic complications of human P. aeruginosa infections. The real-time visualization capabilities and genetic tractability of the zebrafish infection model should enable elucidation of molecular and cellular details of P. aeruginosa pathogenesis in conditions associated with neutropenia or impaired phagocyte function.

The role of the granuloma in expansion and dissemination of early tuberculous infection.

Granulomas, organized aggregates of immune cells, form in response to persistent stimuli and are hallmarks of tuberculosis. Tuberculous granulomas have long been considered host-protective structures formed to contain infection. However, work in zebrafish infected with Mycobacterium marinum suggests that granulomas contribute to early bacterial growth. Here we use quantitative intravital microscopy to reveal distinct steps of granuloma formation and assess their consequence for infection. Intracellular mycobacteria use the ESX-1/RD1 virulence locus to induce recruitment of new macrophages to, and their rapid movement within, nascent granulomas. This motility enables multiple arriving macrophages to efficiently find and phagocytose infected macrophages undergoing apoptosis, leading to rapid, iterative expansion of infected macrophages and thereby bacterial numbers. The primary granuloma then seeds secondary granulomas via egress of infected macrophages. Our direct observations provide insight into how pathogenic mycobacteria exploit the granuloma during the innate immune phase for local expansion and systemic dissemination.

"The very pulse of the machine": the tuberculous granuloma in motion.

What is happening inside the tuberculous granuloma? In this issue of Immunity, Egen et al. (2008) present live images of tuberculous granulomas of the mouse, demonstrating the influx and incessant wandering of T lymphocytes.

Dichotomous role of the macrophage in early Mycobacterium marinum infection of the zebrafish.

In tuberculosis, infecting mycobacteria are phagocytosed by macrophages, which then migrate into deeper tissue and recruit additional cells to form the granulomas that eventually contain infection. Mycobacteria are exquisitely adapted macrophage pathogens, and observations in the mouse model of tuberculosis have suggested that mycobacterial growth is not inhibited in macrophages until adaptive immunity is induced. Using the optically transparent and genetically tractable zebrafish embryo-Mycobacterium marinum model of tuberculosis, we have directly examined early infection in the presence and absence of macrophages. The absence of macrophages led rapidly to higher bacterial burdens, suggesting that macrophages control infection early and are not an optimal growth niche. However, we show that macrophages play a critical role in tissue dissemination of mycobacteria. We propose that residence within macrophages represents an evolutionary trade-off for pathogenic mycobacteria that slows their early growth but provides a mechanism for tissue dissemination.

Zebrafish and frog models of Mycobacterium marinum infection.

Mycobacterium marinum infection of poikilothermic animals, such as fish and frogs, results in chronic granulomatous diseases that bear many similarities to mycobacterioses in mammals, including tuberculosis. This unit describes three animal models of M. marinum infection that can be used to study basic aspects of Mycobacterium-host interactions and granuloma development, as well as trafficking of immune cells in host tissues. Protocols are included that describe intraperitoneal infection of adult leopard frogs (Rana pipiens) and zebrafish (Danio rerio). Protocols also describe subsequent monitoring of the infection by enumeration of bacterial cfu, mean time to death, or visual examination of infected tissue using both conventional histological stains and fluorescence microscopy of fluorescently marked bacteria. Furthermore, protocols are included that describe the infection of embryonic zebrafish and the subsequent analysis of the infection in real time using DIC and fluorescence microscopy.

Real-time visualization of mycobacterium-macrophage interactions leading to initiation of granuloma formation in zebrafish embryos.

Infection of vertebrate hosts with pathogenic Mycobacteria, the agents of tuberculosis, produces granulomas, highly organized structures containing differentiated macrophages and lymphocytes, that sequester the pathogen. Adult zebrafish are naturally susceptible to tuberculosis caused by Mycobacterium marinum. Here, we exploit the optical transparency of zebrafish embryos to image the events of M. marinum infection in vivo. Despite the fact that the embryos do not yet have lymphocytes, infection leads to the formation of macrophage aggregates with pathological hallmarks of granulomas and activation of previously identified granuloma-specific Mycobacterium genes. Thus, Mycobacterium-macrophage interactions can initiate granuloma formation solely in the context of innate immunity. Strikingly, infection can redirect normal embryonic macrophage migration, even recruiting macrophages seemingly committed to their developmentally dictated tissue sites.

Brachyury expression in tailless Molgulid ascidian embryos.

The T-box transcription factor gene Brachyury is important for the differentiation of notochord in all chordates, including the ascidians Halocynthia roretzi and Ciona intestinalis. We isolated Brachyury from molgulid ascidians, which have evolved tailless larvae multiple times independently, and found the genes appear functional by cDNA sequence analyses. We then compared the expression of Mocu-Bra in tailed Molgula oculata embryos to two tailless species, Molgula occulta (Mocc-Bra) and Molgula tectiformis (Mt-Bra). Here we show that both tailless species express Brachyury in the notochord lineage during embryogenesis. Initial expression of Mocu-Bra is normal in tailed M. oculata embryos; 10 precursor notochord cells divide twice to result in 40 notochord cells that converge and extend to make a notochord down the center of the tail. In contrast, in tailless Molgula occulta, Mocc-Bra expression disappears prematurely, and there is only one round of division, resulting in 20 cells in the final notochord lineage that never converge or extend. In M. occulta x M. oculata hybrid embryos, expression of Mocu-Bra is prolonged, and the embryos form a tail with 20 notochord cells that converge and extend normally. However, in Molgula tectiformis, a different tailless ascidian, Mt-Bra was expressed only in the 10 notochord precursor cells, which never divide, converge, or extend. In summary, neither Brachyury function nor the early establishment of the notochord lineage appears to be impaired in tailless embryos. In light of these results, we are continuing to investigate how and why notochord development is lost in tailless molgulid ascidian embryos.