Fungi which invaded cns

In this review, with a focus on Cryptococcus neoformans , we will provide an overview of the application of intravital imaging in fungal infections in the brain, discuss recent findings and speculate on possible future research directions. Infectious meningitis and encephalitis are a major threat to human health, causing high mortality and morbidity throughout the world 1. Following infections, microbes including viruses, bacteria, fungi, and parasites can disseminate from sites of initial infection to the bloodstream.

The circulating pathogens become arrested in the brain vasculature, followed by transmigration into the brain parenchyma across the blood—brain barrier BBB. The BBB is a structural and functional barrier, which maintains the neural microenvironment by regulating the passage of molecules and cells into the brain 2. To date, three mechanisms have been proposed for pathogens to cross the BBB: transcellular migration, paracellular migration, and the Trojan horse mechanism 1.

Once pathogens have translocated to the brain parenchyma, they proliferate and cause brain inflammation, often with devastating consequences. There are three fundamental questions in the field Figure 1 : 1 How are pathogens arrested in the brain vasculature? Figure 1. Possible mechanisms of arrest, transmigration, and resultant host response. The BBB is formed by brain endothelial cells, which are connected by tight junctions, and astrocyte foot processes that surround the endothelial cells and maintain the integrity of the BBB 2 , 3.

A Fungal cells are trapped by vascular constriction with possible sensing and signaling of both cell types 4 , 5. This is followed by transmigration that could be by a trans- or paracellular mechanism paracellular is shown in this panel. Immune and inflammatory cells are recruited to the vascular or extracellular compartment to generate host defense and inflammation.

B Fungal cells adhere directly to the endothelium with possible sensing and signaling of both cell types 6 — This is followed by transmigration that could be by a trans- or paracellular mechanism transcellular is shown in this panel. C Fungal cells are internalized within a host cell Trojan Horse that makes contact with the endothelium, arrests, and generates sensing and signaling of all three cell types 11 , This is followed by transmigration that could be by a trans- or paracellular mechanism.

Modern advances in technology have provided opportunities to better understand host—pathogen interactions. Among them, imaging of organs in living animals, using high-resolution intravital microscopy IVM , represents a major advance in the field. Using this technique, interactions of pathogens with brain endothelial cells, and their transmigration across the BBB can be directly assessed under flow conditions in real time.

In addition, the dynamic interactions of leukocytes with pathogens and their behavior in the brain vasculature and parenchyma can be evaluated in living animals. This is of particular importance, because the extravascular migration of pathogens and their interactions with immune cells are transient and highly dynamic, and investigation of these processes by direct observation using IVM provides insights that cannot be obtained using other techniques.

Of the approximately fungal species that have been reported to be pathogenic to humans 13 , Cryptococcus neoformans, Candida albicans, Histoplasma capsulatum, Coccidioides immitis, Paracoccidioides brasiliensis, Aspergillus spp. In particular, cryptococcal meningoencephalitis is one of the most common infections of the central nervous system and a leading course of HIV-associated mortality globally 16 , 18 , In recent years, much progress has been made to understand migration of pathogens and immune responses induced by the invading pathogens in the brain using IVM.

This review will discuss recent studies that used IVM to address brain infections by a very limited subset of pathogenic fungi Table 1. Intravital microscopy was first employed by Julius Cohnheim in the nineteenth century to visualize leukocyte trafficking in the tongue and mesentery of a frog In the last decade, significant progress has been made in imaging of live animals due to breakthroughs in microscopy. Wide-field microscopy, multiphoton confocal, spinning disk confocal, and multiphoton resonant scanning confocal microscopy have been used to image fungal infection in the brain.

Each imaging system has its advantages or disadvantages depending on whether speed of image acquisition, depth into the tissue, image resolution, photobleaching and phototoxicity, and price are considerations 28 — There are two major surgical methods to make the brain vasculature visible under fluorescent microscopy, i.

Both techniques have advantages and limitations. During imaging through the thinned-skull cranial window, the brain does not need to be superfused with artificial cerebrospinal fluids because the brain tissue is still covered with the skull. It is well suited for observations over long periods of time. However, the skull thickness affects the image quality and achieving optimal and uniform skull thickness requires a high level of surgical proficiency.

By contrast, in an open-skull window, a portion of the skull and dura is removed, and the cortical surface is directly exposed to microscopy. Thus, a better quality of images is usually achieved compared with a thinned-skull window.

However, it is essential to superfuse the brain with artificial cerebrospinal fluid during the period of observation, and great care must be taken to avoid surgical trauma and hemorrhage To facilitate intravital imaging, the organisms, brain microvasculature, and leukocytes can be labeled with fluorochromes.

For example, we labeled C. Two colors allow comparison of two different virulence characteristics or wild-type and mutant strains. However, the yeast cell loses the fluorescent label if it proliferates. This disadvantage might be overcome by using fungi expressing green or red fluorescent proteins if sufficient fluorescent intensity can be achieved 26 , 34 , To label the microvasculature, rat-anti-mouse PECAM-1 [CD31, a molecule expressed on endothelial cells 36 ] can be injected intravenously 37 , Since the tight junctions of endothelial cells express high PECAM-1, this labeling can be used to study interactions of fungi or leukocytes with endothelial tight junctions Alternatively, the vascular compartment can be illuminated by intravenous injection with fluorochrome-conjugated bovine serum albumin or dextran In addition, transgenic mice that express fluorescent proteins in endothelial cells [for example, Tie-2 green fluorescent protein GFP mice 40 ] can be used.

An expanding number of tools are becoming available to study the interactions of fungi with immune and inflammatory cells. To determine the trafficking of leukocytes in the brain, mice can be injected intravenously with rhodamine 6G, which is a cell-permeant dye that is sequestered by active mitochondria 41 , However, to identify the functions of subsets of leukocytes, mAb or transgenic mice can be used. For example, anti-CD45 can be injected intravenously, which labels all leukocytes.

Neutrophils can be labeled in vivo by intravenous injection of anti-Ly6G To image monocytes, mice can be intravenously injected with fluorochrome-labeled anti-CCR2 labels proinflammatory monocytes or anti-CX3CR1 antibody labels patrolling monocytes With time, many more mouse strains are becoming available that have fluorescent reporters linked to other genes that define different subsets of cells and allow us to study the role of those cells in the pathogenesis of infection.

Cryptococcus neoformans is an encapsulated budding yeast that causes a life-threatening illness in immunocompromised individuals, especially in AIDS patients. It is estimated that there are one million cases of cryptococcosis per year and , of these patients will die within 3 months of diagnosis Cryptococcus is found in the environment and enters the body through the respiratory tract. Immunocompetent individuals are usually able to contain C. In the case of an immunocompromised host, the yeast cells cannot be successfully contained and disseminate into the brain via the bloodstream, causing meningoencephalitis 16 , Hematogenous dissemination of C.

Prior to transmigration into the brain parenchyma, circulating C. We became interested in a number of questions related to the pathogenesis of cryptococcal meningoencephalitis. As arrest of C. We demonstrated that C. When first seen, C. The number of yeast cells passing through postcapillary venules was greatest immediately after injection and gradually decreased over time. However, even after 18 h, rare yeast cells could still be seen moving in the brain venules. Interestingly, the yeast cells were arrested in capillaries that appeared to be of the same or smaller diameter than the organism, often at branch points.

Differences in viability, polysaccharide capsule the major virulence factor , and strain failed to affect the deposition of the yeast cells. In particular, there was no significant difference in the behavior and the arrest of polystyrene microspheres of similar size in the brain capillary bed when compared with C. These results suggest that C.

Cryptococcus neoformans transmigrates into the brain parenchyma across the BBB after arrest in the brain capillaries. Previous studies, using in vitro techniques, have shown that C. It was further demonstrated that transcytosis is mediated by interactions between CD44 expressed on endothelium and cryptococcal hyaluronic acids 7 , 8. A secreted fungal metalloprotease 9 , an extracellular phospholipase B1 10 , and brain inositol 50 are critically involved in transcytosis of C.

In addition, it was also reported that C. However, these studies have failed to determine the dynamics of BBB penetration by C. Using IVM, we have recently characterized the transmigration of C. Following arrest in the brain, C. In contrast to trapping, viability, but not replication, was required for C. Urease is critically involved in brain transmigration of the organism.

Accordingly, a urease inhibitor could ameliorate infection of the mouse brain by reducing transmigration of C. Arrest of C. Recently, we addressed this question with the use of IVM Among all subsets of leukocytes in the circulation, neutrophils are the most abundant phagocytes and are usually the first immune cells to be recruited to a site of infection to eliminate pathogens Early work had suggested that human neutrophils kill C.

In particular, the capability of human neutrophils to kill the organism was reported to be even greater than that of monocytes 52 , In vitro , mouse neutrophils appear to move toward C. Complement C5a—C5aR signaling was essential for phagocytosis of C. These in vitro observations encouraged us to address how neutrophils dynamically interact with C.

With the use of IVM, we demonstrated that neutrophils crawled to the yeast cells that had been arrested in the brain microvasculature. Interestingly, crawling neutrophils recognized and interacted with the yeast, resulting in internalization of C. During the interactions of neutrophils with the yeast, morphologic alterations of neutrophils, including deploying pseudopodia, were observed. Internalization of C. Following ingestion of C. Depletion of neutrophils enhanced brain fungal burden 23 , while enhancing the recruitment of neutrophils improved intravascular clearance of C.

Further studies demonstrated that C. Complement C3 was critically involved in the recognition of C. These results revealed that neutrophils are able to remove C. Given that neutrophils are usually considered to kill microorganisms at the infection site, the finding of the direct removal of C. Recently, a live-imaging model based on zebrafish larvae has been established to study the interactions of C. The zebrafish C. It was shown that zebrafish macrophages rapidly phagocytosed the majority of C.

Depletion of macrophages significantly enhanced the fungal burden in zebrafish, demonstrating that macrophages are essential to protect zebrafish from disease progression 24 , However, macrophages preferentially ingested C.

Amphotericin B is the recommended treatment for those patients sick enough to be hospitalized. Those who are less severely ill may be better treated with itraconazole, another anti-fungal. Mucormycosis is one of the most feared neurological infections. When a fungal infection caused by a group of molds called mucomycetes invades the brain or important blood vessels around the brain, the mortality rate is very high.

The fungi that cause these infections, mucomycetes are actually commonly found in nature and all humans are regularly exposed. Like many fungal infections, almost all human cases of invasion occur when the patient is immunocompromised. A mucormycosis infection of the brain usually starts in the nasal sinuses, where the disease initially mimics sinusitis with a headache, congestion, and fever. The fungus kills invaded tissues quickly and can spread from the sinuses directly into the eyes and brain.

Rarely, the fungus can reach the brain through other routes, such as after being injected into the bloodstream with intravenous drugs.

As soon as the diagnosis of mucormycosis is made, a surgeon is required in order to cut away all dead tissue. This surgery can be disfiguring, as the nasal cartilage, the orbit of the eye, and the palate may all have to be removed.

Early initiation of a strong anti-fungal agent such as amphotericin is also critical. Even with aggressive treatment, survival of such invasive cerebral mucormycosis is rare. Most cases of neurological fungal infections occur in people whose immune systems aren't working properly. While a fungus can attack healthy people, such infections are relatively rare.

That said, these infections can be very serious, or even lethal, and need to be recognized and treated as soon as possible. Sign up for our Health Tip of the Day newsletter, and receive daily tips that will help you live your healthiest life. Neuroinfections caused by fungi. Meningitis caused by Candida dubliniensis in a patient with cirrhosis: A case report and review of the literature. Coccidioidomycosis and the skin: a comprehensive review.

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J Fungi Basel. Mucormycosis in immunochallenged patients. J Emerg Trauma Shock. Coccidioidal meningitis. Clinical Infectious Diseases. Voriconazole versus amphotericin B for primary therapy of invasive aspergillosis.

New England Journal of Medicine. Pathogenesis of mucormycosis. Humoral defense mechanisms in cryptococcosis: substances in normal human serum, saliva, and cerebrospinal fluid affecting the growth of Cryptococcus neoformans. The Journal of Infectious Diseases. Segal BH. Histoplasma capsulatum infections of the central nervous system: A clinical review.

Diagnosis and management of central nervous system histoplasmosis. It is likely that the expression tissue factor contributes to the vascular thrombosis at sites of A. Recently, we compared the response of endothelial cells to A. The hyphae invaded both surfaces of the endothelial cells. Kamai, A. Lossinsky, D. Sheppard, and S. Filler, unpublished data. These results suggest that the pathogenesis of angioinvasion during locally invasive aspergillosis may be different from that of hematogenously disseminated aspergillosis.

The A. Zygomycosis, also known as mucormycosis, is caused by fungi of the class Zygomycetes. This disease is usually initiated by inhalation, and the fungus is believed to penetrate the epithelial cell lining of the nasopharynx or pulmonary alveoli [ 59 ]. Like invasive aspergillosis, zygomycosis is characterized by angioinvasion from the abluminal to the luminal surface of the blood vessels [ 60 ].

The Zygomycetes occasionally cause hematogenously disseminated disease. Despite the prominence of angioinvasion in the pathogenesis of zygomycosis, there has only been one report of the interactions of a Zygomycete with endothelial cells. They found that both live and killed R. The endocytosis of live hyphae caused significant endothelial cell damage. Interestingly, killed hyphae induced a similar extent of endothelial cell damage, suggesting that a factor associated with the fungal cell wall is toxic to these cells.

Damage to endothelial cells results in exposure of vascular smooth muscle cells, which can release large quantities of tissue factor and cause intravascular thrombosis [ 62 ]. Thus, induction of endothelial cell damage during angioinvasion may contribute to thrombosis and tissue infarction, which are characteristic of mucormycosis. Sporotrichosis is caused by S. During hematogenous dissemination, it is likely that the organism penetrates the endothelial cell lining of the vasculature to invade the deep tissues.

Unlike other pathogens, endothelial cell invasion does not result in detectable endothelial cell damage. Binding of S. This situation is similar to that which has been seen with Streptococcus pneumoniae, which binds to the platelet-activating factor receptor. This receptor is up-regulated on the surface of activated endothelial cells [ 66 ].

Pulmonary or disseminated histoplasmosis is initiated by the inhalation of H. After initial infection, the organism can persist in the host for years and reactivate when immunity wanes [ 67 ]. It is possible that H. For example, the organism is known to persist in macrophages in vivo and in vitro [ 68 ]. Whether this organism persists in epithelial cells in vivo is currently unknown. Infection with P. These conidia transform into yeast, which cause both pulmonary and disseminated disease [ 71 ].

The yeast have been visualized within the alveolar walls and macrophages during experimental pulmonary infection in mice [ 72 ]. In vitro investigations of the interactions of P. However, there is one report of the interactions of this organism with a type II pneumocyte cell line [ 74 ]: P.

Adherence is mediated in part by two different P. The endocytosis of this fungus requires intact epithelial cell microfilaments and microtubules, and triggers host cell apoptosis [ 74 ]. The identities of the P.

Most pathogenic fungi invade normally non-phagocytic host cells by inducing their own endocytosis. Although the invasion of these host cells is likely central to pathogenesis of disease, there are large gaps in our knowledge about whether invasion is induced by a membrane-ruffling or zipper mechanism. Furthermore, for those fungi that induce their own endocytosis by a zipper mechanism, there is a paucity of knowledge about the fungal surface proteins that induce invasion, the host cell receptor to which these endocytosing-inducing proteins bind, and the host cell signal transduction mechanisms that govern fungal invasion.

However, the tools to efficiently fill these gaps in knowledge have already been developed within the last decade during the study of host cell invasion by viral, bacterial, and protozoal pathogens. Therefore it is highly likely that mechanisms of fungal invasion will be more clearly elucidated in the near future.

We thank the members of the Filler and Sheppard laboratories for stimulating and insightful discussions about this topic. Abstract Many fungi that cause invasive disease invade host epithelial cells during mucosal and respiratory infection, and subsequently invade endothelial cells during hematogenous infection.

Introduction Pathogenic fungi interact with a variety of host cells during the induction of disease. Download: PPT. Candida Species Epithelial cell invasion. Endothelial cell invasion.

Cryptococcus neoformans Epithelial cell invasion. Aspergillus fumigatus Invasion of epithelial cells. Figure 1. Invasion of endothelial cells. Endothelial cells are polarized. Other Fungi Zygomycetes. Sporothrix schenckii. Histoplasma capsulatum. Paracoccidioides brasiliensis. Summary Most pathogenic fungi invade normally non-phagocytic host cells by inducing their own endocytosis. Acknowledgments We thank the members of the Filler and Sheppard laboratories for stimulating and insightful discussions about this topic.

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