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Microbes and Infection 10 (2008) 973e977

Original article

Histoplasma capsulatum at the hostepathogen interface

Joshua D. Nosanchuk a,b,*, Attila Gacser c
Department of Medicine (Infectious Diseases), Albert Einstein College of Medicine, Bronx, NY 10461, USA
Department of Microbiology/Immunology, Albert Einstein College of Medicine, Bronx, NY 10461, USA
Department of Microbiology, University of Szeged, Szeged, Közép fasor 52, H-6726, Hungary

Available online 10 July 2008


Histoplasma capsulatum is the most common cause of invasive fungal pulmonary disease worldwide. The interaction of H. capsulatum with
a host is a complex, dynamic process. Severe disease most commonly occurs in individuals with compromised immunity, and the increasing
utilization of immunomodulators in medicine has revealed significant risks for reactivation disease in patients with latent histoplasmosis.
Fortunately, there are well developed molecular tools and excellent animal models for studying H. capsulatum virulence and numerous recent
advances have been made regarding the pathogenesis of this fungus that will improve our capacity to combat disease.
Ó 2008 Elsevier Masson SAS. All rights reserved.

Keywords: Histoplasma capsulatum; Pathogenesis; Genetics; Vaccine; Antibody

1. Introduction Although the majority of symptomatic infections follow

primary exposures to H. capsulatum, reactivation of latent
Fungal pathogens continue to gain clinical importance infection can result in significant disease, particularly in the
largely due to the increasing number of immunocompromised setting of immunosuppression such as with individuals
individuals worldwide. The dimorphic fungus Histoplasma chronically receiving steroids or patients on chemotherapy [3].
capsulatum var. capsulatum is a model pathogen for the study Individuals with advanced HIV disease are also at significant
of invasive mycotic disease. H. capsulatum is primarily risk for severe infection due to reactivation of latent lesions or
acquired via aerosol exposure with the inhalation of micro- primary disease, and disseminated disease occurs in 95% of
conidia or hyphal fragments. It has been estimated that H. individuals with AIDS [4]. Also, initiation of HAART in
capasulatum is responsible for w500,000 infections in the patients with prior infection with H. capsulatum can result in
USA each year, making it the most prevalent pulmonary an immune reconstitution inflammatory syndrome [5].
fungal pathogen [1]. H. capsulatum produces a broad spectrum Furthermore, reactivation disease can develop in liver trans-
of disease ranging from a mild influenza-like illness to plant recipients with disease originating from latent infections
a disseminated form that may involve virtually any tissue. The in the transplanted organs [6]. Additionally, reactivation
fungus is endemic worldwide, but there are regions with histoplasmosis has increasingly occurred in patients receiving
notably high incidences of infection [2], such as areas along anti-cytokine therapies, especially inhibitors of TNF-
the Ohio and Mississippi River Valleys in the USA and in Rio a (reviewed in [7]). Hence, disease severity and the manifes-
de Janeiro State in southeastern Brazil. tations of histoplasmosis are significantly impacted by the
competence of the host immune response.

2. The hostefungal interface

* Corresponding author. Albert Einstein College of Medicine, 1300 Morris
Park Avenue, Bronx, NY 10461, USA. Tel.: þ1 718 430 3766; fax: þ1 718
430 8701. The outcome of infection with H. capsulatum is dependent
E-mail address: (J.D. Nosanchuk). on dynamic interactions between innate immunity, adaptive

1286-4579/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
974 J.D. Nosanchuk, A. Gacser / Microbes and Infection 10 (2008) 973e977

immunity and fungal virulence factors [8]. Control of H. cap- Table 1

sulatum infection is largely based on activation of cellular Molecular toolbox by date developed for use in H. capsulatum
immunity in concert with innate responses, as progressive Selectable markers
disease with dissemination predominantly occurs in the absence 1988 URA5 [39]
1998 Hygromycin [42]
of intact cellular immunity [9]. Interestingly, there is recent Transformation methods
experimental evidence demonstrating that susceptibility to H. 1990 PEG-LiAc [57]
capsulatum strongly depends on genetic predisposition [10]. 1998 Electroporation [42]
T cells and phagocytes are essential to host resistance 2002 Agrobacterium tumerfaciens [43]
against H. capsulatum [9,11]. Protective immunity is charac- Episomal plasmids
1993 Telomeric plasmid shuttle vector pWU44 [40]
terized by the induction of cytokine production by T cells, Reporter genes
particularly IFN-g and TNF-a, which subsequently activate 1998 lacZ [42]
phagocytic cells. The primary effector cells in host resistance 2000 gfp [58]
to H. capsulatum are macrophages. However, the role of 2006 CRP1 [59]
macrophages in histoplasmosis is complex, since these cells Gene disruption
2000 CBP1 [29]
also provide a protective environment for H. capsulatum as the Microarray
fungus survives and replicates in the phagolysosomes of 2003 Partial genomic microarray [60]
macrophages. In contrast, dendritic cells can kill ingested H. RNA interference
capsulatum yeast cells [12] and dendritic cells presenting H. 2004 AGS1 [30]
capsulatum antigens can stimulate specific CD8þ T cells to 2006 AMY1 [31]
2007 YPS3 [28]
effectively control fungal infection [13]. Single chain variable
TNF-a production is induced rapidly after primary infec- antibody fragments (scFv)
tion and neutralization of TNF-a increases the fungal burden 2007 Non-immune library of scFv of
and mortality of mice infected with H. capsulatum [14,15]. immunoglobulin human genes displayed on
Inhibition of TNF-a results in the generation of antigen the surface of M13 filamentous phages [61]
specific CD4þCD25þ T cells that interfere with effective
immunity in mice [16]. The experimental findings correlate
with the clinical findings that inhibitors of TNF-a greatly studied is the heat shock protein 60 that serves as the ligand
increase the risk for reactivation of latent histoplasmosis for H. capsulatum binding to CR3 on macrophage, which
resulting in severe diseases [7]. initiates the fungus’ intracellular parasitism of these cells [25].
Mice deficient in IFN-g have accelerated mortality [17]. H. capsulatum heat shock proteins are upregulated during the
Similarly, patients with defects in IFN-g signaling are at risk myceliaeyeast transition and are broadly involved in chaper-
for severe histoplasmosis [18]. Adjuvant therapy with IFN-g oning of proteins. The M and H antigens of H. capsulatum
can improve the outcome of murine histoplasmosis [19] and have long been utilized as serological markers of histoplas-
has been used successfully in a child with a defect in his IFN-g mosis [20]. The M antigen, also known as Catalase B, is
receptor [18]. a constitutively expressed protein posited to play a role in
H. capsulatum induces the production of antibodies, which counteracting the oxidative defense reaction mechanism of
historically has provided a means for non-culture based host phagocytic cells [26]. The H antigen is a secreted beta-
methods of diagnosis [20]. Notably, antibody has been shown glucosidases purportedly involved in remodeling of the cell
to affect H. capsulatum pathogenesis in an animal model [21]. wall and nutrient acquisition [27]. Initially identified in
Supporting a role of antibody in histoplasmosis, mice lacking a differential hybridization screen, yeast phase specific protein
B cells have accelerated mortality after experimental reac- 3 (YPS3) is a cell surface and secreted protein of uncertain
tivation histoplasmosis [22]. function that has been associated with virulence since
silencing of the YPS3 gene significantly attenuates virulence
3. H. capsulatum and our molecular toolbox (Table 1) in vitro and during murine infection [28]. H. capsulatum
secretes a calcium binding protein (CBP) during yeast-phase
H. capsulatum is mycelial in the environment, whereas the growth that is essential for growth in calcium limiting condi-
organism exists as a yeast-like, unicellular fungus that repro- tions, such as encountered in vivo, and required for virulence
duces by budding at human physiological temperatures. The during murine infection [29]. Although certain strains of H.
mechanisms controlling the switch from the mycelial to the capsulatum have lost alpha-(1,3)-glucans from their cell
yeast form of H. capsulatum are complex, but are largely surface during microevolution events and have maintained
dependent on the shift in temperature and availability of virulence, strains that display alpha-(1,3)-glucans on their cell
nutrients (reviewed in [23]). The fungus is a prototypical surface are severely attenuated if the production of this glucan
intracellular pathogen that survives within phagolysomes by is disrupted. Also, deletion or silencing of alpha-(1,3)-glucan
regulating the intracellular milieu of macrophages (reviewed synthase [30] or alpha-(1,4)-amylase [31] interferes with
in [24]). alpha-(1,3)-glucans and the resulting mutants have signifi-
In addition to dimorphism, several virulence determinants cantly reduced virulence. Interestingly, alpha-(1,3)-glucans
of H. capsulatum have been characterized. Perhaps the best inhibit the recognition of H. capsulatum from host effector
J.D. Nosanchuk, A. Gacser / Microbes and Infection 10 (2008) 973e977 975

cells by blocking the beta-glucan receptor dectin-1 [32]. H. numerous eukaryotes [30]. In addition to introducing foreign
capsulatum produces melanin in its cell wall [33] that protects DNA by electroporation and biolistic procedures, an Agro-
the fungus from antifungal drugs [34] and the pigment is also bacterium tumefaciens mediated transfection method has
thought to inhibit damage from host defenses, including host proven to be the most efficient method for DNA trans-
derived free radicals and microbicidal peptides [35]. formation in H. capsulatum [43]. In particular, the A. tume-
Several studies have described genetic and/or genomic faciens T-DNA technique readily allows for forward genetic
heterogeneity among H. capsulatum isolates based on screens and can be a powerful approach to identify virulence
restriction fragment length polymorphisms (RFLP), arbitrary- associated genes.
primer PCR analysis, ribosomal DNA sequencing and other
gene sequencing comparisons (reviewed in [36]). Molecular 4. Potential for a vaccine
characterization studies have identified seven genetically
distinguishable phylogenetic species that diverged as much as There is a general consensus among medical mycologist
13 million years ago. For example, there are two discrete that there is sufficient disease due to H. capsulatum to merit
genetic lineages in North America, Histoplasma class I (NAm the development of a vaccine. The knowledge that immuni-
I) and Histoplasma class II (NAm II), that also differ signifi- zation of mice with sublethal inocula of H. capsulatum induces
cantly in virulence [37]. Currently, the sequencing of the protective immunity to subsequent lethal challenge suggests
genome of H. capsulatum is underway at Washington that an effective human vaccine can be achieved. Since the
University for NAm II ( 1970s we have known that immunization with H. capsulatum
cgi?GENOME¼Histoplasma%20capsulatum) and at the ribosomes confers protective responses [44,45]. However,
Broad Institute for NAm I ( major advances in vaccinology for histoplasmosis came with
annotation/genome/histoplasma_capsulatum/Home.html). The the finding that recombinant heat shock protein 60 from H.
sequence data may clarify the genomic basis for the difference capsulatum induces vigorous protective immune responses
in virulence and interactions with the host immune system, that primarily depend on Vb 8.1/8.2þ CD4þ T cells [46,47].
and serves as a rich resource for molecular work on this Interestingly, immunization with a second heat shock protein
pathogen. of 70 kDa does not result in protective cellular responses [48].
The development of molecular genetic tools in H. capsu- More recently, it has been shown that CD8þ T cells can confer
latum is essential for elucidating important questions, such as protection following immunization with heat shock protein 60
the mechanisms for mycelia to yeast phase transition, survival in the absence of CD4þ cells, and that CD8þ T cells can be
in macrophages, and regulation of virulence associated factors. efficiently stimulated by dendritic cells [13]. The findings are
Although, H. capsulatum is a perfect fungus with a character- significant since they indicate that it may be possible to induce
ized sexual cycle, laboratory strains cultured in vitro rapidly protective responses in individuals with altered immunity,
lose the ability to mate [38], significantly complicating the including individuals with advanced HIV infection.
application of classic recombination studies. Also, the sexual Although heat shock protein 60 has been the major focus of
mould form requires BSLIII conditions. The first H. capsu- vaccine development for H. capsulatum, additional targets
latum mutants were URA5 auxotrophs created using UV have been identified. For example, protection from pulmonary
radiation [39]. Subsequent transformation experiments for infection can also be achieved in mice with immunization with
genetic complementation showed that the bacterial plasmid recombinant H antigen [49]. Immunization with purified cell
constructs containing the Podospora anserina URA5 gene free antigen mixtures from H. capsulatum can protect mice
usually integrated randomly, and often tandem amplifications from systemic infection [50]. Protective immunity is also
or rearrangements were present. Analysis of introduced achievable with recombinant H. capsulatum Sec31 [51].
foreign DNA revealed that H. capsulatum actively modifies Furthermore, efforts are underway to define pan-fungal
transforming plasmids, adding guanosine rich hexanucleotide vaccine targets. A hybrid histidine kinase that is a global
repeats to the ends of the linear DNA fragments [40]. Subse- regulator of mycelia-yeast morphogenesis has been identified
quent developments exploited the autonomous replication in the major dimorphic fungi, including H. capsulatum, and
potential of the presented foreign DNA and telomeric shuttle gene deleted strains may be used for vaccines [52]. A second
vectors were constructed for genetic complementation in potential method for a universal fungal vaccine is the utiliza-
uracil auxotrophs [41]. The first gene targeting experiment tion of beta-glucans [53]. Hence, there are several exciting
deleted the URA5 gene in H. capsulatum [42]. The bacterial avenues for the pursuit of a safe and effective vaccine for
hygromycin resistance gene (hph) was used to delete the target histoplasmosis.
gene, allowing positive selection. However, the transformation
experiments showed a very low (1.4  103) homologous 5. Antibody therapy
recombination frequency. To prevent the high frequency of
illegitimate integration, a two-step gene knock-out strategy We have shown that antibody can modify the pathogenesis
has been develop using telomeric linear plasmids that enable of experimental histoplasmosis [21]. Although the protective
the creation of homologous recombinant mutants [29]. More effects of IgM isotype monoclonal antibodies (mAbs) that
recently, double stranded RNA induced RNA interference has target histone 2B on the fungal cell wall were modest, survival
been successfully used for target gene expression silencing in significantly improved when antibody was used concomitantly
976 J.D. Nosanchuk, A. Gacser / Microbes and Infection 10 (2008) 973e977

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