Previous Article | Next Article 
Infection and Immunity, July 1999, p. 3312-3316, Vol. 67, No. 7
Department of Medical Microbiology and
Immunology, University of Wisconsin Medical School, Madison, Wisconsin
53706,1 and Division of Infectious
Diseases, University of Cincinnati College of Medicine, Cincinnati,
Ohio 452672
Received 19 January 1999/Returned for modification 15 March
1999/Accepted 14 April 1999
The H antigen of the dimorphic fungal pathogen Histoplasma
capsulatum was first described over 40 years ago. It is a
secreted glycoprotein that is immunogenic during infection. Recent
cloning of the H antigen gene (HAG1) indicated sequence
homology with genes for fungal Histoplasma capsulatum is
a dimorphic fungal pathogen that is the causative agent of the most
common fungal respiratory infection in the world. Infection with
H. capsulatum usually presents as a mild, perhaps
unrecognized, respiratory illness but may progress into a more serious
systemic disease in immunocompromised individuals. H. capsulatum grows as a saprophytic mold at 25°C but undergoes a
phase change at physiological temperatures within the mammalian host to
form the pathogenic yeast morphotype. During infection, H. capsulatum persists as a facultative intracellular parasite of
host macrophages by thriving inside the phagolysosomal compartment. Internalized H. capsulatum yeasts are thus primed for
dissemination throughout the mononuclear phagocytic system of the
infected individual (6).
Over 40 years ago, two secreted H. capsulatum antigens, H
and M, were identified as prominent components of histoplasmin, a
mycelial culture filtrate. Although the H antigen was originally observed in histoplasmin, the antigen is also present in the yeast phase. The H precipitin band reacts with sera from patients with histoplasmosis in immunodiffusion assays (9). The vigorous immune response generated against the H antigen is thought to be due in
part to its secretion and perhaps early processing by immune effector
cells. Attempts over the years to purify this immunodominant antigen
from histoplasmin resulted in only a rudimentary understanding of this
glycoprotein (2, 8).
The recent cloning of the gene encoding the H antigen revealed sequence
homology to genes for fungal Fungal strains and media.
H. capsulatum G184AR,
G184A-HTE, G184AS, G184ASura5-11, G186AS, G217B, and
G217Bura5-23 have been described previously (10, 12,
19). Strains Downs (ATCC 38904) and UCLA 531S are clinical isolates of RFLP class I (5, 7). G217B and G222B are
clinical isolates (ATCC 26032 and ATCC 26034, respectively) of RFLP
class II. Parental strains G184A and G186A are clinical isolates (ATCC 26027 and ATCC 26029, respectively) of RFLP class III.
G184ASura5-11 and G217Bura5-23 were isolated
after UV mutagenesis and selection with 5-fluoro-orotic acid (12,
19).
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Histoplasma capsulatum Strain Variation
in Both H Antigen Production and
-Glucosidase Activity and
Overexpression of HAG1 from a Telomeric Linear
Plasmid
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
-glucosidases. To understand the
biological role of this immunodominant antigen in H. capsulatum, enzymatic assays were performed to determine whether
H. capsulatum contained a -glucosidase enzyme activity
and whether this activity was encoded by the HAG1 gene.
Substrate gels with H. capsulatum culture supernatants
revealed -glucosidase activity near the predicted mobility of the H
antigen. Quantitative microtiter plate assays revealed marked
differences in secreted -glucosidase activities from three H. capsulatum restriction fragment length polymorphism (RFLP)
classes, with RFLP class II strains displaying high levels of enzyme
activity, in contrast to the low levels of activity exhibited by class
I and III strains. Immunoblotting of culture supernatants with an H
antigen-specific antiserum demonstrated differences in H protein
expression levels between the H. capsulatum classes, with a
correlation between secreted enzyme activity and H protein levels. We
took advantage of these class differences to demonstrate multicopy
plasmid H gene overexpression by transformation of an HAG1
plasmid into H. capsulatum. Both a class II strain (G217Bura5-23) and a class III strain
(G184ASura5-11) transformed with the telomeric
overexpression plasmid pMAD401 displayed increased levels of
-glucosidase enzyme activity and H protein expression compared to
the levels in control transformants containing only the single genomic
copy of HAG1. This is the first demonstration of telomeric
plasmid-mediated protein overexpression in this pathogenic fungus, and
the findings support the identification of the H antigen as a
-glucosidase.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
-glucosidases, a multifunctional class
of enzymes (4). Additionally, evidence for H. capsulatum -glucosidase activity was found in an earlier study
examining secreted hydrolytic enzyme activities directed against fungal cell walls; however, the fungal protein responsible for this enzyme activity was not identified (3). When H. capsulatum was subdivided into two chemotypes based upon glucan
and chitin concentrations, chemotype I displayed approximately 30%
more -glucosidase activity than chemotype II. Currently, H. capsulatum strains may be classified by restriction fragment
length polymorphism (RFLP) analysis (14). For this study, we
examined H. capsulatum isolates from each of the three major
RFLP classes for differences in secreted -glucosidase activities.
Substrate gels and microtiter plate assays of H. capsulatum supernatants indicated appreciable differences in enzyme activity between class II strains and class I and III strains. Here, we present
evidence that the H antigen is responsible for the secreted -glucosidase activity of H. capsulatum and that H antigen
production varies among strains in correlation with this enzyme
activity. Further, we demonstrate a gene copy number effect on protein
expression in this fungus (for the first time, to our knowledge), since
H antigen production and -glucosidase activity were both increased in two strains transformed with a multicopy plasmid containing the H
antigen gene (HAG1). Additionally, the demonstration here of
telomeric plasmid overexpression of a particular gene will be useful
both for functional gene studies and for complementation of H. capsulatum null mutants in order to fulfill Koch's molecular postulates.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
Bacterial strain. Plasmids were propagated in Escherichia coli HB101 (supE44 hsdS20 recA13 ara-14 proA2 lacY1 galK2 rpsL20 xyl-5 mtl-1).
Preparation of H. capsulatum culture supernatants for
detection and quantification of -glucosidase activity.
H.
capsulatum yeasts from late-log-phase cultures (50 to 100 ml) were
pelleted by centrifugation at 1,200 × g for 10 min at 24°C. Supernatants were concentrated 50 to 100-fold by repeated centrifugations (four to six times) (45 min, 8°C, 1,800 × g) and filtration through Ultrafree-15 filter devices (Millipore,
Bedford, Mass.), which should retain H. capsulatum secreted
proteins larger than 5 kDa. Supernatant protein concentrations were
determined according to the manufacturer's directions with a protein
assay kit from Bio-Rad, Hercules, Calif.
Substrate gels and Western blotting.
Polyacrylamide (8%)
gels containing 0.1% sodium dodecyl sulfate (SDS) were prepared as
described previously (13). H. capsulatum concentrated supernatants and control almond -glucosidase (Sigma, St. Louis, Mo.) were mixed with an equal volume of 2× gel loading buffer with dithiothreitol (13) and boiled for 3 min prior
to being loaded on denaturing gels; dithiothreitol and boiling were omitted for nondenaturing conditions. Following electrophoresis, nondenaturing gels were washed for 30 min at room temperature in enzyme
buffer (20 mM Tris-HCl plus 0.6 mM CaCl2 [pH 8.0]) and then immersed in 100 ml of a 10 mM
p-nitrophenyl--D-glucopyranoside (PNPG)
(Sigma) substrate solution for 1 to 2 h at 37°C. Areas of
substrate hydrolysis indicating -glucosidase enzyme activity were
visualized within the gels as discrete yellow bands. Individual substrate gels were scanned with an Agfa scanner.
Microtiter plate -glucosidase assays.
Various amounts of
H. capsulatum supernatant protein were mixed with enzyme
buffer (described above) to a final volume of 110 µl in Eppendorf
tubes and kept on ice. An equivalent amount of 10 mM PNPG was added to
each reaction tube and mixed by vortexing, and 100 µl of the mixture
was added to each of two duplicate wells. Samples were incubated at
37°C for 1.5 h in covered microtiter plates (Nunc). The
absorbance at 405 nm was monitored with a SpectraMax 250 microplate
spectrophotometer (Molecular Devices, Sunnyvale, Calif.) for the
release of the colorimetric p-nitrophenyl leaving group. The
A405 values were compared with a standard curve
constructed by use of almond -glucosidase.
Construction of a HAG1 gene telomeric plasmid.
Plasmid pH is a pBluescript SK(
) derivative containing the H. capsulatum G217B H antigen gene HAG1 (~3.8 kb), more
than 1 kb of 5' and 3' flanking sequences from the genomic locus, and the ampicillin resistance gene for selection in E. coli
(4). To construct plasmid pMAD401 (Fig.
1), we cloned from plasmid pH a 5-kB
NotI/ClaI fragment containing HAG1 and
upstream and downstream H. capsulatum flanking sequences
into pWU45 (12) digested with NotI and
ClaI. The 6-kb pWU45 fragment contains the Podospora
anserina URA5 gene for selection in H. capsulatum. The
resulting 11-kB plasmid, pMAD400, was next digested with
NotI, dephosphorylated with alkaline phosphatase to prevent
vector religation, and ligated with a 1.9-kb NotI kanamycin
resistance cassette (16) to generate the 12.9-kb plasmid
pMAD401. The kanamycin resistance cassette provides additional
selection in E. coli as well as H. capsulatum
telomeric sequences inserted at either end of the cassette. PacI or PmeI digestion of pMAD401 yields an 11-kb
linear molecule with appropriately oriented telomeric termini that
enable autonomous replication of linear plasmids in H. capsulatum (15). Plasmid pWU45, included as a control
plasmid lacking HAG1, was digested with HpaI to
expose telomeric termini prior to electrotransformation of H. capsulatum.
|
Transformation. H. capsulatum (16) and E. coli (13) were electrotransformed as described previously. Selection for transformation of G184ASura5-11 and G217Bura5-23 with URA5 plasmids was done by plating on HMM agarose without uracil.
DNA preparation and Southern analyses.
Plasmids were
prepared from E. coli by use of an alkaline lysis miniprep
protocol (20) or Maxi columns (Qiagen, Santa Clarita, Calif.) according to the manufacturer's recommendations.
Histoplasma genomic DNA was prepared by first enzymatically
digesting H. capsulatum yeast pellets with Zymolyase/Novozym
plus -glucuronidase as described previously (15).
H. capsulatum spheroplasts were next treated with lysis
buffer (800 mM guanidine HCl, 30 mM EDTA, 30 mM Tris-HCl, 5% Tween 20, 0.5% Triton X-100 [pH 8.0]), RNase A, and proteinase K for 90 min at
50°C. Following centrifugation (10 min, 4°C, 4,000 × g), the cleared supernatants were applied to Qiagen Midi-tips and
genomic DNA was isolated following the manufacturer's yeast DNA
isolation protocols. Methods for DNA electrophoresis and Southern blotting have been described previously (15); radiolabeling of DNA probes was performed by random priming. For detection of transformants carrying telomeric linear plasmids, genomic DNA from
pMAD401 or pWU45 transformants was electrophoresed uncut alongside
appropriately digested transforming vectors.
| |
RESULTS AND DISCUSSION |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
|
|---|
-Glucosidase identification by substrate gel
electrophoresis.
We performed substrate gel electrophoresis to
visualize -glucosidase enzyme activity in H. capsulatum
culture supernatants. Areas of substrate hydrolysis in the gel were
represented by yellow bands, and the G217B supernatant displayed a
single band of -glucosidase activity with a mobility similar to that
of the H antigen (Fig. 2A). The band of
-glucosidase activity was excised and boiled in reducing sample
buffer, and both the eluate and the gel slice were placed in a well of
a new gel, reelectrophoresed, and immunoblotted with a polyclonal H
antigen-specific antiserum (Fig. 2B). Immunoblotting demonstrated the
presence of the H antigen within the excised band of -glucosidase
enzyme activity. The control (almond -glucosidase) did not show
serological cross-reactivity with the H antigen-specific antiserum. We
observed a substantial difference in enzyme levels between an H. capsulatum RFLP class II strain (G217B) and an RFLP class III
strain (G184AS) by this technique, since nearly 10-fold more total
class III strain supernatant protein than class II strain supernatant
protein was required to visualize any substrate hydrolysis within the
gel (data not shown).
|
Quantification and comparison of -glucosidase activities between
H. capsulatum RFLP classes.
To assess potential
differences in enzyme activities across H. capsulatum
strains, we also quantified secreted H. capsulatum -glucosidase activity in a colorimetric microtiter plate assay with
almond -glucosidase as a standard. We observed marked differences in
the levels of secreted -glucosidase activity between H. capsulatum RFLP class II (high levels) and H. capsulatum RFLP classes I and III (low levels) (Fig.
3A). At least 10-fold less supernatant protein from class II strains was needed to show activity equivalent to
that of supernatant protein from class I or III strains. Within RFLP
class II, strain G217B displayed high levels of enzyme activity, while
strain G222B exhibited intermediate levels of enzyme activity (although
at least 10-fold higher than those of class I and III strains).
|
-glucosidase activity and
H protein levels in each H. capsulatum strain examined.
Total supernatant protein (100 µg) from the RFLP class I strain Downs displayed a faintly immunoreactive H antigen band, while no H antigen
band was detected when an equivalent amount of supernatant protein from
UCLA 531S was used. Additional immunoblots of even more heavily loaded
gels confirmed the production of a scant amount of H antigen by UCLA
531S (data not shown). The RFLP class III strains G184AS and G186AS
displayed faint H antigen bands with 10 µg of supernatant and
prominent H bands with 100 µg of supernatant. In contrast, 1 µg of
supernatant from class II strains G217B and G222B exhibited strong H
antigen bands. Strain G184AR (from which the variant G184AS was
derived) and strain G184-HTE (a variant selected by growth in hamster
tracheal epithelial cells) were also examined by -glucosidase plate
assays and H antigen immunoblotting. Each strain displayed enzyme and H
antigen levels similar to those of G184AS (data not shown), although
these strains differ in cell wall 
-1,3-glucan expression, colony
morphology, broth growth characteristics, and virulence (5,
10).
By using Western immunoblotting, we found differences in
electrophoretic migration and thus the apparent size of the H antigen in different strains. For example, the G184AS protein showed a smaller
apparent molecular weight than the G217B H antigen. Also, there was
some discordance between Western immunoblotting and enzyme assay
results for some strains. For instance, Downs and UCLA 531S showed
nearly identical enzyme activities, but Downs expressed more H antigen
than UCLA 531S, as detected with the H antigen-specific antiserum. It
is possible that antigenic differences in the proteins from different
strains cause differences in relative levels of detection by Western
immunoblotting with this antiserum, which was raised against a
recombinant form of the G217B protein. Alternatively, we cannot exclude
the possibility that another -glucosidase contributes to enzyme
activity in some strains but is not detectable with this antiserum.
We tested the recombinant H antigen used to generate the antiserum for
-glucosidase and found no significant activity in substrate gels or
the microtiter plate assay (data not shown). The native H. capsulatum H antigen is a secreted glycoprotein, with 10 predicted
N-glycosylation sites. The recombinant antigen was purified from
inclusion bodies in E. coli, requiring denaturation and
renaturation steps. Its lack of enzymatic activity could be due to
incorrect folding, lack of glycosylation, or other inappropriate posttranslational modifications in a prokaryote or could be an effect
of the purification procedures.
Telomeric plasmid overexpression of the HAG1 gene.
To provide further evidence for the association of -glucosidase
enzyme activity with the H antigen and to examine whether low-expression strains such as G184AS are unable to express higher levels, we used recently developed tools for the molecular genetic manipulation of H. capsulatum (16). Linear
plasmids containing terminal telomeric sequences introduced by
transformation replicate as multiple copies episomally (15).
Plasmid pMAD401 (Fig. 1) contains the full G217B HAG1 gene
sequence as well as appropriate selectable markers and telomeric
sequences. This plasmid was transformed into two uracil-auxotrophic
strains of H. capsulatum, G184ASura5-11 and
G217Bura5-23, which display widely disparate levels of
enzyme activity and H antigen production. Plasmid pWU45, lacking the HAG1 gene sequence, was used as a negative control.
Following transformant isolation, genomic DNA was prepared, and the
presence of monomeric, linear plasmids was confirmed by Southern
analysis (data not shown). Concentrated culture supernatants from
transformants and parental H. capsulatum strains were
examined by both quantitative -glucosidase microtiter plate assays
and H antigen immunoblotting. Plasmid pMAD401 transformants of both
uracil-auxotrophic strains of H. capsulatum, regardless of
native levels of the H antigen, could effectively produce more
-glucosidase enzyme and H antigen than control pWU45 transformants,
which exhibited enzyme and antigen levels consistent with those of the
parental strains (Fig. 4). These results
are consistent with a gene copy number effect, with protein
overexpression resulting from supply of the HAG1 gene on a
telomeric linear plasmid. Furthermore, we observed this phenomenon regardless of the native level of expression of the transformation recipient strain. These data indicate the absence of an inherent defect
in expression by G184AS as well as a lack of negative feedback regulation for this gene, at least involving the G217B sequence supplied on the transforming plasmid.
|
-glucosidases have been speculated to function in nutrient
acquisition (by the metabolism of cellulose to acquire glucose)
(17) or in cell wall remodeling events (by the breakdown of
cell wall polymers and carbohydrates) (11). Relevant to the former model, Borok has speculated that the brown mycelial phenotype may be associated with growth on complex carbohydrate media requiring microbial substrate hydrolysis to obtain simple sugars, while the
albino mycelial phenotype is favored when glucose is provided (1). The heavily sporulating brown phenotype and the more
vegetative albino phenotype apply to mycelial cultures and not directly
to the yeast morphotype that we have used, but it should be noted that
both of our high-expression RFLP class II strains (G217B and G222B) are
derived from brown mycelial isolates. With regard to the cell wall
remodeling hypothesis, Kruse and Cole have demonstrated blocking of
arthroconidium-to-spherule-phase transition in the fungal pathogen
Coccidioides immitis by inhibition of -glucosidase activity, suggesting a role for this enzyme in the hydrolysis of fungal
cell walls (11). Definitive assignment of a biological role
for the H antigen and determination of any effect on virulence will
require targeted gene disruption and subsequent complementation to
fulfill Koch's molecular postulates. We are currently attempting HAG1 inactivation by allelic replacement. The telomeric
linear plasmid pMAD401 should be useful for resupplying the gene to a disruptant. Moreover, we have provided here the first demonstration of
protein overexpression based on gene copy number in H. capsulatum. Targeted hag1 mutants from different
H. capsulatum strains may help explain the large differences
in secreted -glucosidase enzyme activities across H. capsulatum RFLP classes, the reasons for which are not immediately
apparent. Although we have shown increased expression of the G217B
HAG1 gene supplied by transformation in different strains,
it remains to be determined whether differential expression of the
native gene of a strain may explain the RFLP class differences that we
observed. Alternatively, there may be a difference at the protein
level, such as secretion, that is obscured by multicopy supply of the
HAG1 gene.
| |
ACKNOWLEDGMENTS |
|---|
This work was supported by Public Health Service grant RO1 HL55949 from the National Heart, Lung, and Blood Institute.
We thank Erik Munson for assistance with the rabbit antiserum.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Department of Medical Microbiology and Immunology, 420 SMI, University of Wisconsin Medical School, 1300 University Ave., Madison, WI 53706-1532. Phone: (608) 265-6292. Fax: (608) 265-6132. E-mail: jpwoods{at}facstaff.wisc.edu.
Editor: T. R. Kozel
| |
REFERENCES |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
|
|---|
| 1. | Borok, R. 1980. The mycelial status and reversibility in Histoplasma capsulatum. Sabouraudia 18:249-253[Medline]. |
| 2. |
Bradley, G.,
L. Pine,
M. W. Reeves, and C. W. Moss.
1974.
Purification, composition, and serological characterization of histoplasmin H and M antigens.
Infect. Immun.
9:870-880 |
| 3. |
Davis, T. E.,
J. E. Domer, and Y.-T. Li.
1977.
Cell wall studies of Histoplasma capsulatum and Blastomyces dermatitidis using autologous and heterologous enzymes.
Infect. Immun.
15:978-987 |
| 4. | Deepe, G. S., Jr., and G. G. Durose. 1995. Immunobiological activity of recombinant H antigen from Histoplasma capsulatum. Infect. Immun. 63:3151-3157[Abstract]. |
| 5. |
Eissenberg, L. G.,
J. L. West,
J. P. Woods, and W. E. Goldman.
1991.
Infection of P388D1 macrophages and respiratory epithelial cells by Histoplasma capsulatum: selection of avirulent variants and their potential role in persistent histoplasmosis.
Infect. Immun.
59:1639-1646 |
| 6. |
Eissenberg, L. G., and W. E. Goldman.
1991.
Histoplasma variation and adaptive strategies for parasitism: new perspectives on histoplasmosis.
Clin. Microbiol. Rev.
4:411-421 |
| 7. |
Gass, M., and G. S. Kobayashi.
1969.
Histoplasmosis. An illustrative case with unusual vaginal and joint involvement.
Arch. Dermatol.
100:724-727 |
| 8. | Graybill, J., M. Patino, and J. Ahrens. 1988. In situ localization of antigens of Histoplasma capsulatum using colloidal gold immune electron microscopy. Mycopathologia 104:181-188[Medline]. |
| 9. |
Heiner, D. C.
1958.
Diagnosis of histoplasmosis using precipitin reactions in agar gel.
Pediatrics
22:616-627 |
| 10. |
Klimpel, K. R., and W. E. Goldman.
1987.
Isolation and characterization of spontaneous avirulent variants of Histoplasma capsulatum.
Infect. Immun.
55:528-533 |
| 11. |
Kruse, D., and G. T. Cole.
1992.
A seroreactive 120-kilodalton -1,3-glucanase of Coccidiodes immitis which may participate in spherule morphogenesis.
Infect. Immun.
60:4350-4363 |
| 12. |
Retallack, D.,
E. L. Heinecke,
R. Gibbons,
G. S. Deepe, Jr., and J. P. Woods.
1999.
The URA5 gene is necessary for Histoplasma capsulatum growth during infection of mouse and human cells.
Infect. Immun.
67:624-629 |
| 13. | Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. |
| 14. |
Vincent, R. D.,
R. Goewert,
W. E. Goldman,
G. S. Kobayashi,
A. M. Lambowitz, and G. Medoff.
1986.
Classification of Histoplasma capsulatum isolates by restriction fragment polymorphisms.
J. Bacteriol.
165:813-818 |
| 15. | Woods, J. P., and W. E. Goldman. 1992. In vivo generation of linear plasmids with addition of telomeric sequences by Histoplasma capsulatum. Mol. Microbiol. 6:3603-3610[Medline]. |
| 16. |
Woods, J. P.,
E. L. Heinecke, and W. E. Goldman.
1998.
Electrotransformation and expression of bacterial genes encoding hygromycin phosphotransferase and -galactosidase in the pathogenic fungus Histoplasma capsulatum.
Infect. Immun.
66:1697-1707 |
| 17. |
Woodward, J., and A. Wiseman.
1982.
Fungal and other -D-glucosidases their properties and applications.
Enzyme Microb. Technol.
4:73-79.
|
| 18. | Worsham, P. L., and W. E. Goldman. 1988. Quantitative plating of Histoplasma capsulatum without addition of conditioned medium or siderophores. J. Med. Vet. Mycol. 26:137-143[Medline]. |
| 19. | Worsham, P. L., and W. E. Goldman. 1988. Selection and characterization of ura5 mutants of Histoplasma capsulatum. Mol. Gen. Genet. 214:348-352[Medline]. |
| 20. | Zhou, C., Y. Yang, and A. Y. Jong. 1990. Mini-prep in ten minutes. BioTechniques 8:172-173[Medline]. |
Infection and Immunity, July 1999, p. 3312-3316, Vol. 67, No. 7
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Bohse, M. L., Woods, J. P.
(2005). Surface Localization of the Yps3p Protein of Histoplasma capsulatum. Eukaryot Cell
4: 685-693
[Abstract] [Full Text] -
Faure, D.
(2002). The Family-3 Glycoside Hydrolases: from Housekeeping Functions to Host-Microbe Interactions. Appl. Environ. Microbiol.
68: 1485-1490
[Full Text] -
Deepe, G. S. Jr., Gibbons, R.
(2001). Protective Efficacy of H Antigen from Histoplasma capsulatum in a Murine Model of Pulmonary Histoplasmosis. Infect. Immun.
69: 3128-3134
[Abstract] [Full Text]
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Copyright © 2009 by the American Society for Microbiology. For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»