Agrobacterium-Mediated Insertional Mutagenesis in Histoplasma capsulatum

Zemska, Olga; Rappleye, Chad A.

doi:10.1007/978-1-61779-539-8_4

Cited by 17 publications

(18 citation statements)

References 15 publications

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“…Histoplasma yeasts were mutagenized using Agrobacterium-mediated transformation (47)(48)(49). Agrobacterium tumefaciens strain LBA1100 harboring plasmid pCM41 was cocultured with WU15 Histoplasma yeasts for 48 h and transferred to HMM plus uracil medium containing 100 g/ml hygromycin to select for Histoplasma transformants.…”

Section: Methodsmentioning

confidence: 99%

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Histoplasma capsulatum Depends on De Novo Vitamin Biosynthesis for Intraphagosomal Proliferation

2014

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During infection of the mammalian host, Histoplasma capsulatum yeasts survive and reside within macrophages of the immune system. Whereas some intracellular pathogens escape into the host cytosol, Histoplasma yeasts remain within the macrophage phagosome. This intracellular Histoplasma-containing compartment imposes nutritional challenges for yeast growth and replication. We identified and annotated vitamin synthesis pathways encoded in the Histoplasma genome and confirmed by growth in minimal medium that Histoplasma yeasts can synthesize all essential vitamins with the exception of thiamine. Riboflavin, pantothenate, and biotin auxotrophs of Histoplasma were generated to probe whether these vitamins are available to intracellular yeasts. Disruption of the RIB2 gene (riboflavin biosynthesis) prevented growth and proliferation of yeasts in macrophages and severely attenuated Histoplasma virulence in a murine model of respiratory histoplasmosis. Rib2-deficient yeasts were not cleared from lung tissue but persisted, consistent with functional survival mechanisms but inability to replicate in vivo. In addition, depletion of Pan6 (pantothenate biosynthesis) but not Bio2 function (biotin synthesis) also impaired Histoplasma virulence. These results indicate that the Histoplasma-containing phagosome is limiting for riboflavin and pantothenate and that Histoplasma virulence requires de novo synthesis of these cofactor precursors. Since mammalian hosts do not rely on vitamin synthesis but instead acquire essential vitamins through diet, vitamin synthesis pathways represent druggable targets for therapeutics.

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Section: Methodsmentioning

confidence: 99%

“…The location of the T-DNA insertion was determined by thermal asymmetric interlaced PCR (TAIL-PCR) (47)(48)(49)(50). Two hundred nanograms of genomic DNA was used as the template for the primary PCR, with a T-DNA left border (LB)-or right border (RB)-specific primer (LB11 or RB9) and one of four semirandom primers (LAD1 to -4).…”

Section: Methodsmentioning

confidence: 99%

Histoplasma capsulatum Depends on De Novo Vitamin Biosynthesis for Intraphagosomal Proliferation

2014

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“…A transfer DNA (T-DNA) insertion at the CFP4 locus was generated using Agrobacterium tumefaciensmediated transformation and recovery from mutant pools (21,22). Briefly, A. tumefaciens strain LBA1100 containing plasmid pCM41 (encoding hygromycin resistance) was used to transform WU8 Histoplasma yeasts (23) and hygromycin-resistant transformants recovered by selection on HMM-uracil (100 g/ml)-hygromycin (150 g/ml).…”

Section: Methodsmentioning

confidence: 99%

Glycosylation and Immunoreactivity of the Histoplasma capsulatum Cfp4 Yeast-Phase Exoantigen

Holbrook

Kemski

Richer³

et al. 2014

Infect Immun

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The yeast phase of Histoplasma capsulatum is the virulent form of this thermally dimorphic fungal pathogen. Among the secreted proteome of Histoplasma, culture filtrate protein 4 (Cfp4) is a heavily glycosylated factor produced abundantly and specifically by Histoplasma yeast cells, suggesting its role in pathogenesis. We have generated three monoclonal antibodies as tools for characterization and detection of Cfp4 and determined the epitope each recognizes. Through site-directed mutagenesis of Cfp4, we identified three asparagines that function as the principal sites of N-linked glycan modification. To test the function of Cfp4 in Histoplasma pathogenesis, we generated Cfp4-deficient strains by insertional mutagenesis and by RNA interference. Cfp4-deficient strains are not attenuated in virulence in human macrophages or during lung infection in a murine model of histoplasmosis. Coinfection of differentially marked Cfp4-producing and Cfp4-deficient strains demonstrates that production of Cfp4 does not confer a fitness advantage to Histoplasma yeasts during murine lung infection. Despite no apparent role in acute virulence in mice, secretion of the Cfp4 glycoprotein by yeast cells is consistent across clinical and laboratory isolates of the North American type 1 and type 2 phylogenetic groups as well as a strain from Panama. In addition, human immune sera recognize the Histoplasma Cfp4 protein, confirming Cfp4 production during infection of human hosts. These results suggest the potential utility of Cfp4 as a diagnostic exoantigen for histoplasmosis.

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“…Agrobacterium tumefaciens was used to mutagenize Histoplasma yeast as described previously (44). Briefly, A. tumefaciens strain LBA1100 harboring plasmid pCM41 .…”

Section: Methodsmentioning

confidence: 99%

“…Such approaches, which enable the discovery of unsuspected or novel virulence factors, require (i) an effective mutagen and (ii) an efficient screen for a phenotype of interest. The development of Agrobacteriummediated transformation techniques for many fungi, including Histoplasma (24,36), has solved the first task; the transfer and integration of transfer DNA (T-DNA) into the fungal genome provides the means of generating insertion mutants that can be quickly mapped with molecular techniques (44).…”

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Discovery of a Role for Hsp82 in Histoplasma Virulence through a Quantitative Screen for Macrophage Lethality

2011

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The application of forward genetics can reveal new factors required for the virulence of intracellular pathogens. To facilitate such virulence screens, we developed macrophage cell lines with which the number of intact host cells following infection with intracellular pathogens can be rapidly and easily ascertained through the expression of a constitutive lacZ transgene. Using known virulence mutants of Francisella novicida and Histoplasma capsulatum, we confirmed the applicability of these host cells for the quantitative assessment of bacterial and fungal virulence, respectively. To identify new genes required for Histoplasma virulence, we employed these transgenic macrophage cells to screen a collection of individual transfer DNA (T-DNA) insertion mutants. Among the mutants showing decreased virulence in macrophages, we identified an insertion in the locus encoding the Histoplasma Hsp82 homolog. The lesion caused by the T-DNA insertion localizes to the promoter region, resulting in significantly decreased HSP82 expression. Reduced HSP82 expression markedly attenuates the virulence of Histoplasma yeast in vivo. While the HSP82 hypomorph grows normally in vitro at 37°C and under acid and salinity stresses, its ability to recover from high-temperature stress is impaired. These results provide genetic proof of the role of stress chaperones in the virulence of a thermally dimorphic fungal pathogen.

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Agrobacterium-Mediated Insertional Mutagenesis in Histoplasma capsulatum

Cited by 17 publications

References 15 publications

Histoplasma capsulatum Depends on De Novo Vitamin Biosynthesis for Intraphagosomal Proliferation

Histoplasma capsulatum Depends on De Novo Vitamin Biosynthesis for Intraphagosomal Proliferation

Glycosylation and Immunoreactivity of the Histoplasma capsulatum Cfp4 Yeast-Phase Exoantigen

Discovery of a Role for Hsp82 in Histoplasma Virulence through a Quantitative Screen for Macrophage Lethality

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