SUMMARY STATEMENT

PROGRAM CONTACT: ( Privileged Communication ) Release Date: 12/15/2013 

Rory Duncan Sr.

Application Number: 1 R03 AI111015-01

Principal Investigator 

RAPPLEYE, CHAD A PHD

Applicant Organization: Ohio State University

Review Group: PTHE

Pathogenic Eukaryotes Study Section

Meeting Date: 12/09/2013 RFA/PA: PA11-262

Council: JAN 2014 PCC: M31

Requested Start: 04/01/2014

Project Title: Forward genetics-based discovery of Histoplasma virulence genes

SRG Action: Impact Score: 

Next Steps: Visit http://grants.nih.gov/grants/next_steps.htm

Human Subjects: 10-No human subjects involved

Animal Subjects: 10-No live vertebrate animals involved for competing appl.

Project Direct Costs Estimated

Year Requested Total Cost 

1 

2

TOTAL

ADMINISTRATIVE BUDGET NOTE: The budget shown is the requested budget and has not been 

adjusted to reflect any recommendations made by reviewers. If an award is planned, the costs will be 

calculated by Institute grants management staff based on the recommendations outlined below in the 

COMMITTEE BUDGET RECOMMENDATIONS section.

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1 R03 AI111015-01 2 PTHE 

RAPPLEYE, C 

1R03AI111015-01 RAPPLEYE, CHAD 

RESUME AND SUMMARY OF DISCUSSION: This application seeks to identify Histoplasma 

capsulatum genes required for survival, replication and destruction of macrophages. The mechanisms 

involved in Histoplasma-macrophage interactions are not well known, thus the work is of high 

significance to expand our knowledge on the pathogenesis of this organism as well as other 

intracellular pathogens. The work benefits from the recent development and validation of a RFPfluorescent Histoplasma strain and a transgenic lac-Z expressing macrophage cell line allowing 

quantitative monitoring of intracellular replication and macrophage destruction. The investigator has an 

excellent track record in the area and should be able to carry out the project successfully. A strong set 

of preliminary data are provided to support the aims of the application. The research plan is feasible 

and well-designed. Although a few minor weaknesses are identified; overall, there is high enthusiasm 

for this application and certainty that novel virulence genes will be identified with the proposed plan. 

DESCRIPTION (provided by applicant): Current understanding of the molecular mechanisms that 

underly Histoplasma pathogenesis remains limited. Unlike opportunistic pathogens, the fungal 

pathogen Histoplasma capsulatum can cause disease even in immunocompetent hosts by parasitizing 

phagocytes of the host. Only a few virulence factors have been identified and characterized to date. In 

this proposal, we will use a forward genetics approach to discover the virulence factors that enable 

Histoplasma to subvert the defenses of the macrophage, Histoplasma's primary host cell. Random 

mutants of Histoplasma yeasts will be created using insertional mutagenesis. Mutants will be screened 

for decreased virulence in macrophages using a transgenic macrophage line and a Histoplasma strain 

that has been engineered with fluorescence to provide high-throughput screening capability. Mutants 

will be classified according to the stage at which Histoplasma pathogenesis is blocked by analysis of 

intramacrophage growth kinetics. The virulence genes represented by each attenuated mutant will be 

identified by mapping of the mutation. The final collection of virulence-defective mutants will be ranked 

according to the severity of their impairment, the classification of their pathogenesis defects, and the 

identity of the virulence gene identities. These rankings will be used to prioritize further characterization 

of the discovered virulence factors in future studies to define their roles in facilitating Histoplasma 

survival and growth in host macrophages. 

PUBLIC HEALTH RELEVANCE: Histoplasmosis, a respiratory and systemic disease caused by 

infections with the fungal pathogen Histoplasma capsulatum, afflicts thousands each year in the United 

States regardless of the host's immune status. The mechanisms that enable Histoplasma to subvert 

immune defenses are poorly understood. This proposal will identify new virulence factors through a 

genetics approach to improve our understanding of Histoplasma pathogenesis. Identification of these 

processes essential to virulence will aid in the development of improved therapeutic options to treat 

histoplasmosis. 

CRITIQUE 1: 

Significance: 2 

Investigator(s): 2 

Innovation: 3 

Approach: 3 

Environment: 2 

Overall Impact: The application developed by this productive and talented investigator centers on a 

forward genetics approach to identify Histoplasma capsulatum genes that are involved in binding to 

macrophages and survival within these cells. The work is supported by strong preliminary data and 

accompanied by an excellent toolbox to perform the experiments in a high throughput manner. There is 
1 R03 AI111015-01 3 PTHE 

RAPPLEYE, C 

little doubt that new data will be forthcoming. One weakness of this approach is that essential genes 

may be involved in these processes and will be missed because mutations will be lethal. For example, 

heat shock protein 60 is a ligand for CD11b and a mutation in the Histoplasma gene might not be 

discovered by this technology since it can be a lethal mutation. A second weakness is that a similar 

approach has been used by others to identify mutant phenotypes. For example, as the investigator 

points out a Cbp1 mutant has a strong phenotype as do several other genes. The investigator has not 

provided a powerful rationale for proceeding to add additional genes to this compendium. 

1. Significance: 

Strengths 

 The identity of genes involved in promoting survival in macrophages is a fundamental query that 

begs to be answered. 

 The findings may be applicable to a variety of fungal and perhaps non-fungal intracellular 

pathogens. In that regard, the data may be highly revealing.

Weaknesses 

Some of this work has been done by others using a similar technology. Therefore, it is not clear 

how this will add to existing progress. 

2. Investigator(s): 

Strengths 

The investigator is talented and highly productive. He has published a number of manuscripts 

that have altered our concepts about Histoplasma. He is capable of performing the experiments. 

Weaknesses 

None noted. 

3. Innovation: 

Strengths 

The toolbox for investigating Histoplasma survival is technically innovative. 

Weaknesses 

The agrobacterium transformation as pointed out by the investigator has been used by others. 

4. Approach: 

Strengths 

The technical approach is sound. It is direct and answers will likely be forthcoming. 

The questions posed are highly relevant to Histoplasma pathogenesis. 

The work is supported by excellent preliminary data including a publication. 

Weaknesses

Essential genes will be missed since mutations may be lethal. 

The accumulation of more genes than those we know is not well-justified. There are numerous 

candidates but little has been done to examine how they promote growth. 
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RAPPLEYE, C 

5. Environment: 

Strengths 

The environment is outstanding. 

Weaknesses 

 None noted. 

Protections for Human Subjects: 

Not Applicable (No Human Subjects) 

Vertebrate Animals: 

Not Applicable (No Vertebrate Animals) 

Biohazards: 

Acceptable 

Budget and Period of Support: 

Recommend as Requested. 

CRITIQUE 2: 

Significance: 1 

Investigator(s): 1 

Innovation: 2 

Approach: 1 

Environment: 1 

Overall Impact: This R03 application proposes an innovative forward genetics screen to identify genes 

required for the survival of H. capsulatum in macrophages. The application is significant because very 

few such genes have been previously identified and it is likely that the insights that arise from studying 

such genes will have implications to other intracellular fungal pathogens. The investigator is 

outstanding and his laboratory is one of only a few in the world that have the expertise to accomplish 

these aims. The screen utilizes a novel screening strategy well-designed to its intended purpose and, 

hence, the application is innovative. There is ample preliminary data and pilot screening results to 

support the feasibility of the proposed screen. The Aims are well-thought out and the screening strategy 

is sound. This application has essentially no identifiable flaws. 

1. Significance: 

Strengths 

Only a small set of virulence genes for Histoplasma have been identified to date.

 Virulence factors identified by this screen are likely to have broader implications for other 

intracellular fungi.
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RAPPLEYE, C 

Weaknesses 

 None noted. 

2. Investigator(s): 

Strengths 

 The investigator is productive and an expert in this area of investigation 

 Few other laboratories are in a position to perform these studies. 

Weaknesses 

 None noted. 

3. Innovation: 

Strengths 

The application describes a novel screening strategy based on a clever combination of reporters 

Weaknesses 

 None noted. 

4. Approach: 

Strengths 

The preliminary data strongly support the feasibility of the proposed screen 

The screening plan is well-thought out and logical 

The hit analysis and prioritization is logical. 

Weaknesses

None identified 

5. Environment: 

Strengths 

The environment is strong. 

Weaknesses 

 None noted. 

Protections for Human Subjects: 

Not Applicable (No Human Subjects) 

Vertebrate Animals: 

Not Applicable (No Vertebrate Animals) 

Biohazards: 

Acceptable 
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RAPPLEYE, C 

Budget and Period of Support: 

Recommend as Requested. 

CRITIQUE 3: 

Significance: 1 

Investigator(s): 1 

Innovation: 1 

Approach: 2 

Environment: 1 

Overall Impact: This is a succinct and well-written R03 application from a productive mid-level 

investigator that proposes to use a forward genetics approach to identify Histoplasma genes required 

for macrophage lysis and intracellular survival/growth. The investigator has developed an 

agrobacterium mutagenesis system, which inserts elements randomly into the genome and has 

developed a macrophage based screening system that can be performed in 96 well plates. Any target 

gene can be sequencing using a nested PCR approach. In aim 1, he proposes to generate a bank of 

40,000 mutants with 2.5X genome coverage. For screening, the investigator has constructed lacZ 

expressing macrophages and RFP expressing Histoplasma to be able to simultaneously monitor in vivo

growth and endpoint macrophage survival. Mutants can then be classified and prioritized based on the 

nature and degree of defect. The investigator has also shown proof of principle for the mutagenesis and 

screening techniques and already identified 21 mutants with these methods. One of these target genes 

has been sequenced and the resulting mutant is defective in virulence in an animal model of infection. 

The investigator has effectively calculated the feasibility of the studies and estimates 400 96 well plates 

will be needed for the screen, with 8-10 plates screened per week. The application is significant 

because there have been very few virulence factors identified in Histoplasma and mechanisms involved 

in survival within macrophages have not been well characterized. There is also a plan to translate 

results from this application into a future R01 application. 

THE FOLLOWING RESUME SECTIONS WERE PREPARED BY THE SCIENTIFIC REVIEW 

OFFICER TO SUMMARIZE THE OUTCOME OF DISCUSSIONS OF THE REVIEW COMMITTEE ON 

THE FOLLOWING ISSUES: 

COMMITTEE BUDGET RECOMMENDATIONS: The budget was recommended as requested. 

NIH has modified its policy regarding the receipt of resubmissions (amended 

applications). See Guide Notice NOT-OD-10-080 at 

http://grants.nih.gov/grants/guide/notice-files/NOT-OD-10-080.html. 

The impact/priority score is calculated after discussion of an application by averaging 

the overall scores (1-9) given by all voting reviewers on the committee and multiplying 

by 10. The criterion scores are submitted prior to the meeting by the individual 

reviewers assigned to an application, and are not discussed specifically at the review 

meeting or calculated into the overall impact score. Some applications also receive a 

percentile ranking. For details on the review process, see 

http://grants.nih.gov/grants/peer_review_process.htm#scoring.