PI: Rappleye, Chad A Title: Forward genetics-based discovery of Histoplasma virulence genes

Received: 06/14/2013 FOA: PA11-262 Council: 01/2014

Competition ID: ADOBE-FORMS-B2 FOA Title: NIH SMALL RESEARCH GRANT PROGRAM (PARENT R03)

1 R03 AI111015-01 Dual: Accession Number: 3599102

IPF: 6218701 Organization: OHIO STATE UNIVERSITY

Former Number: Department: Microbiology

IRG/SRG: PTHE AIDS: N Expedited: N

Subtotal Direct Costs 

(excludes consortium F&A) 

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Senior/Key Personnel: Organization: Role Category:

Chad Rappleye The Ohio State University PD/PI 

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APPLICATION FOR FEDERAL ASSISTANCE

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06/14/2013

The Ohio State University

1960 Kenny Road

Columbus Franklin

43210-1016

Tamara

Dickey

Forward genetics-based discovery of Histoplasma virulence genes

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OH-015

Rappleye

Chad Alan

43210-1292

Franklin

540 Biological Sciences Bldg.

Columbus

484 W. 12th Ave.

Microbiology

Associate Professor

The Ohio State University

OH: Ohio

USA: UNITED STATES

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424 R&R and PHS-398 Specific

Table Of Contents Page Numbers

SF 424 R&R Face Page------------------------------------------------------------------------------------------ 1

Table of Contents--------------------------------------------------------------------------------------------- 3

Performance Sites--------------------------------------------------------------------------------------------- 4

Research & Related Other Project Information------------------------------------------------------------------ 5

 Project Summary/Abstract (Description)---------------------------------------- 6

 Public Health Relevance Statement (Narrative attachment)---------------------------------------- 7

 Facilities & Other Resources---------------------------------------- 8

 Equipment---------------------------------------- 10

Research & Related Senior/Key Person-------------------------------------------------------------------------- 11

 Biographical Sketches for each listed Senior/Key Person---------------------------------------- 12

PHS 398 Specific Cover Page Supplement------------------------------------------------------------------------ 15

PHS 398 Specific Modular Budget------------------------------------------------------------------------------- 17

 Personnel Justification---------------------------------------- 19

PHS 398 Specific Research Plan-------------------------------------------------------------------------------- 20

 Specific Aims---------------------------------------- 21

 Research Strategy---------------------------------------- 22

 Bibliography & References Cited---------------------------------------- 28

PHS 398 Checklist--------------------------------------------------------------------------------------------- 31

 

Principal Investigator/Program Director (Last, first, middle): Rappleye, Chad, Alan 

Table of Contents Page 3
 
Project/Performance Site Location(s)

Project/Performance Site Primary Location

Organization Name:

* Street1:

Street2:

* City: County:

* State:

Province:

* Country:

* ZIP / Postal Code:

The Ohio State University

1960 Kenny Road

Columbus Franklin

OH: Ohio

USA: UNITED STATES

43210-1016

OMB Number: 4040-0010 

Expiration Date: 08/31/2011

I am submitting an application as an individual, and not on behalf of a company, state, 

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I am submitting an application as an individual, and not on behalf of a company, state, 

local or tribal government, academia, or other type of organization. 

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Principal Investigator/Program Director (Last, first, middle): Rappleye, Chad, Alan 

Performance Sites Page 4
 
RESEARCH & RELATED Other Project Information

1. Are Human Subjects Involved?

If no, is the IRB review Pending?

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 environmental impact statement (EIS) been performed?

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6.a. If yes, identify countries:

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7. Project Summary/Abstract

8. Project Narrative

9. Bibliography & References Cited

10. Facilities & Other Resources

11. Equipment

12. Other Attachments

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1.a. If YES to Human Subjects

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5.a. If yes, please explain:

OMB Number: 4040-0001

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Principal Investigator/Program Director (Last, first, middle): Rappleye, Chad, Alan 

Other Information Page 5
 
Project Summary/Abstract

Forward genetics-based discovery of Histoplasma virulence genes

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.

 

Principal Investigator/Program Director (Last, first, middle): Rappleye, Chad, Alan 

Project Description Page 6
 
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.

 

Principal Investigator/Program Director (Last, first, middle): Rappleye, Chad, Alan 

Public Health Relevance Statement Page 7
 
Facilities and Other Resources

Laboratory

The Rappleye lab has 970 ft2 total laboratory space at Ohio State University located on the 5th floor of the 

Biological Sciences Building. This includes a 220 ft2 HEPA-filtered air supply dedicated clean room for BSL-2 

work with fungal pathogens.

Computer

The Rappleye lab has 5 desktop PCs (Pentium IV, i5, and i7 dual and quad core processors) and one laptop 

computer for instrument operation, bioinformatics analyses, data management, and word processing. An HP 

1320n network printer is available for trainees. Access to extensive library resources at OSU is also available 

via the internet.

Office

Dr. Rappleye has 90 ft2 of office space adjacent to his main laboratory.

Other Resources

Plant-Microbe Genomics Facility (http://pmgf.osu.edu/)

Located immediately downstairs from the Rappleye lab is the Plant-Microbe Genomics Facility that provides a 

wealth of “-omics” and sequencing services to OSU researchers at a significant discount. Major services and 

resources relevant to this proposal include:

• DNA sequencing (Sanger and capillary-based): 3730 DNA Analyzer (Applied Biosystems, Inc)

• Quantitative / real-time PCR: CFX96 Real-Time Detection System (BioRad)

• Next-generation / 454 pyrosequencing: GS FLX System (Roche)

• Robotic liquid handling station: Biomek FX 20-deck platform (Beckman)

Scientific Environment

Microbial pathogenesis and microbiology is a significant strength of the research community at Ohio State 

University. Dr. Rappleye has a dual appointment with the Department of Microbiology and the Department of 

Microbial Infection and Immunity. In addition, Dr. Rappleye is a member of the Center for Microbial Interface 

Biology and the Public Health Preparedness for Infectious Diseases initiative. Dr. Rappleye and trainees in his

lab have numerous interactions with members of these departments and centers through weekly seminar 

series and graduate courses, as well as informal meetings.

Department of Microbiology (http://microbiology.osu.edu/)

The Department of Microbiology is comprised of a diverse group of scientists seeking to understand how 

microbes function and influence their environment. Faculty are experts in genetic, molecular genetic, and 

genomic approaches including application to organisms with difficult genetics: Dr. Chuck Daniels (genomics of 

archea bacteria), Tina Henkin (bacterial transcriptional controls), Dr. Mike Ibba (tRNA biology), Dr. Robert 

Munson (bacterial pathogenesis), Dr. John Reeve (molecular biology of archea), Dr. Natacha Ruiz (genetic 

dissection of the bacterial cell envelope), Dr. Tom Santangelo (transcriptional regulation in archea), Dr. Dan 

Wozniak (genetics and pathogenesis of Pseudomonas). In addition, Microbiology faculty provide a wealth of 

 

Principal Investigator/Program Director (Last, first, middle): Rappleye, Chad, Alan 

Facilities Page 8
 
expertise in microbial physiology and biochemistry upon which we can draw for understanding genes 

potentially discovered that are involved in Histoplasma intracellular metabolism: Dr. Birgit Alber (central carbon

metabolism), Dr. Joseph Krzycki (biochemistry of archea), and Dr. Robert Tabita (biochemistry and energy 

pathways in bacteria and archea). The microbiology environment also includes research and expertise with 

eukaryotic microbes which provides unique eukaryotic perspectives that will benefit our research: Dr. Juan 

Alfonzo (Trypanosome biology) and Dr. Abhay Satoskar (Leishmania and immunology of leishmaniasis).

Department of Microbial Infection and Immunity (MII; http://medicine.osu.edu/mii/)

The Department of Microbial Infection and Immunity is part of the College of Medicine at Ohio State University.

Laboratories of the MII are located in the Biomedical Research Tower which is adjacent to the building in which

Dr. Rappleye's laboratory is located. Core members of the MII department include investigators with expertise 

in intracellular pathogens, innate immunity, and phagocyte biology including Dr. Larry Schlesinger 

(Mycobacterium tuberculosis pathogenesis and macrophage biology), Dr. John Gunn (Salmonella and 

Francisella pathogenesis), Dr. Amal Amer (Legionella/Burkholderia pathogenesis and autophagy in 

macrophages), Dr. Mark Drew (Plasmodium pathogenesis), Dr. Stephanie Seveau (Listeria pathogenesis and 

phagocyte cell biology), and Dr. Brian Ahmer (Salmonella genetics and pathogenesis), and Dr. Jordi Torrelles 

(Mycobacterium biochemistry). Members of Dr. Rappleye's lab attend bi-weekly work-in-progress meetings 

with MII faculty and trainees as well as weekly seminars on host-pathogenesis interactions. 

Center for Microbial Interface Biology (CMIB; http://cmib.osu.edu/)

Campus-wide, there are over 70 faculty members in the Center for Microbial Interface Biology. Major themes 

of the CMIB are respiratory infectious diseases, intracellular parasitism, mucosal immunology, biofilms, and 

therapeutics. The CMIB hosts many seminars on topics central to host-pathogen interactions and infectious 

diseases. Access to the diverse areas of expertise among members of the CMIB, which ranges from 

proteomics to animal models of disease, is an invaluable resource for infectious disease work at Ohio State 

University.

 

Principal Investigator/Program Director (Last, first, middle): Rappleye, Chad, Alan 

Facilities Page 9
 
Equipment

Rappleye laboratory

The main laboratory has equipment required for general microbiological, molecular, and biochemical work 

including microcentrifuges, agarose and polyacrylamide electrophoresis equipment, spectrophotometer, 

rocking and shaking platforms, bead-beater cell disruptor, and incubators (shaking and static).

Major equipment relevant to this proposal which are available include:

 Synergy 2 microplate reader (UV, visible, and fluorescence (GFP and RFP) capabilities; BioTek)

 epMotion robotic liquid handling station, 4-deck platform (Eppendorf) for setting up and processing 96-

well plate-based assays

 Nikon Elcipse Ti deconvolution microscope with: 1.4 megapixel CCD camera (HQ2 , Photometrics), 

objectives (40X, 100X) for DIC and fluorescence (FITC/GFP, Texas-red/RFP, and DAPI filter sets)

 Nikon E400 microscope with: objectives (10X, 40X, 60X, 100X) configured for phase-contrast and 

fluorescence microscopy (FITC/GFP, Texas-red/RFP, and DAPI filter sets)

 (2) PCR machines: 96 sample capacity (Applied Biosystems)

 Realplex real-time PCR machine (Eppendorf)

 AlphaImager HP gel documentation and fluorescent imaging system with 1.4 megapixel CCD camera, 

excitation and emission filters for GFP and RFP (for imaging fluorescent colonies)

 4°C, -20°C, -80°C storage units

 AirClean 600 nucleic acid workstation

The Rappleye lab BSL-2 room contains equipment and engineering safety for working with infectious agents:

 (2) class II biological safety cabinets

 (2) water-jacketed 37°C CO2 incubator: 13 cu. ft. total capacity for fungal and tissue culture

 7.5 cu ft CO2-supplied shaking incubator for liquid fungal cultures (Multitron, ATR)

 inverted phase microscope (Olympus, 4X, 10X, 20X objectives)

 upright phase microscope (Leica, 10X, 40X, 100X objectives)

 electroporation apparatus (Bio-rad) for transformation of bacteria and fungi

Other University Resources

As a core member of the Center for Microbial Interface Biology, the Rappleye lab also has access to CMIB 

shared equipment including:

 Biomek robotic liquid handling station: 8-position

 ELISA plate reader (Biotek ELX800)

 Bio-Plex suspension array system (Bio-rad)

 

Principal Investigator/Program Director (Last, first, middle): Rappleye, Chad, Alan 

Equipment Page 10
 
Province:

PROFILE - Project Director/Principal Investigator

Prefix: * First Name: Middle Name:

* Last Name: Suffix:

Organization Name: Division:

Position/Title: Department:

* Street1:

Street2:

* Phone Number: Fax Number:

* E-Mail:

Credential, e.g., agency login:

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RESEARCH & RELATED Senior/Key Person Profile (Expanded)

*Attach Biographical Sketch

Attach Current & Pending Support

Chad Alan

Rappleye

Associate Professor Microbiology

The Ohio State University

484 W. 12th Ave.

Columbus Franklin

540 Biological Sciences Bldg.

OH: Ohio

USA: UNITED STATES 43210-1292

PD/PI

PhD

2002

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Add Attachment Delete Attachment View Attachment

OMB Number: 4040-0001 

Expiration Date: 06/30/2011

Degree Type:

Degree Year:

Province:

PROFILE - Senior/Key Person

Prefix: * First Name: Middle Name:

* Last Name: Suffix:

Organization Name: Division:

Position/Title: Department:

* Street1:

Street2:

* Phone Number: Fax Number:

* E-Mail:

Credential, e.g., agency login:

* Country: * Zip / Postal Code:

* State:

* City: County/ Parish:

USA: UNITED STATES

* Project Role: Other Project Role Category:

1

Degree Type:

Degree Year:

*Attach Biographical Sketch Add Attachment Delete Attachment View Attachment

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Principal Investigator/Program Director (Last, first, middle): Rappleye, Chad, Alan 

Key Personnel Page 11
 
 

Principal Investigator/Program Director (Last, first, middle): Rappleye, Chad, Alan 

BIOGRAPHICAL SKETCH

Provide the following information for the Senior/key personnel and other significant contributors in the order listed on Form Page 2.

Follow this format for each person. DO NOT EXCEED FOUR PAGES.

NAME POSITION TITLE

Chad A. Rappleye Associate Professor of Microbiology

eRA COMMONS USER NAME (credential, e.g., agency login)

EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, include postdoctoral training and 

residency training if applicable.)

DEGREE INSTITUTION AND LOCATION MM/YY FIELD OF STUDY (if applicable)

University of Utah, Salt Lake City, UT B.S. 06/95 Biology

University of California, San Diego, CA Ph.D. 06/02 Biology

Washington University, St. Louis, MO postdoc 08/06 Microbial pathogenesis

A. Personal Statement

My research seeks to understand the biology and pathogenic mechanisms of fungal pathogens. Of particular 

interest are the strategies employed by fungal pathogens to subvert immune defenses and to establish 

permissive replication niches in the host. The majority of my laboratory's efforts are directed at defining the 

molecular mechanisms that contribute to the virulence of the primary fungal pathogen Histoplasma 

capsulatum. To investigate the interactions between Histoplasma and the host, my laboratory uses molecular 

genetics, proteomics, and transcriptomics to identify and characterize factors contributing to Histoplasma's 

ability to infect and grow within host phagocytes. The ability to disrupt gene functions is essential for 

demonstrating the role of candidate factors. We have pioneered techniques to knock-down and knock-out 

genes to facilitate these tests in Histoplasma, an organism notoriously recalcitrant to homologous 

recombination and gene replacement strategies.

My laboratory is one of only a handful of labs with the expertise required to molecularly dissect Histoplasma 

virulence. My work has defined and characterized the role of three of the 5 major virulence factors of 

Histoplasma capsulatum identified to date. These include an α-linked polysaccharide of the yeast cell wall that

acts to conceal yeast from the host, a stress response factor that enables intracellular growth, and an efficient 

extracellular antioxidant system that protects Histoplasma from host-derived antimicrobial metabolites. We 

have identified the constituent proteins comprising the secreted proteome of pathogenic-phase Histoplasma

cells as a basis for understanding how Histoplasma interacts with the host. In addition, we have completed the

first transcriptome analysis of two strains of Histoplasma and comparison of the regulons of pathogenic and 

non-pathogenic phases. These resources will allow us to take advantage of the discoveries made in the 

current proposal and rapidly move from discovery to mechanism studies. We have expertise in physiologically 

relevant infection models including isolation and infection of human and murine phagocytes as well as in vivo 

infections (murine). This allows us to accurately model and investigate histoplasmosis disease.

B. Positions and Honors

Positions and Employment

2002-2006 Postdoctoral Fellow Department of Molecular Microbiology

 Washington University, St. Louis, MO

2006-2012 Assistant Professor Department of Microbiology

PHS 398/2590 (Rev. 06/09) Page 1 Biographical Sketch Format Page

Biosketches Page 12
 
Department of Internal Medicine, Division of Infectious Diseases

 Ohio State University, Columbus OH

2012- Associate Professor Department of Microbiology

Department of Microbial Infection and Immunity

 Ohio State University, Columbus OH

Honors

1997-2002 Howard Hughes Medical Institute Predoctoral Fellowship

2002-2003 NIH/NHLBI Pulmonary Research Training Grant Award, Washington University, St. Louis, MO

2003-2006 Damon Runyon Cancer Research Foundation Postdoctoral Fellowship

2009-2010 Co-chair American Society of Microbiology Conference on Dimorphic Fungal Pathogens

2010-2011 Session convener, American Society of Microbiology General Meeting

C. Selected Peer-reviewed Publications

1. Rappleye CA, Engle JT, and Goldman WE. 2004. RNA interference in Histoplasma capsulatum

demonstrates a role for alpha-(1,3)-glucan in virulence. Mol Microbiol 53(1):153-165. 

2. Marion CL, Rappleye CA, Engle JT, and Goldman WE. 2006. An alpha-(1,4)-amylase is essential for 

alpha-(1,3)-glucan production and virulence in Histoplasma capsulatum. Mol Microbiol 62(4):970-983. 

3. Rappleye CA, and Goldman WE. 2006. Defining virulence genes in the dimorphic fungi. Annu Rev 

Microbiol 60:281-303. 

4. Almeida AJ, Carmona JA, Cunha C, Carvalho A, Rappleye CA, Goldman WE, Hooykaas PJ, Leao C, 

Ludovico P, and Rodrigues F. 2007. Towards a molecular genetic system for the pathogenic fungus 

Paracoccidioides brasiliensis. Fungal Genet Biol 44(12):1387-1398. 

5. Krajaejun T, Gauthier GM, Rappleye CA, Sullivan TD, and Klein BS. 2007. Development and application of

a green fluorescent protein sentinel system for identification of RNA interference in Blastomyces 

dermatitidis illuminates the role of septin in morphogenesis and sporulation. Eukaryot Cell 6(8):1299-1309. 

PMC1951135

6. Rappleye CA, Eissenberg LG, and Goldman WE. 2007. Histoplasma capsulatum alpha-(1,3)-glucan blocks

innate immune recognition by the beta-glucan receptor. Proc Natl Acad Sci U S A 104(4):1366-1370. 

PMC1783108

7. Holbrook ED, and Rappleye CA. 2008. Histoplasma capsulatum pathogenesis: making a lifestyle switch. 

Curr Opin Microbiol 11(4):318-324. 

8. Youseff BH, Dougherty JA, and Rappleye CA. 2009. Reverse genetics through random mutagenesis in 

Histoplasma capsulatum. BMC Microbiol 9:236. PMC2781022

9. Edwards JA, Alore EA, and Rappleye CA. 2011. The yeast-phase virulence requirement for alpha-glucan 

synthase differs among Histoplasma capsulatum chemotypes. Eukaryot Cell 10(1):87-97. PMC3019800

10. Holbrook ED, Edwards JA, Youseff BH, and Rappleye CA. 2011. Definition of the Extracellular Proteome of

Pathogenic-Phase Histoplasma capsulatum. J Proteome Res 10(4):1929-1943. PMC3069693

11. Edwards JA, Zemska O, and Rappleye CA. 2011. Discovery of a Role for Hsp82 in Histoplasma Virulence 

through a Quantitative Screen for Macrophage Lethality. Infect Immun 79(8):3348-3357. PMC3147571

12. Edwards JA, and Rappleye CA. 2011. Histoplasma mechanisms of pathogenesis - one portfolio doesn’t fit 

all. FEMS Microbiol Lett 324(1):1-9. PMC3228276

13. Youseff BH, and Rappleye CA. 2012. RNAi-based gene silencing using a GFP sentinel system in 

Histoplasma capsulatum. In Brand, A, and MacCallum D (eds.), Host-Fungal Interactions: Manipulation of 

Fungal Gene Expression, Methods in Molecular Biology, 845:51-66.

PHS 398/2590 (Rev. 06/09) Page 2 Biographical Sketch Format Page

 

Principal Investigator/Program Director (Last, first, middle): Rappleye, Chad, Alan 

Biosketches Page 13
 
 

Principal Investigator/Program Director (Last, first, middle): Rappleye, Chad, Alan 

14. Zemska O, and Rappleye CA. 2011. Agrobacterium-mediated insertional mutagenesis in Histoplasma 

capsulatum In Brand, A, and MacCallum D (eds.), Host-Fungal Interactions: Manipulation of Fungal Gene 

Expression, Methods in Molecular Biology, 845:151-164.

15. Youseff BH, Holbrook ED, Smolnycki KA, Rappleye CA. 2012. Extracellular Superoxide Dismutase 

Protects Histoplasma Yeast Cells From Host-Derived Oxidative Stress. PLoS Pathogens 8(5):e1002713. 

PMC3355102

16. Kemski MM, Stevens B, and Rappleye CA. 2013. Spectrum of T-DNA Integrations for Insertional 

Mutagenesis of Histoplasma capsulatum. Fungal Biol 117(1):41-51. PMC 3552300

17.

D. Research Support

Ongoing Research Support

R01-AI083335 Rappleye

NIH / NIAID

8/1/2009 – 7/31/2014

Virulence factor discovery in the secreted proteome of Histoplasma capsulatum

The major goals of this project are to define the major factors secreted by pathogenic-phase 

Histoplasma cells and to define their potential roles in promoting Histoplasma pathogenesis.

Role: PI

CCTS Pilot Grant Award: Rappleye / Tjarks

NIH / NCRR (UL1RR0025755 PI: Jackson)

8/17/2102 - 8/16/2013

Lead candidate antifungal drug development

The goal of this project grant is to focus on integrating the traditional biomedical research process 

(basic scientific discovery to translation into clinical research to dissemination into the community for 

improved patient outcomes).

The goal of the pilot grant award, administered through the Center for Clinical and Translational 

Science (CCTS) at OSU, is to advance a hit compound with potent antifungal activity and to establish 

basic structure-activity relationships.

Role: PI

Completed Research Support

PHS 398/2590 (Rev. 06/09) Page 3 Biographical Sketch Format Page

Biosketches Page 14
 
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1. Project Director / Principal Investigator (PD/PI)

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OMB Number: 0925-0001 

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Chad

Alan

Rappleye

No Yes

No Yes

Tamara

Dickey

Sponsored Programs Officer

1960 Kenny Road

Columbus

Franklin

USA: UNITED STATES

OH: Ohio

43210-1016

	


 		

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Principal Investigator/Program Director (Last, first, middle): Rappleye, Chad, Alan 

Clinical Trial & HESC Page 15
 
PHS 398 Cover Page Supplement

4. Human Embryonic Stem Cells

* Does the proposed project involve human embryonic stem cells?

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Principal Investigator/Program Director (Last, first, middle): Rappleye, Chad, Alan 

Clinical Trial & HESC Page 16
 
PHS 398 Modular Budget

OMB Number: 0925-0001 

Budget Period: 1

Start Date: 04/01/2014 End Date: 03/31/2015

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Start Date: 04/01/2015 End Date: 03/31/2016

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B. Indirect Costs

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1. On Campus Organized Research

2. 

3. 

4. 

Cognizant Agency (Agency Name, POC Name and Phone Number) DHHS, Ernest Kinneer, 

Indirect Cost Rate Agreement Date 09/21/2012 Total Indirect Costs

C. Total Direct and Indirect Costs (A + B) Funds Requested ($)

	


 		

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Principal Investigator/Program Director (Last, first, middle): Rappleye, Chad, Alan 

Modular Budget Page 17
 
PHS 398 Modular Budget

Cumulative Budget Information

1. Total Costs, Entire Project Period

Section A, Total Direct Cost less Consortium F&A for Entire Project Period $

Section A, Total Consortium F&A for Entire Project Period $

Section A, Total Direct Costs for Entire Project Period $

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Section C, Total Direct and Indirect Costs (A+B) for Entire Project Period $

2. Budget Justifications

? Personnel Justification Mutant_Screen___Personnel10210 Add Attachment Delete Attachment View Attachment

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Principal Investigator/Program Director (Last, first, middle): Rappleye, Chad, Alan 

Modular Budget Page 18
 
Budget Justification

1. Personnel

Salary calculations are based on actual salaries. Fringe benefits, including mandatory contributions to 

the State of Ohio retirement systems, health insurance, worker’s compensation, Medicare tax, and 

vacation and sick leave costs, as appropriate, are calculated as a percentage of salary and based on 

employment classification.

Chad A. Rappleye, Ph.D.

Principal Investigator (0 academic months, 0.6 summer months)

Dr. Rappleye will be responsible for administration of the project, experimental design, and 

analysis and interpretation of results. Dr. Rappleye provides expertise and experience in 

fungal biology, fungal genetics, and in vitro and in vivo models of infection. Salary support 

equivalent to 5% annual effort for Dr. Rappleye is requested.

Andrew Garfoot, B.S.

Graduate Research Associate (6 academic months)

Mr. Garfoot will be responsible for mutagenesis and for performance of the genetic screen in 

Aim 1. The graduate student will also be responsible for recording and analyzing experimental

data as well as microbial culture of the fungal strains. Mr. Garfoot will conduct the mapping 

experiments in Aim 2. Salary and tuition support equivalent to 50% annual effort is requested.

 Research assistant (to be appointed)

Research Assistant (3.6 calendar months)

A Research Assistant (bachelor degree-holding) will be hired to assist in the preparation and 

execution of the genetic screen (Aim 1). In addition, the research assistant will be responsible 

for maintenance of macrophage lines and set up of virulence assays. Salary support 

equivalent to 30% annual effort for this individual is requested.

2. Variations in modules requested 

There are no variations in budget modules.

 

Principal Investigator/Program Director (Last, first, middle): Rappleye, Chad, Alan 

Personnel Justification Page 19
 
OMB Number: 0925-0001 

PHS 398 Research Plan

1. Application Type:

From SF 424 (R&R) Cover Page. The response provided on that page, regarding the type of application being submitted, is repeated for your 

reference, as you attach the appropriate sections of the Research Plan.

*Type of Application:

1. Introduction to Application 

(for RESUBMISSION or REVISION only)

2. Specific Aims

3. *Research Strategy

Human Subjects Sections

6. Protection of Human Subjects

7. Inclusion of Women and Minorities

8. Targeted/Planned Enrollment Table

9. Inclusion of Children

10. Vertebrate Animals

13. Consortium/Contractual Arrangements

14. Letters of Support

15. Resource Sharing Plan(s)

16. Appendix

2. Research Plan Attachments: 

 Please attach applicable sections of the research plan, below.

Other Research Plan Sections

5. Progress Report Publication List

11. Select Agent Research

12. Multiple PD/PI Leadership Plan

4. Inclusion Enrollment Report

New Resubmission Renewal Continuation Revision

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Principal Investigator/Program Director (Last, first, middle): Rappleye, Chad, Alan 

List of Research Plan Attachments Page 20
 
Specific Aims

The fungal pathogen Histoplasma capsulatum causes an estimated 100,000 infections annually in the United 

States. While most infections are self limiting upon activation of adaptive immunity, thousands each year are 

hospitalized due to acute respiratory disease and life-threatening disseminated histoplasmosis. Unlike 

opportunistic fungal pathogens, Histoplasma causes disease even in immunocompetent individuals. By itself, 

the innate immune system is unable to control Histoplasma yeasts due to Histoplasma's ability to parasitize 

host phagocytes. The mechanisms that enable Histoplasma to survive and replicate with macrophages, 

ultimately leading to destruction of the phagocyte, are only beginning to be defined. 

As the Histoplasma-macrophage interaction is key to pathogenesis, our goal is to better understand the factors

that enable intracellular growth of Histoplasma. Forward genetics is a powerful approach to identify new 

factors if an efficient mutagen and screen are employed. We have optimized and characterized an insertional 

mutagen for Histoplasma based on Agrobacterium-mediated transfer and random integration of T-DNA into 

fungal chromosomes. In addition, we have developed a high-throughput screen to facilitate identification of 

mutants unable to persist in the intramacrophage environment. For this, we developed an RFP-fluorescent 

Histoplasma strain and a transgenic lacZ-expressing macrophage cell line which permits quantitative 

monitoring of both intracellular yeast replication and macrophage destruction, respectively. The combination of

these mutagenesis and screening advances provides the efficiency necessary for forward genetics-based 

discovery of new virulence factors that enable Histoplasma to overcome innate immune defenses and exploit 

the macrophage as its host cell.

Aim 1. Screen Histoplasma T-DNA insertion mutants for attenuated virulence in macrophages.

Aim 1A. Generate a library of T-DNA insertion mutants in Histoplasma yeast.

Agrobacterium-mediated transformation will be used to mutagenize Histoplasma yeasts. Individual 

mutants will be arrayed into 96-well plates to facilitate high-throughput screening and to enable banking

of the mutant collection for long term preservation. A library of 40,000 mutants will be generated 

representing approximately 2.5-fold coverage of the Histoplasma genome.

Aim 1B. Identification of mutants deficient in survival and replication within macrophages.

Macrophages will be infected with individual Histoplasma mutants and the intramacrophage growth of 

yeast monitored non-destructively by measurement of yeast-expressed RFP fluorescence. End point 

macrophage lysis by yeast will be determined by quantifying the remaining macrophage-expressed βgalactosidase activity. Histoplasma mutants will be selected that exhibit at least 50% reduction in 

intramacrophage growth and/or at least 50% decreased ability to lyse macrophages

Aim 2. Determine the identify of genes required for Histoplasma virulence in macrophages.

Aim 2A. Map the disrupted loci in attenuated mutants.

Mutants will be tested by PCR to eliminate those with T-DNA disruption of genes known to be required 

for intramacrophage survival and growth. New virulence genes will be identified by mapping the T-DNA

insertions through hemi-specific PCR techniques (e.g., thermal asymmetric interlaced PCR) and 

sequencing of the amplified regions flanking the T-DNA borders. Disrupted loci will be identified by 

comparison of sequences flanking the insertion to transcriptome-based gene models (best option) or de

novo gene predictions (alternative).

Aim 2B. Classify and prioritize virulence mutants.

Mutants will be classified as: (1) deficient in macrophage entry, (2) impaired survival in macrophages, 

(3) normal survival but impaired replication in macrophages, and (4) normal replication but deficient 

ability to cause macrophage lysis. Candidate factors representing each class will be prioritized by the 

severity of the virulence attenuation, conservation of the factor among intracellular pathogens, and 

increased expression by pathogenic- compared to non-pathogenic-phase cells.

The virulence genes identified will form the basis of future studies to characterize the factors that promote 

Histoplasma pathogenesis in host macrophages.

 

Principal Investigator/Program Director (Last, first, middle): Rappleye, Chad, Alan 

Specific Aims Page 21
 
1. SIGNIFICANCE

athogens. Invasive and systemic fungal infections continue to cause significant morbidity and 

mortality, causing more deaths in the United States than tuberculosis [1,2]. Infection by most fungal pathogens

occurs via the respiratory tract, resulting in varying degrees of respiratory and systemic disease dependent on 

dose, immunological status of the host, and virulence of the pathogen. In contrast to opportunistic fungi such 

as Candida and Aspergillus, certain dimorphic fungal pathogens cause disease even in immunocompetent 

individuals, which is reflected in the fact they constitute the most common pulmonary fungal infection among 

otherwise healthy individuals [3,4]. The majority of hospitalizations due to infections by dimorphic fungal 

infections are caused by Histoplasma capsulatum [4] with up to 90% of individuals residing in endemic regions 

having serological evidence of prior infection with Histoplasma [5]. Whereas Candida and Aspergillus cells are

readily controlled and eliminated by innate immune cells of the immunocompetent host (e.g., phagocytes), the 

innate immune system alone is insufficient to clear infections by Histoplasma [6–8].

Histoplasma as an intracellular pathogen of macrophages. Macrophages provide one of the first lines of 

protection against fungal infections, yet Histoplasma yeasts effectively parasitize these innate immune cells. 

Histoplasma yeast enter into and survive within these normally fungicidal cells. This ability results, in part, from

expression of virulence factors that effectively conceal Histoplasma yeast from pathogen recognition receptors 

of the host and production of defense strategies that enable Histoplasma yeasts to combat antifungal products 

produced by host immune cells (e.g., reactive oxygen compounds). Accomplishment of these tasks is followed

by establishment of an intracellular niche permissive for yeast growth and replication, which includes blocking 

phagosome acidification and activation of lysosomal hydrolases [9]. Replication of intracellular yeasts 

ultimately leads to lysis of the macrophage and spread of Histoplasma to additional phagocytes. Since the 

Histoplasma-macrophage interaction is central to Histoplasma pathogenesis and Histoplasma's ability to 

establish infections in hosts with functional innate immunity, our goal is to better understand the factors that 

bias the outcome of this interaction in Histoplasma's favor.

2. INNOVATION

Current scarcity in defined virulence mechanisms. Although the characteristics of Histoplasma's infection 

of macrophages have been well described, our understanding at a mechanistic level remains quite limited. In 

contrast to fungi with more facile genetics, techniques for manipulating gene expression (e.g., elimination of 

gene function, expression of transgenes, etc.) in Histoplasma are relatively recent developments. Five central 

virulence factors have been defined for Histoplasma: cell wall α-glucan [10,11], the secreted Cbp1 protein [12], 

production of siderophores [13,14], the extracellular Yps3 protein [15], and an extracellular ROS-defense 

system [16,17]. Of these, all were identified through reverse-genetic approaches in which the candidate factor 

was initially selected based on characteristics suggesting a role in virulence. For example, Cbp1, Yps3, and αglucan are specifically produced by the pathogenic-form (i.e., yeast) but not the mycelial form of Histoplasma. 

Siderophores and the superoxide dismutase/catalase antioxidant system are secreted factors that were 

hypothesized to interact with host factors as a consequence of their extracellular localization and putative 

function. Although a profitable approach, reverse genetics relies heavily on initial guesses regarding the 

virulence factors. Once candidates are selected, the difficult genetics of Histoplasma necessitates large efforts

to generate the gene knock-outs or knock-downs required for functional demonstration of their role in virulence.

Forward genetics approach to Histoplasma virulence. Application of forward genetics for dissection of 

Histoplasma pathogenesis provides an excellent approach to discovery of virulence factors. Time and again, 

forward genetics has been instrumental in establishing the molecular components that contribute to a biological

process. From identification of regulators controlling the cell cycle to discovery of genes controlling 

programmed cell death or body patterning, the ability to identify a gene through mutant phenotypes has 

provided key footholds necessary for developing a mechanistic understanding [18–20]. Often these factors 

and the mechanisms to which they contribute have been unsuspected gene products that would otherwise 

have been missed. In addition to the lack of assumptions or hypothesis bias in selecting candidate factors, a 

forward genetics approach immediately provides the mutant strains necessary for further functional tests.

We propose to discover new virulence factors contributing to Histoplasma pathogenesis through a forward 

genetics approach to identify mutants defective for virulence in macrophages. Towards this goal, we have 

Fungal p

 

Principal Investigator/Program Director (Last, first, middle): Rappleye, Chad, Alan 

Research Strategy Page 22
 
optimized insertional mutagenesis methodologies and have developed high-throughput screening techniques 

necessary for detection of intramacrophage virulence phenotypes. These innovations include the generation 

of colorimetric and fluorescent reporter lines of macrophages and yeast, respectively, to efficiently quantify 

intramacrophage yeasts and their ability to lyse host cells. Unlike virulence screens using solid-media plate 

phenotypes or unnatural hosts, we will use the actual host cell (i.e., the macrophage) in our screens. Some 

limited success with forward genetics in Histoplasma has been achieved in defining factors that control the 

morphological switch from mycelia to yeast as well as factors that contribute to environmental and temperature

stresses [21,22]. However, this proposal represents the first large-scale effort to generate a comprehensive 

library of Histoplasma mutants that define the factors required for infection, intracellular survival and growth, 

and lysis of host macrophages.

3. APPROACH

3A. PRELIMINARY STUDIES

T-DNA as an insertional mutagen. The use of a mutagen that provides random mutations as well as rapid 

mapping of the mutations is paramount to the success of a forward genetics screen. Agrobacterium has been 

utilized to transform or mutagenize many different fungal species through transfer and integration of a DNA 

element known as transfer DNA (“T-DNA”; [23,24]). We and others have shown that Agrobacterium-mediated 

transformation can be used as a mutagen for Histoplasma [22,25–29]. Insertional mutagens have the 

advantage of facilitating rapid mapping of the integration site by providing a known sequence anchor that can 

be used to isolate unknown sequences flanking the insertion element (i.e, regions adjacent to the T-DNA).

To optimize the mutagenesis efficiency, we examined different T-DNA elements and different Agrobacterium

strains. Transformation with some T-DNA elements results in a high frequency of carryover of non-T-DNA 

vector sequences beyond the left border (LB) and right border (RB) ends of the T-DNA element [30–32]. As 

mapping of the T-DNA relies upon having Histoplasma genomic sequence immediately adjacent to known ends

of the T-DNA element (see below), transfer of vector backbone or extra sequences frustrates efforts to identify 

the chromosomal site of T-DNA integration. We determined that the T-DNA element carried on plasmid pBHt2 

provides the highest efficiency of transformation (79 ± 20 per 5x107 yeast) and the lowest frequency of vector 

backbone carryover (8%) [26]. In addition, we determined that transfer of the T-DNA element on pBHt2 

preserves the LB and RB T-DNA ends in 87% and 94% of integration events. The T-DNA element on pBHt2 

carries the hygromycin phosphotransferase gene (hph) for hygromycin resistance-based selection of 

transformants in which the T-DNA element has stably integrated (Figure 1).

The precision of T-DNA integration enables the knownsequence of the LB and RB ends to be used as anchors

for mapping of the T-DNA insertions. We have used

thermal asymmetric interlaced PCR (“TAIL-PCR”, [33]), a

hemi-specific PCR technique, to efficiently map the

location of integrated T-DNA in Histoplasma [26,34]. A

nested series of primers matching the LB and RB ends is

combined with random degenerate primers that allow for

PCR-based amplification of sequences immediately

flanking the T-DNA LB and RB borders (Figure 1).

Proximal ends of the recovered sequences are matched

to the T-DNA to ensure the PCR products are anchored in

the T-DNA ends and the remaining sequence is matched

to the Histoplasma genome sequence to provide

information about the chromosomal context. We have

successfully mapped over 100 T-DNA insertions using this

procedure [26,34].

Integration of the T-DNA element from pBHt2 into the Histoplasma chromosome is random, an essential 

characteristic for unbiased mutagenesis. We mapped and examined the individual chromosomal location of 

the T-DNA element in 70 randomly selected Histoplasma transformants to determine the integration 

RB

LB RB

hph

pBHt2

LB

T-DNA

Figure 1. Schematic of the T-DNA element on plasmid pBHt2 

and following integration. The T-DNA element is bounded by 

left- and right border sequences (LB and RB). Short arrows 

indicate the nested LB- and RB-specific and the degenerate PCR 

primers used to amplify the Histoplasma genome sequence (green) 

flanking the T-DNA insertion.

 

Principal Investigator/Program Director (Last, first, middle): Rappleye, Chad, Alan 

Research Strategy Page 23
 
Table 1: Attenuated mutants

mutant attenuation locus

3G3 83% VPS41

4A7 74% VAT1

12B4 71% CBP1

2C10 70% PBS2

8D11 70% HCL1

8G9 66% HYP

7A9 64% PEX10

5D3 60% HYP

5G4 58% PCK1

15C2 57% (tbd)

5C12 56% HYP

5F3 55% HYP

13A6 51% VMA2

16E1 50% HSP82

8B1 44% HYP

2F2 43% PEX14

5E1 43% GPR1

1C8 42% (tbd)

2E10 35% GCN2

4C1 33% DDP1

4A6 31% DSC2

uninfected well infection with wild type (virulent control)

infection with 

attenuated 

mutants

Figure 2. Identification of attenuated mutants using the 

lacZ -macrophage screen . After 6 days of infection with 

different mutants, remaining macrophages were quantified 

by LacZ-based conversion of ONPG to its yellow product. 

Arrows indicate uninfected and wild-type controls as well as 

two attenuated mutants identified by their inability to reduce 

the macrophage popuation (resulting in high LacZ activity)

characteristics [26]. We found no evidence of insertional hotspots in the Histoplasma genome (defined as 

multiple insertions within 5000 base pairs of each other). While T-DNA integration is random relative to 

different genetic loci, the site of integration relative to an individual coding sequence often occurs in the 

promoter region of the gene (36% of integrations; [26]). Nonetheless, we have demonstrated that integrations 

into regions upstream of genes (promoters), as well as integrations into introns, effectively disrupt gene 

function thereby expanding the useful target region for mutagenesis beyond the coding sequence itself 

[22,26,35]. These data validate the use of T-DNA integration as a random insertional mutagen for Histoplasma

and establish the T-DNA element from pBHt2 as the optimal element with good transformation efficiency and 

good fidelity of integration (minimal LB and RB truncation and minimal carryover of backbone sequences 

flanking the LB and RB ends) permitting rapid mapping of T-DNA integrations.

Development of a high-throughput macrophage-based

virulence screen. The second requirement of a forward genetics

approach is a relevant and efficient screen for the phenotype of

interest. The primary limitation of screens for attenuated virulence

is the difficulty of screening large numbers of individual mutants

directly for decreased virulence. Since infection and residence

within macrophages is the central feature of Histoplasma

pathogenesis, we developed technologies to rapidly screen for

mutants defective for intramacrophage growth. The end result of

successful macrophage infection and yeast replication is lysis of

the macrophage. Thus, yeast virulence in macrophages is

inversely proportional to the number of surviving macrophages.

This permits identification of yeasts with attenuated virulence

based on their inability to reduce macrophage numbers following

co-culture of yeasts and macrophages. The following criteria

guided our assay design efforts: (1) the assay must measure only

macrophage numbers in a mixed population of macrophages and yeast, (2) the assay must be sensitive and 

quantitative in order to detect partial defects in virulence, and (3) the assay should require minimal 

manipulations to permit high-throughput screening. To meet these requirements, we engineered macrophages

to constitutively express a lacZ transgene (P388D1-lacZ cells) which can be readily assayed using a one-step 

lysis and assay buffer in combination with a chromogenic (ONPG; o-nitrophenylgalactopyranoside; Figure 2) or fluorescent (FDG; fluorescein digalactoside)

enzyme substrate [22]. Based on the observed assay variability following

infection with wild type Histoplasma yeast, we calculated that the sensitivity of

the assay is sufficient to discriminate 15% decreased rate of macrophage killing

by yeast (i.e., virulence attenuation) with 90% confidence (i.e., 90% statistical

power). As proof of principle, we demonstrated that the virulence attenuation of 

Histoplasma yeasts lacking the known Cbp1 virulence factor is accurately

recapitulated and quantified with LacZ expressing macrophages [22].

Using the macrophage lacZ screen, we have performed pilot screens of T-DNAmutagenized Histoplasma yeasts. Individual yeast transformants were picked

into wells of a 96-well plate and grown for 48 hours. Yeasts were diluted and

used to infect P388D1-lacZ macrophages at a multiplicity of infection (MOI) of

1:1 (yeasts to macrophages) in 96-well plates. We have screened 3478

independent Histoplasma mutants and have identified 21 mutants with

significantly reduced ability to lyse macrophages (at least 30% attenuation

compared to infections by virulent control Histoplasma yeasts; Table 1) and

whose attenuated phenotype was maintained in repeat assays. Among the

mutants isolated to date is an insertion in the promoter of the CBP1 gene 

(mutant 12B4) confirming the mutagenesis and screen will identify mutants with

attenuated virulence. The virulence mutant isolation frequency is 0.76% ±

0.30% (n=8). Mapping of the integration events in these mutants is ongoing as

is work to complement the mutations in order to confirm the phenotype is linked

to the mutation. We have already characterized one mutant isolated from the 

 

Principal Investigator/Program Director (Last, first, middle): Rappleye, Chad, Alan 

Research Strategy Page 24
 
screen with an insertion in the HSP82 promoter and have demonstrated that the 82 kDa heat shock protein is 

requi

ui

red for full Histoplasma virulence [22]. Importantly, this work also confirms that mutation of genes 

req red for virulence in macrophages translates into attenuated virulence in vivo. Notable, even in this small 

collection of mutants, are a number hypothetical or genes of unknown function (“HYP”; Table 1).

Fluorescent reporter yeast to quantify intramacrophage growth. To facilitate more rapid classification of 

the virulence defects associated with mutants identified using the macrophage screen, we developed a nondestructive method for quantifying intramacrophage yeasts. Impaired ability of yeasts to destroy macrophages 

could result from impaired entry, decreased survival in macrophages, defects

in intramacrophage replication, or loss of the ability to trigger macrophage

lysis. The number of intracellular yeast can be used to distinguish between

these classes as low, declining, static, or increasing numbers of intracellular

yeasts, respectively. Typically quantification of intracellular yeasts requires

plating of ma

ver, thi

y due to

res sub

crophage lysates to enumerate yeast colony forming units (CFU).

Howe s is not practical for high-throughput screens as it is an endpoint

assa destruction of the macrophages to recover the yeast and it

requi stantial manual processing of each sample. To overcome these

restrictions, we engineered Histoplasma yeast to constitutively express the

tandom-dimer red-fluorescent protein (tdTomato-RFP) which permits

quantification of intracellular yeasts directly in macrophages based on the total

RFP-fluorescence in wells with infected macrophages (Figure 3; [36]). As the fluorescence can be quantified 

without destruction of the macrophages, the kinetics of intracellular yeast replication can be readily assessed. 

We have confirmed that RFP fluorescence is proportional to yeast cell numbers and that RFP fluorescence 

reports intracellular replication (Figure 4). Most importantly, the RFP-fluorescent Histoplasma strain has 

identical entry rate, intracellular survival, and growth within macrophages as wild-type yeasts (Figure 5). The 

ability to monitor intracellular replication of yeasts and the quantification of macrophage killing by yeasts 

described above combine to create a powerful screen to identify and classify Histoplasma mutants with 

impaired virulence in macrophages.

3B. Experimental Approach

Aim 1. Screen Histoplasma T-DNA insertion mutants for attenuated virulence in macrophages.

Rationale. To discover new factors contributing to Histoplasma virulence in macrophages, we will perform a 

large scale genetic screen designed to identify mutants with attenuated virulence. In preliminary studies, we 

satisfied the two major requirements for our forward genetics approach, namely (1) use of an optimal mutagen 

(i.e., random insertional mutagenesis by T-DNA integration) and (2) development of a relevant phenotypic 

screen, which is amenable to high-throughput through the use of macrophage and yeast reporter backgrounds.

Aim 1A. Generate a library of T-DNA insertion mutants in Histoplasma yeast. Agrobacterium-mediated 

transformation of Histoplasma will be used as the insertional mutagen. The characterized T-DNA element on 

vector pBHt2 will be used as it provides high transformation efficiency (i.e., mutagenesis) and good fidelity of TRFP

Macrophage (DIC) 

Yeasts (Uvitex)

Figure 3. Intramacrophage RFPfluorescent Histoplasma yeasts.

A B yeast density (OD 595) RFP Fluorescence RFP Fluorescence

in vitro intramacrophage

Figure 4. RFP fluorescence can be used as a surrogate means to quantify 

yeasts in vitro and in macrophages. (A) Liquid cultures of RFP-fluorescent yeasts 

were measured for yeast density (turbidity at 595 nm) and RFP fluorescence. (B) 

RFP-fluorescence of intramacrophage yeasts following macrophage infection.

Figure 5. RFP-fluorescent yeasts have equivalent 

survival and growth in macrophages compared to 

wild-type yeasts . Data indicates the viable yeast CFUs 

recovered from macrophages over time.

 

Principal Investigator/Program Director (Last, first, middle): Rappleye, Chad, Alan 

Research Strategy Page 25
 
DNA integration. The RFP-fluorescent Histoplasma yeast strain will be used as the genetic background for the

utagenesis as it provides a means of quantifying intramacrophage yeast replication. Yeast cells will be 

ansformed by co-cultivation of Agrobacterium and Histoplasma yeasts after which yeast transformants will be 

elected on media with hygromycin. Individual transformants will be picked into Histoplasma growth medium 

nd arrayed into 96-well plates. Aliquots of each 96-well plate will be frozen at -80°C for long term 

preservation of the mutant library and for a resource for other researchers to use in other screens. The 

arrayed library will also benefit targeted mutation approaches in which the library can be screened by PCR for 

insertions in specific genes of interest [35].

The purpose of the large scale screen is to recover at least one mutation in each

gene contributing to virulence. To estimate the number of mutants necessary for

2.5-fold coverage of the encoded genes in the Histoplasma genome, we used

parameters on the number and size of genes that were determined from our

recently completed transcriptome project using multiple strains and growth

conditions (Table 2). We estimate the size of the average genetic locus

(promoter plus the gene) to be approximately 2600 base pairs. This number

encompasses the effective target region for gene disruptions since insertions in promoters and introns as well 

as the gene proper are also effective in preventing gene functions. The average gene in Histoplasma consists 

of 3 exons and 2041 base pairs of coding sequence (Table 2). To this, 500 base pairs were added as a 

conservative estimate of a typical promoter based on data from promoter-reporter gene fusions constructed in 

our laboratory (unpublished data). 80% of introns in Histoplasma range from 54 to 170 base pairs based on 

our transcriptome analysis (unpublished data), and an additional 50 base pairs for each of two introns were 

included. Using 2600 base pairs as an effective target region for gene-disrupting T-DNA integrations, a single 

mutation in each 2600 base pair region of the genome requires 15,769 mutants. 39,423 mutants provides for 

2.5-fold coverage. This number is clearly an overestimate of the mutants needed as the number of nonessential genes (and thus recoverable mutations in vitro) is much lower. We plan to generate a library of 

approximately 40,000 mutants. This will require approximately 400 96-well plates which can be feasibly 

accomplished in about 1 year by manually arraying 8 to 10 96-well plates of mutants per week.

Aim 1B. Identification of mutants deficient in survival and replication within macrophages. Recovered 

mutants arrayed into 96-well plates will be used to infect P388D1-lacZ macrophages at an MOI of 1:1. After 2 

hours, the medium will be replaced to remove non-internalized yeast (2 hours is sufficient for internalization of 

>90% of yeast). Yeast replication will be monitored daily over 6 days by scanning wells with infected 

macrophages for Histoplasma yeast-expressed RFP fluorescence. After 6 days, the relative number of 

surviving macrophages will be quantified by measurement of the macrophage-expressed LacZ activity. 

Results will be compared to control wells present on each plate that include (1) uninfected macrophages, (2) 

macrophages infected with non-mutagenized parental RFP-fluorescent yeast (virulent control), and (3) 

macrophages infected with a cbp1 mutant strain (attenuated control). Yeast replication results will be 

compared to the virulent and attenuated control to identify mutants deficient for intracellular survival and 

replication and those showing at least 50% reduction in intramacrophage yeast proliferation will be selected for

secondary screens. Mutants showing at least a 50% reduction in the ability to destroy macrophages compared

to the virulent control will also be selected for follow-up. Presumably, there will be significant overlap in these 

collections. Selected mutants will be recovered from the inoculum plates and the virulence phenotypes 

confirmed by infection of macrophages in triplicate and at multiple MOIs.

Aim 2. Determine the identify of genes required for Histoplasma virulence in macrophages.

Rationale. Based on the frequency of attenuated mutant isolation observed in preliminary studies (0.76%) and 

the proposed number of mutants to be generated (40,000), we expect isolation of approximately 300 mutants. 

These will need to be prioritized for further study and characterization. Prioritization will be based on the 

identify of the genes disrupted and their putative functions as well as classification and ranking of the severity 

of the mutant phenotypes.

m

tr

s

a

genome size 41 Mb

total genes 9359

avg CDS length 2041 bp

avg exons / gene 3.1

gene density / 10 kb 2.3

Table 2: Transcriptome statistics

 

Principal Investigator/Program Director (Last, first, middle): Rappleye, Chad, Alan 

Research Strategy Page 26
 
Aim 2A. Map the disrupted loci in attenuated mutants. The identity of loci required for full virulence in 

macrophages will be determined by mapping of the T-DNA integrations in attenuated mutants. To rapidly 

identify mutants representing known virulence factors, mutants will be initially screened by PCR for T-DNA 

insertions in the CBP1 (Cbp1 virulence factor), SOD3 (extracellular superoxide dismutase), CATB (extracellular

catalase), SID1 (siderophore biosynthesis), SRE1 (siderophore biosynthesis), and YPS3 (secreted factor) 

genes. Mutations in new genes will be mapped by TAIL-PCR using DNA prepared from each mutant and 

primers directed outward from the LB and RB ends of the T-DNA. The PCR products generated, representing 

sequences flanking the T-DNA element, will be matched to the G217B genome sequence [37] using the 

BLAST algorithm. The identify of the disrupted genes (or integration into their respective promoters) will be 

determined by mapping the position of T-DNA integration with that of gene models as determined by 

transcriptome sequencing (unpublished data). This analysis will also enable identification of those mutants 

representing multiple insertions in a single locus. Putative virulence gene functions will be determined by 

BLAST of the disrupted locus to existing protein databases to identify homologous genes as well as GO-term 

annotation of the transcriptome. 

Aim 2B. Classify and prioritize virulence mutants. Attenuated

mutants will be classified based on their intramacrophage growth

phenotype. It is anticipated that attenuated mutants will be deficient to

varying degrees in their ability to cause lysis of the macrophage host cell

and this will be the primary measure of virulence attenuation. Further

classification of these mutants will include: defects in macrophage entry

(low RFP fluorescence) defects in intramacrophage Histoplasma survival

(declining RFP fluorescence during the infection time course), defects in

intramacrophage growth (static but not declining RFP fluorescence), and

defects in causing macrophage lysis (increasing RFP fluorescence yet

lack of significant effects on macrophage survival) (Figure 6).

Secondary ranking of mutants will be done according to the severity of

the defect in intramacrophage yeast growth kinetics as reflected by RFP

fluorescence. For initial rankings, mutants that show impaired growth

outside of macrophages (i.e., in liquid culture) will be lowered in priority.

The most severely attenuated mutants will be further prioritized according to the candidate gene identities and 

their potential connections to virulence. For example, secreted factors (as predicted by the Signal P algorithm 

[38]) will be prioritized since extracellular proteins are strong candidates for factors that directly interact with 

and potentially modulate the host cell. As the goal of this study is to identify unsuspected factors, novel genes 

(or genes without a known function) will also be selected. Two criteria will be used to prioritize novel factors: 

conservation of the factor among intracellular pathogens and preferential expression by pathogenic 

Histoplasma yeast compared to the non-virulent mycelial phase Histoplasma cells. Transcriptional profiles will 

be determined using the Histoplasma yeast- and mycelial phase transcriptomes recently assembled by our 

laboratory (unpublished data). From these criteria we anticipate selecting the top 10% of virulence genes for 

further study.

4. FUTURE DIRECTIONS.

The virulence genes identified through this forward genetics approach will be further characterized in future 

studies to describe the mechanisms that facilitate Histoplasma survival and growth within host macrophages. 

For prioritized genes, immediate efforts will focus on complementation of the mutations to directly link loss of 

virulence to disruption of the candidate locus. In addition, we will confirm that mutants with defects in 

macrophage virulence are similarly attenuated in vivo using a murine inhalational model of respiratory and 

disseminated histoplasmosis. We will subsequently define, with finer resolution, the nature of the virulence 

attenuation through cellular markers of intramacrophage trafficking. Further characterization of the roles of 

new virulence genes will be guided by gene identities and the relevant defects in stages of macrophage 

infection. Our experience and expertise in molecular manipulation and cellular analyses of Histoplasma and 

host cells will enable us to readily move beyond virulence factor identification. These follow up studies will 

provide a mechanistic understanding of how Histoplasma yeasts successfully infect, survive, and replicate 

within macrophages thereby subverting innate immune defenses of the host.

time

RFP fluorescence

time

RFP fluorescence

time

RFP fluorescence

time

RFP fluorescence

entry 

defects

survival 

defects

intramacrophage 

growth defects

lysis 

defects

Figure 6. Predicted intramacrophage yeast 

RFP fluorescence kinetics by mutant class. 

 

Principal Investigator/Program Director (Last, first, middle): Rappleye, Chad, Alan 

Research Strategy Page 27
 
 

Principal Investigator/Program Director (Last, first, middle): Rappleye, Chad, Alan 

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