PI: Raghavan, Rahul Title: Elucidating the evolution of Coxiella to uncover critical metabolic pathways

Received: 06/20/2016 FOA: PA16-200 Council: 01/2017

Competition ID: FORMS-D FOA Title: Academic Research Enhancement Award (Parent R15)

1 R15 AI126385-01A1 Dual: Accession Number: 3949655

IPF: 6297008 Organization: PORTLAND STATE UNIVERSITY

Former Number: Department:

IRG/SRG: ZRG1 IDM-S (81)A AIDS: N Expedited: N

Subtotal Direct Costs 

(excludes consortium F&A) 

Year 1:

Animals: N 

Humans: N 

Clinical Trial: N

Current HS Code: 10

HESC: N

New Investigator: N

Early Stage Investigator: N

Senior/Key Personnel: Organization: Role Category:

Rahul Raghavan PORTLAND STATE UNIVERSITY PD/PI 

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Elucidating the evolution of Coxiella to uncover critical metabolic pathways

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04/01/2017 03/31/2020

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SF 424 (R&R) APPLICATION FOR FEDERAL ASSISTANCE Page 2

14. PROJECT DIRECTOR/PRINCIPAL INVESTIGATOR CONTACT INFORMATION

Prefix: First Name*: Rahul Middle Name: Last Name*: Raghavan Suffix: 

Position/Title: Assistant Professor

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❍ PROGRAM HAS NOT BEEN SELECTED BY STATE FOR

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424 R&R and PHS-398 Specific

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

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

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 Bibliography & References Cited----------------------------------------------------------- 41

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Table of Contents Page 3
	
Contact PD/PI: Raghavan, Rahul 

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Tracking Number: GRANT12198450 Funding Opportunity Number: PA-16-200. Received Date:

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OMB Number: 4040-0001

Expiration Date: 06/30/2016

RESEARCH & RELATED Other Project Information

1. Are Human Subjects Involved?* ❍ Yes ● No

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If YES, check appropriate exemption number: 1 2 3 4 5 6

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Is the IACUC review Pending? ❍ Yes ❍ No

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3. Is proprietary/privileged information included in the application?* ❍ Yes ● No

4.a. Does this project have an actual or potential impact - positive or negative - on the environment?* ❍ Yes ● No

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environmental assessment (EA) or environmental impact statement (EIS) been performed?

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collaborators?*

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7. Project Summary/Abstract* ProjectSummary-R15-2016-

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9. Bibliography & References Cited References-R15-2016-resub-v5.pdf Mime Type: application/pdf

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Contact PD/PI: Raghavan, Rahul 

 Page 5
	
PROJECT SUMMARY/ABSTRACT

Coxiella burnetii’s pathogenicity depends on its ability to grow in a lysosome-derived hostile vacuole within 

human cells. However, metabolic processes critical to C. burnetii’s intracellular growth are largely unknown. 

This lack of knowledge has prevented both the understanding of its basic biology and pathogenesis, and the 

development of better therapeutic agents. The long-term goal is to understand the molecular details of Coxiella’s distinctive physiology, and to apply this knowledge to developing novel therapeutic strategies. The objective of this application is to identify metabolic pathways that are vital to C. burnetii’s intracellular growth. The 

central hypothesis, which was formulated based on preliminary data, is that C. burnetii evolved from a tickassociated ancestor by acquiring critical metabolic genes through horizontal gene transfer (HGT). A novel evolutionary genomics approach will be used to identify metabolic pathways that are critical to C. burnetii’s intracellular growth. The rationale for the proposed research is that once metabolic processes important to C. burnetii’s intracellular growth are identified, pharmacological agents that block these pathways could be developed 

to treat chronic infections more effectively. The objective of this project will be accomplished by three specific 

aims: (1) Identify metabolic pathways that distinguish C. burnetii from tick-associated Coxiella. The working 

hypothesis is that genes critical to C. burnetii’s intracellular physiology will not be present in avirulent tickassociated Coxiella. The genome of a closely related Coxiella from the tick Ornithodoros rostratus will be sequenced and compared to C. burnetii’s genome. (2) Define metabolic pathways that are critical to C. burnetii’s 

intracellular growth. The working hypothesis is that genes acquired via HGT are being maintained in C. burnetii because they are critical to the pathogen’s physiology. Phylogenetic approaches will be used to identify 

HGT-derived genes, and their functions will be validated using RNA-seq and genetic tools. (3) As a proof of 

principle, determine the importance of heme biosynthesis to C. burnetii’s intracellular growth. The working 

hypothesis is that heme biosynthesis is crucial to Coxiella’s growth. Heme production and intracellular growth 

of heme pathway-deficient strains will be assayed. This study is innovative because it (a) uses a novel approach 

that overcomes the current limitations in studying Coxiella at a genome-wide scale, and (b) is based on a novel 

concept that the human pathogen evolved from a tick symbiont via massive HGT. The proposed project is significant because it will (a) uncover metabolic pathways that are critical to the pathogen’s intracellular growth, 

(b) identify new therapeutic targets, for example, HemA and HemL are essential for heme biosynthesis in C. 

burnetii but are not present in humans cells, (c) provide a model approach for identifying genes involved in 

host adaptation, which could be applied broadly to other pathogens such as Francisella and Rickettsia spp. 

where avirulent tick symbionts could be compared to virulent human-specialized strains.

 

Contact PD/PI: Raghavan, Rahul 

Project Summary/Abstract Page 6
	
Relevance to public health

Coxiella burnetii is a zoonotic bacterium that causes Q fever and chronic endocarditis. Metabolic processes

critical to its intracellular growth and pathogenesis are not known. The proposed project will identify 

important metabolic pathways in C. burnetii using a novel evolutionary genomics approach that will 

overcome the current technical difficulties in studying Coxiella at a genome-wide scale. The application will 

also provide a useful model for identifying key metabolic processes in other human pathogens and will 

identify new genes that could be targeted to treat chronic Q fever more effectively.

 

Contact PD/PI: Raghavan, Rahul 

Project Narrative Page 7
	
FACILITIES AND OTHER RESOURCES

Laboratory: 

The PI’s main laboratory is located in the Collaborative Life Sciences building (CLSB), a new academic and 

research facility shared between Portland State University (PSU), Oregon Health and Science University 

(OHSU), and Oregon State University (OSU). The main lab is ~900 sq. ft. with four lab benches and is 

equipped to perform all proposed molecular biology experiments. The lab is part of an open floor plan and is 

flanked by labs from OHSU and OSU, which foster natural collaborations. A separate BSL-2 certified ~200 

sq. ft. room is equipped with biosafety hoods, CO2 incubators, inverted microscope etc. to perform bacterial 

culture and host cell infection assays. Two walk-in cold rooms, a shared equipment room, a microscopy 

room, and a sterilization room equipped with autoclave and dishwasher are located immediately adjacent to 

the main lab. 

Animal: None

Clinical: None

Computer: 

The PI’s laboratory is equipped with two Linux servers (24 core with 256 GB RAM; 12 core with 128 GB 

RAM), one MacPro (64 GB RAM, 2 TB hard drive), two iMac computers, one MacBook Pro, several PCs, and 

a 10 TB RAID storage array. We also have access to three university-wide research servers (12 core with 96 

GB RAM; 16 core with 126 GB RAM; 28 core with 250 GB RAM) and a 23-node, 184-core parallelprocessing Linux cluster. In addition, there is a 20-terminal computer lab in the building. The whole

building has wireless and hard-wired access to the local network and to the Internet. Two full-time IT 

technicians serve the Biology department.

Office: 

The PI has a ~150 sq. ft. office with a MacBook Pro computer. In addition, there is a second ~150 sq. ft. 

office available for postdocs and cubicles with data and phone connections for graduate students.

Other:

The research in the PI’s lab benefits from close collaborations with OHSU and OSU faculty members that 

occupy the shared building (CLSB) where the PI’s lab is located. In addition, the PI’s lab maintains regular 

contact by sharing reagents, performing collaborative experiments, and conducting combined lab meetings

with several research groups at the OHSU main campus located within a 5-minute tram ride from CLSB. 

These research groups include Dr. Richard Goodman’s lab that work on eukaryotic microRNA, Dr. Scott 

Landfear’s group that work with the eukaryotic pathogen Leishmania, Dr. Eric Cambronne’s group that 

works on the pathogenesis of Legionella pneumophila, and Dr. Justin Merritt’s lab that works on the 

genetics of the oral pathogen Streptococcus mutans. In addition, the PI has access to all OHSU core 

facilities (Proteomics, Genomics, Sanger sequencing, Microscopy etc.). 

A profile of the applicant’s institution:

Portland State University is a public state urban university located in downtown Portland, Oregon. It is a 

relatively young school (founded in 1946), but is one of the largest universities in Oregon. A combination of 

targeted outreach programs and proximity to the state’s population and industry results in an unusually 

large number of first generation college students attending PSU. In 2014, approximately 30,000 students 

were enrolled in PSU and 4300 bachelor’s degrees and 1700 graduate degrees were awarded. PSU places 

more students into medical schools than any other university in Oregon, and a large portion of our students 

have gone on to pursue successful research careers.

The Department of Biology has approximately 1000 undergraduate majors and about 70 graduate 

students. Student research is highly encouraged in the department and each PI’s lab typically has three to 

four undergraduate student research trainees at any given time. Undergraduate research training at PSU is 

fostered by regular, one-on-one interactions between students and the PI. Several mechanisms are in place 

 

Contact PD/PI: Raghavan, Rahul 

Facilities & Other Resources Page 8
	
at the Biology department to foster research activity by students, including the Biology Honors program, the 

Forbes Lea Endowed Fund for Student Research, and The David Clark Endowment Fund that supports 

student travel to scientific meetings. Additionally, students are exposed to cutting edge science on a daily 

basis because most biology lectures and labs are held at CLSB, which houses the OHSU Dental School, parts 

of OHSU Medical School, OSU College of Pharmacy, and 30 research labs from OHSU, OSU and PSU.

Impact of an AREA award on student research:

PSU is not a research-intensive university when compared to major academic research institutions in the 

United States. However, PSU is highly committed to growing its research infrastructure. R&D expenditures 

continue to grow, with an average annual growth rate over the past 10 years of 10.6%. PSU’s Science 

Building has recently undergone a major ($45.3 million) renovation and now has improved research and 

teaching labs. Recently PSU in collaboration with OHSU and OSU set up the Collaborative Life Sciences 

building (CLSB), a unique $295 million project that combines the resources of three major universities to 

offer the best possible science education and research opportunities for students. Biology and Chemistry 

teaching labs and four PSU research groups (including the PI’s lab) are now located at CLSB. The Biology 

department has a robust research program. Currently there are 29 externally supported projects (two in PI’s 

lab) among the 20 faculty members. NSF supports most of these projects and NIH funds five. The PI is 

highly committed to student research. The PI has mentored and published with several undergraduate 

students during his graduate and postdoctoral tenures (list provided in biosketch). The PI currently has 

three undergraduate research trainees in the lab and four graduate students. In addition to his research 

program, the PI teaches courses in Microbiology (BI480), and Bioinformatics (BI410/510) that offer the 

opportunity to introduce biomedical research to undergraduates, and to attract students to partake in 

research experience. He therefore has an opportunity to significantly impact the number of graduates at his 

institution that go on to pursue health-related careers. AREA awards are critical to this mission. 

The work proposed here has been designed and structured to provide research opportunities to several 

students over the three-year life of the grant. Jessica Millar, a current graduate student in the lab 

generated most of the preliminary bioinformatics data for this project while she was an undergraduate 

researcher. Abraham Moses started as an undergraduate researcher in the lab and now is a Research 

Associate in the lab. He is adept at Coxiella culture and helped to generate the experimental portion of the 

preliminary data. Currently he is working with Todd Hinsch, an undergraduate researcher to construct 

pJC-CAT vectors to delete Coxiella small RNA genes. Jonathan Gerhart, who is a first year graduate 

student in the lab, is working closely with Melissa Holler an undergraduate researcher to sequence the 

genomes of endosymbionts present in various ticks. 

Institutional investment in the success of the Early Stage Investigator:

The Chair of Biology Department will provide a modest teaching load to the PI to facilitate the research 

outlined in the proposal (please see Letter of Support from the Chair of Biology Department). The PI has 

been assured of continued assignment of space and will be assigned more space if required. The PI has full 

access to a variety of shared equipment, research facilities and cores across PSU and OHSU campuses. In 

addition to providing the PI with a substantial startup package, PSU has supported the PI by providing a 

Research Stimulus Grant and a Faculty Enhancement Grant, and the Biology department supports graduate 

students in PI’s lab through graduate assistantships. Additionally, The Medical Research Foundation at 

OHSU has also supported the PI’s research by providing a New Investigator Grant. Moreover, the PI’s lab is 

part of a consortium of 30 research groups from OHSU, OSU and PSU that collaborate extensively by 

sharing equipment and reagents, and by holding regular meeting and social events.

 

Contact PD/PI: Raghavan, Rahul 

Facilities & Other Resources Page 9
	
MAJOR EQUIPMENT: 

Items within PI’s laboratory:

Eppendorf gradient thermal cycler, ABI thermal cycler, Agilent real-time thermal cycler, New Brunswick Trigas incubator, New Brunswick CO2 incubator, bacterial incubators, shaking incubators, Perkin-Elmer Victor

Multilabel plate reader, Bio-Rad spectrophotometer, BioSpec beadbeater, refrigerators, –20°C freezers, –80°C 

freezer, Eppendorf thermomixer, centrifuges, shaking platforms, gel electrophoresis equipment, Gel 

documentation system, Thermo Scientific Biosafety hoods, NanoDrop, Leica DM750 phase contrast 

microscope, Leica DMIL inverted microscope, Leica TCS SPE Confocal microscope, high performance 

computers.

Common items available to PI: 

Agilent Bio-analyzer, Ultracentrifuges, Li-Cor Odyssey infrared imaging system, Covaris S2 ultrasonicator, 

MALDI-TOF Mass spec, Shimadzu HPLC, scanning electron microscope, transmission electron microscope,

fluorescent microscope, Ion Proton sequencer, cold rooms, high performance computer network and servers. 

 

Contact PD/PI: Raghavan, Rahul 

Equipment Page 10
	
Contact PD/PI: Raghavan, Rahul 

OMB Number: 4040-0001

Expiration Date: 06/30/2016

Tracking Number: GRANT12198450 Funding Opportunity Number: PA-16-200 . Received Date:

2016-06-20T20:28:55.000-04:00

RESEARCH & RELATED Senior/Key Person Profile (Expanded)

PROFILE - Project Director/Principal Investigator

Prefix:  First Name*: Rahul Middle Name  Last Name*: Raghavan Suffix: 

Position/Title*: Assistant Professor

Organization Name*: PORTLAND STATE UNIVERSITY

Department:

Division:

Street1*:

Street2:

City*:

County:

State*:

Province:

Country*:

Zip / Postal Code*:

Phone

Number*: 

Fax Number:  E-Mail*: 

Credential, e.g., agency login: 

Project Role*:  PD/PI Other Project Role Category: 

Degree Type:  PhD Degree Year:  2008

File Name Mime Type

Attach Biographical Sketch*: Biosketch-R15-2016-resub.pdf application/pdf

Attach Current & Pending Support:

 Page 11 
 

Contact PD/PI: Raghavan, Rahul 

BIOGRAPHICAL SKETCH

Provide the following information for the Senior/key personnel and other significant contributors.

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

NAME: Rahul Raghavan

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

POSITION TITLE: Assistant Professor of Biology

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

include postdoctoral training and residency training if applicable. Add/delete rows as necessary.)

INSTITUTION AND LOCATION

DEGREE

(if 

applicable)

Completion 

Date

MM/YYYY

FIELD OF STUDY

University of Calicut, India BSc 08/1995 Biology and Chemistry

Manipal University, India MSc 08/1998 Medical Microbiology

University of Montana, Missoula, MT PhD 08/2008 Molecular Microbiology

Yale University, New Haven, CT Postdoc 08/2012 Microbial Genomics

A. PERSONAL STATEMENT

Even after seven decades of research, most aspects about the origin, basic biology, and pathogenesis of Coxiella 

burnetii remain unknown. One of the biggest gaps is in our understanding of the pathogen’s unique physiology, 

which is critical to its pathogenicity. I propose to use a novel evolutionary-genomic approach to uncover C. 

burnetii’s evolutionary history, and use this knowledge to define metabolic genes critical to its intracellular 

growth and virulence. The outcomes of this project will positively impact the field by providing a set of 

metabolic proteins that could be targeted to develop novel therapeutic agents. I am ideally prepared to 

undertake this project because of my unique combination of expertise in molecular microbiology, 

bioinformatics, high-throughput sequencing, phylogenetics and genomics. I have also setup ongoing

collaborations with establish researchers to ensure the success of the proposed project (see Letters of Support). 

As a doctoral student in Dr. Mike Minnick’s lab (2004-2008), I got trained in working with Coxiella by 

characterizing horizontally acquired selfish genetic elements. I discovered that C. burnetii’s growth is likely 

regulated by introns (ribozymes) through translational inhibition, and showed that by blocking intron splicing 

using Pentamidine, growth of the bacterium could be significantly reduced, thus identifying a novel therapeutic 

strategy. In 2009, I moved to Dr. Howard Ochman’s lab as a postdoc where I was trained in bioinformatics, 

high-throughput sequencing and genome evolution. In my new lab at PSU (since Fall 2012), I have applied my 

knowledge in pathogenomics to explore entirely new areas of Coxiella research: (1) In collaboration with Dr. 

Minnick, we identified the first set of non-coding RNAs in C. burnetii; (2) in collaboration with Drs. Eric 

Cambronne and Scott Landfear at OHSU, we described the first set of microRNAs produced by human 

macrophages in response to C. burnetii infection; (3) in collaboration with Dr. Joseph Gillespie at University of 

Maryland, we sequenced the first genome of a non-pathogenic Coxiella. In total, I have published 14 peerreviewed articles on Coxiella, and have nine publications on various aspects of bacterial genome analyses.

An AREA grant will provide me the opportunity to build on my initial successes to set up a nationally 

recognized research program. Although PSU is not a research-intensive university, I have the full support of my 

department and college to pursue cutting edge research. Moreover, my research group is part of a highly 

interactive and productive research environment where scientists from PSU, Oregon Health and Sciences 

University, and Oregon State University conduct collaborative research. The AREA grant will also help support 

student research in my lab. I currently have three undergraduate researchers in my lab. In the three years at 

PSU, I have mentored 15 undergraduate students, including two students who performed their Honors theses 

in my lab. I also mentor four graduate students at various stages of their career (a student who graduated this 

year, a 2nd year student, and two 1st year students). Two of my graduate students, our lab manager, our

Biosketches Page 12
	
Bioinformatician, and IT manager started out as undergraduate researchers in my lab. This organic approach 

has been very fruitful because all of them are very productive; e.g., my senior grad student (Fenil) has three

publications; my second year student (Jess), who received NSF GRFP, has two publications with another under 

review, and another in development, and a first year student (Jon) has already submitted his first first-author 

paper. In addition, several undergraduate students who worked in my lab were able to find employment as 

research technicians at OHSU and other research centers in Portland. I also encourage students to attend local 

and national conferences (one student received an ASM travel award). I meet with each student regularly and 

the whole lab meets weekly to discuss latest research or new journal articles. Once a month we conduct a 

combined lab meeting with two other labs at OHSU (Dr. Justin Merritt’s and Dr. Eric Cambronne’s labs), 

providing the students with a broad perspective on microbial research. Some of my recent Coxiella papers:

1. Raghavan R. 2016. A repeat motif on a Coxiella effector protein facilitates apoptosis inhibition. 

Virulence 7:369-371. 

2. Millar JA, Valdes R, Kacharia FR, Landfear SM, Cambronne ED, Raghavan R. 2015. Coxiella burnetii

and Leishmania mexicana residing within similar parasitophorous vacuoles elicit disparate host 

responses. Front Microbiol 6:794.

3. Smith TA, Driscoll T, Gillespie JJ, Raghavan R. 2015. A Coxiella-like endosymbiont is a potential 

vitamin source for the Lone Star Tick. Genome Biol Evol 7:831-838. 

4. Warrier I, Hicks LD, Battisti JM, Raghavan R, Minnick MF. 2014. Identification of Novel small RNAs 

and characterization of the 6S RNA of Coxiella burnetii. PLoS ONE 9:e100147.

B. POSITIONS AND HONORS

Positions and Employment

1998-02 Lecturer, School of Health Sciences, University of Calicut, India

2002-04 Research Scientist, St John’s Research Institute, Bangalore, India

2004-08 Graduate Assistant. Division of Biological Sciences, University of Montana

2008-09 Postdoctoral Fellow, Division of Biological Sciences, University of Montana

2009-12 Postdoctoral Associate, Yale University (Started at University of Arizona)

2012-present Assistant Professor, Department of Biology, Portland State University

Teaching Awards 

2005 Outstanding Teaching Assistant. Division of Biological Sciences, University of Montana 

2015 Commendation for excellent teaching from Portland State University College of Liberal Arts and 

Sciences.

Other Awards and Honors 

2006 Travel Award from the American Society for Rickettsiology.

2007 Co-chaired the scientific session on Genetics, Physiology and Metabolism at the 21st Annual Meeting 

of the American Society for Rickettsiology. Colorado Springs, CO. 

2016 Travel Award from the American Society for Rickettsiology.

Professional Membership

American Society for Microbiology 

American Society for Rickettsiology 

Society for Molecular Biology and Evolution 

American Heart Association

Students mentored 

During PhD

Linda Hicks (Undergraduate, University of Montana, Missoula, MT): 2005.

During Postdoc

Ellen DeGennaro (Undergraduate, Vassar College, Poughkeepsie, NY): 2010.

Alan Sage (Undergraduate, Yale University, New Haven, CT): 2010.

Cindy Barlan (MS student, Quinnipiac University, Hamden, CT): 2011.

 

Contact PD/PI: Raghavan, Rahul 

Biosketches Page 13
	
Daniel Phillips (MS student, University of New Haven, West Haven, CT): 2011.

As PI (current students underlined)

Christine Sislak (MS student): 2012-2013.

Niveditha Handral (Undergraduate): 2012.

Jess Millar (Undergraduate, later joined the lab as graduate student): 2012-2016. 

Brian Wendel (Graduate student rotation): 2013.

Abraham Moses (Undergraduate, later joined the lab as Research Associate): 2012-2016. 

Katharine Saucier (Undergraduate): 2012-2014.

Todd Smith (Undergraduate, later joined the lab as Bioinformatician): 2012-2015. 

Savannah Smart (Undergraduate): 2013-2014.

Fenil Kacharia (Undergraduate, later joined the lab as graduate student): 2013-2016. 

Colleen Campbell (Undergraduate): 2013-2014. 

Ashleigh Mustain (Undergraduate): 2013-2014. 

Ali Hourmanish (Undergraduate): 2013-2014. 

Rachel Champaigne (Honors student): 2013-2015

Robyn Reid (Honors student): 2013-2015

Melissa Hollar (Undergraduate): 2014-2016

Todd Hinsch (Undergraduate): 2014-2016

Jim Archuleta (Undergraduate, also works as the lab IT manager: 2015-2016)

Jonathan Gerhart (Graduate student): 2015-2016

Austin Wright (Graduate student): 2015-2016

Selected Presentations at Professional Meetings (students underlined)

1. Moses AS, Millar JA, Bonazzi M, Beare PA, Raghavan R. 2016. Horizontally acquired tRNA and 

metabolic genes fortify Coxiella burnetii’s physiology. National Meeting of the American society for 

Rickettsiology. Big Sky, MT.

2. Millar JA, Raghavan R. 2016. A sewage microbiome is dominated by Arcobacter cryaerophilus that 

expresses multiple drug resistance and virulence genes. ASM Microbe, Boston, MA.

3. Gerhart JG, Raghavan. 2016. A Francisella-like endosymbiont in the Gulf Coast tick evolved from a 

mammalian pathogen. Entomological Society of America North Central Meeting. Cleveland, OH. 

4. Kacharia F, Merritt J, Raghavan R. 2015. Evolution of regulatory RNAs from vestigial bacteriophage 

genes. Regulating with RNA in Bacteria and Archaea Conference. Cancun, Mexico.

5. Kacharia F, Raghavan R. 2015. Quantifying evolution to gauge function. Annual Meeting of the Pacific 

Northwest Quantitative Biology Group. Portland, OR.

6. Smith TA, Raghavan R. 2015. A Coxiella-like endosymbiont is a potential vitamin source for the Lone 

Star Tick. National Meeting of the American society for Rickettsiology, Lake Tahoe, CA.

7. Millar JA, Raghavan R. 2015. Coxiella burnetii and Leishmania mexicana residing within similar 

parasitophorous vacuoles elicit disparate host responses. General Meeting of the American Society for 

Microbiology. New Orleans, LA.

8. Millar JA, Raghavan R. 2014. A horizontally acquired tRNA facilitates Coxiella burnetii adaptation to 

an extreme environment. General Meeting of the American Society for Microbiology. Boston, MA.

C. CONTRIBUTION TO SCIENCE 

1. Genome sequence of an avirulent Coxiella present in the Lone Star tick. We recently sequenced 

the complete genome of a bacterium related to Coxiella burnetii from the Lone star tick Amblyomma 

americanum. We showed that the tick-associated Coxiella does not have any virulence factors but could

compensate for the lack of vitamins and cofactors in the tick’s blood meal. This bacterium is the closest known 

relative of C. burnetii with a fully sequenced genome, thereby facilitating new insights into the evolution of this 

pathogen. Our results are also important for strategies geared towards controlling A. americanum and the 

pathogens it vectors. The lead author in this study was Todd Smith, an undergraduate student in my lab. 

Jonathan Gerhart, a new graduate student in lab is currently assembling the genome of a Francisella

endosymbiont present in the related Gulf Coast tick Amblyomma maculatum (manuscript is currently under 

review). He will sequence and assemble the genome of a Coxiella present in the soft-tick Ornithodoros 

rostratus as part of Specific Aim 1.

 

Contact PD/PI: Raghavan, Rahul 

Biosketches Page 14
	
Smith TA, Driscoll T, Gillespie JJ, Raghavan R. 2015. A Coxiella-like endosymbiont is a potential vitamin 

source for the Lone Star Tick. Genome Biol Evol 7:831-838. 

2. Genome-wide analyses of human macrophage response to C. burnetii infection. Coxiella and 

Leishmania (eukaryote) are the only pathogens that thrive within an acidic lysosome-derived vacuole inside 

human macrophages. To begin to understand the parallel evolution of this unique intracellular niche, Jessica

Millar, a graduate student in my lab infected human macrophages with either pathogen and identified genes, 

mRNA-isoforms and microRNAs (miRNAs) that were differentially expressed between infected and uninfected 

cells. We identified apoptosis-related miRNAs as major regulators during infection, and intriguingly detected 

several isoforms of mRNAs that were specifically expressed during infection. This project was carried out in 

collaboration with Prof. Scott Landfear and Dr. Eric Cambronne at OHSU. We are now following up on these 

clues to identify the molecular mechanisms that promote infections by these intracellular extremophiles.

Interactions between pathogens and host cells at the RNA level is an underexplored area of research. Through 

this project we have now expanded our capability to study ncRNAs and mRNAs in human cells. 

Millar JA, Valdes R, Kacharia FR, Landfear SM, Cambronne ED, Raghavan R. 2015. Coxiella burnetii and 

Leishmania mexicana residing within similar parasitophorous vacuoles elicit disparate host responses. 

Front Microbiol 6:794.

3. Identification of horizontally derived genetic elements in C. burnetii and novel use of a 

pharmacological agent. As part of my doctoral dissertation I studied non-coding RNAs in Coxiella. This 

was an entirely new venture in my advisor’s lab, which I spearheaded because of my interest in understanding 

the evolution and possible functions of RNA-based selfish elements. I discovered a novel Group I intron that 

uses different cofactors for each splicing step, and described two other selfish genetic elements – an 

intervening sequence and an intein – in this pathogen. The introns have to splice out of the rRNAs in order for 

functional ribosomes to form. Exploiting this Achilles’ heel, I used Pentamidine, a splice inhibitor to block 

Coxiella’s growth and showed that this is a good strategy to develop novel anti-Coxiella therapeutic agents. In 

these studies I mentored Linda Hicks, an undergraduate student. 

Raghavan R, Miller SR, Hicks LD, Minnick MF. 2007. The unusual 23S rRNA gene of Coxiella burnetii: 

two self-splicing group I introns flank a 34-base-pair exon and one element lacks the canonical omega 

G. J Bacteriol 189:6572-6579.

Raghavan R, Hicks LD, Minnick MF. 2008. Toxic introns and parasitic intein in Coxiella burnetii: 

legacies of a promiscuous past. J Bacteriol 190:5934-5943. 

Raghavan R, Hicks LD, Minnick MF. 2009. A unique group I intron in Coxiella burnetii is a natural splice 

mutant. J Bacteriol 191:4044-4046. 

Minnick MF, Hicks LD, Battisti JM, Raghavan R. 2010. Pentamidine inhibits Coxiella burnetii 23S rRNA 

intron splicing and intracellular growth. Int J Antimicrob Agents 36: 380-382.

4. Genome-wide identification and characterization of non-coding RNAs in Coxiella, E. coli, 

and Salmonella. During my postdoc, I developed an RNA-seq-based approach to identify novel ncRNAs in

bacteria. This approach allowed us to discover several novel ncRNAs in E. coli, a well-studied model organism. 

On this project, I independently worked out most of the technical details of ncRNA discovery because these 

were completely new areas of research for both my postdoc adviser (Dr. Ochman), and our collaborator Dr. 

Groisman. In addition, I mentored Alan Sage, an undergraduate student. Later, while I was setting up my lab at 

PSU, I used the same approach to identify novel ncRNAs in Coxiella in collaboration with Dr. Minnick. I 

performed all the RNA-seq and bioinformatics analyses for this project, and identified all the ncRNAs. This was 

the first paper to describe ncRNAs in Coxiella. As a project that bridged my postdoc and faculty tenures, my 

students (Fenil Kacharia, Jessica Millar, and Christine Sislak) and I published one of the first clear examples of 

sRNA evolution in bacteria. I conceived this study, and guided my students to perform experiments that 

showed that bacterial sRNAs are created and destroyed by genome rearrangements. 

Raghavan R, Groisman EA, Ochman H. 2011a. Genome-wide detection of novel regulatory RNAs in E. 

coli. Genome Res 21:1487-1497.

Raghavan R, Sage A, Ochman H. 2011b. Genome-wide detection of transcription start sites yield a novel 

thermo-sensing RNA and new CRP-regulated genes in E. coli. J Bacteriol 193:2871-2874.

 

Contact PD/PI: Raghavan, Rahul 

Biosketches Page 15
	
 

Contact PD/PI: Raghavan, Rahul 

Warrier I, Hicks LD, Battisti JM, Raghavan R, Minnick MF. 2014. Identification of Novel small RNAs and 

characterization of the 6S RNA of Coxiella burnetii. PLoS ONE 9:e100147. 

Raghavan R, Kacharia FR, Millar JA, Sislak CD, Ochman H. 2015. Genome rearrangements can make and 

break small RNA genes. Genome Biol Evol 7:557-566. 

5. Evolution of antisense RNAs and bacterial nucleotide composition. The discovery of genomewide production of antisense RNA has upended our traditional understanding of bacterial transcription. I used 

a novel strand-specific sequencing approach to investigate the transcriptomes of E. coli and Salmonella. This 

paper has become a benchmark in the field because it showed for the first time that although antisense 

transcription is pervasive, most individual asRNA are not conserved, indicating that most antisense RNAs are 

likely non-functional. Other labs have extended our study to several other bacteria.

Understanding the significance of the broad range of base compositions (13% to 75% G+C) observed in 

bacteria has been a vexing problem in microbiology. For over 50 years it has been assumed that selection has

no role in determining genomic G+C content. However, we reasoned that because 90% of bacterial genomes 

are composed of protein-coding genes, any small selection occurring on the base composition of all mRNAs 

would influence the genomic G+C content. Using E. coli expressing the same protein encoded by mRNAs with 

varying G+C%, I showed that differences in mRNA base compositions affected bacterial fitness. This study, for 

the first time, revealed a selective force that favors high G+C ratio in bacterial genes. 

Raghavan R, Sloan DB, Ochman H. 2012. Antisense transcription is pervasive but rarely conserved in 

enteric bacteria. mBio 3:e00156-12. 

Raghavan R, Kelkar YD, Ochman H. 2012. A selective force favoring increased G+C content in bacterial 

genes. Proc Natl Acad Sci USA 109:14504-14507.

I have published 23 peer-reviewed articles till date. My complete list of publications is available at: 

http://www.ncbi.nlm.nih.gov/myncbi/browse/collection/41517339/?sort=date&direction=descending

D. RESEARCH SUPPORT

Ongoing Research Support

1. NIH Subaward (11/01/2015-08/31/2016)

This project will analyze the transcriptomes of Streptococcus mutans. 

Role: Collaborator (PI: Justin Merritt)

 

 

4. Portland State University Faculty Enhancement Grant (07/01/2014-06/30/2016)

This project will examine nucleotide modifications in bacterial sRNAs. 

Role: PI

5. Portland State University Startup Fund (09/01/2012-06/30/2017)

This fund helped me setup my new lab and allowed me to generate data and publish papers.

Role: PI

Completed Research Support

 

2. Portland State University Research Stimulus Grant (04/01/2013–03/31/2014)

This grant helped me establish collaborations with research groups at OHSU, and to generate preliminary 

data to apply for NIH grants. 

Role: PI

Biosketches Page 16
	
OMB Number: 4040-0001

Expiration Date: 06/30/2016

Tracking Number: GRANT12198450 Funding Opportunity Number: PA-16-200 . Received Date: 2016-06-20T20:28:55.000-04:00

RESEARCH & RELATED BUDGET - SECTION A & B, Budget Period  1

ORGANIZATIONAL DUNS*:   

Budget Type*: ● Project ❍ Subaward/Consortium

Enter name of Organization: PORTLAND STATE UNIVERSITY

Start Date*: 04-01-2017 End Date*: 03-31-2020 Budget Period: 1

A. Senior/Key Person

# Prefix First Name* Middle

Name

Last Name* Suffix Project Role* Base

Salary ($)

Calendar

Months

Academic

Months

Summer

Months

Requested

Salary ($)*

Fringe

Benefits ($)*

Funds Requested ($)*

1 . Rahul Raghavan PD/PI 9.0

Total Funds Requested for all Senior Key Persons in the attached file

Additional Senior Key Persons: File Name: Total Senior/Key Person

Mime Type:

B. Other Personnel

Number of

Personnel*

Project Role* Calendar Months Academic Months Summer Months Requested Salary ($)* Fringe Benefits* Funds Requested ($)*

Post Doctoral Associates

2 Graduate Students

2 Undergraduate Students

Secretarial/Clerical

1 Research Associate 14.4

5 Total Number Other Personnel Total Other Personnel

Total Salary, Wages and Fringe Benefits (A+B)

RESEARCH & RELATED Budget {A-B} (Funds Requested)

Contact PD/PI: Raghavan, Rahul 

 Page 17
	
Contact PD/PI: Raghavan, Rahul 

Tracking Number: GRANT12198450 Funding Opportunity Number: PA-16-200 . Received Date:

2016-06-20T20:28:55.000-04:00

RESEARCH & RELATED BUDGET - SECTION C, D, & E, Budget Period 1

ORGANIZATIONAL DUNS*: 

Budget Type*: ● Project ❍ Subaward/Consortium

Organization: PORTLAND STATE UNIVERSITY

Start Date*: 04-01-2017 End Date*: 03-31-2020 Budget Period: 1

C. Equipment Description

List items and dollar amount for each item exceeding $5,000

Equipment Item Funds Requested ($)*

Total funds requested for all equipment listed in the attached file

Total Equipment 0.00

Additional Equipment: File Name:

Mime Type:

D. Travel Funds Requested ($)*

1. Domestic Travel Costs ( Incl. Canada, Mexico, and U.S. Possessions)

2. Foreign Travel Costs

Total Travel Cost

E. Participant/Trainee Support Costs Funds Requested ($)*

1. Tuition/Fees/Health Insurance

2. Stipends

3. Travel

4. Subsistence

5. Other: 

 Number of Participants/Trainees Total Participant Trainee Support Costs 0.00

RESEARCH & RELATED Budget {C-E} (Funds Requested)

 Page 18 
Contact PD/PI: Raghavan, Rahul 

Tracking Number: GRANT12198450 Funding Opportunity Number: PA-16-200 . Received Date:

2016-06-20T20:28:55.000-04:00

RESEARCH & RELATED BUDGET - SECTIONS F-K, Budget Period 1

ORGANIZATIONAL DUNS*: 

Budget Type*: ● Project ❍ Subaward/Consortium

Organization: PORTLAND STATE UNIVERSITY

Start Date*: 04-01-2017 End Date*: 03-31-2020 Budget Period: 1

F. Other Direct Costs Funds Requested ($)*

1. Materials and Supplies

2. Publication Costs

3. Consultant Services

4. ADP/Computer Services

5. Subawards/Consortium/Contractual Costs

6. Equipment or Facility Rental/User Fees

7. Alterations and Renovations

Total Other Direct Costs

G. Direct Costs Funds Requested ($)*

Total Direct Costs (A thru F)

H. Indirect Costs

Indirect Cost Type Indirect Cost Rate (%) Indirect Cost Base ($) Funds Requested ($)*

1 . MTDC 48.5

Total Indirect Costs

Cognizant Federal Agency

(Agency Name, POC Name, and POC Phone Number)

DHHS, Jeannette Lu, 415-556-1704

I. Total Direct and Indirect Costs Funds Requested ($)*

Total Direct and Indirect Institutional Costs (G + H)

J. Fee Funds Requested ($)*

K. Budget Justification* File Name: R15-BudgetJustificationjune2016-v1.pdfMime Type: application/pdf

(Only attach one file.)

RESEARCH & RELATED Budget {F-K} (Funds Requested)

 Page 19 
 

Contact PD/PI: Raghavan, Rahul 

Budget Justification 

PERSONNEL

Principal Investigator 

Rahul Raghavan, PhD, will commit 3 summer months of effort to this project each year. He will be 

responsible for the overall administration and direction of the project. He will be actively involved in 

planning experimental approaches, mentoring graduate and undergraduate students, interpretation of 

results, preparation of manuscripts, and dissemination of findings. 

Research Associate 

Abraham Moses, BS, is requesting 4.80 calendar months of salary support each year (0.40 FTE) to 

work on the project. He will concentrate on elucidating the role of heme biosynthesis as described in 

the proposal (Specific Aim 3). He is highly competent in all aspects of the proposed research including

tissue culture, Coxiella culture and molecular techniques. He generated the preliminary data 

pertaining to the importance of heme biosynthesis to Coxiella’s intracellular growth. 

Graduate Research Assistants 

Two graduate students will each work 0.99 summer months each year on the project. 

Jessica Millar, BS, is a second year graduate student in the lab. She generated the preliminary data 

showing the contribution of horizontal gene transfer to Coxiella’s biology. She already has two 

publications from the lab (one as first author), and another paper where she is the first author is under 

review currently. She will concentrate on understanding how horizontally derived genes drive 

Coxiella’s metabolism (Specific Aim 2). This project will be part of her thesis. The requested fund will 

facilitate a timely completion of her graduate degree. 

Jonathan Gerhart, BS, is a first year graduate student in the lab. He is proficient at both 

bioinformatics and genomics. He recently assembled the genomes of a Francisella species present in 

ticks (he is the first author on the paper, which is currently under review). He will concentrate on 

assembling the genome of the Coxiella present in Ornithodoros rostratus (Specific Aim 1). This 

project will be part of his thesis. The requested fund will facilitate a timely completion of his graduate 

degree. 

Undergraduate students 

Melissa Holler and Todd Hinsch are two undergraduate researchers in the lab. They will each work 3 

summer months each year on the project at $12/hr for approximately 69.5 hours per month. They will 

assist with tissue culture, Coxiella culture and molecular techniques such as PCR, RT-qPCR etc. They 

will also be trained on bioinformatics tools required to analyze high-throughput genomic and 

transcriptomic data. The summer support requested will allow the students to be fully immersed in 

the lab experience and not look for other jobs. New students will be hired to replace Melissa and Todd 

when they graduate. 

Fringe Benefits 

Portland State University’s fringe benefit rates are budgeted per budgeting practices as follows: 

- 30% of PI salary 

- 30% of post-doctoral researcher salary 

- 9% of GRAs’ summer salary

- 9% of undergraduate students’ summer salary 

SERVICES & SUPPLIES 

Supplies ($ funds are requested annually for project specific supplies. 

Budget Justification Attachment Page 20
	
 

Contact PD/PI: Raghavan, Rahul 

Genome sequencing library preparation and Illumina HiSeq or NextSeq: $

RNA-seq library preparation and Illumina NextSeq: $

Molecular biology reagents (primers, SYBR green, probes etc.): $

Enzymes and kits (Taq, T7 RNA polymerase, extraction kits etc.): $

Tissue culture and Coxiella culture (plastics, media, fetal calf serum, antibiotics etc.): $

Travel ($ $ is requested annually to support project personnel travel. These funds will 

allow PI or students to attend science meetings related to the proposed research each year, for 

example, the General Meeting of the American Society of Microbiology (ASM) or the Annual Meeting 

of American Society of Rickettsiology (ASR). Yearly meetings for students are a vital part of their 

training. Students will be required to present posters (or short talks when possible) at meetings. Both 

undergraduates and graduate students will be encouraged to attend. 

Publication Costs ($ $ is requested annually to cover page charges for publication of 

results that are generated by this proposal. We will publish the results from this project in high impact 

journals such as Genome Research, Genome Biology Evolution, PLoS Pathogens and mBio. 

Indirect Costs: 

Indirect costs (F&A) at PSU are calculated based on Modified Total Direct Costs (first $ of the 

subcontract, minus tuition, equipment over $ at 48.5% rate over the project period per Portland 

State University’s federally negotiated indirect cost rate agreement.

Budget Justification Attachment Page 21
	
Contact PD/PI: Raghavan, Rahul 

Tracking Number: GRANT12198450 Funding Opportunity Number: PA-16-200 . Received Date:

2016-06-20T20:28:55.000-04:00

RESEARCH & RELATED BUDGET - Cumulative Budget

Totals ($)

Section A, Senior/Key Person

Section B, Other Personnel

Total Number Other Personnel 5

Total Salary, Wages and Fringe Benefits

(A+B)

Section C, Equipment 0.00

Section D, Travel

1. Domestic

2. Foreign 0.00

Section E, Participant/Trainee Support

Costs

0.00

1. Tuition/Fees/Health Insurance 0.00

2. Stipends 0.00

3. Travel 0.00

4. Subsistence 0.00

5. Other 0.00

6. Number of Participants/Trainees 0

Section F, Other Direct Costs

1. Materials and Supplies

2. Publication Costs

3. Consultant Services 0.00

4. ADP/Computer Services 0.00

5. Subawards/Consortium/Contractual

Costs

0.00

6. Equipment or Facility Rental/User

Fees

0.00

7. Alterations and Renovations 0.00

8. Other 1 0.00

9. Other 2 0.00

10. Other 3 0.00

Section G, Direct Costs

(A thru F)

Section H, Indirect Costs

Section I, Total Direct and Indirect Costs

(G + H)

Section J, Fee 0.00

 Page 22 
PHS 398 Cover Page Supplement

OMB Number: 0925-0001

Expiration Date: 10/31/2018

Tracking Number: GRANT12198450 Funding Opportunity Number: PA-16-200 . Received Date:

2016-06-20T20:28:55.000-04:00

1. Human Subjects Section

Clinical Trial? ❍ Yes ● No

*Agency-Defined Phase III Clinical Trial? ❍ Yes ❍ No

2. Vertebrate Animals Section

Are vertebrate animals euthanized? ❍ Yes ● No

If "Yes" to euthanasia

Is the method consistent with American Veterinary Medical Association (AVMA) guidelines?

❍ Yes ❍ No

If "No" to AVMA guidelines, describe method and proved scientific justification

3. *Program Income Section

*Is program income anticipated during the periods for which the grant support is requested?

❍ Yes ● No

If you checked "yes" above (indicating that program income is anticipated), then use the format below to reflect the amount and

source(s). Otherwise, leave this section blank.

*Budget Period *Anticipated Amount ($) *Source(s)

Contact PD/PI: Raghavan, Rahul 

 Page 23
	
PHS 398 Cover Page Supplement

Tracking Number: GRANT12198450 Funding Opportunity Number: PA-16-200 . Received Date:

2016-06-20T20:28:55.000-04:00

4. Human Embryonic Stem Cells Section

*Does the proposed project involve human embryonic stem cells? ❍ Yes ● No

If the proposed project involves human embryonic stem cells, list below the registration number of the specific cell line(s) from the

following list: http://grants.nih.gov/stem_cells/registry/current.htm. Or, if a specific stem cell line cannot be referenced at this time,

please check the box indicating that one from the registry will be used:

❏ Specific stem cell line cannot be referenced at this time. One from the registry will be used.

Cell Line(s) (Example: 0004):

5. Inventions and Patents Section (RENEWAL)

*Inventions and Patents: ❍ Yes ❍ No

If the answer is "Yes" then please answer the following:

*Previously Reported: ❍ Yes ❍ No

6. Change of Investigator / Change of Institution Section

❏ Change of Project Director / Principal Investigator

Name of former Project Director / Principal Investigator

Prefix:

*First Name:

Middle Name:

*Last Name:

Suffix:

❏ Change of Grantee Institution

*Name of former institution:

Contact PD/PI: Raghavan, Rahul 

 Page 24
	
 

PHS 398 Research Plan

OMB Number: 0925-0001

Expiration Date: 10/31/2018

 

Tracking Number: GRANT12198450 Funding Opportunity Number: PA-16-200. Received Date:

2016-06-20T20:28:55.000-04:00

Introduction

1. Introduction to Application

(Resubmission and Revision)

Introduction-R15-2016-resub.pdf

Research Plan Section

2. Specific Aims SpecificAims-R15-2016-resub.pdf

3. Research Strategy* ResearchStrategy-R15-2016-resub-v10.pdf

4. Progress Report Publication List

Human Subjects Section

5. Protection of Human Subjects

6. Data Safety Monitoring Plan

7. Inclusion of Women and Minorities

8. Inclusion of Children

Other Research Plan Section

9. Vertebrate Animals

10. Select Agent Research Select_agents-R15-2016-resub.pdf

11. Multiple PD/PI Leadership Plan

12. Consortium/Contractual Arrangements  

13. Letters of Support LOS_combined.pdf

14. Resource Sharing Plan(s) Resource_sharing-R15-2016-resub.pdf

15. Authentication of Key Biological and/or

Chemical Resources Authentication_key_resources.pdf

Appendix

16. Appendix

Contact PD/PI: Raghavan, Rahul 

 Page 25
	
INTRODUCTION TO APPLICATION

I am grateful to the reviewers for their critiques. I believe the revised proposal has been improved by addressing all their concerns (marked in blue). 

REVIEWER 1

Significance

Small number of annual cases reduces my enthusiasm. It is important to study this select agent because: (1) 

actual rates of C. burnetii infection are not known due to widespread underreporting, (2) C. burnetii causes epidemics such as the recent outbreak in the Netherlands that involved over 4,000 cases, (3) chronic Q fever has 

poor prognosis, and (4) current antimicrobial therapy is protracted (years) and generally ineffective. 

The broader implications of this research are unclear. Our approach could be applied to other bacterial pathogens where human-specialized strains could be compared to avirulent symbionts. E.g., metabolic and virulence 

mechanisms in Francisella and Rickettsia could be compared to that of closely-related tick symbionts. 

The impact of this application on the institution is not clear. This critique runs counter to compliments made 

by reviewer 2. We have addressed this under “Impact of an AREA award on student research” in the Facilities 

and Resources section. Additionally, a student-training plan is detailed at the end of each Aim. 

Innovation

While examining horizontal gene transfer (HGT) is new in Coxiella research, it is a well-understood phenomenon. The methods seem appropriately chosen, but standard practice in the field. While HGT is well studied, our 

project is based on a novel concept that C. burnetii evolved from a tick-associated ancestor through massive 

HGT (>20% of genes). Additionally, our innovative approach will overcome the current difficulty in performing 

genome-scale functional studies in C. burnetii, and will define a novel tRNA-based virulence mechanism.

Approach

Use of KEGG and other databases to confirm the function of genes is overly optimistic. We don’t expect this to 

be a problem because we are not utilizing the databases for identifying specific functions of each gene. Rather, 

we are interested in identifying metabolic pathways that contain pathogen-specific genes. Thus, a lack of functional information or misidentification of a few genes would not adversely impact this project.

Presence of tRNAGlu1 will confound the ability to draw clear conclusions about the essentiality of heme biosynthesis. We don’t expect tRNAGlu1 to impact this project because as shown in Figure 13 (Aim 3), the tRNAGlu2-

deletion strain has a clear growth defect. We will test whether the reduced growth is due to low heme production, and whether it is rectified by complementation, thus revealing the importance of heme biosynthesis.

REVIEWER 2

Significance and Innovation

No clear plan for prioritizing genes identified in Aims 1 and 2. We have added a prioritization plan in the revised application (please see Table 1 under Innovation). We will prioritize: (1) pathways for the synthesis of 

critical metabolites that C. burnetii cannot obtain from host (e.g. heme, biotin), (2) pathways for metabolizing 

critical nutrients such as glutamate, and (3) proteins required for homeostasis (e.g. Na+/H+ antiporters). 

Approach

Will the genes identified in Aim 1 be characterized in Aim 2? We will prioritize pathways that are common to 

both Aim 1 and Aim 2 i.e. pathogen-specific pathways that contain HGT-derived genes (please see Table 1).

How will you differentiate between genes required for metabolism from those required for entry, replication or 

general survival? We expect to identify pathways found only in the human pathogen, which contain HGTderived genes. Of these, we will prioritize metabolic pathways (based on KEGG, MetaCyc etc.), but proteins required for entry, replication etc. will also be analyzed if time and budget permits (please see Table 1).

How will you show that the observed defects specifically manifest during intracellular growth. To identify infection-specific genes, we will compare the genes expressed in C. burnetii grown intracellularly to those grown in 

ACCM-2 medium. Additionally, transposon mutants will be tested in both ACCM-2 and in host cells to identify 

genes that are critical to intracellular growth.

What will be done with hypothetical genes? We expect these genes to be of low priority at this stage of the project. This should not be a problem because we have several high-quality targets that we expect to pursue. 

 

Contact PD/PI: Raghavan, Rahul 

Introduction to Application Page 26
	
SPECIFIC AIMS

The inhospitable environment within a lysosome-derived vacuole is the preferred growth medium for Coxiella burnetii, the etiological agent of human Q fever. The unique ability of C. burnetii to thrive in this acidic 

vacuole that contains noxious cationic peptides, reactive oxygen species, and lysosomal hydrolases is the key 

to its virulence. However, metabolic pathways critical to the pathogen’s intracellular growth are unknown, 

mainly due to the unavailability of appropriate genome-scale approaches. This gap in knowledge is an important problem because it has hindered both the understanding of C. burnetii’s basic biology and pathogenesis, and the development of better therapies. Long-term combinatorial treatment with doxycycline and 

hydroxychloroquine (≥1.5 years) is the best available option; however, strains resistant to this treatment are 

prevalent. This select agent is found worldwide and can occasionally cause epidemics; e.g., a recent outbreak 

in the Netherlands involved ~4,000 human cases and caused the culling of over 50,000 goats (a reservoir). 

Our long-term goal is to understand the molecular details of Coxiella’s distinctive physiology, and to apply 

this knowledge to developing novel therapeutic strategies. Towards attaining this goal, the overall objective

of this application is to identify metabolic pathways that are vital to C. burnetii’s intracellular growth. Our 

central hypothesis is that C. burnetii evolved from a tick-associated ancestor by acquiring critical metabolic 

genes through horizontal gene transfer (HGT). Our hypothesis was formulated based on our previous work 

(Smith et al. 2015), which showed that the closest relatives of C. burnetii are avirulent bacteria with reduced 

metabolic capabilities present in ticks. The work by Duron et al. (2015) also supports this hypothesis. Based 

on these insights, we will use a novel evolutionary genomics approach that will overcome the current technical difficulties to identify metabolic processes critical to C. burnetii. The proposed research will be impactful because once metabolic processes important to C. burnetii’s intracellular growth are identified, new

pharmacological agents that block these pathways can be developed to treat chronic Q fever more effectively. 

To test our central hypothesis, in Specific Aims 1 and 2 we will use two complementary genome-scale approaches to define critical metabolic processes in C. burnetii, and in Specific Aim 3, as a proof of principle, 

we will experimentally validate the importance of heme biosynthesis to Coxiella’s physiology.

1. Identify metabolic pathways that distinguish C. burnetii from tick-associated Coxiella. Because Coxiella species present in ticks do not replicate within a lysosome-derived vacuole, our working hypothesis is that genes critical to C. burnetii’s unique intracellular physiology will not be present in tickassociated Coxiella. We will sequence the complete genome of C. burnetii’s closest known relative—a Coxiella species present in the soft tick Ornithodoros rostratus, and by comparative genome analyses, we will 

identify metabolic genes and pathways unique to C. burnetii. 

2. Define metabolic pathways that are critical to C. burnetii’s intracellular growth. Superfluous

genes are lost from obligate intracellular bacteria such as C. burnetii. This is especially true for genes acquired via HGT, which will be retained only if they improve C. burnetii’s fitness. Consequently, we hypothesize that horizontally derived genes are critical to C. burnetii’s physiology. We will utilize phylogenetic approaches to identify HGT-derived metabolic genes among those identified in Aim 1. Using RNA-seq and 

transposon-insertion libraries, we will validate their importance to C. burnetii’s intracellular growth.

3. Determine the importance of heme biosynthesis to C. burnetii’s intracellular growth. C. 

burnetii’s heme biosynthesis pathway contains HGT-derived genes, and this pathway is not present in Coxiella symbionts, and we established that C. burnetii could not use external heme. We therefore hypothesize

that heme biosynthesis is crucial to C. burnetii’s intracellular growth and can serve as an example for the 

efficacy of our novel approach. We will confirm the importance of heme biosynthesis to Coxiella by assaying 

heme production and intracellular growth in wild-type and heme pathway-deficient strains.

At the successful completion of this project, expected outcomes include elucidation of the evolutionary history of this enigmatic pathogen, and identification of metabolic pathways that are important to Coxiella’s 

intracellular growth and virulence. Results are expected to have a positive impact because it will provide a 

useful model for identifying key metabolic processes in other human pathogens with closely related symbionts such as Rickettsia and Francisella. Additionally, non-redundant proteins in critical metabolic pathways, 

e.g. HemA and HemL in heme biosynthesis, are ideal candidates for developing new anti-Coxiella agents.

 

Contact PD/PI: Raghavan, Rahul 

Specific Aims Page 27
	
RESEARCH STRATEGY

 

(A) SIGNIFICANCE

Importance of the problem: Coxiella burnetii is the etiological agent of a flu-like illness (acute Q fever) 

and a chronic disease commonly manifested as endocarditis (Maurin and Raoult 1999). CDC has classified 

Coxiella as a select agent due to its past use as a bioweapon, environmental stability, aerosol transmission 

and extremely low infectious dose [ID50 of one to ten bacteria in a guinea pig model (Tigertt et al. 1961)]. 

Human Q fever is a nationally notifiable disease in the U.S. since 1999, however the true rate of infection is 

not known due to widespread underreporting (CDC 2002). Untreated chronic Q fever is associated with 

high mortality rate (>60%) (Karakousis et al. 2006), and Coxiella is one of the commonest causes of culturenegative infective endocarditis (Million et al. 2010). C. burnetii is found worldwide, and can cause epidemics, such as the recent one in the Netherlands where thousands of people were infected (van der Hoek et al. 

2012). It has also been detected among U.S. military personnel (Faix et al. 2008), and its high prevalence in 

US environmental samples (Kersh et al. 2010; Loftis et al. 2010) highlight the urgent need to understand its 

biology and virulence mechanisms. Acute infections are 

treatable but even with the recommended 3-week doxycycline treatment, acute Q fever can transform into chronic 

endocarditis (Fenollar et al. 2006). Chronic Coxiella infections are very difficult to treat because it requires prolonged antibiotic regimen: a combination of doxycycline 

and hydroxychloroquine (1.5 to 3 years), or doxycycline 

and fluoroquinolone (3 to 4 years) (Gould et al. 2012). 

However, strains resistant to doxycycline are prevalent 

(Rouli et al. 2012). A critical barrier to understanding 

Coxiella’s basic biology and molecular pathogenesis, and 

to developing more effective therapies is the lack of 

knowledge about metabolic processes that are vital to 

Coxiella’s growth within human cells. 

How the proposed project will advance one or more broad fields: C. burnetii is an intracellular 

pathogen that has a biphasic life cycle (Figure 1). The bacterium survives in the environment as a metabolically quiescent small cell variant (SCV). C. burnetii has occasionally been isolated from ticks, but most human infections occur through inhalation of aerosols contaminated with SCV originating from farm animals. 

Shortly after entering into a host cell (typically an alveolar macrophage in human infections), SCV transforms into a metabolically active large cell variant (LCV) within a lysosome-derived parasitophorous vacuole 

(PV) (Voth and Heinzen 2007). Over the next 5-6 days LCVs divide within the acidic vacuole (pH ~4.5) to 

large numbers and eventually transform into SCVs, which are released upon lysis of the host cell (Minnick 

and Raghavan 2012). Its closest relatives are avirulent bacteria found associated with ticks (Smith et al. 

2015). These Coxiella species do not have the ability to replicate within human cells (Duron et. 2015). It is 

not understood how a tick-associated non-pathogenic ancestor evolved into a human pathogen, and the 

metabolic capabilities that allow C. burnetii to thrive in the harsh conditions within the PV are also not understood (Figure 2). We postulate that elucidating the evolutionary process that resulted in the emergence 

of C. burnetii is the key to unlocking its distinctive physiology and virulence strategy. The proposed research 

is significant because in addition to (1) uncovering the evolutionary history of C. burnetii, we will for the 

first time, (2) identify metabolic processes that are critical to the pathogen’s intracellular growth, thereby 

advancing the field considerably by (3) improving our knowledge about its basic physiology and (4) providing potential therapeutic targets. Importantly, the proposed project provides (5) a model approach for identifying genes involved in host adaptation, which could be applied to other pathogens such as Francisella tularensis or Rickettsia spp. where avirulent symbionts could be compared to virulent human-specialized 

strains. Furthermore, (6) we will examine the importance of heme, which is synthesized from a tRNAGlu

that was acquired horizontally by Coxiella. Because 

other pathogenic bacteria such as Corynebacterium

and Clostridium encode multiple tRNAGlu isoacceptors, 

this novel virulence strategy could be widespread 

among human pathogens. 

Nucleus

Nascent

PV

2 hpi

Early

spacious PV

3 dpi

Mature PV

7 dpi

Autophagosome

Lysosomes

SCV

LCV

Figure 1. Biogenesis of C. burnetii parasitophorous vacuole (PV). 

Entry

Metabolism 

and 

Multiplication

Micro-ecological 

alterations

1 2 3

Figure 2. Stages in C. burnetiiȱȱǻęȱȱ

	ȱŗşŜŖǼǯȱ	ȱȱ ȱȱȱȱǯ

 

Contact PD/PI: Raghavan, Rahul 

Research Strategy Page 28
	
(B) INNOVATION

How the proposed research seeks to shift current research paradigms: Bacterial infection involves three broad stages (Figure 2, previous page). Current research into Coxiella’s pathogenicity is mainly focused on entry (Stage 1), and on manipulation of the host cell environment (Stage 3) (e.g., Weber et al. 

2013; Newton et al. 2014; Larson et al. 2015, Graham et al. 2015). This bias has left a large void in our understanding of Coxiella’s intracellular metabolism (Stage 2), which has been fine-tuned to replicate in the 

harsh environment within a lysosome-derived vacuole. As antibiotic resistance continue to rise among pathogens, it is critical that researchers 

explore unconventional antimicrobial targets. An under-explored opportunity is the simple premise that a bacterium needs to be metabolically active during infection (Brown et al. 2008). Louis Pasteur originally proposed this concept in his model of the body as a culture vessel 

(Pasteur 1878); however, very few studies have explored this concept to 

develop new pharmacological agents that inhibit bacterial growth within 

hosts. Because C. burnetii replicates within an intracellular niche that is 

lethal to all other bacteria, we postulate that uncovering its metabolic 

capabilities is the key to understanding its virulence strategies and to 

developing new therapeutic agents. The proposed research will shift the current paradigm in Coxiella research because we expect to identify, for the first time, the metabolic pathways that are critical to Coxiella’s 

distinct intracellular physiology. 

Novel theoretical concepts, innovative approaches to be developed or used: Currently, no efficient genome-scale methods are available to identify metabolic genes that are important to Coxiella’s intracellular growth. A targeted gene deletion system is available (Beare et al. 2012), but this method is labor intensive and is not suitable to be applied at a genome-wide scale. Random transposon (Tn) insertion mutants 

of C. burnetii have been used to identify genes important for host cell invasion (Martinez et al. 2014, Newton et al. 2014). However, because Tn insertions in essential metabolic genes are likely lethal, it is hard to 

determine whether a gene has not been targeted by Tn because the gene is essential or due of a lack of saturation. Additionally, genetic systems to generate conditional mutants that circumvent the lethality of mutations in essential genes are also not available. To overcome these limitations we have devised an innovative

evolutionary-genomics approach that will identify most metabolic pathways that are vital to C. burnetii’s

intracellular growth. Our approach is based on preliminary data that indicate that C. burnetii evolved from a 

tick-associated ancestor by acquiring genes through horizontal gene transfer (HGT) (Figure 3). This is a 

novel concept in our understanding of bacterial pathogenesis because no other pathogen is known to have 

evolved in this manner. We will compare the genomes of C. burnetii to that of a closely related avirulent 

tick-associated Coxiella to identify pathogen-specific metabolic genes. In parallel, we will identify metabolic 

genes that were acquired via horizontal gene transfer (HGT) (Table 1). By comparing these two sets of 

genes we will generate a list of metabolic pathways that are instrumental in C. burnetii’s ability to grow intracellularly, and are hence ideal candidates for disruption using new therapeutic strategies. As a first step, 

we will examine the importance of heme biosynthesis to Coxiella’s intracellular growth because this pathway 

is unique to the human pathogen and contains horizontally derived genes, including a tRNAGlu. The use of an 

alternate tRNA to promote infection represents a novel 

pathogenic mechanism. Collectively, this project is innovative because it (1) utilizes a novel approach that 

overcomes the current limitations in studying Coxiella

genes at a genome-wide scale, (2) is expected to identify metabolic pathways that are important for Coxiella’s 

intracellular growth, (3) is based on a novel concept 

that the virulent strain evolved via HGT from an avirulent tick-associated ancestor, (4) is expected to make 

new targets available for developing novel therapeutic 

agents, and (5) will reveal a novel tRNA-acquisitionbased virulence mechanism that could be widespread 

among other pathogens. A project overview and our 

prioritization plan are shown in Table 1. 

Environmental ancestor

Tick-associated ancestor

Evolution of human pathogen

$FTXLUHGYLUXOHQFHIDFWRUV

$FTXLUHGPHWDEROLFJHQHV

Figure 3. Emergence of C. burnetii.

 

Contact PD/PI: Raghavan, Rahul 

Research Strategy Page 29
	
(C) APPROACH

Specific Aim 1. Identify metabolic pathways that distinguish C. burnetii from tick-associated 

Coxiella.

Background and Justification

Within macrophages, engulfed pathogens are transported inside phagosomes that later fuse with lysosomes 

to generate the phagolysosome — a very harsh environment due to low pH, high concentration of lysosomal 

hydrolases and presence of cationic peptides (Kinchen and Ravichandran, 2008; Flannagan et al. 2009). 

Pathogenic bacteria have evolved strategies to circumvent the mature lysosome. For instance, Legionella 

pneumophila blocks maturation of phagosomes into phagolysosomes, and Listeria monocytogenes escape 

into the cytoplasm from phagosomes before lysosomal fusion (Flannagan et al., 2009). Unlike other pathogenic bacteria, C. burnetii has evolved the ability to thrive in a parasitophorous vacuole (PV) that is derived 

from the fusion of phagosomes with lysosomes (Voth and Heinzen, 2007; Alix et al., 2011) (Figure 1). The 

only other human pathogen that has evolved this capability is the eukaryotic parasite Leishmania (Real et al. 

2010). We recently showed that C. burnetii and L. mexicana use disparate strategies for growth within their 

respective PVs (Millar et al. 2015). The metabolic pathways that drive C. burnetii’s growth within PV are 

unknown, even though it is fundamental to the bacterium’s physiology and pathogenicity.

The evolutionary origin of C. burnetii is not clearly understood. The closest relatives of C. burnetii

are found in ticks (Smith et al. 2015; Duron et al. 2015) (Figure 4). Coxiella spp. found in ticks are avirulent, cannot infect mammalian cells, and are unable to grow in a culture medium that supports robust 

growth of C. burnetii (Smith et al. 2015; Gottlieb et al. 2015; Duron et al. 2015). These observations suggest 

that despite their close evolutionary relationship, the human pathogen and the tick-associated strains have 

different metabolic capabilities. Our working hypothesis is that genes critical to C. burnetii’s unique lifestyle will not be present in closely related tick-associated coxiellae. To test our hypothesis and thereby to accomplish the objective of this Aim, we will sequence the genome of a Coxiella present in the soft-bodied tick 

Ornithodoros rostratus, which is the closest known relative of C. burnetii (Almeida et al. 2012) (Figure 4). 

We will use a comparative-genomics approach to identify metabolic pathways that are exclusive to C. burnetii. By successfully completing this aim, we expect to reveal the physiological capabilities that facilitated 

the emergence of a human pathogen from a tick-associated ancestor. 

Preliminary Studies and Feasibility 

We are well versed at studying bacterial genomes and 

transcriptomes (Raghavan et al. 2011a, Raghavan et al. 

2011b, Raghavan et al. 2012, Raghavan et al. 2015; 

Warrier et al. 2014; Millar et al. 2015). We recently sequenced the whole genome of a Coxiella-like bacterium from the Lone Star tick Amblyomma americanum (Smith et al. 2015). This was the first genome of a 

tick-associated Coxiella to be sequenced. We will use a 

similar strategy to sequence the genome of the Coxiella

species present in O. rostratus. To prepare for this 

project we procured 35 adult ticks from an O. rostratus

lab colony maintained by Dr. Marcelo Labruna at the 

University of Sao Paulo, Brazil (see Letter of Support). 

Dr. Labruna’s group previously showed that the Coxiella species present in this O. rostratus colony is the closest known relative of C. burnetii (Almeida et al. 2012). 

We have successfully isolated good quality genomic DNA from the ticks and are all set to perform the experiments described below. 

Research Design

(1) Sequence, assemble and annotate the genome of Coxiella present in O. rostratus. 

We will submit genomic DNA (~1μg) extracted from one adult tick to OHSU Massively Parallel Sequencing Shared Resource for high-throughput sequencing (see Letter of Support). We will sequence each 

DNA sample on one lane of Illumina NextSeq (300 cycle, paired-end). We will assemble the genome using 

Velvet and IDBA genome assembly programs (Zerbino and Birney 2008; Peng et al. 2012), and close the 

Figure 4. Two strains from hard ticks have sequenced 

genomes (green). We will sequence the strain from a soft 

tick (in red), which is more closely related to C. burnetii.

Diplorickettsia massiliensis

Rickettsiella grylli

Coxiella burnetii

Legionella pneumophila 

Coxiella in Amblyomma americanum

Coxiella in Ornithodoros rostratus

Coxiella in Rhipicephalus turanicus

 

Contact PD/PI: Raghavan, Rahul 

Research Strategy Page 30
	
gaps using PCR and Sanger sequencing, as we described in Smith et al. (2015). We will annotate the genome 

using the NCBI Prokaryotic Genome Annotation Pipeline (Tatusova et al. 2013), and manually check these 

annotations to produce the final annotated genome. We will deposit the genome sequence in GenBank, and 

the sequencing reads will be made freely available to the scientific community. 

(2) Perform comparative genomics to identify pathogen-specific metabolic genes. Currently, seven completely sequenced genomes of C. burnetii strains are available. We will align the new genome to all seven C. burnetii genomes using Mauve aligner (Darling et al. 2010) to identify pathogenspecific genes (Figure 5). We will also use reciprocal BLAST, as described previously (Raghavan et al. 2012;

Raghavan et al. 2015; Smith et al. 2015) to identify pathogen-specific genes i.e. C. burnetii genes without 

orthologs in tick associated Coxiella. By comparing the C. burnetii genomes, we will identify pathogen specific metabolic genes that are conserved in all strains of C. burnetii. 

To identify the 

metabolic pathways to 

which each gene belongs, 

we will perform a Gene 

Ontology (GO) analysis 

using the Database for Annotation, Visualization and 

Integrated Discovery (DAVID) (Huang et al. 2009). 

This analysis will bin each 

protein into functional

groups based on the GO 

terms associated with each 

one. In parallel, we will use 

the Kyoto Encyclopedia of 

Genes and Genomes 

(KEGG), and the MetaCyc database to corroborate their metabolic functions (Kanehisa et al. 2015; Caspi et 

al. 2012). We have previously utilized these tools (Raghavan et al. 2015; Smith et al. 2015; Millar et al. 2015). 

For instance, in a previous study (Smith et al. 2015), we identified the gene CBU_1152, present in C. burnetii

but not in A. americanum-associated Coxiella, as belonging to tryptophan biosynthesis pathway (Table 2). 

Expected Outcomes

(1) We expect to sequence and annotate the complete genome of a Coxiella spp. present in O. rostratus. The availability of the first Coxiella genome from a soft tick, along with the two genomes from hard ticks 

(Smith et al. 2015; Gottlieb et al. 2015) will be a great resource to understand the evolution of this enigmatic 

pathogen. By studying these three genomes and by comparing them to C. burnetii genomes, we expect to 

uncover the step-wise evolution of the human pathogen by acquisition of genes through HGT (Figure 3

and marked by arrows in Figure 4). 

(2) We expect to define a set of genes that are present only in the pathogenic strains of Coxiella, 

and to identify the metabolic processes that contain these pathogen-specific genes. We will also identify the 

differences in metabolic capabilities of various C. burnetii strains, thereby gaining new insights into strainspecific differences in pathogenicity. 

(3) Student training. Todd Smith, a previous undergraduate student sequenced the genome of the 

Amblyomma-associate Coxiella (Smith et al. 2015). Jonathan Gerhart, a first year graduate student will perform the genome assembly and annotation described in this Aim. I will work with him to achieve the objectives of this Aim in a timely manner. In addition, an undergraduate student Melissa Holler will work with us 

C. burnetii

Coxiella in Amblyomma americanum

0 500,000 1,000,000 1,500,000 2,000,000

0 200,000 400,000 600,000

Figure 5. Alignment of Coxiella genomes. Regions in white in C. burnetii genome (on top) 

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Gene GO term GO description

CBU_1152 GO:0000162 Tryptophan biosynthetic process

 GO:0006568 Tryptophan metabolic process

KEGG Description

KEGG Orthology (KO) [BR:cbu00001]

 Metabolism

 Overview

 01230 Biosynthesis of amino acids

 Amino acid metabolism

 00400 Phenylalanine, tyrosine and tryptophan biosynthesis

Enzymes [BR:cbu01000]

 4. Lyases

 4.1 Carbon-carbon lyases

 4.1.3 Oxo-acid-lyases

 4.1.3.27 anthranilate synthase 

MetaCyc Pathway

Chorisimate L-glutamine

anthranilate

L-tryptophan

CBU_1152

(trpE)

Table 2.ȱęȱȱȱȱȱȬęȱ

 

Contact PD/PI: Raghavan, Rahul 

Research Strategy Page 31
	
to learn programming (e.g. PERL, PYTHON) and the use of bioinformatics tools described above. Melissa 

plans to pursue a Masters degree in Bioinformatics at the joint program offered by PSU and OHSU. 

Potential Problems and Alternative Strategies

We are highly experienced at sequencing genomes and performing comparative genomics to identify species-specific genes (Raghavan et al. 2012; Raghavan et al. 2015; Smith et al 2015; Millar et al. 2015). We 

don’t expect any major problems with this aim; however, couple of minor roadblocks might arise for which 

we have alternate strategies in place:

(1) We expect our genomic DNA samples to be composed mostly of tick DNA. We don’t expect this 

to be a problem because we were able to assemble the genome of the Coxiella in A. americanum using only 

~5% of sequencing reads (Smith et al. 2015). However, if we don’t obtain enough reads to assemble the Coxiella genome from our first attempt, we will resequence the sample in another lane of Illumina NextSeq, 

which should provide us with enough reads to allow efficient assembly of the new Coxiella genome. 

(2) Because bacteria contain repeat sequences that preclude the assembly of genome sequences into 

a single circular chromosome, we expect the initial genome assembly to be composed of multiple fragments 

(contigs). For instance, in our previous study (Smith et al. 2015), we initially had six contigs that were 

stitched together using PCR and Sanger sequencing. If we are unable close the new genome using this approach, we will resequence the DNA using either Illumina Mate-Pair or PacBio technologies that produce

long reads that bridge repeating sequences, thereby allowing us to close the genome. 

(3) Using GO, DAVID, KEGG, and MetaCyc databases, we expect to identify the metabolic pathways 

that contain most of the pathogen-specific genes. However, it is possible that we will not be able to place all

genes into specific pathways (e.g., hypothetical genes), and it is possible that some of the genes might be 

performing functions that are different from those recorded in the databases. However, we don’t expect this 

to be a major hurdle because our goal is to identify metabolic pathways — not specific genes — that are 

unique to the human pathogen. Thus, a lack of functional information or misidentification of a few genes 

would not adversely impact this project. 

Specific Aim 2. Define metabolic pathways that are critical to C. burnetii’s intracellular 

growth.

Background and Justification

When the genomes of C. burnetii were sequenced, it became clear that several genes were acquired via HGT, 

including a Type Four Secretion System (TFSS) — a major virulence factor (Seshadri et al. 2003; Beare et al. 

2009). Interestingly, in addition to TFSS, C. burnetii encodes several genes for a type IV pilus. A related 

type IV pilus enables Acinetobacter baumannii to acquire DNA from the environment with high efficiency 

(Smith et al. 2007). It is unlikely that the type IV pilus has the same function in C. burnetii because some of 

the pil genes in C. burnetii have become pseudogenes (Beare et al. 2009). However, it is plausible that at an 

earlier stage of C. burnetii’s evolution, a functional type IV pilus endowed it with the ability to acquire foreign genes with high efficiency, thereby enabling its shift from a tick-associated bacterium to a zoonotic human pathogen. Although foreign-origin genes are important to C. burnetii’s emergence and virulence, no 

systematic study has been undertaken to understand the origins of all C. burnetii genes. 

Unlike eukaryotic genomes, which contain large fractions of nonfunctional DNA (e.g., >80% of human genome), bacterial genomes are tightly packed with functional genes due to elimination of nonfunctional sequences (Moran 2002). In bacteria there is a bias towards DNA deletion over insertion (Mira et al. 

2001); thus, DNA is retained in a bacterial genome only if selection is acting effectively to preserve it. Many 

biosynthetic genes and pathways are rendered redundant in host-associated bacteria such as C. burnetii because they could obtain the corresponding metabolic intermediates from hosts (Ochman and Moran 2001). 

These superfluous genes will be deleted over time due to lack of selection acting to maintain them, thereby 

resulting in a small genome; e.g., C. burnetii’s genome is only ~2 million bp compared to E. coli’s, which is 

~5 million bp. Consequently, genes acquired through HGT will only be retained in C. burnetii’s genome if 

they improve its fitness (Lawrence 1999). Based on this information, we hypothesize that genes acquired via 

HGT are being retained in C. burnetii’s reduced genome because they play important roles in Coxiella’s biology. We will perform a thorough evolutionary analysis of all protein-coding genes in C. burnetii to determine the ones that are of foreign origin. The HGT-derived genes could be beneficial during C. burnetii’s

growth in it primary reservoir (farm animals), or during its environmental phase, or during its growth with-

 

Contact PD/PI: Raghavan, Rahul 

Research Strategy Page 32
	
in human cells. To identify HGT genes that are functional during C. burnetii’s intracellular growth, we will 

analyze gene expression within human macrophages using RNA-seq. We will also screen Tn-insertion libraries to find strains with Tn-insertions in critical genes. 

Preliminary Studies and Feasibility

(1) Characterization of horizontally acquired parasitic elements in C. burnetii. We have 

a great deal of experience in investigating the evolutionary histories of genes in C. burnetii. We previously 

published several papers that detail the origin and possible functions of group I introns, a homing endonuclease, and an intein present in Coxiella (Raghavan et al. 2007, Raghavan et al. 2008, Raghavan et al. 2009; 

Raghavan and Minnick 2009; Minnick et al. 2010; Hicks et al. 2011). 

(2) Detection of genes that were potentially acquired from distant phyla in C. burnetii.

To identify the closest orthologs of C. burnetii proteins, we performed a bidirectional BLAST analysis with

the NCBI nr database (Wolf and Koonin 2012). To consider a protein to be orthologous, we applied a minimum cutoff of 70% coverage, and 30% amino acid identity, as described previously (Caro-Quintero et al. 

2012). We detected 357 genes in C. burnetii that were putatively acquired from organisms outside of Gammaproteobacteria (Coxiella belongs to this class) (Figure 6A). This is a very conservative list because it 

does not include any known virulence factors such as the TFSS, or any gene that might have been gained 

from another Gammaproteobacteria. Incredibly, even with these omissions, ~20% of C. burnetii’s proteins 

(357/1823) appear to be horizontally derived. This is in contrast to most other bacterial genomes where 

<10% of the proteins are of foreign origin (Koonin et al. 2001). The G+C ratio and codon usage of horizontally acquired genes are typically different from the rest of the genome (Lawrence and Ochman 1997). Our 

analyses showed that in comparison to rest of the genes in C. burnetii, these two measures were significantly

different for the 357 putative HGT genes (Figures 6B and 6C). 

(3) Acquisition of a fatty acid biosynthesis operon from a Spirochete. While the BLAST 

best match approach is a useful first-pass discovery tool, phylogenetic analyses are required to confirm gene 

histories (Koski and Golding 2001). To this end, we analyzed a cluster of genes in C. burnetii that appear to 

have been acquired from a Spirochete. This putative operon contains fatty acid biosynthesis genes 

(fabHAZB) and their closest orthologs are present as a similar operon in Spirochaeta africana (Figure 7A, 

next page). An IS1111A transposase (CBU_0040) is seen next to these genes in C. burnetii suggestive of a 

role for a transposon in the horizontal acquisition of the genes. We confirmed its HGT origin by building a 

Bayesian tree for CBU_0038. As shown in Figure 7B (next page), the C. burnetii gene clustered with genes 

in S. africana and several Deltaproteobacteria, with the exclusion of genes from members of Legionellales

(Coxiella belongs to this order). Furthermore, we confirmed that these genes are functional during intracellular growth by performing RNA-seq on C. burnetii growing within Vero (monkey kidney epithelium) cells 

(Figure 7C, next page). 

Research Design

(1) Define the origins of protein-coding genes in C. burnetii. Both parametric and phylogenetic methods are commonly used to identify horizontally acquired genes in a genome. Parametric methods 

utilize inherent characteristics of a genome sequence such as G+C ratio, oligonucleotide frequency or codon 

usage to detect HGT, whereas phylogenetic methods identify HGT by detecting genes with evolutionary hisAlphaproteobacteria 

Bacilli 

Betaproteo-

-bacteria 

Chlamydiales 

Clostridia 

Deltaproteobacteria Flavobacteriia 

Spirochaetales 

Stigonematales 

Verrucomicrobia 

Zetaproteo-

-bacteria 

environmental

sequences 

Figure 6. (A) Potentail donors of 357 potentially HGT-origin genes. (B) G+C percentage and (C) codon adaptation 

index ȱȱȱȱę¢ȱěȱȱȱȱȱȱǯ

Eukaryota 

HGT genes

Other genes

HGT genes

Other genes

Codon Adaptation Index

GC Ratio

20

40

60

0.40

0.60

0.80

* *

p<0.01

*

p<0.001

A B C

 

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Research Strategy Page 33
	
tories that are inconsistent with the evolutionary history of the organism (Hooper and Berg 2002; Dufraigne

et al. 2005; Vernikos and Parkhill 2006; Ravenhall et al. 2014). As shown in Figure 6 and Figure 7, we 

have the expertise to use both methods to confirm the evolutionary histories of all genes in Coxiella. We will 

align orthologous sequences using MUSCLE (Edgar 2004), with regions of poor alignment masked using 

Gblocks (Talavera and Castresana 2007), and for phylogeny estimation, we will build Maximum-likelihood 

and Bayesian trees utilizing RAxML (Stamatakis et al. 2008) and PhyloBayes (Lartillot et al. 2013), respectively. For parametric analyses we will utilize in-house PERL and PYTHON scripts (Raghavan et al. 2012). 

(2) Identify metabolic pathways that contain horizontally acquired genes. We will identify C. burnetii’s metabolic pathways that contain horizontally acquired genes using GO, DAVID, KEGG and 

MetaCyc as described in Aim 1 (Table 2). By comparing pathogen-specific metabolic pathways identified 

in Aim 1 to those that contain HGT genes (Aim 2), we expect to determine the metabolic processes that are 

critical to C. burnetii’s biology (Table 1).

(3) Confirm the importance of metabolic pathways during intracellular growth using 

RNA-seq and transposon-insertion libraries. To validate the utility of metabolic pathways during intracellular growth, we will analyze the expression of C. burnetii genes within human macrophages using 

RNA-seq, as we have described before (Warrier et al. 2014; Millar et al. 2015). Briefly, we will infect human 

monocyte THP-1 cells (TIB-202; ATCC) differentiated into adherent, macrophage-like cells with C. burnetii

RSA 493 NMII at a multiplicity of infection (MOI) of 25. We will collect total RNA at 3, 5, and 7-day post 

infection and prepare Illumina mRNA sequencing libraries from three independent infections as described 

previously (Warrier et al. 2014; Millar et al. 2015). As a control, we will use C. burnetii grown similarly in ACCM-2 for 3, 

5, and 7 days. Libraries will be pooled and sequenced on a 

lane of Illumina NextSeq, and gene expression will be measured as described previously (Raghavan et al. 2011; Raghavan 

et al. 2012; Raghavan et al. 2015). 

Construction of loss-of-function deletion strains is the 

gold standard approach to validating the utility of a gene. 

However, in C. burnetii, targeted gene deletion is currently 

technically challenging and not expandable to the whole genome. An alternate approach is to use transposon-insertion 

mutants; however, because loss of function of essential metawild-type Tn-hemL

Figure 8. Tn insertion in hemL reduced

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Research Strategy Page 34
	
bolic genes due to transposon insertion will most likely be lethal, we will utilize strains in which a transposon insertion has caused only a partial loss of gene function. For example, we procured a C. burnetii strain 

with a transposon inserted in the 3’ end (1287/1320 bp) of the hemL gene from our collaborator Dr. Matteo 

Bonazzi (CNRS, France). The intracellular growth of this strain is significantly lower than that of the wildtype strain (Figure 8, previous page), indicating that HemL, and hence heme biosynthesis, is important to 

C. burnetii’s physiology (also see Aim 3). We will search transposon-insertion libraries available with Dr. 

Bonazzi and with Dr. James Samuel (Texas A&M University) to identify such Tn-insertion mutants to validate the utility of metabolic pathways to C. burnetii’s intracellular growth (see Letters of Support). 

Expected Outcomes

(1) We expect to identify most horizontally derived genes in C. burnetii. This will be a big step in our 

understanding of the evolution of C. burnetii from a tick-associated ancestor. Our approach could be applied

to investigate the evolution of other human pathogens that have closely related arthropod symbionts, e.g. 

Rickettsia spp., and Francisella tularensis. 

(2) We expect to identify several metabolic pathways that contain horizontally acquired genes. Because these foreign-origin genes are being maintained in the reduced genome of C. burnetii, we expect them

(e.g. heme and fatty acid biosynthesis pathways) to contribute towards organismal fitness, i.e., most of them 

will be important to Coxiella’s biology. 

(3) By analyzing gene expression and Tn-insertion mutants, we expect to validate the importance of 

pathogen-specific, and HGT gene-containing pathways to Coxiella’s intracellular growth. Additionally, by 

comparing the expression of each gene in ACCM-2 vs. THP-1 cells, we will be able to identify genes that are 

induced inside the host cell. Understanding the metabolic pathways that are critical to C. burnetii while 

growing within human cells will be a major breakthrough in Coxiella research. We expect this new 

knowledge to lead to the unraveling of the pathogen’s basic biology and virulence mechanisms, and to provide new targets for the development of novel therapeutic agents. We expect to identify several genes and 

pathways that are potentially important to Coxiella’s intracellular physiology and pathogenicity. For validation, we will prioritize (1) pathways for the synthesis of critical metabolites that C. burnetii cannot obtain 

from host (e.g. heme, biotin), (2) pathways for metabolizing critical nutrients such as glutamate, and (3) 

proteins required for homeostasis (Na+/H+ antiporters) (Table 1). 

(4) Student training. Jessica Millar, a second year graduate student will accomplish the phylogenetic analyses described above. She generated most of the preliminary data that indicated the importance of 

HGT to Coxiella’s physiology. She will also perform the RNA-seq experiments as part of this Aim. She is well 

versed at using high-throughput sequencing to analyze gene expression. We have already published a paper 

that utilized RNA-seq extensively (Millar et al. 2015). Abraham Moses who joined my lab as an undergraduate and is now a Research Associate will perform the analyses on Tn-insertion mutants. He has worked on 

Coxiella for three years and plans to join my lab as a graduate student in couple of years. 

Potential Problems and Alternative Strategies

We are confident about successfully achieving the goals of this aim because we have published several papers using the approaches proposed above (for e.g., Raghavan et al 2007; Raghavan et al. 2008; Raghavan 

et al. 2010; Raghavan et al. 2012; Smith et al. 2015; Millar et al. 2015). From that experience, we expect the 

following potential impediments for which we have alternate strategies in place:

(1) In our preliminary analysis, in addition to the 357 genes potentially acquired from nonGammaproteobacteria, we detected 75 genes that were putatively obtained from members of Gammaproteobacteria. However, the phylogenetic neighborhood that contains Coxiella within Gammaproteobacteria is 

sparsely represented — Legionella, Rickettsiella, and Diplorickettsia are the only bacteria that are somewhat closely related to Coxiella. This lack of data makes it hard to identify genes acquired by C. burnetii

from other Gammaproteobacteria. We will collaborate with Dr. Joseph Gillespie (University of Maryland) to 

try and resolve the evolutionary origins of such genes (see Letter of Support). Dr. Gillespie is an expert in 

identifying gene origins. He was involved in generating the most comprehensive phylogeny of Gammaproteobacteria (Williams et al. 2010), and assembled the database PATRIC (Gillespie et al. 2011). Even if we are 

not able to detect all genes that were acquired from Gammaproteobacteria, we still have enough genes (357) 

that were putatively gained from distant phyla to identify critical metabolic pathways in C. burnetii. 

 

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Research Strategy Page 35
	
(2) Because the intracellular growth of C. burnetii within THP-1 cells is not as high as in Vero cells, 

we might not recover enough bacterial mRNA to accurately analyze gene expression. If that is the case, we 

will sequence without multiplexing, which should provide enough depth to quantify gene expression. 

(3) Both C. burnetii RSA 493 Nine Mile phase I and phase II strains have been shown to have similar growth kinetics within human macrophages (Shannon and Heinzen 2008). However, because the phase 

II strain is an LPS mutant that is not infective to humans, it is possible that the intracellular metabolic activity of this strain is slightly different from that of the phase I strain. To confirm that gene expression in phase 

II and phase I strains are similar, Dr. James Samuel will provide us with RNA extracted from THP-1 cells 

infected with Phase I C. burnetii in his BSL-3 facility (see attached Letter of Support). We will analyze phase 

I RNA samples using RNA-seq, as described above for phase II C. burnetii. 

Specific Aim 3. Determine the importance of heme biosynthesis to C. burnetii’s intracellular 

growth. 

Background and Justification

In order to validate our novel approach to identifying critical metabolic processes in Coxiella, we will examine the importance of heme biosynthesis to Coxiella’s intracellular growth. This biosynthetic pathway was 

chosen because it is unique to the human pathogen, contains horizontally derived genes, and is expressed 

intracellularly, i.e. this is an example of a pathway that we expect to prioritize after concluding Aims 1 and 2.

Coxiella’s genome is less than half the size of E. 

coli’s genome (~2 Mb and ~5 Mb, respectively). Correspondingly, Coxiella has less than half the number of 

tRNAs than in E. coli (42 and 89, respectively). This reduction has occurred due to loss of redundant tRNAs. 

For instance, while E. coli has five copies of tRNAIle, 

Coxiella has only one copy. Against this constant culling 

of redundant tRNAs, Coxiella has acquired, via HGT, an

additional tRNAGlu isoacceptor (tRNAGlu2, anticodon 

CUC) that is not found in any other Gammaproteobacteria (Figure 9). Because tRNAGlu2 is retained in such a 

streamlined genome, and is expressed at very high 

amounts (double that of the other tRNAGlu isoacceptor, 

tRNAGlu1, anticodon UUC) during intracellular growth

(Figure 10), it most likely has a critical contribution to 

bacterial fitness. 

Intriguingly, the major function of tRNAGlu2 is unlikely to be protein synthesis. There are two codons 

for glutamic acid: GAA and 

GAG. In Coxiella proteins, 76% 

of glutamate codons are GAA, 

which tRNAGlu2 (anticodon 

CUC) cannot decode, whereas 

tRNAGlu1 (anticodon UUC) can 

decode both codons efficiently. 

Previous studies have shown 

elevated protein synthesis in 

Coxiella when incubated in a 

glutamate-supplemented medium (Hackstadt and Williams 

1983; Omsland et al. 2008). To 

rule out the possibility that 

Coxiella proteins were enriched for glutamate, and 

hence needed the extra tRNAGlu for efficient translation, we 

determined the total number 

0 

50 

100 

150 

200 

250 

300 

350 

400 

450 

Gene expression value (RNA-seq)

tRNAGlu1

tRNAGlu2

hemA

hemL

hemB

hemC

hemD

hemE

hemF

hemH

Figure 10. Expression of tRNAGlu2 and heme biosynthesis genes within Vero cells. 

Glutamyl-tRNAGlu1 & 2

Glutamate-1-semialdehyde

5-Aminolevulinic acid (ALA)

Heme Biosynthesis Pathway

Heme

hemA

hemL

hemBCDEFH

Francisella tularensis

100

46

Coxiella burnetii

Legionella pneumophila

95

Acinetobacter baumannii

Pseudomonas aeruginosa

Escherichia coli

85

97

Figure 9. A 16S phylogenetic tree of Gammaproteobacteria 

showing the presence (+) or absence (-) of tRNAGlu2.

 

Contact PD/PI: Raghavan, Rahul 

Research Strategy Page 36
	
of Glu codons in Coxiella genes and compared it to E. coli genes. We found that both E. coli and Coxiella

proteins have similar Glu content (~6% of all codons). Together, these data suggest that protein biosynthesis 

is not tRNAGlu2’s major function in C. burnetii. 

Heme is an iron-containing tetrapyrrole that serves multiple cellular functions including in respiration, energy generation, oxidative reactions and signal transduction (Almiron et al. 2001). The universal 

precursor of tetrapyrrole biosynthesis is 5-aminolevulinic acid (ALA). There are two alternate pathways in 

nature for the synthesis of ALA: C5 pathway found in most bacteria, and Shemin pathway present in most

eukaryotes, including humans (Frankenberg et al. 2003). The starting point of the C5 pathway is GlutamyltRNAGlu, which is converted into ALA using two consecutive enzymes HemA and HemL (Figure 10, previous page). In Shemin pathway ALA is synthesized from Succinyl-CoA and glycine by the enzyme ALAS 

(Frankenberg et al. 2003). The rest of the steps are shared between C5 and Shemin pathways. We examined 

the C. burnetii genome and discovered that it contains an intact C5 heme biosynthesis pathway, and by 

RNA-seq we confirmed that these genes are utilized during growth within Vero cells (Figure 10, previous 

page). This pathway is conserved in all C. burnetii strains but is absent in non-pathogenic Coxiella present 

in ticks (Table 3), indicating its importance to the human pathogen. Additionally, a Tn-insertion in the 3’ 

end of hemL caused significant growth inhibition (see Figure 8, Aim 2). Based on these data, our working

hypothesis is that the major function of tRNAGlu2 is as a substrate for heme biosynthesis, which is crucial to 

C. burnetii’s intracellular growth. 

Preliminary Studies and Feasibility

1. C. burnetii cannot use extracellular 

heme as an iron source. C. burnetii encodes a 

transporter system (feoAB) for ferrous iron, which is 

required for heme biosynthesis, but has no known 

transporters for ferric ions or heme. Consequently, we 

reasoned that the pathogen might not be able to utilize 

extracellular heme as its sole source of iron. To test this, 

we inoculated equal amounts of Coxiella into ACCM-2 

medium with FeSO4 (standard recipe) or into ACCM-2 

in which FeSO4 was replaced with equimolar amount 

of hemin. Growth was measured at 7 d pi using 

PicoGreen as described previously (Martinez et al. 

2015). As shown in Figure 11, Coxiella growth in FeSO4-containing ACCM-2 was significantly higher than 

in hemin-containing ACCM-2 (p < 0.001, paired t-test), whereas the fluorescence measurements between 

control samples (ACCM-2 only) and the hemin-containing ACCM-2 samples were not significantly different 

(p > 0.05, paired t-test), indicating that C. burnetii cannot utilize external heme. 

 2. Gabaculine inhibits C. burnetii growth. To test C. burnetii’s 

requirement for heme biosynthesis, we treated C. burnetii with increasing concentrations of gabaculine, an inhibitor of HemL (Wang et al. 

1997). As shown in Figure 11, 1 mM gabaculine significantly inhibited 

the growth of C. burnetii (p < 0.001, paired t-test), confirming the importance of heme biosynthesis to its growth. We were unable to test 

gabaculine against C. burnetii growing in host cells because the drug is 

toxic to mammalian cells. As a follow up to this project, we plan to 

screen small molecule libraries to identify novel chemicals that block C.

burnetii’s heme biosynthesis without causing toxicity in human cells 

(see Future Directions). 

3. Generation of tRNAGlu2-deletion strain. To assay the importance of tRNAGlu2, we generated a tRNAGlu2-deletion strain. This 

was accomplished in collaboration with Dr. Paul Beare at Rocky Mountain Laboratories who developed the Coxiella genetic system (Beare et 

al. 2012) (see Letter of Support). Briefly, C. burnetii was transformed 

with a plasmid (pJC-kan-Glu2-CAT) and a clonal population in which

tRNAGlu2 was replaced by Chloramphenicol acetyltransferase (CAT) 

2000 

4000 

6000 

8000 

Fluorescence 

Figure 11. Hemin in place of FeSo4, 

and 1 mM Gabaculine caused

ęȱ ȱȱ(n=4).

C. burnetii – + + +

Heme – + – –

FeSO4 + – + +

Gabaculine – – – + 

(1 mM)

 Table 3. Heme biosynthesis genes are conserved in all

C. burnetii (blue) but not in CLEAA seen in Amblyomma

americanum and CRt in Rhipicephalus turanicus (red).

RSA

493

RSA

331

DUGWAYQ212 Q154 Z3035 CLEAA CRt

hemA

hemL

hemB

hemC

hemD

hemE

hemF

hemH

+ + + + + + – –

+ + + + + + – –

+ + + + + + – –

+ + + + + + – –

+ + + + + + – – + + + + + + – – + + + + + + – – + + + + + + – – tRNAGlu2 + + + + + + – –

 

Contact PD/PI: Raghavan, Rahul 

Research Strategy Page 37
	
gene was isolated as described previously (Beare et al. 2012) (Figure 12). 

Research Design

1. Complementation of tRNAGlu2-deletion strain. In order 

to confirm that phenotypes displayed by the tRNAGlu2-deletion strain are

not due to aberrant changes, we will construct a complementation strain. 

Briefly, the tRNAGlu2-deletion strain will be transformed with a plasmid 

pTn7-tRNAGlu2, which encodes a transposase that will reintroduce 

tRNAGlu2 along with its promoter into the intergenic region between 

CBU_1787 and CBU_1788, as described previously (Beare et al. 2011; 

Beare et al. 2012). As with the construction of the deletion strain, we will 

collaborate with Dr. Paul Beare to generate the complementation strain (see Letter of Support). 

2. Quantification of heme production. Heme content of wild-type, tRNAGlu2-deletion, and 

complementation strains of Coxiella grown in ACCM-2 will be quantified as described previously (Hughes 

and Macvanin 2010). Briefly, Coxiella cells will be collected by centrifugation and washed in phosphatebuffered saline. A 50 μl aliquot will be mixed with 500 μl of heated 2 M oxalic acid and incubated at 100°C 

for 30 min. This treatment strips iron from heme and the remaining protoporphyrin can be measured by 

fluorescence. After 30 min, 500 μl of phosphate buffer will be added to the mixture and allowed to cool, and 

the fluorescence spectra of the samples will be measured. 

3. Quantification of intracellular growth rate. THP-1 cells differentiated into an adherent, 

macrophage-like morphology will be infected with SCVs of wild-type, tRNAGlu2-deletion and complementation strains of C. burnetii at an MOI of 25, as described in Aim 2. We will collect THP-1 cells every 24 h, disrupt cell pellets using a bead-beater (Bio-Spec) and Phenol:Chloroform (Life technologies) and isolate DNA 

using a Qiagen DNA extraction kit. Quantitative PCR (qPCR) using primers specific to rpoS and dotA genes 

will be used to measure intracellular Coxiella as described previously (Raghavan et al 2008; Omsland et al. 

2011). In parallel to qPCR, we will quantify intracellular Coxiella using florescence microscopy. We will 

transform wild-type, tRNAGlu2-deletion and complementation strains with the vector pKM244 that expresses the mCherry red florescent protein (Chen et al. 2010). Parasitophorous vacuoles formed within THP-1 

cells by C. burnetii strains will be enumerated by florescence imaging, as described previously (Chen et al. 

2010; Beare et al. 2011; Martinez et al. 2014). To prepare for this project, we have procured the plasmid 

pKM244 from Dr. Samuel, and we have a Leica TCS SPE confocal fluorescence microscope. 

Expected Outcomes

(1) We expect both heme production and intracellular growth of the tRNAGlu2-deletion strain to be 

significantly lower than that of the wild-type strain, and expect both measures to be restored in the complementation strain. These data will validate the importance of heme biosynthesis to C. burnetii’s intracellular 

growth, which will be a significant advancement in the field because this will be the first metabolic pathway 

that will be shown to be critical C. burnetii’s intracellular growth. Moreover, we will validate heme biosynthesis as a promising a target for developing new therapeutic agents to treat chronic Q fever, which is the 

next step in accomplishing our long-term goal (see Future Directions).

(2) We expect the horizontally derived tRNAGlu2 to improve the pathogen’s heme production by 

making available more charged tRNAGlu for heme biosynthesis. Having this extra tRNAGlu is expected to free 

up enough tRNAGlu for protein synthesis so that heme production will not impinge upon bacterial growth. 

This discovery will reveal a novel pathogenic mechanism in bacteria whereby an alternate tRNA promotes 

virulence. Just as in Coxiella, several important human pathogens such as Bacteroides, Corynebacterium, 

and Clostridium encode multiple tRNAGlu isoacceptors, and hence might be utilizing a similar pathogenic 

strategy. Our approach of using genome evolution to identify virulence processes and potential antibiotic 

targets could be applied broadly to other pathogens to combat rising antibiotic resistance.

(3) Student training. Abraham Moses, who is currently a Research Associate, produced most of 

the preliminary data described in this Aim while he was working as an undergraduate student in the lab. He 

will perform all the experiments described in this Aim. He plans to join our graduate program in couple of 

years. Todd Hinsch, an undergraduate student in the lab will work with Abraham and me to accomplish the 

goals of this Aim. After graduation, he plans to join the Molecular Microbiology graduate program at OHSU.

 ̈tRNAGlu2

wild-type 3 kb

4 kb

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Contact PD/PI: Raghavan, Rahul 

Research Strategy Page 38
	
Potential Problems and Alternative Strategies

(1) If the heme quantification method described above does not produce consistent results, we will 

utilize an alternate approach described by Levican et al. (2007). In this method, Coxiella cells will be washed 

in 30 mM Hepes, pH 8.0 and suspended in a solution containing 0.5 M NaOH and 2.5% Triton-X. Cells will 

be broken up using sonication and centrifugation, and heme content will be measured at 575 nm absorbance.

Another alternative is a commercially available Heme Assay Kit (Abnova). 

(2) If heme production or intracellular growth (or both) is not impacted by the loss of tRNAGlu2, we 

will compare the proteomes of wild-type and tRNAGlu2 deletion strains. We have prior experience with Coxiella proteomics (Raghavan et al. 2008; Hicks et al. 2010), and the OHSU Proteomics Shared Resources has 

the staff and equipment required for successful proteome analyses of Coxiella strains. Briefly, we will grow C.

burnetii strains in ACCM-2 for 7 days and the bacteria will be pelleted by centrifugation (20,000 x g for 5 

min). Proteins from a total cell lysate will be prepared as described earlier (Hicks et al. 2010; Stead et al. 

2013). Proteins will be separated by SDS-PAGE using 16.5% gel and visualized using SimplyBlue SafeStain 

(Invitrogen), and the lanes will be cut into 10 equal sections and subjected to trypsin digestion. The resulting 

peptides will be sequenced by tandem mass spectrometry (MS/MS) at the OHSU Proteomics Shared Resources using a ThermoFinnigan LTQ mass spectrometer fitted with an IonMax nanospray source. The 

MS/MS spectra will be searched against C. burnetii subset of NCBI nr protein database using Proteome 

Software Scaffold 3.0. The proteomic data is expected to reveal the proteins that were impacted by the loss 

of tRNAGlu2. Genes that encode these proteins will then be interrogated to identify metabolic processes that 

are important to Coxiella’s intracellular growth and virulence. 

(3) Because C. burnetii contains two tRNAGlu isoacceptors, it is possible that the lack of tRNAGlu2 will 

not have any impact on the pathogen. However, this is not a concern because in comparison to wild-type C. 

burnetii, the tRNAGlu2-deletion strain exhibited a severe growth defect in ACCM-2 (Figure 13), indicating 

that tRNAGlu2 is essential for attaining optimum fitness, most likely through augmenting heme production. 

(D) FUTURE DIRECTIONS

At the successful completion of this project, we expect to gain

new insights into the evolution of C. burnetii, and to have identified metabolic pathways that are critical to its intracellular 

growth. Our next steps will be:

(1) As in Aim 3, we will delete specific genes to validate 

the importance of C. burnetii’s metabolic pathways. For example,

we have evidence that biotin biosynthesis is also critical for C.

burnetii’s growth. No transposon-insertion mutants are currently 

available for biotin biosynthesis genes, but we expect to be able 

to generate a C. burnetii strain lacking biotin biosynthesis by 

providing the cofactor in ACCM-2 medium, as shown recently for 

other nutrients (Sandoz et al. 2016). 

(2) The next step towards achieving our long-term goal of 

developing new therapeutic agents will be to identify chemical 

agents that inhibit the function of critical metabolic genes. For 

instance, the small molecule MAC13772 (Maybridge) inhibits biotin biosynthesis in E. coli (Zlitni et al. 2013). 

To identify small molecules that block critical metabolic processes in C. burnetii, we will conduct screens 

using small molecule libraries. In collaboration with Dr. Robert Allen (see Letter of Support) at Oregon 

Translational Research and Development Institute (OTRADI), Austin Wright, a first-year graduate student 

in the lab will perform these experiments. 

(E) RESEARCH TIMELINE. 

This project will take three years to complete. 

Aim 1

Aim 3

Aim 2

A

(

P

(

C

(

Aim 1

Aim 3

Aim 2

Year 1 Year 2 Year 3

0 

20000 

40000 

60000 

wt ǻtRNAglu2 

Fluorescence

Figure 13. C. burnetii growth in ACCM-2 measured 

at 7 d post-inoculation using PicoGreen. p < 0.001 

(paired t-test, n = 3).

 

Contact PD/PI: Raghavan, Rahul 

Research Strategy Page 39
	
SELECT AGENTS

The proposed research involves Coxiella burnetii, a Category B Select Agent. However, all work will use the 

avirulent, exempt Nine Mile Phase II strain (RSA 439), under BSL2 containment. Genes conferring 

resistance to kanamycin and chloramphenicol are approved for Coxiella burnetii genetic transformation by 

the Centers for Disease Control and Prevention Select Agents Program.

 

Contact PD/PI: Raghavan, Rahul 

Select Agent Research Page 40
	
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