SUMMARY STATEMENT

PROGRAM CONTACT: ( Privileged Communication ) Release Date: 11/28/2016

Revised Date:

Application Number: 1 R15 AI126385-01A1

Principal Investigator 

RAGHAVAN, RAHUL

Applicant Organization: PORTLAND STATE UNIVERSITY

Review Group: ZRG1 IDM-S (81)

Center for Scientific Review Special Emphasis Panel

AREA applications in Infectious Diseases and Microbiology

Meeting Date: 11/07/2016 RFA/PA: PA16-200

Council: JAN 2017 PCC: M46C B

Requested Start: 04/01/2017

Project Title: Elucidating the evolution of Coxiella to uncover critical metabolic pathways

SRG Action: Impact Score:

Next Steps: Visithttp://grants.nih.gov/grants/next_steps.htm

Human Subjects: 10-No human subjects involved

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

Project

Year

Direct Costs

Requested

Estimated

Total Cost

1 

TOTAL

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

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

Institute grants management staff based on the recommendations outlined below in the COMMITTEE 

BUDGET RECOMMENDATIONS section.

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1R15AI126385-01A1 Raghavan, Rahul 

RESUME AND SUMMARY OF DISCUSSION: This resubmitted application proposes to identify critical 

metabolic pathways for intracellular growth of Coxiella burnetii using a comparative genomic 

approaches. The results could increase the basic understanding of survival mechanism of C. burnetii in 

the phagosomal vesicles in host cells. The application can also have strong implications on the study of 

pathogenesis of other tick-borne pathogens. The investigator has adequately addressed the previous 

concerns. This submission has a clear prioritization plan. The strong preliminary data, though not 

required, provide the application with a strong premise. The strengths are that the investigator is very 

productive and has a solid publication record; the hypothesis that critical metabolic pathways may be 

acquired through horizontal gene transfer is innovative; the comparative genomic analysis between C. 

burnetii and other nonvirulent tick-associated Coxiella is well described with adequate rigor; a 

transposon-generated mutant library is already available to screen functionally active genes critical for 

intraphagosomal growth of C. burnetii; the potential for student involvement in research is excellent; 

and the environment is appropriate. The overall enthusiasm is very high for this application proposing to 

identify critical metabolic pathways for intracellular survival of C. burnetii. 

DESCRIPTION (provided by applicant): 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 tick-associated 

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 intra- cellular 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 tick-associated 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. 

PUBLIC HEALTH RELEVANCE: Coxiella burnetii is a zoonotic bacterium that causes Q fever and 

chronic endocarditis. Metabolic processes critical to its intracellular growth and pathogenesis are not 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1 R15 AI126385-01A1 3	 ZRG1 IDM-S (81)
 

RAGHAVAN, R 

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. 

CRITIQUE 1: 

Significance: 2 

Investigator(s): 1 

Innovation: 1 

Approach: 1 

Environment: 2 

Overall Impact: This innovative revised application aims to identify the metabolic pathways critical for 

Coxiella burnetii for its intracellular/phagosomal growth. The investigator's hypothesis is that the 

pathogen acquired critical metabolic pathway genes by horizontal gene transfer (HGT). Previous 

studies by the investigator provide a solid premise for this exciting hypothesis. The project has three 

well-focused and independent specific aims. Novel data are expected at the end of this study. Previous 

submission was also considered as highly innovative and that the investigator was identified as wellqualified to carry out the proposed project goals. The investigator responded very well in addressing the 

prior noted minor concerns. Enthusiasm for this revised application remained strong. 

1. Significance: 

Strengths 

	 Coxiella burnetii is an important human pathogen, as it is capable of causing significant
 

morbidity and mortality.
 

	 As very few organisms are capable of causing the disease, Q fever, and that the organism 

survives well in the environment for very long periods of time, this pathogen is listed as a BSL3 

select agent. 

	 The proposed project outcomes may also be valuable in initiating similar studies in 

understanding the molecular basis for pathogenesis of several other tick-borne pathogens 

identified in recent years as causing serious diseases in people. 

Weaknesses 

	 None noted. 

2. Investigator(s): 

Strengths 

	 The investigator is an accomplished young scientist who has the necessary expertise. 

	 His record of productivity up to this point is also excellent and it is directly relevant for studying 

the bacterial genomes and other proposed experiments. 

Weaknesses 

	 None noted. 

3. Innovation: 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Strengths 

	 The project is novel as it aims to identify metabolic pathways critical for Coxiella for its
 

intracellular/phagosomal growth.
 

	 Non-pathogenic endosymbionts also co-exist in ticks along with pathogenic organisms 

belonging to the genera Rickettsia, Ehrlichia and Anaplasma. Comparative genomics proposed 

in the current study on two Coxiella species may open ways for similar research focused on 

understanding how other pathogenic bacteria are evolved in ticks by co-existing with nonpathogenic microbiomes. 

Weaknesses 

	 None noted. 

4. Approach: 

Strengths 

	 This study aims to identify metabolic pathways vital for C. burnetii's intracellular growth. 

Comparative genomics will be performed by analyzing a soft tick-derived endosymbiont species 

of Coxiella and C. burnetii genomes to identify plausible metabolic pathway genes acquired for 

pathogenesis by horizontal gene transfer (HGT) (aim 1). 

	 In aim 2, RNA sequence data from wild type and transposon mutant libraries will be generated 

and evaluated to map functionally active genes critical for the intraphagosomal growth. This 

goal is independent of the first aim and a transposon library is already available to screen. 

	 Prior studies by the investigator’s team already identified heme biosynthesis pathway as having 

HGT genes, which are absent in the Coxiella endosymbionts in ticks. The investigator will 

directly test the hypothesis by evaluating this pathway for its role in intracellular growth (aim 3). 

	 The study is likely to open new research directions in defining how the pathogenic organisms 

are evolved in ticks and in other arthropod vectors. 

	 The proposed project goals in defining the metabolic pathways acquired by HGT in pathogenic 

bacteria are novel. 

	 The study is likely lead to a better understanding of how other disease causing agents are 

evolved. 

	 Alternate methods are well described. 

Weaknesses 

	 None noted. 

5. Environment: 

Strengths 

	 Excellent 

Weaknesses 

	 No concern. 

Protections for Human Subjects: 

Not Applicable 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Vertebrate Animals: 

Not Applicable (No Vertebrate Animals) 

Biohazards: 

No concern 

Resubmission: 

 This is a revised application and the investigator addressed all of the prior concerns. 

Select Agents: 

Acceptable 

Authentication of Key Biological and/or Chemical Resources: 

Acceptable 

Budget and Period of Support: 

Recommend as Requested 

CRITIQUE 2: 

Significance: 3 

Investigator(s): 1 

Innovation: 2 

Approach: 2 

Environment: 1 

Overall Impact: This is a well-written application that aims to use evolutionary genomics approach to 

identify metabolic pathways that are critical to the intracellular growth of Coxiella burnetii. The 

investigator plans to test his hypothesis, which C. burnetii evolved from a tick-associated ancestor by 

acquiring metabolic genes through horizontal gene transfer, by the following three specific aims: 1) 

Identify metabolic pathways that distinguish C. burnetii from the tick-associated Coxiella. 2) Define 

metabolic pathways that are critical to C. burnetii’s intracellular growth. 3) Determine the importance of 

heme biosynthesis to C. burnetii’s intracellular growth. The study, if successful, may give insights on 

metabolic pathways for intracellular growth and new therapeutic targets for C. burnetii. This application 

is likely to produce useful data since the investigator has already obtained some preliminary data and 

collaborated with eight collaborators. The investigator has a track record of manuscript publications and 

training undergraduate and graduate students. 

1. Significance: 

Strengths 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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	 Identification of metabolic genes that are acquired by horizontal gene transfer is important for 

the intracellular growth of C. burnetii. The genome-scale approach of identifying metabolic 

genes using genome sequences from virulent C. burnetii and tick-associated Coxiella symbiont 

could be applied to other tick pathogens, such as Rickettsia and Francisella species. 

	 The tRNA-acquisition-based virulence mechanism studied in this study could serve as a model 

of studying other pathogenic bacteria, including Corynebacterium and Clostridium species. 

Weaknesses 

	 The genome-scale approach of identifying different genes between virulent and symbiotic 

bacteria have been explored in other organisms in the past. 

2. Investigator(s): 

Strengths 

	 The investigator has expertise in bioinformatics, high-throughput sequencing, and microbiology. 

All of them are important to the success of the proposed studies. 

	 The investigator has a track record of manuscript publications. Since the investigator became 

an assistant professor at PSU in 2012, he has published four manuscripts on Coxiella. 

	 There are six support letters from collaborators in this application. All of the collaborators have 

the expertise in the proposed experiments. These are important for the successful completion of 

this research proposal. 

Weaknesses 

	 None noted. 

3. Innovation: 

Strengths 

	 Metabolic pathways that are critical for intracellular growth of C. burnetii have not been
 

explored.
 

	 The concept that virulence factors and metabolic genes of C. burnetii were obtained by
 

horizontal gene transfer is novel.
 

	 The use of a novel tRNA-acquisition-based virulence mechanism has not been explored in C. 

burnetii. 

Weaknesses 

	 Identification of metabolic pathways for intracellular growth by genome comparisons has been 

explored in other organisms. 

	 Figure 3: Although C. burnetii could evolve from a tick-associated ancestor by acquiring genes 

through horizontal gene transfer, C. burnetii could obtain those genes from an environmental 

ancestor by horizontal gene transfer and later became tick-associated symbionts by gene loss. 

4. Approach: 

Strengths 

	 The design and methods of this application is appropriate. 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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	 Preliminary data are available for all three specific aims. The preliminary data support the 

investigator’s hypothesis to be tested and feasibility of the proposed research. 

	 Potential pitfalls and alternative methods are discussed for all three specific aims. 

Weaknesses 

	 The investigator proposed to sequence the genome of a Coxiella species in Ornithodoros 

rostratus. Although the genome sequences will help the investigator to identify metabolic genes 

that are only present in C. burnetii, the specific aim could be carried out with the sequenced 

genomes of Coxiella in Amblyomma americanum and Coxiella in Rhipicephalus turanicus. 

	 The history of O. rostratus lab colony is not known. The genetic elements might be different 

between wild type and lab colony of O. rostratus. 

5. Environment: 

Strengths 

	 The investigator’s lab is located in a new research facility that is shared between Portland State 

University, Oregon Health and Science University, and Oregon State University. The unique 

location maximizes the probability of resource utilization and promotes collaborations among 

faculty members from three universities. 

	 Two Linux servers are available for genome sequencing project and annotation. 

	 The investigator has access to all core facilities in Oregon Health and Science University. 

	 The investigator received a reduced teaching load prior to his evaluation for tenure. 

Weaknesses 

	 None noted 

Protections for Human Subjects: 

Not Applicable (No Human Subjects) 

Vertebrate Animals: 

Not Applicable (No Vertebrate Animals) 

Biohazards: 

Acceptable 

Resubmission: 

	 Appropriate addressed 

Select Agents: 

Acceptable 

Resource Sharing Plans: 

Acceptable 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Authentication of Key Biological and/or Chemical Resources: 

Acceptable 

Budget and Period of Support: 

Recommend as Requested 

CRITIQUE 3: 

Significance: 2 

Investigator(s): 2 

Innovation: 2 

Approach: 2 

Environment: 2 

Overall Impact: The resubmission outlines experiments to understand the evolution and pathogenesis 

of Coxiella burnetii. This intracellular pathogen causes acute flu-like symptoms, and can cause 

endocarditis. Illness is difficult to diagnosis and treat, with antibiotic resistances becoming more 

common. This is a select agent, with bio-terrorism threat. No vaccine is available, and thus significance 

is considerable, though disease is limited in the US. C. burnetii can survive the lysosomal compartment 

as a small cell variant. The closest relative to C. burnetii is found within ticks, and it is proposed that this 

relative evolved to be able to infect humans. The investigator has a solid publication record in the areas 

necessary for carrying out the research. The approach is innovative, made possible by the capabilities 

of nextgen sequencing, RNAseq, and analysis software. The investigator proposes to investigate 

metabolic changes that have allowed for Coxiella virulence in humans to appear. The investigator will 

do whole genome sequencing of the tick-associated Coxiella and two genomes from C. burnetii isolates 

and then compare this to existing C. burnetii genomes, with the hypothesis that metabolic differences 

can, in part, explain human host adaptation. The group will use KEGG and other software analysis, and 

focus on heme metabolism. The have identified a tRNAGlu, acquired by horizontal gene transfer, 

thought to be involved in fitness. The investigator, with a solid track record of publishing, is highly 

experienced in genomic analysis, and the use of software such as KEGG for understanding the 

differences between the Coxiella species is a solid approach. To gain a greater understanding of PV 

survival and the metabolic pathways necessary for pathogenesis, the investigator proposes using 

bioinformatics and then RNAseq to confirm their observations concerning gene expression within 

human macrophage. The experiments proposes are comprehensive, addressing questions from 

multiple angles, and are thus scientifically rigorous. The application would be impactful in training 

undergraduate and graduate students. 

1. Significance: 

Strengths 

 C. burnetii is the causative agent of Q fever 

 Extremely low infectious dose, with increased reported infections in the US 

 Select agent, a bioterrorism threat, no vaccine available 

Weaknesses 

 Somewhat limited disease in the US 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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2. Investigator(s): 

Strengths 

 Productive, early stage investigator
 

 Already has an established track record of mentoring students
 

 Highly experienced in the assembly and analysis of genomic sequences
 

Weaknesses 

	 None noted 

3. Innovation: 

Strengths 

	 Studying the evolutionary history of C. burnetii from a tick-associated relative 

	 Investigating metabolism as it relates to pathogenesis, and how C. burnetii survives within the 

phagolysosome 

	 Chemical biology approaches for targeting heme production in future experimentation offer 

sound approach for intervention 

Weaknesses 

	 Limited, though increasing disease incidence in the US. 

4. Approach: 

Strengths 

	 Comparative genomic analysis to determine the genes required for C. burnetii pathogenesis in 

humans in comparison to the tick-associated O. rostratus most likely will be informative 

	 Collaboration in place for acquiring O. rostratus material and genetic manipulations 

	 Identification of target metabolic pathways through bioinformatics, then testing expression within 

the phagolysosome adds scientific rigor. 

Weaknesses 

	 Construction of genetic mutants using Tn mutagenesis could prove challenging because of 

lethality, etc., though alternate approaches are in place for construction and then testing in the 

macrophage cell line 

	 Not clear how horizontally acquired genes are being identified, except that they are not from 

Gamma proteobacteria. 

5. Environment: 

Strengths 

	 All the resources are in place to carry out the research, with collaborative interaction with 

investigators within and outside the institution 

	 The application would greatly enhance the research capability of the institution 

	 Excellent opportunities for undergraduate and graduate student training 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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	 Partial teaching reduction would allow for focused research effort by the PI 

Weaknesses 

	 None noted. 

Protections for Human Subjects: 

Not Applicable (No Human Subjects) 

Vertebrate Animals: 

Not Applicable (No Vertebrate Animals) 

Biohazards: 

Acceptable 

 BSL-2 facilities and autoclave are in place for the research. 

Resubmission: 

	 The investigator has addressed a number of concerns, both with the significance as well as with 

the approach. The significance is reiterated by the investigator, with implications to other 

intracellular pathogens. For the approach, a number of issues have been addressed, 

concerning how to interpret the data and directions to proceed. The potential impact to the 

institution, in this reviewer's opinion, will be significant. 

Select Agents: 

Acceptable 

	 The investigator states that all work will use the Coxiella burnetii strain to be used for the 

investigation is avirulent and exempt (Nine Mile Phase II strain (RSA 439). Propagation of this 

strain will occur under BSL2 containment. 

Resource Sharing Plans: 

Acceptable 

Authentication of Key Biological and/or Chemical Resources: 

Acceptable 

Budget and Period of Support: 

Recommend as Requested 

THE FOLLOWING SECTIONS WERE PREPARED BY THE SCIENTIFIC REVIEW OFFICER TO 

SUMMARIZE THE OUTCOME OF DISCUSSIONS OF THE REVIEW COMMITTEE, OR REVIEWERS' 

WRITTEN CRITIQUES, ON THE FOLLOWING ISSUES: 
 

 

 

 

 

 

 

 

 

 

 

 

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COMMITTEE BUDGET RECOMMENDATIONS: The budget was recommended as requested. 

Footnotes for 1 R15 AI126385-01A1; PI Name: Raghavan, Rahul 

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

See Guide Notice NOT-OD-14-074 at http://grants.nih.gov/grants/guide/notice-files/NOT-OD14-074.html. The impact/priority score is calculated after discussion of an application by 

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

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

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

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

ranking. For details on the review process, see 

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