Analysis of human microbial communities through the study of microbiomes has begun to elucidate many details of both those organisms that constitute the human body, as well as their contribution to human disease and metabolic processes. While much of the focus of microbiome analysis has been on prokaryote constituents, there has been relatively little attention paid to viruses that inhabit the human environment. Many such viruses are bacteriophages that specifically infect bacteria. Bacteriophages represent the most abundant infectious agents on the planet, and because they have been found wherever bacteria exist, there likely are vast unexplored bacteriophage communities that inhabit the human body. Through either their ability to kill certain bacteria, or to transform bacteria by bringing in new gene-encoded function and thereby providing those bacteria a selective advantage, bacteriophages may have a profound ability to shape human bacterial communities. While much is known about the how bacteriophages interact with their host bacteria in vitro, there is relatively little known about these interactions within complex human ecosystems. One means for improving our understanding of the dynamics of bacteriophage interactions with their hosts in various ecosystems has been the study of CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats). CRISPRs represent adaptive bacterial immune systems against phage predation. They function by incorporating small portions of bacteriophage DNA called spacers between palindromic repeats in the genome of their bacterial host. The result of spacer incorporation is resistance of the host bacterium to previously encountered bacteriophages through a mechanism of RNA interference. In addition, through the incorporation of spacers, the CRISPR locus could be viewed as a log of recently encountered bacteriophages to which resistance has developed. Furthermore, the rate of resistance to newly encountered bacteriophages through the CRISPR mechanism may be ascertained through sampling of CRISPR content over time. There have been no previous comprehensive studies of bacteriophage communities that inhabit the human oral cavity. We propose a multidisciplinary approach combining molecular biology, microbial genomics, and bioinformatics towards understanding the nature of human oral bacteriophage communities, and understanding the implications behind development of resistance to these bacteriophages. The short-term objectives of our compilation of work are to improve our understanding of human oral microbial community dynamics, and to understand the impact that bacteriophages have upon human microbial communities. The long-term objective is to lay a foundation for a career in academic medicine.
Our specific aims for this proposal are as follows:
Aim 1 : To isolate and characterize bacteriophage populations from human saliva and determine whether there is a direct or inverse relationship between bacterial community composition and bacteriophage community members. The experimental approach involves microbiome analysis of bacteriophage and bacterial communities from different human subjects over time.
Aim 2 : To evaluate CRISPR content in certain salivary bacteria in human subjects and determine their relative rate of acquisition of spacers corresponding to salivary bacteriophages. The experimental approach involves analysis of changes in CRISPR loci content in certain oral bacteria in human subjects over time and comparison of CRISPR loci content with the presence or absence of bacteriophages in the community.
Aim 3 : To determine if certain oral bacteria develop resistance in vivo to bacteriophage predation, and ascertain whether the mechanism of resistance is CRISPR mediated. The experimental approach involves isolation of certain oral bacteria and their respective bacteriophages from human subjects over time, examining the ability of phage to lyse their host bacteria, and assessment of changes in CRISPR content. "Microbiome analysis of human oral bacteriophage communities" PI: David T. Pride Our research plan involves providing the first detailed description of bacteriophages in the human oral cavity to gain an improved understanding of the dynamics of complex microbial communities inhabiting the human body. Bacteriophages have a substantial capacity to alter prokaryote communities, and because perturbations in prokaryote communities contribute to disease processes, analysis of bacteriophages may provide important clues as to the development of both periodontitis and dental caries in our human subjects. The implications for disease development render this research relevant to public health, and may provide a new focal point for the development of tests for the diagnosis and prediction of disease associated with human microbial communities.

Public Health Relevance

Microbiome analysis of human oral bacteriophage communities PI: David T. Pride Our research plan involves providing the first detailed description of bacteriophages in the human oral cavity to gain an improved understanding of the dynamics of complex microbial communities inhabiting the human body. Bacteriophages have a substantial capacity to alter prokaryote communities, and because perturbations in prokaryote communities contribute to disease processes, analysis of bacteriophages may provide important clues as to the development of both periodontitis and dental caries in our human subjects. The implications for disease development render this research relevant to public health, and may provide a new focal point for the development of tests for the diagnosis and prediction of disease associated with human microbial communities.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Clinical Investigator Award (CIA) (K08)
Project #
5K08AI085028-03
Application #
8503582
Study Section
Microbiology and Infectious Diseases B Subcommittee (MID)
Program Officer
Park, Eun-Chung
Project Start
2011-08-19
Project End
2014-07-31
Budget Start
2013-08-01
Budget End
2014-07-31
Support Year
3
Fiscal Year
2013
Total Cost
$132,786
Indirect Cost
$9,836
Name
University of California San Diego
Department
Pathology
Type
Schools of Medicine
DUNS #
804355790
City
La Jolla
State
CA
Country
United States
Zip Code
92093
Abeles, Shira R; Robles-Sikisaka, Refugio; Ly, Melissa et al. (2014) Human oral viruses are personal, persistent and gender-consistent. ISME J 8:1753-67
Abeles, Shira R; Pride, David T (2014) Molecular bases and role of viruses in the human microbiome. J Mol Biol 426:3892-906
Ly, Melissa; Abeles, Shira R; Boehm, Tobias K et al. (2014) Altered oral viral ecology in association with periodontal disease. MBio 5:e01133-14
Robles-Sikisaka, Refugio; Naidu, Mayuri; Ly, Melissa et al. (2014) Conservation of streptococcal CRISPRs on human skin and saliva. BMC Microbiol 14:146
Naidu, Mayuri; Robles-Sikisaka, Refugio; Abeles, Shira R et al. (2014) Characterization of bacteriophage communities and CRISPR profiles from dental plaque. BMC Microbiol 14:175
Robles-Sikisaka, Refugio; Ly, Melissa; Boehm, Tobias et al. (2013) Association between living environment and human oral viral ecology. ISME J 7:1710-24