Studying Your Saliva

by Andrew Lum | staff writer | SQ Vol. 10 (2012-2013)

Understanding the Vast Oral Microbiota

Face it­­–we’ve all gone to the dentist with high hopes, only to receive a lecture about the importance of flossing our teeth while they jab at our gums, stirring up pain and guilt. There’s even more of a chance that someone you know has experienced the dreadful effects from one or more forms of gum infection, whether swelling due to mild gingivitis or a case of receding gums that is common with severe periodontal disease. A little blood from flossing may seem minor at first, but an unaddressed infection of the gums can rapidly lead to a long list of serious dental related problems including bone loss, tissue damage, and the worst case scenario­­–tooth loss.

While no one wants to be walking around at an early age with gaps in their smile, vulnerability to the bacterial build up causing periodontal disease can begin as soon as you finish that sandwich you ate for lunch. Much research has been conducted on the pathogenesis and etiology of this bacterial disease but there remains a level of uncertainty about the role of other microbial inhabitants such as viruses.

Currently, Dr. David T. Pride is leading a team of pathologists and immunologists at the UC San Diego School of Medicine in learning how interactions between bacteria and viruses carry a vast amount of information pertaining to human oral cavity conditions and susceptibility to diseases such as periodontitis. The oral cavity is inhabited with pathogenic microorganisms that can be affected by anything that might enter the mouth. As a result, the micro­abrasive surface of our teeth is extremely susceptible to the bacterial colonization and secretion of chemicals which help break down our lunch.

When we forget to brush, or when we put off flossing, the accumulation of bacteria starts to form a bio­film commonly known as plaque. As plaque hardens due to neglect and time, it becomes tartar which is known as calcium build-up. Days turn into weeks, and flossing seems to only make your gums bleed more; so while you put your floss back in the cabinet, tartar forms below your gum line and causes inflammation during the onset of gum infection.

While much is known about the bacterial contribution towards developing periodontal disease, little is known about the role coexisting viruses may have on determining what type of bacteria exist, and how the bacteria might behave in the oral cavity. Viruses are infectious agents that exist in almost all environments, develop continuously into new forms, and thrive wherever bacteria are present. As a result bacteria have become well acquainted with their neighboring infectious viruses.

One way in which the interactions between viruses and bacteria are being studied by Dr. Pride is through the analysis of clustered regularly interspaced short palindromic repeats (CRISPRs), which is a form of an adaptive immunity toward previously encountered viruses and plasmids. Patterns among these sequences, have the potential to reveal composition differences among hosts, as well as the rate of evolution between generations of bacteria, both of which contribute to the overall understanding of the driving force behind periodontal disease.

Analysis of CRISPR Repeats

During their initial study of human salivary viruses in 2010, saliva from four healthy human subjects was collected over an 11­17 month period so that bacterial CRISPR sequences could be analyzed alongside a library of 16S rRNA gene sequences. 16S rRNA is a highly conserved gene among bacteria and archaea that is useful for identifying bacteria. Of additional importance was the evaluation of CRISPR spacers, which are short segments of viral DNA that have been incorporated into the space between each CRISPR. CRISPR repeat loci and spacers analyses were conducted using amplifications of primers specific to sequences present in several common oral bacteria such as Streptococcus mutans.

Collectively, the data is suggestive of a continual interaction between oral bacteria and viruses in order to maintain a stable bacterial population. Differences in spacers between individuals suggests that exposure to different viral populations influence the composition of each CRISPR system.

Dr. Pride and his team have used this information to formulate two important phenomena within the CRISPR system which may contribute to further understanding of the bacteria­virus interactions. The first idea is the presence of a core set of shared CRISPR spacers over time. Preservation of these core units likely reflects the pressure from repeated exposure of the same virus types. The second phenomenon is rapid variation in the CRISPR repeats over time which brings up the possibility of new bacterial strains arising in the oral cavity. As this initial study assessed just a single CRISPR repeat out of many, further analysis will enable them to visualize how a specific household environment can influence an individual’s risk of developing periodontal disease through repeated exposure to certain viral communities.

Virus Identification

In 2011 Dr. Pride revealed the surprising presence of lysogenic bacteriophages in human saliva. Unlike lytic bacteriophages, which result in the death of their infected bacterial host, lysogenic bacteriophages integrate their genetic material into the host DNA before replication occurs. This finding of bacterial viruses was contrasted against their original belief that the oral cavity was primarily host to human viruses.

Furthermore, lysogenic bacteriophages reveal their role in spreading genes among bacteria or a, “Reservoir of bacterial genomic material,” as Dr. Pride phrases it. The composition and role of viruses was determined using a series of blastX analyses which align similar genomic sequences with the purpose of identifying genes against a known library of proteins.This time, saliva from five individuals was collected over a 3 month period for use in virome sequencing and assignment of contigs based on blastX analysis. BlastX analyzes DNA sequencing based on alignment so that contigs, overlapping regions of consensus DNA can be assigned.

Initial comparison of virus composition between subjects indicated the presence of shared homologs between all subjects, throughout the entire experiment. In the oral cavity, several virulence factor homologs, were found among the analyzed viromes. These virulence factors, involved in platelet binding, iron scavenging, lipopolysaccharide biosynthesis, cell wall antigenic variation, and DNA methylation reveal a secondary role for viruses.

Aside from simply targeting bacteria, these viruses represent a bank of infections genes, potentially enabling their target bacteria with pathogenic traits that may contribute to periodontal disease. In addition, each subject revealed 10% of their viral contigs to contain integrase protein homologs, suggesting the presence of lysogenic viruses. This integrase is similar to the enzyme used by retroviruses such as AIDS to incorporate foreign DNA into the host cell genomic material. While viral BlastX analysis determined that there was a complex diversity of viruses present in each subject, bacteriophages were the most abundant. These unique viral qualities differ greatly from the properties that were previously identified in viruses from human respiratory and gastrointestinal tracts, and as a result will require close attention in the future.

CRISPR Sequencing Across Individuals

Dr. Pride’s most recent research on virus­bacteria relationships in the oral cavity has analyzed 13,977 streptococcal CRISPR sequences against 2,588,172 virome reads to further study how bacterial immunity is reflective of current and previously encountered viruses. This study involving saliva from four humans over 17 month periods contributes to his initial findings by concluding that CRISPR spacers and their corresponding viruses coexist in humans at the same time.

In contrast to their first study which compared CRISPR sequences in bacteria to a library of known bacteria, this compared CRISPR sequences and viruses within the same individual. Viruses from saliva were collected and sequenced, while CRISPR spacers were evaluated metagenomically from two repeat sequence families (Streptococcus gordonii and Streptococcus thermophilus), instead of analyzing a single group of bacterial CRISPRs.

Interestingly, there were new CRISPR spacers that arose, which corresponded to specific virome matches. This revealed bacterial ability to adapt to new local viruses. CRISPR spacers were also tested for matches against other virome reads outside of the oral cavity in an individual, but failed to show any significant matches. However, CRISPR spacers did display matches with virome reads of other individuals, indicating the potential for CRISPRs from one individual to neutralize viruses of another individual. While bacteria and their viruses in other ecosystems have been studied extensively, this is the first analysis of CRISPR ­virus relationships in humans that has unearthed critical understandings of what shapes the microbiota existing within humans.

Thus far, data points to the idea that two families of streptococcal oral microbiota contain a developed immune response, designed to deal with the vast oral viral community present in our saliva and encountered in the environment. This has implications for how bacterial communities deal with viruses in the oral cavity, by opening doors for studying the effect of disarming viral communities and how oral microbiota can develop into the disease causing pathogens that lead to periodontal disease. Much of this currently being tested in what Dr. Pride calls, “Household Studies, involving passive resistance” to viral communities.

Dr. Pride hopes his work will someday help reveal the baseline level of microbial factors necessary for developing infections such as periodontal disease. His use of CRISPR sequences to compare virome and 16S rRNA reads have opened up a library of information that was previously unstudied. Although diagnostic tools for marking viruses specific to periodontal disease is a long way off, this research has uncovered a deep viral­bacterial relationship that has major implications for our oral health. While this doesn’t solve the issue of flossing on a daily basis, it sheds light on one of the least understood human ecosystems and provides a target for future diagnostic tests to detect infectious diseases caused by bacteria.

WRITTEN BY ANDREW LUM. Andrew Lum is a Biochemistry and Cell Biology major in Earl Warren College. He will graduate in 2013.