Lyme disease is the fifth most common Nationally Notifiable disease and the most commonly reported vector-borne illness in the United States. It is caused by spirochetes of the bacterial species group Borrelia burgdorferi sensu lato that is obligatorily transmitted by hard-bodied ticks (e.g., Ixodes scapularis in the US). Whereas incidence of Lyme disease in the USA is currently concentrated in the Northeast, Upper Midwest, and West Coast, the disease prevalence continues to rise and the geographic range of pathogen and tick populations continue to expand, with climate changes and declining biodiversity across the global as likely driving factors. Three specific public-health risks have emerged as results of the ongoing geographic expansion of Lyme disease pathogens and vectors. The first risk is the growth of previously rare and isolated pathogen populations ("spill-over"). For example, a new, highly human-virulent species B. mayonii has recently been discovered in the Upper Midwest. The second risk is the colonization of new areas by well-established pathogen populations elsewhere (“invasion”). For example, at least four different types of B. burgdorferi sensu stricto, the main pathogenic species in the U.S., are found in both Europe and North America, likely due to recent cross-Atlantic migration. The third risk is the increasing hybridization between previously segregated species or strains due to recombination (“mixing”). For example, Borrelia populations consists of recombinant genomes in North America and cross-species recombinants in Europe.

Given these heightening Lyme disease risks, there is a pressing need to characterize the full extent of genetic variations of Lyme disease pathogens by whole-genome sequencing. IGS scientists have previously sequenced and published complete genome sequences of over twenty Borrelia bacteria and performed extensive comparative analysis of these genomes. High-quality genome sequences of additional Borrelia strains that span the world-wide range of genetic and epidemiological diversity, including newly discovered Borrelia species and emerging strains of this clinically important bacteria, are currently in the works. Whole-genome sequencing are essential for deducing mechanisms of pathogenesis through comparative analyses, as well as for identifying genetic targets for vaccines, diagnostics, and therapeutics. In addition, high-quality reference genomes representing national and global Borrelia diversity have to potential to provide unprecedented resolution for epidemiological tracking, prediction, and control of the spread of Lyme pathogens.

[PI's: Dr. Sherwood Casjens, U. Of Utah, Dr. Benjamin Luft, Stonybrook U., Dr. Weigang Qiu, Hunter College of U. Of New York, Dr. Steven Schutzer, Rutgers U.]

Abundant lactobacilli in the human vagina are thought to protect against invasion by non-indigenous bacteria, including sexually transmitted infections caused by Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (GC). The means by which this happens are not well understood. It could be that these exclusionary mechanisms are properties of the vaginal microbiome, features of the host immune system and physiology, or some combination of both. The goal of this project is to employ a systems biology approach to identify biomarkers of the vaginal and penile microbiome, the host and the pathogens that are associated with increased or decreased risks of infection by CT, GC or both. Project 3 of this research program will rely on samples collected by the Clinical Core C from STING networks of sex partners who have been exposed to and possibly infected by CT, GC, or both. In these networks we expect that about 20-40% of the participants will have been exposed to, but not infected by these pathogens. This will give us the unique opportunity to assess the role of the microbiome in preventing or facilitating infections by CT and GC. Our overarching hypothesis is that when pathogen transmission does not occur the genetic traits of the infecting pathogen(s) may be insufficient to overcome the host response or the exclusionary mechanisms of the microbiome environment; or that features of the microbiome are protective or induce a protective mucosal environment. In this project, we will build on these findings and use modern ‘omic technologies to identify specific functional features of the vaginal and penile microbiota associated with susceptibility and resistance to infection and co-infection and the importance of host and pathogen genetic variation in this infection process, which will be done in collaboration with Projects 1 & 2. We will achieve these goals by addressing three integrated specific aims: Aim 1. Characterize the genomic variations in CT/GC in participants of the STING networks of sex partners; Aim 2. Use ‘omic approaches and system biology analysis characterize the molecular interactions between the host, the pathogens and the genital microbiota in discordant and concordant couples for CT/GC infections; Aim 3. Validate and explore mechanistic explanations for how the microbiota prevent or facilitate infection by CT/GC using an in vitro three-dimensional model of endocervical epithelial cells. Our long-term goal is to leverage the information generated in this project to develop improved diagnostic methods, identify novel targets for new drug development and develop targeted and effective curative or preventive therapies, and ultimately, promote health, reduce risk to unintended adverse sequelae of STI and improve the quality of life for men and women who are at risk of STIs.

The use of sequencing or chip-based genomic approaches makes it possible to gain insights into the genetic diversity and genome dynamics of bacterial pathogen populations. We study Yersinia pestis, Escherichia coli O157:H7 and diverse Bacillus and Chlamydia species as model systems, all of which have several representative in-house sequenced genome sequences available. A comparative analysis of these populations allows to analyze the types of host variation, selection and adaptation occurring during the time course of a single or multiple outbreaks of human disease and further to elucidate common and unique traits in genome evolution and speciation. To study these subtle but important genetic variations, we have developed a bioinformatics pipeline that facilitates the discovery and validation of rare polymorphisms taking into account the respective genome sequence read coverage and quality. Applying single nucleotide polymorphism (SNP)-based genotyping and re-sequencing methodologies, we are able to reconstitute a detailed evolutionary history of these microbial pathogens and resolve their genetically highly homogenous population structures. Studying the pan-genome, the global gene reservoir of the species, led to an estimate of the degree of reductive evolution and the extent of influx of genetic material via horizontal gene transfer, which aids in the definition of more accurate genetic species borders. The discovery of such genetic alterations in these dynamic bacterial pathogen populations can therefore provide insights into the genome evolution as well as the individual pathogenic potential critical for future forensic, diagnostic and epidemiological studies.

Most of the aerobic Gram-positive spore-forming bacteria were originally placed in the genus Bacillus. As a result, it is a very diverse genus consisting of at least six clades and GC-content ranging from 33-64%. The sequencing of several Bacillus genomes has been completed in the last few years, but has mainly covered genomes of the pathogenic Bacillus thuringiensis-anthracis-cereus group or those closely related to B. subtilis. The ancient nature of B. megaterium makes it of particular importance in understanding the genome evolution, genome dynamics and speciation in Bacillus. The aim of this genomic study is to catalogue the genomic inventory of B. megaterium strain QMB1551 that carries an array of seven plasmids, and the naturally plasmidless isolate DSM319, to study the Bacillus biology through in-depth comparative genomics. The simultaneous sequencing of two prominent B. megaterium strains, deeply rooted in the phylogeny, presents an unique opportunity to study the genomic plasticity and genome evolution of the Bacillus group of organisms and can help to identify unique genetic traits not previously seen in Bacillus. Using the whole genome data of both genomes will allow to predict the species pan-genome and the extent of exchange of genetic material. The potential dissemination of genetic information between chromosomes and plasmids and to other members of the Bacillus group leads to a generation of unique genotype and phenotypes that might be relevant for species-specific niche adaptations.

I have conducted in-depth comparative genomics analyses of isolates of nontypeable Haemophilus influenzae (NTHi) that are critical to the pathogenesis of chronic obstructive pulmonary disease (COPD). We conducted whole-genome sequencing of 269 longitudinally collected cleared and persistent NTHi from a 15-y prospective study of adults with COPD (d). We elucidated the phylogeny of NTHi isolates, identified genomic changes that occur with persistence in the human airways, and evaluated the effect of selective pressure on candidate vaccine antigens. Slipped-strand mispairing, mediated by changes in simple sequence repeats in multiple genes during persistence, was found to regulate expression of critical virulence functions and to be a major mechanism for survival in the hostile environment of the human airways. Our results advanced our understanding of how a bacterial pathogen that plays a critical role in COPD adapts to survival in the human respiratory tract. We are now studying host-pathogen interactions in COPD focusing on NTHi as well as Moraxella catarrhalis (Mcat). Guided by dynamic changes in genomes and transcriptomes in human airways, we will elucidate mechanisms that mediate persistence of NTHi and Mcat using relevant model systems.

[PI: Tim Murphy, U. at Buffalo]

From a common ancestor, Streptococcus pneumoniae and Streptococcus mitis evolved in parallel into one of the most important pathogens and a mutualistic colonizer of humans, respectively. This evolutionary scenario provides a unique basis for studies of both infection-associated properties and properties important for harmonious coexistence with the host. We performed detailed comparisons of 60 genomes of S. pneumoniae, S. mitis, Streptococcus pseudopneumoniae, the three Streptococcus oralis subspecies oralis, tigurinus, and dentisani, and Streptococcus infantis. Nonfunctional remnants of ancestral genes in both S. pneumoniae and in S. mitis support the evolutionary model and the concept that evolutionary changes on both sides were required to reach their present relationship to the host. We used in-depth comparative genomics approaches to identify 224 genes associated with virulence. The striking difference to commensal streptococci was the diversity of regulatory mechanisms, including regulation of capsule production, a significantly larger arsenal of enzymes involved in carbohydrate hydrolysis, and proteins known to interfere with innate immune factors. The exclusive presence of the virulence factors in S. pneumoniae enhances their potential as vaccine components, as a direct impact on beneficial members of the commensal microbiota can be excluded. In addition to loss of these virulence-associated genes, adaptation of S. mitis to a mutualistic relationship with the host apparently required preservation or acquisition of 25 genes lost or absent from S. pneumoniae.

[Not funded, collaborator: Mogens Kilian, Denmark]