Research Interests

The research in our lab lies at the intersection of three topics:

1) Bacterial physiology, genetics, and metabolism,

2) Interaction of intestinal bacteria with human host in health and disease,

3) Use of systems biology, genomics, genetic engineering and bioinformatics tools to understand bacteria and their makeup.

Below we describe current research directions and projects in more detail.

In the healthy adult, there are 10¹¹-10¹⁴ bacteria colonizing the intestine. This outnumbers the total tissue cells in the body by at least an order of magnitude. The composition and activity of this complex microbial system (called microflora or microbiota) have a major influence on health and disease. Commensal microbiota contribute to trophic functions of the gut (producing fermentation products and vitamins that can be used by intestinal epithelial cells), stimulate immune function of the gastrointestinal tract, transform or excrete toxic substances, protect the host against invasion by pathogenic species, and modulate gut motility. At the same time, changes in intestinal microbiota are associated with a variety of GI disorders such as irritable bowel syndrome, inflammatory bowel disease, and colon cancer.

Unfortunately, our knowledge of human intestinal microbiota is far from complete. The relative lack of data has, to a large extent, been due to the difficulty of using standard microbiological techniques to study this complex community. The development of molecular taxonomy tools based on 16S rRNA sequence interrogation has allowed a phylogenetic classification of intestinal bacterial species, and presents an opportunity to design methods for the detection of non-culturable species.

In a recently completed project (Appl. Environ. Microbiol. 2009) we have designed, developed, and validated a custom microarray containing probes to 775 different microbial species of human intestinal microbiota. This microarray is now being used to study the composition of microbiota in healthy childrens in collaboration with Dr. S. Michail, WSU Boonshoft School of Medicine. The microarray analysis will be complemented by the use of other molecular tools such as quantitative PCR. In partnership with computer and statistics scientists at Wright State we are also developing a framework for the computational analyses of such large datsets.

This initial study will be extended to those patients suffering from different intestinal diseases (IBS, IBD, colon cancer) with a hypothesis that clear differences in microbial populations will be detected among these groups of human subjects. Indeed, our preliminary experiments have already identified genus-level differences between healthy children and those diagnosed with Crohn's disease (sub-type of IBD). Using PCA analysis of the abundance of different bacteria in the human gut, we can separate fecal samples between healthy adults (aHLT) and children (kHLT), and we can easily distinguish samples obtained from chidlren suffering from IBD (kIBD).These projects represent an excellent opportunity for synergistic collaborative research between basic and clinical scientists.

In addition, in collaboration with Dr. N. Reo we have also initiated an analysis of metabolic activity of gut microbiota coomunities by using proton NMR measurements of fecal extracts. Similar to our microarray findings, healthy and IBD samples were well separated by their fecal metabolite profiles. Furhter research will profile more human stool samples and we will also consider associations between bacterial presence and intestinal metabolite levels.

Discovery of hypothesized differences in microbial intestinal communities between health and disease can direct the development of new medicines targeted at specific bacterial groups and can lead to the improvement of treatment of the above mentioned diseases.

Escherichia coli is a species of enteric bacteria usually living as commensals in human intestinal tract. Though most of E. coli strains are harmless and even beneficial to their human hosts, a number of different pathogenic strains are also known. Pathogenic E. coli are broadly divided into two major categories: the enteric pathogens (mostly agents of diarrhea and colitis) and extraintestinal pathogens. Among the former are enterohemorrhagic E.coli (EHEC) that are associated with bloody diarrhea and hemolytic uremic syndrome, an example of extraintestinal pathogenic E. coli are uropathogenic strains (collectively called UPEC) that are the predominant group causing urinary tract infections and their extensions such as pyelonephiritis.

Most bacteria including E.coli possess ensembles of transcriptionally regulated genes, commonly called stress or shock response systems, that enable them to adapt to changes in the chemical or physical aspects of their environment, including water activity (osmotic pressure), pH, temperature, and oxygen concentration. Adaptation to most environmental shifts generally involves two stages: a transient or acute phase (shock response) that consists of rapid responses needed to initiate the adaptation to the new conditions, and a continuous or chronic phase (stress response) that consists of responses that are needed to support exponential growth, possibly at a new growth rate, in the altered environment.

We have recently profiled the transcriptional response of commensal E. coli K12 to osmotic and heat stresses (J. Bacteriol. 2008). Several new findings were uncovered with the most interesting one being up-regulation of many genes of oxidative stress regulon by both the heat and an increased medium osmolarity. This can explain the previously noted cross-protection of sub-lethal osmotic stress against further oxidative and heat stresses.

Our current goal is to study osmotic regulation in uropathogenic E. coli. Urinary tract infections usually develop via ascending route, where cells enter the urinary tract through urethra, and move up to bladder and then kidneys. During this path bacterial cells are subjected to significant osmotic pressure from human urine, and UPEC ability to survive and adapt to such environmental stress is important for infection development. Urine contains significant amounts of both urea and inorganic ions, but we do not know to which extent bacterial cells respond differently to the presence of these compounds in the environment. Though uropathogenic E. coli possess a number of known osmotically regulated systems, these have not yet been shown to be necessary for cell survival in human urine, and further studies are needed. Therefore, we will utilize E.coli microarrays, qRT-PCR, and computational approaches to delineate UPEC adapatation to high concentration of urea and inorganic salts in growth environment.

In bacteria, recognition of translational initiation sites by ribosomes involves several features of mRNA molecules, all situated in the immediate upstream region from the start codon and called collectively ribosome binding site (RBS). RBS consists of several different elements; most notably it contains the Shine-Dalgarno (SD) region complementary to the 3’ end of the 16S ribosomal RNA. In the widely accepted model of translation, the 3’ end of 16S rRNA binds to the 5’ end of the mRNA as the first step in translation initiation. Other variable elements of the RBS shown to influence the rate of translation initiation, include the start codon (AUG, GUG, or UUG), a length of the spacer between SD sequence and the start codon, and the GC content of this linker.

There is a significant body of research on the structural biology and biochemistry of protein biosynthesis; however, there is a lack of quantitative understanding of this process, and of protein translation initialization in particular. Despite many research efforts, two gaps still exist in our understanding of the RBS function: i) relative quantitative influence of different RBS components on the resulting efficiency of protein production; and ii) there is no quantitative model of the RBS which would present an estimate of the translation strength of the RBS of a given sequence.

The aim of this project is to carry out a quantitative study of bacterial RBS and their influence on the rate of protein production. Though the main RBS parameters that influence translational efficiency have been revealed, few studies were carried out on the relationship among these parameters, and what is known so far does not allow us to predict the efficiency of an arbitrary RBS sequence well.

This project has three specific objectives: 1) to quantitatively measure dependence of protein production on the nucleotide sequence of RBS in E. coli; 2) to design a quantitative model of the influence of RBS components on protein expression; and 3) to carry out genome-wide studies of the relationship between predicted RBS strength, measured gene expression, and predicted protein expression in E. coli.