Laboratory Research Focus

Structural, Mechanistic, and Evolutionary Studies of Virulence Factors of Streptococcus Genus to Investigate Host-bacteria Interactions
Our laboratory has just determined structures of pneumococcal and S. agalactiae hyaluronate lyase enzymes which degrade hyaluronan, one of the main components of extracellular matrix of tissues, and we have characterized and proposed a mechanism of its catalysis (degradation of hyaluronan). Such degradation facilitates bacterial entry to sites of infection in the human body and enables the spread of the bacteria to various host tissues. This study also allowed the formulation of general mechanisms of degradation of polymeric saccharides by hydrolases and lyases. We are also in the process of determining three-dimensional structures of three additional virulence factors of Streptococcus genus: pneumococcal surface protein A (PspA), pneumolysin (Ply), and autolysin (LytA), and are engaged in further functional and mechanistic studies of hyaluronate lyase. These studies further our understanding of the exact mechanistic properties of these proteins (structure-function relationship) and increase the understanding of the host-pathogen interactions. Our research on PspA lead to the discovery of its function as a bacterial agent protecting pneumococci against the host complement system. As a result, the structural determination of PspA and anti-PspA protective antibodies and studies of their interactions as well as their influence on the pneumococcal disease are well advanced. In addition, novel basic scientific questions are being answered related to, for example, protein (or Ply) interactions with membranes as well as protein properties and behavior in the membranous environment. Our discoveries include changes in protein structure during membrane insertion of these proteins and their ability to form pores in membranes leading to targeted cell lysis. Research on LytA will explain more details on Ply release from the cytoplasm of pneumococci. As a result of this work, there is significant potential for designing novel therapeutic agents, such as vaccines or drugs, against not only streptococci but possibly all Gram-positive organisms.

Additional unknown virulence factors of S. pneumoniae are being identified in our lab and characterized utilizing the novel developing tools of proteomics. We hope to continue this structural/biochemical/molecular biology research by characterizing the functional and structural properties of these newly discovered novel macromolecules. By utilizing the structural/functional genomics (similarly to description below for the spore-forming organisms) we hope to generalize our research in order to make conclusions on a broader evolutionary scale.

Selected publications relevant to this work

Structural, Functional and Evolutionary Genomics of Proteins Involved in Sporulation and Germination/Outgrowth of Spore-forming Bacteria
In order to make our studies of bacteria more complete, we investigate a specific group of Gram-positive organisms that have the ability to make spores. Here we target an important group of proteins involved in the sporulation process and the germination/outgrowth stages of these organisms, especially for the Bacillus species and the Clostridium species. We have already finished structure determination and proposed a mechanism of action/catalysis for a novel B. megaterium germination protease (GPR), a novel, diphosphoglyceric acid independent B. stearothermophilus phosphoglycerate mutase (PGM), and B. subtilis NAD+ synthetase. Structural elucidation of the following proteins are currently underway: B. stearothermophilus zymogen and active forms of GPR; further mechanistic studies of PGM; and B. stearothermophilus penicillin-binding protein related factor A (PrfA). In addition, structural studies of protein products of ger and sleB genes are currently in progress utilizing molecular biology and biochemical methods. Some of these proteins are involved in the supply of various forms of widely understood energy sources for the development of vegetative cells of sporulating organisms. Our goal is to explain these selected essential processes for the sporulation, the germination, and the outgrowth stages of spore formers by relating the structural and functional properties of the above proteins.

Our structural studies of the catalytic mechanism of action of B. subtilis NAD+ synthetase has lead to the structure-based design of inhibitors/antibiotics not only against spore formers but also against most other bacterial pathogens. The necessity of the supply of significant amounts of NAD in cells to carry on multiple biochemical reactions, involving NAD not only as a substrate but also as a cofactor, is essential for living cells. We propose that the inhibitor/antibiotic specificity that can be obtained by using structure-based methods will most likely decrease the toxicity of our new drugs without affecting their potency against bacterial pathogens.

In anticipation of the nearly completed determination of the genomic sequence of B. anthracis (part of this genomic sequence has already been released), we use the knowledge we have gained studying other sporulating bacteria like B. subtilis, B. megaterium, and B. stearothermophilus, and apply it to B. anthracis, the main pathogenic organism of the Bacillus genera. The tools of structural genomics are used to accomplish this goal, especially in connection with the evolution of these proteins or processes.

Selected publications relevant to this work

Structural and Biochemical Studies of Membrane Proteins Involved in the Synthesis of Oligosaccharide Chains
In addition to our major research listed above, we are also involved in other projects which include the following: Saccharomyces cerevisiae dolichol phosphate mannose synthase, a membrane protein, is a key membrane glycosyl transferase enzyme involved in the synthesis of asparagine-linked (N-linked, eukaryotic cells) and O-linked (yeast) oligosaccharide chains. Together with our colleagues at the Russian Academy of Sciences, Moscow, we study structural properties of this glycosyltransferase and study the dolichol-phosphate chemistry in the membranous environment using a wide variety of synthetic chemistry, biochemical, and biophysical methods. A novel, general method to investigate membrane chemistry and membrane proteins' structures predominantly in, but not limited to, the dolichol pathway was developed using fluorescence (FRET) techniques. Our collaborators developed the synthesis of chromophoric groups on the substrate. Availability of such compounds allows for investigations of various aspects of the active site of the enzyme and the placement of the enzyme and its substrate in the membranes of endoplasmic reticulum.

Selected publications relevant to this work

Other Jedrzejas Publications