James M. Musser and his 40-person Laboratory of Human Bacterial Pathogenesis are taking a genome-scale approach to studying the causes of infectious disease. Musser and his team are using genome sequencing, DNA microarrays, and proteomics techniques to understand how bacteria cause disease. Musser hopes to find new gene targets that will lead to the development of new treatments and vaccines. 

Investigators in Musser’s lab are making strides with agents of diseases such as tuberculosis (Mycobacterium tuberculosis), Lyme disease (Borrelia burgdorferi), plague (Yersinia pestis), and staph infections (Staphylococcus aureus). One area of recent progress has been in understanding various aspects of complex Streptococcus pyogenes (or GAS for “group A Streptococcus”) bacteria, the agents responsible for most streptococcal infections. Different strains of S. pyogenes cause strep throat, scarlet fever, and impetigo, as well as more severe infections, such as necrotizing fasciitis. Untreated GAS infections can lead to acute rheumatic fever, which is the number-one cause of preventable pediatric heart disease globally.

Comparing GAS strains using genomic technologies

To find out what makes some strains of GAS more lethal than others, Musser is investigating the various strains from all angles—sequencing multiple genomes, comparing different strains using DNA microarrays, and characterizing their proteins.

Sequencing the bacterial genome reveals the number and general types of genes it possesses but provides only limited information about gene function and expression patterns. DNA microarrays provide a “snapshot” of the genes being expressed at any given time, allowing researchers to compare gene expression between two different strains or between bacteria at rest and those infecting a host. Characterizing proteins expressed by the bacteria can reveal information about bacterial toxins and tools for resisting the immune system.

Using the sequencing, microarray, and protein-characterizing technology available today, Musser’s group is able to identify and characterize previously undescribed genes turned on in response to infection of the host, increasing the number of potential drug targets.

To narrow genes down to likely targets for drugs or vaccines, Musser infected animals such as mice with S. pyogenes, then extracted DNA from the diseased tissue. He created microarrays of thousands of S. pyogenes genes using his own sequencing data. By hybridizing the mRNA from the infected tissue to the microarray, he will be able to see which genes were turned on during the process of infection.

Similar studies are also under way using human tissue culture. Researchers are studying epithelial cells from the upper respiratory tract, to examine how GAS causes pharyngitis, and human leukocytes, to determine how the bacteria evade the host’s immune system.

Isolating epidemic strains

Complicating Musser’s research is the fact that Streptococcus strains vary substantially in gene content. People generally have variable alleles of the same genes, but different strains of GAS may contain different genes altogether, despite being the same species. These genetic differences help define the unique pathology of each strain.

Musser’s lab is also investigating the molecular genetic basis of GAS epidemics. Some strains are much more likely to cause epidemics, while others typically confine themselves to isolated cases. Microarrays of 2,000 open reading frames, derived from his sequencing analysis, allow Musser to characterize each strain and compare differences in the strains’ gene content to find out whether a particular pattern of expression could be a “signature” for organisms capable of causing epidemics.

Zeroing in on proteins

Identifying genes is important, but it is equally important to characterize the proteins they encode. Primarily through systematic characterization of extracellular proteins secreted by GAS, Musser and his team have already identified more than 40 new proteins, and they are working to understand which ones are relevant to infection. Using microarrays, Musser discovered that nearly half of these proteins are upregulated during infection and therefore are good candidates for study. One of the new proteins, homologous to the alpha subunit of human Mac-1, inhibits the normal function of human neutrophils, which attach to and break down foreign cells. Such a protein is likely to assist the bacteria in evading destruction by the immune cells.

Sometimes discerning the function of a protein requires more than just finding a homolog, and solving its structure often provides valuable clues. Musser, along with colleagues in New Zealand, used X-ray diffraction to produce the first solution of a crystal structure for a GAS virulence protein—streptococcal pyrogenic exotoxin B (SpeB), a cysteine protease. The structure—in concert with site-directed mutagenesis studies—has revealed certain functional information about the protein. SpeB is an important enzyme in mediating the severe staph infection necrotizing fasciitis, because it activates a cascade of tissue-destructive processes. Musser discovered that, unlike most cysteine proteases, SpeB’s active site is two amino acids, rather than three. Mutations in either of these amino acids inhibit the protein’s catalytic activity. Musser plans to conduct microarray experiments to determine which proteins SpeB activates in human tissue, with the hope that these proteins will also be potential targets for new drugs or vaccines.

Ongoing fight against disease

Although Musser has been working with group A Streptococcus for more than ten years, his research group at NIAID began the genome-scale approach a little more than a year ago, and Musser plans to continue and expand on that approach. As sequence data come in for different GAS strains, Musser will add the new genes to his microarrays, broadening their usefulness in identifying which genes are related to virulence.

To further study exactly how these bacteria cause disease, Musser plans to move from tissue culture and animal studies to human studies, taking tissue from people with strep infections and, using his microarrays, analyzing the pattern of gene expression characteristic of infection.

Streptococcus, of course, is only one example. Musser and his fellow researchers are exploiting the power of genomic techniques to speed the process of understanding many bacterial diseases, so that one day new drugs or vaccines can be developed to combat them.