CHLAMYDIA – NOT SO GREAT FOR US, BUT UNIQUE AND INTERESTING IN SO MANY WAYS

Chlamydia (klah-MID-e-a) are obligate intracellular bacteria that are propagated and maintained through a phylum defining bi-phasic developmental cycle. The bacteria are transmitted between cells and hosts as small, metabolically inert, Elementary Bodies (EB). After directing entry into a eukaryotic cell, the EBs quickly modifies the early endosome into a Chlamydia specific vessicle termed an inclusion. Within the inclusion, EBs convert into the replicative and metabolically active form termed the Reticulate Body (RB). RBs go through numerous rounds of binary fission before asynchronous conversion of RBs into EBs. Replication and conversion continue until the infected cell is lysed or a portion of the inclusion is extruded away from the host cell, enabling new infection.

The Chlamydia developmental cycle is fascinating and there are many fundamental aspects that are still poorly understood. Some of these unanswered questions include “What are the mechanisms and signals for waking up EBs?”, “What puts them back to sleep?”, “What are the key factors pre-packaged in the EB that enable them to infect a new cell?”, and “Holy hell, how does it manipulate the host cell to get in, stay in, grow, and get out!?!”

While the developmental cycle is intriguing, it is also essential for Chlamydia to cause disease in humans.

Infections by two species of Chlamydia; C. trachomatis and C. pneumoniae, have an immense impact on public health in the US and globally. Chlamydia trachomatis causes both genital tract and ocular diseases. According to the CDC, C. trachomatis has the highest incidence of infection among ALL reportable infectious diseases in the US!! Over a million new infections are reported to the CDC each year, although most infected individuals (~70%!) are asymptomatic (person infected and contagious but haven’t developed symptoms yet to encourage clinical interactions and diagnosis) indicating well over two million new infections annually in the US. These infections can lead to acute (e.g. urethritis), chronic (e.g. pelvic inflammatory disease), and life threatening/preventing disease pathologies (e.g. ectopic pregnancies and sterility). C. trachomatis also is the leading cause of preventable blindness (trachoma) worldwide. This is particularly important in under-developed areas of sub-saharan Africa, Asia, and Central and South America in which over a million persons are blind and 2-3 million have moderate to severely impaired vision.

C. pneumoniae infections are a common cause of community acquired pneumonia. Serological analyses indicate that most of the population is exposed to these organisms (~50% by age of 20 are seropositive). This exposure becomes a greater public health concern as the association of C. pneumoniae with heart disease (athlerosclerosis), the leading cause of death worldwide, is considered. While the association between athlerosclerosis and C. pneumoniae infections is still under investigation, numerous observations support this and include animal studies, shared immuno-pathology, and organism associated with athlerosclerotic lesion. Clearly not the causative component, but a factor that may significantly add to the development of athlerosclerosis.

PRIORITIES AND CHALLENGES IN CHLAMYDIA

Vaccines, Novel Antibiotics, Virulence Factors, and the not so Hypothetical Problem

There are many priorities and challenges associated with Chlamydia research. One of the top priorities in the field is the development of a vaccine. Despite excellent educational and awareness programs, conditional prevention strategies are insufficiently addressing the public health challenge associated with Chlamydia infection rates. Development of a vaccine has been hindered by the lack of vertebrate animal models that more closely mimics human immune responses as well as characterization of the correlates for immunity to human Chlamydia infections. In the absence of a vaccine, a NIH priority is to develop a vaginal delivered microbicide that empowers females for protection against sexually transmitted infections.

A general priority in infectious disease research is the development of new antimicrobials with a preference for those that are pathogen specific. Antibiotic resistance is a major public health threat and while two major classes of antibiotics (e.g., macrolides and tetracyclines) are effective at clearing Chlamydia infections, resistance to one of these has already been observed in pigs strains (yes…there is pig Chlamydia!). Feedstock to human strains is a common path for eventual acquisition in human clincal samples. Moreover, these are broad spectrum antibiotics and have a distorting effect on a patients health and microbiome. Developing pathogen specific antibiotics requires that we have a thorough understanding of molecular mechanisms for pathogenesis that enable us to precisely target candidate virulence factors. Leading to…

Another top priority is identification and characterization of virulence factors! Progress in this area has been hindered by the paucity of genetic tools to evaluate and fulfill ‘Falkow’s postulates‘ for defining chlamydial virulence factors. As described below under ‘Chlamydia Genetics’, many of these barriers have been overcome. It is an exciting time to be in the Chlamydia field and apply these new tools for new discoveries regarding the basic biology and pathogenesis!

Lastly, the Chlamydia protein puzzle is incomplete! Approximately ~35% of the proteins lack sufficient sequence similarity to support functional assignment and have been termed ‘hypothetical’ proteins. This is largely due to the phylogenetic distance of Chlamydia from other ‘model’ bacteria such as B. subtilis (firmicute), C. crescentus (alpha-proteobacteria), and E. coli (gamma-proteobacteria). As a result of this incomplete puzzle, there are substantial gaps in our appreciation of the basic biology of Chlamydia.

TO ADDRESS THESE CHALLENGES AND PRIORITIES, OUR RESEARCH PROGRAM IS ADDRESSING THREE FUNDAMENTAL QUESTIONS :

• What components and mechanisms are used to control the developmental cycle of Chlamydia?
• What are the specific components that are key for Chlamydia pathogenesis?
• What are all of those hypothetical proteins doing?!?

The Hefty Research program uses a combination of innovative and diverse approaches to address these questions

CHLAMYDIA GENETICS

Molecular tools to better understand basic biology and pathogenesis

Chlamydia research has been severely hampered by the paucity of genetic manipulation tools…but no more! Due to the fantastic efforts by Ian Clarke at the University of Southhampton, we now have the ability to introduce DNA into Chlamydia and select for a stably maintained plasmid.

Using these advances, we have developed a system for conditional gene expression to enable the assessment of function and biological role for a candidate gene product. Conditional expression allows us to carefully express a gene product or interfering components to diminish candidate gene expression at specific points in the developmental cycle of Chlamydia and ask “What is the effect on a given phenotype?” These phenotypes can range from aspects of intracellular growth to ascension up the female reproductive tract and resulting tissue pathology. We can add ‘tags’ to proteins to facilitate pulldowns or monitor subcellular localization…in real-time! It truly is an exciting time in Chlamydia to apply these new techniques that have been available in other microbial systems.

GENETICS TO DISCOVER VIRULENCE FACTORS IN CHLAMYDIA

Chlamydia growth is essential for disease (obligate intracellular), but specific factors are likely key for mammalian infection and disease processes.

Unlike many other bacteria that can grow outside of eukaryotic cells, Chlamydia grows only within cells. As such, it is simple to state that if you disrupt growth, you disrupt disease. Most of what the field knows about Chlamydia biology has been done in tissue culture environment.

Now the challenge…separate required growth components from those that are specific for virulence and begin defining them. What factors are needed for colonization and infection of the cervical tissue? What factors are needed to enable ascension up the female reproductive tract?

To facilite these efforts, we have also developed the first transposon system for Chlamydia! This has enabled the random insertion of a Tn into the genome to generate a library of mutant strains for phenotypic analysis. The mutants are being screened for cell culture phenotypes, but we are particularly interested in those mutants with defect in mammalian infection defects.

FUNCTIONAL GENOMICS FOR CHLAMYDIA

Chlamydia species have very similar genomes, yet can have very specific host specificity. We’d like to know what genes contribute to this host specificity, and why they are so critical. We’re mixing and matching regions of genomes from different Chlamydia specifies to discover loci that contain genes associated with host specificity.

Chlamydia genomes can be incredibly similar with strong synteny (overall genomic content and organization). This is evident between C. trachomatis (exclusively human adapted) and C. muridarum (exclusively mouse adapted) with over 98% of the genetic content and organization conserved. While many gene products have distinct differences, and expression patterns may also be different, it is challenging to identify gene candidates that may contribute to host adaptation.

This is where lateral gene transfer and Tn insertions lead the way.

Inside cells, Chlamydia readily shares DNA through lateral gene transfer. In a collaborative project with Drs. Kevin Hybiske (U. Washington) and Daniel Rockey (Oregon State University) and with the extensive expertise of Bob Suchland (U. Washington), we have generated a large library of genomic chimera strains that have regions of C. trachomatis and C. muridarum. The diversity of recombinant chimeric strains were driven by use of different Tn insertions and antibiotic selection. These chimeric strains are being tested to discover gene regions associated with cell culture and mouse infection phenotypes.

STRUCTURAL PROTEOMICS FOR FUNCTIONAL INSIGHTS

Structural similarity can lead to functional understanding

Two proteins that share little to no sequence similarity can adopt very similar three-dimensional structure. As the three-dimensional structure of a protein dictates the protein’s functional role, a protein’s structure can be leveraged to overcome sequence limited functional assignments.

We have leveraged the major technical advances from the Structural Proteomics Centers to close the gap limited information related to functional annotation of hypothetical proteins. Since our efforts began, we have deposited over half of the Chlamydia trachomatis protein structures in the PDB!

We have discovered key proteins for cell division, respiration, and transcription in Chlamydia. Given the paucity of starting information,this has been a ‘box of chocolates’ project…you never know what you’re going to get!

Among the many advantages of this approach is the atomic level details that enable specific dissection of molecular mechanisms of pathogenesis and the development of chemicals to inhibit function (Chemical Biology for Chlamydia). Also, expression of highly purified proteins are analyzed individually and in concert with other recombinant proteins for capability as vaccine candidates in murine challenge studies.

CHEMICAL BIOLOGY FOR CHLAMYDIA

Small molecules to better understand the biology and potential serve as therapeutic tools

Small compounds that specifically inhibit a given protein can be used to carefully analyze the function and biological role. These compounds can potentially be developed into effective pathogen specific antibiotics and used for therapeutic purposes. Our structural proteomics and virulence determinant analyses also provide the key information for rational design of small molecule inhibitors. Complementing this approach is our random screens for small molecules that inhibit growth of Chlamydia. Our screens have consisted of natural products and unique chemically diverse libraries. Moreover, we have use structure-guided approaches to discover small molecules that bind to various protein targets.