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.

Courtesy of Kevin Hybiske

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, and get out!?!"

While the developmental cycle is intruiging, 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 (e.g. condoms) 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 (e.g. IFNg) as well as the inconclusive 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! This priority 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?!?

Developmental Cycle in action!

40 hour live image analysis of GFP labeled C. trachomatis

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.

Defining 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. 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?

Multimeric assembly of CT584

Structure of CT584 (PDB 4MLK) in monomeric, dimeric, trimeric, and then hexameric assembly (Courtesy of Scott Lovell). CT584 is associated with the type III secretion system in Chlamydia.

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 on hypothetical protein ignorance. 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 unknown aspect, 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.

DS-96 dose-dependent treatment of EBs

Chemical Biology of Chlamydia

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 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.

Team Hefty

Principal Investigator

P. Scott Hefty, Ph.D.

Associate Professor

http://molecularbiosciences
.ku.edu/p-scott-hefty

Google Scholar

Doctoral Candidate

Scott LaBrie

B.S. in Environmental Science

Stud Website Developer

Doctoral Candidate

Megan McKinney

B.S. in Biology

Doctoral Candidate

Katelyn Soules

B.S. in Biology

Graduate Researcher

Zoè Dimond

B.S. in Biology

Undergraduate Researcher

Letty Beltran


Undergraduate Researcher

We need YOU! Looking for intelligent persons who are enthusiastic, engaged, driven, ...you get the picture.

Lab Alumni

John Hickey, Ph.D. in Biochemistry (2011)

Senior Research Associate Macromolecule and Vaccine Stabilization Center at the University of Kansas

Ichie Osaka, Ph.D. in Microbiology (2013)

Field Application Scientist, International Distribution

LI-COR Biosciences

Kyle Kemege, Ph.D. in Biochemistry (2013)

Lecturer

Lousiana State University

 

Lindsey Weldon, M.S. in Microbiology (2011)

Qiagen Applied Testing Business Manager

Namita Balwalli, M.S. in Molecular, Cell, and Developmental Biology (2013)

Elsevier - Pharma solutions
Mumbai, India

Keasha Restivo, M.S. in Microbiology (2014)

Thermo-Fisher

 

Lindsay Rutt, M.S.

Research Lead Specialist University of Maryland, Baltimore

Katie Summers, Ph.D.

Animal Biosciences and Biotechnology Laboratory Beltsville Agricultural Research Center

Michael L. Barta, Ph.D.

Scientist
Catalent Pharma Solutions Kansas City, Missouri

Jason Wickstrum, Ph.D.

Okayama University of Science, Okayama, Japan

Kelly Harrison, Ph.D.

Postdoctoral Researcher

Publications

Selected Publications

Hypothetical protein CT398 (CdsZ) interacts with σ(54) (RpoN)-holoenzyme and the type III secretion export apparatus in Chlamydia trachomatis
Barta ML, Battaile KP, Lovell S, Hefty PS.
2015 Oct;24(10):1617-32. doi: 10.1002/pro.2746. Epub 2015 Aug 6.
Protein Sci | PDF

Lipopolysaccharide-binding alkylpolyamine DS-96 inhibits Chlamydia trachomatis infection by blocking attachment and entry
I Osaka, PS Hefty
American Society for Microbiology, 58(6):3245-3254 (2014)
ASM | PDF

Structural and Biochemical Characterization of Chlamydia trachomatis Hypothetical Protein CT263 Supports that Menaquinone Synthesis Occurs through the Futalosine Pathway
Michael L. Barta, Keisha Thomas, Hongling Yuan, Scott Lovell, Kevin P. Battaile, Vern L. Schramm and P. Scott Hefty
J. Biol. Chem. , 10.1074/jbc.M114.594325 (2014)
JBC | PDF

Conditional gene expression in Chlamydia trachomatis using the tet system
Jason Wickstrum, Lindsay R. Sammons, Keasha N. Restivo, P. Scott Hefty*
PLoS ONE, 8(10): e76743 (2013)
Pubmed | PDF

Multimedia

 

 

Live Imaging of Chlamydia

 

Chlamydia

Chlamydia

Research Images

The following images and illustrations are free to download for personal use only (copyright applies). Please contact us for questions regarding use restrictions. Click on thumbnails for larger images and links to download in other formats.


Entertainment

Chlamydia Pop Movie montage

 

Contact Us

The Hefty Lab is with the Department of Molecular Biosciences at the University of Kansas.

Contact Information

Email: pshefty@ku.edu
Lab/office location: 8051 Haworth Hall, 1200 Sunnyside Avenue, Lawrence KS 66045
Office phone: (785) 864-5392

Join The Lab!

The Hefty lab is always interested in hearing from prospective undergraduate and graduate students as well as postdoctoral fellows. We are part of a growing nexus of interdepartmental research in microbial pathogenesis, infectious diseases, and global health at the University of Kansas.

Prospective graduate students
You must apply to (and be accepted by) the Molecular Biosciences PhD Program.

Prospective postdocs and fellows
Send your CV and a detailed statement of why you are interested in joining to: pshefty@ku.edu