Dept. of Microbiology & Immunology

University of Tennessee, Memphis

Biochemistry and Molecular Biology of Chlamydia.  Our laboratory is interested in the biochemistry and molecular biology of the obligately intracellular bacteria of the genus Chlamydia. C. trachomatis is recognized as the most common sexually transmitted agent in the United States. It is responsible for a wide range of human disorders including urethritis, cervicitis, pelvic inflammatory disease, epididymitis, conjunctivitis, infant pneumonia, and in underdeveloped nations, a potentially blinding disease, trachoma. C. pneumoniae is a significant agent of respiratory disease in adolescents and adults and has been detected in atherosclerotic lesions. C. psittaci, a natural parasite of a broad range of mammals and birds, infrequently causes a severe pneumonitis in humans. Chlamydiae not only are important human pathogens but also possess unique and fascinating biological features that include a cell envelope which lacks peptidoglycan, a deficiency in the capacity to generate ATP by cellular metabolism, and an intracellular developmental cycle.

The developmental cycle is initiated when infectious elementary bodies (EBs) attach to and are endocytosed by a suitable eukaryotic host cell. Once within the host cell, EBs reorganize to reticulate bodies (RBs) which multiply by binary fission within a membrane-bound vacuole. Late in the developmental cycle, when almost the entire cytoplasm of the host cell is occupied by the chlamydiae-containing vacuole, RBs reorganize to EBs and the host cell lyses releasing a brood of infectious parasites. EBs are osmotically stable, survive extracellular environments, but are metabolically inert outside of host cells. RBs are noninfectious, osmotically fragile and quickly perish outside of host cells, but are capable of synthesizing macromolecules.

Biochemistry of the chlamydial envelope. The differential stability of EBs and RBs presents an interesting enigma: How is it accomplished—particularly since neither form possesses the bacterial structure, peptidoglycan, normally associated with osmotic stability? The cell envelope of chlamydiae superficially resembles the double lipid bilayer of gram-negative bacteria. Proteins associated with the envelope include the major outer membrane protein (MOMP) and a small and a large cysteine-rich protein (CRP). The MOMP is most actively made during the middle phase of the developmental cycle and spans the outer membrane. The CRPs are synthesized only during the late stage, when RBs reorganize to EBs. To investigate the osmotic properties of chlamydiae, we have cloned and sequenced the genes encoding the MOMP and the CRPs of C. psittaci. The predicted amino acid sequences of the CRPs suggests that neither is directly associated with a membrane, although both proteins possess N-terminal signal sequences associated with membrane translocation. Using isotopic tracers and analytical methods including combined gas chromatography-mass spectrometry, we have shown that the small CRP is a lipoprotein. It appears to be anchored to the outer membrane through its hydrophobic lipid moiety with its hydrophilic peptide component extending into the periplasmic space. Photoaffinity labeling and biophysical studies suggest that the large CRP also is located in the periplasm. We have proposed a model in which the CRPs can form a supramolecular disulfide-bonded complex in the periplasm, perhaps in association with exposed portions of the MOMP. This massive network appears to be the functional equivalent of peptidoglycan, providing osmotic stability to EBs. The absence of CRPs in the periplasm of dividing RBs explains their osmotic fragility.

Genome sequencing has revealed a family of proteins, referred to as POMPs, that are also unique to chlamydiae and predicted to be located in the outer membrane. As many of 9 POMPs may be made in C. trachomatis and 18 may be made by C. pneumoniae. Our laboratory is currently investigating the disulfide cross-linkage of POMPs, their potential surface exposure, and the possible role of POMPs in the infection process.

Nucleotide metabolism. Chlamydiae lack cytochromes and possess only bits and pieces of pathways associated with energy metabolism. How then do chlamydiae obtain the energy required for normal cellular processes? This puzzle was solved when we demonstrated that chlamydiae possess transport mechanisms for directly obtaining host-supplied nucleoside triphosphates. We have exploited this unusual property in a number of ways. Most importantly, we have found that host-free chlamydiae can synthesize stage-specific RNA for brief periods of time when supplied ATP, GTP, UTP, and CTP. This observation has enabled us to generate highly radioactive RNA probes from [a-³²P]NTPs for screening chlamydial genomic libraries for clones expressing stage-specific genes. Using this approach, we have cloned a gene that is preferentially expressed in the early-stage of the developmental cycle and several previously unidentified late-stage genes. The early stage gene, referred to as EUO, is a minor DNA binding protein that prefers AT-rich sequences. A major emphasis of our laboratory is to determine the in vivo binding sites and function of EUO.

Regulation of the chlamydial developmental cycle. Chlamydiae progress through a temporally regulated developmental cycle characterized by the orderly alternation between transcriptionally inactive EBs and metabolically active RBs. All changes take place within a host cell. What cues prompt EBs to awaken? What signals alert RBs that their convenient host is about to die and that preparation for extracellular transit is necessary for survival? By what mechanisms are stage-specific genes turned on and turned off? Answers to these questions are the major goals of our laboratory. Our approach has been to identify and clone stage-specific genes (as described above), sequence the genes and their upstream regions in search of clues to their function and regulation, modify the genes by site-specific mutagenic techniques, and assess the effects of these modifications on gene expression. Assessment of mutations commonly is achieved by classical genetic methods. This avenue is not currently available for chlamydial studies because a genetic system has not been developed. As an alternative, we have developed an in vitro transcription system in which the DNA of cloned genes and mutated variants serve as templates for partially purified chlamydial RNA polymerase and other transcription factors. So far our investigations indicate that most stage-specific genes require the major chlamydial sigma factor for expression and appear to be regulated by conventional methods of activation and repression while others seem to be dependent on more unusual mechanisms of regulation including posttranscriptional processing and antisense RNA. In addition to a major sigma factor, chlamydiae possess two alternative sigma factors. Our laboratory is investigating the time of expression and promoter targets of these alternative sigmas.

Hatch, T. P. 1996. Disulfide cross-linked envelope proteins: the functional equivalent of peptidoglycan in chlamydiae? J. Bacteriol. 78:1-5.

Douglas, A. L., and T. P. Hatch. 1996. Mutagenesis of the P2 promoter of the major outer membrane protein gene of Chlamydia trachomatis. J. Bacteriol. 178:5573 5578.

Zhang, L., A. L. Douglas, and T. P. Hatch. 1998. Characterization of a Chlamydia psittaci DNA binding protein (EUO) synthesized during the early and middle phases of the developmental cycle. Infect. Immun. 66:1167-1173.

Hatch, Thomas P. 1998. Perspectives: Genome Sequencing. Chlamydia: Old Ideas Crushed, New Mysteries Bared. Science 282:638-639.

Zhang, L., M. H. Howe, and T. P. Hatch. Characterization of DNA binding sites of the EUO protein of Chlamydia psittaci. Infect. Immun., submitted.

Douglas, A. L., and T. P. Hatch. Expression of transcripts of the sigma factors and putative sigma factor regulators of Chlamydia trachomatis. J. Bacteriol., submitted.