|About GSBS | FAQ | Job Opportunities | Search UMDNJ|
Niseema Davis Pachikara
Rutgers, the State University of New Jersey
Graduate Program: Physiology & Integrative Biology
Thesis Advisor: Huizhou Fan, MD, PhD
School of Public Health/Research Building
683 Hoes Lane
Friday, February 19, 2010
Chlamydia trachomatis, an obligate intracellular bacterium, is arguably the most prevalent sexually transmitted pathogen world-wide, the major cause of preventable blindness in the developing world, and a common cause of respiratory infection in infants born to genitally infected mothers. The molecular and cellular mechanisms underlying chlamydial parasitism are not well understood. This dissertation research was intended to identify factors that may control chlamydial infection. To this end, we ascertained chlamydial peptide deformylase (PDF) as an essential enzyme for chlamydial infection, and identified several potential mechanisms underlying the regulation of PDF, a potential therapeutic target. In addition, we have examined the interaction between Chlamydia infection and host cell autophagy, and discovered that chlamydial infection leads to alterations in autophagic activities which ensure the survival of chlamydiae.
By characterizing a chlamydial mutant designated GR10, our previous work suggests that hydroxamate-based metalloprotease inhibitors, exemplified by GM6001 and TAPI, inhibit C. trachomatis growth by targeting the bacterial peptide deformylase (PDF), a metal-dependent hydrolase required for the maturation of bacterial neopeptides. Whole genome sequencing of the GM6001 resistant mutant revealed a single point mutation in the PDF promoter region corresponding to increased protein expression in the mutant. Thus, PDF is the sole target of the inhibitors and is essential for chlamydial infection. To study the regulation of this potential anti-chlamydial target, we selected a series of additional GM6001 resistant mutants with various degrees of resistance. Sequencing analysis and biochemical studies suggest that PDF expression and enzyme activity in C. trachomatis are regulated at multiple levels, including those of transcription, mRNA/protein stability and enzymatic catalysis.
Autophagy or “self-eating” is a highly regulated evolutionarily conserved lysosomal degradation process involved in a wide range of physiological functions. While autophagy is a defense mechanism against many microbes, it also serves as growth niche for others. In human cervical carcinoma HeLa cells, chlamydial infection resulted in elevation of the LC3-II/LC3-I ratio. However, this increase of the traditionally referred autophagy marker was not accompanied by an increase in autophagy-dependent degradation of long-lived proteins (LLP). Furthermore, no decrease of p62/SQSTM1, an autophagy substrate, was observed during chlamydial infection. These observations and other data suggest that while chlamydial infection may stimulate the early step(s) of the autophagy pathway, it fails to increase autophagy flux. In macrophage RAW cells, a significant reduction of LC3II/LC1 ratio and LLP degradation, along with a concurrent increase of p62/SQSTM1 level, were detected following chlamydial infection, suggesting that the infection strongly inhibits autophagy. In addition, there were significant reductions of lysosomal enzyme activities in C. trachomatis-infected RAW cells. Furthermore, inhibiting lysosomal acidification using Bafilomycin A, a vacuolar proton ATPase (H+ ATPase) inhibitor, markedly enhanced chlamydial growth in RAW cells. In murine embryonic fibroblasts (MEFs), the status of autophagy had no effect on the cellular ability to support chlamydial growth under normal culture conditions, but autophagy-deficiency renders unresponsiveness to the inhibition by interferon-gamma. Taken together, it appears that the autophagic pathway is differentially regulated among diverse cell types. These findings highlight some of the mechanisms utilized by C. trachomatis to achieve an optimal balance with the host cells.