Biophysical Characterization of Chlamydia trachomatis CT584 Supports Its Potential Role as a Type III Secretion Needle Tip Protein

Abstract

Chlamydia are obligate intracellular bacterial pathogens that cause a variety of diseases. Likemany Gram-negative bacteria, they employ type III secretion systems (T3SS) for invasion, establishing and maintaining their unique intracellular niche, and possibly cellular exit. Computational structure prediction indicated that ORF CT584 is homologous to other T3SS needle tip proteins. Tip proteins have been shown to be localized to the extracellular end of the T3SS needle and play a key role in controlling secretion of effector proteins. We have previously demonstrated that T3SS needle tip proteins from different bacteria share many biophysical characteristics. To support the hypothesis that CT584 is a T3SS needle tip protein, biophysical properties of CT584 were explored as a function of pH and temperature, using spectroscopic techniques. Far-UV circular dichroism, Fourier transform infrared spectroscopy, UV absorbance spectroscopy, ANS extrinsic fluorescence, turbidity, right angle static light scattering, and analytical ultracentrifugation were all employed to monitor the secondary, tertiary, quaternary, and aggregation behavior of this protein. An empirical phase diagram approach is also employed to facilitate such comparisons. These analyses demonstrate that CT584 shares many biophysical characteristics with other T3SS needle tip proteins. These data support the hypothesis that CT584 is a member of the same functional family, although future biologic analyses are required.

Oligomeric states of the Shigella translocator protein IpaB provide structural insights into formation of the type III secretion translocon

“…33 An-60 Ti four-hole rotor and cells with 12 mm charcoal centerpieces and quartz windows were used. The IpaB samples were prepared in OPOE or LDAO and crosslinked using DSP.…”

Biochemical and Localization Analyses of Putative Type III Secretion Translocator Proteins CopB and CopB2 of Chlamydia trachomatis Reveal Significant Distinctions

“…However, the identity and function of some essential components are less obvious. We have previously provided direct evidence that CdsF represents the chlamydial needle protein (8) while biophysical (33) and protein interaction (49) studies suggest that CT584 may be the tip protein. The composition of translocation pores remains incompletely explored.…”

New Frontiers in Type III Secretion Biology: the Chlamydia Perspective

“…Other less wellcharacterized chlamydial class IB T3SCs include the multiple cargo secretion chaperone T3SC, which binds and stabilizes at least two Inc proteins (Cap1 and CT618) (14), and CT584, a potential T3SC that interacts with at least six T3S substrates, including CT082 (34). CT584 secretion is possible since this protein has been suggested to act as a tip complex protein (37) and is capable of binding host lipids in vitro (38). The presence of multiple class IB T3SCs in the Chlamydia T3SS suggests a possible role for these T3SCs in the establishment of a hierarchy of effector protein secretion in these organisms.…”