pH sensitivity of type III secretion system tip proteins

Abstract

Many pathogenic gram-negative bacteria employ type III secretion systems to transport proteins into the host cell membrane and cytoplasm to subvert normal cellular functions. The type III secretion apparatus consists of a basal body spanning the inner and outer bacterial membranes and a needle which extends away from the bacterium. Recent work has found that a special class of proteins localizes to the tip of the needle to control secretion of effector proteins. Five of these tip proteins are IpaD (Shigella flexneri), BipD (Burkholderia pseudomallei), SipD (Salmonella spp.), LcrV (Yersinia spp.), and PcrV (Pseudomonas aeruginosa). In this study, the conformational stability of these proteins was characterized as a function of pH and temperature. Understanding the stability of the proteins in different pH environments is particularly important since they are expected to encounter different pH environments in their passage through the gastrointestinal tract and are exposed to low pH microenvironments near the surface of target cell membranes. Secondary and tertiary structural changes were monitored using the spectroscopic techniques of far-UV circular dichroism, Trp fluorescence, ANS fluorescence, and ultraviolet absorption spectroscopy. Optical density and right angle scattering measurements were also used to evaluate protein association/dissociation. Empirical phase diagrams were then applied to mathematically combine data from the various spectroscopic techniques to provide a global picture of the proteins' structural behavior in solution. The responses of the proteins to changes in temperature and pH conditions reveal two distinct subfamilies in terms of stability. The first is that of IpaD, BipD, and SipD whose corresponding phase diagrams show conformational differences at pH 5-6. The conserved pH dependence in this subfamily suggests possible common mechanistic function. In the second subfamily (LcrV and PcrV), conformational stability is directly related to pH, also indicating mechanistic similarities.

Protein Export According to Schedule: Architecture, Assembly, and Regulation of Type III Secretion Systems from Plant- and Animal-Pathogenic Bacteria

“…Alternatively, the tip complex could also serve as a sensor of the external pH. In this context, it is interesting that the tip complex protein SipD undergoes a conformational change at pH 5 to 6 (346). It remains to be investigated whether pH sensing is also involved in the control of effector protein secretion in other animal-pathogenic bacteria.…”

Studies of the conformational stability of invasion plasmid antigen B from Shigella

“…Far-UV CD, intrinsic fluorescence and static light scattering data were collected and analyzed as previously described. 7 The thermal stability data acquired using spectroscopy techniques was incorporated into EPDs using Matlab software package (The Mathworks, Natick, MA). Details of EPD construction have been discussed previously.…”

“…6 IpaD is an easily purified, stable protein that we have previously studied by biophysical analysis. 7 IpaB, however, is more complex. Purification of large quantities of IpaB in E. coli requires coexpression with its cognate chaperone, IpgC, forming a heterodimer.…”

Formulation and Immunogenicity of a Potential Multivalent Type III Secretion System-Based Protein Vaccine

“…Previous work indicated that all of the putative antigens aggregated significantly at pH 5 and 6 at elevated temperatures. 34 This physical degradation pathway was selected because it could be easily adapted for high throughput screening and reflects a major pathway for the alteration and loss of activity of many proteins. 34 An assay was developed employing 96-well plates at 558C using a SpectraMax plate reader (Molecular Devices, Sunnyvale, CA) which measured the time-dependent optical density increase at 360 nm.…”

“…A 10 mM histidine/5 mM phosphate buffer was selected since this buffer was the initial choice for final formulation based on earlier studies. 34 Although the zero-point charge of the aluminum hydroxide adjuvant in the phosphatecontaining buffer was not measured, it is expected that this buffer still has a zero-point charge that produces electrostatic interaction with the antigen. The concentration of the proteins prior to adjuvant addition was confirmed with an Agilent 8453 diode array spectrophotometer by measuring ultraviolet (UV) absorption spectra from 200 to 400 nm.…”