Anthrax Toxin Receptor Drives Protective Antigen Oligomerization and Stabilizes the Heptameric and Octameric Oligomer by a Similar Mechanism

Anthrax toxin is comprised of protective antigen (PA), lethal factor (LF), and edema factor (EF). These proteins are individually nontoxic; however, when PA assembles with LF and EF, it produces lethal toxin and edema toxin, respectively. Assembly occurs either on cell surfaces or in plasma. In each milieu, PA assembles into a mixture of heptameric and octameric complexes that bind LF and EF. While octameric PA is the predominant form identified in plasma under physiological conditions (pH 7.4, 37°C), heptameric PA is more prevalent on cell surfaces. The difference between these two environments is that the anthrax toxin receptor (ANTXR) binds to PA on cell surfaces. It is known that the extracellular ANTXR domain serves to stabilize toxin complexes containing the PA heptamer by preventing premature PA channel formation--a process that inactivates the toxin. The role of ANTXR in PA oligomerization and in the stabilization of toxin complexes containing octameric PA are not understood.Using a fluorescence assembly assay, we show that the extracellular ANTXR domain drives PA oligomerization. Moreover, a dimeric ANTXR construct increases the extent of and accelerates the rate of PA assembly relative to a monomeric ANTXR construct. Mass spectrometry analysis shows that heptameric and octameric PA oligomers bind a full stoichiometric complement of ANTXR domains. Electron microscopy and circular dichroism studies reveal that the two different PA oligomers are equally stabilized by ANTXR interactions.We propose that PA oligomerization is driven by dimeric ANTXR complexes on cell surfaces. Through their interaction with the ANTXR, toxin complexes containing heptameric and octameric PA oligomers are similarly stabilized. Considering both the relative instability of the PA heptamer and extracellular assembly pathway identified in plasma, we propose a means to regulate the development of toxin gradients around sites of infection during anthrax pathogenesis

Structure-Function Studies of Anthrax Protective Antigen Octamer

AbstractStructure-Function Studies of Anthrax Protective Antigen OctamerbyAlexander Frederick KintzerDoctor of Philosophy in ChemistryUniversity of California, Berkeley Professor Bryan Krantz, ChairThe assembly of bacterial toxins and virulence factors is critical to their function, but the regulation of holotoxin assembly during infection has not been studied. The protective antigen (PA) component of the toxin assembles into ring-shaped homooligomers that bind the two other enzyme components of the toxin, lethal factor (LF) and edema factor (EF), to form toxic complexes. To disrupt the host, these toxic complexes are endocytosed, such that the PA oligomer forms a membrane-spanning channel that LF and EF translocate through to enter the cytosol. We found using single-channel electrophysiology that PA channels contain two populations of conductance states, which correspond with two different PA pre-channel oligomers observed by electron microscopy--the well-described heptamer and a novel octamer. Octameric complexes are likely integral to the PA assembly mechanism, as dimeric and tetrameric intermediates are the intial populated species during assembly. While complexes are functional translocases, a 3.2Å crystal structure suggests that the PA octamer prechannel may be more thermodynamically stable than the heptamer. Indeed, the octamer comprises 20-30% of the oligomers on cells, but predominates (70-80%) during assembly in physiological plasma. To probe the mechanism of octamer stability, we measured the pH-dependence of channel formation using circular dichroism, mass spectrometry, and electron microscopy. We found that octamers form channels at lower pH than heptamers. This results in heptamer inactivation under physiological conditions, while LT complexes containing octameric PA maintain maximal cytotoxic activity. Thus the PA octamer is a physiological, stable, and active assembly state capable of forming lethal toxins that may withstand the hostile conditions encountered in the bloodstream. This assembly mechanism demonstrates a novel means to control cytotoxicity during anthrax infection.Finally, we studied the mechanism of channel formation. We found from structural, thermodynamic, and kinetic studies that two barriers define the prechannel to channel transition--a pH-dependent and independent process. Further analysis revealed that the protonation of the membrane insertion loop, dissociation of domains 2 and 4, and interactions with phenylalanine-clamp comprise the rate-limiting, pH-dependent barrier

Measured^a and theoretical^b molecular masses for msANTXR-PA-LF_N complexes.

a<p>Molecular masses are measured using nanoelectrospray MS according to the method described in Kintzer et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013888#pone.0013888-Kintzer1" target="_blank">[21]</a>.</p>b<p>Theoretical molecular masses are derived using the amino acid sequences of msANTXR, PA₆₃, and LF_N.</p

The formation of SDS-resistant PA₇(LF_N)₃(msANTXR2)₇ complexes is temperature-independent.

<p>SDS-resistance assays <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013888#pone.0013888-Miller1" target="_blank">[25]</a> were performed with PA₇(LF_N)₃(msANTXR2)₇ complexes, which were incubated at the indicated pH at either 25°C or 37°C. The two species of interest on the SDS-PAGE gels are indicated as either the high-molecular-weight, SDS-resistant PA oligomer band (*) or low-molecular-weight SDS-soluble, PA₆₃ monomer band (PA₆₃).</p

The pH dependence of CD-signal changes for PA₇- and PA₈-msANTXR2 complexes.

<p>(<b>A</b>) Time-course records of the CD signal at 222 nm (CD₂₂₂) for either an acid pulse (pH 5.0 final, red trace) or a control with no pH pulse (pH 8.0 final, black trace). (<b>B</b>) The pH-dependence of the CD₂₂₂-signal change for PA₇(LF_N)₃ (black □, data taken from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013888#pone.0013888-Kintzer2" target="_blank">[22]</a>), PA₈(LF_N)₄ (red ○, data taken from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013888#pone.0013888-Kintzer2" target="_blank">[22]</a>), PA₇(LF_N)₃(msANTXR2)₇ (black ▪), PA₈(LF_N)₃(msANTXR2)₈ (red •) complexes. Traces were normalized to the initial and final CD₂₂₂ signals obtained.</p

EM analysis of the stability of PA₇-msANTXR2 and PA₈-msANTXR2 complexes from pH 8.0 to 5.0.

<p>(<b>A</b>) Representative micrographs (49,000×) of PA-msANTXR2 complexes following a 5-minute exposure to 37°C at either pH 8.0 (left) or pH 5.0 (right). A 20-nm scale bar is shown in white for either micrograph. (inset on left) Class-average images of PA₇-msANTXR2 and PA₈-msANTXR2 complexes; a 5-nm scale bar is shown. (<b>B</b>) Quantitative analysis of the number of soluble PA oligomers and the relative proportions of PA₇ and PA₈, identified from electron micrographs at each pH. (left) A plot of the average number of soluble prechannels versus pH for both free PA complexes (□, data taken from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013888#pone.0013888-Kintzer2" target="_blank">[22]</a>) and msANTXR2-bound PA complexes (▪) complexes. Error bars are propagated from the standard deviations of the mean number of particles obtained from at least 10 micrographs for each pH. (right) A plot of the relative proportions of PA₇ (black ▪) and PA₈ (red •) complexes determined using class-average image analysis for both PA-LF_N (open symbols, data taken from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013888#pone.0013888-Kintzer2" target="_blank">[22]</a>) and PA-LF_N-msANTXR2 (filled symbols) complexes.</p

A model for anthrax toxin assembly.

<p>A model for anthrax toxin assembly in plasma and at cells surfaces. (<b>A</b>) In principle, PA components may assemble into a 70∶30 PA₇:PA₈ mixture of toxin complexes in plasma. However, PA₇ readily converts to the channel state and aggregates within 5 minutes under these conditions, leaving PA₈ as the predominant soluble toxin complex capable of infecting cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013888#pone.0013888-Kintzer2" target="_blank">[22]</a>. By contrast, both oligomeric forms are equally stable at the cell surface, where binding to ANTXR2 serves to prevent premature channel formation until PA₇ or PA₈ complexes are properly internalized and the endosomal compartment is acidified to pH values <6. On cell surfaces, PA may also oligomerize into a 70∶30 PA₇:PA₈ mixture <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013888#pone.0013888-Kintzer1" target="_blank">[21]</a>, where assembly is driven through interactions with dimeric ANTXR complexes. These complexes are then able to bind LF and become internalized into cells.</p

A model for the regulation of toxin activity in plasma.

<p>The assembly of LT complexes with different lifetimes may serve as a means to regulate toxin activity in plasma during infection. The reduced lifetime of PA₇ complexes in plasma may limit their cytotoxic effects to local areas in close proximity to the site of <i>B. anthracis</i> infection. By contrast, PA₈, which is produced at lower levels, has a longer lifetime, thereby allowing it to exert cytotoxic effects over longer distances.</p