Thermodynamic properties of the effector domains of MARTX toxins suggest their unfolding for translocation across the host membrane

Abstract

MARTX (multifunctional autoprocessing repeats-in-toxin) family toxins are produced by Vibrio cholerae, Vibrio vulnificus, Aeromonas hydrophila and other Gram-negative bacteria. Effector domains of MARTX toxins cross the cytoplasmic membrane of a host cell through a putative pore formed by the toxin's glycine-rich repeats. The structure of the pore is unknown and the translocation mechanism of the effector domains is poorly understood. We examined the thermodynamic stability of the effector domains of V. cholerae and A. hydrophila MARTX toxins to elucidate the mechanism of their translocation. We found that all but one domain in each toxin are thermodynamically unstable and several acquire a molten globule state near human physiological temperatures. Fusion of the most stable cysteine protease domain to the adjacent effector domain reduces its thermodynamic stability ∼ 1.4-fold (from D G H 2 O 21.8 to 16.1 kJ mol(-1) ). Precipitation of several individual domains due to thermal denaturation is reduced upon their fusion into multi-domain constructs. We speculate that low thermostability of the MARTX effector domains correlates with that of many other membrane-penetrating toxins and implies their unfolding for cell entry. This study extends the list of thermolabile bacterial toxins, suggesting that this quality is essential and could be susceptible for selective targeting of pathogenic toxins.

Human Defensins Facilitate Local Unfolding of Thermodynamically Unstable Regions of Bacterial Protein Toxins

“…In agreement with low thermodynamic stability of ACD Vc (Kudryashova et al, 2014), the toxin’s SID activation yielded two distinct states – a “native” state, with CCS similar to that found in MS analysis, and an “unfolded” state with significantly higher CCS (Figure 4C). Activation of the ACD-defensin complex produced only the “unfolded” state conformation.…”

“…Recently we showed that the majority of MARTX effector domains from V. cholerae and Aeromonas hydrophila are thermodynamically unstable (Kudryashova et al, 2014). To test whether the inhibitory activity of human α-defensin HNP1 extends to MARTX Vc toxin, we assessed its effects on autoprocessing activity of MARTX cysteine protease domain (CPD Vc ) (Prochazkova et al, 2009; Shen et al, 2009) and catalytic activity of the actin crosslinking domain (ACD Vc ) (Kudryashov et al, 2008; Kudryashova et al, 2012).…”

“…We monitored a temperature-induced precipitation (followed by a raise in light scattering) of two ACD orthologues from V. cholerae (ACD Vc ) and A. hydrophila (ACD Ah ) that share 80% sequence similarity, but differ significantly in their thermodynamic stability (Kudryashova et al, 2014). The probability of multivalent tethering of toxin molecules with HNP1 was reduced by keeping both components at equimolar concentrations of 2.5 μM.…”

“…If the above speculations are correct, we should be able to predict a susceptibility of novel toxins, which are not yet recognized as defensin targets, based on their thermodynamic properties. To test this hypothesis, we focused on the effector domains of Vibrio cholerae multifunctional autoprocessing repeats-in-toxin (MARTX) toxin (Satchell, 2011), which have been recently shown to be thermodynamically unstable (Kudryashova et al, 2014). Similarly to many exotoxins, MARTX effector domains have to cross a pore (formed by toxin’s N- and C-terminal glycine-rich repeats) to reach the cytoplasmic domain of a host cell.…”

Revealing the Achilles Heel of Bacterial Toxins

“…This property is linked to conformational plasticity and is indispensable for the toxin's pore formation within membranes and/or for the toxin to pass through a narrow pore. To test this hypothesis, the authors focused on the effector domains of the thermodynamically unstable Vibrio cholera multifunctional autoprocessing repeats-in-toxin (MARTX) toxin (Kudryashova et al, 2014a), which has to cross a pore to reach the cytoplasmic domain of a host cell. To investigate whether the inhibitory activity of a-defensin HNP1 extends to MARTX Vc toxin, the authors assessed its effects on the autoprocessing activity of MARTX cysteine protease domain (CPD Vc ) and the catalytic activity of the actin crosslinking domain (ACD Vc ).…”