1.C.11 The Pore-forming RTX Toxin (RTX-toxin) Family

The RTX-toxin family is a large family of multidomain Gram-negative bacterial pore-forming exotoxins. They are secreted from the bacteria, and after processing, they insert into the membranes of animal cells. They exert both cell type- and species-specific effects (e.g., the leukotoxin of M. haemolytica interacts only with alveolar macrophages, neutrophils, and lymphocytes of ruminants and is believed to promote bacterial proliferation by killing or incapacitating these cells) (Davies et al., 2001). These toxins recognize protein receptors such as the β2-integrins, form pores at high concentrations, and cause cell rupture. Three transmembrane domains are believed to be involved in pore formation which in the E. coli HlyA protein are at residues 299-319, 361-381 and 383-403. However, at low, sublytic concentrations, leukotoxin causes activation of neutrophils, production of inflammatory cytokines, degranulation, generation of oxygen-derived free radicals, and morphologic changes consistent with apoptosis (Davies et al., 2002). Pore-forming mechanisms of various pore-forming toxins (PFTs) based on cryoEM structures have been reviewed (Mondal et al. 2022).

The C-terminal domain of the adenylate cyclase toxin (ACT or CyaA) of Bordetella pertussis forms a small cation-selective channel, disrupting the permeability barrier. This channel probably delivers the N-terminal adenylate cyclase to the host cell cytoplasm. Mutations in residues in an amphipathic α-helix (Glu509 and Glu516) in the pore-forming domain block adenylate cyclase translocation and modulate cation selectivity of the membrane channel (Osickova et al., 1999). ACT does not require a protein receptor and inserts into liposomes. Phosphatidylethanolamine and cholesterol stimulate ACT insertion. ACT also promotes lipid flip-flop suggesting that ACT forms trans-bilayer nonlamellar lipid structures when it inserts into the membrane (Martin et al., 2004). CyaA may form two different types of pore-like structures, dependent on the orientation of the membrane potential and the pH (Knapp et al., 2008).

Members of the RTX superfamily (RTX (1.C.11); CCT (1.C.57), and S-PFT (1.C.75)) contain repeat sequences that are also found in autotransporters (e.g., 1.B.12.10.1 and 1.B.40.1.2) as well as TolA (2.C.1.2.1). These domains probably mediate protein-protein interactions.

The generalized transport reaction proposed for members of the RTX-toxin family is:

Small molecules (in) small molecules (out)



This family belongs to the RTX-toxin Superfamily.

 

References:

Amuategi, J., R. Alonso, and H. Ostolaza. (2022). Four Cholesterol-Recognition Motifs in the Pore-Forming and Translocation Domains of Adenylate Cyclase Toxin Are Essential for Invasion of Eukaryotic Cells and Lysis of Erythrocytes. Int J Mol Sci 23:.

Balashova, N.V., J.A. Crosby, L. Al Ghofaily, and S.C. Kachlany. (2006). Leukotoxin confers β-hemolytic activity to Actinobacillus actinomycetemcomitans. Infect. Immun. 74: 2015-2021.

Benz, R., C. Piselli, and A.A. Potter. (2019). Channel Formation by LktA of in Lipid Bilayer Membranes and Comparison of Channel Properties with Other RTX-Cytolysins. Toxins (Basel) 11:.

Benz, R., E. Maier, S. Bauer, and A. Ludwig. (2014). The deletion of several amino acid stretches of Escherichia coli α-hemolysin (HlyA) suggests that the channel-forming domain contains β-strands. PLoS One 9: e112248.

Bhakdi, S., H. Bayley, A. Valeva, I. Walev, B. Walker, U. Weller, M. Kehoe, and M. Palmer. (1996). Staphylococcal α-toxin, streptolysin-O and Escherichia coli hemolysin: prototypes of pore-forming bacterial cytolysins. Arch. Microbiol. 165: 73-79.

Braun, V. and T. Focareta. (1991). Pore-forming bacterial protein hemolysins (cytolysins). Crit. Rev. Microbiol. 18: 115-158.

Brown, A.C., N.V. Balashova, R.M. Epand, R.F. Epand, A. Bragin, S.C. Kachlany, M.J. Walters, Y. Du, K. Boesze-Battaglia, and E.T. Lally. (2013). Aggregatibacter actinomycetemcomitans leukotoxin utilizes a cholesterol recognition/amino acid consensus site for membrane association. J. Biol. Chem. 288: 23607-23621.

Davies, R.L., S. Campbell, and T.S. Whittam. (2002). Mosaic structure and molecular evolution of the leukotoxin operon (lktCABD) in Mannheimia (Pasteurella) haemolytica, Mannheimia glucosida, and Pasteurella trehalosi. J. Bacteriol. 184: 266-277.

Davies, R.L., T.S. Whittam, and R.K. Selander. (2001). Sequence diversity and molecular evolution of the leukotoxin (lktA) gene in bovine and ovine strains of Mannheimia (Pasteurella) haemolytica. J. Bacteriol. 183: 1394-1404.

Fagerberg, S.K., M.R. Jakobsen, M. Skals, and H.A. Praetorius. (2016). Inhibition of P2X receptors protects human monocytes against damage by leukotoxin from Aggregatibacter actinomycetemcomitans and α-hemolysin from Escherichia coli. Infect. Immun. [Epub: Ahead of Print]

Fiser, R. and I. Konopásek. (2009). Different modes of membrane permeabilization by two RTX toxins: HlyA from Escherichia coli and CyaA from Bordetella pertussis. Biochim. Biophys. Acta. 1788: 1249-1254.

Juntapremjit S., Thamwiriyasati N., Kurehong C., Prangkio P., Shank L., Powthongchin B. and Angsuthanasombat C. (2015). Functional importance of the Gly cluster in transmembrane helix 2 of the Bordetella pertussis CyaA-hemolysin: Implications for toxin oligomerization and pore formation. Toxicon. 106:14-9.

Knapp, O., E. Maier, J. Masín, P. Sebo, and R. Benz. (2008). Pore formation by the Bordetella adenylate cyclase toxin in lipid bilayer membranes: Role of voltage and pH. Biochim. Biophys. Acta. 1778(1): 260-269.

Kristensen BM., Frees D. and Bojesen AM. (2010). GtxA from Gallibacterium anatis, a cytolytic RTX-toxin with a novel domain organisation. Vet Res. 41(3):25.

Kulshrestha, A., S.N. Punnathanam, R. Roy, and K.G. Ayappa. (2023). Cholesterol catalyzes unfolding in membrane-inserted motifs of the pore forming protein cytolysin A. Biophys. J. [Epub: Ahead of Print]

Kurehong, C., C. Kanchanawarin, B. Powthongchin, P. Prangkio, G. Katzenmeier, and C. Angsuthanasombat. (2017). Functional Contributions of Positive Charges in the Pore-Lining Helix 3 of the Bordetella pertussis CyaA-Hemolysin to Hemolytic Activity and Ion-Channel Opening. Toxins (Basel) 9:.

Lu, Y., A. Rafiq, Z. Zhang, F. Aslani, M. Fijak, T. Lei, M. Wang, S. Kumar, J. Klug, M. Bergmann, T. Chakraborty, A. Meinhardt, and S. Bhushan. (2018). Uropathogenic Escherichia coli virulence factor hemolysin A causes programmed cell necrosis by altering mitochondrial dynamics. FASEB J. 32: 4107-4120.

Martín, C., M.-A. Requero, J. Masin, I. Konopasek, F.M. Goñi, P. Sebo, and H. Ostolaza. (2004). Membrane restructuring by Bordetella pertussis adenylate cyclase toxin, a member of the RTX toxin family. J. Bacteriol. 186: 3760-3765.

Mondal, A.K., K. Lata, M. Singh, S. Chatterjee, A. Chauhan, S. Puravankara, and K. Chattopadhyay. (2022). Cryo-EM elucidates mechanism of action of bacterial pore-forming toxins. Biochim. Biophys. Acta. Biomembr 1864: 184013. [Epub: Ahead of Print]

Osickova, A., N. Balashova, J. Masin, M. Sulc, J. Roderova, T. Wald, A.C. Brown, E. Koufos, E.H. Chang, A. Giannakakis, E.T. Lally, and R. Osicka. (2018). Cytotoxic activity of Kingella kingae RtxA toxin depends on post-translational acylation of lysine residues and cholesterol binding. Emerg Microbes Infect 7: 178.

Osickova, A., R. Osicka, E. Maier, R. Benz, and P. Sebo. (1999). An amphipathic α-helix including glutamates 509 and 516 is crucial for membrane translocation of adenylate cyclase toxin and modulates formation and cation selectivity of its membrane channels. J. Biol. Chem. 274: 37644-37650.

Powthongchin, B. and C. Angsuthanasombat. (2009). Effects on haemolytic activity of single proline substitutions in the Bordetella pertussis CyaA pore-forming fragment. Arch. Microbiol. 191: 1-9.

Roderova, J., A. Osickova, A. Sukova, G. Mikusova, R. Fiser, P. Sebo, R. Osicka, and J. Masin. (2019). Residues 529 to 549 participate in membrane penetration and pore-forming activity of the Bordetella adenylate cyclase toxin. Sci Rep 9: 5758.

Soloaga, A., M.P. Veiga, L.M. Garcia-Segura, H. Ostolaza, R. Brasseur, and F.M. Goñi. (1999). Insertion of Escherichia coli α-haemolysin in lipid bilayers as a non-transmembrane integral protein: prediction and experiment. Mol. Microbiol. 31: 1013-1024.

Sukova, A., L. Bumba, P. Srb, V. Veverka, O. Stanek, J. Holubova, J. Chmelik, R. Fiser, P. Sebo, and J. Masin. (2020). Negative charge of the AC-to-Hly linking segment modulates calcium-dependent membrane activities of Bordetella adenylate cyclase toxin. Biochim. Biophys. Acta. Biomembr 1862: 183310.

Svedova, M., J. Masin, R. Fiser, O. Cerny, J. Tomala, M. Freudenberg, L. Tuckova, M. Kovar, G. Dadaglio, I. Adkins, and P. Sebo. (2016). Pore-formation by adenylate cyclase toxoid activates dendritic cells to prime CD8+ and CD4+ T cells. Immunol Cell Biol 94: 322-333.

Wald, T., A. Osickova, J. Masin, P.M. Liskova, I. Petry-Podgorska, T. Matousek, P. Sebo, and R. Osicka. (2016). Transmembrane segments of complement receptor 3 do not participate in cytotoxic activities but determine receptor structure required for action of Bordetella adenylate cyclase toxin. Pathog Dis 74:.

Westrop, G., K. Hormozi, N. da Costa, R. Parton, and J. Coote. (1997). Structure-function studies of the adenylate cyclase toxin of Bordetella pertussis and the leukotoxin of Pasteurella haemolytica by heterologous C protein activation and construction of hybrid proteins. J. Bacteriol. 179: 871-879.

Wiles, T.J. and M.A. Mulvey. (2013). The RTX pore-forming toxin α-hemolysin of uropathogenic Escherichia coli: progress and perspectives. Future Microbiol 8: 73-84.

Examples:

TC#NameOrganismal TypeExample
1.C.11.1.1

Leukotoxin, HlaA or LktA of 955 aas and 2 TMSs. Cytolysin LktA is one of the major pathogenicity factors of Mannheimia haemolytica (formerly Pasteurella haemolytica) that is the cause of pasteurellosis, also known as shipping fever pneumonia, causing substantial loss of sheep and cattle during transport. LktA belongs to the family of RTX-toxins (Repeats in ToXins) that are produced as pathogenicity factors by a variety of Gram-negative bacteria. Sublytic concentrations of LktA cause inflammatory responses of ovine leukocytes while higher concentrations result in formation of transmembrane channels in target cells that may cause cell lysis and apoptosis. Channel formation by LktA occurs in artificial lipid bilayer membranes made of different lipids. LktA channels had a single-channel conductance of about 60 pS in 0.1 M KCl, which is about one tenth of the conductance of most RTX-toxins with the exception of the adenylate cyclase toxin of Bordetella pertussis (Benz et al. 2019). The LktA channels are highly cation-selective, and the channel diameter is around 1.5 nm.

Gram-negative bacteria

HlaA of Mannheimia (Pasteurella) haemolytica

 
1.C.11.1.2RTX-toxin IIA; haemolysin IIA; cytolysin IIA, ClyIIA Gram-negative bacteria ClyIIA of Actinobacillus pleuropneumoniae
 
1.C.11.1.3

Haemolysin A, HlyA (α-haemolysin) (Wiles and Mulvey 2013). The channel-forming domain may contain β-strands, possibly in addition to alpha-helical structures (Benz et al. 2014).  Although homologous, HlyA and CyaA (1.C.11.1.4) exhibit different modes of permeabilization (Fiser and Konopásek 2009). HlyA triggered an increase in mitochondrial Ca2+ levels and manipulated mitochondrial dynamics by causing fragmentation of the mitochondrial network. Alterations in mitochondrial dynamics resulted in severe impairment of mitochondrial functions by loss of membrane potential, increase in reactive oxygen species production, and ATP depletion. HlyA also caused disruption of plasma membrane integrity (Lu et al. 2018). Cholesterol catalyzes unfolding in membrane inserted motifs of the pore forming cytolysin A (Kulshrestha et al. 2023).

Proteobacteria

HlyA of E. coli

 
1.C.11.1.4

Bifunctional adenylate cyclase-haemolysin toxin precursor, CyaA.  Although homologous, HlyA (1.C.11.1.3) and CyaA  exhibit different modes of permeabilization (Fiser and Konopásek 2009).  A pore model comprising three alpha2-loop-alpha3 hairpins suggested that Gly530XXGly533XXXGly537  in TMS2 could function in toxin oligomerization (Juntapremjit et al. 2015).  Structural integrity of TMSs 1, 2, 3 and 5, but not 4, is important for haemolytic activity, particularly for transmembrane helices 2 and 3 that might form the pore (Powthongchin and Angsuthanasombat 2009). CyaA forms small cation-selective membrane pores that permeabilize cells for potassium efflux, contributing to cytotoxicity of CyaA and eventually provoking colloid-osmotic cell lysis (Wald et al. 2016).  The toxin penetrates myeloid phagocytes expressing the complement receptor 3 and delivers into the cytosol its N-terminal adenylate cyclase enzyme domain (~400 residues). In parallel, the ~1300 residue-long RTX hemolysin moiety of CyaA permeabilizes target cell membranes for efflux of cytosolic potassium ions (Svedova et al. 2016).  Positively-charged side-chains substituted at positions Gln574 and Glu581 in the pore-lining alpha3 enhance hemolytic activity and ion-channel opening, mimicing the highly-active RTX (repeat-in-toxin) cytolysins (Kurehong et al. 2017). Residues 529 to 549 participate in membrane penetration and pore-forming activity (Roderova et al. 2019). Two distinct conformers of CyaA appear to accomplish its two parallel activities within target cell membranes. The translocating conformer would deliver the N-terminal adenylyl cyclase domain into the cytosol of cells, while the pore precursor conformer would assemble into oligomeric cation-selective pores and permeabilize cellular membrane. Both toxin activities involve a membrane-interacting 'AC-to-Hly-linking segment' (residues 400 to 500). Two clusters of negatively charged residues within this linking segment (Glu419 to Glu432 and Asp445 to Glu448) regulate the balance between the AC domain translocating and pore-forming capacities of CyaA as a function of the calcium concentration (Sukova et al. 2020). Four cholesterol-recognition motifs in the pore-forming and translocation domains of CyaA are essential for invasion of eukaryotic cells and lysis of erythrocytes (Amuategi et al. 2022).

Proteobacteria

CyaA of Bordetella pertussis

 
1.C.11.1.5

Cytolytic RTX-toxin, GtxA (causes salpingitis and peritonitis in birds (Kristensen et al., 2009)

Gram-negative bacterium

GtxA of Gallibacterium anatis

 
1.C.11.1.6

Enterohemolysin EhxA of 998 aas

Proteobacteria

EhxA of E. coli

 
1.C.11.1.7

Leukotoxin A, LtxA pore-forming toxin of 1055 aas, exhibiting β-hemolytic activity.  Plays a role in immune evasion by lysing human lymphocytes and monocytes. It binds to the LFA-1 integrin on the surface of the host cell and to cholesterol-containing membranes, resulting in large LtxA-LFA-1 clusters in lipid rafts (Balashova et al. 2006; Brown et al. 2013).  Blocking P2X receptors protects monocytes from LtxA (Fagerberg et al. 2016).

LtxA of Aggregatibacter (Actinobacillus) actinomycetemcomitans (Haemophilus actinomycetemcomitans)

 
1.C.11.1.8

Leukotoxin, RtxA or IktA, of 956 aas and 3 or 4 TMSs in a 1 + 1 + 2 TMS arrangement. An interaction between the toxin and cholesterol occurs via two cholesterol recognition/interaction amino acid consensus motifs located in the C-terminal portion of the pore-forming domain of the toxin, and the cytotoxic activity of RtxA depends on post-translational acylation of the K558 and/or K689 residues as well as on the toxin binding to cholesterol in the membrane (Osickova et al. 2018).

RtxA if Kingella kingae

 
1.C.11.1.9

FrpC of 1492 aas and 1 or 2 TMSs, one N-terminal and one at about residue 270. 

FrpC of Vibrio anguillarum