Mike Blackman: Projects

Proteolytic processing and function of apical membrane antigen-1 (AMA1)

AMA1 is a large integral membrane protein that is secreted from micronemes, a set of specialised apically-located secretory organelles, onto the surface of the merozoite just prior to invasion. There AMA1 plays a critical role in invasion and is eventually shed by the action of PfSUB2 (Harris et al., 2005).

AMA1 is of particular interest because antibodies against it can prevent host cell invasion by the parasite. We wish to determine the function of AMA1 and the role of its shedding, as well as defining functionally important regions of the protein that might form part of a sub-unit malaria vaccine (Pizarro et al., 2005). To this end, we have mapped the epitope for one of the most efficient monoclonal antibodies which inhibit invasion (Collins et al., 2007), and shown that this may function by interfering with interactions between AMA1 and partner proteins involved in formation of the moving junction, a transient doughnut-like structure through which the invading merozoite moves as it invades an erythrocyte (Collins et al., 2009). Proteolytic shedding of AMA1 by the subtilisin-like protease SUB2 plays an important role in invasion and evasion of antibodies (Olivieri et al., 2011).

X-ray crystal structure of the AMA1 ectodomain

X-ray crystal structure of the AMA1 ectodomain (Click to view larger image)

The X-ray crystal structure of the AMA1 ectodomain has shown that it comprises three disulphide-constrained domains. Two of these belong to the PAN module superfamily, implicated in protein-protein or protein-carbohydrate interactions. Here, domains I (yellow), II (blue) and III (green) are shown in three orientations. 

Selected publications

Collins, C.R., Withers-Martinez, C., Hackett, F. and Blackman, M.J. (2009) An inhibitory antibody blocks interactions between components of the malarial invasion machinery.  PLoS Pathog 5, e1000273 

Olivieri, A., Collins, C.R., Hackett, F., Withers-Martinez, C., Marshall, J., Flynn, H.R.,Skehel, J.M. and Blackman, M.J. (2011) Juxtamembrane shedding of Plasmodium falciparum AMA1 is sequence independent and essential, and helps evade invasion-inhibitory antibodies.  PLoS Pathog. 7, e1002448.

 

Subtilisin-like proteases of the malaria merozoite

Proteases are known to play important roles during the life cycle of the malaria parasite. We are working on a family of malarial subtilisin-like serine proteases (subtilases) that are involved in host cell invasion and exit.

Molecular structure of the P. falciparum SUB1 catalytic domain.

Molecular structure of the P. falciparum SUB1 catalytic domain. (Click to view larger image)

The predicted catalytic serine, histidine and aspartic acid residues (black) are shown in ball-and-stick representation

SUB1 was the first malarial subtilase to be identified (Blackman et al., 1998). It is highly conserved across the genus and plays a critical role in the blood-stage life cycle.

We have shown that SUB1 has a central regulatory role in release of the parasites from the host cell. SUB1 is discharged from merozoite organelles known as exonemes into the parasitophorous vacuole space, where it is responsible for processing a family of papain-like proteins known as SERA, triggering a cascade of proteolytic events which culminate in host cell rupture and parasite egress (Yeoh et al., 2007).

SUB1 is also involved in remodelling of several essential merozoite surface proteins in preparation for invasion of a new cell (Koussis et al., 2009).

Recent work has also shown that SUB1 is essential for release of merozoites from liver stages of the malarial life cycle (Suarez et al., 2013).

Current investigations of SUB1 are focused on dissecting the regulation, timing and significance of these proteolytic events, and identifying inhibitors of SUB1 that have potential for development as drugs.

PfSUB1: a malaria merozoite serine protease

PfSUB1: a malaria merozoite serine protease (Click to view larger image)

Surface representation of the P. falciparum SUB1 active site, showing the substrate binding cleft with a bound 10 amino acid peptide substrate derived from the physiological substrate SERA5.]

SUB2 (Hackett et al., 1999) is a large membrane-bound subtilase which, like SUB1, is expressed in blood-stage merozoites and is found in all Plasmodium species. SUB2 is the prime candidate for the 'sheddase' which clips important surface proteins such as MSP1 and AMA1 from the surface of the merozoite during erythrocyte invasion (Harris et al., 2005). We are actively pursuing the function of this protease, using biochemical approaches and genetic modification of P. falciparum.

Activity of PfSUB2

Activity of PfSUB2

On the left is an immunofluorescence image showing the surface location of AMA1 (green) on released P. falciparum merozoites. On the right is a schematic depicting proteolytic shedding of AMA1 and another merozoite surface protein complex called MSP1. The protease PfSUB2 mediates release of both proteins by cleaving them at sites close to the membrane.

Selected publications

Withers-Martinez, C., Strath, M., Hackett, F., Haire, L.F., Howell, SA.,Walker, P.A., Christodoulou, E., Dodson, GG and Blackman, M.J. The malaria parasite egress protease SUB1 is a calcium-dependent redox switch subtilisin.  Nat Commun. 5,3726. doi: 10.1038/ncomms4726.

Withers-Martinez, C., Suarez, C., Fulle, S., Kher, S., Penzo, M., Ebejer, J.P., Koussis. K., Hackett, F., Jirgensons, A., Finn, P. and Blackman, M.J. (2012) Plasmodium subtilisin-like protease 1 (SUB1): insights into the active-site structure, specificity and function of a pan-malaria drug targetInt J Parasitol. 42, 597-612.

Suarez, C., Volkmann, K., Gomes, A.R., Billker, O. and Blackman, M.J. (2013) The malarial serine protease SUB1 plays an essential role in parasite liver stage developmentPLoS Pathog. 9, e1003811.

Koussis, K., Withers-Martinez, C., Yeoh, S., Child, M., Hackett, F., Knuepfer, E., Juliano, L., Woehlbier, U., Bujard, H. and Blackman, M.J. (2009) A multifunctional serine protease primes the malaria parasite for red blood cell invasion.  EMBO Journal  28, 725-735 PubMed abstract

The P. falciparum SERA family of papain-like proteins

Upon its discharge in the parasite parasitophorous vacuole, SUB1 comes into contact with a set of vacuolar proteins that include members of a protein family called the serine-rich antigens (SERA). In P. falciparum there are 9 SERA family genes, 8 of which are encoded by a tandem array of genes on chromosome 2. The SERA proteins are unified by the presence of a central domain with sequence and structural homology to the cysteine protease papain.

In asexual blood stages only two SERA family members - SERA5 and SERA6 - appear essential, and we have shown that both of these proteins are cleaved by SUB1 to release the central papain-like domain (Yeoh et al., 2007; Ruecker et al., 2012).

One hypothesis we are pursuing is that cleavage of the SERA proteins by SUB1 activates their function. Whilst SERA6 possess a Cys residue at the position of the canonical nucleophile, and may possess protease activity (Ruecker et al., 2012), SERA5 does not play an enzymatic role in the parasite life cycle (Stallmach et al., 2015).

We want to identify the function(s) and substrates(s) of SERA5 and SSERA6 and to understand the biological significance of their processing by SUB1.

Proteolytic processing of SERA5 by SUB1 releases its central papain-like domain (here labelled P56).

Proteolytic processing of SERA5 by SUB1 releases its central papain-like domain (here labelled P56). (Click to view larger image)

Selected publications

Stallmach, R., Kavishwar, M., Withers-Martinez, C., Hackett, F., Collins, C.R., Howell, S.A., Yeoh, S., Knuepfer, E., Atid, A.J., Holder, A.A. and Blackman, M.J. (2015) Plasmodium falciparum SERA5 plays a non-enzymatic role in the malarial asexual blood-stage lifecycleMol Microbiol. (in press) doi: 10.1111/mmi.12941. [Epub ahead of print]

Ruecker, A., Shea, M., Hackett, F., Suarez, C., Hirst, E.M., Milutinovic, K.,Withers-Martinez. C. and Blackman, M.J. (2012) Proteolytic activation of the essential parasitophorous vacuole cysteine protease SERA6 accompanies malaria parasite egress from its host erythrocyte.  J Biol Chem. 287, 37949-63.

Yeoh, S., O'Donnell, R. A., Koussis, K., Dluzewski, A. R., Ansell, K. H., Osborne, S. A., Hackett, F., Withers-Martinez, C., Mitchell, G. H., Bannister, L. H., Bryans, J. S., Kettleborough, C. A., and Blackman, M.J. (2007) Subcellular discharge of a serine protease mediates release of invasive malaria parasites from host erythrocytes.  Cell 131, 1072-83 PubMed abstract

cGMP-mediated signalling in regulation of malarial egress

Relatively recent work from this laboratory, in collaboration with David Baker's laboratory at the London School of Hygiene and Tropical Medicine, has shown that discharge of SUB1 into the parasitophorous vacuole is regulated by the cyclic nucleotide second messenger cyclic GMP (cGMP), through its activation of a single parasite protein kinase called PKG (Collins et al., 2013a). Selective inhibitors of PKG reversibly block egress, whilst pharmacological agents that artificially upregulate cGMP levels in the parasite can induce premature egress (Collins et al., 2013a; Taylor et al.,2010).

We are currently working with David Baker to further dissect the regulation of the cGMP pathway that controls egress using conditional gene disruption (Collins et al., 2013b), new pharmacological tools, metabolomics and heterologous protein expression.

PKG regulates release of SUB1 and egress

PKG regulates release of SUB1 and egress (Click to view larger image)

Selected publications

Collins, C.R., Hackett, F., Strath, M., Penzo, M., Withers-Martinez, C., Baker, D.A. and Blackman, M.J. (2013a) Malaria parasite cGMP-dependent protein kinase regulates blood stage merozoite secretory organelle discharge and egress.  PLoS Pathog. 9, e1003344.

Collins, C.R., Das, S., Wong, E.H., Andenmatten, N., Stallmach, R., Hackett, F., Herman. J.P., M├╝ller, S, Meissner, M. and Blackman, M.J. (2013b) Robust inducible Cre recombinase activity in the human malaria parasite Plasmodium falciparum enables efficient gene deletion within a single asexual erythrocytic growth cycleMol Microbiol. 88, 687-701.

 

Mike Blackman

Mike Blackman

mike.blackman@crick.ac.uk
+44 (0)20 379 62328

  • Qualifications and history
  • 1981 BSc Microbiology, University of Leeds, UK
  • 1985 MSc Immunology, University of Warwick, UK
  • 1985 RA MRC Laboratories, The Gambia.
  • 1991 PhD MRC-NIMR, London, UK
  • 2000 Group Leader, Medical Research Council National Institute for Medical Research, London, UK
  • 2015 Group Leader, the Francis Crick Institute, London, UK