Biology subjects

Webb, A. I.

Publications and source records attributed to Webb, A. I..

3 recordsLinked to original sources

bioRxiv2018

MSCypher: an integrated database searching and machine learning workflow for multiplexed proteomics.

Improvements in shotgun proteomics approaches are hampered by increases in multiplexed (chimeric) spectra, as improvements in peak capacity, sensitivity or dynamic range all increase the number of co-eluting peptides. This results in diminishing returns using traditional search algorithms, as co-fragmented spectra are known to decrease identification rates. Here we describe MSCypher, a freely available software suite that enables an extensible workflow including a hybrid supervised machine learned strategy that dynamically adjusts to individual datasets. This results in improved identification rates and quantification of low-abundant peptides and proteins. In addition, the integration of peptide de novo sequencing and database searching enables an unbiased view of variants and high-intensity unassigned peptide spectral matches.\n\nHighlightsO_LIOpen-source end-to-end label-free proteomics workflow\nC_LIO_LIIntegrated database searching and machine learning\nC_LIO_LICustomisable and extensible workflow including de novo sequencing\nC_LIO_LIOptimised for multiplexed spectra, challenging proteomics datasets and peptidomics applications\nC_LI

bioinformatics↗

bioRxiv2018

Aspartyl Protease 5 matures virulence factors found at the host-parasite interface in Toxoplasma gondii

Toxoplasma gondii infects approximately 30% of the worlds population, causing disease primarily during pregnancy and in individuals with weakened immune systems. Toxoplasma secretes and exports effector proteins that modulate the host during infection and several of these proteins are processed by the Golgi-associated Aspartyl Protease 5 (ASP5). Here, we identify ASP5 substrates by selectively enriching N-terminally-derived peptides from wildtype and {Delta}asp5 parasites. We reveal over two thousand unique Toxoplasma N-terminal peptides, mapping to both natural N-termini and protease cleavage sites. Several of these peptides mapped directly downstream of the characterised ASP5-cleavage site, arginine-arginine-leucine (RRL). We validate candidates as true ASP5 substrates, revealing they are not processed in parasites lacking ASP5, nor in wild type parasites following mutation of the motif from RRL{longrightarrow}ARL. All new ASP5 substrates are dense granule proteins, and interestingly none appear to be exported, thus differing from the analogous system in related Plasmodium spp., instead revealing that the majority of substrates reside within the parasitophorous vacuole (PV), and its membrane (the PVM), including two kinases and one phosphatase. Furthermore, we show that several of these ASP5-substrates are virulence factors, with their removal leading to attenuation in a mouse model, suggesting that phosphorylation at the host-parasite interface is important for virulence. Collectively, these data constitute the first in-depth analyses of the total list of ASP5 substrates, and shed new light on the role of ASP5 as a maturase of dense granule proteins during the Toxoplasma lytic cycle.

microbiology↗

bioRxiv2018

Protein Kinase A Negatively Regulates Ca2+ signalling in Toxoplasma gondii

The phylum Apicomplexa comprises a group of obligate intracellular parasites that alternate between intracellular replicating forms and actively motile extracellular forms that move through tissue. Parasite cytosolic Ca2+ signalling activates motility, but how this is switched off after invasion is not understood. Here we show that the cAMP-dependent Protein Kinase A catalytic subunit 1 (PKAc1) of Toxoplasma is responsible for suppression of Ca2+ signalling upon host cell invasion. We demonstrate that that PKAc1 is sequestered to the parasite periphery by dual acylation of its regulatory subunit PKAr1. Newly invaded PKAc1-deficient parasites exit host cells shortly thereafter in a perforin-like protein 1 (PLP-1)-dependent fashion. We demonstrate that loss of PKAc1 results in an inability to rapidly downregulate cytosolic Ca2+ levels shortly after invasion. Furthermore, we demonstrate that PKAc1 also specifically negatively regulates resting cytosolic Ca2+ in conditions that mimic intracellularity. We also show that cAMP and cGMP have opposing role in microneme secretion, further supporting evidence that cAMP signalling has a suppressive role during motility. Together, this work provides a new paradigm in understanding how Toxoplasma and related apicomplexan parasites regulate infectivity.

microbiology↗