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Introduction
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December 2011 - December 2014
Publications
Publications (92)
Inflammasomes are important for host defence against pathogens and homeostasis with commensal microbes. Here, we show non-haemolytic enterotoxin (NHE) from the neglected human foodborne pathogen Bacillus cereus is an activator of the NLRP3 inflammasome and pyroptosis. NHE is a non-redundant toxin to haemolysin BL (HBL) despite having a similar mech...
Bacterial autotransporters comprise a C-terminal β-barrel domain, which must be correctly folded and inserted into the outer membrane to facilitate translocation of the N-terminal passenger domain to the cell exterior. Once at the surface, the passenger domains of most autotransporters are folded into an elongated β-helix. In a cellular context, ke...
The outer membranes of gram-negative bacteria contain integral membrane proteins, most of which are of β-barrel structure, and critical for bacterial survival. These β-barrel proteins rely on the β -barrel a ssembly m achinery (BAM) complex for their integration into the outer membrane as folded species. The central and essential subunit of the BAM...
In biological membranes, various protein secretion devices function as nanomachines, and measuring the internal movements of their component parts is a major technological challenge. The translocation and assembly module (TAM) is a nanomachine required for virulence of bacterial pathogens. We have reconstituted a membrane containing the TAM onto a...
Autotransporter (AT) proteins provide a diverse array of important virulence functions to Gram-negative bacterial pathogens, and have also been adapted for protein surface display applications. The “autotransporter” moniker refers to early models that depicted these proteins facilitating their own translocation across the bacterial outer membrane....
Bacterial autotransporters comprise a 12-stranded membrane-embedded β-barrel domain, which must be folded in a process that entraps segments of an N-terminal passenger domain. This first stage of autotransporter folding determines whether subsequent translocation can deliver the N-terminal domain to its functional form on the bacterial cell surface...
Membrane proteins with a β-barrel topology are found in the outer membranes of Gram-negative bacteria and in the plastids and mitochondria of eukaryotic cells. The assembly of these membrane proteins depends on a protein folding reaction (to create the barrel) and an insertion reaction (to integrate the barrel within the outer membrane). Experiment...
Autotransporters are secreted proteins that are assembled into the outer membrane of bacterial cells. The passenger domains of autotransporters are crucial for bacterial pathogenesis, with some remaining attached to the bacterial surface while others are released by proteolysis. An enigma remains as to whether autotransporters should be considered...
(A) The barrel-domain of BigE (shaded grey) shares 40% sequence identity (60% sequence similarity) to that of the protein from Ralstonia. A segment of unknown function from 220–528 (green) is repeated 8 times in BigE. BLAST searches revealed proteins related to the autotransporter from Ralstonia in five other species of Beta-Proteobacteria (YP_0013...
Forty-seven autotransporters dataset.
(PDF)
Phylogenetic analysis of functional (passenger) domains. The active diagram contains all accession information for the 1523 autotransporter sequences. Sub-domain signatures were identified using Pfam analysis of all sequences, and these are represented as radiating coloured symbols. The length of this line is proportional to the number of residues...
Logos characteristic of the β-signal motif. In order to best represent the conserved features inherent in motif 5, a sequence Logo was constructed for the motif 5 sequences from each of the 13 (of 14) classes of barrel-domains shown in Figure 5. Some subtle differences are evident between the classes. In each case, the height of the letter represen...
Autotransporter detection in
E. coli
pathotypes.
(PDF)
It is widely believed that laboratory strains of Escherichia coli, including those used for industrial production of proteins, do not secrete proteins to the extracellular milieu.
Here, we report the development of a generalised module, based on an E. coli autotransporter secretion system, for the production of extracellular recombinant proteins. W...
Figure S2. Nucleotide sequences of the de novo synthesised pet gene and heterologous DNA encoding proteins targeted for secretion.
Figure S3. AT-mediated accumulation of heterologous proteins in the culture medium.
Figure S7. Comparison of the Pic and Pet SPATE proteins.
Table S2. Plasmids used in this study.
Figure S6. Impact of conserved amino acids from the AC domain on secretion of heterologous proteins.
Figure S1. Model of the Pet structure.
Table S1. Mass spectrometry analysis of some recombinant protein fusions with Pet.
Figure S4. Surface localisation of non cleaved Pet and fusion proteins.
Figure S5. Identification of minimal AT module permitting secretion of heterologous proteins to the culture supernatant fraction.
Table S3. Primers used in this study.
Bacteria have mechanisms to export proteins for diverse purposes, including colonization of hosts and pathogenesis. A small number of archetypal bacterial secretion machines have been found in several groups of bacteria and mediate a fundamentally distinct secretion process. Perhaps erroneously, proteins called 'autotransporters' have long been tho...
Autotransporters are a superfamily of proteins that use the type V secretion pathway for their delivery to the surface of Gram-negative bacteria. At first glance, autotransporters look to contain all the functional elements required to promote their own secretion: an amino-terminal signal peptide to mediate translocation across the inner membrane,...
Autotransporters are a superfamily of virulence factors typified by a channel-forming C terminus that facilitates translocation
of the functional N-terminal passenger domain across the outer membrane of Gram-negative bacteria. This final step in the
secretion of autotransporters requires a translocation-competent conformation for the passenger doma...
Salmonella enterica is a major cause of morbidity worldwide and mortality in children and immunocompromised individuals in sub-Saharan Africa.
Outer membrane proteins of Salmonella are of significance because they are at the interface between the pathogen and the host, they can contribute to adherence,
colonization, and virulence, and they are freq...
Autotransporter biogenesis is dependent upon BamA, a central component of the β-barrel assembly machinery (BAM) complex. In
this report, we detail the role of the other BAM components (BamB-E). We identify the importance of BamD in autotransporter
biogenesis and show that BamB, BamC, and BamE are not required.
Clearance of disseminated Salmonella infection requires bacterial-specific Th1 cells and IFN-γ production, and Th1-promoting vaccines are likely to help control these infections. Consequently, vaccine design has focused on developing Th1-polarizing adjuvants or Ag that naturally induce Th1 responses. In this study, we show that, in mice, immunizati...
Enteroaggregative Escherichia coli (EAEC) is a major cause of diarrhoea in developing countries. EAEC 042 is the prototypical strain. EAEC 042 secretes the functionally well-characterized Pet autotransporter toxin that contributes to virulence through its cytotoxic effects on intestinal epithelial cells. Following a global transposon mutagenesis sc...
Insertion of folded proteins into the outer membrane of Gram-negative bacteria is mediated by the essential β-barrel assembly machine (Bam). Here, we report the native structure and mechanism of a core component of this complex, BamE, and show that it is exclusively monomeric in its native environment of the periplasm, but is able to adopt a distin...
Tertiary structural predictions of the Big subdomains comprising the C-terminal passenger domains.
(0.78 MB PDF)
Multiple alignment of passenger subdomain D5.
(0.01 MB PDF)
Multiple alignment of passenger subdomain D7.
(0.01 MB PDF)
Multiple alignment of passenger subdomain D8.
(0.02 MB PDF)
Multiple alignment of C-terminal passenger domains.
(2.82 MB PDF)
Multiple alignment of passenger subdomain D4.
(0.01 MB PDF)
Multiple alignment of passenger subdomain D12.
(0.01 MB PDF)
Multiple alignment of the full-length Int/Inv family members.
(5.07 MB PDF)
Multiple alignment of N-terminal β-barrel domains. Predicted transmembrane β-strands are shaded yellow with predicted β-strands numbered 1 to 16 above the alignment. Fully conserved residues are colored red. Predicted α-helices are shaded blue located between β-strands 12 and 13.
(0.14 MB PDF)
Multiple alignment of passenger subdomain D0.
(0.02 MB PDF)
Multiple alignment of passenger subdomain D6.
(0.01 MB PDF)
Multiple alignment of passenger subdomain D10.
(0.01 MB PDF)
Multiple alignment of passenger subdomain D13.
(0.01 MB PDF)
Multiple alignment of N-terminal β-barrel domains.
(0.37 MB PDF)
The sixty-nine proteins of the Intimin/Invasin (Int/Inv) family included in this study, listed according to phylogenetic cluster and position within that cluster. Cluster designations refer to the clustering patterns in the phylogenetic tree shown in Fig 1A. Protein sizes are presented in numbers of amino acyl residues (aas). Greek letters refer to...
Multiple alignment of passenger subdomain D9.
(0.01 MB PDF)
Multiple alignment of passenger subdomain D14.
(0.01 MB PDF)
Background:
Gram-negative bacteria have developed a limited repertoire of solutions for secreting proteins from the cytoplasmic compartment to the exterior of the cell. Amongst the spectrum of secreted proteins are the intimins and invasins (the Int/Inv family; TC# 1.B.54) which are characterized by an N-terminal β-barrel domain and a C-terminal s...
The plasmid-encoded toxin, Pet, a prototypical member of the serine protease autotransporters of the Enterobacteriaceae, possesses an unusually long signal peptide, which can be divided into five regions termed N1 (charged), H1 (hydrophobic), N2, H2 and C (cleavage site) domains. The N1 and H1 regions correspond to a conserved N-terminal extension...
HIV and Salmonella
HIV-positive individuals who are infected with nontyphoidal strains of Salmonella enterica often succumb to high morbidity and mortality. Why this is the case is unknown. MacLennan et al. (p. 508 ; see the Perspective by Moir and Fauci ) have uncovered a dysregulated antibody response to Salmonella that is the likely culprit. Ser...
Comparison of EAEC 042 and E. coli MG1655 metabolism by phenotype microarray, where EAEC 042 shows greater metabolic activity.
(0.12 MB DOC)
Comparison of EAEC 042 and E. coli MG1655 metabolism by phenotype microarray, where E. coli MG1655 shows greater metabolic activity.
(0.07 MB DOC)
Type 3 secretion system effector genes in the EAEC 042 genome.
(0.05 MB DOC)
Global comparison between EAEC 042 chromosome and those of EHEC, UPEC and K-12. ACT comparison (http://www.sanger.ac.uk/Software/ACT) of amino-acid matches between the complete six-frame translations (computed using TBLASTX) of the whole genome sequences of enterohaemorrhagic E. coli O157:H7 str. Sakai (EHEC; EMBL acc: BA000007), enterohaemorrhagic...
Box-plot showing the estimated core genome size as a function of the number of genomes sequenced. The plot was constructed based on 100 randomly selected permutations of 30 genome sequences. The curve is approaching the limit suggesting that the 2356 conserved genes identified in this study represent the minimal E. coli genome and that this number...
The E. coli pan-genome. A. Box-plot showing the total size of the E. coli pan-genome as a function of the number of genomes sequenced. B. Box-plot showing the number of additional genes discovered with each genome sequenced. The plots were constructed using the same method as Fig. S4. Both indicate an open pan-genome, with ∼360 new genes being iden...
Regions of difference (RODS) in the EAEC 042 genome.
(0.04 MB XLS)
List of EAEC 042 CDS conserved in the other sequenced EAEC genomes (101-1 and 55989), but absent from the genome of the commensal E. coli HS.
(0.20 MB DOC)
Alignment of the EAEC 042 and E. coli MG1655 aga gene clusters. E. coli MG1655 lacks the agaE and agaF genes and has a truncated agaA gene. These truncations result in decreased N-acetyl-D-galactosamine and N-acetyl-D-glucosamine utilisation.
(0.22 MB DOC)
Genomic architecture of the Type II secretion system (T2SS) apparatus of EAEC 042 and representative E. coli strains. The T2SS locus (gsp) appears intact in EAEC 042 and a variety of phylogenetically disparate strains of pathogenic E. coli. In contrast, the locus possesses a lesion in E. coli K12 and is absent in E. coli CFT073. Where the locus is...
Genetic architecture of the capsular polysaccharide locus from EAEC 042. The locus is required for the biosynthesis of a group 2 capsular polysaccharide and appears to contain all the functional genes necessary for capsular production. Three CDS are present in the central region 2 and are likely to be important in the biosynthesis of the particular...
List of genes conserved in all sequenced E. coli genomes.
(1.99 MB DOC)
Genetic similarity of the EAEC 042 prophage. Nine prophage regions, designated 042p1–042p9, were identified in the EAEC 042 genome (see Table 2 in manuscript). The figure represents homology of the phage elements; solid lines indicate complete identity, the absence of lines within the boxes reflect little or no homology and the presence of dashed l...
Phylogenetic relationships among sequenced E. coli and Shigella genomes. The genomes of Escherichia albertii and Escherichia fergusonii are included as outgroup sequences. The tree was obtained by maximum likelihood analysis of a concatenated alignment of 2173 genes, using the general time reversible (GTR/REV) model with the CAT approximation of ra...
Genetic architecture of the region encoding the als locus from E. coli K-12 and the similar region from EAEC 042. The als locus is absent from EAEC 042 explaining the inability of EAEC 042 to utilise allose as a sole carbon source; E. coli K12 is capable of using allose as a sole carbon source. This prediction was confirmed by BioLog PMs.
(0.20 MB...
Sequence alignment of the E. coli K-12 and EAEC 042 porins. All the porins identified in EAEC 042 were aligned with those from E. coli K12 MG1655. NmpC (OmpD) is a pseudogene in E. coli K12 characterised by a C-terminal deletion. Identical residues are marked by asterisks, whereas similar residues are marked by periods and colons. The amino acid se...
Phylogenetic distribution of members of the Type 5 secretion system present in EAEC 042. The Autotransporters (AT-1) are differentially represented amongst the E. coli phylogeny, though several members of this group show phylogenetic clustering. The Two-partner secretion system (TPS) is present in all strains of E. coli suggesting an important phys...
Comparison of the EAEC 042 chromosomal- and plasmid-based copies of the shf loci. The sequences surrounding the loci are not homologous. The ca. 3 kb of nucleotide sequence encompassing shf, rfbU and virK is identical; msbB2 is absent from the plasmid copy but present in the chromosomal copy. The similarity of Shf to the Staphylococcus epidermidis...
Comparison of iron transport / storage genes from MG1655, O157 (Sakai) and O42.
(0.05 MB XLS)
Genetic map of pAA, the large virulence plasmid of EAEC 042. (A) On the basis of nucleotide sequence homology, the plasmid pAA belongs to the IncFIIA family and carries just one identifiable replicon consisting of Ec042-pAA152 (RepA) and Ec042-pAA153 (CopB), which is in contrast to many of the F-family plasmids that have multiple replicons. It poss...
Alignment of the ars operon from of E. coli K12 with EAEC 042. Alignments reveal genetic lesions in the E. coli K12 locus which result in the lack of the arsD and arsA genes. The loss of these genes confer upon E. coli K12 a reduced ability to grow in the presence of arsenite and antimony chloride.
(0.12 MB DOC)
The sor operon of EAEC 042. EAEC 042 possesses the sor operon and has the ability to utilise sorbose as a sole carbon source. E. coli K-12 lacks the operon and can not utilise this carbon source, as confirmed by the BioLog phenotyping arrays.
(0.22 MB DOC)
Comparison of the genomic region from E. coli K-12 and EAEC 042 encoding garD. The EAEC 042 gene is disrupted whereas it is uninterrupted in E. coli K-12. Disruption of garD resulted in the inability of EAEC 042 to utilise D-galactarate as a sole carbon source. E. coli K12 can utilise this substrate.
(0.15 MB DOC)
References for supplementary data.
(0.04 MB DOC)
Genetic architecture and distribution of the Chaperone-Usher systems of EAEC 042. (A) The organisation of the fimbrial loci from EAEC 042 are depicted. CDS flanking the fimbrial loci are depicted by green arrows, CDS encoding regulators are depicted by purple arrows, CDS encoding chaperones are indicated with black arrows, ushers are depicted with...
Genetic architecture of the RTX-like Type 1 secretion locus. An alignment of the genomic regions encompassing the T1SS is depicted, demonstrating the absence of the locus in E. coli K12 and the presence of the locus in EAEC 042 and E. coli O157:H7. In both pathogenic strains the gene encoding the putative secreted RTX-like protein is frameshifted a...
Comparison of three type VI secretion systems (T6SSs) of EAEC 042, and a separate Vgr-encoding region. Genes depicted by the schematic are indicated by the “Ec042” numbers on the right of the figure. Genes are designated by arrows, which are colored by xBASE to correspond to GC content, except for Hcp- and Vgr-homologs, which are shown in purple an...
Escherichia coli can experience a multifaceted life, in some cases acting as a commensal while in other cases causing intestinal and/or extraintestinal disease. Several studies suggest enteroaggregative E. coli are the predominant cause of E. coli-mediated diarrhea in the developed world and are second only to Campylobacter sp. as a cause of bacter...
The nuclear envelope that encloses the nucleus is a significant barrier to non-viral vectors and shrouds the relationship between the trafficking of plasmid DNA to the nucleus and expression of an encoded transgene. Here, we use a novel single cell approach to quantify nuclear import of plasmid DNA following non-viral transfection and correlate thi...
Nucleocytoplasmic trafficking is a major consideration for the design of vehicles for the delivery of drug/DNA cargo to cell nuclei for cancer and gene therapies. Recent data indicate that efficient nuclear import can involve the microtubule (MT)/dynein network, such that nuclear delivery of exogenous cargo could be enhanced by attachment to peptid...
