Mark J. Pallen

The genome sequence of the food-borne pathogen Campylobacter jejuni reveals hypervariable sequences

Campylobacter jejuni, from the delta-epsilon group of proteobacteria, is a microaerophilic, Gram-negative, flagellate, spiral bacterium—properties it shares with the related gastric pathogen Helicobacter pylori. It is the leading cause of bacterial food-borne diarrhoeal disease throughout the world. In addition, infection with C. jejuni is the most frequent antecedent to a form of neuromuscular paralysis known as Guillain–Barré syndrome. Here we report the genome sequence of C. jejuni NCTC11168. C. jejuni has a circular chromosome of 1,641,481 base pairs (30.6% G+C) which is predicted to encode 1,654 proteins and 54 stable RNA species. The genome is unusual in that there are virtually no insertion sequences or phage-associated sequences and very few repeat sequences. One of the most striking findings in the genome was the presence of hypervariable sequences. These short homopolymeric runs of nucleotides were commonly found in genes encoding the biosynthesis or modification of surface structures, or in closely linked genes of unknown function. The apparently high rate of variation of these homopolymeric tracts may be important in the survival strategy of C. jejuni.

Performance comparison of benchtop high-throughput sequencing platforms

Three benchtop high-throughput sequencing instruments are now available. The 454 GS Junior (Roche), MiSeq (Illumina) and Ion Torrent PGM (Life Technologies) are laser-printer sized and offer modest set-up and running costs. Each instrument can generate data required for a draft bacterial genome sequence in days, making them attractive for identifying and characterizing pathogens in the clinical setting. We compared the performance of these instruments by sequencing an isolate of Escherichia coli O104:H4, which caused an outbreak of food poisoning in Germany in 2011. The MiSeq had the highest throughput per run (1.6 Gb/run, 60 Mb/h) and lowest error rates. The 454 GS Junior generated the longest reads (up to 600 bases) and most contiguous assemblies but had the lowest throughput (70 Mb/run, 9 Mb/h). Run in 100-bp mode, the Ion Torrent PGM had the highest throughput (80–100 Mb/h). Unlike the MiSeq, the Ion Torrent PGM and 454 GS Junior both produced homopolymer-associated indel errors (1.5 and 0.38 errors per 100 bases, respectively).

A novel EspA-associated surface organelle of enteropathogenic Escherichia coli involved in protein translocation into epithelial cells.

Enteropathogenic Escherichia coli (EPEC), like many bacterial pathogens, employ a type III secretion system to deliver effector proteins across the bacterial cell. In EPEC, four proteins are known to be exported by a type III secretion system—EspA, EspB and EspD required for subversion of host cell signal transduction pathways and a translocated intimin receptor (Tir) protein (formerly Hp90) which is tyrosine‐phosphorylated following transfer to the host cell to become a receptor for intimin‐mediated intimate attachment and ‘attaching and effacing’ (A/E) lesion formation. The structural basis for protein translocation has yet to be fully elucidated for any type III secretion system. Here, we describe a novel EspA‐containing filamentous organelle that is present on the bacterial surface during the early stage of A/E lesion formation, forms a physical bridge between the bacterium and the infected eukaryotic cell surface and is required for the translocation of EspB into infected epithelial cells.

Type VI secretion: a beginner's guide.

Type VI secretion is a newly described mechanism for protein transport across the cell envelope of Gram-negative bacteria. Components that have been partially characterised include an IcmF homologue, the ATPase ClpV, a regulatory FHA domain protein and the secreted VgrG and Hcp proteins. Type VI secretion is clearly a key virulence factor for some important pathogenic bacteria and has been implicated in the translocation of a potential effector protein into eukaryotic cells by at least one organism (Vibrio cholerae). However, type VI secretion systems (T6SSs) are widespread in nature and not confined to known pathogens. In accordance with the general rule that the expression of protein secretion systems is tightly regulated, expression of type VI secretion is controlled at both transcriptional and post-transcriptional levels.

An extensive repertoire of type III secretion effectors in Escherichia coli O157 and the role of lambdoid phages in their dissemination

Several pathogenic strains of Escherichia coli exploit type III secretion to inject “effector proteins” into human cells, which then subvert eukaryotic cell biology to the bacteriums advantage. We have exploited bioinformatics and experimental approaches to establish that the effector repertoire in the Sakai strain of enterohemorrhagic E. coli (EHEC) O157:H7 is much larger than previously thought. Homology searches led to the identification of >60 putative effector genes. Thirteen of these were judged to be likely pseudogenes, whereas 49 were judged to be potentially functional. In total, 39 proteins were confirmed experimentally as effectors: 31 through proteomics and 28 through translocation assays. At the protein level, the EHEC effector sequences fall into >20 families. The largest family, the NleG family, contains 14 members in the Sakai strain alone. EHEC also harbors functional homologs of effectors from plant pathogens (HopPtoH, HopW, AvrA) and from Shigella (OspD, OspE, OspG), and two additional members of the Map/IpgB family. Genes encoding proven or predicted effectors occur in >20 exchangeable effector loci scattered throughout the chromosome. Crucially, the majority of functional effector genes are encoded by nine exchangeable effector loci that lie within lambdoid prophages. Thus, type III secretion in E. coli is linked to a vast phage “metagenome,” acting as a crucible for the evolution of pathogenicity.

The HtrA family of serine proteases

HtrA, also known as DegP and probably identical to the Do protease, is a heat shock‐induced serine protease that is active in the periplasm of Escherichia coli. Homologues of HtrA have been described in a wide range of bacteria and in eukaryotes. Its chief role is to degrade misfolded proteins in the periplasm. Substrate recognition probably involves the recently described PDZ domains in the C‐terminal half of HtrA and, we suspect, has much in common with the substrate recognition system of the tail‐specific protease, Prc (which also possesses a PDZ domain). The expression of htrA is regulated by a complex set of signal transduction pathways, which includes an alternative sigma factor, RpoE, an anti‐sigma factor, RseA, a two‐component regulatory system, CpxRA, and two phosphoprotein phosphatases, PrpA and PrpB. Mutations in the htrA genes of Salmonella, Brucella and Yersinia cause decreased survival in mice and/or macrophages, and htrA mutants can act as vaccines, as cloning hosts and as carriers of heterologous antigens.

Open-Source Genomic Analysis of Shiga-Toxin-Producing E. coli O104:H4

An outbreak caused by Shiga-toxin–producing Escherichia coli O104:H4 occurred in Germany in May and June of 2011, with more than 3000 persons infected. Here, we report a cluster of cases associated with a single family and describe an open-source genomic analysis of an isolate from one member of the family. This analysis involved the use of rapid, bench-top DNA sequencing technology, open-source data release, and prompt crowd-sourced analyses. In less than a week, these studies revealed that the outbreak strain belonged to an enteroaggregative E. coli lineage that had acquired genes for Shiga toxin 2 and for antibiotic resistance.

The ESAT-6/WXG100 superfamily -- and a new Gram-positive secretion system?

ESAT-6 is a small secreted protein of unknown function from Mycobacterium tuberculosis that is of fundamental importance in virulence and protective immunity. A PSI-BLAST search has identified distant homologues of ESAT-6 in more tractable bacteria, including Bacillus subtilis, Bacillus anthracis, Staphylococcus aureus and Clostridium acetobutylicum. The genes for ESAT-6-like proteins often cluster with genes encoding homologues of B. subtilis YukA. I speculate that the ESAT-6-like and YukA-like proteins form a novel Gram-positive secretion system potentially driven by the FtsK/SpoIIIE ATPase domains in the YukA-like proteins. The way is now open to investigate this hypothesis in organisms that are easier to manipulate than pathogenic mycobacteria.

An embarrassment of sortases – a richness of substrates?

A range of surface proteins is anchored to the cell walls of Gram-positive pathogens such as Staphylococcus aureus by the transpeptidase sortase. Until now, sortase-like proteins and their substrates appeared to be limited mainly to such pathogens. However, by searching for sortase homologues among complete and incomplete genome sequences, we have found them to be present in almost all Gram-positives, a single Gram-negative bacterium and an archaean. There is usually more than one sortase-like protein encoded in each Gram-positive genome, and the genes encoding the sortase-like proteins are often clustered with genes encoding their likely substrates.

Sequencing and analysis of the genome of the Whipple's disease bacterium Tropheryma whipplei

BACKGROUND Whipples disease is a rare multisystem chronic infection, involving the intestinal tract as well as various other organs. The causative agent, Tropheryma whipplei, is a Gram-positive bacterium about which little is known. Our aim was to investigate the biology of this organism by generating and analysing the complete DNA sequence of its genome. METHODS We isolated and propagated T whipplei strain TW08/27 from the cerebrospinal fluid of a patient diagnosed with Whipples disease. We generated the complete sequence of the genome by the whole genome shotgun method, and analysed it with a combination of automatic and manual bioinformatic techniques. FINDINGS Sequencing revealed a condensed 925938 bp genome with a lack of key biosynthetic pathways and a reduced capacity for energy metabolism. A family of large surface proteins was identified, some associated with large amounts of non-coding repetitive DNA, and an unexpected degree of sequence variation. INTERPRETATION The genome reduction and lack of metabolic capabilities point to a host-restricted lifestyle for the organism. The sequence variation indicates both known and novel mechanisms for the elaboration and variation of surface structures, and suggests that immune evasion and host interaction play an important part in the lifestyle of this persistent bacterial pathogen.