Proteomic analysis of Lawsonia intracellularis reveals expression of outer membrane proteins during infection.

Lawsonia intracellularis is the aetiological agent of the commercially significant porcine disease, proliferative enteropathy. Current understanding of host-pathogen interaction is limited due to the fastidious microaerophilic obligate intracellular nature of the bacterium. In the present study, expression of bacterial proteins during infection was investigated using a mass spectrometry approach. LC-ESI-MS/MS analysis of two isolates of L. intracellularis from heavily-infected epithelial cell cultures and database mining using fully annotated L. intracellularis genome sequences identified 19 proteins. According to the Clusters of Orthologous Groups (COG) functional classification, proteins were identified with roles in cell metabolism, protein synthesis and oxidative stress protection; seven proteins with putative or unknown function were also identified. Detailed bioinformatic analyses of five uncharacterised proteins, which were expressed by both isolates, identified domains and motifs common to other outer membrane-associated proteins with important roles in pathogenesis including adherence and invasion. Analysis of recombinant proteins on Western blots using immune sera from L. intracellularis-infected pigs identified two proteins, LI0841 and LI0902 as antigenic. The detection of five outer membrane proteins expressed during infection, including two antigenic proteins, demonstrates the potential of this approach to interrogate L. intracellularis host-pathogen interactions and identify novel targets which may be exploited in disease control.

The LEE1 promoters from both enteropathogenic and enterohemorrhagic Escherichia coli can be activated by PerC-like proteins from either organism.

The PerC protein of enteropathogenic Escherichia coli (EPEC), encoded by the pEAF plasmid, is an activator of the locus of enterocyte effacement (LEE) pathogenicity island via the LEE1 promoter. It has been assumed that the related LEE-containing pathogen enterohemorrhagic E. coli (EHEC) lacks PerC-dependent activation due to utilization of an alternative LEE1 promoter and lack of a perC gene. However, we show here that EPEC PerC can activate both the EPEC and EHEC LEE1 promoters and that the major transcriptional start site is similarly located in both organisms. Moreover, a PerC-like protein family identified from EHEC genome analyses, PerC1 (also termed PchABC), can also activate both promoters in a manner similar to that of EPEC PerC. The perC1 genes are carried by lambdoid prophages, which exist in multiple copies in different EHEC strains, and have a variable flanking region which may affect their expression. Although individual perC1 copies appear to be poorly expressed, the total perC1 expression level from a strain encoding multiple copies approaches that of perC in EPEC and may therefore contribute significantly to LEE1 activation. Alignment of the protein sequences of these PerC homologues allows core regions of the PerC protein to be identified, and we show by site-directed mutagenesis that these core regions are important for function. However, purified PerC protein shows no in vitro binding affinity for the LEE1 promoter, suggesting that other core E. coli proteins may be involved in its mechanism of activation. Our data indicate that the nucleoid-associated protein IHF is one such protein.

Direct and indirect transcriptional activation of virulence genes by an AraC-like protein, PerA from enteropathogenic Escherichia coli.

The plasmid-encoded Per regulatory locus of enteropathogenic Escherichia coli (EPEC) is generally considered to consist of three genes, perA, perB and perC. PerA, a member of the AraC-like family of transcriptional regulators, is known to be an activator of its own promoter (autoactivation) as well as of the plasmid-located bfp operon encoding bundle-forming pili, but its role in activation of the chromosomal locus of enterocyte effacement (LEE) pathogenicity island, which confers the property of intimate adherence on EPEC, requires clarification. Here, we show that PerA is also required for activation of the master regulatory LEE operon, LEE1, but that this activation is indirect, being achieved via autoactivation of the per promoter which ensures sufficient production of the PerC protein to activate LEE1. In contrast, PerA-dependent activation of the per and bfp promoters is direct and does not require the other Per proteins, but is modulated by the nucleoid-associated protein H-NS. The closely related VirF regulator from Shigella flexneri cannot substitute for PerA to activate these promoters, despite being able to bind their upstream regions in vitro. PerA can bind the per and bfp promoter fragments to form multiple complexes, while VirF forms only a single complex. Site-directed mutagenesis of the PerA protein suggests that, like VirF, it may use both of its carboxy-terminal helix-turn-helix motifs for DNA interaction, and may also make direct contacts with RNA polymerase. In addition, we have isolated mutations in the poorly characterized amino-terminal domain of PerA which affect its ability to activate gene expression.

Positive and negative effects of deletions and mutations within the 5' flanking sequences of Plasmodium falciparum DNA polymerase delta.

The DNA polymerase delta gene from Plasmodium falciparum is associated with a 1.76 kb 5' untranslated region and a promoter containing long homopolymeric (dA:dT) tracts and intrinsic DNA curvature. Here, a comprehensive deletion and mutational analysis of the DNA Pol(delta) upstream sequences has been undertaken to define functionally important regions. Removal of promoter-proximal DNA was shown to upregulate luciferase reporter gene expression and the ATG-proximal portion of the 5' untranslated region was required in conjunction with the promoter for reporter activity. In contrast to the ATG-proximal 5' untranslated region, deletion of the central part of the untranslated region had a positive effect on expression. Disruption of a homopolymeric (dA:dT) tract adjacent to the main transcription start site both derepressed gene expression and reduced the intrinsic curvature of DNA fragments containing this sequence.

In vivo DNA-binding and oligomerization properties of the Shigella flexneri AraC-like transcriptional regulator VirF as identified by random and site-specific mutagenesis.

In Shigella flexneri expression of the plasmid-encoded virulence genes is regulated via a complex mechanism involving both environmental signals and specific transactivators. The primary regulator protein, VirF, is a member of the AraC family of transcription factors and shares with other AraC-like proteins a conserved carboxy-terminal domain thought to be important for DNA binding. Random and site-directed mutagenesis of the virF gene encoding VirF yielded a number of mutations along the length of the protein which severely affected the ability of VirF to activate gene expression. The mutant proteins were shown to be affected in their ability to activate the virulence genes virB and icsA, both known to be regulated directly by VirF, as well as the virB-dependent virulence gene mxiC. Mutating key residues predicted to be important for DNA recognition had a significant negative effect, thereby suggesting that VirF interacts with its target sequence via two helix-turn-helix motifs. Two mutants that were dominant negative when coexpressed with the wild-type VirF protein were also isolated, indicating a role for protein-protein oligomerization in normal VirF function.