Megan E. Porter

The Shigella virulence gene regulatory cascade: a paradigm of bacterial gene control mechanisms

Shigella flexneri is the causative agent of bacillary dysentery and is a facultative intracellular pathogen. Its virulence regulon is subject to tight control by several mechanisms involving the products of over 20 genes and an array of environmental signals. The regulon is carried on a plasmid that is prone to instability and to integration into the chromosome, with associated silencing of the virulence genes. Closely related regulons are found in other species of Shigella and in enteroinvasive Escherichia coli. A wealth of detailed information is now available on the Shigella virulence gene control circuits, and it is becoming clear that these share many features with regulatory systems found in other bacterial pathogens. All of this makes the S. flexneri virulence gene control system a very attractive topic for those interested in the nature of gene regulatory networks in bacteria.

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.

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.

Co‐ordinate single‐cell expression of LEE4‐ and LEE5‐encoded proteins of Escherichia coli O157:H7

Escherichia coli O157:H7 is a zoonotic pathogen that can express a type III secretion system (TTSS) considered important for colonization and persistence in ruminants. E. coli O157:H7 strains have been shown to vary markedly in levels of protein secreted using the TTSS and this study has confirmed that a high secretion phenotype is more prevalent among isolates associated with human disease than isolates shed by healthy cattle. The variation in secretion levels is a consequence of heterogeneous expression, being dependent on the proportion of bacteria in a population that are actively engaged in protein secretion. This was demonstrated by indirect immunofluorescence and eGFP fusions that examined the expression of locus of enterocyte effacement (LEE)‐encoded factors in individual bacteria. In liquid media, the expression of EspA, tir::egfp, intimin, but not map::egfp were co‐ordinated in a subpopulation of bacteria. In contrast to E. coli O157:H7, expression of tir::egfp in EPEC E2348/69 was equivalent in all bacteria although the same fusion exhibited variable expression when transformed into an E. coli O157:H7 background. An E. coli O157:H7 strain deleted for the LEE demonstrated weak but variable expression of tir::egfp indicating that the elements controlling the heterogeneous expression lie outside the LEE. The research also demonstrated the rapid induction of tir::egfp and map::egfp on contact with bovine epithelial cells. This control in E. coli O157:H7 may be required to limit exposure of key surface antigens, EspA, Tir and intimin during colonization of cattle but allow their rapid production on contact with bovine gastrointestinal epithelium at the terminal rectum.

DIFFERENTIAL REGULATION OF THE PLASMID-ENCODED GENES IN THE SHIGELLA FLEXNERI VIRULENCE REGULON

Abstract Expression of the Shigella flexneri virulence gene regulon is controlled by multiple environmental signals acting through a regulatory cascade. The primary regulator is VirF, which is a positive regulator of the secondary regulatory gene virB and the structural gene icsA. The product of the virB gene in turn activates transcription of the genes coding for the invasion proteins, and for the type III secretion system which promotes export of the invasion proteins to the bacterial cell surface. The genes making up the regulon were studied in their native locations on the 230-kb virulence plasmid. Transcriptional control was detected at each level of the regulatory cascade. A gearing effect was detected upon thermal induction of transcription in the regulon, with the virF gene being induced by about two fold, virB by 10-fold and the structural genes by 100-fold. In addition, each gene studied displayed individual characteristics in its response to stimuli such as growth medium osmolarity, pH, variations in DNA superhelicity and the presence or absence of H-NS. The primary regulatory gene virF, displayed loose regulation under standard laboratory growth conditions. Regulation was tighter at the secondary regulator virB, while control of structural gene expression was tighter still. It is proposed that this regulatory pattern ensures that energetically wasteful expression of the structural genes under inappropriate conditions is avoided while allowing the regulatory genes to be expressed sufficiently under non-permissive conditions to ensure a rapid response to inducing conditions when these arise. Once induced, fine tuning of the response can be achieved through the different sensitivities of the individual regulon members to external stimuli.

Requirement for the molecular adapter function of StpA at the Escherichia coli bgl promoter depends upon the level of truncated H-NS protein.

Truncated derivatives of the Escherichia coli nucleoid‐associated protein H‐NS that lack the DNA‐binding domain remain competent for silencing of the cryptic bgl operon in vivo. Previous studies have provided evidence for the involvement of either the homologous nucleoid protein StpA or the alternative sigma factor RpoS in this unusual silencing mechanism. Here, we rationalize this apparent discrepancy. We show that two hns alleles (hns‐205::Tn10 and hns60), which produce virtually identical amino‐terminal fragments of H‐NS, have very different requirements for StpA to mediate bgl silencing. The hns60 allele produces a high level of truncated H‐NS, which can overcome the absence of StpA, whereas the lower level expressed by hns‐205::Tn10 requires StpA for silencing. Reversing the relative levels of the two H‐NS fragments reverses their requirement for StpA to silence bgl transcription. This suggests that the amino‐terminal fragment of H‐NS can be targeted to DNA to mediate silencing by multiple protein–protein interactions. The high‐specificity interaction with StpA can function at low levels of truncated H‐NS, whereas an alternative mechanism, perhaps involving lower specificity interactions with another protein(s), is only functional when truncated H‐NS is abundant. These findings have important implications for the involvement of other proteins in H‐NS‐dependent transcriptional repression.

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.

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

Abstract The DNA polymerase δ 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 Polδ 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.

A Novel Lawsonia intracellularis Autotransporter Protein Is a Prominent Antigen

ABSTRACT Investigation of antigenic determinants of the microaerophilic obligate intracellular bacterium Lawsonia intracellularis using a mass spectrometry approach identified a novel bacterial protein present in an extract of cell culture medium aspirated from heavily infected in vitro cell cultures. Western immunoblotting analysis of SDS-PAGE-resolved proteins using immune sera pooled from L. intracellularis-infected pigs revealed the presence of a strongly immunoreactive band of ∼72 kDa. Liquid chromatography-electrospray ionization-tandem mass spectrometry analysis of this component and database mining using a fully annotated L. intracellularis genome sequence and the comprehensive GenBank prokaryotic genomic database highlighted the presence of a protein that shares little sequence similarity with other prokaryotic proteins and appears to be highly species specific. Detailed bioinformatic analyses identified the protein as member of the autotransporter protein family of surface-exposed proteins, and the designation LatA (Lawsonia autotransporter protein A) is suggested. Recognition of recombinant LatA on Western blots by a panel of sera from infected and control pigs corresponded 100% with a commercial serodiagnostic that relies on in vitro culture of this fastidious organism. LatA therefore represents a potential candidate for the development of a rapid and species-specific serodiagnostic reagent.

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.