David George Emslie Smith

Lawsonia intracellularis : getting inside the pathogenesis of proliferative enteropathy

Although proliferative enteropathy (PE) has been recognised for several decades, Lawsonia intracellularis, the aetiological agent, was identified formally in only 1995. This organism is both highly fastidious and obligately intracellular bacterium, characteristics which have inevitably restricted investigations in all aspects of its biology. Despite these limitations, advances have been made in characterising and understanding L. intracellularis-host interaction both in vivo and in vitro. Based upon evidence provided by mainly pathological and histological investigations conducted to date, we review salient features of our current understanding of processes involved throughout the course of infection by this unique pathogen.

Verotoxin 1 binding to intestinal crypt epithelial cells results in localization to lysosomes and abrogation of toxicity

Verotoxins (VTs) are important virulence factors of enterohaemorrhagic Escherichia coli (EHEC), a group of bacteria associated with severe disease sequelae in humans. The potent cytotoxic activity of VTs is important in pathogenicity, resulting in the death of cells expressing receptor Gb3 (globotriaosylceramide). EHEC, particularly serotype O157:H7, frequently colonize reservoir hosts (such as cattle) in the absence of disease, however, the basis to avirulence in this host has been unclear. The objective of this study was assessment of interaction between VT and intestinal epithelium, which represents the major interface between the host and enteric organisms. Bovine intestinal epithelial cells expressed Gb3 in vitro in primary cell cultures, localizing specifically to proliferating crypt cells in corroboration with in situ immunohistological observations on intestinal mucosa. Expression of receptor by these cells contrasts with the absence of Gb3 on human intestinal epithelium in vivo. Despite receptor expression, VT exhibited no cytotoxic activity against bovine epithelial cells. Sub‐cellular localization of VT indicated that this toxin was excluded from endoplasmic reticulum but localized to lysosomes, corresponding with abrogation of cytotoxicity. VT intracellular trafficking was unaffected by treatment of primary cell cultures with methyl‐β‐cyclodextrin, indicating that Gb3 in these cells is not associated with lipid rafts but is randomly distributed in the membrane. The combination of Gb3 isoform, membrane distribution and VT trafficking correlate with observations of other receptor‐positive cells that resist verocytotoxicity. These studies demonstrate that intestinal epithelium is an important determinant in VT interaction with major implications for the differential consequences of EHEC infection in reservoir hosts and humans.

Heterogeneous surface expression of EspA translocon filaments by Escherichia coli O157:H7 is controlled at the posttranscriptional level

ABSTRACT Type III secretion systems of enteric bacteria enable translocation of effector proteins into host cells. Secreted proteins of verotoxigenic Escherichia coli O157 strains include components of a translocation apparatus, EspA, -B, and -D, as well as “effectors” such as the translocated intimin receptor (Tir) and the mitochondrion-associated protein (Map). This research has investigated the regulation of LEE4 translocon proteins, in particular EspA. EspA filaments could not be detected on the bacterial cell surface when E. coli O157:H7 was cultured in M9 minimal medium but were expressed from only a proportion of the bacterial population when cultured in minimal essential medium modified with 25 mM HEPES. The highest proportions of EspA-filamented bacteria were detected in late exponential phase, after which filaments were lost rapidly from the bacterial cell surface. Our previous research had shown that human and bovine E. coli O157:H7 strains exhibit marked differences in EspD secretion levels. Here it is demonstrated that the proportion of the bacterial population expressing EspA filaments was associated with the level of EspD secretion. The ability of individual bacteria to express EspA filaments was not controlled at the level of LEE1-4 operon transcription, as demonstrated by using both β-galactosidase and green fluorescent protein (GFP) promoter fusions. All bacteria, whether expressing EspA filaments or not, showed equivalent levels of GFP expression when LEE1-4 translational fusions were used. Despite this, the LEE4-espADB mRNA was more abundant from populations with a high proportion of nonsecreting bacteria (low secretors) than from populations with a high proportion of secreting and therefore filamented bacteria (high secretors). This research demonstrates that while specific environmental conditions are required to induce LEE1-4 expression, a further checkpoint exists before EspA filaments are produced on the bacterial surface and secretion of effector proteins occurs. This checkpoint in E. coli O157:H7 translocon expression is controlled by a posttranscriptional mechanism acting on LEE4-espADB mRNA. The heterogeneity in EspA filamentation could arise from phase-variable expression of regulators that control this posttranscriptional mechanism.

Analysis of type 1 fimbriae expression in verotoxigenic Escherichia coli: a comparison between serotypes O157 and O26.

Previous research has shown that verotoxin-producing Escherichia coli (VTEC) O157 strains appear unable to express type 1 fimbriae although other serotypes such as O26 and O118 can. This study has investigated the molecular basis of this difference. The study confirmed the presence of a 16 bp deletion within the regulatory region of fimA (fim switch) in 63 VTEC O157 strains but not in other VTEC serotypes tested. The fim switch was shown to be detectable only in the phase off orientation in VTEC O157, but detection of the switch in the phase on orientation correlated with the degree of mannose-sensitive yeast agglutination in VTEC O26. Repair of the 16 bp deletion in the VTEC O157 fim switch region restored phase-variable expression of fimA in a permissive background. Non-O157 VTEC, especially O26 and O118, can be pathogenic in cattle; the role of type 1 fimbriae in this and colonization is discussed.

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.

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.

Infection of cultured rat enterocytes by Ileal symbiont intracellularis depends on host cell function and actin polymerisation.

The mechanisms of entry of Ileal symbiont intracellularis into IEC-18 rat enterocyte cells and subsequent bacterial proliferation were examined in centrifuge-assisted and static infections. Live, oxygen or neomycin damaged, and formalin killed bacteria, each rapidly entered viable cells. Live or damaged bacteria did not enter cells nor proliferate within cells after static infection of cells cooled to 5 degrees C. Infection of cells was greatly reduced at 20 degrees or 32 degrees compared to infection at 37 degrees C. Centrifuge-assisted infection was also reduced by chilling the cells. Cytochalasin D but not B inhibited the entry process indicating an actin-dependent infection, although other pathways may also be involved in centrifuge-assisted infections. Drugs capable of modifying cell membrane charge, heparin receptors or trypsin-labile proteins were all inactive in preventing or enhancing infection. We therefore conclude that infection of enterocytes by IS intracellularis is dependent on host cell activity and actin polymerization, but is independent of bacterial viability.

Consequences of EHEC colonisation in humans and cattle

While many factors have been associated with human EHEC infection, the full role these play in both human and ruminant hosts are not yet clear despite much investigation. It is hoped that the continued intense international research effort into EHEC will provide further insights into the commensal versus pathogenic lifestyles of E. coli and lead to approaches to reduce EHEC carriage in ruminants as well as prevent or treat human disease.

In-vitro interactions of Lawsonia intracellularis with cultured enterocytes

Strains of the obligately intracellular bacterium Lawsonia intracellularis, the etiologic agent of porcine proliferative enteropathy, were co-cultured in rat enterocyte cell cultures (IEC-18) and examined ultrastructurally. No regular surface arrays typical of surface or S-layers were visible on any bacterial strain, with or without Triton-X-100 detergent treatment. In separate experiments, there was no difference in the ability of L. intracellularis to attach and enter enterocytes with or without the presence of added bovine plasma fibronectin, or the peptide Arg-Gly-Ser. Interestingly, there was an increase in the invasiveness of L. intracellularis in the presence of the peptide Arg-Gly-Asp (RGD), in a dose-related manner. A reduction was observed in the ability of L. intracellularis to invade enterocytes in the presence of monovalent fragments of IgG monoclonal antibodies to an outer surface component of L. intracellularis. This neutralization showed an antibody concentration-dependent titration effect and was not apparent with co-cultures incorporating control antibodies. The exact nature of ligand and cell receptor interactions for L. intracellularis remain to be determined.

IgG Antibodies to Gram-negative endotoxin in human sera. I. Lipopolysaccharide (LPS) cross-reactivity due to antibodies to LPS core

A series of 700 blood donor sera were screened for IgG antibodies to the core of Gram-negative bacterial endotoxin with a quantitative enzyme-linked immunosorbent-assay (ELISA), based on a cocktail of incomplete-core R-LPS from four different Gram-negative bacterial species, and further serum samples were obtained from donors exhibiting a range of different reactivity for isolation of serum IgG. Analysis of the different IgG samples by ELISA employing a panel of individual LPS from 31 different Gram-negative bacteria covering a range of species, serotypes and R-LPS chemotyes showed that high-titer sera from the screening ELISA expressed IgG with multiple reactivity to LPS in the complex ELISA. We investigated this multiple reactivity in three serum IgGs by inhibition and absorption of isolated serum IgG ELISA reactivity to R-LPS, employing purified LPS and whole bacteria respectively. In two cases the ELISA reactivity appeared to be predominantly attributable to a single antibody component directed to the inner LPS core structure in the lipid A to KDO region. For the third serum IgG, the results suggested that the cross-reactivity may be attributable to more than one specificity-group of cross-reactive antibodies, although still restricted to the LPS inner core structures.