Prophage induction is enhanced and required for renal disease and lethality in an EHEC mouse model.

Enterohemorrhagic Escherichia coli (EHEC), particularly serotype O157:H7, causes hemorrhagic colitis, hemolytic uremic syndrome, and even death. In vitro studies showed that Shiga toxin 2 (Stx2), the primary virulence factor expressed by EDL933 (an O157:H7 strain), is encoded by the 933W prophage. And the bacterial subpopulation in which the 933W prophage is induced is the producer of Stx2. Using the germ-free mouse, we show the essential role 933W induction plays in the virulence of EDL933 infection. An EDL933 derivative with a single mutation in its 933W prophage, resulting specifically in that phage being uninducible, colonizes the intestines, but fails to cause any of the pathological changes seen with the parent strain. Hence, induction of the 933W prophage is the primary event leading to disease from EDL933 infection. We constructed a derivative of EDL933, SIVET, with a biosensor that specifically measures induction of the 933W prophage. Using this biosensor to measure 933W induction in germ-free mice, we found an increase three logs greater than was expected from in vitro results. Since the induced population produces and releases Stx2, this result indicates that an activity in the intestine increases Stx2 production.

Binding of Plasmodium merozoite proteins RON2 and AMA1 triggers commitment to invasion.

The commitment of Plasmodium merozoites to invade red blood cells (RBCs) is marked by the formation of a junction between the merozoite and the RBC and the coordinated induction of the parasitophorous vacuole. Despite its importance, the molecular events underlying the parasite's commitment to invasion are not well understood. Here we show that the interaction of two parasite proteins, RON2 and AMA1, known to be critical for invasion, is essential to trigger junction formation. Using antibodies (Abs) that bind near the hydrophobic pocket of AMA1 and AMA1 mutated in the pocket, we identified RON2's binding site on AMA1. Abs specific for the AMA1 pocket blocked junction formation and the induction of the parasitophorous vacuole. We also identified the critical residues in the RON2 peptide (previously shown to bind AMA1) that are required for binding to the AMA1 pocket, namely, two conserved, disulfide-linked cysteines. The RON2 peptide blocked junction formation but, unlike the AMA1-specific Ab, did not block formation of the parasitophorous vacuole, indicating that formation of the junction and parasitophorous vacuole are molecularly distinct steps in the invasion process. Collectively, these results identify the binding of RON2 to the hydrophobic pocket of AMA1 as the step that commits Plasmodium merozoites to RBC invasion and point to RON2 as a potential vaccine candidate.

The C-terminus of Toxoplasma RON2 provides the crucial link between AMA1 and the host-associated invasion complex.

Host cell invasion by apicomplexan parasites requires formation of the moving junction (MJ), a ring-like apposition between the parasite and host plasma membranes that the parasite migrates through during entry. The Toxoplasma MJ is a secreted complex including TgAMA1, a transmembrane protein on the parasite surface, and a complex of rhoptry neck proteins (TgRON2/4/5/8) described as host cell-associated. How these proteins connect the parasite and host cell has not previously been described. Here we show that TgRON2 localizes to the MJ and that two short segments flanking a hydrophobic stretch near its C-terminus (D3 and D4) independently associate with the ectodomain of TgAMA1. Pre-incubation of parasites with D3 (fused to glutathione S-transferase) dramatically reduces invasion but does not prevent injection of rhoptry bulb proteins. Hence, the entire C-terminal region of TgRON2 forms the crucial bridge between TgAMA1 and the rest of the MJ complex but this association is not required for rhoptry protein injection.

The moving junction protein RON8 facilitates firm attachment and host cell invasion in Toxoplasma gondii.

The apicomplexan moving junction (MJ) is a highly conserved structure formed during host cell entry that anchors the invading parasite to the host cell and serves as a molecular sieve of host membrane proteins that protects the parasitophorous vacuole from host lysosomal destruction. While recent work in Toxoplasma and Plasmodium has reinforced the composition of the MJ as an important association of rhoptry neck proteins (RONs) with micronemal AMA1, little is known of the precise role of RONs in the junction or how they are targeted to the neck subcompartment. We report the first functional analysis of a MJ/RON protein by disrupting RON8 in T. gondii. Parasites lacking RON8 are severely impaired in both attachment and invasion, indicating that RON8 enables the parasite to establish a firm clasp on the host cell and commit to invasion. The remaining junction components frequently drag in trails behind invading knockout parasites and illustrate a malformed complex without RON8. Complementation of Δron8 parasites restores invasion and reveals a processing event at the RON8 C-terminus. Replacement of an N-terminal region of RON8 with a mCherry reporter separates regions within RON8 that are necessary for rhoptry targeting and complex formation from those required for function during invasion. Finally, the invasion defects in Δron8 parasites seen in vitro translate to radically impaired virulence in infected mice, promoting a model in which RON8 has a crucial and unprecedented task in committing Toxoplasma to host cell entry.

Pathogenesis of renal disease due to enterohemorrhagic Escherichia coli in germ-free mice.

Enterohemorrhagic Escherichia coli (EHEC) is a food-borne pathogen that causes hemorrhagic colitis and acute renal failure. We used a germ-free mouse model to investigate the role of host factors, Shiga toxin 2 (Stx2), and bacterial strain in disease due to EHEC. Germ-free male and female Swiss-Webster mice that were 3 days to 12 weeks old were orally inoculated with 1 of 10 EHEC strains or derivatives of two of these strains with Stx2 deleted. All inoculated mice became infected regardless of the inoculum dose. All bacterial strains colonized the intestines, reaching levels of 10(9) to 10(12) CFU/g of feces by 4 days after inoculation. Seven of the 10 wild-type strains caused disease. However, the two Stx2 deletion mutants, unlike the Stx2(+) parental strains, did not cause disease. The clinical signs of disease in mice included lethargy, dehydration, polyuria, polydypsia, and death. Postmortem examination of affected mice revealed dehydration and luminal cecal fluid accumulation. Histologic examination revealed close adherence of bacteria to the intestinal epithelium in the ileum and cecum but not in the colon. Other lesions included progressive renal tubular necrosis, glomerular fibrin thrombosis, and red blood cell sludging. The severity of disease varied according to the bacterial strain and age, but not sex, of the host. This study demonstrated that EHEC colonizes germ-free mice in large numbers, adheres to the intestinal epithelium, and causes luminal cecal fluid accumulation and progressive renal failure. The disease in mice was Stx2 and bacterial strain dependent. This animal model should be a useful tool for studying the pathogenesis of renal disease secondary to EHEC infection.

Focus on the ringleader: the role of AMA1 in apicomplexan invasion and replication.

Apicomplexan parasites exhibit an unusual mechanism of host cell penetration. A central player in this process is the protein apical membrane antigen 1 (AMA1). Although essential for invasion, the precise functional roles AMA1 plays have been unclear. Several recent studies have provided important functional insight into its role within the multiprotein complex that comprises the moving junction (MJ). Initially formed at the apical tip of the invading parasite, the MJ represents a ring-like region of contact between the surfaces of the invading parasite and the host cell as the invaginated host plasma membrane is forced inward by the penetrating parasite. This review discusses these and other recent insights into AMA1 with particular emphasis on studies conducted in Plasmodium and Toxoplasma.

Characterization of a eukaryotic-like tyrosine protein kinase expressed by the Shiga toxin-encoding bacteriophage 933W.

The Shiga toxin (Stx)-encoding bacteriophage 933W contains an open reading frame, stk, with amino acid sequence similarity to the catalytic domain of eukaryotic serine/threonine (Ser/Thr) protein kinases (PKs). Eukaryotic PKs are related by a common catalytic domain, consisting of invariant and nearly invariant residues necessary for ATP binding and phosphotransfer. We demonstrate that rather than a Ser/Thr kinase, stk encodes a eukaryotic-like tyrosine (Tyr) kinase. An affinity-purified recombinant Stk (rStk) autophosphorylates and catalyzes the phosphorylation of an artificial substrate on Tyr residues and not on Ser or Thr residues. A change of an invariant lysine within the putative catalytic domain abolishes this kinase activity, indicating that Stk uses a phosphotransfer mechanism similar to the mechanism used by eukaryotic PKs. We provide evidence suggesting that stk is cotranscribed with cI from the phage promoter responsible for maintaining CI expression during lysogeny. The stk gene was identified in prophages obtained from independently isolated Stx-producing Escherichia coli clinical isolates, suggesting that selective pressure has maintained the stk gene in these pathogenic bacteria.

The operator and early promoter region of the Shiga toxin type 2-encoding bacteriophage 933W and control of toxin expression.

The genes encoding Shiga toxin (Stx), the major virulence factor of Shiga toxin-producing Escherichia coli, are carried in the genomes of bacteriophages that belong to the lambdoid family of phages. Previous studies demonstrated that induction of prophages encoding stx significantly enhances the production and/or release of Stx from the bacterium. Therefore, factors that regulate the switch between lysogeny and lytic growth, e.g., repressor, operator sites, and associated phage promoters, play important roles in regulating the production and/or release of Stx. We report the results of genetic and biochemical studies characterizing these elements of the Stx-encoding bacteriophage 933W. Like lambda, 933W has three operator repeats in the right operator region (OR), but unlike lambda and all other studied lambdoid phages, which have three operator repeats in the left operator region (OL), 933W only has two operator repeats in OL. As was observed with lambda, the 933W OR and OL regions regulate transcription from the early PR and PL promoters, respectively. A lysogen carrying a 933W derivative encoding a noncleavable repressor fails to produce Stx, unlike a lysogen carrying a 933W derivative encoding a cleavable repressor. This finding provides direct evidence that measurable expression of the stx genes encoded by a 933W prophage requires induction of that prophage with the concomitant initiation of phage gene expression.