Analysis of Dengue Virus Genetic Diversity during Human and Mosquito Infection Reveals Genetic Constraints.

Dengue viruses (DENV) cause debilitating and potentially life-threatening acute disease throughout the tropical world. While drug development efforts are underway, there are concerns that resistant strains will emerge rapidly. Indeed, antiviral drugs that target even conserved regions in other RNA viruses lose efficacy over time as the virus mutates. Here, we sought to determine if there are regions in the DENV genome that are not only evolutionarily conserved but genetically constrained in their ability to mutate and could hence serve as better antiviral targets. High-throughput sequencing of DENV-1 genome directly from twelve, paired dengue patients' sera and then passaging these sera into the two primary mosquito vectors showed consistent and distinct sequence changes during infection. In particular, two residues in the NS5 protein coding sequence appear to be specifically acquired during infection in Ae. aegypti but not Ae. albopictus. Importantly, we identified a region within the NS3 protein coding sequence that is refractory to mutation during human and mosquito infection. Collectively, these findings provide fresh insights into antiviral targets and could serve as an approach to defining evolutionarily constrained regions for therapeutic targeting in other RNA viruses.

The C-terminal 50 amino acid residues of dengue NS3 protein are important for NS3-NS5 interaction and viral replication.

Dengue virus multifunctional proteins NS3 protease/helicase and NS5 methyltransferase/RNA-dependent RNA polymerase form part of the viral replication complex and are involved in viral RNA genome synthesis, methylation of the 5'-cap of viral genome, and polyprotein processing among other activities. Previous studies have shown that NS5 residue Lys-330 is required for interaction between NS3 and NS5. Here, we show by competitive NS3-NS5 interaction ELISA that the NS3 peptide spanning residues 566-585 disrupts NS3-NS5 interaction but not the null-peptide bearing the N570A mutation. Small angle x-ray scattering study on NS3(172-618) helicase and covalently linked NS3(172-618)-NS5(320-341) reveals a rigid and compact formation of the latter, indicating that peptide NS5(320-341) engages in specific and discrete interaction with NS3. Significantly, NS3:Asn-570 to alanine mutation introduced into an infectious DENV2 cDNA clone did not yield detectable virus by plaque assay even though intracellular double-stranded RNA was detected by immunofluorescence. Detection of increased negative-strand RNA synthesis by real time RT-PCR for the NS3:N570A mutant suggests that NS3-NS5 interaction plays an important role in the balanced synthesis of positive- and negative-strand RNA for robust viral replication. Dengue virus infection has become a global concern, and the lack of safe vaccines or antiviral treatments urgently needs to be addressed. NS3 and NS5 are highly conserved among the four serotypes, and the protein sequence around the pinpointed amino acids from the NS3 and NS5 regions are also conserved. The identification of the functionally essential interaction between the two proteins by biochemical and reverse genetics methods paves the way for rational drug design efforts to inhibit viral RNA synthesis.

Flexibility between the protease and helicase domains of the dengue virus NS3 protein conferred by the linker region and its functional implications.

The dengue virus (DENV) NS3 protein is essential for viral polyprotein processing and RNA replication. It contains an N-terminal serine protease region (residues 1-168) joined to an RNA helicase (residues 180-618) by an 11-amino acid linker (169-179). The structure at 3.15 A of the soluble NS3 protein from DENV4 covalently attached to 18 residues of the NS2B cofactor region (NS2B(18)NS3) revealed an elongated molecule with the protease domain abutting subdomains I and II of the helicase (Luo, D., Xu, T., Hunke, C., Grüber, G., Vasudevan, S. G., and Lescar, J. (2008) J. Virol. 82, 173-183). Unexpectedly, using similar crystal growth conditions, we observed an alternative conformation where the protease domain has rotated by approximately 161 degrees with respect to the helicase domain. We report this new crystal structure bound to ADP-Mn(2+) refined to a resolution of 2.2 A. The biological significance for interdomain flexibility conferred by the linker region was probed by either inserting a Gly residue between Glu(173) and Pro(174) or replacing Pro(174) with a Gly residue. Both mutations resulted in significantly lower ATPase and helicase activities. We next increased flexibility in the linker by introducing a Pro(176) to Gly mutation in a DENV2 replicon system. A 70% reduction in luciferase reporter signal and a similar reduction in the level of viral RNA synthesis were observed. Our results indicate that the linker region has evolved to an optimum length to confer flexibility to the NS3 protein that is required both for polyprotein processing and RNA replication.

Tir phosphorylation and Nck/N-WASP recruitment by enteropathogenic and enterohaemorrhagic Escherichia coli during ex vivo colonization of human intestinal mucosa is different to cell culture models.

Tir, the translocated intimin receptor of enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC) and Citrobacter rodentium, is translocated into the host cell by a filamentous type III secretion system. Epithelial cell culture has demonstrated that Tir tyrosine phosphorylation is necessary for attaching effacing (A/E) lesion formation by EPEC and C. rodentium, but is not required by EHEC O157:H7. Recent in vivo work on C. rodentium has reported that Tir translocation, but not its phosphorylation, is necessary for colonization of the mouse colon. In this study we investigated the involvement of Tir and its tyrosine phosphorylation in EPEC and EHEC human intestinal colonization, N-WASP accumulation and F-actin recruitment using in vitro organ culture (IVOC). We showed that both EPEC and EHEC Tir are translocated into human intestinal epithelium during IVOC and that Tir is necessary for ex vivo intestinal colonization by both EPEC and EHEC. EPEC, but not EHEC, Tir is tyrosine phosphorylated but Tir phosphorylation-deficient mutants still colonize intestinal explants. While EPEC Tir recruits the host adaptor protein Nck to initiate N-WASP-Arp2/3-mediated actin polymerization, Tir derivatives deficient in tyrosine phosphorylation recruit N-WASP independently of Nck indicating the presence of a tyrosine phosphorylation-independent mechanism of A/E lesion formation and actin recruitment ex vivo by EPEC in man.

Human intestinal tissue tropism in Escherichia coli O157 : H7--initial colonization of terminal ileum and Peyer's patches and minimal colonic adhesion ex vivo.

Enterohaemorrhagic Escherichia coli (EHEC) are an important cause of diarrhoeal and renal disease in man. Studies of a single prototypic O157 : H7 strain have shown tropism for follicle-associated epithelium (FAE) of distal ileal Peyer's patches without colonization of either small or large intestine. This study determined tropism in a range of Shiga toxin (Stx)-negative EHEC strains and looked for factors that might induce colonic colonization using human in vitro intestinal organ culture (IVOC). An FAE-restricted colonization was confirmed in two strains; four strains additionally colonized ileal villous surfaces, and adhesion to proximal small intestinal FAE was observed. All strains showed minimal adhesion to non-FAE regions of proximal small intestinal and to the transverse colon. Extensive large-bowel IVOC studies using three O157 : H7 strains, an O26 : H11 and an O103 : H2 strain, and tissue from caecum to rectum found colonization and attaching/effacing lesion formation in only 4 of 113 (3.5 %) IVOCs. Colonic adhesion was not enhanced by altering the IVOC technique or environment. Co-incubation of O157 : H7-infected ileal FAE with colonic samples enhanced colonic colonization, producing a novel, non-intimate adhesive phenotype. Thus, in the initial stages of colonization Stx-negative EHEC preferentially infect FAE and villi of the terminal ileal region ex vivo; colonic colonization is infrequently observed as an initial event but may represent a subsequent stage of infection.

Long polar fimbriae and tissue tropism in Escherichia coli O157:H7.

In vitro organ culture has demonstrated the human intestinal tropism of enterohaemorrhagic Escherichia coli O157:H7 for follicle associated epithelium overlying Peyer's patches of the terminal ileum. Long polar (LP) fimbriae are considered to mediate the attachment of Salmonella enterica serovar Typhimurium to Peyer's patch epithelium and, as homologous genes have been identified in O157:H7, we hypothesised that LP fimbriae in O157:H7 may perform the same function. However, mutation of LP fimbriae in O157:H7 strain 85/170 resulted in the novel phenotype of proximal and distal small intestinal colonisation with attaching/effacing lesion formation, while retaining adhesion to follicle associated epithelium. Application of whole genome DNA array technology did not identify changes in known fimbrial genes that could explain the change in tropism, but highlighted several genes that require further investigation. LP fimbrial genes are the first genes to be identified outside the locus of enterocyte effacement pathogenicity island that influence O157:H7 human intestinal tissue tropism.

The lpf gene cluster for long polar fimbriae is not involved in adherence of enteropathogenic Escherichia coli or virulence of Citrobacter rodentium.

Using the enteropathogenic Escherichia coli (EPEC) genome sequence, we found that EPEC E2348/69 has an lpfABCDE gene cluster homologous (about 60% identical at the protein level) to the Salmonella long polar fimbria (LPF) operon. To determine whether this operon is essential for adherence, the lpfABCD(E2)(3) genes were deleted from EPEC strain E2348/69 by allelic exchange. Analysis of the resulting EPECDeltalpfABCD(E23) strain showed no change in adherence to HeLa cells or to human intestinal biopsy cells in the in vitro organ culture (IVOC) system compared to the wild type. Sera from volunteers experimentally infected with E2348/69 showed no antibody response to the major subunit protein, LpfA. These results suggested that the lpf(E23) gene cluster is not necessary for EPEC adherence and attaching/effacing (A/E) lesion formation on human biopsy samples and is not expressed during human infection. We also identified an lpf gene cluster in Citrobacter rodentium strain ICC168 (lpf(cr)). A DeltalpfA(cr) mutant of ICC168 retained wild-type adherence and A/E lesion-forming activity on HeLa cells. C3H/HeJ mice were infected with a wild-type C. rodentium strain and its lpfA(cr) isogenic mutant. Both strains were recovered at high levels in stools, and there were no significant differences between the groups both in terms of the number of CFU/organ (colon and cecum) and in terms of the amount of hyperplasia, as measured by weight. Similar results were observed in a second mouse strain, C57BL/6. These data suggest that in addition to playing no apparent role in EPEC pathogenesis, lpf(cr) is not required for C. rodentium virulence in either the C3H/HeJ or C57BL/6 mouse model.

Distribution of tccP in clinical enterohemorrhagic and enteropathogenic Escherichia coli isolates.

Enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) are diarrheagenic pathogens that colonize the gut through the formation of attaching and effacing lesions, which depend on the translocation of effector proteins via a locus of enterocyte effacement-encoded type III secretion system. Recently, two effector proteins, EspJ and TccP, which are encoded by adjacent genes on prophage CP-933U in EHEC O157:H7, have been identified. TccP consists of a unique N-terminus region and several proline-rich domains. In this project we determined the distribution of tccP in O157:H7, in non-O157 EHEC, and in typical and atypical EPEC isolates. All the EHEC O157:H7 strains tested were tccP(+). Unexpectedly, tccP was also found in non-O157 EHEC, and in typical and atypical EPEC isolates, particularly in strains belonging to serogroups O26 (EHEC), O119 (typical EPEC), and O55 (atypical EPEC). We recorded some variation in the length of tccP, which reflects diversity in the number of the proline-rich repeats. These results show the existence of a class of "attaching and effacing" pathogens which express a combination of EPEC and EHEC virulence determinants.

EspJ is a prophage-carried type III effector protein of attaching and effacing pathogens that modulates infection dynamics.

Enterohemorrhagic Escherichia coli, enteropathogenic E. coli, and Citrobacter rodentium are highly adapted enteropathogens that successfully colonize their host's gastrointestinal tract via the formation of attaching and effacing (A/E) lesions. These pathogens utilize a type III secretion system (TTSS) apparatus, encoded by the locus of enterocyte effacement, to translocate bacterial effector proteins into epithelial cells. Here, we report the identification of EspJ (E. coli-secreted protein J), a translocated TTSS effector that is carried on the 5' end of the cryptic prophage CP-933U. Infection of epithelial cells in culture revealed that EspJ is not required for A/E lesion activity in vivo and ex vivo. However, in vivo studies performed with mice demonstrated that EspJ possesses properties that influence the dynamics of clearance of the pathogen from the host's intestinal tract, suggesting a role in host survival and pathogen transmission.

Distribution of tccP in clinical enterohemorrhagic and enteropathogenic Escherichia coli isolates

Enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) are diarrheagenic pathogens that colonize the gut through the formation of attaching and effacing lesions, which depend on the translocation of effector proteins via a locus of enterocyte effacement-encoded type III secretion system. Recently, two effector proteins, EspJ and TccP, which are encoded by adjacent genes on prophage CP-933U in EHEC O157:H7, have been identified. TccP consists of a unique N-terminus region and several proline-rich domains. In this project we determined the distribution of tccP in O157:H7, in non-O157 EHEC, and in typical and atypical EPEC isolates. All the EHEC O157:H7 strains tested were tccP+. Unexpectedly, tccP was also found in non-O157 EHEC, and in typical and atypical EPEC isolates, particularly in strains belonging to serogroups O26 (EHEC), O119 (typical EPEC), and O55 (atypical EPEC). We recorded some variation in the length of tccP, which reflects diversity in the number of the proline-rich repeats. These results show the existence of a class of “attaching and effacing” pathogens which express a combination of EPEC and EHEC virulence determinants.

TccP is an enterohaemorrhagic Escherichia coli O157:H7 type III effector protein that couples Tir to the actin-cytoskeleton.

Subversion of host cell actin microfilaments is the hallmark of enterohaemorrhagic (EHEC) and enteropathogenic (EPEC) Escherichia coli infections. Both pathogens translocate the trans-membrane receptor protein-translocated intimin receptor (Tir), which links the extracellular bacterium to the cell cytoskeleton. While both converge on neural Wiskott-Aldrich syndrome protein (N-WASP), Tir-mediated actin accretion by EPEC and EHEC differ in that Tir(EPEC) requires both tyrosine phosphorylation and the host adaptor protein Nck, whereas Tir(EHEC) is not phosphorylated and utilizes an unidentified linker. Here we report the identification of Tir-cytoskeleton coupling protein (TccP), a novel EHEC effector that displays an Nck-like coupling activity following translocation into host cells. A tccP mutant did not affect Tir translocation and focusing but failed to recruit alpha-actinin, Arp3, N-WASP and actin to the site of bacterial adhesion. When expressed in EPEC, bacterial-derived TccP restored actin polymerization activity following infection of an Nck-deficient cell line. TccP has a similar biological activity on infected human intestinal explants ex vivo. Purified TccP activates N-WASP stimulating, in the presence of Arp2/3, actin polymerization in vitro. These results show that EHEC translocates both its own receptor (Tir) and an Nck-like protein (TccP) to facilitate actin polymerization.