Jean-Michel Thiberge

Chikungunya virus, southeastern France.

In September 2010, autochthonous transmission of chikungunya virus was recorded in southeastern France, where the Aedes albopictus mosquito vector is present. Sequence analysis of the viral genomes of imported and autochthonous isolates indicated new features for the potential emergence and spread of the virus in Europe.

The Peopling of the Pacific from a Bacterial Perspective

Two prehistoric migrations peopled the Pacific. One reached New Guinea and Australia, and a second, more recent, migration extended through Melanesia and from there to the Polynesian islands. These migrations were accompanied by two distinct populations of the specific human pathogen Helicobacter pylori, called hpSahul and hspMaori, respectively. hpSahul split from Asian populations of H. pylori 31,000 to 37,000 years ago, in concordance with archaeological history. The hpSahul populations in New Guinea and Australia have diverged sufficiently to indicate that they have remained isolated for the past 23,000 to 32,000 years. The second human expansion from Taiwan 5000 years ago dispersed one of several subgroups of the Austronesian language family along with one of several hspMaori clades into Melanesia and Polynesia, where both language and parasite have continued to diverge.

Essential role of Helicobacter pylori gamma-glutamyltranspeptidase for the colonization of the gastric mucosa of mice.

Constitutive expression of γ‐glutamyltranspeptidase (GGT) activity is common to all Helicobacter pylori strains, and is used as a marker for identifying H. pylori isolates. Helicobacter pylori GGT was purified from sonicated extracts of H. pylori strain 85P by anion exchange chromatography. The N‐terminal amino acid sequences of two of the generated endoproteolysed peptides were determined, allowing the cloning and sequencing of the corresponding gene from a genomic H. pylori library. The H. pylori ggt gene consists of a 1681 basepair (bp) open reading frame encoding a protein with a signal sequence and a calculated molecular mass of 61 kDa. Escherichia coli clones harbouring the H. pylori ggt gene exhibited GGT activity at 37°C, in contrast to E. coli host cells (MC1061, HB101), which were GGT negative at 37°C. GGT activity was found to be constitutively expressed by similar genes in Helicobacter felis, Helicobacter canis, Helicobacter bilis, Helicobacter hepaticus and Helicobacter mustelae. Western immunoblots using rabbit antibodies raised against a His‐tagged‐GGT recombinant protein demonstrated that H. pylori GGT is synthesized in both H. pylori and E. coli as a pro‐GGT that is processed into a large and a small subunit. Deletion of a 700 bp fragment within the GGT‐encoding gene of a mouse‐adapted H. pylori strain (SS1) resulted in mutants that were GGT negative yet grew normally in vitro. These mutants, however, were unable to colonize the gastric mucosa of mice when orally administered alone or together (co‐infection) with the parental strain. These results demonstrate that H. pylori GGT activity has an essential role for the establishment of the infection in the mouse model, demonstrating for the first time a physiological role for a bacterial GGT enzyme.

Characterization of the roles of NikR, a nickel-responsive pleiotropic autoregulator of Helicobacter pylori.

The Helicobacter pylori genome contains a gene (hp1338 or nikR) that encodes a nickel‐dependent regulator that is homologous to the Escherichia coli nickel‐responsive regulator, NikR. The H. pylori nikR product acts as a pleiotropic metal‐dependent regulator. We constructed a non‐polar isogenic mutant deleted for the nikR gene. NikR was essential for the survival of H. pylori in the presence of high nickel and cobalt ion concentrations in vitro. We screened a DNA macroarray for genes that were differentially expressed in parental and nikR‐deficient H. pylori strains grown in the presence of excess nickel. We found that H. pylori NikR mediates the expression of nickel‐activated and ‐repressed genes. In the presence of excess nickel, NikR activated the transcription of ureA‐ureB (hp72–73), nixA (hp1077 ), copA2 (hp1072), hpn (hp1427 ) and hpn‐like (hp1432) genes and repressed the expression of genes encoding proteins involved in ferric iron uptake and storage [pfr (hp0653), fur (hp1027 ), frpB4 (hp1512), exbB/exbD (hp1339–1340), ceuE (hp1561)], motility [cheV (hp616), flaA (hp0601), flaB (hp0115 )], stress responses [hrcA‐grpE‐dnaK (hp111–110–109)] and encoding outer‐membrane proteins [omp11(hp0472), omp31 (hp1469), omp32 (hp1501)]. Slot blot DNA/RNA hybridization experiments using RNA from three independent bacterial cultures confirmed the transcriptome data for 10 selected genes. The results of gel shift experiments using purified native NikR, β‐galactosidase assays with the region between nikR and the exbB/exbD divergent operon, and the study of exbB gene expression using a gentamicin/apramycin reporter gene in H. pylori indicated that NikR is an autorepressor that binds to this intergenic region and also controls the expression of the exbB/exbD/tonB operon, which provides energy for ferric iron uptake. Thus, as previously suggested for Fur in H. pylori, NikR appears to be a global regulator of the metabolism of some divalent cations within a highly complex regulated network.

Helicobacter pylori hspA‐hspB heat‐shock gene cluster: nucleotide sequence, expression, putative function and immunogenicity

All Helicobacter pylori isolates synthesize a 54 kDa immunodominant protein that was reported to be associated with the nickel‐dependent urease of H. pylori. This protein was recently recognized as a homologue of the heat‐shock protein of the GroEL class. The gene encoding the GroEL‐like protein of H. pylori (HspB) was cloned (plLL689) and was shown to belong to a bicistronic operon including the hspA and hspB genes. In Escherichia coli. the constitutive expression of the hspA and hspB genes was initiated from a promoter located within an IS5 insertion element that mapped upstream to the two open reading frames (ORFs). IS5 was absent from the H. pylori genome, and was thus acquired during the cosmid cloning process. hspA and hspB encoded polypeptides of 118 and 545 amino acid residues, corresponding to calculated molecular masses of 13.0 and 58.2 kDa, respectively. Amino acid sequence comparison studies revealed that, although H. pylori HspA and HspB proteins were highly similar to their bacterial homologues, the H. pylori HspA featured a striking motif at the C‐terminus. This unique motif consists of a series of cysteine and histidine residues resembling a nickel‐binding domain, which is not present in any of the other bacterial GroES homologues so far characterized. When the plLL689 recombinant plasmid was introduced together with the H. pylori urease gene cluster (plLL763) into an E. coli host strain, an increase of urease activity was observed. This suggested a close interaction between the HspA and HspB proteins and the urease enzyme, and a possible role for HspA in ihe chelation of nickel ions. The genes encoding each of the HspA and HspB polypeptides were cloned, expressed independently as proteins fused to the maltose‐binding protein (WIBP) and purified in large scale. The MBP‐HspA and MBP‐HspB fusion proteins were shown to retain their antigenic properties. Both HspA and HspB represent antigens that are specifically recognized by the sera from H. pylori‐infected patients. Whereas HspB was known to be immunogenic in humans, this is the first demonstration that HspA per se is also immunogenic as proteins fused to the maltose‐binding protein (WIBP) and purified in large scale. The MBP‐HspA and WlBP‐HspB fusion proteins were shown to retain their antigenic properties. Both HspA and HspB represent antigens that are specifically recognized by the sera from H, py/or/‐infected patients. Whereas HspB was known to be immunogenic in humans, this is the first demonstration that HspA per se is also immunogenic.

Reduced activation of inflammatory responses in host cells by mouse‐adapted Helicobacter pylori isolates

Helicobacter pylori strains that harbour the Cag pathogenicity island (Cag PAI) induce interleukin (IL)‐8 secretion in gastric epithelial cells, via the activation of NF‐κB, and are associated with severe inflammation in humans. To investigate the influence of Cag PAI‐mediated inflammatory responses on H. pylori adaptation to mice, a selection of H. pylori clinical isolates (n= 12) was cag PAI genotyped and tested in co‐culture assays with AGS gastric epithelial cells, and in mouse colonization studies. Six isolates were shown to harbour a complete cag PAI and to induce NF‐κB activation and IL‐8 secretion in AGS cells. Of the eight isolates that spontaneously colonized mice, six had a cag PAI– genotype and did not induce pro‐inflammatory responses in these cells. Mouse‐to‐mouse passage of the two cag PAI+‐colonizing strains yielded host‐adapted variants that infected mice with bacterial loads 100‐fold higher than those of the respective parental strains (P= 0.001). These mouse‐adapted variants were affected in their capacity to induce pro‐inflammatory responses in host cells, yet no changes in cag PAI gene content were detected between the strains by DNA microarray analysis. This work provides evidence for in vivo selection of H. pylori bacteria with a reduced capacity to induce inflammatory responses and suggests that such bacteria are better adapted to colonize mice.

Nickel binding and immunological properties of the C‐terminal domain of the Helicobacter pylori GroES homologue (HspA)

Helicobacter pylori synthesizes a heat‐shock protein of the GroES class. The gene encoding this protein (heat‐shock protein A, HspA) was recently cloned and it was shown to be unique in structure. H. pylori HspA consists of two domains: the N‐terminal domain (domain A) homologous with other GroES proteins, and a C‐terminal domain (domain B) corresponding to 27 additional residues resembling a metal‐binding domain. Various recombinant proteins consisting of the entire HspA polypeptide, the A domain, or the B domain were produced independently as proteins fused to maltose‐binding protein (MBP). Comparison of the divalent cation binding properties of the various MBP and MBP‐fused proteins allowed us to conclude that HspA binds nickel ions by means of its C‐terminal domain. HspA exhibited a high and specific affinity for nickel ions in comparison with its affinity for other divalent cations (copper, zinc, cobalt). Equilibrium dialysis experiments revealed that MBP–HspA binds nickel ions with an apparent dissociation constant (Kd) of 1.8 μM and a stoichiometry of 1.9 ions per molecule. The analysis of the deduced HspA amino acid sequences encoded by 35 independent clinical isolates demonstrated the existence of two molecular variants of HspA, i.e. a major and a minor variant present in 89% and 11% of strains, respectively. The two variants differed from each other by the simultaneous substitution of seven amino acids within the B domain, whilst the A domain was highly conserved amongst all the HspA proteins (99–100% identity). On the basis of serological studies, the highly conserved A domain of HspA was found to be the immunodominant domain. Functional complementation experiments were performed to test the properties of the two HspA variants. When co‐expressed together with the H. pylori urease gene cluster in Escherichia coli cells, the two HspA variant‐encoding genes led to a fourfold increase in urease activity, demonstrating that HspA in H. pylori has a specialized function with regard to the nickel metalloenzyme urease.

Local immunoglobulin G antibodies in the stomach may contribute to immunity against Helicobacter infection in mice

BACKGROUND & AIMS Orogastric immunization of mice with Helicobacter antigens, together with a mucosal adjuvant (cholera toxin), has been shown to confer immunity in the Helicobacter felis infection model. The aim of the study was to investigate the humoral immune responses associated with immunity and to compare these with responses in H. felis-infected mice. METHODS Enzyme-linked immunoassays were used to characterize the antibody-secreting cells and antibodies present at mucosal and systemic sites in mice. Animals were immunized orally with either whole-cell H. felis sonicates or Helicobacter pylon urease or heat-shock proteins. RESULTS Infection of mice with H. felis preferentially induced the recruitment of plasma cells committed to immunoglobulin (Ig) A synthesis in salivary gland and gastric tissues. Antigen-specific IgA was the major antibody class detected in mucosal secretions recovered from these tissues. In contrast, immunization of mice against H. felis infection induced the proliferation of large numbers of IgG-secreting cells, as well as the synthesis of local IgG antibodies, in the gastric mucosa of the animals. Protection against H. felis infection occurred in the absence of gastric IgA responses in sonicate-immunized mice. CONCLUSIONS It is proposed that locally synthesized specific IgG antibodies contribute to immunity against gastric Helicobacter infection.

Genetic and transmission analysis of Helicobacter pylori strains within a family.

Point mutations, intragenic recombination, and introduction of foreign alleles enhanced strain diversity within the family.

Distinct lineages of Ebola virus in Guinea during the 2014 West African epidemic

An epidemic of Ebola virus disease of unprecedented scale has been ongoing for more than a year in West Africa. As of 29 April 2015, there have been 26,277 reported total cases (of which 14,895 have been laboratory confirmed) resulting in 10,899 deaths. The source of the outbreak was traced to the prefecture of Guéckédou in the forested region of southeastern Guinea. The virus later spread to the capital, Conakry, and to the neighbouring countries of Sierra Leone, Liberia, Nigeria, Senegal and Mali. In March 2014, when the first cases were detected in Conakry, the Institut Pasteur of Dakar, Senegal, deployed a mobile laboratory in Donka hospital to provide diagnostic services to the greater Conakry urban area and other regions of Guinea. Through this process we sampled 85 Ebola viruses (EBOV) from patients infected from July to November 2014, and report their full genome sequences here. Phylogenetic analysis reveals the sustained transmission of three distinct viral lineages co-circulating in Guinea, including the urban setting of Conakry and its surroundings. One lineage is unique to Guinea and closely related to the earliest sampled viruses of the epidemic. A second lineage contains viruses probably reintroduced from neighbouring Sierra Leone on multiple occasions, while a third lineage later spread from Guinea to Mali. Each lineage is defined by multiple mutations, including non-synonymous changes in the virion protein 35 (VP35), glycoprotein (GP) and RNA-dependent RNA polymerase (L) proteins. The viral GP is characterized by a glycosylation site modification and mutations in the mucin-like domain that could modify the outer shape of the virion. These data illustrate the ongoing ability of EBOV to develop lineage-specific and potentially phenotypically important variation.