Molecular and Phylogeographic Analysis of Human Immuno-deficiency Virus Type 1 Strains Infecting Treatment-naive Patients from Kigali, Rwanda

This study aimed at describing the genetic subtype distribution of HIV-1 strains circulating in Kigali and their epidemiological link with the HIV-1 strains from the five countries surrounding Rwanda. One hundred and thirty eight pol (RT and PR) sequences from 116 chronically- and 22 recently-infected antiretroviral therapy (ART)-naïve patients from Kigali were generated and subjected to HIV drug resistance (HIV-DR), phylogenetic and recombinant analyses in connection with 366 reference pol sequences from Rwanda, Burundi, Kenya, Democratic Republic of Congo, Tanzania and Uganda (Los Alamos database). Among the Rwandan samples, subtype A1 predominated (71.7%), followed by A1/C recombinants (18.1%), subtype C (5.8%), subtype D (2.9%), one A1/D recombinant (0.7%) and one unknown subtype (0.7%). Thirteen unique and three multiple A1/C recombinant forms were identified. No evidence for direct transmission events was found within the Rwandan strains. Molecular characteristics of HIV-1 were similar between chronically and recently-infected individuals and were not significantly associated with demographic or social factors. Our report suggests that the HIV-1 epidemic in Kigali is characterized by the emergence of A1/C recombinants and is not phylogenetically connected with the HIV-1 epidemic in the five neighboring countries. The relatively low level of transmitted HIV-DR mutations (2.9%) reported here indicates the good performance of the ART programme in Rwanda. However, the importance of promoting couples'' counseling, testing and disclosure during HIV prevention strategies is highlighted.     

Invasive and adherent bacterial pathogens co-Opt host clathrin for infection

Infection by the bacterium Listeria monocytogenes depends on host cell clathrin. To determine whether this requirement is widespread, we analyzed infection models using diverse bacteria. We demonstrated that bacteria that enter cells following binding to cellular receptors (termed "zippering" bacteria) invade in a clathrin-dependent manner. In contrast, bacteria that inject effector proteins into host cells in order to gain entry (termed "triggering" bacteria) invade in a clathrin-independent manner. Strikingly, enteropathogenic Escherichia coli (EPEC) required clathrin to form actin-rich pedestals in host cells beneath adhering bacteria, even though this pathogen remains extracellular. Furthermore, clathrin accumulation preceded the actin rearrangements necessary for Listeria entry. These data provide evidence for a clathrin-based entry pathway allowing internalization of large objects (bacteria and ligand-coated beads) and used by "zippering" bacteria as part of a general mechanism to invade host mammalian cells. We also revealed a nonendocytic role for clathrin required for extracellular EPEC infections.     

A role for septin 2 in Drp1‐mediated mitochondrial fission

Mitochondria are essential eukaryotic organelles often forming intricate networks. The overall network morphology is determined by mitochondrial fusion and fission. Among the multiple mechanisms that appear to regulate mitochondrial fission, the ER and actin have recently been shown to play an important role by mediating mitochondrial constriction and promoting the action of a key fission factor, the dynamin‐like protein Drp1. Here, we report that the cytoskeletal component septin 2 is involved in Drp1‐dependent mitochondrial fission in mammalian cells. Septin 2 localizes to a subset of mitochondrial constrictions and directly binds Drp1, as shown by immunoprecipitation of the endogenous proteins and by pulldown assays with recombinant proteins. Depletion of septin 2 reduces Drp1 recruitment to mitochondria and results in hyperfused mitochondria and delayed FCCP‐induced fission. Strikingly, septin depletion also affects mitochondrial morphology in Caenorhabditis elegans, strongly suggesting that the role of septins in mitochondrial dynamics is evolutionarily conserved.     

Enzyme diversity of the cellulolytic system produced by Clostridium cellulolyticum explored by two-dimensional analysis: identification of seven genes encoding new dockerin-containing proteins

The enzyme diversity of the cellulolytic system produced by Clostridium cellulolyticum grown on crystalline cellulose as a sole carbon and energy source was explored by two-dimensional electrophoresis. The cellulolytic system of C. cellulolyticum is composed of at least 30 dockerin-containing proteins (designated cellulosomal proteins) and 30 noncellulosomal components. Most of the known cellulosomal proteins, including CipC, Cel48F, Cel8C, Cel9G, Cel9E, Man5K, Cel9M, and Cel5A, were identified by using two-dimensional Western blot analysis with specific antibodies, whereas Cel5N, Cel9J, and Cel44O were identified by using N-terminal sequencing. Unknown enzymes having carboxymethyl cellulase or xylanase activities were detected by zymogram analysis of two-dimensional gels. Some of these enzymes were identified by N-terminal sequencing as homologs of proteins listed in the NCBI database. Using Trap-Dock PCR and DNA walking, seven genes encoding new dockerin-containing proteins were cloned and sequenced. Some of these genes are clustered. Enzymes encoded by these genes belong to glycoside hydrolase families GH2, GH9, GH10, GH26, GH27, and GH59. Except for members of family GH9, which contains only cellulases, the new modular glycoside hydrolases discovered in this work could be involved in the degradation of different hemicellulosic substrates, such as xylan or galactomannan.     

Ac His1 [D-Phe2, K15, R16, L27] VIP (3-7)/GRF (8-27)--a VPAC1 receptor antagonist--is an inverse agonist on two constitutively active truncated VPAC1 receptors

C-terminally truncated human VPAC(1) receptors were constructed and stably transfected in Chinese hamster ovary (CHO) cells. Selected clones expressing comparable receptor densities were studied for ligand's binding properties, basal and stimulated adenylate cyclase activity. The wild-type (1-457) receptor served as reference. The binding properties of all the constructions were preserved. As judged by the intrinsic activity of the partial agonist Q(3)-VIP, the shortest receptors have a moderate impairment of the coupling efficacy to G(alpha s) protein. Cells expressing the VPAC(1) (1-436) and (1-441) truncated receptors had a two- to three-fold higher basal adenylate cyclase activity than those expressing the wild-type or the VPAC(1) (1-444), (1-433), (1-429), (1-421) and (1-398) receptor. The stimulatory effect of VIP and other agonist was preserved. This suggested that VPAC(1) (1-436) and (1-441) receptors had a constitutive activity. The selective VPAC(1) receptor antagonist Ac His(1) [D-Phe(2), K(15), R(16), L(27)] VIP (3-7)/GRF (8-27) reduced by 60% the basal activity with an EC(50) value of 3 nM comparable to its IC(50) value for binding. This agonist behaved thus like an inverse agonist on the constitutively active VPAC(1) receptors generated by C-terminal truncation and expressed in CHO cells.     

Celiac disease association with CD8+ T cell responses: identification of a novel gliadin-derived HLA-A2-restricted epitope

One of the diagnostic hallmarks of the histological lesions associated with celiac disease is the extensive infiltration of the small intestinal epithelium by CD8(+) T cells of unknown Ag specificity. In this study, we report recognition of the gliadin-derived peptide (A-gliadin 123-132) by CD8(+) T lymphocytes from celiac patients. A-gliadin 123-132-specific IFN-gamma production and cytotoxic activity were detected in PBMCs derived from patients on gluten-free diet, but not from either celiac patients on gluten-containing diet or healthy controls. In contrast, A-gliadin 123-132-specific cells were isolated from small intestine biopsies of patients on either gluten-free or gluten-containing diets. Short-term T cell lines derived from the small intestinal mucosa and specific for the 123-132 epitope recognized human APC pulsed with either whole recombinant alpha-gliadin or a partial pepsin-trypsin gliadin digest. Finally, we speculate on a possible mechanism leading to processing and presentation of class I-restricted gliadin-derived epitopes in celiac disease patients.