Both HLA-B*57 and Plasma HIV RNA Levels Contribute to the HIV-Specific CD8+ T Cell Response in HIV Controllers

CD8⁺ T cell responses are thought to play an important role during HIV infection, particularly in HIV controllers (HIC) in whom viral replication is spontaneously controlled without any treatment. We have demonstrated that CD8⁺ T cells from these subjects are able to suppress viral replication in vitro. In parallel, HIV-specific CD8⁺ responses were shown to be strong and of high quality, with proliferative abilities and cytotoxic capacities, in HIC. The HLA-B*57 allele, which is associated with a better clinical outcome in HIV infection, is overrepresented in HIC. However, we showed that these patients constitute a heterogeneous group that includes subjects who present weak suppression of viral replication in vitro and HIV-specific responses. We performed an extensive study of 101 HIC (49 HLA-B*57⁺ and 52 HLA-B*57⁻) to determine the impact of HLA-B*57 on the HIV-specific CD8⁺ response. The HLA-B*57-restricted response displayed better qualitative features, such as higher functional avidity, higher proliferation capacity, and a higher level of cytokine production, than responses not restricted by HLA-B*57. However, the highest frequencies of HIV-specific CD8⁺ T cells were observed only in a subset of HLA-B*57⁺ subjects. They were tightly associated with the ability to suppress viral replication in vitro. In contrast, the subset of HLA-B*57⁺ subjects with a weak ability to suppress viral replication had significantly lower ultrasensitive viral loads than all the other groups of controllers. In conclusion, both HLA-B*57 and the amount of ultrasensitive viral load seem to play a role in HIV-specific CD8⁺ T cell responses in HIC.     

Radical-induced oxidation of trans-resveratrol

trans-Resveratrol (RVT) (3,5,4'-trihydroxystilbene), a polyphenolic constituent of red wine, is thought to be beneficial in reducing the incidence of cardiovascular diseases, partly via its antioxidant properties. However, the mechanism of action by which trans-resveratrol displays its antioxidant effect has not been totally unravelled. This study aimed at establishing a comprehensive scheme of the reaction mechanisms of the direct scavenging of HO(*) and O(2)(*-) radicals generated by water gamma radiolysis. Aerated aqueous solutions of trans-RVT (from 10 to 100μmolL(-1)) were irradiated with increasing radiation doses (from 25 to 400Gy) and further analyzed by UV-visible absorption spectrophotometry for detection of trans-RVT oxidation products. Separation and quantification of RVT and its four oxidation products previously identified by mass spectrometry, i.e., piceatannol (PCT), 3,5-dihydroxybenzoic acid (3,5-DHBA), 3,5-dihydroxybenzaldehyde (3,5-DHB) and para-hydroxybenzaldehyde (PHB), were performed by HPLC/UV-visible spectrophotometry. Determination of the radiolytic yields of trans-RVT consumption and oxidation product formation has allowed us to establish balance between trans-RVT disappearance and the sum of oxidation products formation. Under our conditions, O(2)(-) radicals seemed to poorly initiate oxidation of trans-RVT, whereas the latter, whatever its initial concentration, quantitatively reacted with HO() radicals, via a dismutation mechanism. Two reaction pathways involving HO()-induced trans-RVT primary radicals have been proposed to explain the formation of the oxidation end-products of trans-RVT.     

Modifications of the chemical structure of terpenes in antiplasmodial and antifungal drug research

Pure natural monoterpenes were evaluated in vitro for their antiplasmodial activities against Plasmodium falciparum. Chemically modified terpenes were also tested to see whether the introduction of an alkyne, a cyclopropane, a diene, or a cyclopentenone moiety had an influence on the biological activity. The IC(50) obtained on a chloroquine-resistant strain of Plasmodium (FcM29-Cameroon) showed moderate activity, but with the alkyne and the cyclopentenone derivatives showing a promising enhancement of activity compared with the parent molecules. On the contrary, no antifungal activity was found in vitro using Candida albicans. Given the observed antiplasmodial activity of some of these modified monoterpenes, new monoterpene derivatives could be the basis for new antimalarial drugs to be researched.     

Differential Role of Autophagy in CD4 T Cells and Macrophages during X4 and R5 HIV-1 Infection

Background

HIV-1 can infect and replicate in both CD4 T cells and macrophages. In these cell types, HIV-1 entry is mediated by the binding of envelope glycoproteins (gp120 and gp41, Env) to the receptor CD4 and a coreceptor, principally CCR5 or CXCR4, depending on the viral strain (R5 or X4, respectively). Uninfected CD4 T cells undergo X4 Env-mediated autophagy, leading to their apoptosis, a mechanism now recognized as central to immunodeficiency.

Methodology/Principal Findings

We demonstrate here that autophagy and cell death are also induced in the uninfected CD4 T cells by HIV-1 R5 Env, while autophagy is inhibited in productively X4 or R5-infected CD4 T cells. In contrast, uninfected macrophages, a preserved cell population during HIV-1 infection, do not undergo X4 or R5 Env-mediated autophagy. Autophagosomes, however, are present in macrophages exposed to infectious HIV-1 particles, independently of coreceptor use. Interestingly, we observed two populations of autophagic cells: one highly autophagic and the other weakly autophagic. Surprisingly, viruses could be detected in the weakly autophagic cells but not in the highly autophagic cells. In addition, we show that the triggering of autophagy in macrophages is necessary for viral replication but addition of Bafilomycin A1, which blocks the final stages of autophagy, strongly increases productive infection.

Conclusions/Significance

Taken together, our data suggest that autophagy plays a complex, but essential, role in HIV pathology by regulating both viral replication and the fate of the target cells.     

Virucidal Efficacy of Glutaraldehyde against Enteroviruses Is Related to the Location of Lysine Residues in Exposed Structures of the VP1 Capsid Protein

Glutaraldehyde (GTA) is a potent virucidal disinfectant whose exact mode of action against enteroviruses is not understood. Earlier reports showed that GTA reacts preferentially with the VP1 capsid protein of echovirus 25 and poliovirus 1 and that GTA has affinity for exposed lysine residues on proteins. To investigate further the inactivation of enteroviruses by GTA, seven strains were selected on the basis of differences in their overall number and the positions of lysine residues in the amino acid sequences of the VP1 polypeptide. Inactivation kinetics experiments were performed with 0.10% GTA. The viruses grouped into three clusters and exhibited significantly different levels of sensitivity to GTA. The results were analyzed in the light of current knowledge of the three-dimensional structure of enteroviruses and the viral life cycle. The differences observed in sensitivity to GTA were related to the number of lysine residues and their locations in the VP1 protein. The overall findings suggest that the BC and DE loops, which cluster at the fivefold axis of symmetry and are the most exposed on the outer surface of the virions, are primary reactive sites for GTA.     

The Grey Mouse Lemur Uses Season-Dependent Fat or Protein Sparing Strategies to Face Chronic Food Restriction

During moderate calorie restriction (CR) the heterotherm Microcebus murinus is able to maintain a stable energy balance whatever the season, even if only wintering animals enter into torpor. To understand its energy saving strategies to respond to food shortages, we assessed protein and energy metabolisms associated with wintering torpor expression or summering torpor avoidance. We investigated body composition, whole body protein turnover, and daily energy expenditure (DEE), during a graded (40 and 80%) 35-day CR in short-days (winter; SD40 and SD80, respectively) and long-days (summer; LD40 and LD80, respectively) acclimated animals. LD40 animals showed no change in fat mass (FM) but a 12% fat free mass (FFM) reduction. Protein balance being positive after CR, the FFM loss was early and rapid. The 25% DEE reduction, in LD40 group was mainly explained by FFM changes. LD80 animals showed a steady body mass loss and were excluded from the CR trial at day 22, reaching a survival-threatened body mass. No data were available for this group. SD40 animals significantly decreased their FM level by 21%, but maintained FFM. Protein sparing was achieved through a 35 and 39% decrease in protein synthesis and catabolism (protein turnover), respectively, overall maintaining nitrogen balance. The 21% reduction in energy requirement was explained by the 30% nitrogen flux drop but also by torpor as DEE FFM-adjusted remained 13% lower compared to ad-libitum. SD80 animals were unable to maintain energy and nitrogen balances, losing both FM and FFM. Thus summering mouse lemurs equilibrate energy balance by a rapid loss of active metabolic mass without using torpor, whereas wintering animals spare protein and energy through increased torpor expression. Both strategies have direct fitness implication: 1) to maintain activities at a lower body size during the mating season and 2) to preserve an optimal wintering muscle mass and function.     

Association of Hemolytic Activity of Pseudomonas entomophila,a Versatile Soil Bacterium,with Cyclic Lipopeptide Production

Pseudomonas entomophila is an entomopathogenic bacterium that is able to infect and kill Drosophila melanogaster upon ingestion. Its genome sequence suggests that it is a versatile soil bacterium closely related to Pseudomonas putida. The GacS/GacA two-component system plays a key role in P. entomophila pathogenicity, controlling many putative virulence factors and AprA, a secreted protease important to escape the fly immune response. P. entomophila secretes a strong diffusible hemolytic activity. Here, we showed that this activity is linked to the production of a new cyclic lipopeptide containing 14 amino acids and a 3-C₁₀OH fatty acid that we called entolysin. Three nonribosomal peptide synthetases (EtlA, EtlB, EtlC) were identified as responsible for entolysin biosynthesis. Two additional components (EtlR, MacAB) are necessary for its production and secretion. The P. entomophila GacS/GacA two-component system regulates entolysin production, and we demonstrated that its functioning requires two small RNAs and two RsmA-like proteins. Finally, entolysin is required for swarming motility, as described for other lipopeptides, but it does not participate in the virulence of P. entomophila for Drosophila. While investigating the physiological role of entolysin, we also uncovered new phenotypes associated with P. entomophila, including strong biocontrol abilities.Pseudomonas entomophila is a recently isolated Pseudomonas species that is closely related to the saprophytic soil bacterium Pseudomonas putida. It was initially characterized as a natural pathogen of Drosophila (63). Indeed, P. entomophila was first isolated from flies sampled in Guadeloupe, and it is highly pathogenic for Drosophila larvae and adults. P. entomophila can also effectively kill members of other insect orders (e.g., Bombyx mori, Anopheles gambiae), which makes it a new entomopathogenic bacterium. Its ability to infect and kill Drosophila melanogaster very efficiently after ingestion makes it an appropriate model for the study of host-pathogen interactions (38, 62, 63).In order to unravel features contributing to the entomopathogenic properties of P. entomophila, its genome was sequenced. The results suggest that this strain is a ubiquitous, metabolically versatile bacterium that may colonize diverse habitats, including soil, rhizosphere, and aquatic systems, as shown for P. putida KT2440 (62). However, in contrast to the P. putida genome, the P. entomophila genome contains many genes that are predicted to be important for virulence toward insects. Notably, P. entomophila could secrete many degradative enzymes (proteases and lipases), putative toxins, and secondary metabolites (62). Similar factors have been shown to play a key role in the virulence of other entomopathogenic bacteria like Photorhabdus and Xenorhabdus sp. (27, 29).Insertional mutagenesis allowed the identification of several P. entomophila genes required to infect and/or kill Drosophila. This analysis demonstrated that P. entomophila virulence is under the control of the GacS/GacA two-component system (62, 63), a global regulatory system which is known to control secondary metabolite production, protein secretion, and pathogenic abilities in gammaproteobacteria (37, 65). Another study indicates that P. entomophila can counteract the Drosophila gut immune response as a result of the secretion of an abundant protease, AprA, which degrades antimicrobial peptides produced by gut epithelia and thereby promotes bacterial persistence (38). However, an AprA-deficient mutant remains virulent to some extent, indicating that P. entomophila virulence is multifactorial, AprA being one virulence factor among others.The secretion of virulence factors is a common mechanism employed by pathogens to compromise host defenses. Several entomopathogenic bacteria (e.g., Photorhabdus luminescens) secrete toxins that allow them to impair host function (8). The starting point of this study was the observation that, in contrast to several other Pseudomonas strains, P. entomophila secretes a strong diffusible hemolytic activity (which is also controlled by the Gac system). This raises the possibility of a link between this hemolytic activity and the pathogenicity of P. entomophila for Drosophila. Indeed, bacterial hemolysins are exotoxins that attack blood cell membranes and cause cell rupture by poorly defined mechanisms. It was conceivable that this hemolytic activity could be a readout for the ability of P. entomophila to damage the epithelial cells of the Drosophila gut, which plays a crucial role in its virulence (10, 33, 63).In this study, the P. entomophila hemolytic factor was identified as a cyclic lipopeptide (CLP) whose structure was elucidated. CLPs are versatile molecules with antimicrobial, cytotoxic, and surfactant properties that are produced by members of the genera Bacillus, Serratia, Burkholderia, and Pseudomonas (31, 41, 43, 50). They are produced by a ribosome-independent mechanism that utilizes multifunctional enzymes called nonribosomal peptide synthetases (NRPSs) (42, 59). These NRPSs are composed of repeated amino acid activation modules containing domains for condensation, aminoacyl adenylation, and thiolation. Modules are responsible for activation and incorporation of amino acids into the growing peptide. A large number of prokaryotic and some eukaryotic organisms synthesize peptide metabolites via this nonribosomal mechanism of biosynthesis (42, 47).Several genes involved in P. entomophila lipopeptide production were identified, three of them encoding NRPSs. The physiological role of this lipopeptide was also investigated, and it does not seem to play a role in the process of virulence towards Drosophila and Dictyostelium or in the P. entomophila biocontrol activity that was uncovered by this study. This suggests that the lifestyle of this newly identified bacterium is probably quite versatile and that lipopeptide production could be required only under specific circumstances.