An inventory of the bacterial macromolecular components and their spatial organization.

Formerly regarded as small 'bags' of nucleic acids with randomly diffusing enzymes, bacteria are organized by a sophisticated and tightly regulated molecular machinery. Here, we review qualitative and quantitative data on the intracellular organization of bacteria and provide a detailed inventory of macromolecular structures such as the divisome, the degradosome and the bacterial 'nucleolus'. We discuss how these metabolically active structures manage the spatial organization of the cell and how macromolecular crowding influences them. We present for the first time a visualization program, lifeexplorer, that can be used to study the interplay between metabolism and spatial organization of a prokaryotic cell.

Densovirus infectious pathway requires clathrin-mediated endocytosis followed by trafficking to the nucleus.

Junonia coenia densovirus (JcDNV) is an ambisense insect parvovirus highly pathogenic for lepidopteran pests at larval stages. The potential use of DNVs as biological control agents prompted us to reinvestigate the host range and cellular mechanisms of infection. In order to understand the early events of infection, we set up a functional infection assay in a cell line of the pest Lymantria dispar to determine the intracellular pathway undertaken by JcDNV to infect a permissive lepidopteran cell line. Our results show that JcDNV particles are rapidly internalized into clathrin-coated vesicles and slowly traffic within early and late endocytic compartments. Blocking late-endocytic trafficking or neutralizing the pH with drugs inhibited infection. During internalization, disruption of the cytoskeleton, and inhibition of phosphatidylinositol 3-kinase blocked the movement of vesicles containing the virus to the nucleus and impaired infection. In summary, our results define for the first time the early endocytic steps required for a productive DNV infection.

Growth deficiencies of Neisseria meningitidis pfs and luxS mutants are not due to inactivation of quorum sensing.

The activated methyl cycle (AMC) is a central metabolic pathway used to generate (and recycle) several important metabolites and enable methylation. Pfs and LuxS are considered integral components of this pathway because they convert S-adenosylhomocysteine (SAH) to S-ribosylhomocysteine (SRH) and S-ribosylhomocysteine to homocysteine (HCY), respectively. The latter reaction has a second function since it also generates the precursor of the quorum-sensing molecule autoinducer 2 (AI-2). By demonstrating that there was a complete lack of AI-2 production in pfs mutants of the causative agent of meningitis and septicemia, Neisseria meningitidis, we showed that the Pfs reaction is the sole intracellular source of the AI-2 signal. Analysis of lacZ reporters and real-time PCR experiments indicated that pfs is expressed constitutively from a promoter immediately upstream, and careful study of the pfs mutants revealed a growth defect that could not be attributed to a lack of AI-2. Metabolite profiling of the wild type and of a pfs mutant under various growth conditions revealed changes in the concentrations of several AMC metabolites, particularly SRH and SAH and under some conditions also HCY. Similar studies established that an N. meningitidis luxS mutant also has metabolite pool changes and growth defects in line with the function of LuxS downstream of Pfs in the AMC. Thus, the observed growth defect of N. meningitidis pfs and luxS mutants is not due to quorum sensing but is probably due to metabolic imbalance and, in the case of pfs inactivation, is most likely due to toxic accumulation of SAH.

Making 'sense' of metabolism: autoinducer-2, LuxS and pathogenic bacteria.

Bacteria exploit many mechanisms to communicate with each other and their surroundings. Mechanisms using small diffusible signals to coordinate behaviour with cell density (quorum sensing) frequently contribute to pathogenicity. However, pathogens must also be able to acquire nutrients and replicate to successfully invade their host. One quorum-sensing system, based on the possession of LuxS, bears the unique feature of contributing directly to metabolism, and therefore has the potential to influence both gene regulation and bacterial fitness. Here, we discuss the influence that LuxS and its product, autoinducer-2, have on virulence, relating the current evidence to the preferred niche of the pathogen and the underlying mechanisms involved.

The ability of chloroquine to prevent tat-induced cytokine secretion by monocytes is implicated in its in vivo anti-human immunodeficiency virus type 1 activity.

Hydroxychloroquine at 1 microM reduces the load of human immunodeficiency virus type 1 (HIV-1) in patients, whereas chloroquine (CQ) concentrations above 3 microM are required for inhibition of HIV-1 replication in peripheral blood mononuclear cells. Exogenous HIV-1 Tat reaches the cytosol of T cells by using low endosomal pH, and endosome neutralization by CQ prevents Tat from entering and affecting T cells. We show here that 0.6 microM CQ inhibits cytokine secretion induced by Tat in monocytes without affecting lipopolysaccharide-triggered cytokine release. This finding suggests that the in vivo anti-HIV-1 effect of CQ results not from a direct effect on the infected cell but rather from the capacity of CQ to prevent Tat from perturbing the cytokine balance.

HIV-1 Tat enters T cells using coated pits before translocating from acidified endosomes and eliciting biological responses.

The HIV-1 Tat protein is secreted by infected cells. Extracellular Tat can affect bystander uninfected T cells and induce numerous biological responses such as apoptosis and cytokine secretion. Tat is likely involved in several immune disorders during AIDS. Nevertheless, it is not known whether Tat triggers cell responses directly upon binding to signaling receptors at the plasma membrane or after delivery to the cytosol. The pathway that enables Tat to reach the cytosol is also unclear. Here we visualized Tat within T-cell-coated pits and endosomes. Moreover, inhibitors of clathrin/AP-2-mediated uptake such as chlorpromazine, activated RhoA, or dominant-negative mutants of Eps15, intersectin, dynamin, or rab5 impaired Tat delivery to the cytosol by preventing its endocytosis. Molecules neutralizing low endosomal pH or Hsp90 inhibitors abolished Tat entry at a later stage by blocking its endosomal translocation, as directly shown using a cell-free translocation assay. Finally, endosomal pH neutralization prevented Tat from inducing T-cell responses such as NF-kappaB activation, apoptosis, and interleukin secretion, indicating that cytosolic delivery is required for Tat signaling. Hence, Tat enters T cells essentially like diphtheria toxin, using clathrin-mediated endocytosis before low-pH-induced and Hsp90-assisted endosomal translocation. Cell responses are then induced from the cytosol.