Stéphanie Guadagnini

Assembly and role of pili in group B streptococci

Streptococcus agalactiae[group B streptococcus (GBS)] is the leading cause of neonatal pneumonia, sepsis and meningitis. An in silico genome analysis indicated that GBS strain NEM316 encodes five putative sortases, including the major class A sortase enzyme and four class C sortases. The genes encoding the class C sortases are tandemly arranged in two different loci, srtC1‐C2 and srtC3‐C4, with a similar genetic organization and are thought to be involved in pilus biosynthesis. Each pair of sortase genes is flanked by LPXTG protein encoding genes, two upstream and one downstream, and a divergently transcribed regulatory gene located upstream from this locus. We demonstrated that strain NEM316 expresses only the srtC3‐C4 locus, which encodes three surface proteins (Gbs1474, Gbs1477 and Gbs1478) that polymerize to form appendages resembling pili. Structural and functional analysis of this locus revealed that: (i) the transcriptional activator RogB is required for expression of the srtC3‐C4 operon; (ii) Gbs1477, and either SrtC3 or SrtC4 are absolutely required for pilus biogenesis; and (iii) GBS NEM316 pili are composed of three surface proteins, Gbs1477, the bona fide pilin which is the major component, Gbs1474, a minor associated component, and Gbs1478, a pilus‐associated adhesin. Surprisingly, pilus‐like structures can be formed in the absence of the two minor components, i.e. the putative anchor Gbs1474 or the adhesin Gbs1478. Adherence assays showed that Gbs1478 confers adhesive capacity to the pilus. This study provides the first evidence that adhesive pili are also present in Gram‐positive pathogens.

HIV‐1 DNA Flap formation promotes uncoating of the pre‐integration complex at the nuclear pore

The HIV‐1 central DNA Flap acts as a cis‐acting determinant of HIV‐1 genome nuclear import. Indeed, DNA‐Flap re‐insertion within lentiviral‐derived gene transfer vectors strongly stimulates gene transfer efficiencies. In this study, we sought to understand the mechanisms by which the central DNA Flap mediates HIV‐1 nuclear import. Here, we show that reverse transcription (RT°) occurs within an intact capsid (CA) shell, independently of the routing process towards the nuclear membrane, and that uncoating is not an immediate post‐fusion event, but rather occurs at the nuclear pore upon RT° completion. We provide the first observation with ultrastructural resolution of intact intracellular HIV‐1 CA shells by scanning electron microscopy. In the absence of central DNA Flap formation, uncoating is impaired and linear DNA remains trapped within an integral CA shell precluding translocation through the nuclear pore. These data show that DNA Flap formation, the very last event of HIV‐1 RT°, acts as a viral promoting element for the uncoating of HIV‐1 at the nuclear pore.

Simultaneous Cell-to-Cell Transmission of Human Immunodeficiency Virus to Multiple Targets through Polysynapses

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) efficiently propagates through cell-to-cell contacts, which include virological synapses (VS), filopodia, and nanotubes. Here, we quantified and characterized further these diverse modes of contact in lymphocytes. We report that viral transmission mainly occurs across VS and through “polysynapses,” a rosette-like structure formed between one infected cell and multiple adjacent recipients. Polysynapses are characterized by simultaneous HIV clustering and transfer at multiple membrane regions. HIV Gag proteins often adopt a ring-like supramolecular organization at sites of intercellular contacts and colocalize with CD63 tetraspanin and raft components GM1, Thy-1, and CD59. In donor cells engaged in polysynapses, there is no preferential accumulation of Gag proteins at contact sites facing the microtubule organizing center. The LFA-1 adhesion molecule, known to facilitate viral replication, enhances formation of polysynapses. Altogether, our results reveal an underestimated mode of viral transfer through polysynapses. In HIV-infected individuals, these structures, by promoting concomitant infection of multiple targets in the vicinity of infected cells, may facilitate exponential viral growth and escape from immune responses.

An extracellular matrix glues together the aerial-grown hyphae of Aspergillus fumigatus

Pulmonary infections due to Aspergillus fumigatus result from the development of a colony of tightly associated hyphae in contact with the air, either in the alveoli (invasive aspergillosis) or in an existing cavity (aspergilloma). The fungal ball observed in vivo resembles an aerial colony obtained in agar medium in vitro more than a mycelial mass obtained in liquid shaken conditions that have been classically used to date to study A. fumigatus physiology. For this reason, we embarked on an analysis of the characteristics of A. fumigatus colonies grown in aerial static conditions. (i) Under static aerial conditions, mycelial growth is greater than in shaken, submerged conditions. (ii) The colony surface of A. fumigatus revealed the presence of an extracellular hydrophobic matrix that acts as a cohesive linkage bonding hyphae into a contiguous sheath. (iii) The extracellular matrix is composed of galactomannan, α1,3 glucans, monosaccharides and polyols, melanin and proteins including major antigens and hydrophobins. (iv) A. fumigatus colonies were more resistant to polyenes than shake, submerged mycelium. This is the first analysis of the three dimensional structure of a mycelial colony. Knowledge of this multicellular organization will impact our future understanding of the pathobiology of aerial mold pathogens.

Extracellular bacterial pathogen induces host cell surface reorganization to resist shear stress.

Bacterial infections targeting the bloodstream lead to a wide array of devastating diseases such as septic shock and meningitis. To study this crucial type of infection, its specific environment needs to be taken into account, in particular the mechanical forces generated by the blood flow. In a previous study using Neisseria meningitidis as a model, we observed that bacterial microcolonies forming on the endothelial cell surface in the vessel lumen are remarkably resistant to mechanical stress. The present study aims to identify the molecular basis of this resistance. N. meningitidis forms aggregates independently of host cells, yet we demonstrate here that cohesive forces involved in these bacterial aggregates are not sufficient to explain the stability of colonies on cell surfaces. Results imply that host cell attributes enhance microcolony cohesion. Microcolonies on the cell surface induce a cellular response consisting of numerous cellular protrusions similar to filopodia that come in close contact with all the bacteria in the microcolony. Consistent with a role of this cellular response, host cell lipid microdomain disruption simultaneously inhibited this response and rendered microcolonies sensitive to blood flow–generated drag forces. We then identified, by a genetic approach, the type IV pili component PilV as a triggering factor of plasma membrane reorganization, and consistently found that microcolonies formed by a pilV mutant are highly sensitive to shear stress. Our study shows that bacteria manipulate host cell functions to reorganize the host cell surface to form filopodia-like structures that enhance the cohesion of the microcolonies and therefore blood vessel colonization under the harsh conditions of the bloodstream.

Colonization of the salivary glands of Naucoris cimicoides by Mycobacterium ulcerans requires host plasmatocytes and a macrolide toxin, mycolactone.

Mycobacterium ulcerans was first identified as the causative agent of Buruli ulcer; this cutaneous tissue‐destructive process represents the third most important mycobacterial disease in humans after tuberculosis and leprosy. More recently other life traits were documented. M. ulcerans is mainly detected in humid tropical zones as part of a complex ecosystem comprising algae, aquatic insect predators of the genus Naucoris, and very likely their vegetarian preys. Coelomic plasmatocytes could be the first cells of Naucoris cimicoides to be involved in the infection process, acting as shuttle cells that deliver M. ulcerans to the salivary glands as suggested by both in vitro and in vivo approaches. Furthermore, a key element for the early and long‐term establishment of M. ulcerans in Naucoridae is demonstrated by the fact that only mycolactone toxin‐producing M. ulcerans isolates are able to invade the salivary glands, a site where they proliferate. Later, the raptorial legs of Naucoris are covered by M. ulcerans‐containing material that displays features of biofilms.

Role of AmiA in the morphological transition of Helicobacter pylori and in immune escape.

The human gastric pathogen Helicobacter pylori is responsible for peptic ulcers and neoplasia. Both in vitro and in the human stomach it can be found in two forms, the bacillary and coccoid forms. The molecular mechanisms of the morphological transition between these two forms and the role of coccoids remain largely unknown. The peptidoglycan (PG) layer is a major determinant of bacterial cell shape, and therefore we studied H. pylori PG structure during the morphological transition. The transition correlated with an accumulation of the N-acetyl-D-glucosaminyl-β(1,4)-N-acetylmuramyl-L-Ala–D-Glu (GM-dipeptide) motif. We investigated the molecular mechanisms responsible for the GM-dipeptide motif accumulation, and studied the role of various putative PG hydrolases in this process. Interestingly, a mutant strain with a mutation in the amiA gene, encoding a putative PG hydrolase, was impaired in accumulating the GM-dipeptide motif and transforming into coccoids. We investigated the role of the morphological transition and the PG modification in the biology of H. pylori. PG modification and transformation of H. pylori was accompanied by an escape from detection by human Nod1 and the absence of NF-κB activation in epithelial cells. Accordingly, coccoids were unable to induce IL-8 secretion by AGS gastric epithelial cells. amiA is, to our knowledge, the first genetic determinant discovered to be required for this morphological transition into the coccoid forms, and therefore contributes to modulation of the host response and participates in the chronicity of H. pylori infection.

Septins Regulate Bacterial Entry into Host Cells

Background Septins are conserved GTPases that form filaments and are required in many organisms for several processes including cytokinesis. We previously identified SEPT9 associated with phagosomes containing latex beads coated with the Listeria surface protein InlB. Methodology/Principal Findings Here, we investigated septin function during entry of invasive bacteria in non-phagocytic mammalian cells. We found that SEPT9, and its interacting partners SEPT2 and SEPT11, are recruited as collars next to actin at the site of entry of Listeria and Shigella. SEPT2-depletion by siRNA decreased bacterial invasion, suggesting that septins have roles during particle entry. Incubating cells with InlB-coated beads confirmed an essential role for SEPT2. Moreover, SEPT2-depletion impaired InlB-mediated stimulation of Met-dependent signaling as shown by FRET. Conclusions/Significance Together these findings highlight novel roles for SEPT2, and distinguish the roles of septin and actin in bacterial entry.

A M23B family metallopeptidase of Helicobacter pylori required for cell shape, pole formation and virulence

The molecular basis of the regulation of specific shapes and their role for the bacterial fitness remain largely unknown. We focused in this study on the Gram‐negative and spiral‐shaped Helicobacter pylori. To colonize its unique niche, H. pylori needs to reach quickly the human gastric mucosa, by swimming to and through the mucus layer. For that reason, the specific shape of H. pylori is predicted to be necessary for optimal motility in vivo, and consequently for its colonization ability. Here, we describe the involvement of a PG‐modifying enzyme, HdpA (HP0506), in the mouse colonization ability of this bacterium, by regulating its shape. Indeed, the inactivation of the hp0506 gene led to a stocky and branched phenotype, affecting H. pylori colonization capacity despite a normal motility phenotype in vitro. In contrast, the overexpression of the hp0506 gene induced the transformation of H. pylori from rod to dividing cocci shaped bacteria. Furthermore, we demonstrated by PG analysis and enzymology, that HdpA carried both d,d‐carboxypeptidase and d,d‐endopeptidase activities. Thus, HdpA is the first enzyme belonging to the M23‐peptidase family able to perform the d,d‐carboxypeptidation and regulate cell shape.

ActA Promotes Listeria monocytogenes Aggregation, Intestinal Colonization and Carriage

Listeria monocytogenes (Lm) is a ubiquitous bacterium able to survive and thrive within the environment and readily colonizes a wide range of substrates, often as a biofilm. It is also a facultative intracellular pathogen, which actively invades diverse hosts and induces listeriosis. So far, these two complementary facets of Lm biology have been studied independently. Here we demonstrate that the major Lm virulence determinant ActA, a PrfA-regulated gene product enabling actin polymerization and thereby promoting its intracellular motility and cell-to-cell spread, is critical for bacterial aggregation and biofilm formation. We show that ActA mediates Lm aggregation via direct ActA-ActA interactions and that the ActA C-terminal region, which is not involved in actin polymerization, is essential for aggregation in vitro. In mice permissive to orally-acquired listeriosis, ActA-mediated Lm aggregation is not observed in infected tissues but occurs in the gut lumen. Strikingly, ActA-dependent aggregating bacteria exhibit an increased ability to persist within the cecum and colon lumen of mice, and are shed in the feces three order of magnitude more efficiently and for twice as long than bacteria unable to aggregate. In conclusion, this study identifies a novel function for ActA and illustrates that in addition to contributing to its dissemination within the host, ActA plays a key role in Lm persistence within the host and in transmission from the host back to the environment.