Dual Role for Pilus in Adherence to Epithelial Cells and Biofilm Formation in Streptococcus agalactiae

Streptococcus agalactiae is a common human commensal and a major life-threatening pathogen in neonates. Adherence to host epithelial cells is the first critical step of the infectious process. Pili have been observed on the surface of several gram-positive bacteria including S. agalactiae. We previously characterized the pilus-encoding operon gbs1479-1474 in strain NEM316. This pilus is composed of three structural subunit proteins: Gbs1478 (PilA), Gbs1477 (PilB), and Gbs1474 (PilC), and its assembly involves two class C sortases (SrtC3 and SrtC4). PilB, the bona fide pilin, is the major component; PilA, the pilus associated adhesin, and PilC, are both accessory proteins incorporated into the pilus backbone. We first addressed the role of the housekeeping sortase A in pilus biogenesis and showed that it is essential for the covalent anchoring of the pilus fiber to the peptidoglycan. We next aimed at understanding the role of the pilus fiber in bacterial adherence and at resolving the paradox of an adhesive but dispensable pilus. Combining immunoblotting and electron microscopy analyses, we showed that the PilB fiber is essential for efficient PilA display on the surface of the capsulated strain NEM316. We then demonstrated that pilus integrity becomes critical for adherence to respiratory epithelial cells under flow-conditions mimicking an in vivo situation and revealing the limitations of the commonly used static adherence model. Interestingly, PilA exhibits a von Willebrand adhesion domain (VWA) found in many extracellular eucaryotic proteins. We show here that the VWA domain of PilA is essential for its adhesive function, demonstrating for the first time the functionality of a prokaryotic VWA homolog. Furthermore, the auto aggregative phenotype of NEM316 observed in standing liquid culture was strongly reduced in all three individual pilus mutants. S. agalactiae strain NEM316 was able to form biofilm in microtiter plate and, strikingly, the PilA and PilB mutants were strongly impaired in biofilm formation. Surprisingly, the VWA domain involved in adherence to epithelial cells was not required for biofilm formation

Viral to metazoan marine plankton nucleotide sequences from the Tara Oceans expedition

A unique collection of oceanic samples was gathered by the Tara Oceans expeditions (2009-2013), targeting plankton organisms ranging from viruses to metazoans, and providing rich environmental context measurements. Thanks to recent advances in the field of genomics, extensive sequencing has been performed for a deep genomic analysis of this huge collection of samples. A strategy based on different approaches, such as metabarcoding, metagenomics, single-cell genomics and metatranscriptomics, has been chosen for analysis of size-fractionated plankton communities. Here, we provide detailed procedures applied for genomic data generation, from nucleic acids extraction to sequence production, and we describe registries of genomics datasets available at the European Nucleotide Archive (ENA, www.ebi.ac.uk/ena). The association of these metadata to the experimental procedures applied for their generation will help the scientific community to access these data and facilitate their analysis. This paper complements other efforts to provide a full description of experiments and open science resources generated from the Tara Oceans project, further extending their value for the study of the world's planktonic ecosystems

Alternative Neisseria spp. type IV pilin glycosylation with a glyceramido acetamido trideoxyhexose residue

The importance of protein glycosylation in the interaction of pathogenic bacteria with their host is becoming increasingly clear. Neisseria meningitidis, the etiological agent of cerebrospinal meningitis, crosses cellular barriers after adhering to host cells through type IV pili. Pilin glycosylation genes (pgl) are responsible for the glycosylation of PilE, the major subunit of type IV pili, with the 2,4-diacetamido-2,4,6-trideoxyhexose residue. Nearly half of the clinical isolates, however, display an insertion in the pglBCD operon, which is anticipated to lead to a different, unidentified glycosylation. Here the structure of pilin glycosylation was determined in such a strain by “top-down” MS approaches. MALDI-TOF, nanoelectrospray ionization Fourier transform ion cyclotron resonance, and nanoelectrospray ionization quadrupole TOF MS analysis of purified pili preparations originating from N. meningitidis strains, either wild type or deficient for pilin glycosylation, revealed a glycan mass inconsistent with 2,4-diacetamido-2,4,6-trideoxyhexose or any sugar in the databases. This unusual modification was determined by in-source dissociation of the sugar from the protein followed by tandem MS analysis with collision-induced fragmentation to be a hexose modified with a glyceramido and an acetamido group. We further show genetically that the nature of the sugar present on the pilin is determined by the carboxyl-terminal region of the pglB gene modified by the insertion in the pglBCD locus. We thus report a previously undiscovered monosaccharide involved in posttranslational modification of type IV pilin subunits by a MS-based approach and determine the molecular basis of its biosynthesis

Metagenomic shotgun sequencing of blood to identify bacteria and viruses in leukemic febrile neutropenia.

Despite diagnostic advances in microbiology, the etiology of neutropenic fever remains elusive in most cases. In this study, we evaluated the utility of a metagenomic shotgun sequencing based assay for detection of bacteria and viruses in blood samples of patients with febrile neutropenia. We prospectively enrolled 20 acute leukemia patients and obtained blood from these patients at three time points: 1) anytime from onset of neutropenia until before development of neutropenic fever, 2) within 24 hours of onset of neutropenic fever, 3) 5-7 days after onset of neutropenic fever. Blood samples underwent sample preparation, sequencing and analysis using the iDTECT® Dx Blood v1® platform (PathoQuest, Paris, France). Clinically relevant viruses or bacteria were detected in three cases each by metagenomic shotgun sequencing and blood cultures, albeit with no concordance between the two. Further optimization of sample preparation methods and sequencing platforms is needed before widespread adoption of this technology into clinical practice