Penicillin Binding Proteins as Danger Signals: Meningococcal Penicillin Binding Protein 2 Activates Dendritic Cells through Toll-Like Receptor 4

Neisseria meningitidis is a human pathogen responsible for life-threatening inflammatory diseases. Meningococcal penicillin-binding proteins (PBPs) and particularly PBP2 are involved in bacterial resistance to β-lactams. Here we describe a novel function for PBP2 that activates human and mouse dendritic cells (DC) in a time and dose-dependent manner. PBP2 induces MHC II (LOGEC50 = 4.7 µg/ml±0.1), CD80 (LOGEC50 = 4.88 µg/ml±0.15) and CD86 (LOGEC50 = 5.36 µg/ml±0.1). This effect was abolished when DCs were co-treated with anti-PBP2 antibodies. PBP2-treated DCs displayed enhanced immunogenic properties in vitro and in vivo. Furthermore, proteins co-purified with PBP2 showed no effect on DC maturation. We show through different in vivo and in vitro approaches that this effect is not due to endotoxin contamination. At the mechanistic level, PBP2 induces nuclear localization of p65 NF-kB of 70.7±5.1% cells versus 12±2.6% in untreated DCs and needs TLR4 expression to mature DCs. Immunoprecipitation and blocking experiments showed tha

A Secreted NlpC/P60 Endopeptidase from Photobacterium damselae subsp. piscicida Cleaves the Peptidoglycan of Potentially Competing Bacteria

Peptidoglycan (PG) is a major component of the bacterial cell wall, forming a mesh-like structure enwrapping the bacteria that is essential for maintaining structural integrity and providing support for anchoring other components of the cell envelope. PG biogenesis is highly dynamic and requires multiple enzymes, including several hydrolases that cleave glycosidic or amide bonds in the PG. This work describes the structural and functional characterization of an NlpC/P60-contain-ing peptidase from Photobacterium damselae subsp. piscicida (Phdp), a Gram-negative bacterium that causes high mortality of warm-water marine fish with great impact for the aquaculture industry. PnpA (Photobacterium NlpC-like protein A) has a four-domain structure with a hydrophobic and narrow access to the catalytic center and specificity for the γ-D-glutamyl-meso-diaminopimelic acid bond. However, PnpA does not cleave the PG of Phdp or PG of several Gram-negative and Gram-positive bacterial species. Interestingly, it is secreted by the Phdp type II secretion system and degrades the PG of Vibrio anguillarum and Vibrio vulnificus. This suggests that PnpA is used by Phdp to gain an advantage over bacteria that compete for the same resources or to obtain nutrients in nutrient-scarce environments. Comparison of the muropeptide composition of PG susceptible and resistant to the catalytic activity of PnpA showed that the global content of muropeptides is similar, suggesting that susceptibility to PnpA is determined by the three-dimensional organization of the muropeptides in the PG. IMPORTANCE Peptidoglycan (PG) is a major component of the bacterial cell wall formed by long chains of two alternating sugars interconnected by short peptides, generating a mesh-like structure that enwraps the bacterial cell. Although PG provides structural integrity and support for anchoring other components of the cell envelope, it is constantly being remodeled through the action of specific enzymes that cleave or join its components. Here, it is shown that Photobacterium damselae subsp. piscicida, a bacterium that causes high mortality in warm-water marine fish, produces PnpA, an enzyme that is secreted into the environment and is able to cleave the PG of potentially competing bacteria, either to gain a competitive advantage and/or to obtain nutrients. The specificity of PnpA for the PG of some bacteria and its inability to cleave others may be explained by differences in the structure of the PG mesh and not by different muropeptide composition.Europeu de Desenvolvimento Regional (FEDER) funds through the COMPETE 2020 Operacional Program for Competitiveness and Internationalization (POCI), Portugal 2020, and by Portuguese funds through Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior and Fundação para a Ciência e a Tecnologia (FCT), I.P., within the scope of the Norma Transitória - DL57/2016/CP1355/CT0010. This work had also support from the State Agency for Research (AEI) of Spain cofunded by the FEDER Program from the European Union (grants AGL2016-79738-R and BIO2016-77639-P

From array-based hybridization of Helicobacter pylori isolates to the complete genome sequence of an isolate associated with MALT lymphoma

<p>Abstract</p> <p>Background</p> <p><it>elicobacter pylori </it>infection is associated with several gastro-duodenal inflammatory diseases of various levels of severity. To determine whether certain combinations of genetic markers can be used to predict the clinical source of the infection, we analyzed well documented and geographically homogenous clinical isolates using a comparative genomics approach.</p> <p>Results</p> <p>A set of 254 <it>H. pylori </it>genes was used to perform array-based comparative genomic hybridization among 120 French <it>H. pylori </it>strains associated with chronic gastritis (n = 33), duodenal ulcers (n = 27), intestinal metaplasia (n = 17) or gastric extra-nodal marginal zone B-cell MALT lymphoma (n = 43). Hierarchical cluster analyses of the DNA hybridization values allowed us to identify a homogeneous subpopulation of strains that clustered exclusively with <it>cag</it>PAI minus MALT lymphoma isolates. The genome sequence of B38, a representative of this MALT lymphoma strain-cluster, was completed, fully annotated, and compared with the six previously released <it>H. pylori </it>genomes (i.e. J99, 26695, HPAG1, P12, G27 and Shi470). B38 has the smallest <it>H. pylori </it>genome described thus far (1,576,758 base pairs containing 1,528 CDSs); it contains the <it>vacA</it>s2m2 allele and lacks the genes encoding the major virulence factors (absence of <it>cag</it>PAI, <it>bab</it>B, <it>bab</it>C, <it>sab</it>B, and <it>hom</it>B). Comparative genomics led to the identification of very few sequences that are unique to the B38 strain (9 intact CDSs and 7 pseudogenes). Pair-wise genomic synteny comparisons between B38 and the 6 <it>H. pylori </it>sequenced genomes revealed an almost complete co-linearity, never seen before between the genomes of strain Shi470 (a Peruvian isolate) and B38.</p> <p>Conclusion</p> <p>These isolates are deprived of the main <it>H. pylori </it>virulence factors characterized previously, but are nonetheless associated with gastric neoplasia.</p

Standardized Whole-Blood Transcriptional Profiling Enables the Deconvolution of Complex Induced Immune Responses

SummarySystems approaches for the study of immune signaling pathways have been traditionally based on purified cells or cultured lines. However, in vivo responses involve the coordinated action of multiple cell types, which interact to establish an inflammatory microenvironment. We employed standardized whole-blood stimulation systems to test the hypothesis that responses to Toll-like receptor ligands or whole microbes can be defined by the transcriptional signatures of key cytokines. We found 44 genes, identified using Support Vector Machine learning, that captured the diversity of complex innate immune responses with improved segregation between distinct stimuli. Furthermore, we used donor variability to identify shared inter-cellular pathways and trace cytokine loops involved in gene expression. This provides strategies for dimension reduction of large datasets and deconvolution of innate immune responses applicable for characterizing immunomodulatory molecules. Moreover, we provide an interactive R-Shiny application with healthy donor reference values for induced inflammatory genes

A Commensal Helicobacter sp. of the Rodent Intestinal Flora Activates TLR2 and NOD1 Responses in Epithelial Cells

Helicobacter spp. represent a proportionately small but significant component of the normal intestinal microflora of animal hosts. Several of these intestinal Helicobacter spp. are known to induce colitis in mouse models, yet the mechanisms by which these bacteria induce intestinal inflammation are poorly understood. To address this question, we performed in vitro co-culture experiments with mouse and human epithelial cell lines stimulated with a selection of Helicobacter spp., including known pathogenic species as well as ones for which the pathogenic potential is less clear. Strikingly, a member of the normal microflora of rodents, Helicobacter muridarum, was found to be a particularly strong inducer of CXC chemokine (Cxcl1/KC, Cxcl2/MIP-2) responses in a murine intestinal epithelial cell line. Time-course studies revealed a biphasic pattern of chemokine responses in these cells, with H. muridarum lipopolysaccharide (LPS) mediating early (24–48 h) responses and live bacteria seeming to provoke later (48–72 h) responses. H. muridarum LPS per se was shown to induce CXC chemokine production in HEK293 cells stably expressing Toll-like receptor 2 (TLR2), but not in those expressing TLR4. In contrast, live H. muridarum bacteria were able to induce NF-κB reporter activity and CXC chemokine responses in TLR2–deficient HEK293 and in AGS epithelial cells. These responses were attenuated by transient transfection with a dominant negative construct to NOD1, and by stable expression of NOD1 siRNA, respectively. Thus, the data suggest that both TLR2 and NOD1 may be involved in innate immune sensing of H. muridarum by epithelial cells. This work identifies H. muridarum as a commensal bacterium with pathogenic potential and underscores the potential roles of ill-defined members of the normal flora in the initiation of inflammation in animal hosts. We suggest that H. muridarum may act as a confounding factor in colitis model studies in rodents

Mammalian PGRPs in the Spotlight

Peptidoglycan-recognition proteins (PGRPs) play a central role in the insect innate immune response to bacteria. In this issue of Cell Host & Microbe, Saha et al. (2009) report that the mammalian PGRP, PGLYRP-2, functions as a cytokine-like molecule in a PG-induced arthritis model

Validation des hydrolases du peptidoglycane comme nouvelles cibles thérapeutiques contre l'infection à Helicobacter pylori

L infection à Helicobacter pylori touche près de 50 % de la population mondiale, et peut être associée à des pathologies sérieuses. De plus, l émergence de résistances bactériennes ne cesse d accroître le taux d échec thérapeutique, justifiant pleinement la recherche de nouvelles stratégies thérapeutiques. Dans ce sens, ce travail de thèse a eu pour objet de rechercher de nouvelles cibles thérapeutiques contre H. pylori, via la caractérisation d acteurs du métabolisme du peptidoglycane et de leur rôle dans la virulence bactérienne. Ainsi, nos travaux ont permis d une part, la caractérisation d une nouvelle peptidase bifonctionnelle du PG, HdpA, impliquée dans la virulence de H. pylori, via la régulation de sa forme bâtonnet, et d autre part, la mise en évidence de l action inhibitrice de la bulgécine sur la transglycosylase lytique Slt, engendrant une altération de la mobilité de H. pylori, ainsi qu une forte réduction de sa croissance par action synergique avec l amoxicilline.Helicobacter pylori is a major pathogen since it colonizes around 50 % of the world population and it can be associated to serious diseases. Moreover the emergence of bacterial resitances leads to more and more therapeutic failures, requiring the search for new therapeutic strategies. Consequently, the aim of that PhD work was to identify new therapeutic targets against H. pylori, via the characterization of peptidoglycan metabolism proteins, and their role in the bacterial virulence. Thereby, our work led firstly, to the characterization of a novel PG peptidase, involved in the virulence of H. pylori via its shape regulation, and secondly, to the study of the bulgecin, an inhibitor of the lytic transglycosylase Slt, provoking a bacterial motility defect and a strong growth decrease in presence of amoxicillin.CHATENAY M.-PARIS 11-BU Pharma. (920192101) / SudocSudocFranceF

Bacteria and MAMP-induced morphogenesis of the immune system.

To metazoans, bacteria are more than just potential pathogens. Many examples now document the role of bacterial symbionts in complementing the host for its full development and increasing its digestive and protective functions. Here, we discuss the role of Gram-negative bacteria in the development of intestinal lymphoid tissues and the impact of Segmented Filamentous Bacteria and Bacteroides on intestinal immunity and homeostasis. Furthermore, we discuss the potentially beneficial role of Helicobacter pylori on immunity of the stomach and beyond, even though this bacteria is primarily known for its pathogenicity. Altogether, these and other symbiotic bacteria may manipulate the host and the host immune system through the shedding of MAMPs that cross not only the epithelial barriers, but also permeate the circulation and impact every tissue and function of the host

Rôle des transglycosylases lytiques dans la mobilité chez Helicobacter pylori

La mobilité est essentielle à la survie de H. pylori. Elle repose sur des flagelles qui traversent le peptidoglycane (PG) pour leur assemblage. Le PG se compose de chaînes de sucres liées par des peptides. Il est modelé entre autres, par des transglycosylases lytiques (TGLs) qui sont deux chez H. pylori, Slt et MltD. Les mutants slt et mltD sont affectés en mobilité mais sont dotés de flagelles. Lors de cette thèse, nous avons caractérisé ce phénotype, et tenté d élucider le rôle de Slt et MltD dans la mobilité. Nous avons confirmé que l inactivation de slt et/ou mltD entraînait une perte de mobilité, mais sans affecter l assemblage du flagelle (ils sont intacts, bien localisés, en nombre similaire à la souche parentale). Selon la souche, Slt ou MltD joue un rôle prépondérant, qui ne peut être expliqué par des différences d expression. Nos données indiquent que leur activité même est impliquée dans la mobilité, et est conservée selon le fond génétique. Ce phénotype original nous suggère que Slt et MltD participeraient à la bonne fixation supposée de MotB (protéine du moteur flagellaire) au PG : par leur activité, ils créeraient les motifs sur lesquels se fixerait MotB. Ceci est conforté par la faible mobilité du mutant mltD en milieu liquide. L analyse du mutant aut de L. monocytogenes (Auto clive aussi les chaînes de sucres) révèle qu il est fortement affecté en mobilité malgré aussi des flagelles intacts. Nous proposons que Slt et MltD permettraient la génération de muropeptides comme ligands pour MotB. Ce rôle inédit pourrait être universel et serait tenu par Auto, une glucosaminidase, chez L. monocytogenes, en accord avec l absence d activité TGL chez cette espècePARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

The hidden base of the iceberg: gut peptidoglycome dynamics is foundational to its influence on the host

International audienceThe intestinal microbiota of humans includes a highly diverse range of bacterial species. All these bacteria possess a cell wall, composed primarily of the macromolecule peptidoglycan. As such, the gut also harbors an abundant and varied peptidoglycome. A remarkable range of host physiological pathways are regulated by peptidoglycan fragments that originate from the gut microbiota and enter the host system. Interactions between the host system and peptidoglycan can influence physiological development and homeostasis, promote health, or contribute to inflammatory disease. Underlying these effects is the interplay between microbiota composition and enzymatic processes that shape the intestinal peptidoglycome, dictating the types of peptidoglycan generated, that subsequently cross the gut barrier. In this review, we highlight and discuss the hidden and emerging functional aspects of the microbiome, i.e. the hidden base of the iceberg, that modulate the composition of gut peptidoglycan, and how these fundamental processes are drivers of physiological outcomes for the host.</div