The essential genome of Streptococcus agalactiae.

BACKGROUND: Next-generation sequencing of transposon-genome junctions from a saturated bacterial mutant library (Tn-seq) is a powerful tool that permits genome-wide determination of the contribution of genes to fitness of the organism under a wide range of experimental conditions. We report development, testing, and results from a Tn-seq system for use in Streptococcus agalactiae (group B Streptococcus; GBS), an important cause of neonatal sepsis. METHODS: Our method uses a Himar1 mini-transposon that inserts at genomic TA dinucleotide sites, delivered to GBS on a temperature-sensitive plasmid that is subsequently cured from the bacterial population. In order to establish the GBS essential genome, we performed Tn-seq on DNA collected from three independent mutant libraries-with at least 135,000 mutants per library-at serial 24 h time points after outgrowth in rich media. RESULTS: After statistical analysis of transposon insertion density and distribution, we identified 13.5 % of genes as essential and 1.2 % as critical, with high levels of reproducibility. Essential and critical genes are enriched for fundamental cellular housekeeping functions, such as acyl-tRNA biosynthesis, nucleotide metabolism, and glycolysis. We further validated our system by comparing fitness assignments of homologous genes in GBS and a close bacterial relative, Streptococcus pyogenes, which demonstrated 93 % concordance. Finally, we used our fitness assignments to identify signal transduction pathway components predicted to be essential or critical in GBS. CONCLUSIONS: We believe that our baseline fitness assignments will be a valuable tool for GBS researchers and that our system has the potential to reveal key pathogenesis gene networks and potential therapeutic/preventative targets.This work was supported by NIH/NIAID R01 AI092743, R33 AI098654, and R21 AI11020 to A.J.R.; NIH/NICHD K23 HD065844 to T.M.R.; John M. Driscoll, Jr., M.D. Children’s Fund (Columbia University Department of Pediatrics) and the Pediatric Scientist Development Program (NIH/NICHD K12 HD000850) to T.A.H

Rational manipulation of mRNA folding free energy allows rheostat control of pneumolysin production by Streptococcus pneumoniae

Rational manipulation of mRNA folding free energy allows rheostat control of pneumolysin production by Streptococcus pneumoniaeThe contribution of specific factors to bacterial virulence is generally investigated through creation of genetic "knockouts" that are then compared to wild-type strains or complemented mutants. This paradigm is useful to understand the effect of presence vs. absence of a specific gene product but cannot account for concentration-dependent effects, such as may occur with some bacterial toxins. In order to assess threshold and dose-response effects of virulence factors, robust systems for tunable expression are required. Recent evidence suggests that the folding free energy (?G) of the 5' end of mRNA transcripts can have a significant effect on translation efficiency and overall protein abundance. Here we demonstrate that rational alteration of 5' mRNA folding free energy by introduction of synonymous mutations allows for predictable changes in pneumolysin (PLY) expression by Streptococcus pneumoniae without the need for chemical inducers or heterologous promoters. We created a panel of isogenic S. pneumoniae strains, differing only in synonymous (silent) mutations at the 5' end of the PLY mRNA that are predicted to alter ?G. Such manipulation allows rheostat-like control of PLY production and alters the cytotoxicity of whole S. pneumoniae on primary and immortalized human cells. These studies provide proof-of-principle for further investigation of mRNA ?G manipulation as a tool in studies of bacterial pathogenesis.National Institutes of Health (www.nih.gov) (R01 AI092743 and R21 AI111020 to A.J.R.). F.E.A. was supported by the Portuguese Foundation for Science and Technology (www.fct.pt) SFRH/BD/33901/2009 and the Luso-American Development Foundation (www.flad.pt). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Generation of recombinant single-chain antibodies neutralizing the cytolytic activity of vaginolysin, the main virulence factor of Gardnerella vaginalis

Generated scFvs is the first example of recombinant single-chain antibodies with VLY-neutralizing activity produced in prokaryote expression system. G. vaginalis caused infections continue to be a world-wide problem, therefore neutralizing recombinant antibodies may provide novel therapeutic agents useful in the treatment of bacterial vaginosis and other diseases caused by G. vaginalis

Lipid analogs reveal features critical for hemolysis and diminish granadaene mediated Group B Streptococcus infection

Although certain microbial lipids are toxins, the structural features important for cytotoxicity remain unknown. Increased functional understanding is essential for developing therapeutics against toxic microbial lipids. Group B Streptococci (GBS) are bacteria associated with preterm births, stillbirths, and severe infections in neonates and adults. GBS produce a pigmented, cytotoxic lipid, known as granadaene. Despite its importance to all manifestations of GBS disease, studies towards understanding granadaene’s toxic activity are hindered by its instability and insolubility in purified form. Here, we report the synthesis and screening of lipid derivatives inspired by granadaene, which reveal features central to toxin function, namely the polyene chain length. Furthermore, we show that vaccination with a non-toxic synthetic analog confers the production of antibodies that inhibit granadaene-mediated hemolysis ex vivo and diminish GBS infection in vivo. This work provides unique structural and functional insight into granadaene and a strategy to mitigate GBS infection, which will be relevant to other toxic lipids encoded by human pathogens.This work was supported by funding from the National Institutes of Health Grants R01AI112619, R01AI133976, R01AI100989, and R21AI125907 and seed funds from Seattle Childrens Research Institute to L.

A mouse model reproducing the pathophysiology of neonatal group B streptococcal infection

Group B streptococcal (GBS) meningitis remains a devastating disease. The absence of an animal model reproducing the natural infectious process has limited our understanding of the disease and, consequently, delayed the development of effective treatments. We describe here a mouse model in which bacteria are transmitted to the offspring from vaginally colonised pregnant females, the natural route of infection. We show that GBS strain BM110, belonging to the CC17 clonal complex, is more virulent in this vertical transmission model than the isogenic mutant BM110∆cylE, which is deprived of hemolysin/cytolysin. Pups exposed to the more virulent strain exhibit higher mortality rates and lung inflammation than those exposed to the attenuated strain. Moreover, pups that survive to BM110 infection present neurological developmental disability, revealed by impaired learning performance and memory in adulthood. The use of this new mouse model, that reproduces key steps of GBS infection in newborns, will promote a better understanding of the physiopathology of GBS-induced meningitis.The authors gratefully acknowledge the help of Encarnaca̧ ̃o Ribeiro for excellent technical assistance, Joana Tavares for assisting with IVIS Lumina LT, Susana Roque for the luminex instrument experiments, the Molecular Microbiology group at i3S for microscope use, and the Portuguese architect and artist Gil Ferreira da Silva for the artworks included in the last figure. This work was supported by funds from Foundation for Science and Technology (FCT), European Regional Development Fund (FEDER) and Compete under project POCI-01-0145-FEDER-016607 (PTDC/IMI-MIC/1049/2014) and from the project NORTE-01-0145-FEDER-000012, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). T.S. and A.M. were supported by Investigador FCT (IF/00875/2012 and IF/00753/2014), POPH and Fundo Social Europeu. E.B.A. and C.C.P. hold postdoctoral fellowships from FCT (PTDC/IMI-MIC/1049/2014 and SFRH/BPD/91962/2012). Ar.F. and P.T.C. were supported by Laboratoire d’Excellence (LABEX) Integrative Biology of Emerging Infectious Diseases (grant ANR-10-LABX-62-IBEID).info:eu-repo/semantics/publishedVersio

Human-specific bacterial pore-forming toxins induce programmed necrosis in erythrocytes.

UNLABELLED: A subgroup of the cholesterol-dependent cytolysin (CDC) family of pore-forming toxins (PFTs) has an unusually narrow host range due to a requirement for binding to human CD59 (hCD59), a glycosylphosphatidylinositol (GPI)-linked complement regulatory molecule. hCD59-specific CDCs are produced by several organisms that inhabit human mucosal surfaces and can act as pathogens, including Gardnerella vaginalis and Streptococcus intermedius. The consequences and potential selective advantages of such PFT host limitation have remained unknown. Here, we demonstrate that, in addition to species restriction, PFT ligation of hCD59 triggers a previously unrecognized pathway for programmed necrosis in primary erythrocytes (red blood cells [RBCs]) from humans and transgenic mice expressing hCD59. Because they lack nuclei and mitochondria, RBCs have typically been thought to possess limited capacity to undergo programmed cell death. RBC programmed necrosis shares key molecular factors with nucleated cell necroptosis, including dependence on Fas/FasL signaling and RIP1 phosphorylation, necrosome assembly, and restriction by caspase-8. Death due to programmed necrosis in RBCs is executed by acid sphingomyelinase-dependent ceramide formation, NADPH oxidase- and iron-dependent reactive oxygen species formation, and glycolytic formation of advanced glycation end products. Bacterial PFTs that are hCD59 independent do not induce RBC programmed necrosis. RBC programmed necrosis is biochemically distinct from eryptosis, the only other known programmed cell death pathway in mature RBCs. Importantly, RBC programmed necrosis enhances the growth of PFT-producing pathogens during exposure to primary RBCs, consistent with a role for such signaling in microbial growth and pathogenesis. IMPORTANCE: In this work, we provide the first description of a new form of programmed cell death in erythrocytes (RBCs) that occurs as a consequence of cellular attack by human-specific bacterial toxins. By defining a new RBC death pathway that shares important components with necroptosis, a programmed necrosis module that occurs in nucleated cells, these findings expand our understanding of RBC biology and RBC-pathogen interactions. In addition, our work provides a link between cholesterol-dependent cytolysin (CDC) host restriction and promotion of bacterial growth in the presence of RBCs, which may provide a selective advantage to human-associated bacterial strains that elaborate such toxins and a potential explanation for the narrowing of host range observed in this toxin family

Loss of phosphoinositide 3-kinase γ decreases migration and activation of phagocytes but not T cell activation in antigen-induced arthritis

<p>Abstract</p> <p>Background</p> <p>Phosphoinositide 3-kinase γ (PI3Kγ) has been depicted as a major regulator of inflammatory processes, including leukocyte activation and migration towards several chemokines. This study aims to explore the role of PI3Kγ in the murine model of antigen-induced arthritis (AIA).</p> <p>Methods</p> <p>Development of AIA was investigated in wildtype and PI3Kγ-deficient mice as well as in mice treated with a specific inhibitor of PI3Kγ (AS-605240) in comparison to untreated animals. Inflammatory reactions of leukocytes, including macrophage and T cell activation, and macrophage migration, were studied <it>in vivo </it>and <it>in vitro</it>.</p> <p>Results</p> <p>Genetic deletion or pharmacological inhibition of PI3Kγ induced a marked decrease of clinical symptoms in early AIA, together with a considerably diminished macrophage migration and activation (lower production of NO, IL-1β, IL-6). Also, macrophage and neutrophil infiltration into the knee joint were impaired <it>in vivo</it>. However, T cell functions, measured by cytokine production (TNFα, IFNγ, IL-2, IL-4, IL-5, IL-17) <it>in vitro </it>and DTH reaction <it>in vivo </it>were not altered, and accordingly, disease developed normally at later timepoints</p> <p>Conclusion</p> <p>PI3Kγ specifically affects phagocyte function in the AIA model but has no impact on T cell activation.</p