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1.
FEBS J ; 290(11): 2968-2992, 2023 06.
Article in English | MEDLINE | ID: mdl-36629470

ABSTRACT

Cyclic di-AMP is an essential signalling molecule in Gram-positive bacteria. This second messenger regulates the osmotic pressure of the cell by interacting directly with the regulatory domains, either RCK_C or CBS domains, of several potassium and osmolyte uptake membrane protein systems. Cyclic di-AMP also targets stand-alone CBS domain proteins such as DarB in Bacillus subtilis and CbpB in Listeria monocytogenes. We show here that the CbpB protein of Group B Streptococcus binds c-di-AMP with a very high affinity. Crystal structures of CbpB reveal the determinants of binding specificity and significant conformational changes occurring upon c-di-AMP binding. Deletion of the cbpB gene alters bacterial growth in low potassium conditions most likely due to a decrease in the amount of ppGpp caused by a loss of interaction between CbpB and Rel, the GTP/GDP pyrophosphokinase.


Subject(s)
Carrier Proteins , Streptococcus agalactiae , Streptococcus agalactiae/genetics , Streptococcus agalactiae/metabolism , Guanosine Pentaphosphate , Guanosine Tetraphosphate , Bacterial Proteins/metabolism , Cyclic AMP , Dinucleoside Phosphates/metabolism , Potassium/metabolism
2.
Front Microbiol ; 13: 881549, 2022.
Article in English | MEDLINE | ID: mdl-35572655

ABSTRACT

Group B Streptococcus (GBS) is a Gram-positive bacterium able to switch from a harmless commensal of healthy adults to a pathogen responsible for invasive infections in neonates. The signals and regulatory mechanisms governing this transition are still largely unknown. CodY is a highly conserved global transcriptional regulator that links nutrient availability to the regulation of major metabolic and virulence pathways in low-G+C Gram-positive bacteria. In this work, we investigated the role of CodY in BM110, a GBS strain representative of a hypervirulent lineage associated with the majority of neonatal meningitis. Deletion of codY resulted in a reduced ability of the mutant strain to cause infections in neonatal and adult animal models. The observed decreased in vivo lethality was associated with an impaired ability of the mutant to persist in the blood, spread to distant organs, and cross the blood-brain barrier. Notably, the codY null mutant showed reduced adhesion to monolayers of human epithelial cells in vitro and an increased ability to form biofilms, a phenotype associated with strains able to asymptomatically colonize the host. RNA-seq analysis showed that CodY controls about 13% of the genome of GBS, acting mainly as a repressor of genes involved in amino acid transport and metabolism and encoding surface anchored proteins, including the virulence factor Srr2. CodY activity was shown to be dependent on the availability of branched-chain amino acids, which are the universal cofactors of this regulator. These results highlight a key role for CodY in the control of GBS virulence.

3.
PLoS Genet ; 17(9): e1009761, 2021 09.
Article in English | MEDLINE | ID: mdl-34491998

ABSTRACT

Virulence of the neonatal pathogen Group B Streptococcus is under the control of the master regulator CovR. Inactivation of CovR is associated with large-scale transcriptome remodeling and impairs almost every step of the interaction between the pathogen and the host. However, transcriptome analyses suggested a plasticity of the CovR signaling pathway in clinical isolates leading to phenotypic heterogeneity in the bacterial population. In this study, we characterized the CovR regulatory network in a strain representative of the CC-17 hypervirulent lineage responsible of the majority of neonatal meningitis. Transcriptome and genome-wide binding analysis reveal the architecture of the CovR network characterized by the direct repression of a large array of virulence-associated genes and the extent of co-regulation at specific loci. Comparative functional analysis of the signaling network links strain-specificities to the regulation of the pan-genome, including the two specific hypervirulent adhesins and horizontally acquired genes, to mutations in CovR-regulated promoters, and to variability in CovR activation by phosphorylation. This regulatory adaptation occurs at the level of genes, promoters, and of CovR itself, and allows to globally reshape the expression of virulence genes. Overall, our results reveal the direct, coordinated, and strain-specific regulation of virulence genes by the master regulator CovR and suggest that the intra-species evolution of the signaling network is as important as the expression of specific virulence factors in the emergence of clone associated with specific diseases.


Subject(s)
Bacterial Proteins/physiology , Gene Regulatory Networks , Streptococcus agalactiae/pathogenicity , Virulence Factors/physiology , Virulence/genetics , Bacterial Proteins/genetics , Chromosomes, Bacterial , Genes, Bacterial , Host-Pathogen Interactions , Humans , Promoter Regions, Genetic , Prophages/genetics , Streptococcus agalactiae/genetics , Transcription, Genetic/physiology , Virulence Factors/genetics
4.
Am J Trop Med Hyg ; 105(5): 1339-1346, 2021 08 30.
Article in English | MEDLINE | ID: mdl-34460418

ABSTRACT

Maternal group B Streptococcus (GBS) colonization is a major risk factor for neonatal GBS infection. However, data on GBS are scarce in low- and middle-income countries. Using sociodemographic data and vaginal swabs collected from an international cohort of mothers and newborns, this study aimed to estimate the prevalence of GBS colonization among pregnant women in Madagascar (n = 1,603) and Senegal (n = 616). The prevalence was 5.0% (95% CI, 3.9-6.1) and 16.1% (95% CI, 13.1-19.0) in Madagascar and Senegal, respectively. No factors among sociodemographic characteristics, living conditions, and obstetric history were found to be associated independently with GBS colonization in both countries. This community-based study provides one of the first estimates of maternal GBS colonization among pregnant women from Madagascar and Senegal.


Subject(s)
Maternal Exposure/statistics & numerical data , Mothers/statistics & numerical data , Pregnancy Complications, Infectious/diagnosis , Pregnancy Complications, Infectious/epidemiology , Streptococcal Infections/diagnosis , Streptococcal Infections/epidemiology , Streptococcus/isolation & purification , Adult , Cohort Studies , Female , Humans , Infant, Newborn , Madagascar/epidemiology , Population Surveillance , Pregnancy , Pregnant Women , Prevalence , Senegal/epidemiology
5.
Sci Rep ; 8(1): 14322, 2018 09 25.
Article in English | MEDLINE | ID: mdl-30254272

ABSTRACT

Streptococcus agalactiae (Group B Streptococcus or GBS) is a frequent cause of serious disease in newborns and adults. Epidemiological evidence indicates a strong association between GBS strains belonging to the hypervirulent CC17 clonal complex and the occurrence of meningitis in neonates. We investigate here the role of PbsP, a cell wall plasminogen binding protein, in colonization of the central nervous system by CC17 GBS. Deletion of pbsP selectively impaired the ability of the CC17 strain BM110 to colonize the mouse brain after intravenous challenge, despite its unchanged capacity to persist at high levels in the blood and to invade the kidneys. Moreover, immunization with a recombinant form of PbsP considerably reduced brain infection and lethality. In vitro, pbsP deletion markedly decreased plasmin-dependent transmigration of BM110 through brain microvascular endothelial cells. Although PbsP was modestly expressed in bacteria grown under standard laboratory conditions, pbsP expression was markedly upregulated during in vivo infection or upon contact with cultured brain endothelial cells. Collectively, our studies indicate that PbsP is a highly conserved Plg binding adhesin, which is functionally important for invasion of the central nervous system by the hypervirulent CC17 GBS. Moreover, this antigen is a promising candidate for inclusion in a universal GBS vaccine.


Subject(s)
Bacterial Proteins/metabolism , Brain/microbiology , Streptococcus agalactiae/metabolism , Streptococcus agalactiae/pathogenicity , Animals , Brain/cytology , Cell Movement , Endothelial Cells/cytology , Fibrinolysin/metabolism , Gene Expression Regulation, Bacterial , Mice , Streptococcus agalactiae/genetics , Virulence
6.
Nat Commun ; 9(1): 3138, 2018 08 07.
Article in English | MEDLINE | ID: mdl-30087335

ABSTRACT

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.


Subject(s)
Disease Models, Animal , Infectious Disease Transmission, Vertical , Streptococcal Infections/physiopathology , Animals , Animals, Newborn , Behavior, Animal , Body Weight , Female , Hemolysin Proteins/chemistry , Inflammation , Male , Maze Learning , Meningitis/microbiology , Meningitis, Bacterial , Mice , Mice, Inbred BALB C , Perforin/chemistry , Pregnancy , Pregnancy, Animal , Streptococcal Infections/transmission , Streptococcus agalactiae/pathogenicity , Vagina/microbiology
7.
Mol Microbiol ; 110(1): 82-94, 2018 10.
Article in English | MEDLINE | ID: mdl-30030946

ABSTRACT

Binding of microbial pathogens to host vitronectin (Vtn) is a common theme in the pathogenesis of invasive infections. In this study, we characterized the role of Vtn in the invasion of mucosal epithelial cells by Streptococcus agalactiae (i.e. group B streptococcus or GBS), a frequent human pathogen. Moreover, we identified PbsP, a previously described plasminogen-binding protein of GBS, as a dual adhesin that can also interact with human Vtn through its streptococcal surface repeat (SSURE) domains. Deletion of the pbsP gene decreases both bacterial adhesion to Vtn-coated inert surfaces and the ability of GBS to interact with epithelial cells. Bacterial adherence to and invasion of epithelial cells were either inhibited or enhanced by cell pretreatment with, respectively, anti-Vtn antibodies or Vtn, confirming the role of Vtn as a GBS ligand on host cells. Finally, antibodies directed against the integrin αv subunit inhibited Vtn-dependent cell invasion by GBS. Collectively, these results indicate that Vtn acts as a bridge between the SSURE domains of PbsP on the GBS surface and host integrins to promote bacterial invasion of epithelial cells. Therefore, inhibition of interactions between PbsP and extracellular matrix components could represent a viable strategy to prevent colonization and invasive disease by GBS.


Subject(s)
Bacterial Proteins/metabolism , Integrin alphaV/metabolism , Streptococcal Infections/microbiology , Streptococcus agalactiae/metabolism , Streptococcus agalactiae/pathogenicity , Vitronectin/metabolism , A549 Cells , Bacterial Adhesion/genetics , Bacterial Proteins/genetics , Caco-2 Cells , Cell Wall/metabolism , Epithelial Cells/microbiology , Humans , Integrin alphaV/genetics , Protein Domains , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Streptococcus agalactiae/genetics , Vitronectin/genetics
8.
PLoS Genet ; 14(4): e1007342, 2018 04.
Article in English | MEDLINE | ID: mdl-29659565

ABSTRACT

Cyclic nucleotides are universally used as secondary messengers to control cellular physiology. Among these signalling molecules, cyclic di-adenosine monophosphate (c-di-AMP) is a specific bacterial second messenger recognized by host cells during infections and its synthesis is assumed to be necessary for bacterial growth by controlling a conserved and essential cellular function. In this study, we sought to identify the main c-di-AMP dependent pathway in Streptococcus agalactiae, the etiological agent of neonatal septicaemia and meningitis. By conditionally inactivating dacA, the only diadenyate cyclase gene, we confirm that c-di-AMP synthesis is essential in standard growth conditions. However, c-di-AMP synthesis becomes rapidly dispensable due to the accumulation of compensatory mutations. We identified several mutations restoring the viability of a ΔdacA mutant, in particular a loss-of-function mutation in the osmoprotectant transporter BusAB. Identification of c-di-AMP binding proteins revealed a conserved set of potassium and osmolyte transporters, as well as the BusR transcriptional factor. We showed that BusR negatively regulates busAB transcription by direct binding to the busAB promoter. Loss of BusR repression leads to a toxic busAB expression in absence of c-di-AMP if osmoprotectants, such as glycine betaine, are present in the medium. In contrast, deletion of the gdpP c-di-AMP phosphodiesterase leads to hyperosmotic susceptibility, a phenotype dependent on a functional BusR. Taken together, we demonstrate that c-di-AMP is essential for osmotic homeostasis and that the predominant mechanism is dependent on the c-di-AMP binding transcriptional factor BusR. The regulation of osmotic homeostasis is likely the conserved and essential function of c-di-AMP, but each species has evolved specific c-di-AMP mechanisms of osmoregulation to adapt to its environment.


Subject(s)
Dinucleoside Phosphates/metabolism , Osmoregulation/physiology , Streptococcus agalactiae/metabolism , ATP-Binding Cassette Transporters/genetics , ATP-Binding Cassette Transporters/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Genes, Bacterial , Homeostasis/physiology , Host-Pathogen Interactions/physiology , Humans , Mutation , Osmoregulation/genetics , Phosphorus-Oxygen Lyases/genetics , Phosphorus-Oxygen Lyases/metabolism , Potassium/metabolism , Second Messenger Systems/physiology , Streptococcus agalactiae/genetics , Streptococcus agalactiae/growth & development
9.
Curr Opin Microbiol ; 41: 21-28, 2018 02.
Article in English | MEDLINE | ID: mdl-29169058

ABSTRACT

Cyclic di-AMP (c-di-AMP) is a bacterial signaling nucleotide synthesized by several human pathogens. This widespread and specific bacterial product is recognized by infected host cells to trigger an innate immune response. Detection of c-di-AMP in the host cytosol leads primarily to the induction of type I interferon via the STING-cGAS signaling axis, while being also entangled in the activation of the NF-κB pathway. During their long-standing interaction, host and pathogens have co-evolved to control c-di-AMP activation of innate immunity. On the bacterial side, the quantity of c-di-AMP released inside cells allows to manipulate the host response to exacerbate infection by avoiding immune recognition or, at the opposite, by overloading the STING-cGAS pathway.


Subject(s)
Bacteria/metabolism , Cyclic AMP/metabolism , Host-Pathogen Interactions/physiology , Animals , Bacteria/growth & development , Bacteria/immunology , Bacteria/pathogenicity , Cyclic AMP/biosynthesis , Host-Pathogen Interactions/immunology , Humans , Immunity, Innate , Macrophages/immunology , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Nucleotidyltransferases/metabolism , Signal Transduction/immunology , Signal Transduction/physiology
10.
Cell Host Microbe ; 20(1): 49-59, 2016 Jul 13.
Article in English | MEDLINE | ID: mdl-27414497

ABSTRACT

Induction of type I interferon (IFN) in response to microbial pathogens depends on a conserved cGAS-STING signaling pathway. The presence of DNA in the cytoplasm activates cGAS, while STING is activated by cyclic dinucleotides (cdNs) produced by cGAS or from bacterial origins. Here, we show that Group B Streptococcus (GBS) induces IFN-ß production almost exclusively through cGAS-STING-dependent recognition of bacterial DNA. However, we find that GBS expresses an ectonucleotidase, CdnP, which hydrolyzes extracellular bacterial cyclic-di-AMP. Inactivation of CdnP leads to c-di-AMP accumulation outside the bacteria and increased IFN-ß production. Higher IFN-ß levels in vivo increase GBS killing by the host. The IFN-ß overproduction observed in the absence of CdnP is due to the cumulative effect of DNA sensing by cGAS and STING-dependent sensing of c-di-AMP. These findings describe the importance of a bacterial c-di-AMP ectonucleotidase and suggest a direct bacterial mechanism that dampens activation of the cGAS-STING axis.


Subject(s)
Dinucleoside Phosphates/metabolism , Immune Evasion , Interferon Type I/metabolism , Membrane Proteins/metabolism , Pyrophosphatases/metabolism , Streptococcus agalactiae/immunology , Streptococcus agalactiae/metabolism , Biotransformation , Streptococcus agalactiae/enzymology
12.
Mol Microbiol ; 101(1): 27-41, 2016 07.
Article in English | MEDLINE | ID: mdl-26888569

ABSTRACT

Streptococcus agalactiae (Group B Streptococcus or GBS) is a leading cause of invasive infections in neonates whose virulence is dependent on its ability to interact with cells and host components. We here characterized a surface protein with a critical function in GBS pathophysiology. This adhesin, designated PbsP, possesses two Streptococcal Surface Repeat domains, a methionine and lysine-rich region, and a LPXTG cell wall-anchoring motif. PbsP mediates plasminogen (Plg) binding both in vitro and in vivo and we showed that cell surface-bound Plg can be activated into plasmin by tissue plasminogen activator to increase the bacterial extracellular proteolytic activity. Absence of PbsP results in a decreased bacterial transmigration across brain endothelial cells and impaired virulence in a murine model of infection. PbsP is conserved among the main GBS lineages and is a major plasminogen adhesin in non-CC17 GBS strains. Importantly, immunization of mice with recombinant PbsP confers protective immunity. Our results indicate that GBS have evolved different strategies to recruit Plg which indicates that the ability to acquire cell surface proteolytic activity is essential for the invasiveness of this bacterium.


Subject(s)
Adhesins, Bacterial/metabolism , Plasminogen/metabolism , Streptococcus agalactiae/metabolism , Amino Acid Sequence , Animals , Bacterial Adhesion/physiology , Cell Wall/metabolism , Endothelial Cells/metabolism , Fibrinolysin/metabolism , Humans , Mice , Protein Binding , Streptococcal Infections/microbiology , Streptococcus/metabolism , Streptococcus agalactiae/genetics , Streptococcus agalactiae/pathogenicity , Virulence
13.
J Clin Microbiol ; 54(1): 75-82, 2016 01.
Article in English | MEDLINE | ID: mdl-26491182

ABSTRACT

Group B Streptococcus (GBS) is a common commensal bacterium in adults, but is also the leading cause of invasive bacterial infections in neonates in developed countries. The ß-hemolysin/cytolysin (ß-h/c), which is always associated with the production of an orange-to-red pigment, is a major virulence factor that is also used for GBS diagnosis. A collection of 1,776 independent clinical GBS strains isolated in France between 2006 and 2013 was evaluated on specific medium for ß-h/c activity and pigment production. The genomic sequences of nonhemolytic and nonpigmented (NH/NP) strains were analyzed to identify the molecular basis of this phenotype. Gene deletions or complementations were carried out to confirm the genotype-phenotype association. Sixty-three GBS strains (3.5%) were NH/NP, and 47 of these (74.6%) originated from invasive infections, including bacteremia and meningitis, in neonates or adults. The mutations are localized predominantly in the cyl operon, encoding the ß-h/c pigment biosynthetic pathway and, in the abx1 gene, encoding a CovSR regulator partner. In conclusion, although usually associated with GBS virulence, ß-h/c pigment production is not absolutely required to cause human invasive infections. Caution should therefore be taken in the use of hemolysis and pigmentation as criteria for GBS diagnosis in routine clinical laboratory settings.


Subject(s)
Hemolysin Proteins/analysis , Pigments, Biological/analysis , Streptococcal Infections/microbiology , Streptococcus agalactiae/genetics , Streptococcus agalactiae/isolation & purification , Adult , Bacteriological Techniques , Culture Media/chemistry , France/epidemiology , Gene Deletion , Genetic Association Studies , Genetic Complementation Test , Genome, Bacterial , Humans , Infant, Newborn , Sequence Analysis, DNA , Streptococcal Infections/epidemiology , Streptococcus agalactiae/classification
14.
PLoS Pathog ; 10(12): e1004542, 2014 Dec.
Article in English | MEDLINE | ID: mdl-25502890

ABSTRACT

Biofilm formation is an important virulence trait of the pathogenic yeast Candida albicans. We have combined gene overexpression, strain barcoding and microarray profiling to screen a library of 531 C. albicans conditional overexpression strains (∼10% of the genome) for genes affecting biofilm development in mixed-population experiments. The overexpression of 16 genes increased strain occupancy within a multi-strain biofilm, whereas overexpression of 4 genes decreased it. The set of 16 genes was significantly enriched for those encoding predicted glycosylphosphatidylinositol (GPI)-modified proteins, namely Ihd1/Pga36, Phr2, Pga15, Pga19, Pga22, Pga32, Pga37, Pga42 and Pga59; eight of which have been classified as pathogen-specific. Validation experiments using either individually- or competitively-grown overexpression strains revealed that the contribution of these genes to biofilm formation was variable and stage-specific. Deeper functional analysis of PGA59 and PGA22 at a single-cell resolution using atomic force microscopy showed that overexpression of either gene increased C. albicans ability to adhere to an abiotic substrate. However, unlike PGA59, PGA22 overexpression led to cell cluster formation that resulted in increased sensitivity to shear forces and decreased ability to form a single-strain biofilm. Within the multi-strain environment provided by the PGA22-non overexpressing cells, PGA22-overexpressing cells were protected from shear forces and fitter for biofilm development. Ultrastructural analysis, genome-wide transcript profiling and phenotypic analyses in a heterologous context suggested that PGA22 affects cell adherence through alteration of cell wall structure and/or function. Taken together, our findings reveal that several novel predicted GPI-modified proteins contribute to the cooperative behaviour between biofilm cells and are important participants during C. albicans biofilm formation. Moreover, they illustrate the power of using signature tagging in conjunction with gene overexpression for the identification of novel genes involved in processes pertaining to C. albicans virulence.


Subject(s)
Biofilms/growth & development , Candida albicans/physiology , Cell Wall/physiology , Fungal Proteins/physiology , Proteome/physiology , Candida albicans/cytology , Cell Adhesion/physiology , Fungal Proteins/genetics , Gene Expression Regulation, Fungal/physiology , Phenotype , Proteome/genetics , Shear Strength/physiology , Transcriptome/physiology
15.
J Biol Chem ; 289(30): 21003-21015, 2014 Jul 25.
Article in English | MEDLINE | ID: mdl-24904056

ABSTRACT

Streptococcus agalactiae (group B Streptococcus or GBS) is a common cause of invasive infections in newborn infants and adults. The ability of GBS to bind human fibrinogen is of crucial importance in promoting colonization and invasion of host barriers. We characterized here a novel fibrinogen-binding protein of GBS, designated FbsC (Gbs0791), which is encoded by the prototype GBS strain NEM316. FbsC, which bears two bacterial immunoglobulin-like tandem repeat domains and a C-terminal cell wall-anchoring motif (LPXTG), was found to be covalently linked to the cell wall by the housekeeping sortase A. Studies using recombinant FbsC indicated that it binds fibrinogen in a dose-dependent and saturable manner, and with moderate affinity. Expression of FbsC was detected in all clinical GBS isolates, except those belonging to the hypervirulent lineage ST17. Deletion of fbsC decreases NEM316 abilities to adhere to and invade human epithelial and endothelial cells, and to form biofilm in vitro. Notably, bacterial adhesion to fibrinogen and fibrinogen binding to bacterial cells were abolished following fbsC deletion in NEM316. Moreover, the virulence of the fbsC deletion mutant and its ability to colonize the brain were impaired in murine models of infection. Finally, immunization with recombinant FbsC significantly protected mice from lethal GBS challenge. In conclusion, FbsC is a novel fibrinogen-binding protein expressed by most GBS isolates that functions as a virulence factor by promoting invasion of epithelial and endothelial barriers. In addition, the protein has significant immunoprotective activity and may be a useful component of an anti-GBS vaccine.


Subject(s)
Bacterial Proteins/immunology , Fibrinogen/immunology , Host-Pathogen Interactions/immunology , Streptococcal Infections/immunology , Streptococcus agalactiae/physiology , Virulence Factors/immunology , Animals , Bacterial Adhesion/genetics , Bacterial Adhesion/immunology , Bacterial Proteins/genetics , Caco-2 Cells , Disease Models, Animal , Endothelial Cells/immunology , Endothelial Cells/microbiology , Endothelial Cells/pathology , Epithelial Cells/immunology , Epithelial Cells/microbiology , Epithelial Cells/pathology , Fibrinogen/genetics , Humans , Mice , Protein Binding/genetics , Protein Binding/immunology , Streptococcal Infections/genetics , Streptococcal Vaccines/genetics , Streptococcal Vaccines/immunology , Virulence Factors/genetics
16.
PLoS Pathog ; 10(6): e1004211, 2014 Jun.
Article in English | MEDLINE | ID: mdl-24945925

ABSTRACT

The opportunistic fungal pathogen Candida glabrata is a frequent cause of candidiasis, causing infections ranging from superficial to life-threatening disseminated disease. The inherent tolerance of C. glabrata to azole drugs makes this pathogen a serious clinical threat. To identify novel genes implicated in antifungal drug tolerance, we have constructed a large-scale C. glabrata deletion library consisting of 619 unique, individually bar-coded mutant strains, each lacking one specific gene, all together representing almost 12% of the genome. Functional analysis of this library in a series of phenotypic and fitness assays identified numerous genes required for growth of C. glabrata under normal or specific stress conditions, as well as a number of novel genes involved in tolerance to clinically important antifungal drugs such as azoles and echinocandins. We identified 38 deletion strains displaying strongly increased susceptibility to caspofungin, 28 of which encoding proteins that have not previously been linked to echinocandin tolerance. Our results demonstrate the potential of the C. glabrata mutant collection as a valuable resource in functional genomics studies of this important fungal pathogen of humans, and to facilitate the identification of putative novel antifungal drug target and virulence genes.


Subject(s)
Antifungal Agents/pharmacology , Azoles/pharmacology , Candida glabrata/genetics , Drug Resistance, Fungal/genetics , Echinocandins/pharmacology , Biofilms/growth & development , Candida glabrata/growth & development , Candidiasis/drug therapy , Caspofungin , Cell Wall/drug effects , Cell Wall/genetics , Fungal Proteins/genetics , Gene Deletion , Gene Knockout Techniques , Gene Library , Lipopeptides , Microbial Sensitivity Tests , Osmotic Pressure , Phenotype
17.
J Biol Chem ; 289(20): 13701-5, 2014 May 16.
Article in English | MEDLINE | ID: mdl-24692555

ABSTRACT

The inflammatory cytokine IL-1ß is critical for host responses against many human pathogens. Here, we define Group B Streptococcus (GBS)-mediated activation of the Nod-like receptor-P3 (NLRP3) inflammasome in macrophages. NLRP3 activation requires GBS expression of the cytolytic toxin, ß-hemolysin, lysosomal acidification, and leakage. These processes allow the interaction of GBS RNA with cytosolic NLRP3. The present study supports a model in which GBS RNA, along with lysosomal components including cathepsins, leaks out of lysosomes and interacts with NLRP3 to induce IL-1ß production.


Subject(s)
Bacterial Proteins/metabolism , Carrier Proteins/metabolism , Hemolysin Proteins/metabolism , Inflammasomes/metabolism , Interleukin-1beta/biosynthesis , Macrophages/metabolism , RNA, Bacterial/metabolism , Streptococcus agalactiae/physiology , Animals , Humans , Interleukin-1beta/metabolism , Lysosomes/metabolism , Lysosomes/microbiology , Macrophages/cytology , Macrophages/microbiology , Mice , NLR Family, Pyrin Domain-Containing 3 Protein , Phagosomes/metabolism , Phagosomes/microbiology , Streptococcus agalactiae/metabolism
18.
J Biol Chem ; 289(9): 5479-89, 2014 Feb 28.
Article in English | MEDLINE | ID: mdl-24429288

ABSTRACT

Streptococcus agalactiae (Group B Streptococcus) is a commensal of the human intestine and vagina of adult women but is the leading cause of invasive infection in neonates. This Gram-positive bacterium displays a set of virulence-associated surface proteins involved in the interaction with the host, such as adhesion to host cells, invasion of tissues, or subversion of the immune system. In this study, we characterized a cell wall-localized protein as an ecto-5'-nucleoside diphosphate phosphohydrolase (NudP) involved in the degradation of extracellular nucleotides which are central mediators of the immune response. Biochemical characterization of recombinant NudP revealed a Mn(2+)-dependent ecto-5'-nucleotidase activity on ribo- and deoxyribonucleoside 5'-mono- and 5'-diphosphates with a substrate specificity different from that of known orthologous enzymes. Deletion of the gene coding the housekeeping enzyme sortase A led to the release of NudP into the culture supernatant, confirming that this enzyme is anchored to the cell wall by its non-canonical LPXTN motif. The NudP ecto-5'-nucleotidase activity is reminiscent of the reactions performed by the mammalian ectonucleotidases CD39 and CD73 involved in regulating the extracellular level of ATP and adenosine. We further demonstrated that the absence of NudP activity decreases bacterial survival in mouse blood, a process dependent on extracellular adenosine. In vivo assays in animal models of infection showed that NudP activity is critical for virulence. These results demonstrate that Group B Streptococcus expresses a specific ecto-5'-nucleotidase necessary for its pathogenicity and highlight the diversity of reactions performed by this enzyme family. These results suggest that bacterial pathogens have developed specialized strategies to subvert the mammalian immune response controlled by the extracellular nucleotide signaling pathways.


Subject(s)
Adenosine/metabolism , Microbial Viability , N-Glycosyl Hydrolases/metabolism , Streptococcus agalactiae/enzymology , Adenosine/genetics , Amino Acid Motifs , Animals , Female , Humans , Mice , Mice, Inbred BALB C , N-Glycosyl Hydrolases/genetics , N-Glycosyl Hydrolases/immunology , Rats , Rats, Sprague-Dawley , Signal Transduction/physiology , Streptococcus agalactiae/genetics , Streptococcus agalactiae/immunology
19.
J Biol Chem ; 288(19): 13387-96, 2013 May 10.
Article in English | MEDLINE | ID: mdl-23508952

ABSTRACT

BACKGROUND: SUN proteins are involved in yeast morphogenesis, but their function is unknown. RESULTS: SUN protein plays a role in the Aspergillus fumigatus morphogenesis. Biochemical properties of recombinant SUN proteins were elucidated. CONCLUSION: Both Candida albicans and Aspergillus fumigatus sun proteins show a ß-(1,3)-glucanase activity. SIGNIFICANCE: The mode of action of SUN proteins on ß-(1,3)-glucan is unique, new, and original. In yeasts, the family of SUN proteins has been involved in cell wall biogenesis. Here, we report the characterization of SUN proteins in a filamentous fungus, Aspergillus fumigatus. The function of the two A. fumigatus SUN genes was investigated by combining reverse genetics and biochemistry. During conidial swelling and mycelial growth, the expression of AfSUN1 was strongly induced, whereas the expression of AfSUN2 was not detectable. Deletion of AfSUN1 negatively affected hyphal growth and conidiation. A closer examination of the morphological defects revealed swollen hyphae, leaky tips, intrahyphal growth, and double cell wall, suggesting that, like in yeast, AfSun1p is associated with cell wall biogenesis. In contrast to AfSUN1, deletion of AfSUN2 either in the parental strain or in the AfSUN1 single mutant strain did not affect colony and hyphal morphology. Biochemical characterization of the recombinant AfSun1p and Candida albicans Sun41p showed that both proteins had a unique hydrolysis pattern: acting on ß-(1,3)-oligomers from dimer up to insoluble ß-(1,3)-glucan. Referring to the CAZy database, it is clear that fungal SUN proteins represent a new family of glucan hydrolases (GH132) and play an important morphogenetic role in fungal cell wall biogenesis and septation.


Subject(s)
Aspergillus fumigatus/enzymology , Fungal Proteins/metabolism , Glycoside Hydrolases/metabolism , Hyphae/enzymology , Morphogenesis , Spores, Fungal/enzymology , Amino Acid Sequence , Aspergillus fumigatus/genetics , Aspergillus fumigatus/growth & development , Candida albicans/enzymology , Fungal Proteins/chemistry , Fungal Proteins/genetics , Gene Expression , Gene Expression Regulation, Fungal , Glycoproteins/metabolism , Glycoside Hydrolases/chemistry , Glycoside Hydrolases/genetics , Glycosylation , Hydrolysis , Hyphae/genetics , Hyphae/growth & development , Molecular Sequence Data , Oligosaccharides/chemistry , Protein Binding , Protein Processing, Post-Translational , Sequence Homology, Amino Acid , Spores, Fungal/genetics , Spores, Fungal/growth & development
20.
PLoS Pathog ; 9(2): e1003179, 2013 Feb.
Article in English | MEDLINE | ID: mdl-23436996

ABSTRACT

Group B Streptococcus (GBS), a common commensal of the female genital tract, is the leading cause of invasive infections in neonates. Expression of major GBS virulence factors, such as the hemolysin operon cyl, is regulated directly at the transcriptional level by the CovSR two-component system. Using a random genetic approach, we identified a multi-spanning transmembrane protein, Abx1, essential for the production of the GBS hemolysin. Despite its similarity to eukaryotic CaaX proteases, the Abx1 function is not involved in a post-translational modification of the GBS hemolysin. Instead, we demonstrate that Abx1 regulates transcription of several virulence genes, including those comprising the hemolysin operon, by a CovSR-dependent mechanism. By combining genetic analyses, transcriptome profiling, and site-directed mutagenesis, we showed that Abx1 is a regulator of the histidine kinase CovS. Overexpression of Abx1 is sufficient to activate virulence gene expression through CovS, overcoming the need for an additional signal. Conversely, the absence of Abx1 has the opposite effect on virulence gene expression consistent with CovS locked in a kinase-competent state. Using a bacterial two-hybrid system, direct interaction between Abx1 and CovS was mapped specifically to CovS domains involved in signal processing. We demonstrate that the CovSR two-component system is the core of a signaling pathway integrating the regulation of CovS by Abx1 in addition to the regulation of CovR by the serine/threonine kinase Stk1. In conclusion, our study reports a regulatory function for Abx1, a member of a large protein family with a characteristic Abi-domain, which forms a signaling complex with the histidine kinase CovS in GBS.


Subject(s)
Bacterial Proteins/genetics , Gene Expression Regulation, Bacterial , Signal Transduction , Streptococcal Infections/microbiology , Streptococcus agalactiae/genetics , Amino Acid Sequence , Animals , Bacterial Proteins/metabolism , Epistasis, Genetic , Female , Gene Expression Profiling , Hemolysis , Histidine Kinase , Humans , Models, Biological , Molecular Sequence Data , Mutation , Oligonucleotide Array Sequence Analysis , Phosphoprotein Phosphatases/genetics , Phosphoprotein Phosphatases/metabolism , Pigments, Biological/metabolism , Protein Interaction Mapping , Protein Kinases/genetics , Protein Kinases/metabolism , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Protein Structure, Tertiary , Rats , Sequence Alignment , Streptococcus agalactiae/metabolism , Streptococcus agalactiae/pathogenicity , Virulence/genetics , Virulence Factors/genetics
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