scholarly journals Genome-Wide fitness analysis of group B Streptococcus in human amniotic fluid reveals a transcription factor that controls multiple virulence traits

2021 ◽  
Vol 17 (3) ◽  
pp. e1009116
Author(s):  
Allison N. Dammann ◽  
Anna B. Chamby ◽  
Andrew J. Catomeris ◽  
Kyle M. Davidson ◽  
Hervé Tettelin ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Amniotic Fluid ◽  
Deletion Strain ◽  
Growth Defect ◽  
Wild Type ◽  
Human Amniotic Fluid ◽  
Wide Range ◽  
Pregnant Mice ◽  
Group B

Streptococcus agalactiae (group B Streptococcus; GBS) remains a dominant cause of serious neonatal infections. One aspect of GBS that renders it particularly virulent during the perinatal period is its ability to invade the chorioamniotic membranes and persist in amniotic fluid, which is nutritionally deplete and rich in fetal immunologic factors such as antimicrobial peptides. We used next-generation sequencing of transposon-genome junctions (Tn-seq) to identify five GBS genes that promote survival in the presence of human amniotic fluid. We confirmed our Tn-seq findings using a novel CRISPR inhibition (CRISPRi) gene expression knockdown system. This analysis showed that one gene, which encodes a GntR-class transcription factor that we named MrvR, conferred a significant fitness benefit to GBS in amniotic fluid. We generated an isogenic targeted deletion of the mrvR gene, which had a growth defect in amniotic fluid relative to the wild type parent strain. The mrvR deletion strain also showed a significant biofilm defect in vitro. Subsequent in vivo studies showed that while the mutant was able to cause persistent murine vaginal colonization, pregnant mice colonized with the mrvR deletion strain did not develop preterm labor despite consistent GBS invasion of the uterus and the fetoplacental units. In contrast, pregnant mice colonized with wild type GBS consistently deliver prematurely. In a sepsis model the mrvR deletion strain showed significantly decreased lethality. In order to better understand the mechanism by which this newly identified transcription factor controls GBS virulence, we performed RNA-seq on wild type and mrvR deletion GBS strains, which revealed that the transcription factor affects expression of a wide range of genes across the GBS chromosome. Nucleotide biosynthesis and salvage pathways were highly represented among the set of differentially expressed genes, suggesting that MrvR may be involved in regulating nucleotide availability.

scholarly journals MrvR, a Group B Streptococcus Transcription Factor that Controls Multiple Virulence Traits

2020 ◽  
Author(s):  
Allison N. Dammann ◽  
Anna B. Chamby ◽  
Andrew J. Catomeris ◽  
Kyle M. Davidson ◽  
Hervé Tettelin ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Amniotic Fluid ◽  
Self Regulation ◽  
Growth Defect ◽  
Wild Type ◽  
Knockout Strain ◽  
Virulence Traits ◽  
Wide Range ◽  
Pregnant Mice ◽  
Group B

AbstractStreptococcus agalactiae (group B Streptococcus; GBS) remains a dominant cause of serious neonatal infections. One aspect of GBS that renders it particularly virulent during the perinatal period is its ability to invade the chorioamniotic membranes and persist in amniotic fluid, which is nutritionally deplete and rich in fetal immunologic factors such as antimicrobial peptides. We used next-generation sequencing of transposon-genome junctions (Tn-seq) to identify five GBS genes that promote survival in the presence of human amniotic fluid. We confirmed our Tn-seq findings using a novel CRISPR inhibition (CRISPRi) gene expression knockdown system. This analysis showed that one gene, which encodes a GntR-class transcription factor that we named MrvR, conferred a significant fitness benefit to GBS in amniotic fluid. We generated an isogenic targeted knockout of the mrvR gene, which we found to have a growth defect in amniotic fluid relative to the wild type parent strain. In addition to growing poorly in amniotic fluid, the knockout also showed a significant biofilm defect in vitro. Subsequent in vivo studies showed that, while the knockout was able to cause persistent murine vaginal colonization, pregnant mice colonized with the knockout strain did not develop preterm labor despite consistent GBS invasion of the uterus and the fetoplacental units. In contrast, pregnant mice colonized with wild type GBS consistently deliver prematurely. Similarly, in a sepsis model in which 87% of mice infected with wild type GBS died within three days, none of the mice infected with the knockout strain died. In order to better understand the mechanism by which this newly identified transcription factor controls GBS virulence, we performed electrophoresis mobility shift assays with recombinant MrvR and whole-genome transcriptomic analysis on the knockout and wild type strains. We show that MrvR binds to its own promoter region, suggesting likely self-regulation. RNA-seq revealed that the transcription factor affects expression of a wide range of genes across the GBS chromosome. Nucleotide biosynthesis and salvage pathways were highly represented among the set of differentially expressed genes, suggesting a linkage between purine or pyrimidine availability and activity of MrvR in multiple GBS virulence traits.

scholarly journals Enterohemorrhagic Escherichia coli O157:H7 gal Mutants Are Sensitive to Bacteriophage P1 and Defective in Intestinal Colonization

2006 ◽  
Vol 75 (4) ◽  
pp. 1661-1666 ◽  
Author(s):  
Theresa Deland Ho ◽  
Matthew K. Waldor
Keyword(s):  
Escherichia Coli ◽  
Genetic Manipulation ◽  
Enterohemorrhagic Escherichia Coli ◽  
Deletion Strain ◽  
Growth Defect ◽  
Intestinal Colonization ◽  
Wild Type ◽  
E Coli ◽  
O Antigen

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC), especially E. coli O157:H7, is an emerging cause of food-borne illness. Unfortunately, E. coli O157 cannot be genetically manipulated using the generalized transducing phage P1, presumably because its extensive O antigen obscures the P1 receptor, the lipopolysaccharide (LPS) core subunit. The GalE, GalT, GalK, and GalU proteins are necessary for modifying galactose before it can be assembled into the repeating subunit of the O antigen. Here, we constructed E. coli O157:H7 gal mutants which presumably have little or no O antigen. These strains were able to adsorb P1. P1 lysates grown on the gal mutant strains could be used to move chromosomal markers between EHEC strains, thereby facilitating genetic manipulation of E. coli O157:H7. The gal mutants could easily be reverted to a wild-type Gal+ strain using P1 transduction. We found that the O157:H7 galETKM::aad-7 deletion strain was 500-fold less able to colonize the infant rabbit intestine than the isogenic Gal+ parent, although it displayed no growth defect in vitro. Furthermore, in vivo a Gal+ revertant of this mutant outcompeted the galETKM deletion strain to an extent similar to that of the wild type. This suggests that the O157 O antigen is an important intestinal colonization factor. Compared to the wild type, EHEC gal mutants were 100-fold more sensitive to a peptide derived from bactericidal permeability-increasing protein, a bactericidal protein found on the surface of intestinal epithelial cells. Thus, one way in which the O157 O antigen may contribute to EHEC intestinal colonization is to promote resistance to host-derived antimicrobial polypeptides.

Proliferation of group B streptococci in human amniotic fluid in vitro

1987 ◽  
Vol 156 (1) ◽  
pp. 95-99 ◽  
Author(s):  
Iffath A. Abbasi ◽  
Val G. Hemming ◽  
Gary S. Eglinton ◽  
Timothy R.B. Johnson
Keyword(s):  
Amniotic Fluid ◽  
Human Amniotic Fluid ◽  
Group B Streptococci ◽  
Group B

Rapid in vitro Replication of Group B Streptococcus in Term Human Amniotic Fluid

1985 ◽  
Vol 19 (3) ◽  
pp. 124-129 ◽  
Author(s):  
V.G. Hemming ◽  
K. Nagarajan ◽  
L.W. Hess ◽  
G.W. Fischer ◽  
S.R. Wilson ◽  
...  
Keyword(s):  
Amniotic Fluid ◽  
Group B Streptococcus ◽  
Human Amniotic Fluid ◽  
In Vitro Replication ◽  
Group B

Effects of alterations of zinc-to-phosphorus ratios and meconium content on Group B Streptococcus growth in human amniotic fluid in vitro

1987 ◽  
Vol 157 (3) ◽  
pp. 770-773 ◽  
Author(s):  
Iffath Abbasi Hoskins ◽  
Val G. Hemming ◽  
Timothy R.B. Johnson ◽  
Craig A. Winkel
Keyword(s):  
Amniotic Fluid ◽  
Group B Streptococcus ◽  
Human Amniotic Fluid ◽  
Group B

scholarly journals Production of the Siderophore 2,3-Dihydroxybenzoic Acid Is Required for Wild-Type Growth of Brucella abortus in the Presence of Erythritol under Low-Iron Conditions In Vitro

2003 ◽  
Vol 71 (5) ◽  
pp. 2927-2832 ◽  
Author(s):  
Bryan H. Bellaire ◽  
Philip H. Elzer ◽  
Cynthia L. Baldwin ◽  
R. Martin Roop
Keyword(s):  
Brucella Abortus ◽  
Reproductive Tract ◽  
Iron Acquisition ◽  
Energy Sources ◽  
Growth Defect ◽  
Wild Type ◽  
Dihydroxybenzoic Acid ◽  
Experimental Findings ◽  
The Relationship

ABSTRACT Production of the siderophore 2,3-dihyroxybenzoic acid (2,3-DHBA) is required for the wild-type virulence of Brucella abortus in cattle. A possible explanation for this requirement was uncovered when it was determined that a B. abortus dhbC mutant (BHB1) defective in 2,3-DHBA production displays marked growth restriction in comparison to its parent strain, B. abortus 2308, when cultured in the presence of erythritol under low-iron conditions. This phenotype is not displayed when these strains are cultured under low-iron conditions in the presence of other readily utilizable carbon and energy sources. The addition of either exogenous 2,3-DHBA or FeCl3 relieves this growth defect, suggesting that the inability of the B. abortus dhbC mutant to display wild-type growth in the presence of erythritol under iron-limiting conditions is due to a defect in iron acquisition. Restoring 2,3-DHBA production to the B. abortus dhbC mutant by genetic complementation abolished the erythritol-specific growth defect exhibited by this strain in low-iron medium, verifying the relationship between 2,3-DHBA production and efficient growth in the presence of erythritol under low-iron conditions. The positive correlation between 2,3-DHBA production and growth in the presence of erythritol was further substantiated by the observation that the addition of erythritol to low-iron cultures of B. abortus 2308 stimulated the production of 2,3-DHBA by increasing the transcription of the dhbCEBA operon. Correspondingly, the level of exogenous iron needed to repress dhbCEBA expression in B. abortus 2308 was also greater when this strain was cultured in the presence of erythritol than that required when it was cultured in the presence of any of the other readily utilizable carbon and energy sources tested. The tissues of the bovine reproductive tract are rich in erythritol during the latter stages of pregnancy, and the ability to metabolize erythritol is thought to be important to the virulence of B. abortus in pregnant ruminants. Consequently, the experimental findings presented here offer a plausible explanation for the attenuation of the B. abortus 2,3-DHBA-deficient mutant BHB1 in pregnant ruminants.

scholarly journals Structure and inhibition of Cryptococcus neoformans sterylglucosidase to develop antifungal agents

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Nivea Pereira de Sa ◽  
Adam Taouil ◽  
Jinwoo Kim ◽  
Timothy Clement ◽  
Reece M. Hoffmann ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Rational Design ◽  
Antifungal Agents ◽  
Pathogenic Fungus ◽  
Pathogenic Fungi ◽  
Growth Defect ◽  
Wild Type ◽  
Secondary Infections ◽  
Heavy Burden

AbstractPathogenic fungi exhibit a heavy burden on medical care and new therapies are needed. Here, we develop the fungal specific enzyme sterylglucosidase 1 (Sgl1) as a therapeutic target. Sgl1 converts the immunomodulatory glycolipid ergosterol 3β-D-glucoside to ergosterol and glucose. Previously, we found that genetic deletion of Sgl1 in the pathogenic fungus Cryptococcus neoformans (Cn) results in ergosterol 3β-D-glucoside accumulation, renders Cn non-pathogenic, and immunizes mice against secondary infections by wild-type Cn, even in condition of CD4+ T cell deficiency. Here, we disclose two distinct chemical classes that inhibit Sgl1 function in vitro and in Cn cells. Pharmacological inhibition of Sgl1 phenocopies a growth defect of the Cn Δsgl1 mutant and prevents dissemination of wild-type Cn to the brain in a mouse model of infection. Crystal structures of Sgl1 alone and with inhibitors explain Sgl1’s substrate specificity and enable the rational design of antifungal agents targeting Sgl1.

In vitro survival of Listeria monocytogenes in human amniotic fluid

1999 ◽  
Vol 202 (5) ◽  
pp. 377-382 ◽  
Author(s):  
Clelia Altieri ◽  
Giuseppe Maruotti ◽  
Costanzo Natale ◽  
Salvatore Massa
Keyword(s):  
Listeria Monocytogenes ◽  
Amniotic Fluid ◽  
Human Amniotic Fluid ◽  
In Vitro Survival

scholarly journals Identification of Zinc-Dependent Mechanisms Used by Group B Streptococcus To Overcome Calprotectin-Mediated Stress

mBio ◽  
2020 ◽  
Vol 11 (6) ◽  
Author(s):  
Lindsey R. Burcham ◽  
Yoann Le Breton ◽  
Jana N. Radin ◽  
Brady L. Spencer ◽  
Liwen Deng ◽  
...  
Keyword(s):  
Knockout Mice ◽  
Metal Ion ◽  
Invasive Disease ◽  
Female Reproductive Tract ◽  
Zinc Uptake ◽  
Wild Type ◽  
Metal Transporters ◽  
High Concentrations ◽  
Group B

ABSTRACT Nutritional immunity is an elegant host mechanism used to starve invading pathogens of necessary nutrient metals. Calprotectin, a metal-binding protein, is produced abundantly by neutrophils and is found in high concentrations within inflammatory sites during infection. Group B Streptococcus (GBS) colonizes the gastrointestinal and female reproductive tracts and is commonly associated with severe invasive infections in newborns such as pneumonia, sepsis, and meningitis. Although GBS infections induce robust neutrophil recruitment and inflammation, the dynamics of GBS and calprotectin interactions remain unknown. Here, we demonstrate that disease and colonizing isolate strains exhibit susceptibility to metal starvation by calprotectin. We constructed a mariner transposon (Krmit) mutant library in GBS and identified 258 genes that contribute to surviving calprotectin stress. Nearly 20% of all underrepresented mutants following treatment with calprotectin are predicted metal transporters, including known zinc systems. As calprotectin binds zinc with picomolar affinity, we investigated the contribution of GBS zinc uptake to overcoming calprotectin-imposed starvation. Quantitative reverse transcriptase PCR (qRT-PCR) revealed a significant upregulation of genes encoding zinc-binding proteins, adcA, adcAII, and lmb, following calprotectin exposure, while growth in calprotectin revealed a significant defect for a global zinc acquisition mutant (ΔadcAΔadcAIIΔlmb) compared to growth of the GBS wild-type (WT) strain. Furthermore, mice challenged with the ΔadcAΔadcAIIΔlmb mutant exhibited decreased mortality and significantly reduced bacterial burden in the brain compared to mice infected with WT GBS; this difference was abrogated in calprotectin knockout mice. Collectively, these data suggest that GBS zinc transport machinery is important for combatting zinc chelation by calprotectin and establishing invasive disease. IMPORTANCE Group B Streptococcus (GBS) asymptomatically colonizes the female reproductive tract but is a common causative agent of meningitis. GBS meningitis is characterized by extensive infiltration of neutrophils carrying high concentrations of calprotectin, a metal chelator. To persist within inflammatory sites and cause invasive disease, GBS must circumvent host starvation attempts. Here, we identified global requirements for GBS survival during calprotectin challenge, including known and putative systems involved in metal ion transport. We characterized the role of zinc import in tolerating calprotectin stress in vitro and in a mouse model of infection. We observed that a global zinc uptake mutant was less virulent than the parental GBS strain and found calprotectin knockout mice to be equally susceptible to infection by wild-type (WT) and mutant strains. These findings suggest that calprotectin production at the site of infection results in a zinc-limited environment and reveals the importance of GBS metal homeostasis to invasive disease.

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