scholarly journals Increased Excess Intracellular Cyclic di-AMP Levels Impair Growth and Virulence of Bacillus anthracis

2020 ◽  
Vol 202 (9) ◽  
Author(s):  
Jia Hu ◽  
Gaobo Zhang ◽  
Leiqin Liang ◽  
Chengfeng Lei ◽  
Xiulian Sun
Keyword(s):  
Bacillus Anthracis ◽  
Bacterial Growth ◽  
Bacterial Pathogen ◽  
Bacterial Toxin ◽  
Biological Processes ◽  
Content Type ◽  
Host Pathogen Interaction ◽  
Factor Expression ◽  
Increased Sensitivity ◽  
Deadly Disease

ABSTRACT Cyclic di-AMP (c-di-AMP) is a recently identified bacterial second messenger that regulates biological processes. In this study, we found that inactivation of two c-di-AMP phosphodiesterases (PDEs), GdpP and PgpH, resulted in accumulation of 3.8-fold higher c-di-AMP levels than in the parental strain Sterne in Bacillus anthracis and inhibited bacterial growth. Moreover, excess c-di-AMP accumulation decreased bacterial toxin expression, increased sensitivity to osmotic stress and detergent, and attenuated virulence in both C57BL/6J and A/J mice. Complementation of the PDE mutant with a plasmid carrying gdpP or pgpH in trans from a Pspac promoter restored bacterial growth, virulence factor expression, and resistance to detergent. Our results indicate that c-di-AMP is a pleiotropic signaling molecule in B. anthracis that is important for host-pathogen interaction. IMPORTANCE Anthrax is an ancient and deadly disease caused by the spore-forming bacterial pathogen Bacillus anthracis. Vegetative cells of this species produce anthrax toxin proteins and S-layer components during infection of mammalian hosts. So far, how the expression of these virulence factors is regulated remains largely unknown. Our results suggest that excess elevated c-di-AMP levels inhibit bacterial growth and reduce expression of S-layer components and anthracis toxins as well as reduce virulence in a mouse model of disease. These results indicate that c-di-AMP signaling plays crucial roles in B. anthracis biology and disease.

scholarly journals Glutamate Racemase Mutants of Bacillus anthracis

2015 ◽  
Vol 197 (11) ◽  
pp. 1854-1861 ◽  
Author(s):  
So-Young Oh ◽  
Stefan G. Richter ◽  
Dominique M. Missiakas ◽  
Olaf Schneewind
Keyword(s):  
Glutamic Acid ◽  
Bacillus Anthracis ◽  
Causative Agent ◽  
Bacterial Growth ◽  
Bacterial Infections ◽  
Content Type ◽  
Antibiotic Development ◽  
Drug Candidates ◽  
Glutamate Racemase ◽  
Cell Shapes

ABSTRACTd-Glutamate is an essential component of bacterial peptidoglycan and a building block of the poly-γ-d-glutamic acid (PDGA) capsule ofBacillus anthracis, the causative agent of anthrax. Earlier work suggested that two glutamate racemases, encoded byracE1andracE2, are each essential for growth ofB. anthracis, supplyingd-glutamic acid for the synthesis of peptidoglycan and PDGA capsule. Earlier work could not explain, however, why two enzymes that catalyze the same reaction may be needed for bacterial growth. Here, we report that deletion ofracE1orracE2did not prevent growth ofB. anthracisSterne (pXO1+pXO2−), the noncapsulating vaccine strain, or ofB. anthracisAmes (pXO1+pXO2+), a fully virulent, capsulating isolate. While mutants with deletions inracE1andracE2were not viable,racE2deletion delayed vegetative growth ofB. anthracisfollowing spore germination and caused aberrant cell shapes, phenotypes that were partially restored by exogenousd-glutamate. Deletion ofracE1orracE2fromB. anthracisAmes did not affect the production or stereochemical composition of the PDGA capsule. A model is presented wherebyB. anthracis, similar toBacillus subtilis, utilizes two functionally redundant racemase enzymes to synthesized-glutamic acid for peptidoglycan synthesis.IMPORTANCEGlutamate racemases, enzymes that convertl-glutamate tod-glutamate, are targeted for antibiotic development. Glutamate racemase inhibitors may be useful for the treatment of bacterial infections such as anthrax, where the causative agent,B. anthracis, requiresd-glutamate for the synthesis of peptidoglycan and poly-γ-d-glutamic acid (PDGA) capsule. Here we show thatB. anthracispossesses two glutamate racemase genes that can be deleted without abolishing either bacterial growth or PDGA synthesis. These data indicate that drug candidates must inhibit both glutamate racemases, RacE1 and RacE2, in order to blockB. anthracisgrowth and achieve therapeutic efficacy.

scholarly journals The ClpCP Complex Modulates Respiratory Metabolism inStaphylococcus aureusand Is Regulated in a SrrAB-Dependent Manner

2019 ◽  
Vol 201 (15) ◽  
Author(s):  
Ameya A. Mashruwala ◽  
Brian J. Eilers ◽  
Amanda L. Fuchs ◽  
Javiera Norambuena ◽  
Carly A. Earle ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Energy Metabolism ◽  
Energy Homeostasis ◽  
Protein Complexes ◽  
Present Report ◽  
Bacterial Pathogen ◽  
Dependent Manner ◽  
Content Type ◽  
Fermentative Growth ◽  
Increased Sensitivity

ABSTRACTThestaphylococcalrespiratoryregulator (SrrAB) modulates energy metabolism inStaphylococcus aureus. Studies have suggested that regulated protein catabolism facilitates energy homeostasis. Regulated proteolysis inS. aureusis achieved through protein complexes composed of a peptidase (ClpQ or ClpP) in association with an AAA+family ATPase (typically, ClpC or ClpX). In the present report, we tested the hypothesis that SrrAB regulates a Clp complex to facilitate energy homeostasis inS. aureus. Strains deficient in one or more Clp complexes were attenuated for growth in the presence of puromycin, which causes enrichment of misfolded proteins. A ΔsrrABstrain had increased sensitivity to puromycin. Epistasis experiments suggested that the puromycin sensitivity phenotype of the ΔsrrABstrain was a result of decreased ClpC activity. Consistent with this, transcriptional activity ofclpCwas decreased in the ΔsrrABmutant, and overexpression ofclpCsuppressed the puromycin sensitivity of the ΔsrrABstrain. We also found that ClpC positively influenced respiration and that it did so upon association with ClpP. In contrast, ClpC limited fermentative growth, while ClpP was required for optimal fermentative growth. Metabolomics studies demonstrated that intracellular metabolic profiles of the ΔclpCand ΔsrrABmutants were distinct from those of the wild-type strain, supporting the notion that both ClpC and SrrAB affect central metabolism. We propose a model wherein SrrAB regulates energy homeostasis, in part, via modulation of regulated proteolysis.IMPORTANCEOxygen is used as a substrate to derive energy by the bacterial pathogenStaphylococcus aureusduring infection; however,S. aureuscan also grow fermentatively in the absence of oxygen. To successfully cause infection,S. aureusmust tailor its metabolism to take advantage of respiratory activity. Different proteins are required for growth in the presence or absence of oxygen; therefore, when cells transition between these conditions, several proteins would be expected to become unnecessary. In this report, we show that regulated proteolysis is used to modulate energy metabolism inS. aureus. We report that the ClpCP protein complex is involved in specifically modulating aerobic respiratory growth but is dispensable for fermentative growth.

scholarly journals The Aminoglycoside Antibiotic Kanamycin Damages DNA Bases in Escherichia coli: Caffeine Potentiates the DNA-Damaging Effects of Kanamycin while Suppressing Cell Killing by Ciprofloxacin in Escherichia coli and Bacillus anthracis

2012 ◽  
Vol 56 (6) ◽  
pp. 3216-3223 ◽  
Author(s):  
Tina Manzhu Kang ◽  
Jessica Yuan ◽  
Angelyn Nguyen ◽  
Elinne Becket ◽  
Hanjing Yang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Bacillus Anthracis ◽  
Gene Knockout ◽  
Aminoglycoside Antibiotic ◽  
Double Strand Breaks ◽  
Repair Capacity ◽  
Content Type ◽  
Strand Breaks ◽  
Knockout Mutants ◽  
Increased Sensitivity

ABSTRACTThe distribution of mutants in the Keio collection ofEscherichia coligene knockout mutants that display increased sensitivity to the aminoglycosides kanamycin and neomycin indicates that damaged bases resulting from antibiotic action can lead to cell death. Strains lacking one of a number of glycosylases (e.g., AlkA, YzaB, Ogt, KsgA) or other specific repair proteins (AlkB, PhrB, SmbC) are more sensitive to these antibiotics. Mutants lacking AlkB display the strongest sensitivity among the glycosylase- or direct lesion removal-deficient strains. This perhaps suggests the involvement of ethenoadenine adducts, resulting from reactive oxygen species and lipid peroxidation, since AlkB removes this lesion. Other sensitivities displayed by mutants lacking UvrA, polymerase V (Pol V), or components of double-strand break repair indicate that kanamycin results in damaged base pairs that need to be removed or replicated past in order to avoid double-strand breaks that saturate the cellular repair capacity. Caffeine enhances the sensitivities of these repair-deficient strains to kanamycin and neomycin. The gene knockout mutants that display increased sensitivity to caffeine (dnaQ,holC,holD, andpriAknockout mutants) indicate that caffeine blocks DNA replication, ultimately leading to double-strand breaks that require recombinational repair by functions encoded byrecA,recB, andrecC, among others. Additionally, caffeine partially protects cells of bothEscherichia coliandBacillus anthracisfrom killing by the widely used fluoroquinolone antibiotic ciprofloxacin.

scholarly journals Intracellularly Induced Cyclophilins Play an Important Role in Stress Adaptation and Virulence of Brucella abortus

2012 ◽  
Vol 81 (2) ◽  
pp. 521-530 ◽  
Author(s):  
Mara S. Roset ◽  
Lucía García Fernández ◽  
Vito G. DelVecchio ◽  
Gabriel Briones
Keyword(s):  
Host Cell ◽  
Brucella Abortus ◽  
Intracellular Localization ◽  
Bacterial Pathogen ◽  
Host Cells ◽  
Content Type ◽  
Double Deletion ◽  
Proteomic Investigation ◽  
Increased Sensitivity ◽  
Lower Temperature

ABSTRACTBrucellais an intracellular bacterial pathogen that causes the worldwide zoonotic disease brucellosis.Brucellavirulence relies on its ability to transition to an intracellular lifestyle within host cells. Thus, this pathogen must sense its intracellular localization and then reprogram gene expression for survival within the host cell. A comparative proteomic investigation was performed to identify differentially expressed proteins potentially relevant forBrucellaintracellular adaptation. Two proteins identified as cyclophilins (CypA and CypB) were overexpressed in the intracellular environment of the host cell in comparison to laboratory-grownBrucella. To define the potential role of cyclophilins inBrucellavirulence, a double-deletion mutant was constructed and its resulting phenotype was characterized. TheBrucella abortusΔcypABmutant displayed increased sensitivity to environmental stressors, such as oxidative stress, pH, and detergents. In addition, theB. abortusΔcypABmutant strain had a reduced growth rate at lower temperature, a phenotype associated with defective expression of cyclophilins in other microorganisms. TheB. abortusΔcypABmutant also displays reduced virulence in BALB/c mice and defective intracellular survival in HeLa cells. These findings suggest that cyclophilins are important forBrucellavirulence and survival in the host cells.

scholarly journals The Bacillus anthracis Cell Envelope: Composition, Physiological Role, and Clinical Relevance

2020 ◽  
Vol 8 (12) ◽  
pp. 1864
Author(s):  
Alice Chateau ◽  
Sander E. Van der Verren ◽  
Han Remaut ◽  
Antonella Fioravanti
Keyword(s):  
Bacillus Anthracis ◽  
Environmental Conditions ◽  
Clinical Relevance ◽  
Cell Envelope ◽  
Bacterial Pathogen ◽  
Physiological Role ◽  
The Novel ◽  
Dynamic Composition ◽  
Deadly Disease ◽  
Novel Therapeutic

Anthrax is a highly resilient and deadly disease caused by the spore-forming bacterial pathogen Bacillus anthracis. The bacterium presents a complex and dynamic composition of its cell envelope, which changes in response to developmental and environmental conditions and host-dependent signals. Because of their easy to access extracellular locations, B. anthracis cell envelope components represent interesting targets for the identification and development of novel therapeutic and vaccine strategies. This review will focus on the novel insights regarding the composition, physiological role, and clinical relevance of B. anthracis cell envelope components.

scholarly journals The PerR-Regulated P1B-4-Type ATPase (PmtA) Acts as a Ferrous Iron Efflux Pump in Streptococcus pyogenes

2017 ◽  
Vol 85 (6) ◽  
Author(s):  
Andrew G. Turner ◽  
Cheryl-lynn Y. Ong ◽  
Karrera Y. Djoko ◽  
Nicholas P. West ◽  
Mark R. Davies ◽  
...  
Keyword(s):  
Streptococcus Pyogenes ◽  
Ferrous Iron ◽  
Deletion Mutant ◽  
Efflux Pump ◽  
Bacterial Pathogen ◽  
Efflux System ◽  
Content Type ◽  
Metal Sequestration ◽  
Group A ◽  
Increased Sensitivity

ABSTRACT Streptococcus pyogenes (group A Streptococcus [GAS]) is an obligate human pathogen responsible for a broad spectrum of human disease. GAS has a requirement for metal homeostasis within the human host and, as such, tightly modulates metal uptake and efflux during infection. Metal acquisition systems are required to combat metal sequestration by the host, while metal efflux systems are essential to protect against metal overload poisoning. Here, we investigated the function of PmtA (PerR-regulated metal transporter A), a P1B-4-type ATPase efflux pump, in invasive GAS M1T1 strain 5448. We reveal that PmtA functions as a ferrous iron [Fe(II)] efflux system. In the presence of high Fe(II) concentrations, the 5448ΔpmtA deletion mutant exhibited diminished growth and accumulated 5-fold-higher levels of intracellular Fe(II) than did the wild type and the complemented mutant. The 5448ΔpmtA deletion mutant also showed enhanced susceptibility to killing by the Fe-dependent antibiotic streptonigrin as well as increased sensitivity to hydrogen peroxide and superoxide. We suggest that the PerR-mediated control of Fe(II) efflux by PmtA is important for bacterial defense against oxidative stress. PmtA represents an exemplar for an Fe(II) efflux system in a host-adapted Gram-positive bacterial pathogen.

scholarly journals Genes Required for Aerial Growth, Cell Division, and Chromosome Segregation Are Targets of WhiA before Sporulation in Streptomyces venezuelae

mBio ◽  
2013 ◽  
Vol 4 (5) ◽  
Author(s):  
Matthew J. Bush ◽  
Maureen J. Bibb ◽  
Govind Chandra ◽  
Kim C. Findlay ◽  
Mark J. Buttner
Keyword(s):  
Cell Division ◽  
Chromosome Segregation ◽  
Regulatory Networks ◽  
Liquid Culture ◽  
Biological Processes ◽  
Streptomyces Venezuelae ◽  
Content Type ◽  
Master Regulators ◽  
Aerial Growth ◽  
Genes Encoding

ABSTRACTWhiA is a highly unusual transcriptional regulator related to a family of eukaryotic homing endonucleases. WhiA is required for sporulation in the filamentous bacteriumStreptomyces, but WhiA homologues of unknown function are also found throughout the Gram-positive bacteria. To better understand the role of WhiA inStreptomycesdevelopment and its function as a transcription factor, we identified the WhiA regulon through a combination of chromatin immunoprecipitation-sequencing (ChIP-seq) and microarray transcriptional profiling, exploiting a new model organism for the genus,Streptomyces venezuelae, which sporulates in liquid culture. The regulon encompasses ~240 transcription units, and WhiA appears to function almost equally as an activator and as a repressor. Bioinformatic analysis of the upstream regions of the complete regulon, combined with DNase I footprinting, identified a short but highly conserved asymmetric sequence, GACAC, associated with the majority of WhiA targets. Construction of a null mutant showed thatwhiAis required for the initiation of sporulation septation and chromosome segregation inS. venezuelae, and several genes encoding key proteins of theStreptomycescell division machinery, such asftsZ,ftsW, andftsK, were found to be directly activated by WhiA during development. Several other genes encoding proteins with important roles in development were also identified as WhiA targets, including the sporulation-specific sigma factor σWhiGand the diguanylate cyclase CdgB. Cell division is tightly coordinated with the orderly arrest of apical growth in the sporogenic cell, andfilP, encoding a key component of the polarisome that directs apical growth, is a direct target for WhiA-mediated repression during sporulation.IMPORTANCESince the initial identification of the genetic loci required forStreptomycesdevelopment, all of thebldandwhidevelopmental master regulators have been cloned and characterized, and significant progress has been made toward understanding the cell biological processes that drive morphogenesis. A major challenge now is to connect the cell biological processes and the developmental master regulators by dissecting the regulatory networks that link the two. Studies of these regulatory networks have been greatly facilitated by the recent introduction ofStreptomyces venezuelaeas a new model system for the genus, a species that sporulates in liquid culture. Taking advantage ofS. venezuelae, we have characterized the regulon of genes directly under the control of one of these master regulators, WhiA. Our results implicate WhiA in the direct regulation of key steps in sporulation, including the cessation of aerial growth, the initiation of cell division, and chromosome segregation.

scholarly journals Failure To Recruit Anti-Inflammatory CD103+Dendritic Cells and a Diminished CD4+Foxp3+Regulatory T Cell Pool in Mice That Display Excessive Lung Inflammation and Increased Susceptibility to Mycobacterium tuberculosis

2012 ◽  
Vol 80 (3) ◽  
pp. 1128-1139 ◽  
Author(s):  
Chaniya Leepiyasakulchai ◽  
Lech Ignatowicz ◽  
Andrzej Pawlowski ◽  
Gunilla Källenius ◽  
Markus Sköld
Keyword(s):  
Immune Response ◽  
Dendritic Cells ◽  
Mycobacterium Tuberculosis ◽  
T Cell ◽  
Bacterial Growth ◽  
Lung Inflammation ◽  
Chronic Infection ◽  
Host Immune Response ◽  
Content Type ◽  
Tnf Α

Susceptibility toMycobacterium tuberculosisis characterized by excessive lung inflammation, tissue damage, and failure to control bacterial growth. To increase our understanding of mechanisms that may regulate the host immune response in the lungs, we characterized dendritic cells expressing CD103 (αEintegrin) (αE-DCs) and CD4+Foxp3+regulatory T (Treg) cells duringM. tuberculosisinfection. In resistant C57BL/6 and BALB/c mice, the number of lung αE-DCs increased dramatically duringM. tuberculosisinfection. In contrast, highly susceptible DBA/2 mice failed to recruit αE-DCs even during chronic infection. Even though tumor necrosis factor alpha (TNF-α) is produced by multiple DCs and macrophage subsets and is required for control of bacterial growth, αE-DCs remained TNF-α negative. Instead, αE-DCs contained a high number of transforming growth factor beta-producing cells in infected mice. Further, we show that Tregcells in C57BL/6 and DBA/2 mice induce gamma interferon during pulmonary tuberculosis. In contrast to resistant mice, the Tregcell population was diminished in the lungs, but not in the draining pulmonary lymph nodes (PLN), of highly susceptible mice during chronic infection. Tregcells have been reported to inhibitM. tuberculosis-specific T cell immunity, leading to increased bacterial growth. Still, despite the reduced number of lung Tregcells in DBA/2 mice, the bacterial load in the lungs was increased compared to resistant animals. Our results show that αE-DCs and Tregcells that may regulate the host immune response are increased inM. tuberculosis-infected lungs of resistant mice but diminished in infected lungs of susceptible mice.

scholarly journals Changing Molecular Markers of Antimalarial Drug Sensitivity across Uganda

2018 ◽  
Vol 63 (3) ◽  
Author(s):  
Victor Asua ◽  
Joanna Vinden ◽  
Melissa D. Conrad ◽  
Jennifer Legac ◽  
Simon P. Kigozi ◽  
...  
Keyword(s):  
Antimalarial Drug ◽  
Drug Sensitivity ◽  
Artemisinin Resistance ◽  
Wild Type ◽  
National Treatment ◽  
Antimalarial Drug Resistance ◽  
Content Type ◽  
Increased Sensitivity ◽  
Low Prevalence ◽  
Poor Efficacy

ABSTRACT The potential spread of antimalarial drug resistance to Africa, in particular for artemisinins and key partner drugs, is a major concern. We surveyed Plasmodium falciparum genetic markers associated with drug sensitivity on 3 occasions at ∼6-month intervals in 2016 and 2017 at 10 sites representing a range of epidemiological settings in Uganda. For putative drug transporters, we found continued evolution toward wild-type sequences associated with increased sensitivity to chloroquine. For pfcrt K76T, by 2017 the prevalence of the wild type was >60% at all sites and >90% at 6 sites. For the pfmdr1 N86Y and D1246Y alleles, wild type prevalence ranged from 80 to 100%. We found low prevalence of K13 propeller domain mutations, which are associated with artemisinin resistance in Asia, but one mutation previously identified in northern Uganda, 675V, was seen in 2.0% of samples, including 5.5% of those from the 3 northernmost sites. Amplification of the pfmdr1 and plasmepsin2 genes, associated elsewhere with decreased sensitivity to lumefantrine and piperaquine, respectively, was seen in <1% of samples. For the antifolate targets pfdhfr and pfdhps, 5 mutations previously associated with resistance were very common, and the pfdhfr 164L and pfdhps 581G mutations associated with higher-level resistance were seen at multiple sites, although prevalence did not clearly increase over time. Overall, changes were consistent with the selective pressure of the national treatment regimen, artemether-lumefantrine, with increased sensitivity to chloroquine, and with poor efficacy of antifolates. Strong evidence for resistance to artemisinins was not seen. Continued surveillance of markers that predict antimalarial drug sensitivity is warranted.

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