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 Cryo-electron Microscopy Structure of the Acinetobacter baumannii 70S Ribosome and Implications for New Antibiotic Development

mBio ◽  
2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Christopher E. Morgan ◽  
Wei Huang ◽  
Susan D. Rudin ◽  
Derek J. Taylor ◽  
James E. Kirby ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Acinetobacter Baumannii ◽  
Bacterial Infections ◽  
Bound States ◽  
Antimicrobial Agents ◽  
Gram Negative ◽  
Content Type ◽  
Antibiotic Development ◽  
Cryo Electron Microscopy ◽  
70S Ribosome

ABSTRACT Antimicrobial resistance is a major health threat as it limits treatment options for infection. At the forefront of this serious issue is Acinetobacter baumannii, a Gram-negative opportunistic pathogen that exhibits the remarkable ability to resist antibiotics through multiple mechanisms. As bacterial ribosomes represent a target for multiple distinct classes of existing antimicrobial agents, we here use single-particle cryo-electron microscopy (cryo-EM) to elucidate five different structural states of the A. baumannii ribosome, including the 70S, 50S, and 30S forms. We also determined interparticle motions of the 70S ribosome in different tRNA bound states using three-dimensional (3D) variability analysis. Together, our structural data further our understanding of the ribosome from A. baumannii and other Gram-negative pathogens and will enable structure-based drug discovery to combat antibiotic-resistant bacterial infections. IMPORTANCE Acinetobacter baumannii is a severe nosocomial threat largely due to its intrinsic antibiotic resistance and remarkable ability to acquire new resistance determinants. The bacterial ribosome serves as a major target for modern antibiotics and the design of new therapeutics. Here, we present cryo-EM structures of the A. baumannii 70S ribosome, revealing several unique species-specific structural features that may facilitate future drug development to combat this recalcitrant bacterial pathogen.

scholarly journals Subpopulations of Stressed Yersinia pseudotuberculosis Preferentially Survive Doxycycline Treatment within Host Tissues

mBio ◽  
2020 ◽  
Vol 11 (4) ◽  
Author(s):  
Jasmine Ramirez Raneses ◽  
Alysha L. Ellison ◽  
Bessie Liu ◽  
Kimberly M. Davis
Keyword(s):  
Bacterial Growth ◽  
Intraperitoneal Injection ◽  
Bacterial Infections ◽  
Yersinia Pseudotuberculosis ◽  
Antibiotic Administration ◽  
Bacterial Cells ◽  
Fluorescent Reporter ◽  
Content Type ◽  
Drug Concentrations ◽  
Host Tissues

ABSTRACT Severe systemic bacterial infections result in colonization of deep tissues, which can be very difficult to eliminate with antibiotics. It remains unclear if this is because antibiotics are not reaching inhibitory concentrations within tissues, if subsets of bacteria are less susceptible to antibiotics, or if both contribute to limited treatment efficacy. To detect exposure to doxycycline (Dox) present in deep tissues following treatment, we generated a fluorescent transcriptional reporter derived from the tet operon to specifically detect intracellular tetracycline exposure at the single bacterial cell level. Dox exposure was detected in the spleen 2 h after intraperitoneal injection, and by 4 h postinjection, this treatment resulted in a significant decrease in viable Yersinia pseudotuberculosis bacteria in the spleen. Nitric oxide-stressed bacteria preferentially survived treatment, suggesting that stress was sufficient to alter Dox susceptibility. Many bacteria (∼10%) survived a single dose of Dox, and the antibiotic accumulated at the periphery of microcolonies to growth inhibitory concentrations until 48 h posttreatment. After this time point, antibiotic concentrations decreased and bacterial growth resumed. Dox-treated mice eventually succumbed to the infection, albeit with significantly prolonged survival relative to that of untreated mice. These results indicate that Dox delivery by intraperitoneal injection results in rapid diffusion of inhibitory concentrations of antibiotic into the spleen, but stressed cells preferentially survive drug treatment, and bacterial growth resumes once drug concentrations decrease. This fluorescent reporter strategy for antibiotic detection could easily be modified to detect the concentration of additional antimicrobial compounds within host tissues following drug administration. IMPORTANCE Bacterial infections are very difficult to treat when bacteria spread into the bloodstream and begin to replicate within deep tissues, such as the spleen. Subsets of bacteria can survive antibiotic treatment, but it remains unclear if this survival is because of limited drug diffusion into tissues, or if there are changes within the bacteria, promoting survival of some bacterial cells. Here, we have developed a fluorescent reporter to detect doxycycline (Dox) diffusion into host tissues, and we show that Dox impacts the bacterial population within hours of administration and inhibits bacterial growth for 48 h. However, bacterial growth resumes when antibiotic concentrations decrease. Subsets of bacteria, stressed by the host response to infection, survive Dox treatment at a higher rate. These results provide critical information about the dynamics that occur within deep tissues following antibiotic administration and suggest that subsets of bacteria are predisposed to survive inhibitory concentrations of antibiotic before exposure.

scholarly journals The Poly-γ-d-Glutamic Acid Capsule of Bacillus anthracis Enhances Lethal Toxin Activity

2011 ◽  
Vol 79 (9) ◽  
pp. 3846-3854 ◽  
Author(s):  
Jeyoun Jang ◽  
Minhui Cho ◽  
Jeong-Hoon Chun ◽  
Min-Hee Cho ◽  
Jungchan Park ◽  
...  
Keyword(s):  
Glutamic Acid ◽  
Bacillus Anthracis ◽  
Protective Antigen ◽  
Immune Surveillance ◽  
Lethal Toxin ◽  
Mitogen Activated Protein Kinase ◽  
Content Type ◽  
Mitogen Activated Protein ◽  
Anthrax Infection ◽  
Processed Form

ABSTRACTThe poly-γ-d-glutamic acid (PGA) capsule is one of the major virulence factors ofBacillus anthracis, which causes a highly lethal infectious disease. The PGA capsule disguisesB. anthracisfrom immune surveillance and allows its unimpeded growth in the host. The PGA capsule recently was reported to be associated with lethal toxin (LT) in the blood of experimentally infected animals (M. H. Cho, et al., Infect. Immun. 78:387-392, 2010). The effect of PGA, either alone or in combination with LT, on macrophages, which play an important role in the progression of anthrax disease, has not been thoroughly investigated. In this study, we investigated the effect of PGA on LT cytotoxicity using the mouse macrophage cell line J774A.1. PGA produced a concentration-dependent enhancement of the cytotoxicity of LT on J774A.1 cells through an enhancement in the binding and accumulation of protective antigen to its receptors. The increase of LT activity was confirmed using Western blot analysis, which showed that the combination of PGA and LT produced a greater degree of degradation of mitogen-activated protein kinase kinases and an increased level of the activation of the proform of caspase-1 to its processed form compared to the effects of LT alone. In addition, mice that received a tail vein injection of both PGA and LT had a significantly increased rate of death compared to that of mice injected with LT alone. PGA had no effect when added to cultures or administered to mice in the absence of LT. These results emphasize the importance of PGA in the pathogenesis of anthrax infection.

scholarly journals Deletion of nudB Causes Increased Susceptibility to Antifolates in Escherichia coli and Salmonella enterica

2017 ◽  
Vol 61 (5) ◽  
Author(s):  
Kun Li ◽  
Ting Li ◽  
Shan-shan Yang ◽  
Xu-De Wang ◽  
Lei-xin Gao ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Bacterial Growth ◽  
Salmonella Enterica ◽  
Bacterial Infections ◽  
Synergistic Effects ◽  
Fixed Dose ◽  
Biosynthesis Pathway ◽  
Content Type ◽  
Folate Biosynthesis ◽  
Increased Susceptibility

ABSTRACT Co-trimoxazole, a fixed-dose combination of sulfamethoxazole (SMX) and trimethoprim (TMP), has been used for the treatment of bacterial infections since the 1960s. Since it has long been assumed that the synergistic effects between SMX and TMP are the consequence of targeting 2 different enzymes of bacterial folate biosynthesis, 2 genes (pabB and nudB) involved in the folate biosynthesis of Escherichia coli were deleted, and their effects on the susceptibility to antifolates were tested. The results showed that the deletion of nudB resulted in a lag of growth in minimal medium and increased susceptibility to both SMX and TMP. Moreover, deletion of nudB also greatly enhanced the bactericidal effect of TMP. To elucidate the mechanism of how the deletion of nudB affects the bacterial growth and susceptibility to antifolates, 7,8-dihydroneopterin and 7,8-dihydropteroate were supplemented into the growth medium. Although those metabolites could restore bacterial growth, they had no effect on susceptibilities to the antifolates. Reverse mutants of the nudB deletion strain were isolated to further study the mechanism of how the deletion of nudB affects susceptibility to antifolates. Targeted sequencing and subsequent genetic studies revealed that the disruption of the tetrahydromonapterin biosynthesis pathway could reverse the phenotype caused by the nudB deletion. Meanwhile, overexpression of folM could also lead to increased susceptibility to both SMX and TMP. These data suggested that the deletion of nudB resulted in the excess production of tetrahydromonapterin, which then caused the increased susceptibility to antifolates. In addition, we found that the deletion of nudB also resulted in increased susceptibility to both SMX and TMP in Salmonella enterica. Since dihydroneopterin triphosphate hydrolase is an important component of bacterial folate biosynthesis and the tetrahydromonapterin biosynthesis pathway also exists in a variety of bacteria, it will be interesting to design new compounds targeting dihydroneopterin triphosphate hydrolase, which may inhibit bacterial growth and simultaneously potentiate the antimicrobial activities of antifolates targeting other components of folate biosynthesis.

scholarly journals Screening a Commercial Library of Pharmacologically Active Small Molecules against Staphylococcus aureus Biofilms

2016 ◽  
Vol 60 (10) ◽  
pp. 5663-5672 ◽  
Author(s):  
Nelson S. Torres ◽  
Johnathan J. Abercrombie ◽  
Anand Srinivasan ◽  
Jose L. Lopez-Ribot ◽  
Anand K. Ramasubramanian ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Antimicrobial Activity ◽  
Bacterial Infections ◽  
Drug Repurposing ◽  
Attractive Alternative ◽  
Chemical Library ◽  
Content Type ◽  
Antibiotic Development ◽  
Pharmaceutical Industries ◽  
Pharmacologically Active

ABSTRACTIt is now well established that bacterial infections are often associated with biofilm phenotypes that demonstrate increased resistance to common antimicrobials. Further, due to the collective attrition of new antibiotic development programs by the pharmaceutical industries, drug repurposing is an attractive alternative. In this work, we screened 1,280 existing commercially available drugs in the Prestwick Chemical Library, some with previously unknown antimicrobial activity, againstStaphylococcus aureus, one of the commonly encountered causative pathogens of burn and wound infections. From the primary screen of the entire Prestwick Chemical Library at a fixed concentration of 10 μM, 104 drugs were found to be effective against planktonicS. aureusstrains, and not surprisingly, these were mostly antimicrobials and antiseptics. The activity of 18 selected repurposing candidates, that is, drugs that show antimicrobial activity that are not already considered antimicrobials, observed in the primary screen was confirmed in dose-response experiments. Finally, a subset of nine of these drug candidates was tested against preformed biofilms ofS. aureus. We found that three of these drugs, niclosamide, carmofur, and auranofin, possessed antimicrobial activity against preformed biofilms, making them attractive candidates for repurposing as novel antibiofilm therapies.

scholarly journals Poly-γ-d-Glutamic Acid Capsule Interferes with Lytic Infection of Bacillus anthracis by B. anthracis-Specific Bacteriophages

2012 ◽  
Vol 79 (2) ◽  
pp. 714-717 ◽  
Author(s):  
David Negus ◽  
Jane Burton ◽  
Angela Sweed ◽  
Romuald Gryko ◽  
Peter W. Taylor
Keyword(s):  
Glutamic Acid ◽  
Bacillus Anthracis ◽  
Outer Layer ◽  
Lytic Infection ◽  
Content Type

ABSTRACTThe poly-γ-d-glutamic acid capsule ofBacillus anthracisis a barrier to infection byB. anthracis-specific bacteriophages. Capsule expression was found to completely inhibit lytic infection by γ phage, an observation supported by the demonstration that this phage does not elaborate a hydrolase that would facilitate penetration through the protective capsule outer layer.

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 Draft Genome Sequences of Five Historical Bacillus anthracis Strains

2020 ◽  
Vol 9 (1) ◽  
Author(s):  
Daniel D. Sommer ◽  
Shashikala Ratnayake ◽  
Diana Radune ◽  
Kisha Parker ◽  
Sana Enke ◽  
...  
Keyword(s):  
Bacillus Anthracis ◽  
Causative Agent ◽  
Draft Genome ◽  
Genome Sequences ◽  
Content Type

Bacillus anthracis is the causative agent of anthrax, a disease of livestock, wildlife, and humans. Here, we present the draft genome sequences of five historical B. anthracis strains that were preserved as lyophilates in glass vials for decades.

scholarly journals The Poly-γ-d-Glutamic Acid Capsule Surrogate of the Bacillus anthracis Capsule Is a Novel Toll-Like Receptor 2 Agonist

2015 ◽  
Vol 83 (10) ◽  
pp. 3847-3856 ◽  
Author(s):  
Jun Ho Jeon ◽  
Hae-Ri Lee ◽  
Min-Hee Cho ◽  
Ok-Kyu Park ◽  
Jungchan Park ◽  
...  
Keyword(s):  
Glutamic Acid ◽  
Bacillus Anthracis ◽  
Map Kinases ◽  
Chinese Hamster Ovary Cells ◽  
Mouse Bone Marrow ◽  
Dependent Manner ◽  
Toll Like Receptor ◽  
Content Type ◽  
Tnf Α ◽  
Toll Like Receptor 2

Bacillus anthracisis a pathogenic Gram-positive bacterium that causes a highly lethal infectious disease, anthrax. The poly-γ-d-glutamic acid (PGA) capsule is one of the major virulence factors ofB. anthracis, along with exotoxins. PGA enablesB. anthracisto escape phagocytosis and immune surveillance. Our previous study showed that PGA activates the human macrophage cell line THP-1 and human dendritic cells, resulting in the production of the proinflammatory cytokine interleukin-1β (IL-1β) (M. H. Cho et al., Infect Immun 78:387–392, 2010,http://dx.doi.org/10.1128/IAI.00956-09). Here, we investigated PGA-induced cytokine responses and related signaling pathways in mouse bone marrow-derived macrophages (BMDMs) usingBacillus licheniformisPGA as a surrogate forB. anthracisPGA. Upon exposure to PGA, BMDMs produced proinflammatory mediators, including tumor necrosis factor alpha (TNF-α), IL-6, IL-12p40, and monocyte chemoattractant protein 1 (MCP-1), in a concentration-dependent manner. PGA stimulated Toll-like receptor 2 (TLR2) but not TLR4 in Chinese hamster ovary cells expressing either TLR2 or TLR4. The ability of PGA to induce TNF-α and IL-6 was retained in TLR4−/−but not TLR2−/−BMDMs. Blocking experiments with specific neutralizing antibodies for TLR1, TLR6, and CD14 showed that TLR6 and CD14 also were necessary for PGA-induced inflammatory responses. Furthermore, PGA enhanced activation of mitogen-activated protein (MAP) kinases and nuclear factor-kappa B (NF-κB), which are responsible for expression of proinflammatory cytokines. Additionally, PGA-induced TNF-α production was abrogated not only in MyD88−/−BMDMs but also in BMDMs pretreated with inhibitors of MAP kinases and NF-κB. These results suggest that immune responses induced by PGA occur via TLR2, TLR6, CD14, and MyD88 through activation of MAP kinase and NF-κB pathways.

scholarly journals Galactosylation of the Secondary Cell Wall Polysaccharide ofBacillus anthracisand Its Contribution to Anthrax Pathogenesis

2017 ◽  
Vol 200 (5) ◽  
Author(s):  
Alice Chateau ◽  
Justin Mark Lunderberg ◽  
So Young Oh ◽  
Teresa Abshire ◽  
Arthur Friedlander ◽  
...  
Keyword(s):  
Cell Wall ◽  
Glutamic Acid ◽  
Bacillus Anthracis ◽  
Secondary Cell Wall ◽  
Cell Wall Polysaccharide ◽  
Wild Type ◽  
Content Type ◽  
Bacterial Inoculum ◽  
Associated Proteins

ABSTRACTBacillus anthracis, the causative agent of anthrax disease, elaborates a secondary cell wall polysaccharide (SCWP) that is essential for bacterial growth and cell division.B. anthracisSCWP is comprised of trisaccharide repeats with the structure, [→4)-β-ManNAc-(1→4)-β-GlcNAc(O3-α-Gal)-(1→6)-α-GlcNAc(O3-α-Gal,O4-β-Gal)-(1→]6-12. The genes whose products promote the galactosylation ofB. anthracisSCWP are not yet known. We show here that the expression ofgalE1, encoding a UDP-glucose 4-epimerase necessary for the synthesis of UDP-galactose, is required forB. anthracisSCWP galactosylation. ThegalE1mutant assembles surface (S) layer and S layer-associated proteins that associate with ketal-pyruvylated SCWP via their S layer homology domains similarly to wild-typeB. anthracis, but the mutant displays a defect in γ-phage murein hydrolase binding to SCWP. Furthermore, deletion ofgalE1diminishes the capsulation ofB. anthraciswith poly-d-γ-glutamic acid (PDGA) and causes a reduction in bacterial virulence. These data suggest that SCWP galactosylation is required for the physiologic assembly of theB. anthraciscell wall envelope and for the pathogenesis of anthrax disease.IMPORTANCEUnlike virulentBacillus anthracisisolates,B. anthracisstrain CDC684 synthesizes secondary cell wall polysaccharide (SCWP) trisaccharide repeats without galactosyl modification, exhibits diminished growthin vitroin broth cultures, and is severely attenuated in an animal model of anthrax. To examine whether SCWP galactosylation is a requirement for anthrax disease, we generated variants ofB. anthracisstrains Sterne 34F2 and Ames lacking UDP-glucose 4-epimerase by mutating the genesgalE1andgalE2. We identifiedgalE1as necessary for SCWP galactosylation. Deletion ofgalE1decreased the poly-d-γ-glutamic acid (PDGA) capsulation of the vegetative form ofB. anthracisand increased the bacterial inoculum required to produce lethal disease in mice, indicating that SCWP galactosylation is indeed a determinant of anthrax disease.

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