scholarly journals A Nonoligomerizing Mutant Form of Helicobacter pylori VacA Allows Structural Analysis of the p33 Domain

2016 ◽  
Vol 84 (9) ◽  
pp. 2662-2670 ◽  
Author(s):  
Christian González-Rivera ◽  
Anne M. Campbell ◽  
Stacey A. Rutherford ◽  
Tasia M. Pyburn ◽  
Nora J. Foegeding ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Limited Proteolysis ◽  
Helical Structure ◽  
Water Soluble ◽  
Host Cells ◽  
Mutant Form ◽  
Terminal Portion ◽  
Toxic Activity ◽  
Content Type ◽  
Amino Terminal

Helicobacter pylorisecretes a pore-forming VacA toxin that has structural features and activities substantially different from those of other known bacterial toxins. VacA can assemble into multiple types of water-soluble flower-shaped oligomeric structures, and most VacA activities are dependent on its capacity to oligomerize. The 88-kDa secreted VacA protein can undergo limited proteolysis to yield two domains, designated p33 and p55. The p33 domain is required for membrane channel formation and intracellular toxic activities, and the p55 domain has an important role in mediating VacA binding to cells. Previous studies showed that the p55 domain has a predominantly β-helical structure, but no structural data are available for the p33 domain. We report here the purification and analysis of a nonoligomerizing mutant form of VacA secreted byH. pylori. The nonoligomerizing 88-kDa mutant protein retains the capacity to enter host cells but lacks detectable toxic activity. Analysis of crystals formed by the monomeric protein reveals that the β-helical structure of the p55 domain extends into the C-terminal portion of p33. Fitting the p88 structural model into an electron microscopy map of hexamers formed by wild-type VacA (predicted to be structurally similar to VacA membrane channels) reveals that p55 and the β-helical segment of p33 localize to peripheral arms but do not occupy the central region of the hexamers. We propose that the amino-terminal portion of p33 is unstructured when VacA is in a monomeric form and that it undergoes a conformational change during oligomer assembly.

Functional properties of oligomeric and monomeric forms of Helicobacter pylori VacA toxin

2021 ◽  
Author(s):  
Georgia C. Caso ◽  
Mark S. McClain ◽  
Amanda L. Erwin ◽  
Mandy D. Truelock ◽  
Anne M. Campbell ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Water Soluble ◽  
Host Cells ◽  
Acid Activation ◽  
Mutant Form ◽  
Acidic Ph ◽  
Wild Type ◽  
Oligomeric State ◽  
Mutant Proteins ◽  
Contact Sites

Helicobacter pylori VacA is a secreted toxin that assembles into water-soluble oligomeric structures and forms anion-selective membrane channels. Acidification of purified VacA enhances its activity in cell culture assays. Sites of protomer-protomer contact within VacA oligomers have been identified by cryo-EM, and in the current study we validated several of these interactions by chemical cross-linking and mass spectrometry. We then mutated amino acids at these contact sites and analyzed the effect of the alterations on VacA oligomerization and activity. VacA proteins with amino acid charge reversals at interprotomer contact sites retained the capacity to assemble into water-soluble oligomers and retained cell-vacuolating activity. Introduction of paired cysteine substitutions at these sites resulted in formation of disulfide bonds between adjacent protomers. Negative stain electron microscopy and single particle 2D class analysis revealed that wild-type VacA oligomers disassemble when exposed to acidic pH, whereas the mutant proteins with paired cysteine substitutions retain an oligomeric state at acidic pH. Acid-activated wild-type VacA caused vacuolation of cultured cells, whereas acid-activated mutant proteins with paired cysteine substitutions lacked cell-vacuolating activity. Treatment of these mutant proteins with both low pH and a reducing agent resulted in VacA binding to cells, VacA internalization and cell vacuolation. Internalization of a non-oligomerizing mutant form of VacA by host cells was detected without a requirement for acid-activation. Collectively, these results enhance our understanding of the molecular interactions required for VacA oligomerization and support a model in which toxin activity depends on interactions of monomeric VacA with host cells.

scholarly journals Toxicity and SidJ-Mediated Suppression of Toxicity Require Distinct Regions in the SidE Family of Legionella pneumophila Effectors

2015 ◽  
Vol 83 (9) ◽  
pp. 3506-3514 ◽  
Author(s):  
James C. Havey ◽  
Craig R. Roy
Keyword(s):  
Central Region ◽  
Legionella Pneumophila ◽  
Genetic Interaction ◽  
Terminal Region ◽  
Effector Proteins ◽  
Host Cells ◽  
Toxic Activity ◽  
Content Type ◽  
Amino Terminal ◽  
Low Levels

Intracellular bacteria use a variety of strategies to evade degradation and create a replicative niche.Legionella pneumophilais an intravacuolar pathogen that establishes a replicative niche through the secretion of more than 300 effector proteins. The function of most effectors remains to be determined. Toxicity in yeast has been used to identify functional domains and elucidate the biochemical function of effectors. A library ofL. pneumophilaeffectors was screened using an expression plasmid that produces low levels of each protein. This screen identified the effector SdeA as a protein that confers a strong toxic phenotype that inhibits yeast replication. The toxicity of SdeA was suppressed in cells producing the effector SidJ. The effector SdeA is a member of the SidE family ofL. pneumophilaeffector proteins. All SidE orthologs encoded by the Philadelphia isolate ofLegionella pneumophilawere toxic to yeast, and SidJ suppressed the toxicity of each. We identified a conserved central region in the SidE proteins that was sufficient to mediate yeast toxicity. Surprisingly, SidJ did not suppress toxicity when this central region was produced in yeast. We determined that the amino-terminal region of SidE was essential for SidJ-mediated suppression of toxicity. Thus, there is a genetic interaction that links the activity of SidJ and the amino-terminal region of SidE, which is required to modulate the toxic activity displayed by the central region of the SidE protein. This suggests a complex mechanism by which theL. pneumophilaeffector SidJ modulates the function of the SidE proteins after translocation into host cells.

scholarly journals The Helicobacter pylori Autotransporter ImaA Tempers the Bacterium's Interaction with α5β1 Integrin

2016 ◽  
Vol 85 (1) ◽  
Author(s):  
William E. Sause ◽  
Daniela Keilberg ◽  
Soufiane Aboulhouda ◽  
Karen M. Ottemann
Keyword(s):  
Helicobacter Pylori ◽  
Host Cell ◽  
Type Iv Secretion ◽  
Host Cells ◽  
Wild Type ◽  
Content Type ◽  
Type Iv ◽  
H Pylori ◽  
Α5β1 Integrin ◽  
Cell Interface

ABSTRACT The human pathogen Helicobacter pylori uses the host receptor α5β1 integrin to trigger inflammation in host cells via its cag pathogenicity island (cag PAI) type IV secretion system (T4SS). Here, we report that the H. pylori ImaA protein (HP0289) decreases the action of the cag PAI T4SS via tempering the bacterium's interaction with α5β1 integrin. Previously, imaA-null mutants were found to induce an elevated inflammatory response that was dependent on the cag PAI T4SS; here we extend those findings to show that the elevated response is independent of the CagA effector protein. To understand how ImaA could be affecting cag PAI T4SS activity at the host cell interface, we utilized the Phyre structural threading program and found that ImaA has a region with remote homology to bacterial integrin-binding proteins. This region was required for ImaA function. Unexpectedly, we observed that imaA mutants bound higher levels of α5β1 integrin than wild-type H. pylori, an outcome that required the predicted integrin-binding homology region of ImaA. Lastly, we report that ImaA directly affected the amount of host cell β1 integrin but not other cellular integrins. Our results thus suggest a model in which H. pylori employs ImaA to regulate interactions between integrin and the T4SS and thus alter the host inflammatory strength.

scholarly journals The Helicobacter pylori CZB Cytoplasmic Chemoreceptor TlpD Forms an Autonomous Polar Chemotaxis Signaling Complex That Mediates a Tactic Response to Oxidative Stress

2016 ◽  
Vol 198 (11) ◽  
pp. 1563-1575 ◽  
Author(s):  
Kieran D. Collins ◽  
Tessa M. Andermann ◽  
Jenny Draper ◽  
Lisa Sanders ◽  
Susan M. Williams ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Reactive Oxygen Species ◽  
Helicobacter Pylori ◽  
Molecular Mechanisms ◽  
Reactive Oxygen ◽  
Host Cells ◽  
Oxygen Species ◽  
Content Type ◽  
Signaling Complex ◽  
H Pylori

ABSTRACTCytoplasmic chemoreceptors are widespread among prokaryotes but are far less understood than transmembrane chemoreceptors, despite being implicated in many processes. One such cytoplasmic chemoreceptor isHelicobacter pyloriTlpD, which is required for stomach colonization and drives a chemotaxis response to cellular energy levels. Neither the signals sensed by TlpD nor its molecular mechanisms of action are known. We report here that TlpD functions independently of the other chemoreceptors. When TlpD is the sole chemoreceptor, it is able to localize to the pole and recruits CheW, CheA, and at least two CheV proteins to this location. It loses the normal membrane association that appears to be driven by interactions with other chemoreceptors and with CheW, CheV1, and CheA. These results suggest that TlpD can form an autonomous signaling unit. We further determined that TlpD mediates a repellent chemotaxis response to conditions that promote oxidative stress, including being in the presence of iron, hydrogen peroxide, paraquat, and metronidazole. Last, we found that all testedH. pyloristrains express TlpD, whereas other chemoreceptors were present to various degrees. Our data suggest a model in which TlpD coordinates a signaling complex that responds to oxidative stress and may allowH. pylorito avoid areas of the stomach with high concentrations of reactive oxygen species.IMPORTANCEHelicobacter pylorisenses its environment with proteins called chemoreceptors. Chemoreceptors integrate this sensory information to affect flagellum-based motility in a process called chemotaxis. Chemotaxis is employed during infection and presumably aidsH. pyloriin encountering and colonizing preferred niches. A cytoplasmic chemoreceptor named TlpD is particularly important in this process, and we report here that this chemoreceptor is able to operate independently of other chemoreceptors to organize a chemotaxis signaling complex and mediate a repellent response to oxidative stress conditions.H. pyloriencounters and must cope with oxidative stress during infection due to oxygen and reactive oxygen species produced by host cells. TlpD's repellent response may allow the bacteria to escape niches experiencing inflammation and elevated reactive oxygen species (ROS) production.

scholarly journals Natural Competence Promotes Helicobacter pylori Chronic Infection

2012 ◽  
Vol 81 (1) ◽  
pp. 209-215 ◽  
Author(s):  
Marion S. Dorer ◽  
Ilana E. Cohen ◽  
Tate H. Sessler ◽  
Jutta Fero ◽  
Nina R. Salama
Keyword(s):  
Helicobacter Pylori ◽  
Chronic Infection ◽  
Acute Infection ◽  
Host Cells ◽  
High Rate ◽  
Natural Competence ◽  
Content Type ◽  
Type Iv ◽  
Competence Factor ◽  
H Pylori

Animal models are important tools for studies of human disease, but developing these models is a particular challenge with regard to organisms with restricted host ranges, such as the human stomach pathogenHelicobacter pylori. In most cases,H. pyloriinfects the stomach for many decades before symptoms appear, distinguishing it from many bacterial pathogens that cause acute infection. To model chronic infection in the mouse, a human clinical isolate was selected for its ability to survive for 2 months in the mouse stomach, and the resulting strain, MSD132, colonized the mouse stomach for at least 28 weeks. During selection, thecagYcomponent of the Cag type IV secretion system was mutated, disrupting a key interaction with host cells. Increases in both bacterial persistence and bacterial burden occurred prior to this mutation, and a mixed population ofcagY+andcagYmutant cells was isolated from a single mouse, suggesting that mutations accumulate during selection and that factors in addition to the Cag apparatus are important for murine adaptation. Diversity in both alleles and genes is common inH. pyloristrains, and natural competence mediates a high rate of interstrain genetic exchange. Mutations of the Com apparatus, a membrane DNA transporter, and DprA, a cytosolic competence factor, resulted in reduced persistence, although initial colonization was normal. Thus, exchange of DNA between genetically heterogeneousH. pyloristrains may improve chronic colonization. The strains and methods described here will be important tools for defining both the spectrum of mutations that promote murine adaptation and the genetic program of chronic infection.

scholarly journals Identification and Characterization of the Binding Site of the Respiratory Syncytial Virus Phosphoprotein to RNA-Free Nucleoprotein

2015 ◽  
Vol 89 (7) ◽  
pp. 3484-3496 ◽  
Author(s):  
Marie Galloux ◽  
Gaëlle Gabiane ◽  
Julien Sourimant ◽  
Charles-Adrien Richard ◽  
Patrick England ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Rna Polymerase ◽  
Rna Synthesis ◽  
Polymerase Activity ◽  
Viral Rna ◽  
Free Form ◽  
Mutant Form ◽  
Content Type ◽  
Amino Terminal ◽  
Syncytial Virus

ABSTRACTThe RNA genome of respiratory syncytial virus (RSV) is constitutively encapsidated by the viral nucleoprotein N, thus forming a helical nucleocapsid. Polymerization of N along the genomic and antigenomic RNAs is concomitant to replication and requires the preservation of an unassembled monomeric nucleoprotein pool. To this end, and by analogy withParamyxoviridaeandRhabdoviridae, it is expected that the viral phosphoprotein P acts as a chaperone protein, forming a soluble complex with the RNA-free form of N (N0-P complex). Here, we have engineered a mutant form of N that is monomeric, is unable to bind RNA, still interacts with P, and could thus mimic the N0monomer. We used this N mutant, designated Nmono, as a substitute for N0in order to characterize the P regions involved in the N0-P complex formation. Using a series of P fragments, we determined by glutathioneS-transferase (GST) pulldown assays that the N and C termini of P are able to interact with Nmono. We analyzed the functional role of amino-terminal residues of P by site-directed mutagenesis, using an RSV polymerase activity assay based on a human RSV minireplicon, and found that several residues were critical for viral RNA synthesis. Using GST pulldown and surface plasmon resonance assays, we showed that these critical residues are involved in the interaction between P[1-40] peptide and Nmonoin vitro. Finally, we showed that overexpression of the peptide P[1-29] can inhibit the polymerase activity in the context of the RSV minireplicon, thus demonstrating that targeting the N0-P interaction could constitute a potential antiviral strategy.IMPORTANCERespiratory syncytial virus (RSV) is the leading cause of lower respiratory tract illness in infants. Since no vaccine or efficient antiviral treatment is available against RSV, it is essential to better understand how the viral machinery functions in order to develop new antiviral strategies. RSV phosphoprotein P, the main RNA polymerase cofactor, is believed to function as a chaperon protein, maintaining N as a nonassembled, RNA-free protein (N0) competent for RNA encapsidation. In this paper, we provide the first evidence, to our knowledge, that the N terminus of P contains a domain that binds specifically to this RNA-free form of N. We further show that overexpression of a small peptide spanning this region of P can inhibit viral RNA synthesis. These findings extend our understanding of the function of RSV RNA polymerase and point to a new target for the development of drugs against this virus.

scholarly journals Chemokines and Antimicrobial Peptides Have acag-Dependent Early Response to Helicobacter pylori Infection in Primary Human Gastric Epithelial Cells

2014 ◽  
Vol 82 (7) ◽  
pp. 2881-2889 ◽  
Author(s):  
Pascale Mustapha ◽  
Isabelle Paris ◽  
Magali Garcia ◽  
Cong Tri Tran ◽  
Julie Cremniter ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Epithelial Cells ◽  
Inflammatory Response ◽  
Antimicrobial Peptides ◽  
Virulence Factors ◽  
Inflammatory Mediator ◽  
Host Cells ◽  
Gastric Epithelial Cells ◽  
Content Type ◽  
H Pylori

ABSTRACTHelicobacter pyloriinfection systematically causes chronic gastric inflammation that can persist asymptomatically or evolve toward more severe gastroduodenal pathologies, such as ulcer, mucosa-associated lymphoid tissue (MALT) lymphoma, and gastric cancer. Thecagpathogenicity island (cagPAI) ofH. pyloriallows translocation of the virulence protein CagA and fragments of peptidoglycan into host cells, thereby inducing production of chemokines, cytokines, and antimicrobial peptides. In order to characterize the inflammatory response toH. pylori, a new experimental protocol for isolating and culturing primary human gastric epithelial cells was established using pieces of stomach from patients who had undergone sleeve gastrectomy. Isolated cells expressed markers indicating that they were mucin-secreting epithelial cells. Challenge of primary epithelial cells withH. pyloriB128 underscored early dose-dependent induction of expression of mRNAs of the inflammatory mediators CXCL1 to -3, CXCL5, CXCL8, CCL20, BD2, and tumor necrosis factor alpha (TNF-α). In AGS cells, significant expression of only CXCL5 and CXCL8 was observed following infection, suggesting that these cells were less reactive than primary epithelial cells. Infection of both cellular models withH. pyloriB128ΔcagM, acagPAI mutant, resulted in weak inflammatory-mediator mRNA induction. At 24 h after infection of primary epithelial cells withH. pylori, inflammatory-mediator production was largely due tocagPAI substrate-independent virulence factors. Thus,H. pyloricagPAI substrate appears to be involved in eliciting an epithelial response during the early phases of infection. Afterwards, other virulence factors of the bacterium take over in development of the inflammatory response. Using a relevant cellular model, this study provides new information on the modulation of inflammation duringH. pyloriinfection.

scholarly journals Nuclease Activity of Legionella pneumophila Cas2 Promotes Intracellular Infection of Amoebal Host Cells

2014 ◽  
Vol 83 (3) ◽  
pp. 1008-1018 ◽  
Author(s):  
Felizza F. Gunderson ◽  
Celeste A. Mallama ◽  
Stephanie G. Fairbairn ◽  
Nicholas P. Cianciotto
Keyword(s):  
Legionella Pneumophila ◽  
Acanthamoeba Castellanii ◽  
Nuclease Activity ◽  
Infection Process ◽  
Host Cells ◽  
Mutant Form ◽  
Content Type ◽  
Intracellular Infection ◽  
Short Palindromic Repeat ◽  
Naegleria Lovaniensis

Legionella pneumophila, the primary agent of Legionnaires' disease, flourishes in both natural and man-made environments by growing in a wide variety of aquatic amoebae. Recently, we determined that the Cas2 protein ofL. pneumophilapromotes intracellular infection ofAcanthamoeba castellaniiandHartmannella vermiformis, the two amoebae most commonly linked to cases of disease. The Cas2 family of proteins is best known for its role in the bacterial and archeal clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated protein (Cas) system that constitutes a form of adaptive immunity against phage and plasmid. However, the infection event mediated byL. pneumophilaCas2 appeared to be distinct from this function, becausecas2mutants exhibited infectivity defects in the absence of added phage or plasmid and since mutants lacking the CRISPR array or any one of the othercasgenes were not impaired in infection ability. We now report that the Cas2 protein ofL. pneumophilahas both RNase and DNase activities, with the RNase activity being more pronounced. By characterizing a catalytically deficient version of Cas2, we determined that nuclease activity is critical for promoting infection of amoebae. Also, introduction of Cas2, but not its catalytic mutant form, into a strain ofL. pneumophilathat naturally lacks a CRISPR-Cas locus caused that strain to be 40- to 80-fold more infective for amoebae, unequivocally demonstrating that Cas2 facilitates the infection process independently of any other component encoded within the CRISPR-Cas locus. Finally, acas2mutant was impaired for infection ofWillaertia magnabut notNaegleria lovaniensis, suggesting that Cas2 promotes infection of most but not all amoebal hosts.

scholarly journals Uptake of Helicobacter pylori Vesicles Is Facilitated by Clathrin-Dependent and Clathrin-Independent Endocytic Pathways

mBio ◽  
2014 ◽  
Vol 5 (3) ◽  
Author(s):  
Annelie Olofsson ◽  
Lars Nygård Skalman ◽  
Ikenna Obi ◽  
Richard Lundmark ◽  
Anna Arnqvist
Keyword(s):  
Helicobacter Pylori ◽  
Host Cell ◽  
Host Cells ◽  
Gastric Epithelial Cells ◽  
Host Cell Membrane ◽  
Content Type ◽  
Effector Molecules ◽  
Chemical Inhibition ◽  
H Pylori ◽  
Endocytic Pathways

ABSTRACTBacteria shed a diverse set of outer membrane vesicles that function as transport vehicles to deliver effector molecules and virulence factors to host cells.Helicobacter pyloriis a gastric pathogen that infects half of the world’s population, and in some individuals the infection progresses into peptic ulcer disease or gastric cancer. Here we report that intact vesicles fromH. pyloriare internalized by clathrin-dependent endocytosis and further dynamin-dependent processes, as well as in a cholesterol-sensitive manner. We analyzed the uptake ofH. pylorivesicles by gastric epithelial cells using a method that we refer to as quantification of internalized substances (qIS). The qIS assay is based on a near-infrared dye with a cleavable linker that enables the specific quantification of internalized substances after exposure to reducing conditions. Both chemical inhibition and RNA interference in combination with the qIS assay showed thatH. pylorivesicles enter gastric epithelial cells via both clathrin-mediated endocytosis and additional endocytic processes that are dependent on dynamin. Confocal microscopy revealed thatH. pylorivesicles colocalized with clathrin and dynamin II and with markers of subsequent endosomal and lysosomal trafficking. Interestingly, however, knockdown of components required for caveolae had no significant effect on internalization and knockdown of components required for clathrin-independent carrier (CLIC) endocytosis increased internalization ofH. pylorivesicles. Furthermore, uptake of vesicles by both clathrin-dependent and -independent pathways was sensitive to depletion, but not sequestering, of cholesterol in the host cell membrane suggesting that membrane fluidity influences the efficiency ofH. pylorivesicle uptake.IMPORTANCEBacterial vesicles act as long-distance tools to deliver toxins and effector molecules to host cells. Vesicles can cause a variety of host cell responses via cell surface-induced cell signaling or internalization. Vesicles of diverse bacterial species enter host cells via different endocytic pathways or via membrane fusion. With the combination of a fluorescence-based quantification assay that quantifies internalized vesicles in a large number of cells and either chemical inhibition or RNA interference, we show that clathrin-mediated endocytosis is the major pathway for uptake ofHelicobacter pylorivesicles and that lipid microdomains of the host cell membrane affect uptake of vesicles via clathrin-independent pathways. Our results provide important insights about membrane fluidity and its important role in the complex process that directs theH. pylorivesicle to a specific endocytic pathway. Understanding the mechanisms that operate in vesicle-host interactions is important to fully recognize the impact of vesicles in pathogenesis.

scholarly journals Preclinical Studies of Amixicile, a Systemic Therapeutic Developed for Treatment of Clostridium difficile Infections That Also Shows Efficacy against Helicobacter pylori

2014 ◽  
Vol 58 (8) ◽  
pp. 4703-4712 ◽  
Author(s):  
Paul S. Hoffman ◽  
Alexandra M. Bruce ◽  
Igor Olekhnovich ◽  
Cirle A. Warren ◽  
Stacey L. Burgess ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Helicobacter Pylori ◽  
Clostridium Difficile ◽  
Maximum Concentration ◽  
Water Soluble ◽  
Thiamine Pyrophosphate ◽  
Escape Mutation ◽  
Rat Liver Microsomes ◽  
Cancer Resistance ◽  
Content Type

ABSTRACTAmixicile shows efficacy in the treatment ofClostridium difficileinfections (CDI) in a mouse model, with no recurrence of CDI. Since amixicile selectively inhibits the action of a B vitamin (thiamine pyrophosphate) cofactor of pyruvate:ferredoxin oxidoreductase (PFOR), it may both escape mutation-based drug resistance and spare beneficial probiotic gut bacteria that do not express this enzyme. Amixicile is a water-soluble derivative of nitazoxanide (NTZ), an antiparasitic therapeutic that also shows efficacy against CDI in humans. In comparative studies, amixicile showed no toxicity to hepatocytes at 200 μM (NTZ was toxic above 10 μM); was not metabolized by human, dog, or rat liver microsomes; showed equivalence or superiority to NTZ in cytochrome P450 assays; and did not activate efflux pumps (breast cancer resistance protein, P glycoprotein). A maximum dose (300 mg/kg) of amixicile given by the oral or intraperitoneal route was well tolerated by mice and rats. Plasma exposure (rats) based on the area under the plasma concentration-time curve was 79.3 h · μg/ml (30 mg/kg dose) to 328 h · μg/ml (100 mg/kg dose), the maximum concentration of the drug in serum was 20 μg/ml, the time to the maximum concentration of the drug in serum was 0.5 to 1 h, and the half-life was 5.6 h. Amixicile did not concentrate in mouse feces or adversely affect gut populations ofBacteroidesspecies,Firmicutes, segmented filamentous bacteria, orLactobacillusspecies. Systemic bioavailability was demonstrated through eradication ofHelicobacter pyloriin a mouse infection model. In summary, the efficacy of amixicile in treating CDI and other infections, together with low toxicity, an absence of mutation-based drug resistance, and excellent drug metabolism and pharmacokinetic metrics, suggests a potential for broad application in the treatment of infections caused by PFOR-expressing microbial pathogens in addition to CDI.

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