Edwardsiella tarda Ivy, a Lysozyme Inhibitor That Blocks the Lytic Effect of Lysozyme and Facilitates Host Infection in a Manner That Is Dependent on the Conserved Cysteine Residue

ABSTRACTEdwardsiella tardais a Gram-negative bacterial pathogen with a broad host range that includes fish and humans. In this study, we examined the activity and function of the lysozyme inhibitor Ivy (named IvyEt) identified in the pathogenicE. tardastrain TX01. IvyEtpossesses the Ivy signature motif CKPHDC in the form of82CQPHNC87and contains several highly conserved residues, including a tryptophan (W55). For the purpose of virulence analysis, an isogenic TX01 mutant, TXivy, was created. TXivy bears an in-frame deletion of theivyEtgene. A live infection study in a turbot (Scophthalmus maximus) model showed that, compared to TX01, TXivy exhibited attenuated overall virulence, reduced tissue dissemination and colonization capacity, an impaired ability to replicate in host macrophages, and decreased resistance against the bactericidal effect of host serum. To facilitate functional analysis, recombinant IvyEt(rIvy) and three mutant proteins, i.e., rIvyW55A, rIvyC82S, and rIvyH85D, which bear Ala, Ser, and Asp substitutions at W55, C82, and H85, respectively, were prepared.In vitrostudies showed that rIvy, rIvyW55A, and rIvyH85D were able to block the lytic effect of lysozyme on a Gram-positive bacterium, whereas rIvyC82S could not do so. Likewise, rIvy, but not rIvyC82S, inhibited the serum-facilitated killing effect of lysozyme onE. tarda.In vivoanalysis showed that rIvy, but not rIvyC82S, restored the lost pathogenicity of TXivy and enhanced the infectivity of TX01. Together these results indicate that IvyEtis a lysozyme inhibitor and a virulence factor that depends on the conserved C82 for biological activity.

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Activity of the Pore-Forming Virulence Factor Listeriolysin O Is Reversibly Inhibited by Naturally Occurring S-Glutathionylation

Infection and Immunity ◽

10.1128/iai.00959-16 ◽

2017 ◽

Vol 85 (4) ◽

Cited By ~ 15

Author(s):

Jonathan L. Portman ◽

Qiongying Huang ◽

Michelle L. Reniere ◽

Anthony T. Iavarone ◽

Daniel A. Portnoy

Keyword(s):

Protein Function ◽

Inhibitory Effect ◽

Cysteine Residue ◽

Infection Model ◽

Listeriolysin O ◽

Content Type ◽

Pore Forming Proteins

ABSTRACT Cholesterol-dependent cytolysins (CDCs) represent a family of homologous pore-forming proteins secreted by many Gram-positive bacterial pathogens. CDCs mediate membrane binding partly through a conserved C-terminal undecapeptide, which contains a single cysteine residue. While mutational changes to other residues in the undecapeptide typically have severe effects, mutation of the cysteine residue to alanine has minor effects on overall protein function. Thus, the role of this highly conserved reactive cysteine residue remains largely unknown. We report here that the CDC listeriolysin O (LLO), secreted by the facultative intracellular pathogen Listeria monocytogenes, was posttranslationally modified by S-glutathionylation at this conserved cysteine residue and that either endogenously synthesized or exogenously added glutathione was sufficient to form this modification. When recapitulated with purified protein in vitro, this modification completely ablated the activity of LLO, and this inhibitory effect was fully reversible by treatment with reducing agents. A cysteine-to-alanine mutation in LLO rendered the protein completely resistant to inactivation by S-glutathionylation, and a mutant expressing this mutation retained full hemolytic activity. A mutant strain of L. monocytogenes expressing the cysteine-to-alanine variant of LLO was able to infect and replicate within bone marrow-derived macrophages indistinguishably from the wild type in vitro, yet it was attenuated 4- to 6-fold in a competitive murine infection model in vivo. This study suggests that S-glutathionylation may represent a mechanism by which CDC-family proteins are posttranslationally modified and regulated and help explain an evolutionary pressure to retain the highly conserved undecapeptide cysteine.

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Symbiotic Relationship between Streptococcus mutans and Candida albicans Synergizes Virulence of Plaque BiofilmsIn Vivo

Infection and Immunity ◽

10.1128/iai.00087-14 ◽

2014 ◽

Vol 82 (5) ◽

pp. 1968-1981 ◽

Cited By ~ 216

Author(s):

Megan L. Falsetta ◽

Marlise I. Klein ◽

Punsiri M. Colonne ◽

Kathleen Scott-Anne ◽

Stacy Gregoire ◽

...

Keyword(s):

Candida Albicans ◽

Dental Caries ◽

Streptococcus Mutans ◽

Bacterial Pathogen ◽

Single Species ◽

Content Type ◽

3 Dimensional ◽

Biofilm Development

ABSTRACTStreptococcus mutansis often cited as the main bacterial pathogen in dental caries, particularly in early-childhood caries (ECC).S. mutansmay not act alone;Candida albicanscells are frequently detected along with heavy infection byS. mutansin plaque biofilms from ECC-affected children. It remains to be elucidated whether this association is involved in the enhancement of biofilm virulence. We showed that the ability of these organisms together to form biofilms is enhancedin vitroandin vivo. The presence ofC. albicansaugments the production of exopolysaccharides (EPS), such that cospecies biofilms accrue more biomass and harbor more viableS. mutanscells than single-species biofilms. The resulting 3-dimensional biofilm architecture displays sizeableS. mutansmicrocolonies surrounded by fungal cells, which are enmeshed in a dense EPS-rich matrix. Using a rodent model, we explored the implications of this cross-kingdom interaction for the pathogenesis of dental caries. Coinfected animals displayed higher levels of infection and microbial carriage within plaque biofilms than animals infected with either species alone. Furthermore, coinfection synergistically enhanced biofilm virulence, leading to aggressive onset of the disease with rampant carious lesions. Ourin vitrodata also revealed that glucosyltransferase-derived EPS is a key mediator of cospecies biofilm development and that coexistence withC. albicansinduces the expression of virulence genes inS. mutans(e.g.,gtfB,fabM). We also found thatCandida-derived β1,3-glucans contribute to the EPS matrix structure, while fungal mannan and β-glucan provide sites for GtfB binding and activity. Altogether, we demonstrate a novel mutualistic bacterium-fungus relationship that occurs at a clinically relevant site to amplify the severity of a ubiquitous infectious disease.

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Edwardsiella tarda Eta1, anIn Vivo-Induced Antigen That Is Involved in Host Infection

Infection and Immunity ◽

10.1128/iai.00063-12 ◽

2012 ◽

Vol 80 (8) ◽

pp. 2948-2955 ◽

Cited By ~ 30

Author(s):

Yun Sun ◽

Wen-Jiang Zheng ◽

Yong-Hua Hu ◽

Bo-Guang Sun ◽

Li Sun

Keyword(s):

Early Stage ◽

Paralichthys Olivaceus ◽

Cellular Level ◽

Host Cells ◽

Edwardsiella Tarda ◽

Pcr Analysis ◽

Logarithmic Phase ◽

Content Type

ABSTRACTEdwardsiella tarda, a Gram-negative bacterium, is a severe fish pathogen that can also infect humans. In this study, we identified, viain vivo-induced antigen technology, anE. tardaantigen, Eta1, and analyzed its function in a Japanese flounder (Paralichthys olivaceus) model. Eta1 is composed of 226 residues and shares homology with putative bacterial adhesins. Quantitative real-time reverse transcriptase (RT)-PCR analysis indicated that when culturedin vitro,eta1expression was growth phase dependent and reached maximum at mid-logarithmic phase. During infection of flounder lymphocytes,eta1expression was drastically increased at the early stage of infection. Compared to the wild type, theeta1-defective mutant, TXeta1, was unaffected in growth but exhibited attenuated overall virulence, reduced tissue dissemination and colonization capacity, and impaired ability to invade flounder lymphocytes and to block the immune response of host cells. The lost virulence of TXeta1 was restored when a functionaleta1gene was reintroduced into the strain. Western blot and immunodetection analyses showed that Eta1 is localized to the outer membrane and exposed on the surface ofE. tardaand that recombinant Eta1 (rEta1) was able to interact with flounder lymphocytes. Consistent with these observations, antibody blocking of Eta1 inhibitedE. tardainfection at the cellular level. Furthermore, when used as a subunit vaccine, rEta1 induced strong protective immunity in flounder against lethalE. tardachallenge. Taken together, these results indicate that Eta1 is anin vivo-induced antigen that mediates pathogen-host interaction and, as a result, is required for optimal bacterial infection.

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A SilencedvanAGene Cluster on a Transferable Plasmid Caused an Outbreak of Vancomycin-Variable Enterococci

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00286-16 ◽

2016 ◽

Vol 60 (7) ◽

pp. 4119-4127 ◽

Cited By ~ 16

Author(s):

Audun Sivertsen ◽

Torunn Pedersen ◽

Kjersti Wik Larssen ◽

Kåre Bergh ◽

Torunn Gresdal Rønning ◽

...

Keyword(s):

Gene Cluster ◽

Molecular Mechanisms ◽

Genetic Difference ◽

Brain Heart Infusion ◽

Broad Host Range ◽

S1 Nuclease ◽

Content Type ◽

Transcription Profiles

ABSTRACTWe report an outbreak of vancomycin-variablevanA+enterococci (VVE) able to escape phenotypic detection by current guidelines and demonstrate the molecular mechanisms forin vivoswitching into vancomycin resistance and horizontal spread of thevanAcluster. Forty-eightvanA+Enterococcus faeciumisolates and oneEnterococcus faecalisisolate were analyzed for clonality with pulsed-field gel electrophoresis (PFGE), and theirvanAgene cluster compositions were assessed by PCR and whole-genome sequencing of six isolates. The susceptible VVE strains were cultivated in brain heart infusion broth containing vancomycin at 8 μg/ml forin vitrodevelopment of resistant VVE. The transcription profiles of susceptible VVE and their resistant revertants were assessed using quantitative reverse transcription-PCR. Plasmid content was analyzed with S1 nuclease PFGE and hybridizations. Conjugative transfer ofvanAwas assessed by filter mating. The only genetic difference between thevanAclusters of susceptible and resistant VVE was an ISL3-family element upstream ofvanHAX, which silencedvanHAXgene transcription in susceptible VVE. Furthermore, the VVE had an insertion of IS1542betweenorf2andvanRthat attenuated the expression ofvanHAX. Growth of susceptible VVE occurred after 24 to 72 h of exposure to vancomycin due to excision of the ISL3-family element. ThevanAgene cluster was located on a transferable broad-host-range plasmid also detected in outbreak isolates with different pulsotypes, including oneE. faecalisisolate. Horizontally transferable silencedvanAable to escape detection and revert into resistance during vancomycin therapy represents a new challenge in the clinic. Genotypic testing of invasive vancomycin-susceptible enterococci byvanA-PCR is advised.

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Transcriptome Analysis of Paralichthys olivaceus Erythrocytes Reveals Profound Immune Responses Induced by Edwardsiella tarda Infection

International Journal of Molecular Sciences ◽

10.3390/ijms21093094 ◽

2020 ◽

Vol 21 (9) ◽

pp. 3094

Author(s):

Bin Sun ◽

Xuepeng Li ◽

Xianhui Ning ◽

Li Sun

Keyword(s):

Immune Response ◽

Transcriptome Analysis ◽

Antigen Processing ◽

Paralichthys Olivaceus ◽

Bacterial Pathogen ◽

Infected Fish ◽

Edwardsiella Tarda ◽

Kegg Pathways

Unlike mammalian red blood cells (RBCs), fish RBCs are nucleated and thus capable of gene expression. Japanese flounder (Paralichthys olivaceus) is a species of marine fish with important economic values. Flounder are susceptible to Edwardsiella tarda, a severe bacterial pathogen that is able to infect and survive in flounder phagocytes. However, the infectivity of and the immune response induced by E. tarda in flounder RBCs are unclear. In the present research, we found that E. tarda was able to invade and replicate inside flounder RBCs in both in vitro and in vivo infections. To investigate the immune response induced by E. tarda in RBCs, transcriptome analysis of the spleen RBCs of flounder challenged with E. tarda was performed. Six sequencing libraries were constructed, and an average of 43 million clean reads per library were obtained, with 85% of the reads being successfully mapped to the genome of flounder. A total of 1720 differentially expressed genes (DEGs) were identified in E. tarda-infected fish. The DEGs were significantly enriched in diverse Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, especially those associated with immunity, disease, and infection. Ninety-one key DEGs involved in 12 immune-related pathways were found to form extensive interaction networks. Twenty-one genes that constituted the hub of the networks were further identified, which were highly regulated by E. tarda and involved in a number of immune processes, notably pathogen recognition and signal transduction, antigen processing and presentation, inflammation, and splicing. These results provide new insights into the immune role of flounder RBCs during bacterial infection.

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Caspase-1 and Caspase-11 Mediate Pyroptosis, Inflammation, and Control ofBrucellaJoint Infection

Infection and Immunity ◽

10.1128/iai.00361-18 ◽

2018 ◽

Vol 86 (9) ◽

Cited By ~ 21

Author(s):

Carolyn A. Lacey ◽

William J. Mitchell ◽

Alexis S. Dadelahi ◽

Jerod A. Skyberg

Keyword(s):

Interleukin 1 ◽

Joint Infection ◽

Bacterial Pathogen ◽

Joint Inflammation ◽

Dependent Manner ◽

Content Type ◽

Caspase 1 ◽

And Control

ABSTRACTBrucellosis, caused by the intracellular bacterial pathogenBrucella, is a zoonotic disease for which arthritis is the most common focal complication in humans. Here we investigated the role of inflammasomes and their effectors, including interleukin-1 (IL-1), IL-18, and pyroptosis, on inflammation and control of infection duringBrucella-induced arthritis. Early in infection, both caspase-1 and caspase-11 were found to initiate joint inflammation and proinflammatory cytokine production. However, by 1 week postinfection, caspase-1 and caspase-11 also contributed to control ofBrucellajoint infection. Inflammasome-dependent restriction ofBrucellajoint burdens did not require AIM2 (absent in melanoma 2) or NLRP3 (NLR family, pyrin domain containing 3). IL-1R had a modest effect onBrucella-induced joint swelling, but mice lacking IL-1R were not impaired in their ability to control infection of the joint byBrucella. In contrast, IL-18 contributed to the initiation of joint swelling and control of jointBrucellainfection. Caspase1/11-dependent cell death was observedin vivo, andin vitrostudies demonstrated that both caspase-1 and caspase-11 induce pyroptosis, which limitedBrucellainfection in macrophages.Brucellalipopolysaccharide alone was also able to induce caspase-11-dependent pyroptosis. Collectively, these data demonstrate that inflammasomes induce inflammation in an IL-18-dependent manner and that inflammasome-dependent IL-18 and pyroptosis restrictBrucellainfection.

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Verapamil Targets Membrane Energetics inMycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02107-17 ◽

2018 ◽

Vol 62 (5) ◽

Cited By ~ 27

Author(s):

Chao Chen ◽

Susana Gardete ◽

Robert Sander Jansen ◽

Annanya Shetty ◽

Thomas Dick ◽

...

Keyword(s):

Bacterial Pathogen ◽

Membrane Vesicles ◽

Pharmacokinetic Interaction ◽

Drug Accumulation ◽

Membrane Function ◽

Content Type ◽

Mechanistic Basis ◽

Emergence Of Resistance

ABSTRACTMycobacterium tuberculosiskills more people than any other bacterial pathogen and is becoming increasingly untreatable due to the emergence of resistance. Verapamil, an FDA-approved calcium channel blocker, potentiates the effect of several antituberculosis (anti-TB) drugsin vitroandin vivo. This potentiation is widely attributed to inhibition of the efflux pumps ofM. tuberculosis, resulting in intrabacterial drug accumulation. Here, we confirmed and quantified verapamil's synergy with several anti-TB drugs, including bedaquiline (BDQ) and clofazimine (CFZ), but found that the effect is not due to increased intrabacterial drug accumulation. We show that, consistent with itsin vitropotentiating effects on anti-TB drugs that target or require oxidative phosphorylation, the cationic amphiphile verapamil disrupts membrane function and induces a membrane stress response similar to those seen with other membrane-active agents. We recapitulated these activitiesin vitrousing inverted mycobacterial membrane vesicles, indicating a direct effect of verapamil on membrane energetics. We observed bactericidal activity against nonreplicating “persister”M. tuberculosisthat was consistent with such a mechanism of action. In addition, we demonstrated a pharmacokinetic interaction whereby human-equivalent doses of verapamil caused a boost of rifampin exposure in mice, providing a potential explanation for the observed treatment-shortening effect of verapamil in mice receiving first-line drugs. Our findings thus elucidate the mechanistic basis for verapamil's potentiation of anti-TB drugsin vitroandin vivoand highlight a previously unrecognized role for the membrane ofM. tuberculosisas a pharmacologic target.

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Edwardsiella tarda MliC, a Lysozyme Inhibitor That Participates in Pathogenesis in a Manner That Parallels Ivy

Infection and Immunity ◽

10.1128/iai.02473-14 ◽

2014 ◽

Vol 83 (2) ◽

pp. 583-590 ◽

Cited By ~ 27

Author(s):

Mo-Fei Li ◽

Chong Wang ◽

Li Sun

Keyword(s):

Bacterial Pathogen ◽

Structural Features ◽

Edwardsiella Tarda ◽

Fish Tissues ◽

Farmed Fish ◽

Double Knockout ◽

Content Type ◽

Bacterial Dissemination ◽

Lysozyme Inhibitor ◽

Single Knockout

Edwardsiella tarda, a bacterial pathogen to farmed fish as well as humans, possesses the genes of two lysozyme inhibitors,ivyandmliC(ivyEtandmliCEt). We recently studied IvyEtand found it to be implicated inE. tardavirulence. In the present study, we characterized MliCEtin comparison with IvyEtin a turbot model. MliCEtcontains the FWSKG motif and two cysteines (C33 and C98) that are highly conserved in subgroup 1 MliCs but are of unknown functional importance. To examine the essentialness of these conserved structural features, recombinant MliCEt(rMliC) and its mutants bearing C33S and W79A (of the FWSKG motif) substitutions were prepared. Subsequent analysis showed that rMliC (i) inhibited lysozyme-induced lysis of a Gram-positive bacterium, (ii) reduced serum-facilitated lysozyme killing ofE. tarda, and (iii) when introduced into turbot, promoted bacterial dissemination in fish tissues. The C33S mutation had no influence on the activity of rMliC, while the W79A mutation slightly but significantly enhanced the activity of rMliC. Knockout strains of eithermliCEtorivyEtwere severely attenuated for the ability of tissue invasion, host lethality, serum survival, and intracellular replication. The lost virulence of themliCtransformant (TXΔmliC) was restored by complementation with an introducedmliCEtgene. Compared to the ΔivyEtor ΔmliCEtsingle-knockout strains, the ΔmliCEtΔivyEtdouble-knockout strain was significantly impaired in most of the virulence features. Together, these results provide the first evidence that the conserved cysteine is functionally dispensable to a subgroup 1 MliC and that as a virulence factor, MliCEtmost likely works in a concerted and parallel manner with Ivy.

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Phosphatidylinositol-(3,4,5)-Trisphosphate Induces Phagocytosis of NonmotilePseudomonas aeruginosa

Infection and Immunity ◽

10.1128/iai.00215-18 ◽

2018 ◽

Vol 86 (8) ◽

Cited By ~ 5

Author(s):

Sally Demirdjian ◽

Daniel Hopkins ◽

Hector Sanchez ◽

Michael Libre ◽

Scott A. Gerber ◽

...

Keyword(s):

Pathogenic Bacteria ◽

Bacterial Pathogen ◽

Human Neutrophils ◽

Resistant Bacteria ◽

Flagellar Motility ◽

Chronic Infections ◽

Gram Negative ◽

Content Type

ABSTRACTPathogenic bacteria that establish chronic infections in immunocompromised patients frequently undergo adaptation or selection for traits that are advantageous for their growth and survival. Clinical isolates ofPseudomonas aeruginosa, a Gram-negative, opportunistic bacterial pathogen, exhibit a temporal transition from a motile to a nonmotile phenotype through loss of flagellar motility during the course of chronic infection. This progressive loss of motility is associated with increased resistance to both antibiotic and immune clearance. We have previously shown that loss of bacterial motility enablesP. aeruginosato evade phagocytic clearance bothin vitroandin vivoand fails to activate the phosphatidylinositol 3-kinase (PI3K)/Akt-dependent phagocytic pathway. Therefore, we tested the hypothesis that clearance of phagocytosis-resistant bacteria could be induced by exogenously pretreating innate immune cells with the Akt-activating molecule phosphatidylinositol-(3,4,5)-trisphosphate (PIP3). Here, we demonstrate that PIP3induces the uptake of nonmotileP. aeruginosaby primary human neutrophils >25-fold, and this effect is phenocopied with the use of murine phagocytes. However, surprisingly, mechanistic studies revealed that the induction of phagocytosis by PIP3occurs because polyphosphoinositides promote bacterial binding by the phagocytes rather than bypassing the requirement for PI3K. Moreover, this induction was selective since the uptake of other nonmotile Gram-negative, but not Gram-positive, bacteria can also be induced by PIP3. Since there is currently no treatment that effectively eradicates chronicP. aeruginosainfections, these findings provide novel insights into a potential methodology by which to induce clearance of nonmotile pathogenic bacteria and into the endogenous determinants of phagocytic recognition ofP. aeruginosa.

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Uptake and Metabolism of Antibiotics Roseoflavin and 8-Demethyl-8-Aminoriboflavin in Riboflavin-Auxotrophic Listeria monocytogenes

Journal of Bacteriology ◽

10.1128/jb.00388-16 ◽

2016 ◽

Vol 198 (23) ◽

pp. 3233-3243 ◽

Cited By ~ 19

Author(s):

Andreas Matern ◽

Danielle Pedrolli ◽

Stephanie Großhennig ◽

Jörgen Johansson ◽

Matthias Mack

Keyword(s):

Listeria Monocytogenes ◽

Bacterial Pathogen ◽

Antibiotic Activity ◽

Flavin Mononucleotide ◽

Vitamin B ◽

Content Type ◽

Riboflavin Transport ◽

Uptake And Metabolism

ABSTRACTThe riboflavin analogs roseoflavin (RoF) and 8-demethyl-8-aminoriboflavin (AF) are produced by the bacteriaStreptomyces davawensisandStreptomyces cinnabarinus. Riboflavin analogs have the potential to be used as broad-spectrum antibiotics, and we therefore studied the metabolism of riboflavin (vitamin B2), RoF, and AF in the human pathogenListeria monocytogenes, a bacterium which is a riboflavin auxotroph. We show that theL. monocytogenesprotein Lmo1945 is responsible for the uptake of riboflavin, RoF, and AF. Following import, these flavins are phosphorylated/adenylylated by the bifunctional flavokinase/flavin adenine dinucleotide (FAD) synthetase Lmo1329 and adenylylated by the unique FAD synthetase Lmo0728, the first monofunctional FAD synthetase to be described in bacteria. Lmo1329 generates the cofactors flavin mononucleotide (FMN) and FAD, whereas Lmo0728 produces FAD only. The combined activities of Lmo1329 and Lmo0728 are responsible for the intracellular formation of the toxic cofactor analogs roseoflavin mononucleotide (RoFMN), roseoflavin adenine dinucleotide (RoFAD), 8-demethyl-8-aminoriboflavin mononucleotide (AFMN), and 8-demethyl-8-aminoriboflavin adenine dinucleotide (AFAD).In vivoreporter gene assays andin vitrotranscription/translation experiments show that theL. monocytogenesFMN riboswitch Rli96, which controls expression of the riboflavin transport genelmo1945, is negatively affected by riboflavin/FMN and RoF/RoFMN but not by AF/AFMN. Treatment ofL. monocytogeneswith RoF or AF leads to drastically reduced FMN/FAD levels. We suggest that the reduced flavin cofactor levels in combination with concomitant synthesis of inactive cofactor analogs (RoFMN, RoFAD, AFMN, and AFAD) explain why RoF and AF contribute to antibiotic activity inL. monocytogenes.IMPORTANCEThe riboflavin analogs roseoflavin (RoF) and 8-demethyl-8-aminoriboflavin (AF) are small molecules which are produced byStreptomyces davawensisandStreptomyces cinnabarinus. RoF and AF were reported to have antibacterial activity, and we studied how these compounds are metabolized by the human bacterial pathogenListeria monocytogenes. We found that theL. monocytogenesprotein Lmo1945 mediates uptake of AF and RoF and that the combined activities of the enzymes Lmo1329 and Lmo0728 are responsible for the conversion of AF and RoF to toxic cofactor analogs. Comparative studies with RoF and AF (a weaker antibiotic) suggest that the reduction in FMN/FAD levels and the formation of inactive FMN/FAD analogs explain to a large extent the antibiotic activity of AF and RoF.

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