Engineered Biosynthesis of Disaccharide-Modified Polyene Macrolides

ABSTRACTRecent work has uncovered genes for two glycosyltransferases that are thought to catalyze mannosylation of mycosaminyl sugars of polyene macrolides. These two genes arenypYfromPseudonocardiasp. strain P1 andpegAfromActinoplanes caeruleus. Here we analyze these genes by heterologous expression in various strains ofStreptomyces nodosus, producer of amphotericins, and inStreptomyces albidoflavus, which produces candicidins. The NypY glycosyltransferase converted amphotericins A and B and 7-oxo-amphotericin B to disaccharide-modified formsin vivo. The enzyme did not act on amphotericin analogs lacking exocyclic carboxyl or mycosamine amino groups. Both NypY and PegA acted on candicidins. This work confirms the functions of these glycosyltransferases and provides insights into their acceptor substrate tolerance. Disaccharide-modified polyenes may have potential as less toxic antibiotics.

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MetR-Regulated Vibrio cholerae Metabolism Is Required for Virulence

mBio ◽

10.1128/mbio.00236-12 ◽

2012 ◽

Vol 3 (5) ◽

Cited By ~ 28

Author(s):

Ryan W. Bogard ◽

Bryan W. Davies ◽

John J. Mekalanos

Keyword(s):

Amino Acid ◽

Vibrio Cholerae ◽

Virulence Factor ◽

Current Knowledge ◽

Intestinal Colonization ◽

Wild Type ◽

Pathogenic Potential ◽

Content Type ◽

Mouse Intestine

ABSTRACTLysR-type transcriptional regulators (LTTRs) are the largest, most diverse family of prokaryotic transcription factors, with regulatory roles spanning metabolism, cell growth and division, and pathogenesis. Using a sequence-defined transposon mutant library, we screened a panel ofV. choleraeEl Tor mutants to identify LTTRs required for host intestinal colonization. Surprisingly, out of 38 LTTRs, only one severely affected intestinal colonization in the suckling mouse model of cholera: the methionine metabolism regulator, MetR. Genetic analysis of genes influenced by MetR revealed thatglyA1andmetJwere also required for intestinal colonization. Chromatin immunoprecipitation of MetR and quantitative reverse transcription-PCR (qRT-PCR) confirmed interaction with and regulation ofglyA1, indicating that misregulation ofglyA1is likely responsible for the colonization defect observed in themetRmutant. TheglyA1mutant was auxotrophic for glycine but exhibited wild-type trimethoprim sensitivity, making folate deficiency an unlikely cause of its colonization defect. MetJ regulatory mutants are not auxotrophic but are likely altered in the regulation of amino acid-biosynthetic pathways, including those for methionine, glycine, and serine, and this misregulation likely explains its colonization defect. However, mutants defective in methionine, serine, and cysteine biosynthesis exhibited wild-type virulence, suggesting that these amino acids can be scavenged in vivo. Taken together, our results suggest that glycine biosynthesis may be required to alleviate an in vivo nutritional restriction in the mouse intestine; however, additional roles for glycine may exist. Irrespective of the precise nature of this requirement, this study illustrates the importance of pathogen metabolism, and the regulation thereof, as a virulence factor.IMPORTANCEVibrio choleraecontinues to be a severe cause of morbidity and mortality in developing countries. Identification ofV. choleraefactors critical to disease progression offers the potential to develop or improve upon therapeutics and prevention strategies. To increase the efficiency of virulence factor discovery, we employed a regulator-centric approach to multiplex our in vivo screening capabilities and allow whole regulons inV. choleraeto be interrogated for pathogenic potential. We identified MetR as a new virulence regulator and serine hydroxymethyltransferase GlyA1 as a new MetR-regulated virulence factor, both required byV. choleraeto colonize the infant mouse intestine. Bacterial metabolism is a prerequisite to virulence, and current knowledge of in vivo metabolism of pathogens is limited. Here, we expand the known role of amino acid metabolism and regulation in virulence and offer new insights into the in vivo metabolic requirements ofV. choleraewithin the mouse intestine.

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Interactions and Localization of Escherichia coli Error-Prone DNA Polymerase IV after DNA Damage

Journal of Bacteriology ◽

10.1128/jb.00101-15 ◽

2015 ◽

Vol 197 (17) ◽

pp. 2792-2809 ◽

Cited By ~ 20

Author(s):

Sarita Mallik ◽

Ellen M. Popodi ◽

Andrew J. Hanson ◽

Patricia L. Foster

Keyword(s):

Dna Damage ◽

Dna Polymerase ◽

Dna Helicase ◽

Dna Lesions ◽

Replication Forks ◽

Content Type ◽

Pol Iv ◽

Dna Polymerase Iv ◽

Stalled Replication Forks

ABSTRACTEscherichia coli's DNA polymerase IV (Pol IV/DinB), a member of the Y family of error-prone polymerases, is induced during the SOS response to DNA damage and is responsible for translesion bypass and adaptive (stress-induced) mutation. In this study, the localization of Pol IV after DNA damage was followed using fluorescent fusions. After exposure ofE. colito DNA-damaging agents, fluorescently tagged Pol IV localized to the nucleoid as foci. Stepwise photobleaching indicated ∼60% of the foci consisted of three Pol IV molecules, while ∼40% consisted of six Pol IV molecules. Fluorescently tagged Rep, a replication accessory DNA helicase, was recruited to the Pol IV foci after DNA damage, suggesting that thein vitrointeraction between Rep and Pol IV reported previously also occursin vivo. Fluorescently tagged RecA also formed foci after DNA damage, and Pol IV localized to them. To investigate if Pol IV localizes to double-strand breaks (DSBs), an I-SceI endonuclease-mediated DSB was introduced close to a fluorescently labeled LacO array on the chromosome. After DSB induction, Pol IV localized to the DSB site in ∼70% of SOS-induced cells. RecA also formed foci at the DSB sites, and Pol IV localized to the RecA foci. These results suggest that Pol IV interacts with RecAin vivoand is recruited to sites of DSBs to aid in the restoration of DNA replication.IMPORTANCEDNA polymerase IV (Pol IV/DinB) is an error-prone DNA polymerase capable of bypassing DNA lesions and aiding in the restart of stalled replication forks. In this work, we demonstratein vivolocalization of fluorescently tagged Pol IV to the nucleoid after DNA damage and to DNA double-strand breaks. We show colocalization of Pol IV with two proteins: Rep DNA helicase, which participates in replication, and RecA, which catalyzes recombinational repair of stalled replication forks. Time course experiments suggest that Pol IV recruits Rep and that RecA recruits Pol IV. These findings providein vivoevidence that Pol IV aids in maintaining genomic stability not only by bypassing DNA lesions but also by participating in the restoration of stalled replication forks.

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Exogenous Alginate Protects Staphylococcus aureus from Killing by Pseudomonas aeruginosa

Journal of Bacteriology ◽

10.1128/jb.00559-19 ◽

2019 ◽

Vol 202 (8) ◽

Cited By ~ 2

Author(s):

Courtney E. Price ◽

Dustin G. Brown ◽

Dominique H. Limoli ◽

Vanessa V. Phelan ◽

George A. O’Toole

Keyword(s):

Staphylococcus Aureus ◽

Pseudomonas Aeruginosa ◽

Physical Space ◽

Late Time ◽

Protective Effects ◽

Content Type ◽

Alginate Production ◽

The Impact

ABSTRACT Cystic fibrosis (CF) patients chronically infected with both Pseudomonas aeruginosa and Staphylococcus aureus have worse health outcomes than patients who are monoinfected with either P. aeruginosa or S. aureus. We showed previously that mucoid strains of P. aeruginosa can coexist with S. aureus in vitro due to the transcriptional downregulation of several toxic exoproducts typically produced by P. aeruginosa, including siderophores, rhamnolipids, and HQNO (2-heptyl-4-hydroxyquinoline N-oxide). Here, we demonstrate that exogenous alginate protects S. aureus from P. aeruginosa in both planktonic and biofilm coculture models under a variety of nutritional conditions. S. aureus protection in the presence of exogenous alginate is due to the transcriptional downregulation of pvdA, a gene required for the production of the iron-scavenging siderophore pyoverdine as well as the downregulation of the PQS (Pseudomonas quinolone signal) (2-heptyl-3,4-dihydroxyquinoline) quorum sensing system. The impact of exogenous alginate is independent of endogenous alginate production. We further demonstrate that coculture of mucoid P. aeruginosa with nonmucoid P. aeruginosa strains can mitigate the killing of S. aureus by the nonmucoid strain of P. aeruginosa, indicating that the mechanism that we describe here may function in vivo in the context of mixed infections. Finally, we investigated a panel of mucoid clinical isolates that retain the ability to kill S. aureus at late time points and show that each strain has a unique expression profile, indicating that mucoid isolates can overcome the S. aureus-protective effects of mucoidy in a strain-specific manner. IMPORTANCE CF patients are chronically infected by polymicrobial communities. The two dominant bacterial pathogens that infect the lungs of CF patients are P. aeruginosa and S. aureus, with ∼30% of patients coinfected by both species. Such coinfected individuals have worse outcomes than monoinfected patients, and both species persist within the same physical space. A variety of host and environmental factors have been demonstrated to promote P. aeruginosa-S. aureus coexistence, despite evidence that P. aeruginosa kills S. aureus when these organisms are cocultured in vitro. Thus, a better understanding of P. aeruginosa-S. aureus interactions, particularly mechanisms by which these microorganisms are able to coexist in proximal physical space, will lead to better-informed treatments for chronic polymicrobial infections.

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Dynamics ofMycobacterium tuberculosisAg85B Revealed by a Sensitive Enzyme-Linked Immunosorbent Assay

mBio ◽

10.1128/mbio.00611-19 ◽

2019 ◽

Vol 10 (2) ◽

Cited By ~ 2

Author(s):

Joel D. Ernst ◽

Amber Cornelius ◽

Miriam Bolz

Keyword(s):

Protein Secretion ◽

Immunosorbent Assay ◽

Bacterial Population ◽

Enzyme Linked Immunosorbent Assay ◽

Bacterial Protein ◽

Gram Positive ◽

Gram Negative ◽

Content Type ◽

Bacterial Proteins

ABSTRACTSecretion of specific proteins contributes to pathogenesis and immune responses in tuberculosis and other bacterial infections, yet the kinetics of protein secretion and fate of secreted proteinsin vivoare poorly understood. We generated new monoclonal antibodies that recognize theMycobacteriumtuberculosissecreted protein Ag85B and used them to establish and characterize a sensitive enzyme-linked immunosorbent assay (ELISA) to quantitate Ag85B in samples generatedin vitroandin vivo. We found that nutritional or culture conditions had little impact on the secretion of Ag85B and that there is considerable variation in Ag85B secretion by distinct strains in theM. tuberculosiscomplex: compared with the commonly used H37Rv strain (lineage 4),Mycobacteriumafricanum(lineage 6) secretes less Ag85B, and two strains from lineage 2 secrete more Ag85B. We also used the ELISA to determine that the rate of secretion of Ag85B is 10- to 100-fold lower than that of proteins secreted by Gram-negative and Gram-positive bacteria, respectively. ELISA quantitation of Ag85B in lung homogenates ofM. tuberculosisH37Rv-infected mice revealed that although Ag85B accumulates in the lungs as the bacterial population expands, the amount of Ag85B per bacterium decreases nearly 10,000-fold at later stages of infection, coincident with the development of T cell responses and arrest of bacterial population growth. These results indicate that bacterial protein secretionin vivois dynamic and regulated, and quantitation of secreted bacterial proteins can contribute to the understanding of pathogenesis and immunity in tuberculosis and other infections.IMPORTANCEBacterial protein secretion contributes to host-pathogen interactions, yet the process and consequences of bacterial protein secretion during infection are poorly understood. We developed a sensitive ELISA to quantitate a protein (termed Ag85B) secreted byM. tuberculosisand used it to find that Ag85B secretion occurs with slower kinetics than for proteins secreted by Gram-positive and Gram-negative bacteria and that accumulation of Ag85B in the lungs is markedly regulated as a function of the bacterial population density. Our results demonstrate that quantitation of bacterial proteins during infection can reveal novel insights into host-pathogen interactions.

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Pharmacokinetics and Pharmacodynamics of the Nitroimidazole DNDI-0690 in Mouse Models of Cutaneous Leishmaniasis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00829-19 ◽

2019 ◽

Vol 63 (9) ◽

Cited By ~ 5

Author(s):

Gert-Jan Wijnant ◽

Simon L. Croft ◽

Raul de la Flor ◽

Mo Alavijeh ◽

Vanessa Yardley ◽

...

Keyword(s):

Cutaneous Leishmaniasis ◽

Mouse Models ◽

Drug Exposure ◽

Oral Treatment ◽

Drug Application ◽

Skin Tissue ◽

Drug Candidate ◽

Topical Formulation ◽

Content Type

ABSTRACT The nitroimidazole DNDI-0690 is a clinical drug candidate for visceral leishmaniasis (VL) that also shows potent in vitro and in vivo activity against cutaneous leishmaniasis (CL). To support further development of this compound into a patient-friendly oral or topical formulation for the treatment of CL, we investigated the free drug exposure at the dermal site of infection and subsequent elimination of the causative Leishmania pathogen. This study evaluates the pharmacokinetics (PK) and pharmacodynamics (PD) of DNDI-0690 in mouse models of CL. Skin microdialysis and Franz diffusion cell permeation studies revealed that DNDI-0690 permeated poorly (<1%) into the skin lesion upon topical drug application (0.063% [wt/vol], 30 μl). In contrast, a single oral dose of 50 mg/kg of body weight resulted in the rapid and nearly complete distribution of protein-unbound DNDI-0690 from the plasma into the infected dermis (ratio of the area under the curve [0 to 6 h] of the free DNDI-0690 concentration in skin tissue to blood [fAUC0-6 h, skin tissue/fAUC0-6 h, blood] is greater than 80%). Based on in vivo bioluminescence imaging, two doses of 50 mg/kg DNDI-0690 were sufficient to reduce the Leishmania mexicana parasite load by 100-fold, while 6 such doses were needed to achieve similar killing of L. major; this was confirmed by quantitative PCR. The combination of rapid accumulation and potent activity in the Leishmania-infected dermis indicates the potential of DNDI-0690 as a novel oral treatment for CL.

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In Vitro Synergy and In Vivo Activity of Tigecycline-Ciprofloxacin Combination Therapy against Vibrio vulnificus Sepsis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00310-19 ◽

2019 ◽

Vol 63 (10) ◽

Author(s):

Seong Eun Kim ◽

Hee Kyung Kim ◽

Su-Mi Choi ◽

Yohan Yu ◽

Uh Jin Kim ◽

...

Keyword(s):

Survival Rate ◽

Mortality Rate ◽

Combination Therapy ◽

Combination Treatment ◽

Vibrio Vulnificus ◽

Antibiotic Regimen ◽

Content Type ◽

Time Kill

ABSTRACT The mortality rate associated with Vibrio vulnificus sepsis remains high. An in vitro time-kill assay revealed synergism between tigecycline and ciprofloxacin. The survival rate was significantly higher in mice treated with tigecycline plus ciprofloxacin than in mice treated with cefotaxime plus minocycline. Thus, combination treatment with tigecycline-ciprofloxacin may be an effective novel antibiotic regimen for V. vulnificus sepsis.

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ZN148 Is a Modular Synthetic Metallo-β-Lactamase Inhibitor That Reverses Carbapenem Resistance in Gram-Negative Pathogens In Vivo

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02415-19 ◽

2020 ◽

Vol 64 (6) ◽

Cited By ~ 3

Author(s):

Ørjan Samuelsen ◽

Ove Alexander Høgmoen Åstrand ◽

Christopher Fröhlich ◽

Adam Heikal ◽

Susann Skagseth ◽

...

Keyword(s):

Active Site ◽

Therapeutic Potential ◽

Treatment Options ◽

Carbapenem Resistance ◽

Functional Space ◽

Bactericidal Effect ◽

Gram Negative ◽

Content Type

ABSTRACT Carbapenem-resistant Gram-negative pathogens are a critical public health threat and there is an urgent need for new treatments. Carbapenemases (β-lactamases able to inactivate carbapenems) have been identified in both serine β-lactamase (SBL) and metallo-β-lactamase (MBL) families. The recent introduction of SBL carbapenemase inhibitors has provided alternative therapeutic options. Unfortunately, there are no approved inhibitors of MBL-mediated carbapenem-resistance and treatment options for infections caused by MBL-producing Gram-negatives are limited. Here, we present ZN148, a zinc-chelating MBL-inhibitor capable of restoring the bactericidal effect of meropenem and in vitro clinical susceptibility to carbapenems in >98% of a large international collection of MBL-producing clinical Enterobacterales strains (n = 234). Moreover, ZN148 was able to potentiate the effect of meropenem against NDM-1-producing Klebsiella pneumoniae in a murine neutropenic peritonitis model. ZN148 showed no inhibition of the human zinc-containing enzyme glyoxylase II at 500 μM, and no acute toxicity was observed in an in vivo mouse model with cumulative dosages up to 128 mg/kg. Biochemical analysis showed a time-dependent inhibition of MBLs by ZN148 and removal of zinc ions from the active site. Addition of exogenous zinc after ZN148 exposure only restored MBL activity by ∼30%, suggesting an irreversible mechanism of inhibition. Mass-spectrometry and molecular modeling indicated potential oxidation of the active site Cys221 residue. Overall, these results demonstrate the therapeutic potential of a ZN148-carbapenem combination against MBL-producing Gram-negative pathogens and that ZN148 is a highly promising MBL inhibitor that is capable of operating in a functional space not presently filled by any clinically approved compound.

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hfqPlays Important Roles in Virulence and Stress Adaptation in Cronobacter sakazakii ATCC 29544

Infection and Immunity ◽

10.1128/iai.03161-14 ◽

2015 ◽

Vol 83 (5) ◽

pp. 2089-2098 ◽

Cited By ~ 21

Author(s):

Seongok Kim ◽

Hyelyeon Hwang ◽

Kwang-Pyo Kim ◽

Hyunjin Yoon ◽

Dong-Hyun Kang ◽

...

Keyword(s):

Pathogenic Bacteria ◽

Bacterial Species ◽

Soft Agar ◽

Host Cells ◽

Neonatal Meningitis ◽

Animal Cells ◽

Content Type ◽

Flagellar Biosynthesis

Cronobacterspp. are opportunistic pathogens that cause neonatal meningitis and sepsis with high mortality in neonates. Despite the peril associated withCronobacterinfection, the mechanisms of pathogenesis are still being unraveled. Hfq, which is known as an RNA chaperone, participates in the interaction with bacterial small RNAs (sRNAs) to regulate posttranscriptionally the expression of various genes. Recent studies have demonstrated that Hfq contributes to the pathogenesis of numerous species of bacteria, and its roles are varied between bacterial species. Here, we tried to elucidate the role of Hfq inC. sakazakiivirulence. In the absence ofhfq,C. sakazakiiwas highly attenuated in disseminationin vivo, showed defects in invasion (3-fold) into animal cells and survival (103-fold) within host cells, and exhibited low resistance to hydrogen peroxide (102-fold). Remarkably, the loss ofhfqled to hypermotility on soft agar, which is contrary to what has been observed in other pathogenic bacteria. The hyperflagellated bacteria were likely to be attributable to the increased transcription of genes associated with flagellar biosynthesis in a strain lackinghfq. Together, these data strongly suggest thathfqplays important roles in the virulence ofC. sakazakiiby participating in the regulation of multiple genes.

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Lysyl oxidase activity and elastin/glycosaminoglycan interactions in growing chick and rat aortas.

The Journal of Cell Biology ◽

10.1083/jcb.105.3.1463 ◽

1987 ◽

Vol 105 (3) ◽

pp. 1463-1469 ◽

Cited By ~ 60

Author(s):

C Fornieri ◽

M Baccarani-Contri ◽

D Quaglino ◽

I Pasquali-Ronchetti

Keyword(s):

Hydrophobic Interactions ◽

Isonicotinic Acid ◽

Lysyl Oxidase ◽

Acid Hydrazide ◽

Elastic Fibers ◽

Amino Groups ◽

Lysine Residues ◽

Oxidase Inhibition

Hydrophobic tropoelastin molecules aggregate in vitro in physiological conditions and form fibers very similar to natural ones (Bressan, G. M., I. Pasquali Ronchetti, C. Fornieri, F. Mattioli, I. Castellani, and D. Volpin, 1986, J. Ultrastruct. Molec. Struct. Res., 94:209-216). Similar hydrophobic interactions might be operative in in vivo fibrogenesis. Data are presented suggesting that matrix glycosaminoglycans (GAGs) prevent spontaneous tropoelastin aggregation in vivo, at least up to the deamination of lysine residues on tropoelastin by matrix lysyl oxidase. Lysyl oxidase inhibitors beta-aminopropionitrile, aminoacetonitrile, semicarbazide, and isonicotinic acid hydrazide were given to newborn chicks, to chick embryos, and to newborn rats, and the ultrastructural alterations of the aortic elastic fibers were analyzed and compared with the extent of the enzyme inhibition. When inhibition was greater than 65% all chemicals induced alterations of elastic fibers in the form of lateral aggregates of elastin, which were always permeated by cytochemically and immunologically recognizable GAGs. The number and size of the abnormal elastin/GAGs aggregates were proportional to the extent of lysyl oxidase inhibition. The phenomenon was independent of the animal species. All data suggest that, upon inhibition of lysyl oxidase, matrix GAGs remain among elastin molecules during fibrogenesis by binding to positively charged amino groups on elastin. Newly synthesized and secreted tropoelastin has the highest number of free epsilon amino groups, and, therefore, the highest capability of binding to GAGs. These polyanions, by virtue of their great hydration and dispersing power, could prevent random spontaneous aggregation of hydrophobic tropoelastin in the extracellular space.

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Arsenic Directly Binds to and Activates the Yeast AP-1-Like Transcription Factor Yap8

Molecular and Cellular Biology ◽

10.1128/mcb.00842-15 ◽

2015 ◽

Vol 36 (6) ◽

pp. 913-922 ◽

Cited By ~ 17

Author(s):

Nallani Vijay Kumar ◽

Jianbo Yang ◽

Jitesh K. Pillai ◽

Swati Rawat ◽

Carlos Solano ◽

...

Keyword(s):

Transcription Factor ◽

Transcriptional Activation ◽

Transcriptional Response ◽

Transcriptional Regulator ◽

Yeast Protein ◽

Content Type ◽

Arsenic Tolerance ◽

Arsenic Sensing

The AP-1-like transcription factor Yap8 is critical for arsenic tolerance in the yeastSaccharomyces cerevisiae. However, the mechanism by which Yap8 senses the presence of arsenic and activates transcription of detoxification genes is unknown. Here we demonstrate that Yap8 directly binds to trivalent arsenite [As(III)]in vitroandin vivoand that approximately one As(III) molecule is bound per molecule of Yap8. As(III) is coordinated by three sulfur atoms in purified Yap8, and our genetic and biochemical data identify the cysteine residues that form the binding site as Cys132, Cys137, and Cys274. As(III) binding by Yap8 does not require an additional yeast protein, and Yap8 is regulated neither at the level of localization nor at the level of DNA binding. Instead, our data are consistent with a model in which a DNA-bound form of Yap8 acts directly as an As(III) sensor. Binding of As(III) to Yap8 triggers a conformational change that in turn brings about a transcriptional response. Thus, As(III) binding to Yap8 acts as a molecular switch that converts inactive Yap8 into an active transcriptional regulator. This is the first report to demonstrate how a eukaryotic protein couples arsenic sensing to transcriptional activation.

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