scholarly journals Mechanistic Features of the Enterococcal pCF10 Sex Pheromone Response and the Biology ofEnterococcus faecalisin Its Natural Habitat

2018 ◽  
Vol 200 (14) ◽  
pp. e00733-17 ◽  
Author(s):  
Rebecca J. Breuer ◽  
Helmut Hirt ◽  
Gary M. Dunny
Keyword(s):  
Opportunistic Infections ◽  
Natural Habitat ◽  
Test Tube ◽  
Gi Tract ◽  
Pheromone Response ◽  
Content Type ◽  
Pheromone Signaling ◽  
Control Transfer

ABSTRACTConjugative transfer of plasmids in enterococci is promoted by intercellular communication using peptide pheromones. The regulatory mechanisms that control transfer have been extensively studiedin vitro. However, the complicated systems that regulate the spread of these plasmids did not evolve in the laboratory test tube, and remarkably little is known about this form of signaling in the intestinal tract, the primary niche of these organisms. Because the evolution ofEnterococcus faecalisstrains and their coresident pheromone-inducible plasmids, such as pCF10, have occurred in the gastrointestinal (GI) tract, it is important to consider the functions controlled by pheromones in light of this ecology. This review summarizes our current understanding of the pCF10-encoded pheromone response. We consider how selective pressures in the natural environment may have selected for the complex and very tightly regulated systems controlling conjugation, and we pay special attention to the ecology of enterococci and the pCF10 plasmid as a gut commensal. We summarize the results of recent studies of the pheromone response at the single-cell level, as well as those of the first experiments demonstrating a role for pheromone signaling in plasmid transfer and in GI tract competitive fitness. These results will serve as a foundation for furtherin vivostudies that could lead to novel interventions to reduce opportunistic infections and the spread of antibiotic resistance.

scholarly journals Chemokine CCL28 Is a Potent Therapeutic Agent for Oropharyngeal Candidiasis

2020 ◽  
Vol 64 (8) ◽  
Author(s):  
Jie He ◽  
Monica A. Thomas ◽  
Jaime de Anda ◽  
Michelle W. Lee ◽  
Emma Van Why ◽  
...  
Keyword(s):  
Therapeutic Agent ◽  
Opportunistic Infections ◽  
Membrane Disruption ◽  
Oropharyngeal Candidiasis ◽  
Wild Type ◽  
Content Type ◽  
Immunodeficient Mice ◽  
Drug Classes

ABSTRACT Candida albicans is a commensal organism that causes life-threatening or life-altering opportunistic infections. Treatment of Candida infections is limited by the paucity of antifungal drug classes. Naturally occurring antimicrobial peptides are promising agents for drug development. CCL28 is a CC chemokine that is abundant in saliva and has in vitro antimicrobial activity. In this study, we examine the in vivo Candida killing capacity of CCL28 in oropharyngeal candidiasis as well as the spectrum and mechanism of anti-Candida activity. In the mouse model of oropharyngeal candidiasis, application of wild-type CCL28 reduces oral fungal burden in severely immunodeficient mice without causing excessive inflammation or altering tissue neutrophil recruitment. CCL28 is effective against multiple clinical strains of C. albicans. Polyamine protein transporters are not required for CCL28 anti-Candida activity. Both structured and unstructured CCL28 proteins show rapid and sustained fungicidal activity that is superior to that of clinical antifungal agents. Application of wild-type CCL28 to C. albicans results in membrane disruption as measured by solute movement, enzyme leakage, and induction of negative Gaussian curvature on model membranes. Membrane disruption is reduced in CCL28 lacking the functional C-terminal tail. Our results strongly suggest that CCL28 can exert antifungal activity in part via membrane permeation and has potential for development as an anti-Candida therapeutic agent without inflammatory side effects.

scholarly journals In VivoandIn VitroAcquisition of Resistance to Voriconazole by Candida krusei

2014 ◽  
Vol 58 (8) ◽  
pp. 4604-4611 ◽  
Author(s):  
Elisabete Ricardo ◽  
Isabel M. Miranda ◽  
Isabel Faria-Ramos ◽  
Raquel M. Silva ◽  
Acácio Gonçalves Rodrigues ◽  
...  
Keyword(s):  
Opportunistic Infections ◽  
Efflux Pump ◽  
Synergistic Effects ◽  
Candida Krusei ◽  
Clinical Isolates ◽  
Content Type ◽  
Synonymous Mutations ◽  
Resistant Strains

ABSTRACTCandida kruseiis an important agent of opportunistic infections that often displays resistance to several antifungals. We describe here thein vivoacquisition of resistance to voriconazole (VRC) byC. kruseiisolates recovered from a leukemia patient during a long period of VRC therapy. In order to mimic thein vivodevelopment of VRC resistance, a susceptibleC. kruseiisolate was exposed daily to 1 μg/ml of VRCin vitro. Interestingly, after 5 days of exposure to VRC, a MIC of 4 μg/ml was achieved; this value remained constant after 25 additional days of treatment with VRC and also after 30 consecutive days of incubation in VRC-free medium. Our objective was to determine the associated molecular resistance mechanisms, such as expression of efflux pump genes andERG11gene mutations, among the resistant strains. Synergistic effects between the efflux blocker tacrolimus (FK506) and VRC were found in all of the resistant strains. Moreover,ABC1gene expression increased over time in both thein vivo- andin vitro-induced resistant strains, in contrast to theABC2andERG11genes, whose expression was invariably lower and constant.ERG11gene sequencing showed two different types of mutations, i.e., heterozygosity at T1389T/C, corresponding to synonymous mutations, inC. kruseistrains and a missense mutation at position T418C, resulting in a change from Tyr to His, among resistantC. kruseiclinical isolates. This study highlights the relevance of ATP-dependent efflux pump (namely, Abc1p) activity in VRC resistance and describes new mutations in theERG11gene among resistantC. kruseiclinical isolates.

scholarly journals Exogenous Alginate Protects Staphylococcus aureus from Killing by Pseudomonas aeruginosa

2019 ◽  
Vol 202 (8) ◽  
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.

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

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.

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

2020 ◽  
Vol 64 (6) ◽  
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.

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

2015 ◽  
Vol 36 (6) ◽  
pp. 913-922 ◽  
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.

scholarly journals Surface-Engineered Dendrimeric Nanoconjugates for Macrophage-Targeted Delivery of Amphotericin B: Formulation Development andIn VitroandIn VivoEvaluation

2015 ◽  
Vol 59 (5) ◽  
pp. 2479-2487 ◽  
Author(s):  
Keerti Jain ◽  
Ashwni Kumar Verma ◽  
Prabhat Ranjan Mishra ◽  
Narendra Kumar Jain
Keyword(s):  
Drug Release ◽  
Amphotericin B ◽  
Human Erythrocytes ◽  
Antileishmanial Activity ◽  
Cell Uptake ◽  
Antiparasitic Activity ◽  
Content Type ◽  
Drug Localization

ABSTRACTThe present study aimed to develop an optimized dendrimeric delivery system for amphotericin B (AmB). Fifth-generation (5.0G) poly(propylene imine) (PPI) dendrimers were synthesized, conjugated with mannose, and characterized by use of various analytical techniques, including Fourier transform infrared spectroscopy (FTIR),1H nuclear magnetic resonance (1H-NMR) spectroscopic analysis, and atomic force microscopy (AFM). Mannose-conjugated 5.0G PPI (MPPI) dendrimers were loaded with AmB and evaluated for drug loading efficiency,in vitrodrug release profile, stability, hemolytic toxicity to human erythrocytes, cytotoxicity to and cell uptake by J774A.1 macrophage cells, antiparasitic activity against intracellularLeishmania donovaniamastigotes,in vivopharmacokinetic and biodistribution profiles, drug localization index, toxicity, and antileishmanial activity. AFM showed the nanometric size of the MPPI dendrimers, with a nearly globular architecture. The conjugate showed a good entrapment efficiency for AmB, along with pH-sensitive drug release. Highly significant reductions in toxicity toward human erythrocytes and macrophage cells, without compromising the antiparasitic activity of AmB, were observed. The dendrimeric formulation of AmB showed a significant enhancement of the parasiticidal activity of AmB toward intramacrophagicL. donovaniamastigotes. In thein vitrocell uptake studies, the formulation showed selectivity toward macrophages, with significant intracellular uptake. Further pharmacokinetic and organ distribution studies elucidated the controlled delivery behavior of the formulation. The drug localization index was found to increase significantly in macrophage-rich organs.In vivostudies showed a biocompatible behavior of MPPIA, with negligible toxicity even at higher doses, and promising antileishmanial activity. From the results, we concluded that surface-engineered dendrimers may serve as optimized delivery vehicles for AmB with enhanced activity and low or negligible toxicity.

scholarly journals Application of the 1-µsec pulsed-dye laser to the treatment of experimental cerebral vasospasm

1991 ◽  
Vol 75 (2) ◽  
pp. 271-276 ◽  
Author(s):  
Atsushi Teramura ◽  
Robert Macfarlane ◽  
Christopher J. Owen ◽  
Ralph de la Torre ◽  
Kenton W. Gregory ◽  
...  
Keyword(s):  
Cerebral Vasospasm ◽  
Basilar Artery ◽  
Laser Energy ◽  
Autologous Blood ◽  
Preliminary Investigation ◽  
Dye Laser ◽  
Pulsed Dye Laser ◽  
Content Type

✓ Laser energy of 480 nm was applied in 1-µsec pulses varying between 2.2 and 10 mJ to in vitro and in vivo models of cerebral vasospasm. First, the pulsed-dye laser was applied intravascularly via a 320-µm fiber to basilar artery segments from six dogs. The segments were mounted in a vessel-perfusion apparatus and constricted to, on average, 70% of resting diameter by superfusion with dog hemolysate. Immediate increase in basilar artery diameter occurred to a mean of 83% of control. In a second model, the basilar artery was exposed transclivally in the rabbit. In three normal animals, superfusion of the artery with rabbit hemolysate resulted in a reduction of mean vessel diameter to 81% of control. Following extravascular application of the laser, vessels returned to an average of 106% of the resting state. In six rabbits, the basilar artery was constricted by two intracisternal injections of autologous blood, 3 days apart. Two to 4 days after the second injection, the basilar artery was exposed. Extravascular laser treatment from a quartz fiber placed perpendicular to the vessel adventitia resulted in an immediate 53% average increase in caliber to an estimated 107% of control. No reconstriction was observed over a period of up to 5 hours. Morphologically, damage to the arterial wall was slight. This preliminary investigation suggests that the 1-µsec pulsed-dye laser may be of benefit in the treatment of cerebral vasospasm.

scholarly journals Use of an Endogenous Plasmid Locus for Stable intransComplementation in Borrelia burgdorferi

2014 ◽  
Vol 81 (3) ◽  
pp. 1038-1046 ◽  
Author(s):  
Irene N. Kasumba ◽  
Aaron Bestor ◽  
Kit Tilly ◽  
Patricia A. Rosa
Keyword(s):  
Borrelia Burgdorferi ◽  
Shuttle Vector ◽  
Targeted Mutagenesis ◽  
Multiple Cloning Site ◽  
Stable Gene ◽  
Content Type ◽  
Shuttle Vectors ◽  
Stable Maintenance

ABSTRACTTargeted mutagenesis and complementation are important tools for studying genes of unknown function in the Lyme disease spirocheteBorrelia burgdorferi. A standard method of complementation is reintroduction of a wild-type copy of the targeted gene on a shuttle vector. However, shuttle vectors are present at higher copy numbers thanB. burgdorferiplasmids and are potentially unstable in the absence of selection, thereby complicating analyses in the mouse-tick infectious cycle.B. burgdorferihas over 20 plasmids, with some, such as linear plasmid 25 (lp25), carrying genes required by the spirochetein vivobut relatively unstable duringin vitrocultivation. We propose that complementation on an endogenous plasmid such as lp25 would overcome the copy number andin vivostability issues of shuttle vectors. In addition, insertion of a selectable marker on lp25 could ensure its stable maintenance by spirochetes in culture. Here, we describe the construction of a multipurpose allelic-exchange vector containing a multiple-cloning site and either of two selectable markers. This suicide vector directs insertion of the complementing gene into thebbe02locus, a site on lp25 that was previously shown to be nonessential during bothin vitroandin vivogrowth. We demonstrate the functional utility of this strategy by restoring infectivity to anospCmutant through complementation at this site on lp25 and stable maintenance of theospCgene throughout mouse infection. We conclude that this represents a convenient and widely applicable method for stable gene complementation inB. burgdorferi.

scholarly journals The Novel Arylamidine T-2307 MaintainsIn VitroandIn VivoActivity against Echinocandin-Resistant Candida albicans

2014 ◽  
Vol 59 (2) ◽  
pp. 1341-1343 ◽  
Author(s):  
Nathan P. Wiederhold ◽  
Laura K. Najvar ◽  
Annette W. Fothergill ◽  
Rosie Bocanegra ◽  
Marcos Olivo ◽  
...  
Keyword(s):  
Body Weight ◽  
Candida Albicans ◽  
Invasive Candidiasis ◽  
Placebo Control ◽  
The Novel ◽  
Content Type ◽  
Fungal Burden ◽  
Potential Use

ABSTRACTWe evaluated thein vitroandin vivoactivities of the investigational arylamidine T-2307 against echinocandin-resistantCandida albicans. T-2307 demonstrated potentin vitroactivity, and daily subcutaneous doses between 0.75 and 6 mg/kg of body weight significantly improved survival and reduced fungal burden compared to placebo control and caspofungin (10 mg/kg/day) in mice with invasive candidiasis caused by an echinocandin-resistant strain. Thus, T-2307 may have potential use in the treatment of echinocandin-resistantC. albicansinfections.

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