The Persistence-Inducing Toxin HokB Forms Dynamic Pores That Cause ATP Leakage

ABSTRACTBacterial populations harbor a small fraction of cells that display transient multidrug tolerance. These so-called persister cells are extremely difficult to eradicate and contribute to the recalcitrance of chronic infections. Several signaling pathways leading to persistence have been identified. However, it is poorly understood how the effectors of these pathways function at the molecular level. In a previous study, we reported that the conserved GTPase Obg induces persistence inEscherichia colivia transcriptional upregulation of the toxin HokB. In the present study, we demonstrate that HokB inserts in the cytoplasmic membrane where it forms pores. The pore-forming capacity of the HokB peptide is demonstrated byin vitroconductance measurements on synthetic and natural lipid bilayers, revealing an asymmetrical conductance profile. Pore formation is directly linked to persistence and results in leakage of intracellular ATP. HokB-induced persistence is strongly impeded in the presence of a channel blocker, thereby providing a direct link between pore functioning and persistence. Furthermore, the activity of HokB pores is sensitive to the membrane potential. This sensitivity presumably results from the formation of either intermediate or mature pore types depending on the membrane potential. Taken together, these results provide a detailed view on the mechanistic basis of persister formation through the effector HokB.IMPORTANCEThere is increasing awareness of the clinical importance of persistence. Indeed, persistence is linked to the recalcitrance of chronic infections, and evidence is accumulating that persister cells constitute a pool of viable cells from which resistant mutants can emerge. Unfortunately, persistence is a poorly understood process at the mechanistic level. In this study, we unraveled the pore-forming activity of HokB inE. coliand discovered that these pores lead to leakage of intracellular ATP, which is correlated with the induction of persistence. Moreover, we established a link between persistence and pore activity, as the number of HokB-induced persister cells was strongly reduced using a channel blocker. The latter opens opportunities to reduce the number of persister cells in a clinical setting.

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Effects of Methanogenic Inhibitors on Methane Production and Abundances of Methanogens and Cellulolytic Bacteria inIn VitroRuminal Cultures

Applied and Environmental Microbiology ◽

10.1128/aem.02779-10 ◽

2011 ◽

Vol 77 (8) ◽

pp. 2634-2639 ◽

Cited By ~ 82

Author(s):

Zhenming Zhou ◽

Qingxiang Meng ◽

Zhongtang Yu

Keyword(s):

Methane Production ◽

Volatile Fatty Acids ◽

Denaturing Gradient Gel Electrophoresis ◽

Cellulolytic Bacteria ◽

Bacterial Populations ◽

Content Type ◽

Specific Pcr ◽

Methanogen Population ◽

Propynoic Acid

ABSTRACTThe objective of this study was to systematically evaluate and compare the effects of select antimethanogen compounds on methane production, feed digestion and fermentation, and populations of ruminal bacteria and methanogens usingin vitrocultures. Seven compounds, including 2-bromoethanesulphonate (BES), propynoic acid (PA), nitroethane (NE), ethyltrans-2-butenoate (ETB), 2-nitroethanol (2NEOH), sodium nitrate (SN), and ethyl-2-butynote (EB), were tested at a final concentration of 12 mM. Ground alfalfa hay was included as the only substrate to simulate daily forage intake. Compared to no-inhibitor controls, PA, 2NEOH, and SN greatly reduced the production of methane (70 to 99%), volatile fatty acids (VFAs; 46 to 66%), acetate (30 to 60%), and propionate (79 to 82%), with 2NEOH reducing the most. EB reduced methane production by 23% without a significant effect on total VFAs, acetate, or propionate. BES significantly reduced the propionate concentration but not the production of methane, total VFAs, or acetate. ETB or NE had no significant effect on any of the above-mentioned measurements. Specific quantitative-PCR (qPCR) assays showed that none of the inhibitors significantly affected total bacterial populations but that they did reduce theFibrobacter succinogenespopulation. SN reduced theRuminococcus albuspopulation, while PA and 2NEOH increased the populations of bothR. albusandRuminococcus flavefaciens. Archaeon-specific PCR-denaturing gradient gel electrophoresis (DGGE) showed that all the inhibitors affected the methanogen population structure, while archaeon-specific qPCR revealed a significant decrease in methanogen population in all treatments. These results showed that EB, ETB, NE, and BES can effectively reduce the total population of methanogens but that they reduce methane production to a lesser extent. The results may guide futureinvivostudies to develop effective mitigation of methane emission from ruminants.

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Within-Host Evolution of Pseudomonas aeruginosa Reveals Adaptation toward Iron Acquisition from Hemoglobin

mBio ◽

10.1128/mbio.00966-14 ◽

2014 ◽

Vol 5 (3) ◽

Cited By ~ 88

Author(s):

Rasmus Lykke Marvig ◽

Søren Damkiær ◽

S. M. Hossein Khademi ◽

Trine M. Markussen ◽

Søren Molin ◽

...

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Iron Acquisition ◽

Chronic Infections ◽

Content Type ◽

Mortality And Morbidity ◽

Airway Infections ◽

Host Evolution ◽

Promoter Mutations

ABSTRACTPseudomonas aeruginosaairway infections are a major cause of mortality and morbidity of cystic fibrosis (CF) patients. In order to persist,P. aeruginosadepends on acquiring iron from its host, and multiple different iron acquisition systems may be active during infection. This includes the pyoverdine siderophore and thePseudomonasheme utilization (phu) system. While the regulation and mechanisms of several iron-scavenging systems are well described, it is not clear whether such systems are targets for selection during adaptation ofP. aeruginosato the host environment. Here we investigated the within-host evolution of the transmissibleP. aeruginosaDK2 lineage. We found positive selection for promoter mutations leading to increased expression of thephusystem. By mimicking conditions of the CF airwaysin vitro, we experimentally demonstrate that increased expression ofphuRconfers a growth advantage in the presence of hemoglobin, thus suggesting thatP. aeruginosaevolves toward iron acquisition from hemoglobin. To rule out that this adaptive trait is specific to the DK2 lineage, we inspected the genomes of additionalP. aeruginosalineages isolated from CF airways and found similar adaptive evolution in two distinct lineages (DK1 and PA clone C). Furthermore, in all three lineages,phuRpromoter mutations coincided with the loss of pyoverdine production, suggesting that within-host adaptation toward heme utilization is triggered by the loss of pyoverdine production. Targeting heme utilization might therefore be a promising strategy for the treatment ofP. aeruginosainfections in CF patients.IMPORTANCEMost bacterial pathogens depend on scavenging iron within their hosts, which makes the battle for iron between pathogens and hosts a hallmark of infection. Accordingly, the ability of the opportunistic pathogenPseudomonas aeruginosato cause chronic infections in cystic fibrosis (CF) patients also depends on iron-scavenging systems. While the regulation and mechanisms of several such iron-scavenging systems have been well described, not much is known about how the within-host selection pressures act on the pathogens’ ability to acquire iron. Here, we investigated the within-host evolution ofP. aeruginosa, and we found evidence thatP. aeruginosaduring long-term infections evolves toward iron acquisition from hemoglobin. This adaptive strategy might be due to a selective loss of other iron-scavenging mechanisms and/or an increase in the availability of hemoglobin at the site of infection. This information is relevant to the design of novel CF therapeutics and the development of models of chronic CF infections.

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Daptomycin Pore Formation and Stoichiometry Depend on Membrane Potential of Target Membrane

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01589-18 ◽

2018 ◽

Vol 63 (1) ◽

Cited By ~ 8

Author(s):

Gabriela Seydlová ◽

Albert Sokol ◽

Petra Lišková ◽

Ivo Konopásek ◽

Radovan Fišer

Keyword(s):

Membrane Potential ◽

Lipid Bilayers ◽

Pore Formation ◽

Content Type ◽

Calcium Dependent ◽

Screening Experiment ◽

Serious Infections ◽

Fluorescence Methods ◽

Initial Membrane ◽

Target Membrane

ABSTRACT Daptomycin is a calcium-dependent lipodepsipeptide antibiotic clinically used to treat serious infections caused by Gram-positive pathogens. Its precise mode of action is somewhat controversial; the biggest issue is daptomycin pore formation, which we directly investigated here. We first performed a screening experiment using propidium iodide (PI) entry to Bacillus subtilis cells and chose the optimum and therapeutically relevant conditions (10 µg/ml daptomycin and 1.25 mM CaCl2) for the subsequent analyses. Using conductance measurements on planar lipid bilayers, we show that daptomycin forms nonuniform oligomeric pores with conductance ranging from 120 pS to 14 nS. The smallest conductance unit is probably a dimer; however, tetramers and pentamers occur in the membrane most frequently. Moreover, daptomycin pore-forming activity is exponentially dependent on the applied membrane voltage. We further analyzed the membrane-permeabilizing activity in B. subtilis cells using fluorescence methods [PI and DiSC3(5)]. Daptomycin most rapidly permeabilizes cells with high initial membrane potential and dissipates it within a few minutes. Low initial membrane potential hinders daptomycin pore formation.

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Stochastic Variation in Expression of the Tricarboxylic Acid Cycle Produces Persister Cells

mBio ◽

10.1128/mbio.01930-19 ◽

2019 ◽

Vol 10 (5) ◽

Cited By ~ 24

Author(s):

Eliza A. Zalis ◽

Austin S. Nuxoll ◽

Sylvie Manuse ◽

Geremy Clair ◽

Lauren C. Radlinski ◽

...

Keyword(s):

Antibiotic Treatment ◽

Bacterial Infections ◽

Tricarboxylic Acid Cycle ◽

Tricarboxylic Acid ◽

Resistant Bacteria ◽

Chronic Infections ◽

Persister Cells ◽

Stochastic Variation ◽

Content Type ◽

Atp Levels

ABSTRACT Chronic bacterial infections are difficult to eradicate, though they are caused primarily by drug-susceptible pathogens. Antibiotic-tolerant persisters largely account for this paradox. In spite of their significance in the recalcitrance of chronic infections, the mechanism of persister formation is poorly understood. We previously reported that a decrease in ATP levels leads to drug tolerance in Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. We reasoned that stochastic fluctuation in the expression of tricarboxylic acid (TCA) cycle enzymes can produce cells with low energy levels. S. aureus knockouts in glutamate dehydrogenase, 2-oxoketoglutarate dehydrogenase, succinyl coenzyme A (CoA) synthetase, and fumarase have low ATP levels and exhibit increased tolerance of fluoroquinolone, aminoglycoside, and β-lactam antibiotics. Fluorescence-activated cell sorter (FACS) analysis of TCA genes shows a broad Gaussian distribution in a population, with differences of over 3 orders of magnitude in the levels of expression between individual cells. Sorted cells with low levels of TCA enzyme expression have an increased tolerance of antibiotic treatment. These findings suggest that fluctuations in the levels of expression of energy-generating components serve as a mechanism of persister formation. IMPORTANCE Persister cells are rare phenotypic variants that are able to survive antibiotic treatment. Unlike resistant bacteria, which have specific mechanisms to prevent antibiotics from binding to their targets, persisters evade antibiotic killing by entering a tolerant nongrowing state. Persisters have been implicated in chronic infections in multiple species, and growing evidence suggests that persister cells are responsible for many cases of antibiotic treatment failure. New antibiotic treatment strategies aim to kill tolerant persister cells more effectively, but the mechanism of tolerance has remained unclear until now.

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Efficacy of Guanabenz Combination Therapy against Chronic Toxoplasmosis across Multiple Mouse Strains

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00539-20 ◽

2020 ◽

Vol 64 (9) ◽

Cited By ~ 1

Author(s):

Jennifer Martynowicz ◽

J. Stone Doggett ◽

William J. Sullivan

Keyword(s):

Limit Of Detection ◽

Acute Infection ◽

Antagonistic Effect ◽

Mouse Strains ◽

Chronic Infections ◽

Content Type ◽

Brain Cyst ◽

The Brain

ABSTRACT Toxoplasma gondii, an obligate intracellular parasite that can cause life-threatening acute disease, differentiates into a quiescent cyst stage to establish lifelong chronic infections in animal hosts, including humans. This tissue cyst reservoir, which can reactivate into an acute infection, is currently refractory to clinically available therapeutics. Recently, we and others have discovered drugs capable of significantly reducing the brain cyst burden in latently infected mice, but not to undetectable levels. In this study, we examined the use of novel combination therapies possessing multiple mechanisms of action in mouse models of latent toxoplasmosis. Our drug regimens included combinations of pyrimethamine, clindamycin, guanabenz, and endochin-like quinolones (ELQs) and were administered to two different mouse strains in an attempt to eradicate brain tissue cysts. We observed mouse strain-dependent effects with these drug treatments: pyrimethamine-guanabenz showed synergistic efficacy in C57BL/6 mice yet did not improve upon guanabenz monotherapy in BALB/c mice. Contrary to promising in vitro results demonstrating toxicity to bradyzoites, we observed an antagonistic effect between guanabenz and ELQ-334 in vivo. While we were unable to completely eliminate the brain cyst burden, we found that a combination treatment with ELQ-334 and pyrimethamine impressively reduced the brain cyst burden by 95% in C57BL/6 mice, which approached the limit of detection. These analyses highlight the importance of evaluating anti-infective drugs in multiple mouse strains and will help inform further preclinical studies of cocktail therapies designed to treat chronic toxoplasmosis.

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The Formation of Streptococcus mutans Persisters Induced by the Quorum-Sensing Peptide Pheromone Is Affected by the LexA Regulator

Journal of Bacteriology ◽

10.1128/jb.02496-14 ◽

2015 ◽

Vol 197 (6) ◽

pp. 1083-1094 ◽

Cited By ~ 23

Author(s):

Vincent Leung ◽

Dragana Ajdic ◽

Stephanie Koyanagi ◽

Céline M. Lévesque

Keyword(s):

Quorum Sensing ◽

Streptococcus Mutans ◽

Bacterial Species ◽

Regulatory System ◽

Bacterial Survival ◽

Chronic Infections ◽

Persister Cells ◽

Gene Encoding ◽

Content Type ◽

Peptide Pheromone

The presence of multidrug-tolerant persister cells within microbial populations has been implicated in the resiliency of bacterial survival against antibiotic treatments and is a major contributing factor in chronic infections. The mechanisms by which these phenotypic variants are formed have been linked to stress response pathways in various bacterial species, but many of these mechanisms remain unclear. We have previously shown that in the cariogenic organismStreptococcus mutans, the quorum-sensing peptide CSP (competence-stimulating peptide) pheromone was a stress-inducible alarmone that triggered an increased formation of multidrug-tolerant persisters. In this study, we characterized SMU.2027, a CSP-inducible gene encoding a LexA ortholog. We showed that in addition to exogenous CSP exposure, stressors, including heat shock, oxidative stress, and ofloxacin antibiotic, were capable of triggering expression oflexAin an autoregulatory manner akin to that of LexA-like transcriptional regulators. We demonstrated the role of LexA and its importance in regulating tolerance toward DNA damage in a noncanonical SOS mechanism. We showed its involvement and regulatory role in the formation of persisters induced by the CSP-ComDE quorum-sensing regulatory system. We further identified key genes involved in sugar and amino acid metabolism, the clustered regularly interspaced short palindromic repeat (CRISPR) system, and autolysin from transcriptomic analyses that contribute to the formation of quorum-sensing-induced persister cells.

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Horizontal Spread of Rhodococcus equi Macrolide Resistance Plasmid pRErm46 across Environmental Actinobacteria

Applied and Environmental Microbiology ◽

10.1128/aem.00108-20 ◽

2020 ◽

Vol 86 (9) ◽

Author(s):

Sonsiray Álvarez-Narváez ◽

Steeve Giguère ◽

Londa J. Berghaus ◽

Cody Dailey ◽

José A. Vázquez-Boland

Keyword(s):

Bacterial Species ◽

Low Frequency ◽

Rhodococcus Equi ◽

Macrolide Resistance ◽

Conjugative Plasmid ◽

Bacterial Populations ◽

Prolonged Incubation ◽

Content Type ◽

Host Restriction

ABSTRACT Conjugation is one of the main mechanisms involved in the spread and maintenance of antibiotic resistance in bacterial populations. We recently showed that the emerging macrolide resistance in the soilborne equine and zoonotic pathogen Rhodococcus equi is conferred by the erm(46) gene carried on the 87-kb conjugative plasmid pRErm46. Here, we investigated the conjugal transferability of pRErm46 to 14 representative bacteria likely encountered by R. equi in the environmental habitat. In vitro mating experiments demonstrated conjugation to different members of the genus Rhodococcus as well as to Nocardia and Arthrobacter spp. at frequencies ranging from ∼10−2 to 10−6. pRErm46 transfer was also observed in mating experiments in soil and horse manure, albeit at a low frequency and after prolonged incubation at 22 to 30°C (environmental temperatures), not 37°C. All transconjugants were able to transfer pRErm46 back to R. equi. Conjugation could not be detected with Mycobacterium or Corynebacterium spp. or several members of the more distant phylum Firmicutes such as Enterococcus, Streptococcus, or Staphylococcus. Thus, the pRErm46 host range appears to span several actinobacterial orders with certain host restriction within the Corynebacteriales. All bacterial species that acquired pRErm46 expressed increased macrolide resistance with no significant deleterious impact on fitness, except in the case of Rhodococcus rhodnii. Our results indicate that actinobacterial members of the environmental microbiota can both acquire and transmit the R. equi pRErm46 plasmid and thus potentially contribute to the maintenance and spread of erm(46)-mediated macrolide resistance in equine farms. IMPORTANCE This study demonstrates the efficient horizontal transfer of the Rhodococcus equi conjugative plasmid pRErm46, recently identified as the cause of the emerging macrolide resistance among equine isolates of this pathogen, to and from different environmental Actinobacteria, including a variety of rhodococci as well as Nocardia and Arthrobacter spp. The reported data support the notion that environmental microbiotas may act as reservoirs for the endemic maintenance of antimicrobial resistance in an antibiotic pressurized farm habitat.

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d-Amino Acids Enhance the Activity of Antimicrobials against Biofilms of Clinical Wound Isolates of Staphylococcus aureus and Pseudomonas aeruginosa

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02468-14 ◽

2014 ◽

Vol 58 (8) ◽

pp. 4353-4361 ◽

Cited By ~ 57

Author(s):

Carlos J. Sanchez ◽

Kevin S. Akers ◽

Desiree R. Romano ◽

Ronald L. Woodbury ◽

Sharanda K. Hardy ◽

...

Keyword(s):

Amino Acids ◽

Staphylococcus Aureus ◽

Pseudomonas Aeruginosa ◽

Clinical Isolates ◽

Chronic Infections ◽

Content Type ◽

Log Reduction ◽

Viable Bacteria ◽

Biofilm Bacteria

ABSTRACTWithin wounds, microorganisms predominantly exist as biofilms. Biofilms are associated with chronic infections and represent a tremendous clinical challenge. As antibiotics are often ineffective against biofilms, use of dispersal agents as adjunctive, topical therapies for the treatment of wound infections involving biofilms has gained interest. We evaluatedin vitrothe dispersive activity ofd-amino acids (d-AAs) on biofilms from clinical wound isolates ofStaphylococcus aureusandPseudomonas aeruginosa; moreover, we determined whether combinations ofd-AAs and antibiotics (clindamycin, cefazolin, oxacillin, rifampin, and vancomycin forS. aureusand amikacin, colistin, ciprofloxacin, imipenem, and ceftazidime forP. aeruginosa) enhance activity against biofilms.d-Met,d-Phe, andd-Trp at concentrations of ≥5 mM effectively dispersed preformed biofilms ofS. aureusandP. aeruginosaclinical isolates, an effect that was enhanced when they were combined as an equimolar mixture (d-Met/d-Phe/d-Trp). When combined withd-AAs, the activity of rifampin was significantly enhanced against biofilms of clinical isolates ofS. aureus, as indicated by a reduction in the minimum biofilm inhibitory concentration (MBIC) (from 32 to 8 μg/ml) and a >2-log reduction of viable biofilm bacteria compared to treatment with antibiotic alone. The addition ofd-AAs was also observed to enhance the activity of colistin and ciprofloxacin against biofilms ofP. aeruginosa, reducing the observed MBIC and the number of viable bacteria by >2 logs and 1 log at 64 and 32 μg/ml in contrast to antibiotics alone. These findings indicate that the biofilm dispersal activity ofd-AAs may represent an effective strategy, in combination with antimicrobials, to release bacteria from biofilms, subsequently enhancing antimicrobial activity.

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Burkholderia Species Are Major Inhabitants of White Lupin Cluster Roots

Applied and Environmental Microbiology ◽

10.1128/aem.05845-11 ◽

2011 ◽

Vol 77 (21) ◽

pp. 7715-7720 ◽

Cited By ~ 36

Author(s):

Laure Weisskopf ◽

Stefanie Heller ◽

Leo Eberl

Keyword(s):

Burkholderia Cepacia ◽

Carbon Sources ◽

White Lupin ◽

Cluster Roots ◽

Bacterial Populations ◽

Content Type ◽

Significant Enrichment ◽

Branched Structure ◽

Successful Colonization

ABSTRACTThe formation of cluster roots by plants represents a highly efficient strategy for acquisition of sparingly available phosphate. This particular root type is characterized by a densely branched structure and high exudation of organic acids and protons, which are likely to influence the resident bacterial community. Until now, the identity of the bacterial populations living in cluster roots has not been investigated. We applied cultivation-dependent and cultivation-independent methods to characterize the dominant bacterial genera inhabiting the growing cluster roots of white lupin. We observed a high relative abundance ofBurkholderiaspecies (up to 58% of all isolated strains and 44% of all retrieved 16S rRNA sequences) and a significant enrichment with increasing cluster root age. Most of the sequences retrieved clustered together with known plant- or fungus-associatedBurkholderiaspecies, while only one of 98 sequences was affiliated with theBurkholderia cepaciacomplex.In vitroassays revealed thatBurkholderiastrains were much more tolerant to low pH than non-Burkholderiastrains. Moreover, many strains produced large amounts of siderophores and were able to utilize citrate and oxalate as carbon sources. These features seem to represent important traits for the successful colonization and maintenance ofBurkholderiaspecies in white lupin cluster roots.

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Assessment of Intracellular Calcium and Plasmalemmal Membrane Potential in Cryopreserved Metaphase II Mouse Oocytes

10.21203/rs.3.rs-47525/v2 ◽

2020 ◽

Author(s):

Omar Farhan Ammar ◽

Therishnee Moodley

Keyword(s):

Membrane Potential ◽

Xenopus Oocytes ◽

Channel Blocker ◽

Oocyte Activation ◽

Human Oocytes ◽

Mouse Oocytes ◽

Failed Fertilization ◽

Intracellular Ca ◽

In Vitro Ageing

Abstract Objectives: Ca2+ is critical for normal oocyte activation and fertilization, and any alteration to the Ca2+ homeostasis may lead to failed fertilization or even cell death. It has been shown that intracellular Ca2+ is increased in bovine and human oocytes when cultured in vitro. Additionally, ATP sensitive potassium channels have been characterised recently in human and Xenopus oocytes. Glibenclamide a KATP channel blocker was shown to protect human oocytes from Ca+2 overloading via inhibition of plasmalemmal KATP channels. This research note aims to demonstrate the effects of oxidative stress and in vitro ageing on the intracellular Ca+2 and plasmalemmal membrane potential dynamics in cryopreserved metaphase II (MII) mouse oocytes. Also, this study aims to show if glibenclamide (a KATP channel blocker ) has a role in regulating intracellular Ca+2 and plasmalemmal membrane potential through KATP channels in cryopreserved metaphase II mouse oocytes.Results: our data did not show an increase in intracellular Ca2+ in untreated cryopreserved mouse oocytes loaded with Fluo-3 AM dye. However, an increase in the plasmalemmal membrane potential was noticed (hyperpolarization). Glibenclamide has shown no significant effect on Ca2+ and plasmalemmal membrane potential.

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