scholarly journals Daptomycin Resistance and Tolerance Due to Loss of Function inStaphylococcus aureusdsp1andasp23

2018 ◽  
Vol 63 (1) ◽  
Author(s):  
Elaine M. Barros ◽  
Melissa J. Martin ◽  
Elizabeth M. Selleck ◽  
François Lebreton ◽  
Jorge Luiz M. Sampaio ◽  
...  
Keyword(s):  
Cell Wall ◽  
Antimicrobial Peptides ◽  
Multidrug Resistant ◽  
Loss Of Function ◽  
Cationic Antimicrobial Peptides ◽  
Content Type ◽  
Transposon Insertion ◽  
Transposon Mutants ◽  
A Cell ◽  
Transposon Insertions

ABSTRACTLipopeptide daptomycin is a last-line cell-membrane-targeting antibiotic to treat multidrug-resistantStaphylococcus aureus. Alarmingly, daptomycin-resistantS. aureusisolates have emerged. The mechanisms underlying daptomycin resistance are diverse and share similarities with resistances to cationic antimicrobial peptides and other lipopeptides, but they remain to be fully elucidated. We selected mutants with increased resistance to daptomycin from a library of transposon insertions in sequent type 8 (ST8)S. aureusHG003. Insertions conferring increased daptomycin resistance were localized to two genes, one coding for a hypothetical lipoprotein (SAOUHSC_00362, Dsp1), and the other for an alkaline shock protein (SAOUHSC_02441, Asp23). Markerless loss-of-function mutants were then generated for comparison. All transposon mutants and knockout strains exhibited increased daptomycin resistance compared to those of wild-type and complemented strains. Null and transposon insertion mutants also exhibited increased resistance to cationic antimicrobial peptides. Interestingly, theΔdsp1mutant also showed increased resistance to vancomycin, a cell-wall-targeting drug with a different mode of action. Null mutations in bothdsp1andasp23resulted in increased tolerance as reflected by reduced killing to both daptomycin and vancomycin, as well as an increased tolerance to surfactant (Triton X-100). Neither mutant exhibited increased resistance to lysostaphin, a cell-wall-targeting endopeptidase. These findings identified two genes core to theS. aureusspecies that make previously uncharacterized contributions to antimicrobial resistance and tolerance inS. aureus.

scholarly journals Rational Design of Engineered Cationic Antimicrobial Peptides Consisting Exclusively of Arginine and Tryptophan, and Their Activity against Multidrug-Resistant Pathogens

2013 ◽  
Vol 57 (6) ◽  
pp. 2511-2521 ◽  
Author(s):  
Berthony Deslouches ◽  
Jonathan D. Steckbeck ◽  
Jodi K. Craigo ◽  
Yohei Doi ◽  
Timothy A. Mietzner ◽  
...  
Keyword(s):  
Antimicrobial Peptides ◽  
Rational Design ◽  
Antimicrobial Agents ◽  
Divalent Cations ◽  
Multidrug Resistant ◽  
Resistance Mechanisms ◽  
Broth Culture ◽  
Cationic Antimicrobial Peptides ◽  
Content Type ◽  
Amphipathic Helices

ABSTRACTThe emergence of multidrug-resistant (MDR) pathogens underscores the need for new antimicrobial agents to overcome the resistance mechanisms of these organisms. Cationic antimicrobial peptides (CAPs) provide a potential source of new antimicrobial therapeutics. We previously characterized a lytic base unit (LBU) series of engineered CAPs (eCAPs) of 12 to 48 residues demonstrating maximum antibacterial selectivity at 24 residues. Further, Trp substitution in LBU sequences increased activity against bothP. aeruginosaandS. aureusunder challenging conditions (e.g., saline, divalent cations, and serum). Based on these findings, we hypothesized that the optimal length and, therefore, the cost for maximum eCAP activity under physiologically relevant conditions could be significantly reduced using only Arg and Trp arranged to form idealized amphipathic helices. Hence, we developed a novel peptide series, composed only of Arg and Trp, in a sequence predicted and verified by circular dichroism to fold into optimized amphipathic helices. The most effective antimicrobial activity was achieved at 12 residues in length (WR12) against a panel of both Gram-negative and Gram-positive clinical isolates, including extensively drug-resistant strains, in saline and broth culture and at various pH values. The results demonstrate that the rational design of CAPs can lead to a significant reduction in the length and the number of amino acids used in peptide design to achieve optimal potency and selectivity against specific pathogens.

scholarly journals Bordetella pertussis Lipid A Glucosamine Modification Confers Resistance to Cationic Antimicrobial Peptides and Increases Resistance to Outer Membrane Perturbation

2014 ◽  
Vol 58 (8) ◽  
pp. 4931-4934 ◽  
Author(s):  
Nita R. Shah ◽  
Robert E. W. Hancock ◽  
Rachel C. Fernandez
Keyword(s):  
Immune System ◽  
Antimicrobial Peptides ◽  
Outer Membrane ◽  
Bordetella Pertussis ◽  
Lipid A ◽  
Whooping Cough ◽  
Human Immune System ◽  
Cationic Antimicrobial Peptides ◽  
Content Type ◽  
Membrane Perturbation

ABSTRACTBordetella pertussis, the causative agent of whooping cough, has many strategies for evading the human immune system. Lipopolysaccharide (LPS) is an important Gram-negative bacterial surface structure that activates the immune system via Toll-like receptor 4 and enables susceptibility to cationic antimicrobial peptides (CAMPs). We show modification of the lipid A region of LPS with glucosamine increased resistance to numerous CAMPs, including LL-37. Furthermore, we demonstrate that this glucosamine modification increased resistance to outer membrane perturbation.

scholarly journals Emergence of Epidemic Multidrug-Resistant Enterococcus faecium from Animal and Commensal Strains

mBio ◽  
2013 ◽  
Vol 4 (4) ◽  
Author(s):  
François Lebreton ◽  
Willem van Schaik ◽  
Abigail Manson McGuire ◽  
Paul Godfrey ◽  
Allison Griggs ◽  
...  
Keyword(s):  
Enterococcus Faecium ◽  
Sequence Data ◽  
Mobile Element ◽  
Multidrug Resistant ◽  
Human Infection ◽  
Animal Origin ◽  
Loss Of Function ◽  
Content Type ◽  
Hospital Acquired Infection ◽  
Hospital Acquired

ABSTRACTEnterococcus faecium, natively a gut commensal organism, emerged as a leading cause of multidrug-resistant hospital-acquired infection in the 1980s. As the living record of its adaptation to changes in habitat, we sequenced the genomes of 51 strains, isolated from various ecological environments, to understand howE. faeciumemerged as a leading hospital pathogen. Because of the scale and diversity of the sampled strains, we were able to resolve the lineage responsible for epidemic, multidrug-resistant human infection from other strains and to measure the evolutionary distances between groups. We found that the epidemic hospital-adapted lineage is rapidly evolving and emerged approximately 75 years ago, concomitant with the introduction of antibiotics, from a population that included the majority of animal strains, and not from human commensal lines. We further found that the lineage that included most strains of animal origin diverged from the main human commensal line approximately 3,000 years ago, a time that corresponds to increasing urbanization of humans, development of hygienic practices, and domestication of animals, which we speculate contributed to their ecological separation. Each bifurcation was accompanied by the acquisition of new metabolic capabilities and colonization traits on mobile elements and the loss of function and genome remodeling associated with mobile element insertion and movement. As a result, diversity within the species, in terms of sequence divergence as well as gene content, spans a range usually associated with speciation.IMPORTANCEEnterococci, in particular vancomycin-resistantEnterococcus faecium, recently emerged as a leading cause of hospital-acquired infection worldwide. In this study, we examined genome sequence data to understand the bacterial adaptations that accompanied this transformation from microbes that existed for eons as members of host microbiota. We observed changes in the genomes that paralleled changes in human behavior. An initial bifurcation within the species appears to have occurred at a time that corresponds to the urbanization of humans and domestication of animals, and a more recent bifurcation parallels the introduction of antibiotics in medicine and agriculture. In response to the opportunity to fill niches associated with changes in human activity, a rapidly evolving lineage emerged, a lineage responsible for the vast majority of multidrug-resistantE. faeciuminfections.

scholarly journals Characterization of RelA in Acinetobacter baumannii

2020 ◽  
Vol 202 (12) ◽  
Author(s):  
María Pérez-Varela ◽  
Aimee R. P. Tierney ◽  
Ju-Sim Kim ◽  
Andrés Vázquez-Torres ◽  
Philip Rather
Keyword(s):  
Acinetobacter Baumannii ◽  
Galleria Mellonella ◽  
Multidrug Resistant ◽  
Cell Physiology ◽  
Content Type ◽  
Content Model ◽  
Transposon Insertion ◽  
Downstream Effectors

ABSTRACT In response to nutrient depletion, the RelA and SpoT proteins generate the signaling molecule (p)ppGpp, which then controls a number of downstream effectors to modulate cell physiology. In Acinetobacter baumannii strain AB5075, a relA ortholog (ABUW_3302) was identified by a transposon insertion that conferred an unusual colony phenotype. An in-frame deletion in relA (ΔrelA) failed to produce detectable levels of ppGpp when amino acid starvation was induced with serine hydroxamate. The ΔrelA mutant was blocked from switching from the virulent opaque colony variant (VIR-O) to the avirulent translucent colony variant (AV-T), but the rate of AV-T to VIR-O switching was unchanged. In addition, the ΔrelA mutation resulted in a pronounced hypermotile phenotype on 0.35% agar plates. This hypermotility was dependent on the activation of a LysR regulator ABUW_1132, which was required for expression of AbaR, a LuxR family quorum-sensing regulator. In the ΔrelA mutant, ABUW_1132 was also required for the increased expression of an operon composed of the ABUW_3766-ABUW_3773 genes required for production of the surfactant-like lipopeptide acinetin 505. Additional phenotypes identified in the ΔrelA mutant included (i) cell elongation at high density, (ii) reduced formation of persister cells tolerant to colistin and rifampin, and (iii) decreased virulence in a Galleria mellonella model. IMPORTANCE Acinetobacter baumannii is a pathogen of worldwide importance. Due to the increasing prevalence of antibiotic resistance, these infections are becoming increasingly difficult to treat. New therapies are required to combat multidrug-resistant isolates. The role of RelA in A. baumannii is largely unknown. This study demonstrates that like in other bacteria, RelA controls a variety of functions, including virulence. Strategies to inhibit the activity of RelA and the resulting production of ppGpp could inhibit virulence and may represent a new therapeutic approach.

scholarly journals Complex Response of the CpxAR Two-Component System to β-Lactams on Antibiotic Resistance and Envelope Homeostasis in Enterobacteriaceae

2020 ◽  
Vol 64 (6) ◽  
Author(s):  
Muriel Masi ◽  
Elizabeth Pinet ◽  
Jean-Marie Pagès
Keyword(s):  
Efflux Pump ◽  
Multidrug Resistant ◽  
Diaminopimelic Acid ◽  
Single Amino Acid ◽  
Dependent Manner ◽  
Content Type ◽  
Promising Therapeutic Target ◽  
Abnormal Accumulation ◽  
A Cell ◽  
Mutational Activation

ABSTRACT The Cpx stress response is widespread among Enterobacteriaceae. We previously reported a mutation in cpxA in a multidrug-resistant strain of Klebsiella aerogenes isolated from a patient treated with imipenem. This mutation yields a single-amino-acid substitution (Y144N) located in the periplasmic sensor domain of CpxA. In this work, we sought to characterize this mutation in Escherichia coli by using genetic and biochemical approaches. Here, we show that cpxAY144N is an activated allele that confers resistance to β-lactams and aminoglycosides in a CpxR-dependent manner, by regulating the expression of the OmpF porin and the AcrD efflux pump, respectively. We also demonstrate the effect of the intimate interconnection between the Cpx system and peptidoglycan integrity on the expression of an exogenous AmpC β-lactamase by using imipenem as a cell wall-active antibiotic or by inactivating penicillin-binding proteins. Moreover, our data indicate that the Y144N substitution abrogates the interaction between CpxA and CpxP and increases phosphotransfer activity on CpxR. Because the addition of a strong AmpC inducer such as imipenem is known to cause abnormal accumulation of muropeptides (disaccharide-pentapeptide and N-acetylglucosamyl-1,6-anhydro-N-acetylmuramyl-l-alanyl-d-glutamy-meso-diaminopimelic-acid-d-alanyl-d-alanine) in the periplasmic space, we propose these molecules activate the Cpx system by displacing CpxP from the sensor domain of CpxA. Altogether, these data could explain why large perturbations to peptidoglycans caused by imipenem lead to mutational activation of the Cpx system and bacterial adaptation through multidrug resistance. These results also validate the Cpx system, in particular, the interaction between CpxA and CpxP, as a promising therapeutic target.

scholarly journals Artificial Selection for Pathogenicity Mutations inStaphylococcus aureusIdentifies Novel Factors Relevant to Chronic Infection

2019 ◽  
Vol 87 (4) ◽  
Author(s):  
Kathryn McLean ◽  
Elizabeth A. Holmes ◽  
Kelsi Penewit ◽  
Duankun K. Lee ◽  
Samantha R. Hardy ◽  
...  
Keyword(s):  
Artificial Selection ◽  
Chronic Infection ◽  
Loss Of Function ◽  
Diverse Range ◽  
Content Type ◽  
Transposon Mutants ◽  
Genetic Mechanisms ◽  
Functionally Diverse ◽  
Chromosomal Mutations ◽  
Virulence Regulator

ABSTRACTAdaptation ofStaphylococcus aureusto host microenvironments during chronic infection involves spontaneous mutations, yet changes underlying adaptive phenotypes remain incompletely explored. Here, we employed artificial selection and whole-genome sequencing to better characterize spontaneous chromosomal mutations that alter two pathogenicity phenotypes relevant to chronic infection inS. aureus: intracellular invasiveness and intracellular cytotoxicity. We identified 23 genes whose alteration coincided with enhanced virulence, 11 that were previously known and 12 (52%) that had no previously described role inS. aureuspathogenicity. Using precision genome editing, transposon mutants, and gene complementation, we empirically assessed the contributions of individual genes to the two virulence phenotypes. We functionally validated 14 of 21 genes tested as measurably influencing invasion and/or cytotoxicity, including 8 newly implicated by this study. We identified inactivating mutations (murA,ndhC, and a hypothetical membrane protein) and gain-of-function mutations (aroEThr182Ile,yhcFThr74Ile, and Asp486Glu in a hypothetical peptidase) in previously unrecognizedS. aureusvirulence genes that enhance pathogenesis when introduced into a clean genetic background, as well as a novel activating mutation in the known virulence regulator genesaeS(Ala106Thr). Investigation of potentially epistatic interactions identified atufAmutation (Ala271Val) that enhances virulence only in the context of purine operon repressor gene (purR) inactivation. This project reveals a functionally diverse range of genes affected by gain- or loss-of-function mutations that contribute toS. aureusadaptive virulence phenotypes. More generally, the work establishes artificial selection as a means to determine the genetic mechanisms underlying complex bacterial phenotypes relevant to adaptation during infection.

scholarly journals Cationic Antimicrobial Peptides and Biogenic Silver Nanoparticles Kill Mycobacteria without Eliciting DNA Damage and Cytotoxicity in Mouse Macrophages

2013 ◽  
Vol 57 (8) ◽  
pp. 3688-3698 ◽  
Author(s):  
Soumitra Mohanty ◽  
Prajna Jena ◽  
Ranjit Mehta ◽  
Rashmirekha Pati ◽  
Birendranath Banerjee ◽  
...  
Keyword(s):  
Dna Damage ◽  
Silver Nanoparticles ◽  
Antimicrobial Peptides ◽  
Fluorescein Isothiocyanate ◽  
Antibacterial Activities ◽  
Multidrug Resistant ◽  
Content Type ◽  
Biogenic Silver Nanoparticles ◽  
Transmission Electron ◽  
Mouse Macrophages

ABSTRACTWith the emergence of multidrug-resistant mycobacterial strains, better therapeutic strategies are required for the successful treatment of the infection. Although antimicrobial peptides (AMPs) and silver nanoparticles (AgNPs) are becoming one of the popular antibacterial agents, their antimycobacterial potential is not fully evaluated. In this study, we synthesized biogenic-silver nanoparticles using bacterial, fungal, and plant biomasses and analyzed their antibacterial activities in combination with AMPs against mycobacteria.Mycobacterium smegmatiswas found to be more susceptible to AgNPs compared toM. marinum. We found that NK-2 showed enhanced killing effect with NP-1 and NP-2 biogenic nanoparticles at a 0.5-ppm concentration, whereas LLKKK-18 showed antibacterial activity only with NP-2 at 0.5-ppm dose againstM. smegmatis. In case ofM. marinumNK-2 did not show any additive activity with NP-1 and NP-2 and LLKKK-18 alone completely inhibited the bacterial growth. Both NP-1 and NP-2 also showed increased killing ofM. smegmatisin combination with the antituberculosis drug rifampin. The sizes and shapes of the AgNPs were determined by transmission electron microscopy and dynamic light scattering. AgNPs showed no cytotoxic or DNA damage effects on macrophages at the mycobactericidal dose, whereas treatment with higher doses of AgNPs caused toxicity and micronuclei formation in cytokinesis blocked cells. Macrophages actively endocytosed fluorescein isothiocyanate-labeled AgNPs resulting in nitric oxide independent intracellular killing ofM. smegmatis. Apoptosis and cell cycle studies showed that treatment with higher dose of AgNPs arrested macrophages at the G1-phase. In summary, our data suggest the combined effect of biogenic-AgNPs and antimicrobial peptides as a promising antimycobacterial template.

scholarly journals Identification of a Virulence-Associated Determinant, Dihydrolipoamide Dehydrogenase (lpd), in Mycoplasma gallisepticum through In Vivo Screening of Transposon Mutants

2006 ◽  
Vol 74 (2) ◽  
pp. 931-939 ◽  
Author(s):  
P. Hudson ◽  
T. S. Gorton ◽  
L. Papazisi ◽  
K. Cecchini ◽  
S. Frasca ◽  
...  
Keyword(s):  
Insertion Site ◽  
Mycoplasma Gallisepticum ◽  
Dihydrolipoamide Dehydrogenase ◽  
Gene Encoding ◽  
Transposon Insertion ◽  
Transposon Mutants ◽  
Genomic Regions ◽  
Transposon Insertions ◽  
Biologic Function

ABSTRACT To effectively analyze Mycoplasma gallisepticum for virulence-associated determinants, the ability to create stable genetic mutations is essential. Global M. gallisepticum mutagenesis is currently limited to the use of transposons. Using the gram-positive transposon Tn4001mod, a mutant library of 110 transformants was constructed and all insertion sites were mapped. To identify transposon insertion points, a unique primer directed outward from the end of Tn4001mod was used to sequence flanking genomic regions. By comparing sequences obtained in this manner to the annotated M. gallisepticum genome, the precise locations of transposon insertions were discerned. After determining the transposon insertion site for each mutant, unique reverse primers were synthesized based on the specific sequences, and PCR was performed. The resultant amplicons were used as unique Tn4001mod mutant identifiers. This procedure is referred to as signature sequence mutagenesis (SSM). SSM permits the comprehensive screening of the M. gallisepticum genome for the identification of novel virulence-associated determinants from a mixed mutant population. To this end, chickens were challenged with a pool of 27 unique Tn4001mod mutants. Two weeks postinfection, the birds were sacrificed, and organisms were recovered from respiratory tract tissues and screened for the presence or absence of various mutants. SSM is a negative-selection screening technique whereby those mutants possessing transposon insertions in genes essential for in vivo survival are not recovered from the host. We have identified a virulence-associated gene encoding dihydrolipoamide dehydrogenase (lpd). A transposon insertion in the middle of the coding sequence resulted in diminished biologic function and reduced virulence of the mutant designated Mg 7.

scholarly journals Role of the LytSR Two-Component Regulatory System in Adaptation to Cationic Antimicrobial Peptides in Staphylococcus aureus

2013 ◽  
Vol 57 (8) ◽  
pp. 3875-3882 ◽  
Author(s):  
Soo-Jin Yang ◽  
Yan Q. Xiong ◽  
Michael R. Yeaman ◽  
Kenneth W. Bayles ◽  
Wessam Abdelhady ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Antimicrobial Peptides ◽  
Regulatory System ◽  
Target Tissue ◽  
Adaptive Responses ◽  
Cationic Antimicrobial Peptide ◽  
Cationic Antimicrobial Peptides ◽  
Content Type ◽  
Response System ◽  
Two Component

ABSTRACTMany host defense cationic antimicrobial peptides (HDPs) perturb the staphylococcal cell membrane (CM) and alter transmembrane potential (ΔΨ) as key parts of their lethal mechanism. Thus, a sense-response system for detecting and mediating adaptive responses to such stresses could impact organism survival; theStaphylococcus aureusLytSR two-component regulatory system (TCRS) may serve as such a ΔΨ sensor. One well-known target of this system is thelrgABoperon, which, along with the relatedcidABCoperon, has been shown to be a regulator in the control of programmed cell death and lysis. We used an isogenic set ofS. aureusstrains: (i) UAMS-1, (ii) its isogenic ΔlytSand ΔlrgABmutants, and (iii) plasmid-complemented ΔlytSRand ΔlrgABmutants. The ΔlytSstrain displayed significantly increasedin vitrosusceptibilities to all HDPs tested (neutrophil-derived human neutrophil peptide 1 [hNP-1], platelet-derived thrombin-induced platelet microbicidal proteins [tPMPs], and the tPMP-mimetic peptide RP-1), as well as to calcium-daptomycin (DAP), a cationic antimicrobial peptide (CAP). In contrast, the ΔlrgABstrain exhibited no significant changes in susceptibilities to these cationic peptides, indicating that althoughlytSRpositively regulates transcription oflrgAB, increased HDP/CAP susceptibilities in the ΔlytSmutant werelrgABindependent. Further, parental UAMS-1 (but not the ΔlytSmutant) became more resistant to hNP-1 and DAP following pretreatment with carbonyl cyanidem-chlorophenylhydrazone (CCCP) (a CM-depolarizing agent). Of note,lytSR-dependent survival against CAP/HDP killing was not associated with changes in either surface positive charge, expression ofmprFanddlt, or CM fluidity. The ΔlytSstrain (but not the ΔlrgABmutant) displayed a significant reduction in target tissue survival in an endocarditis model during DAP treatment. Collectively, these results suggest that thelytSRTCRS plays an important role in adaptive responses ofS. aureusto CM-perturbing HDPs/CAPs, likely by functioning as a sense-response system for detecting subtle changes in ΔΨ.

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