scholarly journals Overexpression of SmeDEF Efflux Pump Decreases Aminoglycoside Resistance in Stenotrophomonas maltophilia

2017 ◽  
Vol 61 (5) ◽  
Author(s):  
Yi-Wei Huang ◽  
Cheng-Wen Lin ◽  
Hsiao-Chen Ning ◽  
Yi-Tsung Lin ◽  
Yi-Chih Chang ◽  
...  
Keyword(s):  
Binding Site ◽  
Nalidixic Acid ◽  
Stenotrophomonas Maltophilia ◽  
Deletion Mutant ◽  
Efflux Pump ◽  
Regulatory System ◽  
Aminoglycoside Resistance ◽  
Content Type ◽  
Regulatory Circuit ◽  
Two Component

ABSTRACT The SmeDEF pump of Stenotrophomonas maltophilia is negatively regulated by SmeT. In this study, strains KJΔT (smeT deletion mutant) and KJT-Dm (mutant with a defective SmeT-binding site) showed increased resistance to chloramphenicol/nalidixic acid/macrolides and susceptibility to aminoglycoside. Overexpression of the SmeDEF pump, in either KJΔT or KJT-Dm, downregulated smeYZ expression, which is responsible for the reduced aminoglycoside resistance. Furthermore, the SmeRySy two-component regulatory system was downregulated in response to SmeDEF overexpression, which supports its involvement in the regulatory circuit.

scholarly journals Interplay among Membrane-Bound Lytic Transglycosylase D1, the CreBC Two-Component Regulatory System, the AmpNG-AmpDI-NagZ-AmpR Regulatory Circuit, and L1/L2 β-Lactamase Expression in Stenotrophomonas maltophilia

2015 ◽  
Vol 59 (11) ◽  
pp. 6866-6872 ◽  
Author(s):  
Yi-Wei Huang ◽  
Chao-Jung Wu ◽  
Rouh-Mei Hu ◽  
Yi-Tsung Lin ◽  
Tsuey-Ching Yang
Keyword(s):  
Stenotrophomonas Maltophilia ◽  
Regulatory System ◽  
Underlying Mechanism ◽  
Dependent Manner ◽  
Content Type ◽  
Lytic Transglycosylases ◽  
Lytic Transglycosylase ◽  
Regulatory Circuit ◽  
Two Component ◽  
Membrane Bound

ABSTRACTLytic transglycosylases (LTs) are an important class of enzymes involved in peptidoglycan (PG) cleavage, with the concomitant formation of an intramolecular 1,6-anhydromuramoyl reaction product. There are six annotated LT genes in theStenotrophomonas maltophiliagenome, including genes for five membrane-bound LTs (mltA,mltB1,mltB2,mltD1, andmltD2) and a gene for soluble LT (slt). Six LTs ofS. maltophiliaKJ were systematically mutated, yielding the ΔmltA, ΔmltB1, ΔmltB2, ΔmltD1, ΔmltD2, and Δsltmutants. Inactivation ofmltD1conferred a phenotype of elevated uninduced β-lactamase activity. The underlying mechanism responsible for this phenotype was elucidated by the construction of several mutants and determination of β-lactamase activity. The expression of the genes assayed was assessed by quantitative reverse transcriptase PCR and a promoter transcription fusion assay. The results demonstrate that ΔmltD1mutant-mediated L1/L2 β-lactamase expression involved thecreBCtwo-component regulatory system (TCS) and theampNG-ampDI-nagZ-ampRregulatory circuit. The inactivation ofmltD1resulted inmltB1andmltD2upexpression in acreBC- andampNG-dependent manner. The overexpressed MltB1 and MltD2 activity contributed to the expression of the L1/L2 β-lactamase genes via theampNG-ampDI-nagZ-ampRregulatory circuit. These findings reveal, for the first time, a linkage between LTs, the CreBC TCS, theampNG-ampDI-nagZ-ampRregulatory circuit, and L1/L2 β-lactamase expression inS. maltophilia.

scholarly journals Novel Mechanisms of Efflux-Mediated Levofloxacin Resistance and Reduced Amikacin Susceptibility in Stenotrophomonas maltophilia

2020 ◽  
Vol 65 (1) ◽  
pp. e01284-20
Author(s):  
Punyawee Dulyayangkul ◽  
Karina Calvopiña ◽  
Kate J. Heesom ◽  
Matthew B. Avison
Keyword(s):  
Abc Transporter ◽  
Abc Transporters ◽  
Stenotrophomonas Maltophilia ◽  
Efflux Pump ◽  
Regulatory System ◽  
Fluoroquinolone Resistance ◽  
Content Type ◽  
Antimicrobial Drug Resistance ◽  
Regulatory Systems ◽  
Selection Of

ABSTRACTFluoroquinolone resistance in Stenotrophomonas maltophilia is multifactorial, but the most significant factor is overproduction of efflux pumps, particularly SmeDEF, following mutation. Here, we report that mutations in the glycosyl transferase gene smlt0622 in S. maltophilia K279a mutant K M6 cause constitutive activation of SmeDEF production, leading to elevated levofloxacin MIC. Selection of a levofloxacin-resistant K M6 derivative, K M6 LEVr, allowed identification of a novel two-component regulatory system, Smlt2645/6 (renamed SmaRS). The sensor kinase Smlt2646 (SmaS) is activated by mutation in K M6 LEVr causing overproduction of two novel ABC transporters and the known aminoglycoside efflux pump SmeYZ. Overproduction of one ABC transporter, Smlt1651-4 (renamed SmaCDEF), causes levofloxacin resistance in K M6 LEVr. Overproduction of the other ABC transporter, Smlt2642/3 (renamed SmaAB), and SmeYZ both contribute to the elevated amikacin MIC against K M6 LEVr. Accordingly, we have identified two novel ABC transporters associated with antimicrobial drug resistance in S. maltophilia and two novel regulatory systems whose mutation causes resistance to levofloxacin, clinically important as a promising drug for monotherapy against this highly resistant pathogen.

scholarly journals Mutations in Ribosomal Protein RplA or Treatment with Ribosomal Acting Antibiotics Activates Production of Aminoglycoside Efflux Pump SmeYZ in Stenotrophomonas maltophilia

2019 ◽  
Vol 64 (2) ◽  
Author(s):  
Karina Calvopiña ◽  
Punyawee Dulyayangkul ◽  
Matthew B. Avison
Keyword(s):  
Ribosomal Protein ◽  
Stenotrophomonas Maltophilia ◽  
Efflux Pump ◽  
Clinical Isolates ◽  
Efflux System ◽  
Aminoglycoside Resistance ◽  
Content Type ◽  
Ribosomal Protein L1 ◽  
Significant Mechanism

ABSTRACT Aminoglycoside resistance in Stenotrophomonas maltophilia is multifactorial, but the most significant mechanism is overproduction of the SmeYZ efflux system. By studying laboratory-selected mutants and clinical isolates, we show here that damage to the 50S ribosomal protein L1 (RplA) activates SmeYZ production. We also show that gentamicin and minocycline, which target the ribosome, induce expression of smeYZ. These findings explain the role of SmeYZ in both intrinsic and mutationally acquired aminoglycoside resistance.

scholarly journals RND-Type Efflux Pumps in Multidrug-Resistant Clinical Isolates of Acinetobacter baumannii: Major Role for AdeABC Overexpression and AdeRS Mutations

2013 ◽  
Vol 57 (7) ◽  
pp. 2989-2995 ◽  
Author(s):  
Eun-Jeong Yoon ◽  
Patrice Courvalin ◽  
Catherine Grillot-Courvalin
Keyword(s):  
Acinetobacter Baumannii ◽  
Efflux Pump ◽  
Regulatory System ◽  
Multidrug Resistant ◽  
Clinical Isolates ◽  
Clinical Settings ◽  
Two Component System ◽  
Content Type ◽  
High Incidence ◽  
Two Component

ABSTRACTIncreased expression of chromosomal genes for resistance-nodulation-cell division (RND)-type efflux systems plays a major role in the multidrug resistance (MDR) ofAcinetobacter baumannii. However, the relative contributions of the three most prevalent pumps, AdeABC, AdeFGH, and AdeIJK, have not been evaluated in clinical settings. We have screened 14 MDR clinical isolates shown to be distinct on the basis of multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) for the presence and overexpression of the three Ade efflux systems and analyzed the sequences of the regulators AdeRS, a two-component system, for AdeABC and AdeL, a LysR-type regulator, for AdeFGH. GeneadeBwas detected in 13 of 14 isolates, andadeGand the intrinsicadeJgene were detected in all strains. Significant overexpression ofadeBwas observed in 10 strains, whereas only 7 had moderately increased levels of expression of AdeFGH, and none overexpressed AdeIJK. Thirteen strains had reduced susceptibility to tigecycline, but there was no correlation between tigecycline MICs and the levels of AdeABC expression, suggesting the presence of other mechanisms for tigecycline resistance. No mutations were found in the highly conserved LysR regulator of the nine strains expressing AdeFGH. In contrast, functional mutations were found in conserved domains of AdeRS in all the strains that overexpressed AdeABC with two mutational hot spots, one in AdeS near histidine 149 suggesting convergent evolution and the other in the DNA binding domain of AdeR compatible with horizontal gene transfer. This report outlines the high incidence of AdeABC efflux pump overexpression in MDRA. baumanniias a result of a variety of single mutations in the corresponding two-component regulatory system.

scholarly journals Regulation of Sporangium Formation, Spore Dormancy, and Sporangium Dehiscence by a Hybrid Sensor Histidine Kinase in Actinoplanes missouriensis: Relationship with the Global Transcriptional Regulator TcrA

2020 ◽  
Vol 202 (21) ◽  
Author(s):  
Yuichiro Hashiguchi ◽  
Takeaki Tezuka ◽  
Yoshihiro Mouri ◽  
Kenji Konishi ◽  
Azusa Fujita ◽  
...  
Keyword(s):  
Histidine Kinase ◽  
Deletion Mutant ◽  
Response Regulator ◽  
Regulatory System ◽  
Morphological Development ◽  
Sequencing Analysis ◽  
Content Type ◽  
Rare Actinomycetes ◽  
Two Component ◽  
Hybrid Histidine Kinase

ABSTRACT The rare actinomycete Actinoplanes missouriensis forms terminal sporangia containing a few hundred flagellated spores. In response to water, the sporangia open and release the spores into external environments. The orphan response regulator TcrA functions as a global transcriptional activator during sporangium formation and dehiscence. Here, we report the characterization of an orphan hybrid histidine kinase, HhkA. Sporangia of an hhkA deletion mutant contained many distorted or ectopically germinated spores and scarcely opened to release the spores under sporangium dehiscence-inducing conditions. These phenotypic changes are quite similar to those observed in a tcrA deletion mutant. Comparative RNA sequencing analysis showed that genes controlled by HhkA mostly overlap TcrA-regulated genes. The direct interaction between HhkA and TcrA was suggested by a bacterial two-hybrid assay, but this was not conclusive. The phosphorylation of TcrA using acetyl phosphate as a phosphate donor markedly enhanced its affinity for the TcrA box sequences in the electrophoretic mobility shift assay. Taking these observations together with other results, we proposed that HhkA and TcrA compose a cognate two-component regulatory system, which controls the transcription of the genes involved in many aspects of morphological development, including sporangium formation, spore dormancy, and sporangium dehiscence in A. missouriensis. IMPORTANCE Actinoplanes missouriensis goes through complex morphological differentiation, including formation of flagellated spore-containing sporangia, sporangium dehiscence, swimming of zoospores, and germination of zoospores to filamentous growth. Although the orphan response regulator TcrA globally activates many genes required for sporangium formation, spore dormancy, and sporangium dehiscence, its partner histidine kinase remained unknown. Here, we analyzed the function of an orphan hybrid histidine kinase, HhkA, and proposed that HhkA constitutes a cognate two-component regulatory system with TcrA. That HhkA and TcrA homologues are highly conserved among the genus Actinoplanes and several closely related rare actinomycetes indicates that this possible two-component regulatory system is employed for complex morphological development in sporangium- and/or zoospore-forming rare actinomycetes.

scholarly journals Mutation-Driven Evolution ofPseudomonas aeruginosain the Presence of either Ceftazidime or Ceftazidime-Avibactam

2018 ◽  
Vol 62 (10) ◽  
Author(s):  
Fernando Sanz-García ◽  
Sara Hernando-Amado ◽  
José Luis Martínez
Keyword(s):  
Experimental Evolution ◽  
Efflux Pump ◽  
Multidrug Resistant ◽  
Two Component System ◽  
Aminoglycoside Resistance ◽  
Content Type ◽  
Chromosomal Deletions ◽  
Two Component ◽  
Main Driver ◽  
Selection Of

ABSTRACTCeftazidime-avibactam is a combination of β-lactam/β-lactamase inhibitor, the use of which is restricted to some clinical cases, including cystic fibrosis patients infected with multidrug-resistantPseudomonas aeruginosa, in which mutation is the main driver of resistance. This study aims to predict the mechanisms of mutation-driven resistance that are selected for whenP. aeruginosais challenged with either ceftazidime or ceftazidime-avibactam. For this purpose,P. aeruginosaPA14 was submitted to experimental evolution in the absence of antibiotics and in the presence of increasing concentrations of ceftazidime or ceftazidime-avibactam for 30 consecutive days. Final populations were analyzed by whole-genome sequencing. All evolved populations reached similar levels of ceftazidime resistance. In addition, they were more susceptible to amikacin and produced pyomelanin. A first event in this evolution was the selection of large chromosomal deletions containinghmgA(involved in pyomelanin production),galU(involved in β-lactams resistance), andmexXY-oprM(involved in aminoglycoside resistance). Besides mutations inmplanddacBthat regulate β-lactamase expression, mutations related to MexAB-OprM overexpression were prevalent. Ceftazidime-avibactam challenge selected mutants in the putative efflux pumpPA14_45890andPA14_45910and in a two-component system (PA14_45870andPA14_45880), likely regulating its expression. All populations produced pyomelanin and were more susceptible to aminoglycosides, likely due to the selection of large chromosomal deletions. Since pyomelanin-producing mutants presenting similar deletions are regularly isolated from infections, the potential aminoglycoside hypersusceptiblity and reduced β-lactam susceptibility of pyomelanin-producingP. aeruginosashould be taken into consideration for treating infections caused by these isolates.

scholarly journals The MisR Response Regulator Is Necessary for Intrinsic Cationic Antimicrobial Peptide and Aminoglycoside Resistance in Neisseria gonorrhoeae

2016 ◽  
Vol 60 (8) ◽  
pp. 4690-4700 ◽  
Author(s):  
Justin L. Kandler ◽  
Concerta L. Holley ◽  
Jennifer L. Reimche ◽  
Vijaya Dhulipala ◽  
Jacqueline T. Balthazar ◽  
...  
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Lipid A ◽  
Efflux Pump ◽  
Response Regulator ◽  
Membrane Integrity ◽  
Regulatory System ◽  
Phagocytic Cells ◽  
Aminoglycoside Resistance ◽  
Cationic Antimicrobial Peptide ◽  
Content Type

ABSTRACTDuring infection, the sexually transmitted pathogenNeisseria gonorrhoeae(the gonococcus) encounters numerous host-derived antimicrobials, including cationic antimicrobial peptides (CAMPs) produced by epithelial and phagocytic cells. CAMPs have both direct and indirect killing mechanisms and help link the innate and adaptive immune responses during infection. Gonococcal CAMP resistance is likely important for avoidance of host nonoxidative killing systems expressed by polymorphonuclear granulocytes (e.g., neutrophils) and intracellular survival. Previously studied gonococcal CAMP resistance mechanisms include modification of lipid A with phosphoethanolamine by LptA and export of CAMPs by the MtrCDE efflux pump. In the related pathogenNeisseria meningitidis, a two-component regulatory system (2CRS) termed MisR-MisS has been shown to contribute to the capacity of the meningococcus to resist CAMP killing. We report that the gonococcal MisR response regulator but not the MisS sensor kinase is involved in constitutive and inducible CAMP resistance and is also required for intrinsic low-level resistance to aminoglycosides. The 4- to 8-fold increased susceptibility ofmisR-deficient gonococci to CAMPs and aminoglycosides was independent of phosphoethanolamine decoration of lipid A and the levels of the MtrCDE efflux pump and seemed to correlate with a general increase in membrane permeability. Transcriptional profiling and biochemical studies confirmed that expression oflptAandmtrCDEwas not impacted by the loss of MisR. However, several genes encoding proteins involved in membrane integrity and redox control gave evidence of being MisR regulated. We propose that MisR modulates the levels of gonococcal susceptibility to antimicrobials by influencing the expression of genes involved in determining membrane integrity.

scholarly journals Target (MexB)- and Efflux-Based Mechanisms Decreasing the Effectiveness of the Efflux Pump Inhibitor D13-9001 in Pseudomonas aeruginosa PAO1: Uncovering a New Role for MexMN-OprM in Efflux of β-Lactams and a Novel Regulatory Circuit (MmnRS) Controlling MexMN Expression

2018 ◽  
Vol 63 (2) ◽  
pp. e01718-18 ◽  
Author(s):  
Srijan Ranjitkar ◽  
Adriana K. Jones ◽  
Mina Mostafavi ◽  
Zachary Zwirko ◽  
Oleg Iartchouk ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Efflux Pump ◽  
Response Regulator ◽  
Heavy Metal Resistance ◽  
Metal Resistance ◽  
Rna Seq ◽  
Efflux Pump Inhibitor ◽  
Content Type ◽  
Regulatory Circuit ◽  
Outer Membrane Channel

ABSTRACT Efflux pumps contribute to antibiotic resistance in Gram-negative pathogens. Correspondingly, efflux pump inhibitors (EPIs) may reverse this resistance. D13-9001 specifically inhibits MexAB-OprM in Pseudomonas aeruginosa. Mutants with decreased susceptibility to MexAB-OprM inhibition by D13-9001 were identified, and these fell into two categories: those with alterations in the target MexB (F628L and ΔV177) and those with an alteration in a putative sensor kinase of unknown function, PA1438 (L172P). The alterations in MexB were consistent with reported structural studies of the D13-9001 interaction with MexB. The PA1438L172P alteration mediated a >150-fold upregulation of MexMN pump gene expression and a >50-fold upregulation of PA1438 and the neighboring response regulator gene, PA1437. We propose that these be renamed mmnR and mmnS for MexMN regulator and MexMN sensor, respectively. MexMN was shown to partner with the outer membrane channel protein OprM and to pump several β-lactams, monobactams, and tazobactam. Upregulated MexMN functionally replaced MexAB-OprM to efflux these compounds but was insusceptible to inhibition by D13-9001. MmnSL172P also mediated a decrease in susceptibility to imipenem and biapenem that was independent of MexMN-OprM. Expression of oprD, encoding the uptake channel for these compounds, was downregulated, suggesting that this channel is also part of the MmnSR regulon. Transcriptome sequencing (RNA-seq) of cells encoding MmnSL172P revealed, among other things, an interrelationship between the regulation of mexMN and genes involved in heavy metal resistance.

scholarly journals Identification of a Second Two-Component Signal Transduction System That Controls Fosfomycin Tolerance and Glycerol-3-Phosphate Uptake

2014 ◽  
Vol 197 (5) ◽  
pp. 861-871 ◽  
Author(s):  
Kumiko Kurabayashi ◽  
Yuko Hirakawa ◽  
Koichi Tanimoto ◽  
Haruyoshi Tomita ◽  
Hidetada Hirakawa
Keyword(s):  
Phosphate Uptake ◽  
Response Regulator ◽  
Anaerobic Respiration ◽  
Regulatory System ◽  
Carbon Substrate ◽  
Two Component System ◽  
Component System ◽  
Content Type ◽  
Two Component ◽  
Biological Cost

Particular interest in fosfomycin has resurfaced because it is a highly beneficial antibiotic for the treatment of refractory infectious diseases caused by pathogens that are resistant to other commonly used antibiotics. The biological cost to cells of resistance to fosfomycin because of chromosomal mutation is high. We previously found that a bacterial two-component system, CpxAR, induces fosfomycin tolerance in enterohemorrhagicEscherichia coli(EHEC) O157:H7. This mechanism does not rely on irreversible genetic modification and allows EHEC to relieve the fitness burden that results from fosfomycin resistance in the absence of fosfomycin. Here we show that another two-component system, TorSRT, which was originally characterized as a regulatory system for anaerobic respiration utilizing trimethylamine-N-oxide (TMAO), also induces fosfomycin tolerance. Activation of the Tor regulatory pathway by overexpression oftorR, which encodes the response regulator, or addition of TMAO increased fosfomycin tolerance in EHEC. We also show that phosphorylated TorR directly represses the expression ofglpT, a gene that encodes a symporter of fosfomycin and glycerol-3-phosphate, and activation of the TorR protein results in the reduced uptake of fosfomycin by cells. However, cells in which the Tor pathway was activated had an impaired growth phenotype when cultured with glycerol-3-phosphate as a carbon substrate. These observations suggest that the TorSRT pathway is the second two-component system to reversibly control fosfomycin tolerance and glycerol-3-phosphate uptake in EHEC, and this may be beneficial for bacteria by alleviating the biological cost. We expect that this mechanism could be a potential target to enhance the utility of fosfomycin as chemotherapy against multidrug-resistant pathogens.

Sign in / Sign up

Export Citation Format

Share Document