scholarly journals Novel Expansions of the Gene Encoding Dihydropteroate Synthase in Trimethoprim-Sulfamethoxazole-ResistantStreptococcus pneumoniae

1999 ◽  
Vol 43 (9) ◽  
pp. 2225-2230 ◽  
Author(s):  
Thanugarani Padayachee ◽  
Keith P. Klugman
Keyword(s):  
South African ◽  
Specific Region ◽  
Site Directed Mutagenesis ◽  
Clinical Isolates ◽  
Wild Type ◽  
Gene Encoding ◽  
Dihydropteroate Synthase ◽  
Sulfonamide Resistance ◽  
Resistant Strains ◽  
Chromosomal Mutations

ABSTRACT A study of eight sulfonamide-resistant clinical isolates ofStreptococcus pneumoniae revealed chromosomal mutations within the gene encoding dihydropteroate synthase that play a role in conferring resistance to sulfamethoxazole. The presence of thesuld mutation, found previously only in a laboratory mutant, was shown to occur in three of the wild-type clinical isolates. The duplication of Ser61, the other previously defined mutation in the dihydropteroate synthase gene of S. pneumoniae, was observed in only one of the isolates characterized. We report two previously unidentified amino acid alterations, namely, a duplication of Arg58 and Pro59 and an insertion of an arginine residue between Gly60 and Ser61 in trimethoprim-sulfamethoxazole-resistant strains. The significance of these mutations was confirmed by site-directed mutagenesis and by the transformation of a susceptible strain of S. pneumoniae to sulfamethoxazole resistance. Two resistant isolates did not contain any mutations within the gene encoding dihydropteroate synthase. The results presented suggest the independent generation of resistant mutations among South African clinical isolates. It is also proposed that the mechanism of sulfonamide resistance in S. pneumoniae involves the expansion of a specific region within dihydropteroate synthase, which probably forms part of the sulfonamide binding site.

scholarly journals Amino Acid Repetitions in the Dihydropteroate Synthase of Streptococcus pneumoniae Lead to Sulfonamide Resistance with Limited Effects on SubstrateKm

2001 ◽  
Vol 45 (3) ◽  
pp. 805-809 ◽  
Author(s):  
Ylva Haasum ◽  
Katrin Ström ◽  
Rahma Wehelie ◽  
Vicki Luna ◽  
Marilyn C. Roberts ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Washington State ◽  
Site Directed Mutagenesis ◽  
Clinical Isolates ◽  
Directed Mutagenesis ◽  
Aminobenzoic Acid ◽  
Kinetic Measurements ◽  
Dihydropteroate Synthase ◽  
Gene Coding ◽  
Sulfonamide Resistance

ABSTRACT Sulfonamide resistance in Streptococcus pneumoniae is due to changes in the chromosomal folP (sulA) gene coding for dihydropteroate synthase (DHPS). The first reported laboratory-selected sulfonamide-resistant S. pneumoniaeisolate had a 6-bp repetition, the sul-d mutation, leading to a repetition of the amino acids Ile66 and Glu67 in the gene product DHPS. More recently, clinical isolates showing this and other repetitions have been reported. WA-5, a clinical isolate from Washington State, contains a 6-bp repetition in the folP gene, identical to the sul-d mutation. The repetition was deleted by site-directed mutagenesis. Enzyme kinetic measurements showed that the deletion was associated with a 35-fold difference in Ki for sulfathiazole but changed the Km for p-aminobenzoic acid only 2.5-fold and did not significantly change theKm for 2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine pyrophosphate. The enzyme characteristics of the deletion variant were identical to those of DHPS from a sulfonamide-susceptible strain. DHPS from clinical isolates with repetitions of Ser61 had very similar enzyme characteristics to the DHPS from WA-5. The results confirm that the repetitions are sufficient for development of a resistant enzyme and suggest that the fitness cost to the organism of developing resistance may be very low.

Mechanism of sulfonamide resistance in clinical isolates of Streptococcus pneumoniae.

1997 ◽  
Vol 41 (10) ◽  
pp. 2121-2126 ◽  
Author(s):  
J P Maskell ◽  
A M Sefton ◽  
L M Hall
Keyword(s):  
Streptococcus Pneumoniae ◽  
Genetic Basis ◽  
Laboratory Strain ◽  
Clinical Isolates ◽  
Gene Exchange ◽  
Gene Encoding ◽  
Dihydropteroate Synthase ◽  
Loss Of Resistance ◽  
Sulfonamide Resistance

The genetic basis of sulfonamide resistance in six clinical isolates of Streptococcus pneumoniae was demonstrated to be 3- or 6-bp duplications within sulA, the chromosomal gene encoding dihydropteroate synthase. The duplications all result in repetition of one or two amino acids in the region from Arg58 to Tyr63, close to but distinct from the sul-d mutation, a duplication previously reported in a resistant laboratory strain (P. Lopez, M. Espinosa, B. Greenberg, and S. A. Lacks, J. Bacteriol. 169:4320-4326, 1987). Six sulfonamide-susceptible clinical isolates lacked such duplications. The role of the duplications in conferring sulfonamide resistance was confirmed by transforming 319- or 322-bp PCR fragments into the chromosome of a susceptible recipient. Two members of a clone of serotype 9V, one susceptible and one resistant to sulfonamide, which are highly related by other criteria, were shown to have sulA sequences that differ in 7.2% of nucleotides in addition to the duplication responsible for resistance. It is postulated that horizontal gene exchange has been involved in the acquisition (or loss) of resistance within this clone. However, five of the six resistant isolates have distinct duplications and other sequence polymorphisms, suggesting that resistance has arisen independently on many occasions.

scholarly journals Mutations of folC cause increased susceptibility to sulfamethoxazole in Mycobacterium tuberculosis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ruiqi Wang ◽  
Kun Li ◽  
Jifang Yu ◽  
Jiaoyu Deng ◽  
Yaokai Chen
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Type Strain ◽  
Wild Type Strain ◽  
Clinical Isolates ◽  
Aminobenzoic Acid ◽  
Wild Type ◽  
Dihydropteroate Synthase ◽  
Expression Levels ◽  
Encoding Gene ◽  
Increased Susceptibility

AbstractPrevious studies showed that mutation of folC caused decreased expression of the dihydropteroate synthase encoding gene folP2 in Mycobacterium tuberculosis (M. tuberculosis). We speculated that mutation of folC in M. tuberculosis might affect the susceptibility to sulfamethoxazole (SMX). To prove this, 53 clinical isolates with folC mutations were selected and two folC mutants (I43A, I43T) were constructed based on M. tuberculosis H37Ra. The results showed that 42 of the 53 clinical isolates (79.2%) and the two lab-constructed folC mutants were more sensitive to SMX. To probe the mechanism by which folC mutations make M. tuberculosis more sensitive to SMX, folP2 was deleted in H37Ra, and expression levels of folP2 were compared between H37Ra and the two folC mutants. Although deletion of folP2 resulted in increased susceptibility to SMX, no difference in folP2 expression was observed. Furthermore, production levels of para-aminobenzoic acid (pABA) were compared between the folC mutants and the wild-type strain, and results showed that folC mutation resulted in decreased production of pABA. Taken together, we show that folC mutation leads to decreased production of pABA in M. tuberculosis and thus affects its susceptibility to SMX, which broadens our understanding of mechanisms of susceptibilities to antifolates in this bacterium.

scholarly journals A Regulator of G Protein Signaling-containing Kinase Is Important for Chemotaxis and Multicellular Development inDictyostelium

2003 ◽  
Vol 14 (4) ◽  
pp. 1727-1743 ◽  
Author(s):  
Binggang Sun ◽  
Richard A. Firtel
Keyword(s):  
Protein Kinase ◽  
G Protein ◽  
Kinase Activity ◽  
Site Directed Mutagenesis ◽  
Membrane Localization ◽  
Pleckstrin Homology Domain ◽  
G Protein Signaling ◽  
Protein Signaling ◽  
Wild Type ◽  
Gene Encoding

We have identified a gene encoding RGS domain-containing protein kinase (RCK1), a novel regulator of G protein signaling domain-containing protein kinase. RCK1 mutant strains exhibit strong aggregation and chemotaxis defects. rck1 null cells chemotax ∼50% faster than wild-type cells, suggesting RCK1 plays a negative regulatory role in chemotaxis. Consistent with this finding, overexpression of wild-type RCK1 reduces chemotaxis speed by ∼40%. On cAMP stimulation, RCK1 transiently translocates to the membrane/cortex region with membrane localization peaking at ∼10 s, similar to the kinetics of membrane localization of the pleckstrin homology domain-containing proteins CRAC, Akt/PKB, and PhdA. RCK1 kinase activity also increases dramatically. The RCK1 kinase activity does not rapidly adapt, but decreases after the cAMP stimulus is removed. This is particularly novel considering that most other chemoattractant-activated kinases (e.g., Akt/PKB, ERK1, ERK2, and PAKa) rapidly adapt after activation. Using site-directed mutagenesis, we further show that both the RGS and kinase domains are required for RCK1 function and that RCK1 kinase activity is required for the delocalization of RCK1 from the plasma membrane. Genetic evidence suggests RCK1 function lies downstream from Gα2, the heterotrimeric G protein that couples to the cAMP chemoattractant receptors. We suggest that RCK1 might be part of an adaptation pathway that regulates aspects of chemotaxis in Dictyostelium.

scholarly journals A Novel Cecropin D-Derived Short Cationic Antimicrobial Peptide Exhibits Antibacterial Activity Against Wild-Type and Multidrug-Resistant Strains of Klebsiella pneumoniae and Pseudomonas aeruginosa

2020 ◽  
Vol 16 ◽  
pp. 117693432093626
Author(s):  
Iván Darío Ocampo-Ibáñez ◽  
Yamil Liscano ◽  
Sandra Patricia Rivera-Sánchez ◽  
José Oñate-Garzón ◽  
Ashley Dayan Lugo-Guevara ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Antibacterial Activity ◽  
Klebsiella Pneumoniae ◽  
Multidrug Resistant ◽  
Clinical Isolates ◽  
Health Concern ◽  
Wild Type ◽  
Cationic Antimicrobial Peptide ◽  
Promising Alternative ◽  
Resistant Strains

Infections caused by multidrug-resistant (MDR) Pseudomonas aeruginosa and Klebsiella pneumoniae are a serious worldwide public health concern due to the ineffectiveness of empirical antibiotic therapy. Therefore, research and the development of new antibiotic alternatives are urgently needed to control these bacteria. The use of cationic antimicrobial peptides (CAMPs) is a promising candidate alternative therapeutic strategy to antibiotics because they exhibit antibacterial activity against both antibiotic susceptible and MDR strains. In this study, we aimed to investigate the in vitro antibacterial effect of a short synthetic CAMP derived from the ΔM2 analog of Cec D-like (CAMP-CecD) against clinical isolates of K pneumoniae (n = 30) and P aeruginosa (n = 30), as well as its hemolytic activity. Minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) of CAMP-CecD against wild-type and MDR strains were determined by the broth microdilution test. In addition, an in silico molecular dynamic simulation was performed to predict the interaction between CAMP-CecD and membrane models of K pneumoniae and P aeruginosa. The results revealed a bactericidal effect of CAMP-CecD against both wild-type and resistant strains, but MDR P aeruginosa showed higher susceptibility to this peptide with MIC values between 32 and >256 μg/mL. CAMP-CecD showed higher stability in the P aeruginosa membrane model compared with the K pneumoniae model due to the greater number of noncovalent interactions with phospholipid 1-Palmitoyl-2-oleyl-sn-glycero-3-(phospho-rac-(1-glycerol)) (POPG). This may be related to the boosted effectiveness of the peptide against P aeruginosa clinical isolates. Given the antibacterial activity of CAMP-CecD against wild-type and MDR clinical isolates of P aeruginosa and K pneumoniae and its nonhemolytic effects on human erythrocytes, CAMP-CecD may be a promising alternative to conventional antibiotics.

scholarly journals In VitroCross-Resistance Profile of Nucleoside Reverse Transcriptase Inhibitor (NRTI) BMS-986001 against Known NRTI Resistance Mutations

2013 ◽  
Vol 57 (11) ◽  
pp. 5500-5508 ◽  
Author(s):  
Zhufang Li ◽  
Brian Terry ◽  
William Olds ◽  
Tricia Protack ◽  
Carol Deminie ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Nucleoside Reverse Transcriptase Inhibitor ◽  
Transcriptase Inhibitor ◽  
Fold Change ◽  
Site Directed Mutagenesis ◽  
Clinical Isolates ◽  
Cross Resistance ◽  
Wild Type ◽  
Resistance Profile ◽  
Reverse Transcriptase Inhibitor

ABSTRACTBMS-986001 is a novel HIV nucleoside reverse transcriptase inhibitor (NRTI). To date, little is known about its resistance profile. In order to examine the cross-resistance profile of BMS-986001 to NRTI mutations, a replicating virus system was used to examine specific amino acid mutations known to confer resistance to various NRTIs. In addition, reverse transcriptases from 19 clinical isolates with various NRTI mutations were examined in the Monogram PhenoSense HIV assay. In the site-directed mutagenesis studies, a virus containing a K65R substitution exhibited a 0.4-fold change in 50% effective concentration (EC50) versus the wild type, while the majority of viruses with the Q151M constellation (without M184V) exhibited changes in EC50versus wild type of 0.23- to 0.48-fold. Susceptibility to BMS-986001 was also maintained in an L74V-containing virus (0.7-fold change), while an M184V-only-containing virus induced a 2- to 3-fold decrease in susceptibility. Increasing numbers of thymidine analog mutation pattern 1 (TAM-1) pathway mutations correlated with decreases in susceptibility to BMS-986001, while viruses with TAM-2 pathway mutations exhibited a 5- to 8-fold decrease in susceptibility, regardless of the number of TAMs. A 22-fold decrease in susceptibility to BMS-986001 was observed in a site-directed mutant containing the T69 insertion complex. Common non-NRTI (NNRTI) mutations had little impact on susceptibility to BMS-986001. The results from the site-directed mutants correlated well with the more complicated genotypes found in NRTI-resistant clinical isolates. Data from clinical studies are needed to determine the clinically relevant resistance cutoff values for BMS-986001.

scholarly journals Dihydropteroate Synthase of Mycobacterium leprae and Dapsone Resistance

2000 ◽  
Vol 44 (6) ◽  
pp. 1530-1537 ◽  
Author(s):  
Diana L. Williams ◽  
Laynette Spring ◽  
Eugene Harris ◽  
Paul Roche ◽  
Thomas P. Gillis
Keyword(s):  
Dna Sequences ◽  
Inhibitory Concentration ◽  
Mycobacterium Leprae ◽  
Clinical Isolates ◽  
Missense Mutations ◽  
Wild Type ◽  
Knockout Mutant ◽  
Dihydropteroate Synthase ◽  
E Coli ◽  
Bacterial Lysates

ABSTRACT Two Mycobacterium leprae genes, folP1 andfolP2, encoding putative dihydropteroate synthases (DHPS), were studied for enzymatic activity and for the presence of mutations associated with dapsone resistance. Each gene was cloned and expressed in a folP knockout mutant of Escherichia coli(C600ΔfolP::Kmr). Expression ofM. leprae folP1 in C600ΔfolP::Kmr conferred growth on a folate-deficient medium, and bacterial lysates exhibited DHPS activity. This recombinant displayed a 256-fold-greater sensitivity to dapsone (measured by the MIC) than wild-type E. coli C600, and 50-fold less dapsone was required to block (expressed as the 50% inhibitory concentration [IC50]) the DHPS activity of this recombinant. When the folP1 genes of several dapsone-resistant M. leprae clinical isolates were sequenced, two missense mutations were identified. One mutation occurred at codon 53, substituting an isoleucine for a threonine residue (T53I) in the DHPS-1, and a second mutation occurred in codon 55, substituting an arginine for a proline residue (P55R). Transformation of the C600ΔfolP::Kmr knockout with plasmids carrying either the T53I or the P55R mutant allele did not substantially alter the DHPS activity compared to levels produced by recombinants containing wild-type M. leprae folP1. However, both mutations increased dapsone resistance, with P55R having the greatest affect on dapsone resistance by increasing the MIC 64-fold and the IC50 68-fold. These results prove that thefolP1 of M. leprae encodes a functional DHPS and that mutations within this gene are associated with the development of dapsone resistance in clinical isolates of M. leprae. Transformants created with M. leprae folP2 did not confer growth on the C600ΔfolP::Kmrknockout strain, and DNA sequences of folP2 from dapsone-susceptible and -resistant M. leprae strains were identical, indicating that this gene does not encode a functional DHPS and is not involved in dapsone resistance in M. leprae.

scholarly journals Sulfonamide Resistance in Streptococcus pyogenes Is Associated with Differences in the Amino Acid Sequence of Its Chromosomal Dihydropteroate Synthase

1998 ◽  
Vol 42 (5) ◽  
pp. 1062-1067 ◽  
Author(s):  
Göte Swedberg ◽  
Signe Ringertz ◽  
Ola Sköld
Keyword(s):  
Amino Acid ◽  
Streptococcus Pyogenes ◽  
Gtp Cyclohydrolase ◽  
Gene Encoding ◽  
Additional Indication ◽  
Kinetic Measurements ◽  
Dihydropteroate Synthase ◽  
Development Of Resistance ◽  
Sulfonamide Resistance ◽  
Genes Encoding

ABSTRACT Sulfonamide resistance in recent isolates of Streptococcus pyogenes was found to be associated with alterations of the chromosomally encoded dihydropteroate synthase (DHPS). There were 111 different nucleotides (13.8%) in the genes found in susceptible and resistant isolates, respectively, resulting in 30 amino acid changes (11.3%). These substantial changes suggested the possibility of a foreign origin of the resistance gene, in parallel to what has already been found for sulfonamide resistance in Neisseria meningitidis. The gene encoding DHPS was linked to at least three other genes encoding enzymes of the folate pathway. These genes were in the order GTP cyclohydrolase, dihydropteroate synthase, dihydroneopterin aldolase, and hydroxymethyldihydropterin pyrophosphokinase. The nucleotide differences in genes from resistant and susceptible strains extended from the beginning of the GTP cyclohydrolase gene to the end of the gene encoding DHPS, an additional indication for gene transfer in the development of resistance. Kinetic measurements established different affinities for sulfathiazole for DHPS enzymes isolated from resistant and susceptible strains.

scholarly journals A Role for Ubiquitination in Mitochondrial Inheritance in Saccharomyces cerevisiae

1999 ◽  
Vol 145 (6) ◽  
pp. 1199-1208 ◽  
Author(s):  
Harold A. Fisk ◽  
Michael P. Yaffe
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Site Directed Mutagenesis ◽  
Temperature Sensitive ◽  
Mitochondrial Inheritance ◽  
Wild Type ◽  
Gene Encoding ◽  
Yeast Saccharomyces Cerevisiae ◽  
Protein Ubiquitination ◽  
Ubiquitin Ligase Activity ◽  
Mitochondrial Distribution

The smm1 mutation suppresses defects in mitochondrial distribution and morphology caused by the mdm1-252 mutation in the yeast Saccharomyces cerevisiae. Cells harboring only the smm1 mutation themselves display temperature-sensitive growth and aberrant mitochondrial inheritance and morphology at the nonpermissive temperature. smm1 maps to RSP5, a gene encoding an essential ubiquitin-protein ligase. The smm1 defects are suppressed by overexpression of wild-type ubiquitin but not by overexpression of mutant ubiquitin in which lysine-63 is replaced by arginine. Furthermore, overexpression of this mutant ubiquitin perturbs mitochondrial distribution and morphology in wild-type cells. Site-directed mutagenesis revealed that the ubiquitin ligase activity of Rsp5p is essential for its function in mitochondrial inheritance. A second mutation, smm2, which also suppressed mdm1-252 defects, but did not cause aberrant mitochondrial distribution and morphology, mapped to BUL1, encoding a protein interacting with Rsp5p. These results indicate that protein ubiquitination mediated by Rsp5p plays an essential role in mitochondrial inheritance, and reveal a novel function for protein ubiquitination.

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