Crystal structures of FolM alternative dihydrofolate reductase 1 from Brucella suis and Brucella canis

Members of the bacterial genus Brucella cause brucellosis, a zoonotic disease that affects both livestock and wildlife. Brucella are category B infectious agents that can be aerosolized for biological warfare. As part of the structural genomics studies at the Seattle Structural Genomics Center for Infectious Disease (SSGCID), FolM alternative dihydrofolate reductases 1 from Brucella suis and Brucella canis were produced and their structures are reported. The enzymes share ∼95% sequence identity but have less than 33% sequence identity to other homologues with known structure. The structures are prototypical NADPH-dependent short-chain reductases that share their highest tertiary-structural similarity with protozoan pteridine reductases, which are being investigated for rational therapeutic development.

Repurposing the Trypanosomatidic GSK Kinetobox for the Inhibition of Parasitic Pteridine and Dihydrofolate Reductases

Three open-source anti-kinetoplastid chemical boxes derived from a whole-cell phenotypic screening by GlaxoSmithKline (Tres Cantos Anti-Kinetoplastid Screening, TCAKS) were exploited for the discovery of a novel core structure inspiring new treatments of parasitic diseases targeting the trypansosmatidic pteridine reductase 1 (PTR1) and dihydrofolate reductase (DHFR) enzymes. In total, 592 compounds were tested through medium-throughput screening assays. A subset of 14 compounds successfully inhibited the enzyme activity in the low micromolar range of at least one of the enzymes from both Trypanosoma brucei and Lesihmania major parasites (pan-inhibitors), or from both PTR1 and DHFR-TS of the same parasite (dual inhibitors). Molecular docking studies of the protein–ligand interaction focused on new scaffolds not reproducing the well-known antifolate core clearly explaining the experimental data. TCMDC-143249, classified as a benzenesulfonamide derivative by the QikProp descriptor tool, showed selective inhibition of PTR1 and growth inhibition of the kinetoplastid parasites in the 5 μM range. In our work, we enlarged the biological profile of the GSK Kinetobox and identified new core structures inhibiting selectively PTR1, effective against the kinetoplastid infectious protozoans. In perspective, we foresee the development of selective PTR1 and DHFR inhibitors for studies of drug combinations.

Pressure Adaptations in Deep-Sea Moritella Dihydrofolate Reductases: Compressibility versus Stability

Proteins from “pressure-loving” piezophiles appear to adapt by greater compressibility via larger total cavity volume. However, larger cavities in proteins have been associated with lower unfolding pressures. Here, dihydrofolate reductase (DHFR) from a moderate piezophile Moritella profunda (Mp) isolated at ~2.9 km in depth and from a hyperpiezophile Moritella yayanosii (My) isolated at ~11 km in depth were compared using molecular dynamics simulations. Although previous simulations indicate that MpDHFR is more compressible than a mesophile DHFR, here the average properties and a quasiharmonic analysis indicate that MpDHFR and MyDHFR have similar compressibilities. A cavity analysis also indicates that the three unique mutations in MyDHFR are near cavities, although the cavities are generally similar in size in both. However, while a cleft overlaps an internal cavity, thus forming a pathway from the surface to the interior in MpDHFR, the unique residue Tyr103 found in MyDHFR forms a hydrogen bond with Leu78, and the sidechain separates the cleft from the cavity. Thus, while Moritella DHFR may generally be well suited to high-pressure environments because of their greater compressibility, adaptation for greater depths may be to prevent water entry into the interior cavities.

Adaptations for Pressure and Temperature in Dihydrofolate Reductases

Enzymes from extremophilic microbes that live in extreme conditions are generally adapted so that they function under those conditions, although adaptations for extreme temperatures and pressures can be difficult to unravel. Previous studies have shown mutation of Asp27 in Escherichia coli dihydrofolate reductase (DHFR) to Glu27 in Moritella profunda (Mp). DHFR enhances activity at higher pressures, although this may be an adaptation for cold. Interestingly, MpDHFR unfolds at ~70 MPa, while Moritella yayanosii (My) was isolated at depths corresponding to ~110 MPa, indicating that MyDHFR might be adapted for higher pressures. Here, these adaptations are examined using molecular dynamics simulations of DHFR from different microbes in the context of not only experimental studies of activity and stability of the protein but also the evolutionary history of the microbe. Results suggest Tyr103 of MyDHFR may be an adaptation for high pressure since Cys103 in helix F of MpDHFR forms an intra-helix hydrogen bond with Ile99 while Tyr103 in helix F of MyDHFR forms a hydrogen bond with Leu78 in helix E. This suggests the hydrogen bond between helices F and E in MyDHFR might prevent distortion at higher pressures.

The Bacterial Genomic Context of Highly Trimethoprim-Resistant DfrB Dihydrofolate Reductases Highlights an Emerging Threat to Public Health

Type B dihydrofolate reductase (dfrb) genes were identified following the introduction of trimethoprim in the 1960s. Although they intrinsically confer resistance to trimethoprim (TMP) that is orders of magnitude greater than through other mechanisms, the distribution and prevalence of these short (237 bp) genes is unknown. Indeed, this knowledge has been hampered by systematic biases in search methodologies. Here, we investigate the genomic context of dfrbs to gain information on their current distribution in bacterial genomes. Upon searching publicly available databases, we identified 61 sequences containing dfrbs within an analyzable genomic context. The majority (70%) of those sequences also harbor virulence genes and 97% of the dfrbs are found near a mobile genetic element, representing a potential risk for antibiotic resistance genes. We further identified and confirmed the TMP-resistant phenotype of two new members of the family, dfrb10 and dfrb11. Dfrbs are found both in Betaproteobacteria and Gammaproteobacteria, a majority (59%) being in Pseudomonas aeruginosa. Previously labelled as strictly plasmid-borne, we found 69% of dfrbs in the chromosome of pathogenic bacteria. Our results demonstrate that the intrinsically TMP-resistant dfrbs are a potential emerging threat to public health and justify closer surveillance of these genes.

Identification and characterization of two novel ISCR1-associated genes dfrA42 and dfrA43 encoding trimethoprim resistant dihydrofolate reductases

Two novel ISCR1-associated dfr genes, dfrA42 and dfrA43, were identified from trimethoprim (TMP)-resistant Proteus strains and were shown to confer high level TMP resistance (MIC ≥ 1024 mg/L) when cloned into Escherichia coli. These genes were hosted by complex class 1 integrons suggesting their potentials for dissemination. Analysis of enzymatic parameters and TMP affinity were performed, suggesting that the mechanism of TMP resistance for these novel DHFRs is the reduction of binding with TMP.

Genetic and phenotypic determinants of resistance to antibiotics in Aeromonas spp., strains isolated from pediatric patients

Introduction: Intestinal and extraintestinal infections by Aeromonas spp., remain controversial, due to the existence of healthy carriers of Aeromonas spp. In children under five years old, the diarrhea of infectious origin constitutes the second cause of mortality and remains a major concern for public health. The aim of this work was to detect the pheno/genotype of β-lactamases and class 1 integrons in Aeromonas spp., strains isolated from pediatric patients in a tertiary referral hospital in Mexico. Methodology: Sixty-six strains of Aeromonas spp., were isolated from clinical samples of pediatric origin and were identified by RFLP-PCR 16S rRNA. Resistance phenotype according to CLSI, genetic and phenotypic detection of extended-spectrum β-lactamases (ESBL) and metallo-b-lactamases (MBL) was performed. Finally, characterization of class 1 integrons was performed. Results: Aeromonas spp., strains of diarrheic origin were more predominant. A wide heterogeneity was detected, where A. caviae was the predominant specie. Second-generation cephalosporins, fluoroquinolones, and nitrofurans had best antimicrobial activity; moreover, antibiotics of the β-lactamic and lincosamides families showed lower inhibitory activity. Phenotypically, prevalences of 4.55% and 3.03% were detected for MBL (intestinal origin) and ESBL (extraintestinal origin), respectively. blaIMIS-cphA and blaTEM-1 genes, and nineteen class 1 integrons carrying two variants of cassettes corresponding to adenylyl transferases (aadA), and dihydrofolate reductases (dfrA). Monogenic array with aadA1 cassette was predominantly. Conclusions: ESBL and class 1 integrons, in Aeromonas collected from pediatric patients, determines a major detection challenge for the clinical microbiology laboratory and represents a remarkable epidemiological risk of nosocomial spread of multidrug-resistant determinants.

Commensal and virulent Escherichia coli strains of vaginal origin are reservoirs of resistance cassettes in class 1 integrons

Introduction: Antimicrobial resistance in Escherichia coli, one of the causal agents of aerobic vaginitis, leads to the persistence of the infection. The investigation of integrons acquires relevance, since they are elements that are responsible for the acquisition of resistance to antibiotics. The aim of this work was to describe the structural diversity of class 1 integrons in virulent and commensal strains of E. coli isolated from patients with vaginal infection. Methodology: Ninety-two strains of E. coli were isolated from patients with aerobic vaginitis. Resistance profile against 19 antibiotics and class 1 integrons were detected by PCR. The identity and arrangement of cassettes was determined by sequencing. ERIC-PCR assays were carried out in strains with identical arrays. Finally, genotyping by Clermont algorithm and serotyping were performed. Seventeen strains showed integrons located in plasmids. Results: Ten different gene cassette arrays were identified in 30 strains of E. coli. Cassettes corresponding to genes coding for adenylyltransferases (aadA), dihydrofolate reductases (dfrA), and oxacillinases (blaOXA) were detected. Array dfrA17-aadA5 was predominantly prevalent over the other arrays identified. Phylogenetic group A was the most predominant, followed by group B2 and D. Conclusions: This study demonstrates the presence of E. coli of vaginal origin carrying class 1 integrons, which are main genetic elements of capture of resistance genes, with the possibility of capturing new resistance cassettes. These evidences should serve for the modification of protocols in the diagnosis and treatment of aerobic vaginitis, and the development of policies for the rational use of antimicrobials.