The acquisition of clinically relevant amoxicillin resistance in Streptococcus pneumoniae requires ordered horizontal gene transfer of four loci

Understanding how antimicrobial resistance spreads is critical for optimal application of new treatments. In the naturally competent human pathogen Streptococcus pneumoniae, resistance to β-lactam antibiotics is mediated by recombination events in genes encoding the target proteins, resulting in reduced drug binding affinity. However, for the front-line antibiotic amoxicillin, the exact mechanism of resistance still needs to be elucidated. Through successive rounds of transformation with genomic DNA from a clinically resistant isolate, we followed amoxicillin resistance development. Using whole genome sequencing, we showed that multiple recombination events occurred at different loci during one round of transformation. We found examples of non-contiguous recombination, and demonstrated for the first time that this can occur through multiple D-loop formation from one donor DNA molecule or by the uptake of multiple DNA fragments. We also show that the final minimum inhibitory concentration differs depending on recipient genome, and the sampled fitness landscape. Finally, through back transformations of mutant alleles and fluorescently labelled penicillin (bocillin-FL) binding assays, we show that pbp1a, pbp2b, pbp2x, and murM are the main resistance determinants for amoxicillin resistance, and that the order of allele uptake matters. We conclude that recombination events are complex, and that this complexity contributes to the highly diverse genotypes of amoxicillin-resistant pneumococcal isolates.

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Single Amino Acid Based Self-Assemblies of Cysteine and Methionine

10.26434/chemrxiv.5972173.v1 ◽

2018 ◽

Author(s):

Nidhi Gour ◽

Bharti Koshti ◽

Chandra Kanth P. ◽

Dhruvi Shah ◽

Vivek Shinh Kshatriya ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Self Assembly ◽

Aromatic Amino Acids ◽

Neutral Condition ◽

Single Amino Acid ◽

Binding Assays ◽

Human Neuroblastoma ◽

Cytotoxicity Assays ◽

First Time

We report for the very first time self-assembly of Cysteine and Methionine to discrenible strucutres under neutral condition. To get insights into the structure formation, thioflavin T and Congo red binding assays were done which revealed that aggregates may not have amyloid like characteristics. The nature of interactions which lead to such self-assemblies was purported by coincubating assemblies in urea and mercaptoethanol. Further interaction of aggregates with short amyloidogenic dipeptide diphenylalanine (FF) was assessed. While cysteine aggregates completely disrupted FF fibres, methionine albeit triggered fibrillation. The cytotoxicity assays of cysteine and methionine structures were performed on Human Neuroblastoma IMR-32 cells which suggested that aggregates are not cytotoxic in nature and thus, may not have amyloid like etiology. The results presented in the manuscript are striking, since to the best of our knowledge,this is the first report which demonstrates that even non-aromatic amino acids (cysteine and methionine) can undergo spontaneous self-assembly to form ordered aggregates.

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The Assessment of IL-21 and IL-22 at the mRNA Level in Tumor Tissue and Protein Concentration in Serum and Peritoneal Fluid in Patients with Ovarian Cancer

Journal of Clinical Medicine ◽

10.3390/jcm10143058 ◽

2021 ◽

Vol 10 (14) ◽

pp. 3058

Author(s):

Aleksandra Mielczarek-Palacz ◽

Celina Kruszniewska-Rajs ◽

Marta Smycz-Kubańska ◽

Jarosław Strzelczyk ◽

Wojciech Szanecki ◽

...

Keyword(s):

Ovarian Cancer ◽

Peritoneal Fluid ◽

Tumor Tissue ◽

Mrna Level ◽

Enzyme Linked Immunosorbent Assay ◽

Control Group ◽

Expression Of Genes ◽

Genes Encoding ◽

First Time ◽

Ovarian Serous Cancer

The aim of the analysis was for the first time to assess the expression of genes encoding IL-21 and IL-22 at the mRNA level in ovarian tumor specimens and the concentration of these parameters in serum and peritoneal fluid in patients with ovarian serous cancer. The levels of IL-21 and IL-22 transcripts were evaluated with the use of the real-time RT-qPCR. Enzyme-linked immunosorbent assay (ELISA) was used to determine the concentration of proteins. Quantitative analysis of IL-21 gene mRNA in the tumor tissue showed the highest activity in the G1 degree of histopathological differentiation and was higher in G1 compared to the control group. The concentration of IL-21 and IL-22 in the serum and in the peritoneal fluid of women with ovarian cancer varied depending on the degree of histopathological differentiation of the cancer and showed statistical variability compared to controls. The conducted studies have shown that the local and systemic changes in the immune system involving IL-21 and IL-22 indicate the participation of these parameters in the pathogenesis of ovarian cancer, and modulation in the IL-21/IL-22 system may prove useful in the development of new diagnostic and therapeutic strategies used in patients, which require further research.

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Expression of BARD1 β Isoform in Selected Pediatric Tumors

Genes ◽

10.3390/genes12020168 ◽

2021 ◽

Vol 12 (2) ◽

pp. 168

Author(s):

Anna Jasiak ◽

Natalia Krawczyńska ◽

Mariola Iliszko ◽

Katarzyna Czarnota ◽

Kamil Buczkowski ◽

...

Keyword(s):

Pediatric Cancer ◽

Genome Stability ◽

Cell Cycle Control ◽

Reverse Transcriptase Gene ◽

Neoplastic Tissue ◽

Adult Type ◽

Qpcr Method ◽

New Treatments ◽

First Time ◽

Work Supports

Currently, many new possible biomarkers and mechanisms are being searched and tested to analyse pathobiology of pediatric tumours for the development of new treatments. One such candidate molecular factor is BARD1 (BRCA1 Associated RING Domain 1)—a tumour-suppressing gene involved in cell cycle control and genome stability, engaged in several types of adult-type tumours. The data on BARD1 significance in childhood cancer is limited. This study determines the expression level of BARD1 and its isoform beta (β) in three different histogenetic groups of pediatric cancer—neuroblastic tumours, and for the first time in chosen germ cell tumours (GCT), and rhabdomyosarcoma (RMS), using the qPCR method. We found higher expression of beta isoform in tumour compared to healthy tissue with no such changes concerning BARD1 full-length. Additionally, differences in expression of BARD1 β between histological types of neuroblastic tumours were observed, with higher levels in ganglioneuroblastoma and ganglioneuroma. Furthermore, a higher expression of BARD1 β characterized yolk sac tumours (GCT type) and RMS when comparing with non-neoplastic tissue. These tumours also showed a high expression of the TERT (Telomerase Reverse Transcriptase) gene. In two RMS cases we found deep decrease of BARD1 β in post-chemotherapy samples. This work supports the oncogenicity of the beta isoform in pediatric tumours, as well as demonstrates the differences in its expression depending on the histological type of neoplasm, and the level of maturation in neuroblastic tumours.

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Designing P. aeruginosa synthetic phages with reduced genomes

Scientific Reports ◽

10.1038/s41598-021-81580-2 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Diana P. Pires ◽

Rodrigo Monteiro ◽

Dalila Mil-Homens ◽

Arsénio Fialho ◽

Timothy K. Lu ◽

...

Keyword(s):

Galleria Mellonella ◽

Antibacterial Properties ◽

Genetically Engineered ◽

In Vivo Studies ◽

Infection Model ◽

Promising Therapeutic Approach ◽

Genes Encoding ◽

First Time

AbstractIn the era where antibiotic resistance is considered one of the major worldwide concerns, bacteriophages have emerged as a promising therapeutic approach to deal with this problem. Genetically engineered bacteriophages can enable enhanced anti-bacterial functionalities, but require cloning additional genes into the phage genomes, which might be challenging due to the DNA encapsulation capacity of a phage. To tackle this issue, we designed and assembled for the first time synthetic phages with smaller genomes by knocking out up to 48% of the genes encoding hypothetical proteins from the genome of the newly isolated Pseudomonas aeruginosa phage vB_PaeP_PE3. The antibacterial efficacy of the wild-type and the synthetic phages was assessed in vitro as well as in vivo using a Galleria mellonella infection model. Overall, both in vitro and in vivo studies revealed that the knock-outs made in phage genome do not impair the antibacterial properties of the synthetic phages, indicating that this could be a good strategy to clear space from phage genomes in order to enable the introduction of other genes of interest that can potentiate the future treatment of P. aeruginosa infections.

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Genetic Relatedness of the Streptococcus pneumoniae Capsular Biosynthetic Loci

Journal of Bacteriology ◽

10.1128/jb.00836-07 ◽

2007 ◽

Vol 189 (21) ◽

pp. 7841-7855 ◽

Cited By ~ 84

Author(s):

Angeliki Mavroidi ◽

David M. Aanensen ◽

Daniel Godoy ◽

Ian C. Skovsted ◽

Margit S. Kaltoft ◽

...

Keyword(s):

Streptococcus Pneumoniae ◽

Genetic Relatedness ◽

Pneumococcal Serotypes ◽

Major Cluster ◽

Genes Encoding ◽

Pneumococcal Serotype ◽

Streptococcal Polysaccharide ◽

Single Cluster ◽

Dependent Pathway ◽

Sequence Similarities

ABSTRACT Streptococcus pneumoniae (the pneumococcus) produces 1 of 91 capsular polysaccharides (CPS) that define the serotype. The cps loci of 88 pneumococcal serotypes whose CPS is synthesized by the Wzy-dependent pathway were compared with each other and with additional streptococcal polysaccharide biosynthetic loci and were clustered according to the proportion of shared homology groups (HGs), weighted for the sequence similarities between the genes encoding the shared HGs. The cps loci of the 88 pneumococcal serotypes were distributed into eight major clusters and 21 subclusters. All serotypes within the same serogroup fell into the same major cluster, but in six cases, serotypes within the same serogroup were in different subclusters and, conversely, nine subclusters included completely different serotypes. The closely related cps loci within a subcluster were compared to the known CPS structures to relate gene content to structure. The Streptococcus oralis and Streptococcus mitis polysaccharide biosynthetic loci clustered within the pneumococcal cps loci and were in a subcluster that also included the cps locus of pneumococcal serotype 21, whereas the Streptococcus agalactiae cps loci formed a single cluster that was not closely related to any of the pneumococcal cps clusters.

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Phosphorylation of MgrA and Its Effect on Expression of the NorA and NorB Efflux Pumps of Staphylococcus aureus

Journal of Bacteriology ◽

10.1128/jb.00018-10 ◽

2010 ◽

Vol 192 (10) ◽

pp. 2525-2534 ◽

Cited By ~ 55

Author(s):

Que Chi Truong-Bolduc ◽

David C. Hooper

Keyword(s):

Staphylococcus Aureus ◽

Multidrug Resistance ◽

Virulence Factors ◽

Double Mutant ◽

Efflux Pumps ◽

Efflux Transporters ◽

Global Regulator ◽

Binding Assays ◽

Genes Encoding ◽

Gel Shift

ABSTRACT MgrA is a global regulator in Staphylococcus aureus that controls the expression of diverse genes encoding virulence factors and multidrug resistance (MDR) efflux transporters. We identified pknB, which encodes the (Ser/Thr) kinase PknB, in the S. aureus genome. PknB was able to autophosphorylate as well as phosphorylate purified MgrA. We demonstrated that rsbU, which encodes a Ser/Thr phosphatase and is involved in the activation of the SigB regulon, was able to dephosphorylate MgrA-P but not PknB-P. Serines 110 and 113 of MgrA were found to be phosphorylated, and Ala substitutions at these positions resulted in reductions in the level of phosphorylation of MgrA. DNA gel shift binding assays using norA and norB promoters showed that MgrA-P was able to bind the norB promoter but not the norA promoter, a pattern which was the reverse of that for unphosphorylated MgrA. The double mutant MgrAS110A-S113A bound to the norA promoter but not the norB promoter. The double mutant led to a 2-fold decrease in norA transcripts and a 2-fold decrease in the MICs of norfloxacin and ciprofloxacin in strain RN6390. Thus, phosphorylation of MgrA results in loss of binding to the norA promoter, but with a gain of the ability to bind the norB promoter. Loss of the ability to phosphorylate MgrA by Ala substitution resulted in increased repression of norA expression and in reductions in susceptibilities to NorA substrates.

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Protein binding to a single termination-associated sequence in the mitochondrial DNA D-loop region.

Molecular and Cellular Biology ◽

10.1128/mcb.13.4.2162 ◽

1993 ◽

Vol 13 (4) ◽

pp. 2162-2171 ◽

Cited By ~ 66

Author(s):

C S Madsen ◽

S C Ghivizzani ◽

W W Hauswirth

Keyword(s):

Mitochondrial Dna ◽

Protein Binding ◽

Loop Formation ◽

Sequence Element ◽

Conserved Sequence ◽

Loop Region ◽

In Organello ◽

Close Proximity ◽

D Loop

A methylation protection assay was used in a novel manner to demonstrate a specific bovine protein-mitochondrial DNA (mtDNA) interaction within the organelle (in organello). The protected domain, located near the D-loop 3' end, encompasses a conserved termination-associated sequence (TAS) element which is thought to be involved in the regulation of mtDNA synthesis. In vitro footprinting studies using a bovine mitochondrial extract and a series of deleted mtDNA templates identified a approximately 48-kDa protein which binds specifically to a single TAS element also protected within the mitochondrion. Because other TAS-like elements located in close proximity to the protected region did not footprint, protein binding appears to be highly sequence specific. The in organello and in vitro data, together, provide evidence that D-loop formation is likely to be mediated, at least in part, through a trans-acting factor binding to a conserved sequence element located 58 bp upstream of the D-loop 3' end.

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Non-denaturing bisulfite treatment and single-molecule real-time sequencing reveals d-loop footprints, their length, position and distribution

10.1101/2020.05.20.106617 ◽

2020 ◽

Cited By ~ 1

Author(s):

Shanaya Shital Shah ◽

Stella Hartono ◽

Frédéric Chédin ◽

Wolf-Dietrich Heyer

Keyword(s):

Real Time ◽

Single Molecule ◽

Detection Methods ◽

Loop Formation ◽

Bisulfite Treatment ◽

Loop Detection ◽

D Loop ◽

Donor Choice ◽

The Relationship

ABSTRACTDisplacement loops (D-loops) are signature intermediates formed during homologous recombination. Numerous factors regulate D-loop formation and disruption, thereby influencing crucial aspects of DNA repair, including donor choice and the possibility of a crossover outcome. While D-loop detection methods exist, it is currently unfeasible to assess the relationship between D-loop editors and D-loop characteristics such as length and position. Here, we developed a novel in vitro assay to characterize the length and position of individual D-loop with base-pair resolution and deep coverage, while also revealing their distribution in a population. Non-denaturing bisulfite treatment modifies the cytosines on the displaced strand of the D-loop to uracil, leaving a permanent signature for the displaced strand. Subsequent single-molecule real-time sequencing uncovers the cytosine conversion patch as a D-loop footprint, revealing D-loop characteristics at unprecedented resolution. The D-loop Mapping Assay is widely applicable with different substrates and donor types and can be used to study factors that influence D-loop properties.

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THE PRODUCTION OF MONOCLONAL ANTIBODIES TO TETRASACCHARIDE - SYNTHETIC ANALOGUE OF THE CAPSULAR POLYSACCHARIDE OF STREPTOCOCCUS PNEUMONIAE OF SEROTYPE 14 AND THEIR IMMUNOCHEMICAL CHARACTERIZATION

Journal of microbiology epidemiology immunobiology ◽

10.36233/0372-9311-2018-5-26-31 ◽

2018 ◽

pp. 26-31

Author(s):

I. V. Yakovleva ◽

E. A. Kurbatova ◽

E. A. Akhmatova ◽

E. V. Sukhova ◽

D. V. Yashunsky ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Streptococcus Pneumoniae ◽

Capsular Polysaccharide ◽

Solid Phase ◽

Synthetic Analogue ◽

Immunochemical Characterization ◽

Carbohydrate Epitopes ◽

First Time

Aim. Production of monoclonal antibodies (mAb) to synthetic tetrasaccharide - repeating unit of the capsular polysaccharide (CP) of Streptococcus pneumoniae serotype 14 and their immunochemical characterization. Materials and methods. In order to generate the hybridoma producing mAb, mice were immunized with synthetic tetrasaccharide conjugated with bovine serum albumin (BSA) with following hybridization of B lymphocytes with mouse myeloma cells. Antibodies were obtained in vitro andin vivo. Immunochemical characterization of mAb to tetrasaccharide was carried out using a variety of ELISA options. Results. For the first time obtained mouse hybridoma, producing IgM to tetrasacchride. The IgM titer of anti-tetrasacharide antibodies in supernatants of clones and in the ascitic fluid of mice in ELISA detected by biotinylated tetrasaccharide and synthetic CP adsorbed on the solid phase was higher compared to the use of bacterial CP as well cover antigen. In the reaction of inhibition of the ELISA, the mAb recognized the corresponding carbohydrate epitopes of the bacterial CP of S. pneumoniae serotype 14 dissolved in the liquid phase better than tetrasaccharide ligand and synthetic CP. Conclusion. To detect mAb to tetrasaccharide in ELISA preferably to use synthetic analogues of the CP as solid phase antigens. The obtained mAb to tetrasaccharide can be used to determine the representation of the protective tetrasaccharide epitope of CP in the development of pneumococcal vaccines.

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Ribosome profiling in Streptococcus pneumoniae reveals the role of methylation of 23S rRNA nucleotide G748 on ribosome stalling

10.1101/2020.12.21.423859 ◽

2020 ◽

Author(s):

Tatsuma Shoji ◽

Akiko Takaya ◽

Yoko Kusuya ◽

Hiroki Takahashi ◽

Hiroto Kawashima

Keyword(s):

Streptococcus Pneumoniae ◽

Ribosome Profiling ◽

23S Rrna ◽

Ribosome Stalling ◽

70S Ribosome ◽

Exit Tunnel ◽

Species Specific ◽

Peptidyltransferase Center ◽

First Time

2.Abstract(1) BackgroundMany nucleotides in 23S rRNA are methylated post-transcriptionally by methyltransferases and cluster around the peptidyltransferase center (PTC) and the nascent peptidyl exit tunnel (NPET) located in 50S subunit of 70S ribosome. Biochemical interactions between a nascent peptide and the tunnel may stall ribosome movement and affect expression levels of the protein. However, no studies have shown a role for NPET on ribosome stalling using an NPET mutant.(2) ResultsA ribosome profiling assay in Streptococcus pneumoniae demonstrates for the first time that an NPET mutant exhibits completely different ribosome occupancy compared to wild-type. We demonstrate, using RNA footprinting, that changes in ribosome occupancy correlate with changes in ribosome stalling. Further, statistical analysis shows that short peptide sequences that cause ribosome stalling are species-specific and evolutionarily selected. NPET structure is required to realize these specie-specific ribosome stalling.(3) ConclusionsResults support the role of NPET on ribosome stalling. NPET structure is required to realize the species-specific and evolutionary conserved ribosome stalling. These findings clarify the role of NPET structure on the translation process.

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