RNase HI depletion strongly potentiates cell killing by rifampicin in mycobacteria

Multidrug resistant (MDR) tuberculosis (TB) is defined by the resistance of Mycobacterium tuberculosis, the causative organism, to the first-line antibiotics rifampicin and isoniazid. Mitigating or reversing resistance to these drugs offers a means of preserving and extending their use in TB treatment. R-loops are RNA/DNA hybrids that are formed in the genome during transcription, and can be lethal to the cell if not resolved. RNase HI is an enzyme that removes R-loops, and this activity is essential in M. tuberculosis: knockouts of rnhC, the gene encoding RNase HI, are non-viable. This essentiality supports it as a candidate target for the development of new antibiotics. In the model organism Mycolicibacterium smegmatis, RNase HI activity is provided by two RNase HI enzymes, RnhA and RnhC. We show that the partial depletion of RNase HI activity in M. smegmatis, by knocking out either of the genes encoding RnhA or RnhC, led to the accumulation of R-loops. The sensitivity of the knockout strains to the antibiotics moxifloxacin, streptomycin and rifampicin was increased, with sensitivity to the transcriptional inhibitor rifampicin strikingly increased by nearly 100-fold. We also show that R-loop accumulation accompanies partial transcriptional inhibition, suggesting a mechanistic basis for the synergy between RNase HI depletion and transcriptional inhibition. A model of how transcriptional inhibition can potentiate R-loop accumulation is presented. Finally, we identified four small molecules that inhibit recombinant RnhC activity and that also potentiated rifampicin activity in whole-cell assays against M. tuberculosis, supporting an on-target mode of action, and providing the first step in developing a new class of anti-mycobacterial drug.

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The fliK Gene Is Required for the Resistance of Bacillus thuringiensis to Antimicrobial Peptides and Virulence in Drosophila melanogaster

Frontiers in Microbiology ◽

10.3389/fmicb.2020.611220 ◽

2020 ◽

Vol 11 ◽

Author(s):

Zaynoun Attieh ◽

Carine Mouawad ◽

Agnès Rejasse ◽

Isabelle Jehanno ◽

Stéphane Perchat ◽

...

Keyword(s):

Bacillus Thuringiensis ◽

Antimicrobial Peptides ◽

Random Mutagenesis ◽

Essential Element ◽

Multidrug Resistant ◽

Gene Encoding ◽

Genes Encoding ◽

Molecular Ruler ◽

Flagella Assembly ◽

Bacteria Resistance

Antimicrobial peptides (AMPs) are essential effectors of the host innate immune system and they represent promising molecules for the treatment of multidrug resistant microbes. A better understanding of microbial resistance to these defense peptides is thus prerequisite for the control of infectious diseases. Here, using a random mutagenesis approach, we identify the fliK gene, encoding an internal molecular ruler that controls flagella hook length, as an essential element for Bacillus thuringiensis resistance to AMPs in Drosophila. Unlike its parental strain, that is highly virulent to both wild-type and AMPs deficient mutant flies, the fliK deletion mutant is only lethal to the latter’s. In agreement with its conserved function, the fliK mutant is non-flagellated and exhibits highly compromised motility. However, comparative analysis of the fliK mutant phenotype to that of a fla mutant, in which the genes encoding flagella proteins are interrupted, indicate that B. thuringiensis FliK-dependent resistance to AMPs is independent of flagella assembly. As a whole, our results identify FliK as an essential determinant for B. thuringiensis virulence in Drosophila and provide new insights on the mechanisms underlying bacteria resistance to AMPs.

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Adaptation of Porphyromonas gingivalis to microaerophilic conditions involves increased consumption of formate and reduced utilization of lactate

Microbiology ◽

10.1099/mic.0.027953-0 ◽

2009 ◽

Vol 155 (11) ◽

pp. 3758-3774 ◽

Cited By ~ 40

Author(s):

Janina P. Lewis ◽

Divya Iyer ◽

Cecilia Anaya-Bergman

Keyword(s):

Gene Expression ◽

Porphyromonas Gingivalis ◽

Model Organism ◽

Oxygen Metabolism ◽

Exponential Growth Phase ◽

Transcriptional Level ◽

Gene Encoding ◽

Aerobic Conditions ◽

Small Proteins ◽

Genes Encoding

Porphyromonas gingivalis, previously classified as a strict anaerobe, can grow in the presence of low concentrations of oxygen. Microarray analysis revealed alteration in gene expression in the presence of 6 % oxygen. During the exponential growth phase, 96 genes were upregulated and 79 genes were downregulated 1.4-fold. Genes encoding proteins that play a role in oxidative stress protection were upregulated, including alkyl hydroperoxide reductase (ahpCF), superoxide dismutase (sod) and thiol peroxidase (tpx). Significant changes in gene expression of proteins that mediate oxidative metabolism, such as cytochrome d ubiquinol oxidase-encoding genes, cydA and cydB, were detected. The expression of genes encoding formate uptake transporter (PG0209) and formate tetrahydrofolate ligase (fhs) was drastically elevated, which indicates that formate metabolism plays a major role under aerobic conditions. The concomitant reduction of expression of a gene encoding the lactate transporter PG1340 suggests decreased utilization of this nutrient. The concentrations of both formate and lactate were assessed in culture supernatants and cells, and they were in agreement with the results obtained at the transcriptional level. Also, genes encoding gingipain protease secretion/maturation regulator (porR) and protease transporter (porT) had reduced expression in the presence of oxygen, which also correlated with reduced protease activities under aerobic conditions. In addition, metal transport was affected, and while iron-uptake genes such as the genes encoding the haemin uptake locus (hmu) were downregulated, expression of manganese transporter genes, such as feoB2, was elevated in the presence of oxygen. Finally, genes encoding putative regulatory proteins such as extracellular function (ECF) sigma factors as well as small proteins had elevated expression levels in the presence of oxygen. As P. gingivalis is distantly related to the well-studied model organism Escherichia coli, results from our work may provide further understanding of oxygen metabolism and protection in other related bacteria belonging to the phylum Bacteroidetes.

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Co-Occurrence of Plasmid-Mediated AmpC β-Lactamase Activity Among Klebsiella pneumoniae and Escherichia Coli

The Open Microbiology Journal ◽

10.2174/1874285801711010195 ◽

2017 ◽

Vol 11 (1) ◽

pp. 195-202 ◽

Cited By ~ 6

Author(s):

Abdulaziz Zorgani ◽

Hiyam Daw ◽

Najib Sufya ◽

Abdullah Bashein ◽

Omar Elahmer ◽

...

Keyword(s):

Escherichia Coli ◽

Klebsiella Pneumoniae ◽

Multidrug Resistant ◽

Control Measures ◽

Gene Encoding ◽

E Coli ◽

Infection Control Measures ◽

Genes Encoding ◽

Polymerase Chain ◽

Variant Enzyme

Introduction: Extended-spectrum β-lactamases (ESBLs), including the AmpC type, are important mechanisms of resistance among Klebsiella pneumoniae and Escherichia coli isolates. Objective: The aim of the study was to investigate the occurrence of AmpC-type β-lactamase producers isolated from two hospitals in Tripoli, Libya. Methods: All clinical isolates (76 K. pneumoniae and 75 E. coli) collected over two years (2013-2014) were evaluated for susceptibility to a panel of antimicrobials and were analyzed phenotypically for the ESBL and AmpC phenotype using E-test and ESBL and AmpC screen disc test. Both ESBL and AmpC-positive isolates were then screened for the presence of genes encoding plasmid-mediated AmpC β-lactamases by polymerase chain reaction (PCR). Results: Of the K. pneumoniae and E. coli tested, 75% and 16% were resistant to gentamicin, 74% and 1.3% to imipenem, 71% and 12% to cefoxitin, 80% and 12% to cefepime, 69% and 22.6% to ciprofloxacin, respectively. None of the E. coli isolates were multidrug resistant compared with K. pneumoniae (65.8%). K. pneumoniae ESBL producers were significantly higher (85.5%) compared with (17.3%) E. coli isolates (P <0.0001, OR=4.93). Plasmid-mediated AmpC genes were detected in 7.9% of K. pneumoniae, and 4% E. coli isolates. There was low agreement between phenotypic and genotypic methods, phenotypic testing underestimated detection of AmpC enzyme and did not correlate well with molecular results. The gene encoding CMY enzyme was the most prevalent (66.6%) of AmpC positive isolates followed by MOX, DHA and EBC. Only one AmpC gene was detected in 5/9 isolates, i.e, blaCMY (n=3), bla MOX (n=1), blaDHA (n=1). However, co-occurrence of AmpC genes were evident in 3/9 isolates with the following distribution: bla CMY and blaEBC (n=1), and blaCMY and blaMOX (n=2). Neither blaFOX nor blaACC was detected in all tested isolates. All AmpC positive strains were resistant to cefoxitin and isolated from patients admitted to intensive care units. Conclusion: Further studies are needed for detection of other AmpC variant enzyme production among such isolates. Continued surveillance and judicious antibiotic usage together with the implementation of efficient infection control measures are absolutely required.

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Screening of Antimicrobial Resistance Genes and Epidemiological Features in Hospital and Community-Associated Carbapenem Resistant Pseudomonas aeruginosa Infections

10.21203/rs.3.rs-103545/v1 ◽

2020 ◽

Author(s):

ayse erturk ◽

Ayşegül Çopur ÇİÇEK ◽

Nebahat EJDER ◽

Uğur KOSTAKOĞLU ◽

İlknur Esen YILDIZ ◽

...

Keyword(s):

Resistance Genes ◽

Multidrug Resistant ◽

Gene Encoding ◽

Pfge Typing ◽

Carbapenem Resistant ◽

Antimicrobial Resistance Genes ◽

Genes Encoding ◽

Pcr Method ◽

Vitek 2 ◽

Hospital Acquired

Abstract Background: Researching carbapenem-resistant isolates and the use of antibiotics and following infection control policies enable the identification of carbapenemase-producing bacteria and prevent their spread.Methods: P. aeruginosa isolates were recovered from Medicine Faculty of Recep Tayyip Erdoğan University between April 2015 and October 2016 and identified by conventional methods and the automated Vitek 2 Compact (BioMerieux, France) system. Antimicrobial susceptibility experiments were performed in accordance with CLSI criteria and the automated Vitek 2 Compact system. The PCR method was investigated for the presence of β-lactamase resistance genes. PFGE typing was performed to show clonal relation among samples.Results: Seventy P. aeruginosa strains were isolated from seventy patients. The median age of 70 cases was found 66 with minimum 17 and maximum 92 years old. 67.1% of the patients had contact with the health service in the last 90 days and 75.7% of the patients had received antimicrobial therapy in the previous 90 days. The most common comorbidity was cardiovascular diseases. Twenty-four (34.3%) strains were carbapenem resistant, 2 strains were multidrug-resistant except colistin, and none of the samples had colistin resistance. The gene encoding β-lactamase or metallo-β-lactamase was found in a total of 36 strains. The bla VEB gene was identified in only 1 strain alone, but in combination with other resistance genes in a total of 17 strains. While the bla PER gene was detected in 5 samples alone, it was found in 13 samples in combination with other genes. Among the genes encoding metallo-β-lactamase, the most bla NDM positive was detected (n=22), followed by 14 positive samples of bla KPC . bla IMP and bla VIM were detected in 5 and 1 samples, respectively. Also, the association of bla VEB - bla PER and bla VEB - bla KPC - bla NDM was found to be very high. Much more resistance genes and associations were detected in hospital-acquired samples than community-acquired samples, both proportionally and in terms of co-occurrence. Most of the community-associated strains were collected in the F2 clade, while most of the hospital-associated strains were collected in the G1 clade. However, no difference was found between the community and hospital-associated strains according to PFGE results. Simultaneously, other microorganisms were also isolated from patients from which these 6 P. aeruginosa strains were isolated. Of these patients, 5 patients died, except the number 70.Conclusions: The median length of stay (days) was found to be significantly higher in the group with HAI than in the group with CAI. Compared to sample 28 and 37, which carried 5 β-lactamase coding genes, the death of these 5 patients with fewer or no resistance genes showed that the coexistence of other factors - especially other microorganisms in addition to resistance genes, was important.

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Biodegradation of the Organic Disulfide 4,4′-Dithiodibutyric Acid by Rhodococcus spp.

Applied and Environmental Microbiology ◽

10.1128/aem.02059-15 ◽

2015 ◽

Vol 81 (24) ◽

pp. 8294-8306 ◽

Cited By ~ 16

Author(s):

Heba Khairy ◽

Jan Hendrik Wübbeler ◽

Alexander Steinbüchel

Keyword(s):

Carbon Source ◽

Sole Carbon Source ◽

Rhodococcus Erythropolis ◽

Model Organism ◽

Hypothetical Protein ◽

Gene Encoding ◽

Content Type ◽

Gene Coding ◽

Initial Cleavage ◽

Genes Encoding

ABSTRACTFourRhodococcusspp. exhibited the ability to use 4,4′-dithiodibutyric acid (DTDB) as a sole carbon source for growth. The most important step for the production of a novel polythioester (PTE) using DTDB as a precursor substrate is the initial cleavage of DTDB. Thus, identification of the enzyme responsible for this step was mandatory. BecauseRhodococcus erythropolisstrain MI2 serves as a model organism for elucidation of the biodegradation of DTDB, it was used to identify the genes encoding the enzymes involved in DTDB utilization. To identify these genes, transposon mutagenesis ofR. erythropolisMI2 was carried out using transposon pTNR-TA. Among 3,261 mutants screened, 8 showed no growth with DTDB as the sole carbon source. In five mutants, the insertion locus was mapped either within a gene coding for a polysaccharide deacetyltransferase, a putative ATPase, or an acetyl coenzyme A transferase, 1 bp upstream of a gene coding for a putative methylase, or 176 bp downstream of a gene coding for a putative kinase. In another mutant, the insertion was localized between genes encoding a putative transcriptional regulator of the TetR family (noxR) and an NADH:flavin oxidoreductase (nox). Moreover, in two other mutants, the insertion loci were mapped within a gene encoding a hypothetical protein in the vicinity ofnoxRandnox. The interruption mutant generated,R. erythropolisMI2noxΩtsr, was unable to grow with DTDB as the sole carbon source. Subsequently,noxwas overexpressed and purified, and its activity with DTDB was measured. The specific enzyme activity of Nox amounted to 1.2 ± 0.15 U/mg. Therefore, we propose that Nox is responsible for the initial cleavage of DTDB into 2 molecules of 4-mercaptobutyric acid (4MB).

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The Prevalence of Virulence Determinants and Antibiotic Resistance Patterns in Methicillin—Resistant Staphylococcus aureus in a Nursing Home in Poland

Pathogens ◽

10.3390/pathogens10040427 ◽

2021 ◽

Vol 10 (4) ◽

pp. 427

Author(s):

Martyna Kasela ◽

Agnieszka Grzegorczyk ◽

Bożena Nowakowicz-Dębek ◽

Anna Malm

Keyword(s):

Staphylococcus Aureus ◽

Methicillin Resistant Staphylococcus Aureus ◽

Variable Number Tandem Repeat ◽

Multidrug Resistant ◽

Variable Number ◽

Pulse Field ◽

Virulence Determinants ◽

Gene Encoding ◽

Methicillin Resistant ◽

Pulse Field Gel Electrophoresis

Nursing homes (NH) contribute to the regional spread of methicillin-resistant Staphylococcus aureus (MRSA). Moreover, residents are vulnerable to the colonization and subsequent infection of MRSA etiology. We aimed at investigating the molecular and phenotypic characteristics of 21 MRSA collected from the residents and personnel in an NH (Lublin, Poland) during 2018. All MRSA were screened for 20 genes encoding virulence determinants (sea-see, eta, etb, tst, lukS-F-PV, eno, cna, ebpS, fib, bbp, fnbA, fnbB, icaADBC) and for resistance to 18 antimicrobials. To establish the relatedness and clonal complexes of MRSA in NH we applied multiple-locus variable-number tandem-repeat fingerprinting (MLVF), pulse field gel electrophoresis (PFGE), multilocus sequence typing (MLST) and staphylococcal cassette chromosome mec (SCCmec) typing. We identified four sequence types (ST) among two clonal complexes (CC): ST (CC22) known as EMRSA-15 as well as three novel STs—ST6295 (CC8), ST6293 (CC8) and ST6294. All tested MRSA were negative for sec, eta, etb, lukS-F-PV, bbp and ebpS genes. The most prevalent gene encoding toxin was sed (52.4%; n = 11/21), and adhesins were eno and fnbA (100%). Only 9.5% (n = 2/21) of MRSA were classified as multidrug-resistant. The emergence of novel MRSA with a unique virulence and the presence of epidemic clone EMRSA-15 creates challenges for controlling the spread of MRSA in NH.

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DIPG-41. DISSECTING THE MECHANISTIC BASIS FOR ACVR1 AND PIK3CA MUTATION CO-OCCURRENCE IN DIFFUSE MIDLINE GLIOMAS USING GENETICALLY ENGINEERED MOUSE MODELS

Neuro-Oncology ◽

10.1093/neuonc/noaa222.088 ◽

2020 ◽

Vol 22 (Supplement_3) ◽

pp. iii295-iii295

Author(s):

Annette Wu ◽

Tak Mak ◽

Jerome Fortin

Keyword(s):

Mouse Models ◽

Pik3ca Mutation ◽

Cell Lineage ◽

Gene Expression Signature ◽

Genetically Engineered ◽

Genetically Engineered Mouse Models ◽

Dismal Prognosis ◽

Human Pathology ◽

Genes Encoding ◽

Mechanistic Basis

Abstract Diffuse midline gliomas (DMGs) are aggressive childhood brain tumors with a dismal prognosis. Most of these tumors carry K27M mutations in histone H3-encoding genes, particularly H3F3A and HIST1H3B. In addition, activating mutations in ACVR1 and PIK3CA co-occur in a subset of DMGs. To understand how these lesions drive the development of DMGs, we generated genetically engineered mouse models in which Acvr1G328V, Hist1h3bK27M, and Pik3caH1047R are targeted to the OLIG2-expressing cell lineage. Animals carrying Acvr1G328V and Pik3caH1047R, with (“AHPO”) or without (“APO”) Hist1h3bK27M, developed high-grade diffuse gliomas involving midline and forebrain regions. Neither Acvr1G328V nor Pik3caH1047R drove tumorigenesis by themselves, but Acvr1G328V was sufficient to cause oligodendroglial differentiation arrest, pointing to a role in the earliest stages of gliomas formation. Transcriptomic analyses of AHPO and APO tumors indicated a predominantly proneural and oligodendrocyte precursor-like gene expression signature, consistent with the corresponding human pathology. Genes encoding transcription factors (TFs) with dual roles in controlling glial and neuronal differentiation were upregulated in tumors. Some of these genes were mildly induced by Acvr1G328V alone. Functional experiments using CRISPR/Cas9-mediated gene editing in patient-derived cell lines confirmed a role for some of these TFs in controlling DMG cell fitness. Overall, our results suggest that Pik3caH1047R consolidates Acvr1G328V-induced glial differentiation arrest to drive DMG development and progression.

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Expression and function of the cdgD gene, encoding a CHASE–PAS-DGC-EAL domain protein, in Azospirillum brasilense

Scientific Reports ◽

10.1038/s41598-020-80125-3 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

José Francisco Cruz-Pérez ◽

Roxana Lara-Oueilhe ◽

Cynthia Marcos-Jiménez ◽

Ricardo Cuatlayotl-Olarte ◽

María Luisa Xiqui-Vázquez ◽

...

Keyword(s):

Azospirillum Brasilense ◽

Type Strain ◽

Wild Type Strain ◽

Soil Conditions ◽

Wild Type ◽

Gene Encoding ◽

Wheat Roots ◽

Genes Encoding ◽

In The Wild ◽

Plant Growth Promoting

AbstractThe plant growth-promoting bacterium Azospirillum brasilense contains several genes encoding proteins involved in the biosynthesis and degradation of the second messenger cyclic-di-GMP, which may control key bacterial functions, such as biofilm formation and motility. Here, we analysed the function and expression of the cdgD gene, encoding a multidomain protein that includes GGDEF-EAL domains and CHASE and PAS domains. An insertional cdgD gene mutant was constructed, and analysis of biofilm and extracellular polymeric substance production, as well as the motility phenotype indicated that cdgD encoded a functional diguanylate protein. These results were correlated with a reduced overall cellular concentration of cyclic-di-GMP in the mutant over 48 h compared with that observed in the wild-type strain, which was recovered in the complemented strain. In addition, cdgD gene expression was measured in cells growing under planktonic or biofilm conditions, and differential expression was observed when KNO3 or NH4Cl was added to the minimal medium as a nitrogen source. The transcriptional fusion of the cdgD promoter with the gene encoding the autofluorescent mCherry protein indicated that the cdgD gene was expressed both under abiotic conditions and in association with wheat roots. Reduced colonization of wheat roots was observed for the mutant compared with the wild-type strain grown in the same soil conditions. The Azospirillum-plant association begins with the motility of the bacterium towards the plant rhizosphere followed by the adsorption and adherence of these bacteria to plant roots. Therefore, it is important to study the genes that contribute to this initial interaction of the bacterium with its host plant.

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Structure, expression, chromosomal location and product of the gene encoding Adh2 in Petunia.

Genetics ◽

10.1093/genetics/133.4.999 ◽

1993 ◽

Vol 133 (4) ◽

pp. 999-1007

Author(s):

R G Gregerson ◽

L Cameron ◽

M McLean ◽

P Dennis ◽

J Strommer

Keyword(s):

Chromosomal Location ◽

Higher Plants ◽

Gene Encoding ◽

Genomic Libraries ◽

Regulatory Strategy ◽

Adh1 Gene ◽

Adh Genes ◽

Genes Encoding ◽

Adh Gene ◽

Polymerase Chain

Abstract In most higher plants the genes encoding alcohol dehydrogenase comprise a small gene family, usually with two members. The Adh1 gene of Petunia has been cloned and analyzed, but a second identifiable gene was not recovered from any of three genomic libraries. We have therefore employed the polymerase chain reaction to obtain the major portion of a second Adh gene. From sequence, mapping and northern data we conclude this gene encodes ADH2, the major anaerobically inducible Adh gene of Petunia. The availability of both Adh1 and Adh2 from Petunia has permitted us to compare their structures and patterns of expression to those of the well-studied Adh genes of maize, of which one is highly expressed developmentally, while both are induced in response to hypoxia. Despite their evolutionary distance, evidenced by deduced amino acid sequence as well as taxonomic classification, the pairs of genes are regulated in strikingly similar ways in maize and Petunia. Our findings suggest a significant biological basis for the regulatory strategy employed by these distant species for differential expression of multiple Adh genes.

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Non-Syndromic Dentinogenesis Imperfecta Caused by Mild Mutations in COL1A2

Journal of Personalized Medicine ◽

10.3390/jpm11060526 ◽

2021 ◽

Vol 11 (6) ◽

pp. 526

Author(s):

Yejin Lee ◽

Youn Jung Kim ◽

Hong-Keun Hyun ◽

Jae-Cheoun Lee ◽

Zang Hee Lee ◽

...

Keyword(s):

Collagen Type ◽

Molecular Genetic ◽

Collagen Type I ◽

Type I ◽

Dentinogenesis Imperfecta ◽

Gene Encoding ◽

Korean Families ◽

Whole Exome ◽

Genes Encoding ◽

Candidate Gene Sequencing

Hereditary dentin defects can be categorized as a syndromic form predominantly related to osteogenesis imperfecta (OI) or isolated forms without other non-oral phenotypes. Mutations in the gene encoding dentin sialophosphoprotein (DSPP) have been identified to cause dentinogenesis imperfecta (DGI) Types II and III and dentin dysplasia (DD) Type II. While DGI Type I is an OI-related syndromic phenotype caused mostly by monoallelic mutations in the genes encoding collagen type I alpha 1 chain (COL1A1) and collagen type I alpha 2 chain (COL1A2). In this study, we recruited families with non-syndromic dentin defects and performed candidate gene sequencing for DSPP exons and exon/intron boundaries. Three unrelated Korean families were further analyzed by whole-exome sequencing due to the lack of the DSPP mutation, and heterozygous COL1A2 mutations were identified: c.3233G>A, p.(Gly1078Asp) in Family 1 and c.1171G>A, p.(Gly391Ser) in Family 2 and 3. Haplotype analysis revealed different disease alleles in Families 2 and 3, suggesting a mutational hotspot. We suggest expanding the molecular genetic etiology to include COL1A2 for isolated dentin defects in addition to DSPP.

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