Biology subjects

Puhar, A.

Publications and source records attributed to Puhar, A..

6 recordsLinked to original sources

bioRxiv2024

Antibacterial Compounds Against Non-Growing and Intracellular Bacteria

Slow- and non-growing bacterial populations, along with intracellular pathogens, often evade standard antibacterial treatments and are linked to persistent and recurrent infections. This necessitates the development of therapies specifically targeting nonproliferating bacteria. To identify compounds active against non-growing uropathogenic Escherichia coli (UPEC) we performed a drug-repurposing screen of 6,454 approved drugs and drug candidates. Using dilution-regrowth assays, we identified 39 compounds that either kill non-growing UPEC or delay its regrowth post-treatment. The hits include fluoroquinolones, macrolides, rifamycins, biguanide disinfectants, a pleuromutilin, and anti-cancer agents. 29 of the hits have not previously been recognized as active against non-growing bacteria. The hits were further tested against non-growing Pseudomonas aeruginosa and Staphylococcus aureus. Ten compounds - solithromycin, rifabutin, mitomycin C, and seven fluoroquinolones - have strong bactericidal activity against non-growing P. aeruginosa, killing >4 log10 of bacteria at 2.5 {micro}M. Solithromycin, valnemulin, evofosfamide, and satraplatin are unique in their ability to selectively target non-growing bacteria, exhibiting poor efficacy against growing bacteria. Finally, 31 hit compounds inhibit the growth of intracellular Shigella flexneri in a human enterocyte infection model, indicating their ability to permeate the cytoplasm of host cells. The identified compounds hold potential for treating persistent infections, warranting further comparative studies with current standard-of-care antibiotics.

microbiology↗

bioRxiv2024

Extracellular ATP is a signalling molecule in bacteria

In animals and plants extracellular ATP (eATP) functions as signal and regulates the immune response. During inflammation intestinal bacteria are exposed to elevated eATP originating from the mucosa. However, whether bacteria respond to eATP is unclear. Here we show that non-pathogenic Escherichia coli responds to eATP by modifying its transcriptional and metabolic landscapes. A genome-scale promoter library showed that the response is time-, concentration-, and medium-dependent and ATP-specific. The second messenger cAMP and genes related to metabolism, quorum sensing, and envelope stress were regulated downstream of eATP. Metabolomics confirmed that eATP triggers enrichment of compounds with bioactive properties on the host or bacteria. Combined genome-scale modelling revealed global metabolic and biomass building blocks modifications. Consequently, eATP altered the sensitivity to antibiotics and antimicrobial peptides. Finally, in pathogens eATP controlled virulence factor expression. Our results indicate that eATP is an environmental cue in prokaryotes which broadly regulates physiology, antimicrobial resistance, and virulence.

microbiology↗

bioRxiv2022

Parallel CRISPR-Cas guided mutagenesis of chromosome and virulence plasmid in Shigella flexneri by cytosine base editing

Shigella is a Gram-negative bacterium that invades the human gut epithelium. The resulting infection, shigellosis, is the deadliest bacterial diarrheal disease. Much of the information about the genes dictating the pathophysiology of Shigella, both on the chromosome and the virulence plasmid, was obtained by classical reverse genetics. However, technical limitations of the prevalent mutagenesis techniques restrict the generation of mutants in a single reaction to a small number, preventing large scale targeted mutagenesis of Shigella and the subsequent assessment of phenotype. We adopted a CRISPR-Cas dependent approach, where a nickase Cas9 and cytidine deaminase fusion is guided by sgRNA to introduce targeted C[->]T transitions, resulting in internal STOP codons and premature termination of translation. In proof-of-principle experiments using an mCherry fluorescent reporter, we were able to generate loss-of-function mutants in both E. coli and Shigella with up to 100% efficacy. Using a modified fluctuation assay, we determined that under optimized conditions, the frequency of untargeted mutations introduced by the Cas9-deaminase fusion is in the same range as spontaneous mutations, making our method a safe choice for bacterial mutagenesis. Further, we programmed the method to mutate well-characterized chromosomal and plasmid-borne Shigella genes and found the mutant phenotype to be similar to that of the reported gene deletion mutants, with no apparent polar effects at the phenotype level. This method can be used in a 96-well plate format to increase the throughput and generate an array of targeted loss-of-function mutants in a few days. ImportanceLoss-of-function mutagenesis is critical in understanding the physiological role of genes. Therefore, high-throughput techniques to generate such mutants are important for facilitating the assessment of gene function at a pace that matches system biology approaches. However, to our knowledge, no such method was available for generating an array of single gene mutants in an important enteropathogen - Shigella. This pathogen causes high morbidity and mortality in children and antibiotic resistant strains are quickly emerging. Therefore, determination of the function of unknown Shigella genes is of utmost importance to develop effective strategies to control infections. Our present work will bridge this gap by providing a rapid method for generating loss-of-function mutants. The highly effective and specific method has the potential to be programmed to generated multiple mutants in a single, massively parallel, reaction. By the virtue of plasmid compatibility, this method can be extended to other members of Enterobacteriaceae.

microbiology↗

bioRxiv2020

The Vibrio cholerae cytotoxin MakA induces noncanonical autophagy resulting in the spatial inhibition of canonical autophagy

Autophagy plays an essential role in the defence against many microbial pathogens as a regulator of both innate and adaptive immunity. Among some pathogens, sophisticated mechanisms have evolved that promote their ability to evade or subvert host autophagy. Here, we describe a novel mechanism of autophagy subversion mediated by the recently discovered Vibrio cholerae cytotoxin, MakA. pH-dependent endocytosis of MakA by host cells resulted in the formation of a cholesterol-rich endolysosomal membrane aggregate in the perinuclear region. Aggregate formation induced the noncanonical autophagy pathway driving unconventional LC3 lipidation on endolysosomal membranes. Subsequent sequestration of the ATG12-ATG5-ATG16L1 E3-like enzyme complex required for LC3 lipidation at the membranous aggregate resulted in an inhibition of both canonical autophagy and autophagy-related processes including the unconventional secretion of IL-1β. These findings identify a novel mechanism of host autophagy subversion and immune modulation employed by V. cholerae during bacterial infection.Competing Interest StatementThe authors have declared no competing interest.View Full Text

cell biology↗

bioRxiv2019

The generation of plasma cells and CD27-IgD- B cells during Hantavirus infection are associated with distinct pathological findings

Human hantavirus infections can cause hemorrhagic fever with renal syndrome (HFRS), major signs of the disease being thrombocytopenia and transient kidney dysfunction. By a comprehensive and longitudinal study of circulating B cells, we demonstrate that these two pathologies associate with distinct effects on the humoral immune system during HFRS. Low thrombocyte counts strongly associated with an abnormal frequency of plasmablasts in circulation, whereas kidney dysfunction was indicative of an accumulation of CD27- B cells and plasmablasts. Finally, we provide evidence that high levels of extracellular ATP in circulation during HFRS correlates with shedding of surface CD27 on B cells via a metallomatrix proteinase-8-mediated mechanism. Since extracellular ATP is known to regulate kidney function, our study reveals a link between kidney dysfunction and the generation of CD27-IgD- B cells, and a potential molecular target for treatment of the symptomatic phase of HFRS.

immunology↗

bioRxiv2019

Complete genome sequence and annotation of the laboratory reference strain Shigella flexneri serovar 5a M90T and genome-wide transcription start site determination

BackgroundShigella is a Gram-negative facultative intracellular bacterium that causes bacillary dysentery in humans. Shigella invades cells of the colonic mucosa owing to its virulence plasmid-encoded Type 3 Secretion System (T3SS), and multiplies in the target cell cytosol. Although the laboratory reference strain S. flexneri serotype 5a M90T has been extensively used to understand the molecular mechanisms of pathogenesis, its complete genome sequence is not available, thereby greatly limiting studies employing high-throughput sequencing and systems biology approaches.\n\nResultsWe have sequenced, assembled, annotated and manually curated the full genome of S. flexneri 5a M90T. This yielded two complete circular contigs, the chromosome and the virulence plasmid (pWR100). To obtain the genome sequence, we have employed long-read PacBio DNA sequencing followed by polishing with Illumina RNA-seq data. This provides a new pipeline to prepare gapless, highly accurate genome sequences. Furthermore, we have performed genome-wide analysis of transcriptional start sites and determined the length of 5 untranslated regions (5-UTRs) at typical culture conditions for the inoculum of in vitro infection experiments. We identified 6,723 primary TSS (pTSS) and 7,328 secondary TSS (sTSS). The S. flexneri 5a M90T annotated genome sequence and the transcriptional start sites are integrated into RegulonDB (http://regulondb.ccg.unam.mx) and RSAT (http://embnet.ccg.unam.mx/rsat/) to use its analysis tools in S. flexneri 5a M90T genome.\n\nConclusionsWe provide the first complete genome for S. flexneri serotype 5a, specifically the laboratory reference strain M90T. Our work opens the possibility of employing S. flexneri M90T in high-quality systems biology studies such as transcriptomic and differential expression analyses or in genome evolution studies. Moreover, the catalogue of TSS that we report here can be used in molecular pathogenesis studies as a resource to know which genes are transcribed before infection of host cells. The genome sequence, together with the analysis of transcriptional start sites, is also a valuable tool for precise genetic manipulation of S. flexneri 5a M90T. The hybrid pipeline that we report here combining genome sequencing with long-reads technology and polishing with RNAseq data defines a powerful strategy for genome assembly, polishing and annotation in any type of organism.

microbiology↗