Biology subjects

Givskov, M.

Publications and source records attributed to Givskov, M..

9 recordsLinked to original sources

bioRxiv2025

Colorectal Cancer-Associated Streptococcus gallolyticus: A Hidden Diversity Exposed

Streptococcus gallolyticus subsp. gallolyticus (SGG) is a bacterial pathogen implicated in bacteremia and endocarditis, and is often associated with colon tumors in elderly individuals. The development of colorectal cancer (CRC) has been linked to intestinal dysbiosis, characterized by increased proportions of SGG and other intestinal microbes. In this study, we present the complete nucleotide sequence of five novel clinical isolates of SGG associated with colorectal cancer, revealing unexpected genetic diversity. Sequencing an additional 30 SGG clinical isolates provided a more comprehensive description of this genetic diversity. We did not identify a pathogenicity island specific to CRC-associated SGG isolates. Most of these human-derived SGG isolates exhibit resistance to multiple antibiotics. Our findings also offer additional insights into multilocus sequence typing (MLST), capsular loci, and pilus organization. Analysis of the repertoire of surface proteins reveals high potential for binding and foraging complex polysaccharides. Finally, comparative genomics with the phylogenetically closest non-pathogenic subspecies S. gallolyticus subps. macedonicus, confirmed that SGG pathogenicity-associated factors mostly rely on a large repertoire of surface proteins involved in host colonization, presence of C5a peptidase to avoid innate immunity, bile salt hydrolase to persist in the gut, and of specific bacteriocin and type VII-dependent effectors to colonize the host colon. Additionally, the presence of extracellular polysaccharides in SGG probably helps the bacterium survive in harsher conditions. ImportanceStreptococcus gallolyticus subsp. gallolyticus (SGG) was the first intestinal bacterium associated with colorectal cancer. It is now widely accepted that colonic microbiota dysbiosis contributes to oncogenesis, with a higher relative abundance of several potentially pro-carcinogenic bacteria. For example, the oncogenic role of Escherichia coli pks+ and enterotoxinogenic Bacteroides fragilis in colorectal cancer have been well established identifying the role of genetic loci encoding toxins. Through the sequencing and analysis of 11 clinical SGG isolates from CRC patients and comparisons with non-CRC isolates, we uncovered a significant diversity among CRC-associated strains. Our findings suggest that SGG association with CRC is complex and is not linked to a specific strain or pathogenicity island, thus highlighting the opportunistic and versatile nature of SGG.

microbiology↗

bioRxiv2023

Inhibition of Pseudomonas aeruginosa quorum sensing by chemical induction of the MexEF-oprN efflux pump

The cell-to-cell communication system quorum sensing (QS), used by various pathogenic bacteria to synchronize gene expression and increase host invasion potentials, is studied as a potential target for persistent infection control. To search for novel molecules targeting the QS system in the Gram-negative opportunistic pathogen Pseudomonas aeruginosa a chemical library consisting of 3280 small compounds from LifeArc was screened. A series of ten conjugated phenones that have not previously been reported to target bacteria were identified as inhibitors of QS in P. aeruginosa. Two lead compounds (ethylthio enynone and propylthio enynone) were resynthesized for verification of activity and further elucidation of the mode of action. The isomeric pure Z-ethylthio enynone was used for RNA sequencing revealing a strong inhibitor of QS-regulated genes and the QS-regulated virulence factors rhamnolipid and pyocyanin were significantly decreased by treatment with the compounds. A transposon mutagenesis screen performed in a newly constructed lasB-gfp monitor strain identified the target of Z-ethylthio enynone in P. aeruginosa to be the MexEF-OprN efflux pump, which was further established using defined mex knockout mutants. Our data indicate that the QS inhibitory capabilities of Z-ethylthio enynone were caused by the drainage of intracellular signal molecules as a response to chemical-induced stimulation of the MexEF-oprN efflux pump thereby inhibiting the auto-generated positive feedback and its enhanced signal-molecule synthesis.

microbiology↗

bioRxiv2022

Characterization of TelD, an LXG effector of Streptococcus gallolyticus, antagonized by a non-canonical immunity protein

Streptococcus gallolyticus subsp. gallolyticus (SGG) is an opportunistic bacterial pathogen strongly associated with colorectal cancer. Here, through comparative genomics analysis, we demonstrated that the genetic locus encoding the Type VIIb Secretion System (T7SSb) machinery is uniquely present in SGG in two different arrangements. SGG UCN34 carrying the most prevalent T7SSb genetic arrangement was chosen as the reference strain. To identify the effectors secreted by this secretion system, we inactivated the essC gene encoding the motor of this machinery. Comparison of the proteins secreted by UCN34 WT and its isogenic {Delta}essC mutant revealed six T7SSb effector proteins, including the expected WXG effector EsxA and three LXG-containing proteins. In this work, we characterized an LXG-family toxin named herein TelE displaying pore-forming activity. Seven homologs of TelE harboring a conserved glycine zipper motif at the C-terminus were identified in different SGG isolates. Scanning mutagenesis of this motif showed that the glycine residue at position 470 was crucial for TelE pore-forming activity. Unlike other pore-forming toxins commonly antagonized by a membrane protein, TelE activity was antagonized by a small protein TipE belonging to the DUF5085 family. Overall, we report herein a unique SGG T7SSb effector exhibiting a pore-forming activity against non-immune bacteria. IMPORTANCEIn this study, 38 clinical isolates of Streptococcus gallolyticus subsp. gallolyticus (SGG) were sequenced and a genetic locus encoding the Type VIIb secretion system (T7SSb) was found conserved and absent from 16 genomes of the closely related S. gallolyticus subsp. pasteurianus (SGP). The T7SSb is a bona fide pathogenicity island. Here, we report that the model organism SGG strain UCN34 secretes six T7SSb effectors. One of the six effectors named TelE displayed a strong toxicity when overexpressed in Escherichia coli. Our results indicate that TelE is a pore forming toxin whose activity can be antagonized by a non-canonical immunity protein named TipE. Overall, we report a unique toxin-immunity protein pair and our data expand the range of effectors secreted through T7SSb.

microbiology↗

bioRxiv2021

Disruption of the Pseudomonas aeruginosa Tat system perturbs PQS-dependent quorum sensing and biofilm maturation through loss of the Rieske cytochrome bc1 sub-unit

Extracellular DNA (eDNA) is a major constituent of the extracellular matrix of Pseudomonas aeruginosa biofilms and its release is regulated via the pseudomonas quinolone signal (PQS) dependent quorum sensing (QS). By screening a P. aeruginosa transposon library to identify factors required for DNA release, mutants with insertions in the twin-arginine translocation (Tat) pathway were identified as exhibiting reduced eDNA release, and defective biofilm architecture with enhanced susceptibility to tobramycin. P. aeruginosa tat mutants showed substantial reductions in pyocyanin, rhamnolipid and membrane vesicle (MV) production consistent with perturbation of 2-heptyl-3-hydroxy-4-quinolone (PQS) dependent QS as demonstrated by changes in pqsA expression and 2-alkyl-4-quinolone (AQ) production. Provision of exogenous PQS to the tat mutants did not return pqsA, rhlA or phzA1 expression or pyocyanin production to wild type levels. However, transformation of the tat mutants with the AQ-independent pqs effector pqsE restored phzA1 expression and pyocyanin production. Since mutation or inhibition of Tat prevented PQS-driven auto-induction, we sought to identify the Tat secretion substrate responsible. A pqsA::lux fusion was introduced into each of 34 validated P. aeruginosa Tat substrate deletion mutants. Analysis of each mutant for reduced bioluminescence revealed that the signalling defect was associated with the Rieske iron-sulfur subunit of the cytochrome bc1 complex. In common with the parent strain, a Rieske mutant exhibited defective PQS signalling, AQ production, rhlA expression and eDNA release that could be restored by genetic complementation. Thus, lack of the Rieske sub-unit export is clearly responsible for the Tat-mediated perturbation of PQS-dependent QS, the loss of virulence factor production, biofilm eDNA and the tobramycin tolerance of P. aeruginosa biofilms. Author SummaryPseudomonas aeruginosa is a highly adaptable human pathogen responsible for causing chronic biofilm-associated infections. Biofilms are highly refractory to host defences and antibiotics and thus difficult to eradicate. The biofilm extracellular matrix incorporates extracellular DNA (eDNA). This stabilizes biofilm architecture and helps confer tolerance to antibiotics. Since mechanisms that control eDNA release are not well understood, we screened a P. aeruginosa mutant bank for strains with defects in eDNA release and discovered a role for the twin-arginine translocation (Tat) pathway that exports folded proteins across the cytoplasmic membrane. Perturbation of the Tat pathway resulted in defective biofilms susceptible to antibiotic treatment as a consequence of perturbed pseudomonas quinolone (PQS) signalling. This resulted in the failure to produce or release biofilm components including eDNA, phenazines and rhamnolipids as well as microvesicles. Furthermore, we discovered that perturbation of PQS signalling was a consequence of the inability of tat mutants to translocate the Rieske subunit of the cytochrome bc1 complex involved in electron transfer and energy transduction. Given the importance of PQS signalling and the Tat system to virulence and biofilm maturation in P. aeruginosa, our findings underline the potential of the Tat system as a drug target for novel antimicrobial agents.

microbiology↗

bioRxiv2020

Pseudomonas aeruginosa isolates co-incubated with Acanthamoeba castellanii exhibit phenotypes similar to chronic cystic fibrosis isolates

The opportunistic pathogen, Pseudomonas aeruginosa, is ubiquitous in the environment, and in humans is capable of causing acute and chronic infections. P. aeruginosa, when co-incubated with the bacterivorous amoeba, Acanthamoeba castellanii, for extended periods, produced genetic and phenotypic variants. Sequencing of late-stage amoeba-adapted P. aeruginosa isolates demonstrated single nucleotide polymorphisms within genes that encode known virulence factors, and this correlated with a reduction in expression of virulence traits. Virulence towards the nematode, Caenorhabditis elegans, was attenuated in late-stage amoeba-adapted P. aeruginosa compared to early stage amoeba-adapted and non-adapted counterparts. Late-stage amoeba-adapted P. aeruginosa lost competitive fitness compared to non-adapted counterparts when grown in nutrient rich media. However, non-adapted P. aeruginosa were rapidly cleared by amoeba predation, whereas late-stage amoeba-adapted isolates remained in higher numbers 24 h after ingestion by amoeba. In addition, there was reduced uptake by macrophage of amoeba-adapted isolates and reduced uptake by human neutrophils as well as increased survival in the presence of neutrophils. Our findings indicate that the selection imposed by amoeba on P. aeruginosa resulted in reduced virulence over time. Importantly, the genetic and phenotypic traits possessed by late-stage amoeba-adapted P. aeruginosa are similar to what is observed for isolates obtained from chronic cystic fibrosis infections. This notable overlap in adaptation to different host types suggests similar selection pressures among host cell types. Author SummaryPseudomonas aeruginosa is an opportunistic pathogen that causes both acute infections in plants and animals, including humans and also causes chronic infections in immune compromised and cystic fibrosis patients. This bacterium is commonly found in soils and water where bacteria are constantly under threat of being consumed by the bacterial predators, protozoa. To escape being killed, bacteria have evolved a suite of mechanisms that protect them from being consumed or digested. Here we examined the effect of long-term predation on the genotype and phenotypes expressed by P. aeruginosa. We show that long-term co-incubation with protozoa resulted in mutations in the bacteria that made them less pathogenic. This is particularly interesting as we see similar mutations arise in bacteria associated with chronic infections. Thus, predation by protozoa and long term colonization of the human host may represent similar environments that select for similar losses in gene functions.

microbiology↗

bioRxiv2017

Auranofin inhibits virulence in Pseudomonas aeruginosa

Pseudomonas aeruginosa is widely attributed as the leading cause of hospital-acquired infections. Due to intrinsic antibiotic resistance mechanisms and the ability to form biofilms, P. aeruginosa infections are challenging to treat. P. aeruginosa employs multiple virulence mechanisms to establish infections, many of which are controlled by the global virulence regulator Vfr. An attractive strategy to combat P. aeruginosa infections is thus the use of anti-virulence compounds. Here, we report the discovery that FDA-approved drug auranofin attenuates virulence in P. aeruginosa. We demonstrate that auranofin acts by targeting Vfr, which in turn leads to inhibition of quorum sensing (QS) and Type IV pili (TFP). Consistent with inhibition of QS and TFP expression, we show that auranofin attenuates biofilm maturation, and when used in combination with colistin, displays strong synergy in eradicating P. aeruginosa biofilms. Auranofin may have immediate applications as an anti-virulence drug against P. aeruginosa infections.

microbiology↗

bioRxiv2017

Synergy of Quorum Quenching Enzyme and Quorum Sensing Inhibitor in Inhibiting P. aeruginosa Quorum Sensing

The threat of antibiotic resistant bacteria has called for alternative antimicrobial strategies that would mitigate the increase of classical resistance mechanism. Many bacteria employ quorum sensing (QS) to govern the production of virulence genes and formation of drug-resistance biofilms. Blocking QS mechanisms have proven to be a functional alternative to conventional antibiotic control of infections. The concepts of quorum sensing inhibitors (QSI) and quorum quenching enzymes (QQ) have been investigated separately. In this study however, we simulated the synergistic effect of QQ and QSI in blocking bacterial QS. This effect was validated by experiments using AiiA and G1 as QQ and QSI respectively on Pseudomonas aeruginosa LasR/I and RhlR/I QS circuits. The combination of a QQ and a QSI almost completely blocked the P. aeruginosa QS las and rhl system. Our findings provided a potential application strategy for bacterial QS disruption.

microbiology↗

bioRxiv2017

The Acquisition of Resistance to Carbapenem and Macrolide-mediated Quorum Sensing Inhibition by Pseudomonas aeruginosa via a Novel Integrative and Conjugative Element ICETn43716385

Pseudomonas aeruginosa can cause persistant and life-threatening infections in immunocompromised patients. Carbapenems are the first-line agents to treat P. aeruginosa infections; therefore, the emergence of carbapenem-resistant P. aeruginosa strains has greatly challenged effective antibiotic therapy. In this study, we characterised the full-length genomes of two carbapenem resistant P. aeruginosa clinical isolates that produce the carbapebemase New Delhi metallo-{beta}-lactamase-1 (NDM-1). We found that the blaNDM-1 gene is encoded by a novel intergrative and conjugative element (ICE) ICETn43716385, which also carries the macrolide resistance gene msr(E) and the florfenicol resistance gene floR. The msr(E) gene has rarely been described in P. aeruginosa genomes. To investigate the functional roles of msr(E) in P. aeruginosa, we exogeneously expressed this gene in P. aeruginosa laboratory strains and found that the acquisition of msr(E) could abolish the azithromycin-mediated quorum sensing inhibition in vitro and the anti-Pseudomonas effect of azithromycin in vivo. In addition, the expression of msr(E) almost completely restored the azithromycin-affected P. aeruginosa transcriptome, as shown by our RNA sequencing analysis. We present the first evidence of blaNDM-1 to be carried by intergrative and conjugative elements, and the first evidence of co-transfer of carbapenem resistance and the resistance to macrolide-mediated quorum sensing inhibition into P. aeruginosa genomes.\n\nImportanceCarbapenem resistant P. aeruginosa has recently been listed as the top three most dangerous superbugs by World Health Organisation. The transmission of blaNDM-1 gene into P. aeruginosa can cause extreme resistance to carbapenems and fourth generation cephalosporins, which greatly compromises the effectiveness of these antibiotics against Pseudomonas infections. However, the lack of complete genome sequence of NDM-1-producing P. aeruginosa has limited our understanding of the transmisibility of blaNDM-1 in this organism. Here we showed the co-transfer of blaNDM-1 and msr(E) into P. aeruginosa genome by a novel integrative and conjugative element (ICE). The acquisition of these two genes confers P. aeruginosa with resistance to carbapenem and macrolide-mediated quorum sensing inhibition, both of which are important treatment stretagies for P. aeruginosa infections. Our findings highlight the potential of ICEs in transmitting carbapenem resistance, and that the anti-virulence treatment of P. aeruginosa infections by macrolides can be challenged by horizontal gene transfer.

genomics↗

bioRxiv2017

Cyclic-di-GMP is required for corneal infection by Pseudomonas aeruginosa and modulates host immunity

Biofilms are extremely tolerant toward antimicrobial treatment and host immune clearance due to their distinct physiology and protection by extracellular polymeric substances. Bis-(3{acute}-5{acute})-cyclic dimeric guanosine monophosphate (c-di-GMP) is an essential messenger that regulates biofilm formation by a wide range of bacteria. However, there is a lack of physiological characterization of biofilms in vivo as well as the roles of c-di-GMP signaling in mediating host-biofilm interactions. Here, we employed dual RNA-Seq to characterize the host and pathogen transcriptomes during Pseudomonas aeruginosa infection using a mouse keratitis model. In vivo P. aeruginosa biofilms maintained a distinct physiology compared with in vitro P. aeruginosa biofilms, with enhanced virulence and iron uptake capacity. C-di-GMP synthesis was enhanced in P. aeruginosa cells in vivo, potentially due to down-regulation of the expression of several phosphodiesterases (e.g., DipA, NbdA). Increased intracellular c-di-GMP levels were required for long-term ocular colonization of P. aeruginosa and impaired host innate immunity.

microbiology↗