Biology subjects

Janssen, A. B.

Publications and source records attributed to Janssen, A. B..

10 recordsLinked to original sources

bioRxiv2026

A functional genetic landscape of antibiotic sensitivity across the pneumococcal pangenome reveals conserved and lineage-specific vulnerabilities

The large pangenome of Streptococcus pneumoniae enables this opportunistic pathogen to adapt and evade antibiotic treatment. Effective treatment of pneumococcal infections requires a better understanding of the genes that modulate susceptibility to antibiotics across the pangenome. Using CRISPRi-seq, we identified genes that contribute to antibiotic sensitivity against a panel of clinically relevant antibiotics across nine pneumococcal strains with diverse resistance profiles, serotypes, and lineages. The here-generated chemical-genetics atlas revealed distinct genome-wide signatures of antibiotic stress that were specific to the antibiotic mode of action and showed both strain-specific and conserved signatures. This allowed us to identify conserved genes involved in antibiotic vulnerability and assign functions to previously uncharacterized genes. For instance, deletion of mutS2, which may act as a ribosome collision sensor and spv_1295, a conserved gene of unknown function, resulted in increased sensitivity to the macrolide azithromycin across strains, including a macrolide resistant strain, and could be potential targets for global sensitizing therapies. This work establishes a pangenome-wide framework for understanding antibiotic stress responses in S. pneumoniae, providing a foundation for the rational development of therapies that exploit conserved and strain-specific vulnerabilities.

microbiology↗

bioRxiv2025

Strain-resolved CRISPRi-Seq reveals conserved antibiotic vulnerabilities in Staphylococcus aureus

Staphylococcus aureus remains a major clinical threat due to rising antibiotic resistance and high rates of treatment failure. Deciphering the genetic responses to antibiotic pressure and identifying conserved vulnerabilities are essential steps toward developing broadly effective therapies. Here, we constructed strain-resolved CRISPR interference (CRISPRi) libraries targeting all genes in four clinically relevant S. aureus strains spanning major clonal complexes. CRISPRi-seq screens enabled high-resolution mapping of their fitness landscapes and the definition of a core essentialome representing robust targets for antimicrobial intervention. Exposure of the CRISPRi libraries to four mechanistically distinct antibiotics revealed genome-wide susceptibility profiles, identifying both strain-dependent and conserved susceptibility signatures shaped by the drug mode of action and genetic background. Analysis of these conserved vulnerabilities provided insight into antibiotic-specific stress responses and resistance mechanisms. Among the core determinants of vancomycin vulnerability, we identified several previously uncharacterized genes, including a conserved membrane-associated operon, here designated EsrABC, whose disruption markedly increases vancomycin sensitivity in the four strains. Our study provides a genome-wide atlas of S. aureus fitness and conditional vulnerabilities, fully explorable in the here-developed online AureoBrowse platform (https://aureobrowse.veeninglab.com/), revealing candidates for synergistic therapies and potential therapeutic targets.

microbiology↗

bioRxiv2025

CRISPRi-seq in Haemophilus influenzae reveals genome-wide and medium-specific growth determinants

Work in the human pathobiont Haemophilus influenzae has pioneered functional genomics in bacteria such as genome-wide transposon mutagenesis combined with deep sequencing. These approaches unveiled a large set of likely essential genes, but functional studies are hampered due to a limited molecular toolbox. To bridge this gap, we engineered a titratable anhydrotetracycline (aTc)-inducible CRISPRi (Clustered Regularly Interspaced Short Palindromic Repeats interference) platform for efficient regulation of gene expression in H. influenzae. Genome-wide fitness analyses in two different in vitro culture media by CRISPRi-seq revealed growth medium-dependent fitness cost for a panel of H. influenzae genes. We demonstrated that CRISPRi-programmed fitness defects can be rescuable, and refined previous Tn-seq based essentialome studies. Finally, we introduce HaemoBrowse, an extensive user-friendly online resource for visual inspection of H. influenzae genome annotations, including sgRNA spacers. The inducible CRISPRi platform described here represents a valuable tool enabling functional genomics and the study of essential genes, thereby contributing to the identification of therapeutic targets for developing drugs and vaccines against H. influenzae. ImportanceCRISPRi-seq is a robust method to study bacterial gene fitness and essentiality via relative quantification and comparison of sgRNA abundance at a genome-wide scale. Here, we present a novel CRISPRi system for individual genes or pooled libraries knockdown in Haemophilus influenzae. A genome-wide CRISPRi library designed to cover 99.27% of all total genetic features in the genome of RdKW20 strain was constructed and screened in two laboratory growth media through CRISPRi-seq, uncovering growth medium-dependent fitness cost, further confirmed with individual knockdown/knockout mutants. We also introduce HaemoBrowse (https://HaemoBrowse.VeeningLab.com), through which genome annotations and sgRNA design on H. influenzae genomes can be readily inspected. This platform provides a valuable tool for gene function and essentiality analyses in a notorious human pathobiont.

microbiology↗

bioRxiv2024

PneumoBrowse 2: An integrated visual platform for curated genome annotation and multiomics data analysis of Streptococcus pneumoniae

Streptococcus pneumoniae is an opportunistic human pathogen responsible for high morbidity and mortality rates. Extensive genome sequencing revealed its large pangenome, serotype diversity, and provided insight into genome dynamics. However, functional genome analysis has lagged behind, as that requires detailed and time-consuming manual curation of genome annotations, and integration of genomic and phenotypic data. To remedy this, PneumoBrowse was presented in 2018; a user-friendly interactive online platform, which provided the detailed annotation of the S. pneumoniae D39V genome, alongside transcriptomic data. Since 2018, many new studies on S. pneumoniae genome biology and protein functioning have been performed. Here, we present PneumoBrowse 2 (https://veeninglab.com/pneumobrowse), fully rebuilt in JBrowse 2. We updated annotations for transcribed and transcriptional regulatory features in the D39V genome. We added genome-wide data tracks for high-resolution chromosome conformation capture (Hi-C) data, chromatin immunoprecipitation coupled to high-throughput sequencing (ChIP-Seq), ribosome profiling, CRISPRi-seq gene essentiality data and more. Additionally, we included 18 phylogenetically diverse S. pneumoniae genomes and their annotations. By providing easy access to diverse high-quality genome annotations, and links to other databases (including UniProt and AlphaFold), PneumoBrowse 2 will further accelerate research and development into preventive and treatment strategies, through increased understanding of the pneumococcal genome. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC="FIGDIR/small/606308v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@1a38ce5org.highwire.dtl.DTLVardef@773428org.highwire.dtl.DTLVardef@1368aorg.highwire.dtl.DTLVardef@1a39f0d_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2024

Make-or-break prime editing for bacterial genome engineering

CRISPR-Cas9 has revolutionized genome engineering by allowing precise introductions of DNA double-strand breaks (DSBs). However, genome engineering in bacteria is still a complex, multi-step process requiring a donor DNA template for repair of DSBs. Prime editing circumvents this need as the repair template is indirectly provided within the prime editing guide RNA (pegRNA). Here, we developed make-or-break Prime Editing (mbPE) that allows for precise and effective genetic engineering in the opportunistic human pathogen Streptococcus pneumoniae. In contrast to traditional prime editing in which a nicking Cas9 is employed, mbPE harnesses wild type Cas9 in combination with a pegRNA that destroys the seed region or protospacer adjacent motif. Since most bacteria poorly perform template-independent end joining, correctly genome-edited clones are selectively enriched during mbPE. We show that mbPE is RecA-independent and can be used to introduce point mutations, deletions and targeted insertions, including protein tags such as a split luciferase, at selection efficiencies of over 93%. mbPE enables sequential genome editing, is scalable, and can be used to generate pools of mutants in a high-throughput manner. The mbPE system and pegRNA design guidelines described here will ameliorate future bacterial genome editing endeavors.

microbiology↗

bioRxiv2023

Klebsiella pneumoniae OmpR facilitates lung infection through transcriptional regulation of key virulence factors

Bacteria must adapt to the stresses of specific environmental conditions to survive. This adaptation is often achieved by altering gene expression through two-component regulatory systems (TCSs). In Gram-negative bacteria, the response to environmental changes in osmolarity and pH are primarily mediated by the EnvZ/OmpR TCS. Although the functioning of EnvZ/OmpR has been well characterized in Escherichia coli, Salmonella enterica, and the Yersinia genus, the importance of EnvZ/OmpR TCS in the opportunistic human pathogen Klebsiella pneumoniae has been limitedly studied. Here, we investigated the importance of EnvZ/OmpR in K. pneumoniae for fitness, gene regulation, virulence, and infection. Through the generation of a markerless ompR-deletion mutant, we show that overall fitness of K. pneumoniae is not impacted in vitro. Using dual RNA-seq of K. pneumoniae co-incubated with human lung epithelial cells we demonstrate that the K. pneumoniae OmpR regulon includes important virulence factors, but shows otherwise limited overlap with the regulons of other Gram-negative bacteria. In addition, we show that deletion of ompR in K. pneumoniae leads to a stronger antibacterial transcriptional response in human lung epithelial cells. Lastly, we show that OmpR is crucial for K. pneumoniae virulence and infection through a murine lung infection model. As the adaptation of commensal bacteria to specific niches is mediated by TCSs, we show that EnvZ/OmpR plays a crucial role in successful lung infection, as well as in virulence. These results suggest that OmpR is an interesting target for anti-virulence drug discovery programs. ImportanceBacteria use two-component regulatory systems (TCSs) to adapt to changes in their environment by changing their gene expression. In this study, we show that the EnvZ/OmpR TCS of Klebsiella pneumoniae plays an important role in successfully establishing lung infection, and virulence. In addition, we discern the transcriptional response that OmpR facilitates within this clinically relevant opportunistic pathogen, and within the host. This work suggests that K. pneumoniae OmpR might be a promising target for innovative anti-infectives.

microbiology↗

bioRxiv2022

Colistin resistance mutations in phoQ sensitize Klebsiella pneumoniae to IgM-mediated complement killing

The Gram-negative bacterium Klebsiella pneumoniae is notorious for a strong increase of infections with antibiotic resistant strains. To treat infections with antibiotic resistant K. pneumoniae, clinicians increasingly need to use the last resort antibiotic colistin. K. pneumoniae can develop colistin resistance by modifying its membranes. During infection the membranes of Gram-negative bacteria are also targeted by the human immune system via the complement system. Gram-negative bacteria have an outer and inner membrane separated by a thin cell wall. Activation of the complement system leads to the formation of the membrane attack complex (MAC), a pore that inserts into the outer membrane, and ultimately leads to lysis of the bacterium. As both colistin and the MAC interact with the outer membrane of Gram-negative bacteria, we wondered if developing colistin resistance influences MAC-mediated killing of K. pneumoniae. Using clinical isolates that developed colistin resistance, we found that the strain Kp209_CSTR became more sensitive to MAC-mediated killing compared to the wild-type strain. MAC-mediated membrane permeabilization of Kp209_CSTR required antibody dependent activation of the classical complement pathway. Strikingly, Kp209_CSTR was bound by IgM in human serum that did not recognise the wild-type strain. Depletion of Kp209_CSTR-specific antibodies from serum prevented MAC-mediated membrane permeabilization, which was restored by adding back IgM. Genomic sequence comparison revealed that Kp209_CSTR has a deletion in the phoQ gene. RNAseq analysis suggested that this mutation locks PhoQ in a constitutively active state. These results indicate that PhoQ activation in Kp209_CSTR leads to both colistin resistance and sensitivity to MAC-mediated killing. Together, our results show that developing colistin resistance can sensitize K. pneumoniae to killing by the immune system.

microbiology↗

bioRxiv2020

In vitro evolution of colistin resistance in the Klebsiella pneumoniae complex follows multiple evolutionary trajectories with variable effects on fitness and virulence characteristics.

The increasing prevalence of multidrug-resistant Gram-negative opportunistic pathogens, including Klebsiella pneumoniae, has led to a resurgence in the use of colistin as a last-resort drug. Colistin is a cationic lipopeptide antibiotic that selectively acts on Gram-negative bacteria through electrostatic interactions with anionic phosphate groups of the lipid A moiety of lipopolysaccharides (LPS). Colistin resistance in K. pneumoniae is mediated through loss of these phosphate groups, or modification with cationic groups (e.g. 4-amino-4-deoxy-L-arabinose (L-Ara4N), or phosphoethanolamine), but also hydroxylation of acyl-groups of lipid A. Here, we study the in vitro evolutionary trajectories towards colistin resistance in clinical K. pneumoniae complex strains (three K. pneumoniae sensu stricto strains and one K. variicola subsp. variicola strain) and their impact on fitness and virulence characteristics. Through population sequencing during the in vitro evolution experiment, we found that resistance develops through a combination of single nucleotide polymorphisms (SNPs), insertion and deletions (indels), and the integration of insertion sequence (IS) elements, affecting genes associated with LPS biosynthesis and modification, and capsule structures. The development of colistin resistance decreased the maximum growth rate of one K. pneumoniae sensu stricto strain, but not in the other three K. pneumoniae sensu lato strains. Colistin-resistant strains had lipid A modified through hydroxylation, palmitoylation, and L-Ara4N addition. Colistin-resistant K. pneumoniae sensu stricto strains exhibited cross-resistance to LL-37, in contrast to the K. variicola subsp. variicola strain that did not change in susceptibility to LL-37. Virulence, as determined in a Caenorhabditis elegans survival assay, was higher in two colistin-resistant strains. Our study suggests that nosocomial K. pneumoniae complex strains can rapidly develop colistin resistance de novo through diverse evolutionary trajectories upon exposure to colistin. This effectively shortens the lifespan of this last-resort antibiotic for the treatment of infections with multidrug-resistant Klebsiella. Author summaryBacteria that frequently cause infections in hospitalised patients are becoming increasingly resistant to antibiotics. Colistin is a positively charged antibiotic that is used for the treatment of infections with multidrug-resistant Gram-negative bacteria. Colistin acts by specifically interacting with the negatively charged LPS molecule in the outer membrane of Gram-negative bacteria. Colistin resistance is mostly mediated through modification of LPS to reduce its negative charge. Here, we use a laboratory evolution experiment to show that strains belonging to the Klebsiella pneumoniae complex, a common cause of multidrug-resistant hospital-acquired infections, can rapidly accumulate mutations that reduce the negative charge of LPS without an appreciable loss of fitness. Colistin resistance can lead to cross-resistance to an antimicrobial peptide of the human innate immune system, but can increase susceptibility to serum, and virulence in a nematode model. These findings show that extensively resistant K. pneumoniae complex strains may rapidly develop resistance to the last-resort antibiotic colistin via different evolutionary trajectories, while retaining their ability to cause infections.

microbiology↗

bioRxiv2020

Microevolution of acquired colistin resistance in Enterobacteriaceae from ICU patients receiving selective decontamination of the digestive tract.

Colistin is an antibiotic that targets the lipopolysaccharides present in the membranes of Gram-negative bacteria. It is used as last-resort drug to treat infections with multidrug-resistant strains. Colistin is also used in selective decontamination of the digestive tract (SDD), a prophylactic therapy used in patients hospitalised in intensive care units (ICUs) to selectively eradicate opportunistic pathogens in the oropharyngeal and gut microbiota. In this study, we aimed to unravel the mechanisms of acquired colistin resistance in Gram-negative opportunistic pathogens obtained from SDD-treated patients. Routine surveillance of 428 SDD-treated patients resulted in thirteen strains with acquired colistin resistance (Escherichia coli n=9; Klebsiella aerogenes, n=3; Enterobacter asburiae, n=1) from five patients. Genome sequence analysis showed that these isolates represented multiple distinct colistin-resistant clones, but that within the same patients, colistin-resistant strains were clonally related. We identified previously described mechanisms that lead to colistin resistance, i.e. a G53 substitution in the response regulator PmrA/BasR, and the acquisition of the mobile colistin resistance gene mcr-1.1, but we also observed novel variants of basR with an 18-bp deletion, and a G19E substitution in the sensor histidine kinase BasS. We experimentally confirmed these variants to contribute to reduced colistin susceptibility. In a single patient, we observed that colistin resistance in a single E. coli clone evolved through two unique variants in basRS. We show that prophylactic use of colistin during SDD can select for colistin resistance in species that are not intrinsically colistin-resistant. This highlights the importance of continued surveillance for the emergence of colistin resistance in patients treated with SDD.

microbiology↗

bioRxiv2019

Mechanisms of colistin resistance in Escherichia strains isolated from bloodstream infections

Infections by multidrug-resistant Gram-negative bacteria are increasingly common, prompting the renewed interest in the use of colistin. Colistin specifically targets Gram-negative bacteria by interacting with the anionic lipid A moieties of lipopolysaccharides, leading to membrane destabilization and cell death. Here, we aimed to uncover the mechanisms of colistin resistance in nine colistin-resistant Escherichia coli strains and one E. albertii strain. These were the only colistin-resistant strains out of 1140 bloodstream Escherichia isolates collected in a tertiary hospital over a ten-year period (2006 - 2015). Core genome phylogenetic analysis showed that each patient was colonised by a unique strain, suggesting that colistin resistance was acquired independently in each strain. All colistin-resistant strains had lipid A that was modified with phosphoethanolamine. In addition, two E. coli strains had hepta-acylated lipid A species, containing an additional palmitate compared to the canonical hexa-acylated E. coli lipid A. One E. coli strain carried the mobile colistin resistance (mcr) gene mcr-1.1 on an IncX4-type plasmid. Through construction of chromosomal transgene integration mutants, we experimentally determined that mutations in basRS, encoding a two-component signal transduction system, contributed to colistin resistance in four strains. We confirmed these observations by reversing the mutations in basRS to the sequences found in reference strains, resulting in loss of colistin resistance. While the mcr-genes have become a widely studied mechanism of colistin resistance in E. coli, sequence variation in basRS is another, potentially more prevalent but relatively underexplored, cause of colistin resistance in this important nosocomial pathogen. ImportanceMultidrug resistance among Gram-negative bacteria has led to the use of colistin as a last-resort drug. The cationic colistin kills Gram-negative bacteria through electrostatic interaction with the anionic lipid A moiety of lipopolysaccharides. Due to increased use in clinical and agricultural settings, colistin resistance has recently started to emerge. In this study, we used a combination of whole genome sequence analysis and experimental validation to characterise the mechanisms through which E. coli strains from bloodstream infections can develop colistin resistance. We found no evidence of direct transfer of colistin-resistant isolates between patients. The lipid A of all isolates was modified by the addition of phosphoethanolamine. In four isolates, colistin resistance was experimentally verified to be caused by mutations in the basRS genes, encoding a two-component regulatory system. Our data show that chromosomal mutations are an important cause of colistin resistance among clinical E. coli isolates.

microbiology↗