Biology subjects

Veening, J.-W.

Publications and source records attributed to Veening, J.-W..

At least 19 recordsLinked to original sources

bioRxiv2026

Genome-wide CRISPRi maps pneumococcal attachment and intracellular survival during single infection and Influenza co-infection

Streptococcus pneumoniae remains a leading cause of bacterial pneumonia and frequently causes severe secondary infections following influenza A virus (IAV) infection. Yet, the genetic requirements underlying pneumococcal pathogenicity during viral co-infection remain incompletely understood. Here, we employed an inducible genome-wide CRISPR interference (CRISPRi) library to systematically investigate pneumococcal gene fitness during epithelial attachment and intracellular survival in macrophages under both single-infection and IAV co-infection conditions. Screening across multiple host cell types and viral strains identified 42 pneumococcal genes affecting host-cell attachment and 63 genes influencing intracellular survival. While global patterns of gene fitness were largely conserved between single infection and co-infection, genes involved in cell envelope biogenesis, including the undecaprenyl pyrophosphate phosphatase uppP, became increasingly important during co-infection. Pharmacological inhibition of cell wall biosynthesis recapitulated several genetic phenotypes and revealed enhanced sensitivity to bacitracin in virally altered host environments. Integration of CRISPRi screening with untargeted metabolomics uncovered extensive remodeling of nucleotide metabolism during infection. Disruption of nucleoside transport altered bacterial fitness, morphology, capsule expression, and host-cell attachment. Uridine availability emerged as a key regulator linking metabolic adaptation to virulence-associated traits, highlighting a trade-off between bacterial growth and adherence. Taken together, our study provides a genome-scale resource of pneumococcal fitness determinants during infection and demonstrates that influenza co-infection selectively reshapes bacterial genetic dependencies rather than globally altering pathogenicity programs. These findings identify cell envelope homeostasis and nucleotide metabolism as central regulators of pneumococcal virulence and reveal potential targets for future antimicrobial and anti-virulence strategies.

microbiology↗

bioRxiv2026

Genome-wide interrogation of genetic requirements for root colonization by CRISPRi-seq in Bacillus subtilis

Bacillus subtilis is a known plant growth promoting rhizobacterium, yet, the genetic basis of effective root colonization remains understudied. Here, we applied genome-wide CRISPR interference sequencing (CRISPRi-seq) to systematically identify genes required for early Arabidopsis thaliana root colonization by the B. subtilis strain 3610 . Our genome-wide inducible CRISPRi library was used to screen for bacterial fitness during hydroponic root colonization. A complementary RNA-seq in the same conditions further revealed a distinct transcriptional state of root-associated cells. We identified 249 genes impacting root colonization. We reveal a critical role of cell envelope remodeling during early root association, specifically wall teichoic acid synthesis and D-alanylation. Moreover, we observed a metabolic shift toward TCA-cycle-driven aerobic growth with uptake of fructose, active repression of sporulation, competence and prophage programs, as well as induction of secondary metabolite biosynthesis. Our work establishes CRISPRi-seq as a powerful tool for dissecting bacterial-plant interactions relevant to sustainable agriculture.

microbiology↗

bioRxiv2026

Staphylococcus aureus Mnh1 cation/proton antiporter promotes phagocytic uptake and intracellular survival in human monocytes

While Staphylococcus aureus is predominantly an extracellular human commensal, it can also exhibit an intracellular lifestyle that enables its persistence within diverse host cell types, including monocytes and macrophages. This capacity complicates treatment, contributing to chronic infections and therapeutic failure. Employing combined flow cytometry and microscopy, we show that monocyte internalization is associated with markedly increased antibiotic survival of intracellular S. aureus. To identify staphylococcal genetic determinants important for its intracellular lifestyle, we utilized a genome-wide CRISPRi-seq screening strategy using a co-infection model with human-derived THP-1 cells. This screen revealed a set of genes specifically required for intracellular fitness, confirming established virulence factors as well as identifying novel contributors to uptake and survival within host phagocytes. Among these, we identified the cation-proton antiporter Mnh1 and demonstrated that it is particularly important for phagocytic entry. Time-lapse microscopy demonstrated that its depletion significantly reduced uptake by THP-1 cells leading to reduced intracellular bacterial load. Functional assays further showed that the Mnh1 operon plays a critical role in the intracellular survival of multiple S. aureus lineages in THP-1 cells. Consistent with this finding, levofloxacin treatment of infected monocytes cleared knockdown strains more efficiently than the control strain. Our findings underscore the intracellular niche as a critical reservoir for bacteria and suggest that selective inhibition of Mnh1, in combination with clinically relevant antibiotics, offers a promising therapeutic approach for the treatment of intracellular S. aureus infections.

microbiology↗

bioRxiv2026

Epithelial innate immune sensing of pneumococci is inherently restricted to a small cellular minority across species and infection niches

Streptococcus pneumoniae colonises the nasopharynx asymptomatically yet causes life-threatening invasive disease. How it navigates early epithelial immune surveillance to cause disease remains unclear. Conventional innate immune models predict coordinated, population-wide epithelial responses to bacterial infection. Using single-cell RNA sequencing, RNA fluorescence in situ hybridization and in vivo mouse and zebrafish models, pneumococcal infection is instead shown to activate innate immune genes including chemokine, NF-{kappa}B regulatory, and prostaglandin pathway genes, in only 1-4% of lung epithelial cells. This restriction is seemingly pneumococcal-specific as Escherichia coli triggers responses in over 40% of the same cells. Strikingly, nasopharyngeal epithelial cells show complete immune silence to pneumococci while responding robustly to E. coli and Staphylococcus aureus, suggesting niche-specific immune evasion. Additionally, pharmacological inhibition of COX-2 significantly increased mortality in a zebrafish meningitis model, identifying prostaglandin signalling as a protective host response during invasive disease. Competence-associated surface remodelling contributes modestly and incrementally to immune restriction, while the predominant dampening is competence-independent. These findings challenge canonical epithelial immunity models against bacterial infection and provide a cellular framework for understanding pneumococcal commensalism and pathogenesis. Significance statementClassical innate immune models predict that bacterial infection triggers coordinated, population-wide transcriptional responses across the epithelium. Using single-cell RNA sequencing, RNA fluorescence in situ hybridization, and in vivo zebrafish and mouse models, we show that Streptococcus pneumoniae, responsible for over one million deaths annually, activates innate immune genes in only 1-4% of lung epithelial cells. Escherichia coli triggers responses in over 40% of the same cells, demonstrating this restriction is pneumococcal-specific. Nasopharyngeal epithelial cells, the bacteriums primary colonization niche, show complete immune silence to pneumococci, suggesting niche-specific evolutionary adaptation. The prostaglandin pathway is identified as a protective host response during invasive disease. These findings challenge canonical models of epithelial immunity and provide a cellular framework for pneumococcal commensalism and pathogenesis.

microbiology↗

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↗

bioRxiv2026

Mechanistic basis of teichoic acid transport by a gatekeeper flippase

The cell wall is a complex structure that protects bacteria from environmental threats. Phosphocholine-containing teichoic acids are key cell wall biopolymers critical for host colonization, immune evasion, competence, and persistence in Streptococcus pneumoniae. The flippase TacF, a member of the multidrug/oligosaccharide-lipid/polysaccharide (MOP) superfamily, monitors the phosphocholine content of teichoic acids during transport, yet the underlying mechanism of this process remains unknown. We present a cryo-EM structure of S. pneumoniae TacF in lipid nanodiscs. In vivo complementation assays and molecular dynamics simulations reveal key residues involved in teichoic acid recognition and transport, while coevolutionary and conservation analyses delineate common mechanistic elements among MOP flippases, indicating a shared mechanism for polyprenyl-diphosphate-linked oligosaccharide lipid transport. Our findings provide mechanistic insights into an essential flippase involved in S. pneumoniae pathogenesis and a potential drug target.

biochemistry↗

bioRxiv2025

Systematic mapping of bacteriophage gene essentiality with HIDEN-SEQ

The constant arms race of bacteriophages and their bacterial hosts has inspired major breakthroughs in biotechnology and shaped phages as fierce predators with great clinical potential to fight multidrug-resistant bacterial pathogens. However, the vast amount of genomic "dark matter" composed of genes of unknown function in phage genomes remains a major obstacle for the molecular understanding of phage-host interactions. Here we present HIDEN-SEQ, a transposon-insertion sequencing method for phages that systematically links viral genes to selectable phenotypes. Using model phage T4, we show that HIDEN-SEQ readily reproduces the gene essentiality map established over decades of research. Subsequently, we show that our method is easily portable to different phages far beyond classical laboratory models. Across a panel of bacterial hosts and growth conditions, HIDEN-SEQ reveals many conditionally essential phage genes, including previously unknown viral anti-defense factors that we could match to specific antiviral defenses of the respective hosts. Compared to analogous techniques, HIDEN-SEQ provides unprecedented depth and near base-pair resolution as well as great ease of use and portability. We therefore anticipate that HIDEN-SEQ will accelerate discoveries in phage biology by uncovering functions of viral dark matter with direct relevance for microbial ecology, biotechnology, and improvements of phage therapy.

microbiology↗

bioRxiv2025

In vivo genome-wide CRISPRi-Seq in Streptococcus pneumoniae reveals host-adaptive pathways essential for meningitis and identifies new therapeutic targets

Streptococcus pneumoniae is an important human pathogen that causes invasive diseases and remains the leading cause of bacterial meningitis. However, the bacterial determinants required for survival within the central nervous system (CNS) are poorly understood. Here, we performed a genome-wide CRISPR interference screen (CRISPRi-Seq) in an in vivo zebrafish model to systematically map pneumococcal fitness determinants during meningitis. Using this approach, we investigated essential and non-essential genes contributing to the pathogenesis of pneumococcal meningitis. The screen identified 244 loci whose repression significantly reduced bacterial fitness in vivo, representing pathways involved in virulence, metabolism, cell-envelope biogenesis, translation, and stress adaptation. Comparative analysis with in vitro datasets showed that metabolic genes such as purA, proABC, thrC, glyA, and manLMN, as well as those involved in peptidoglycan and capsule biosynthesis (pbp3, cps2 operon) and oxidative stress response (nox, dpr), become selectively more essential in vivo. Functional validation confirmed that these pathways are critical for virulence, nutrient-dependent growth, and oxidative stress response during meningitis. Untargeted metabolomics of infected zebrafish corroborate several observed bacterial genetic sensitivities in the CNS with altered levels of key nutrients such as glucose and 4-aminobenzoic acid. Integration of the CRISPRi-Seq dataset with antibiotic-target annotations revealed both established and previously unrecognised vulnerabilities, including aminoacyl-tRNA synthetases such as leucyl-tRNA synthetase (LeuRS), whose inhibition by the benzoxaborole epetraborole improved host survival, highlighting its potential as a new therapeutic strategy for multidrug-resistant (MDR) S. pneumoniae. Together, these findings demonstrate the power of in vivo CRISPRi-Seq to define the pneumococcal essentialome under physiological conditions and reveal that metabolic adaptation, cell-envelope maintenance, and the oxidative stress response are central to bacterial survival in the CNS. This fitness map advances our understanding of pneumococcal adaptation in the CNS and identifies promising targets for developing therapies against S. pneumoniae.

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↗

bioRxiv2025

Multi-omics profiling reveals atypical sugar utilization and identifies a key membrane composition regulator in Streptococcus pneumoniae

The human body comprises many different microenvironments, each with their own challenges for microorganisms to overcome in order to survive, and possibly cause infection. The human pathogen Streptococcus pneumoniae is notoriously flexible in this regard, and can adapt to a wide range of host niches, including the nasopharynx, lungs, and cerebrospinal fluid. However, the molecular and genetic underpinnings of this ability remain largely obscure. In this work, using infection-mimicking growth conditions we demonstrate that niche adaptation imposes genome-wide changes on multiple levels, including gene essentiality, expression and membrane lipid composition. In general, we show that gene expression and fitness profiling couple orthogonal sets of genes to environmental stimuli. For instance, N-acetylglucosamine (GlcNAc) import (manLMN) and catabolism (nagAB) genes were required for growth on this sugar, but not differentially expressed in its presence, whereas other amino sugar metabolism pathways were upregulated, but not essential. Surprisingly, we found that pneumococci do not necessarily prefer glucose over GlcNAc and that uptake of GlcNAc in absence of subsequent catabolism was toxic. Moreover, we identified a previously overlooked fatty acid saturation regulator, FasR, controlling membrane composition, rendering it important during heat stress. A fundamental understanding of how genes contribute to bacterial niche adaptation, including nutrient availability or temperature fluctuations, is crucial for understanding successful antibiotic therapy and vaccination strategies and the development of novel anti-infectives.

microbiology↗

bioRxiv2025

A Modular Genetic Toolbox for Precise Gene Regulation and Multi-Color Imaging in Streptococci

Fluorescent labeling is a powerful tool in microbiology allowing live cell imaging and providing insights into dynamic cellular processes, quantification of gene expression and protein subcellular localization. Although multicolor imaging is widely used in Streptococcus pneumoniae and S. mutans, variants other than the green fluorescent protein (GFP) have rarely been applied in other streptococcal species. To address this gap in the streptococcal molecular toolbox, we benchmarked five different fluorescent proteins. The various fluorescent proteins were fused to the C-terminus of S. pneumoniae HlpA, a small non-specific DNA binding histone-like protein. These reporters, combined with four different antibiotic resistance genes, were engineered with various expression systems (inducible or constitutive) to form versatile cassettes. We provide methods to transfer these cassettes to different streptococcal species including S. salivarius and S. thermophilus. As a proof of concept, we generated a triple labeled S. salivarius strain in which HlpA, FtsZ and DivIVA were fused to three spectrally-distinct compatible fluorescent proteins. Multiple fluorescent labeling has broad applications for deciphering a wide range of scientific problems, from cellular processes to infectious disease mechanisms. The availability of these cassettes should allow for a wider use of single-cell labeling strategies in the Streptococcus clade and other closely related bacteria.

microbiology↗

bioRxiv2025

MAIT cells protect in severe pneumococcal pneumonia by regulating neutrophil/macrophage antimicrobial activities

Mucosal-Associated Invariant T (MAIT) cells populate the lung tissue where they contribute to defense against respiratory infections. While MAIT cells have been implicated in host resistance to infections caused by Gram-negative bacteria, their contribution in immunity against Gram-positive bacteria-driven pneumonia is still enigmatic. Here, we demonstrate that both mouse and human MAIT cells are activated during severe infection caused by Streptococcus pneumoniae, the major cause of community-acquired bacterial pneumonia. Upon infection, lung MAIT cells undergo a transcriptional reprogramming associated with acquisition of potent antimicrobial properties. MAIT cell-deficient mice are more susceptible to pneumococcal pneumonia, including higher mortality, uncontrolled bacterial growth and dissemination, and impaired neutrophil and interstitial macrophage activity. Moreover, prophylactic stimulation of MAIT cells using cognate antigen protects from pneumococcus-induced lethal pneumonia. These findings demonstrate that MAIT cells are key cellular actors during Gram-positive bacterial infections.

immunology↗

bioRxiv2024

A bacterial cell wall repair and modification system to resist host antibacterial factors

Pathogenic bacteria have acquired the ability to resist antibacterial defense mechanisms of the host. Streptococci are common in animal microbiota and include opportunistic pathogens like Group A Streptococcus (GAS) and Streptococcus pneumoniae (pneumococcus). While the conserved streptococcal S protein has been identified as a key factor in GAS virulence, its exact function is unclear. Here, we show that the pneumococcal S protein is crucial for resisting against host-derived antimicrobials by coordinating cell wall modification and repair. Specifically, we show that S proteins are septally localized through their transmembrane domain and contain an extracellular peptidoglycan (PG) binding LysM domain which is required for its function. Protein-protein and genetic interaction studies demonstrate that the pneumococcal S protein directly interacts with a PG synthase, class A penicillin binding protein PBP1a, and the PG deacetylase PgdA. Single-molecule experiments reveal that the fraction of circumferentially moving PBP1a molecules is reduced in the absence of S protein. Consistent with an impaired PBP1a function, streptococci lacking S protein exhibit increased susceptibility to cell wall targeting antibiotics and altered cell morphologies. PG analysis showed reduced N-deacetylation of glycans in the S. pneumoniae S protein mutant, indicating reduced PgdA activity. We show that pneumococci lacking the S protein cannot persist transient penicillin treatment, are more susceptible to the human antimicrobial peptide LL-37 and to lysozyme, and show decreased virulence in zebrafish and mice. Our data support a model in which S proteins regulate PBP1a activity and play a key role in coordinating PG repair and modification. This cell wall sentinel control system provides defense against host-derived and environmental antimicrobial attack.

microbiology↗

bioRxiv2024

Genome-wide analysis of fitness determinants of Staphylococcus aureus during growth in milk

Staphylococcus aureus is a major concern in the dairy industry due to its significance as a pathogen causing bovine mastitis as well as a source of food poisoning. The nutrient-rich milk environment supports bacterial growth, but the specific genetic determinants that facilitate S. aureus proliferation and persistence in milk are poorly understood. In this study, we conducted a genome-wide CRISPR interference sequencing (CRISPRi-seq) screen to identify fitness determinants essential for S. aureus growth and survival in milk. We identified 282 milk-essential genes, including those with key roles in DNA replication, protein synthesis, and metabolism. Comparative analysis with brain heart infusion (BHI) as growth medium, revealed 79 genes with differential fitness, highlighting specific adaptations required for growth in milk. Notably, we found that purine biosynthesis, folate cycle pathways, and metal acquisition were particularly important in this environment. Based on this, we further demonstrate that S. aureus is more sensitive to the folate inhibitors trimethoprim-sulfamethoxazole (TMP-SMX) in milk and identify several genes whose knockdown results in hypersensitivity to TMP-SMX in milk. Additionally, our analysis showed a relatively reduced importance of cell wall components, such as teichoic acids, for S. aureus fitness in milk, which is also reflected in reduced efficiency of antimicrobials targeting teichoic acids. Together, these findings provide new insights into the genetic basis of S. aureus fitness and antibiotic susceptibility in milk, offering directions for novel treatment strategies against bovine mastitis.

microbiology↗

bioRxiv2024

Genome-wide antibiotic-CRISPRi profiling identifies LiaR activation as a strategy to resensitize fluoroquinolone-resistant Streptococcus pneumoniae

Streptococcus pneumoniae is a human pathogen that has become increasingly resistant to the synthetic fluoroquinolone antibiotics that target bacterial topoisomerases. To identify pathways that are essential under fluoroquinolone stress and thus might represent novel targets to revitalize the use of this class of antibiotics, we performed genome-wide CRISPRi-seq screens to determine antibiotic-gene essentiality signatures. As expected, genes involved in DNA recombination and repair become more important under fluoroquinolone-induced DNA damage, such as recA, recJ, recF, recO, rexAB and ruvAB. Surprisingly, we also found that specific downregulation of the gene encoding the histidine kinase liaS caused fluoroquinolone hypersensitivity. LiaS is part of the LiaFSR (VraTSR) three-component regulatory system involved in cell envelope homeostasis. We show that LiaS keeps the response regulator LiaR inactive, and that deletion of liaS causes hyperphosphorylation of LiaR and subsequent upregulation of the LiaR regulon. RNA-seq was used to refine the LiaR regulon, highlighting the role of the heat-shock response and the pleiotropic regulator SpxA2 in fluoroquinolone sensitivity. Activating the LiaR-regulon by the cell envelope-targeting antibiotic bacitracin synergized with ciprofloxacin and levofloxacin. This synergistic antibiotic combination restored sensitivity in fluoroquinolone-resistant strains in vitro. Importantly, bacitracin/levofloxacin combination therapy was also effective in vivo and improved the treatment of fluoroquinolone-resistant S. pneumoniae infection in a zebrafish meningitis model. Together, the approaches and findings presented here provides a starting point for identification and validation of potent combination therapies that could be used in the clinic to treat antibiotic-resistant pneumococcal infections.

microbiology↗

bioRxiv2024

HU promotes higher-order chromosome organisation and influences DNA replication rates in Streptococcus pneumoniae

Nucleoid-associated proteins (NAPs) are crucial for maintaining chromosomal compaction and architecture and are actively involved in DNA replication, recombination, repair, and gene regulation. In the opportunistic pathogen Streptococcus pneumoniae, HU is the only identified NAP, and its role in chromosome conformation and other essential processes has not yet been investigated. Here, we use a multi-scale approach to explore the role of HU in chromosome conformation and segregation dynamics. By combining superresolution microscopy and whole-genome binding analysis, we describe the nucleoid as a dynamic structure where HU binds transiently across the entire nucleoid, with a preference for the origin of replication over the terminus. Reducing cellular HU levels impacts nucleoid maintenance and disrupts robust nucleoid scaling with cell size. This effect is similar to the distortion caused by fluoroquinolone-antibiotics, supporting earlier observations that HU is essential for maintaining DNA supercoiling. Furthermore, in cells lacking HU, the replication machinery is misplaced, and cells are unable to initiate and proceed with on-going replication. Chromosome conformation capture (Hi-C) experiments revealed that HU is required to maintain cohesion between the two chromosomal arms, in a similar way to the structural maintenance of the chromosome complex SMC. Together, we show that by promoting long-range chromosome interactions and supporting the architecture of the domain encompassing the origin, HU is fundamental for chromosome integrity and the intimately related processes of chromosome replication and segregation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=101 SRC="FIGDIR/small/615122v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@10cfa9eorg.highwire.dtl.DTLVardef@119ae12org.highwire.dtl.DTLVardef@f35cd4org.highwire.dtl.DTLVardef@1537e7b_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2024

Genome-wide CRISPRi-seq identified ferredoxin-NADP reductase FprB as a synergistic target for gallium therapy in Pseudomonas aeruginosa

With the rise of antibiotic-resistant bacteria, non-antibiotic therapies like gallium are increasingly gaining attention. Gallium ions exhibit potent activity against multidrug-resistant bacteria and intravenous gallium nitrite is under phase 2 clinical trials to treat chronic Pseudomonas aeruginosa infections in cystic fibrosis patients. However, its clinical efficacy is constrained by the achievable peak concentration in human tissue. To address this limitation, we applied a genome-wide CRISPR interference approach (CRISPRi-seq), to identify potential synergistic targets with gallium. Through the systemic screening, we classified the essential genes by response time and growth reduction, pinpointing the most vulnerable therapeutic targets in this species. In addition, we identified a highly conserved gene fprB, encoding a ferredoxin-NADP+ reductase, the downregulation of which dramatically sensitized the cells to gallium. Using a null mutant, we confirmed the loss of fprB lowers the minimum inhibitory concentration of gallium from 320 {micro}M to 10 {micro}M and shifted galliums mode of action from bacteriostatic to bactericidal. Further investigation revealed that FprB plays a critical role in modulating oxidative stress induced by gallium, via control of the iron homeostasis and reactive oxygen species accumulation. Deleting fprB also enhanced galliums efficacy against biofilms formation and improved outcomes in murine lung infection model of P. aeruginosa, suggesting FprB as a promising drug target in combination with gallium. Overall, our data showed CRISPRi-seq as a powerful tool for systematic genetic analysis of P. aeruginosa, advancing identification of novel therapeutic targets.

microbiology↗