Biology subjects

Veening, J.-W.

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

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Dual CRISPRi-Seq for genome-wide genetic interaction studies identifies key genes involved in the pneumococcal cell cycle

A major goal in biology is to uncover the relationship between genotype and phenotype. However, identifying gene function is often hampered by genetic redundancy. For example, under standard laboratory conditions, three-quarters of the genes in the human pathogen Streptococcus pneumoniae are non-essential. A powerful approach to unravel genetic redundancy is by identifying gene-gene interactions. To uncover genetic interactions (GIs) in S. pneumoniae on a genome-wide scale, a generally applicable dual CRISPRi-Seq method and associated analysis pipeline was developed. Specifically, we created a library of 869 dual sgRNAs targeting high-confidence operons that encode essential and non-essential genes, covering over 70% of the genetic elements in the pneumococcal genome. Testing these 378,015 unique combinations, 4,026 significant GIs were identified, including 1,935 negative and 2,091 positive interactions. Besides known GIs, we found and confirmed previously unknown interactions involving genes responsible for fundamental cellular processes such as cell division, cell shape maintenance, and chromosome segregation. The presented methods and bioinformatic approaches can serve as a roadmap for genome-wide gene interaction studies in other organisms. Lastly, all interactions are available for exploration via the Pneumococcal Genetic Interaction Network (PneumoGIN) at https://veeninglab.shinyapps.io/PneumoGIN, which can serve as a starting point for new biological discoveries and translational research.

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↗

bioRxiv2024

The Streptococcus pneumoniae transcriptome in patient cerebrospinal fluid identifies novel virulence factors required for meningitis

To better understand Streptococcus pneumoniae pathogenesis we performed RNA sequencing on cerebrospinal fluid (CSF) from meningitis patients to identify bacterial genes expressed during invasion of the central nervous system. Comparison to transcriptome data for serotype 1 S. pneumoniae cultured in ex vivo human CSF defined a subset of 57 genes with high expression during human meningitis. Deletion of two of the most highly expressed genetic loci, bgaA (encodes for a {beta}-galactosidase) or the SP_1801-5 putative stress response operon, resulted in S. pneumoniae strains still able to transmigrate the blood brain barrier but which were more susceptible to complement opsonisation and unable to maintain brain infection in a murine meningitis model. In 1144 meningitis patients, infection with bgaA containing S. pneumoniae strains was associated with a higher mortality (22% versus 14% p=0.02). These data demonstrate that direct bacterial RNAseq from CSF can identify previously undescribed S. pneumoniae virulence factors required for meningitis pathogenesis.

microbiology↗

bioRxiv2024

An unsupervised deep learning framework encodes super-resolved image features to decode bacterial cell cycle

Super-resolution microscopy can resolve cellular features at the nanoscale. However, increased spatial resolution comes with increased phototoxicity, and reduced temporal resolution. As a result, studies that require the highest spatial resolutions often rely on static or fixed images, lacking dynamic information. This is particularly true of bacteria, whose lateral dimensions approach the scale of the diffraction limit. In this work, we present Enso, a method based on unsupervised machine learning to recover bacterial cell cycle and cell type information from static single molecule localization microscopy (SMLM) images, whilst retaining nanoscale spatial resolution. Enso uses single-cell images as input, and orders cells according to their spatial pattern progression, ultimately linked to the cell cycle. Our method requires no a priori knowledge or categories, and is validated on both simulated and user-annotated experimental data.

bioinformatics↗

bioRxiv2024

An efficient in vivo-inducible CRISPR interference system for group A Streptococcus genetic analysis and pathogenesis studies

While genome-wide transposon mutagenesis screens have identified numerous essential genes in the significant human pathogen Streptococcus pyogenes (group A Streptococcus or GAS), many of their functions remain elusive. This knowledge gap is attributed in part to the limited molecular toolbox for controlling GAS gene expression and the bacteriums poor genetic transformability. CRISPR interference (CRISPRi), using catalytically inactive GAS Cas9 (dCas9), is a powerful approach to specifically repress gene expression in both bacteria and eukaryotes, but ironically has never been harnessed for controlled gene expression in GAS. In this study, we present a highly transformable and fully virulent serotype M1T1 GAS strain and introduce a doxycycline-inducible CRISPRi system for efficient repression of bacterial gene expression. We demonstrate highly efficient, oligo-based sgRNA cloning directly to GAS, enabling the construction of a gene knockdown strain in just two days, in contrast to the several weeks typically required. The system is shown to be titratable and functional both in vitro and in vivo using a murine model of GAS infection. Furthermore, we provide direct in vivo evidence that the expression of the conserved cell division gene ftsZ is essential for GAS virulence, highlighting its promise as a target for emerging FtsZ-inhibitors. Finally, we introduce SpyBrowse (https://veeninglab.com/SpyBrowse), a comprehensive and user-friendly online resource for visually inspecting and exploring GAS genetic features. The tools and methodologies described in this work are poised to facilitate fundamental research in GAS, contribute to vaccine development, and aid in the discovery of antibiotic targets. Significance statementWhile GAS remains a predominant cause of bacterial infections worldwide, there are limited genetic tools available to study its basic cell biology. Here, we bridge this gap by creating a highly transformable, fully virulent M1T1 GAS strain. In addition, we established a tight and titratable doxycycline-inducible system and developed CRISPR interference for controlled gene expression in GAS. We show that CRISPRi is functional in vivo in a mouse infection model. Additionally, we present SpyBrowse, an intuitive and accessible genome browser (https://veeninglab.com/SpyBrowse). Overall, this work overcomes significant technical challenges of working with GAS, and together with SpyBrowse, represents a valuable resource for researchers in the GAS field.

microbiology↗

bioRxiv2023

The function of CozE proteins is linked to lipoteichoic acid biosynthesis in Staphylococcus aureus

To maintain cell integrity and facilitate cell division in Staphylococcus aureus, a well-coordinated interplay between membrane biogenesis, peptidoglycan formation, and teichoic acid synthesis is crucial. However, the molecular mechanisms and regulatory pathways that underpin their coordination are still poorly understood. CozE constitute a conserved family of membrane proteins implicated in cell division via regulation of penicillin binding proteins. It has been shown that the two staphylococcal cozE genes (cozEa and cozEb) constitute a synthetic lethal gene pair. Depletion of CozEa and CozEb simultaneously in S. aureus resulted in severely defective cell division phenotypes, reminiscent of cell lacking lipoteichoic acid (LTA). Indeed, we demonstrate that there is an intricate interplay between CozE, biosynthesis of LTA, and membrane homeostasis in S. aureus. By screening for potential genetic links, we establish that there is synthetic lethal relationship between CozE and UgtP, the enzyme synthesizing the LTA glycolipid anchor Glc2DAG. On the contrary, in cells lacking LtaA, the flippase of Glc2DAG, the essentiality of CozEa and CozEb was alleviated. Furthermore, by immunoblotting, we found that CozEb plays a unique role in controlling LTA polymer length and stability. Using reconstituted proteoliposomes, we also demonstrated that CozE proteins modulate the glycolipid flipping activity of LtaA in vitro. Together, the results demonstrate a new function of CozE proteins, facilitating proper membrane homeostasis and LTA biosynthesis in S. aureus.

microbiology↗

bioRxiv2023

Genome-wide CRISPRi screens reveal the essentialome and determinants for susceptibility to dalbavancin in Staphylococcus aureus

Antibiotic resistance and tolerance remain a major problem for treatment of staphylococcal infections. Knowing genes that influence antibiotic susceptibility could open the door to novel antimicrobial strategies, including targets for new synergistic drug combinations. Here, we developed a genome-wide CRISPR interference library for Staphylococcus aureus, demonstrated its use by quantifying the essentialome in different strains through CRISPRi-seq, and used it to identify genes that modulate susceptibility to the lipoglycopeptide dalbavancin. By exposing the library to sublethal concentrations of dalbavancin using both CRISPRi-seq and direct selection methods, we found genes previously reported to be involved in antibiotic susceptibility, but also identified genes thus far unknown to affect antibiotic tolerance. Importantly, some of these genes could not have been detected by more conventional knock-out approaches because they are essential for growth, stressing the complementary value of CRISPRi-based methods. Notably, knockdown of a gene encoding the uncharacterized protein KapB specifically sensitizes the cells to dalbavancin, but not to other antibiotics of the same class, while knockdown of the Shikimate pathway surprisingly has the opposite effect. The results presented here demonstrate the potential of CRISPRi-seq screens to identify genes and pathways involved in antibiotic susceptibility and pave the way to explore alternative antimicrobial treatments through these insights.

microbiology↗

bioRxiv2023

The ClpX chaperone and a hypermorphic FtsA variant with impaired self-interaction are mutually compensatory for coordinating Staphylococcus aureus cell division

Bacterial cell division requires the coordinated assembly and disassembly of a large protein complex called the divisome, however, the exact role of molecular chaperones in this critical process remains unclear. In the important pathogenic bacterium Staphylococcus aureus, the ClpX chaperone is essential for growth at 30{degrees}C and microscopic analyses suggested that ClpX plays a temperature-dependent role in cell division. We here provide genetic evidence that ClpX unfoldase activity is a determinant for proper coordination of cell division by showing that a spontaneous G325V substitution in the ATP-binding domain of the essential FtsA cell division protein rescues growth and septum synthesis in a Staphylococcus aureus clpX mutant. The polymerization state of FtsA is thought to control initiation of bacterial septum synthesis and, while restoring the aberrant FtsA dynamics in clpX cells, the FtsAG325V variant displayed reduced ability to interact with itself and other cell division proteins. In wild-type cells, the ftsAG325V allele shared phenotypes with E. coli superfission ftsA mutants and accelerated the cell cycle, increased the risk of daughter cell lysis, and conferred sensitivity to heat and antibiotics inhibiting cell wall synthesis. Strikingly, lethality was mitigated by spontaneous mutations that inactivate ClpX. Taken together, our results suggest that ClpX promotes septum synthesis by antagonizing FtsA interactions and illuminates the critical role of a protein unfoldase in coordinating bacterial cell division. IMPORTANCEEssential biological processes, such as cell division, are performed by multiple proteins working together in dynamic functional complexes. In eukaryotic cells, the disassembly of such molecular machines is often assisted by molecular chaperones capable of unfolding proteins. The ClpX unfoldase is conserved from bacteria to humans, however, the roles of ClpX in bacterial cell biology remain relatively unexplored. By combining genetic methods with super-resolution microscopy techniques, we show here that ClpX and a mutant variant of the essential cell division protein FtsA mutually compensate for each other in controlling cell division of the pathogenic bacterium Staphylococcus aureus. The selected FtsAG325V variant has diminished self-interactions and restored the aberrant FtsA dynamics in clpX cells suggesting that ClpX promotes cell division by antagonizing FtsA protein interactions. This study, for the first time, illuminates the important role of protein unfoldases in the functioning of the divisome, a multienzyme complex fundamental to bacterial reproduction.

molecular biology↗

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↗

bioRxiv2023

A conserved antigen induces respiratory Th17-mediated serotype-independent protection against pneumococcal superinfection

Several vaccines targeting bacterial pathogens show reduced efficacy in the context of intercurrent viral infection indicating a new vaccinology approach is required to protect against such superinfections. To find antigens for the human pathogen Streptococcus pneumoniae that are effective following influenza infection, we performed CRISPRi-seq in a murine model of superinfection and identified the highly conserved lafB gene as virulence factor. We show that LafB is a membrane-associated, intracellular protein that catalyzes the formation of galactosyl-glucosyl-diacylglycerol, a glycolipid we show is important for cell wall homeostasis. Respiratory vaccination with recombinant LafB, in contrast to subcutaneous vaccination, was highly protective against all serotypes in a murine model. In contrast to standard pneumococcal capsule-based conjugate vaccines, protection did not require LafB-specific antibodies but was dependent on airway CD4+ T helper 17 cells. Healthy human individuals can elicit LafB-specific immune responses, suggesting its merit as a universal pneumococcal vaccine antigen that remains effective following influenza infection. One-Sentence SummaryDiscovery of a universal pneumococcal vaccine protective during superinfection.

microbiology↗

bioRxiv2023

Rewiring capsule production by CRISPRi-based genetic oscillators demonstrates a functional role of phenotypic variation in pneumococcal-host interactions

Phenotypic variation is the phenomenon in which clonal cells display different traits even under identical environmental conditions. This plasticity is thought to be important for processes including bacterial virulence1-8, but direct evidence for its relevance is often lacking. For instance, variation in capsule production in the human pathogen Streptococcus pneumoniae has been linked to different clinical outcomes9-14, but the exact relationship between variation and pathogenesis is not well understood due to complex natural regulation15-20. In this study, we used synthetic oscillatory gene regulatory networks (GRNs) based on CRISPR interference together with live cell microscopy and cell tracking within microfluidics devices to mimic and test the biological function of bacterial phenotypic variation. We provide a universally applicable approach for engineering intricate GRNs using only two components: dCas9 and extended sgRNAs (ext-sgRNAs). Our findings demonstrate that variation in capsule production is beneficial for pneumococcal fitness in traits associated with pathogenesis providing conclusive evidence for this longstanding question.

microbiology↗

bioRxiv2023

CRISPRi-TnSeq: A genome-wide high-throughput tool for bacterial essential-nonessential genetic interaction mapping

Genetic interaction networks can help identify functional connections between genes and pathways, which can be leveraged to establish (new) gene function, drug targets, and fill pathway gaps. Since there is no optimal tool that can map genetic interactions across many different bacterial strains and species, we develop CRISPRi-TnSeq, a genome-wide tool that maps genetic interactions between essential genes and nonessential genes through the knockdown of a targeted essential gene (CRISPRi) and the simultaneous knockout of individual nonessential genes (Tn-Seq). CRISPRi-TnSeq thereby identifies, on a genome-wide scale, synthetic and suppressor-type relationships between essential and nonessential genes, enabling the construction of essential-nonessential genetic interaction networks. To develop and optimize CRISPRi-TnSeq, CRISPRi strains were obtained for 13 essential genes in Streptococcus pneumoniae, involved in different biological processes including metabolism, DNA replication, transcription, cell division and cell envelope synthesis. Transposon-mutant libraries were constructed in each strain enabling screening of [~]24,000 gene-gene pairs, which led to the identification of 1,334 genetic interactions, including 754 negative and 580 positive genetic interactions. Through extensive network analyses and validation experiments we identify a set of 17 pleiotropic genes, of which a subset tentatively functions as genetic capacitors, dampening phenotypic outcomes and protecting against perturbations. Furthermore, we focus on the relationships between cell wall synthesis, integrity and cell division and highlight: 1) how essential gene knockdown can be compensated by rerouting flux through nonessential genes in a pathway; 2) the existence of a delicate balance between Z-ring formation and localization, and septal and peripheral peptidoglycan (PG) synthesis to successfully accomplish cell division; 3) the control of c-di-AMP over intracellular K+ and turgor, and thereby modulation of the cell wall synthesis machinery; 4) the dynamic nature of cell wall protein CozEb and its effect on PG synthesis, cell shape morphology and envelope integrity; 5) functional dependency between chromosome decatenation and segregation, and the critical link with cell division, and cell wall synthesis. Overall, we show that CRISPRi-TnSeq uncovers genetic interactions between closely functionally linked genes and pathways, as well as disparate genes and pathways, highlighting pathway dependencies and valuable leads for gene function. Importantly, since both CRISPRi and Tn-Seq are widely used tools, CRISPRi-TnSeq should be relatively easy to implement to construct genetic interaction networks across many different microbial strains and species.

genetics↗

bioRxiv2022

Competence remodels the pneumococcal cell wall providing resistance to fratricide and surface exposing key virulence factors

Competence development in the human pathogen Streptococcus pneumoniae controls several features such as genetic transformation, biofilm formation and virulence. Competent bacteria produce so called fratricins such as CbpD, that kill non-competent siblings by cleaving peptidoglycan (PGN). CbpD is a choline-binding protein (CBP) that binds to phosphorylcholine residues found on wall- and lipoteichoic acids (WTA and LTA) that together with PGN are major constituents of the pneumococcal cell wall. Competent pneumococci are protected against fratricide by producing the immunity protein ComM. How competence and fratricide contribute to virulence is unknown. Here, using a genome-wide CRISPRi-seq screen, we show that genes involved in teichoic acid biosynthesis are essential during competence. We demonstrate that LytR is the major enzyme mediating the final step in WTA formation, and that, together with ComM, is essential for immunity against CbpD. Importantly, we show that key virulence factors PspA and PspC become more surface-exposed at midcell during competence, in a CbpD-dependent manner. Together, our work supports a model in which activation of competence is crucial for host adherence by increased surface exposure of its various CBPs.

microbiology↗

bioRxiv2022

A genome-wide CRISPRi screen reveals a StkP-mediated connection between cell-wall integrity and competence in Streptococcus salivarius

Competence is one of the most efficient bacterial evolutionary and adaptative strategies by synchronizing production of antibacterial compounds and integration of DNA released by dead cells. In most streptococci, this tactic is orchestrated by the ComRS system, a pheromone communication device providing a sharp time window of activation in which only part of the population is responsive. Understanding how this developmental process integrates multiple inputs to fine-tune the adequate response is a long-standing question. However, essential genes involved in the regulation of ComRS have been challenging to study. In this work, we built a conditional mutant library using CRISPR-interference and performed three complementary screens to investigate competence genetic regulation in the human commensal Streptococus salivarius. We show that initiation of competence increases upon cell-wall impairment, suggesting a connection between cell envelope stress and competence activation. Notably, we report a key role for StkP, a serine-threonine kinase known to regulate cell-wall homeostasis. We show that StkP controls competence by a mechanism that reacts to peptidoglycan fragments. Together, our data suggest a key cell-wall sensing mechanism coupling competence to cell envelope integrity. IMPORTANCESurvival of human commensal streptococci in the digestive tract requires efficient strategies which must be tightly and collectively controlled for responding to competitive pressure and drastic environmental changes. In this context, the autocrine signaling system ComRS controlling competence for natural transformation and predation in salivarius streptococci could be seen as a multi-input device integrating a variety of environmental stimuli. In this work, we revealed novel positive and negative competence modulators by using a genome-wide CRISPR- interference strategy. Notably, we highlighted an unexpected connection between bacterial envelope integrity and competence activation that involves several cell-wall sensors. Together, these results showcase how commensal streptococci can fine-tune the pheromone-based competence system by responding to multiple inputs affecting their physiological status in order to calibrate an appropriate collective behavior.

microbiology↗

bioRxiv2022

Pneumolysin promotes host cell necroptosis and bacterial competence during pneumococcal meningitis as shown by whole animal dual RNA-seq

Pneumolysin is a major virulence factor of Streptococcus pneumoniae that plays a key role in interaction with the host during invasive disease. How pneumolysin influences these dynamics between host and pathogen interaction during early phase of central nervous system infection in pneumococcal meningitis remains unclear. Using a whole animal in vivo dual RNA-seq approach, we identified pneumolysin-specific transcriptional responses in both S. pneumoniae and zebrafish (Danio rerio) during early pneumococcal meningitis. By functional enrichment analysis we identified host pathways known to be activated by pneumolysin, and discovered the importance of necroptosis for host survival. Inhibition of this pathway using the drugs necrostatin-5 or GSK872 increased host mortality during pneumococcal meningitis. On the pathogens side, we find that pneumolysin-dependent competence activation is crucial for intra-host replication and virulence and that not all bacteria activate competence at the same time. Altogether, this study provides new insights into pneumolysin-specific transcriptional responses and identifies key pathways involved in pneumococcal meningitis. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/479878v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1214d1aorg.highwire.dtl.DTLVardef@14d0e8corg.highwire.dtl.DTLVardef@1f6133aorg.highwire.dtl.DTLVardef@685955_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LIPneumolysin-specific host and bacterial responses as identified by whole animal dual RNA-seq, available at https://veeninglab.com/dual-danio C_LIO_LIDiscovery of a functional necroptosis or necroptosis-like pathway in zebrafish C_LIO_LIHeterogeneity in competence development during infection C_LIO_LICompetence development is an important virulence determinant C_LI

microbiology↗

bioRxiv2021

2FAST2Q: A general-purpose sequence search and counting program for FASTQ files

The increasingly widespread use of next generation sequencing protocols has brought the need for the development of user-friendly raw data processing tools. Here, we present 2FAST2Q, a versatile and intuitive standalone program capable of extracting and counting feature occurrences in FASTQ files. 2FAST2Q can be used in any experimental setup that requires feature extraction from raw reads, being able to quickly handle mismatch alignments, nucleotide wise Phred score filtering, custom read trimming, and sequence searching within a single program. Using published CRISPRi datasets in which Escherichia coli and Mycobacterium tuberculosis gene essentiality, as well as host-cell sensitivity towards SARS-CoV2 infectivity were tested, we demonstrate that 2FAST2Q efficiently recapitulates the output in read counts per provided feature as with traditional pipelines. Moreover, we show how different FASTQ read filtering parameters impact downstream analysis, and suggest a default usage protocol. 2FAST2Q has a familiar user interface and uses a custom sequence mismatch search algorithm, taking advantage of Pythons numba module JIT runtime speeds. It is thus easier to use and faster than currently available tools, efficiently processing large CRISPRi-Seq or random-barcode sequencing datasets on any up-to-date laptop. 2FAST2Q is available as an executable file for all current operating systems without installation and as a Python3 module on the PyPI repository (available at https://veeninglab.com/2fast2q). We expect that 2FAST2Q will not only be useful for people working in microbiology but also for other fields in which amplicon sequencing data is generated.

bioinformatics↗

bioRxiv2021

The acquisition of clinically relevant amoxicillin resistance in Streptococcus pneumoniae requires ordered horizontal gene transfer of four loci

Understanding how antimicrobial resistance spreads is critical for optimal application of new treatments. In the naturally competent human pathogen Streptococcus pneumoniae, resistance to {beta}-lactam antibiotics is mediated by recombination events in genes encoding the target proteins, resulting in reduced drug binding affinity. However, for the front-line antibiotic amoxicillin, the exact mechanism of resistance still needs to be elucidated. Through successive rounds of transformation with genomic DNA from a clinically resistant isolate, we followed amoxicillin resistance development. Using whole genome sequencing, we showed that multiple recombination events occurred at different loci during one round of transformation. We found examples of non-contiguous recombination, and demonstrated that this could occur either through multiple D-loop formation from one donor DNA molecule, or by the integration of multiple DNA fragments. We also show that the final minimum inhibitory concentration (MIC) differs depending on recipient genome, explained by differences in the extent of recombination at key loci. Finally, through back transformations of mutant alleles and fluorescently labelled penicillin (bocillin-FL) binding assays, we confirm that pbp1a, pbp2b, pbp2x, and murM are the main resistance determinants for amoxicillin resistance, and that the order of allele uptake is important for successful resistance evolution. We conclude that recombination events are complex, and that this complexity contributes to the highly diverse genotypes of amoxicillin-resistant pneumococcal isolates.

microbiology↗