Biology subjects

de Bakker, V.

Publications and source records attributed to de Bakker, V..

18 recordsLinked to original sources

bioRxiv2026

A TmaT-AftD interaction is required for the CmpL4 mycomembrane biogenesis pathway in Corynebacterium glutamicum.

Bacteria in the Mycobacteriales order like mycobacteria and corynebacteria surround themselves with a multilayered cell envelope. Their cytoplasmic membrane is fortified by a peptidoglycan cell wall that is decorated with branched arabinogalactan (AG) polymers. The AG glycans are further modified with mycolic acids to form an outer membrane. Biogenesis of this mycomembrane requires the transport of mycolates from their site of synthesis in the cytoplasmic membrane to the cell surface. How mycolate transport is controlled and coordinated with AG synthesis has remained unclear. Mycolate transport is mediated by the essential RND-family transporter MmpL3 in mycobacteria and a pair of related, partially redundant transporters called CmpL1 and CmpL4 in corynebacteria. The acetyltransferase TmaT has also been implicated in mycolate transport in both types of bacteria. In corynebacteria, it is required for the production of acetylated mycolates, and this modification has been proposed to promote mycolate transport via both CmpL transporters in the model organism Corynebacterium glutamicum (Cglu). Here, we reinvestigated the function of TmaT in Cglu and found that it and several factors encoded in the tmaT locus are specifically required for mycolate transport via the CmpL4 transporter pathway. Notably, one of these additional genes encodes the arabinosyltransferase AftD involved in AG biogenesis. TmaT and AftD were found to interact, and our results indicate that this interaction is required for acetylated mycolate production, mycolate transport via the CmpL4 pathway, and normal arabinan synthesis. Thus, the TmaT-AftD interaction may serve as a regulatory link connecting mycolate transport with AG biogenesis. SIGNIFICANCEMycobacteriales bacteria, including pathogens like Mycobacterium tuberculosis (Mtb), have a complex cell surface comprising an inner membrane, a cell wall modified with arabinogalactan (AG), and an outer mycomembrane made of mycolic acids linked to AG polymers. Because these surface biogenesis pathways are targeted by frontline anti-Mtb drugs, there is great interest in elucidating their underlying mechanisms. Here, we identify an interaction between factors involved in mycomembrane (TmaT) and AG biosynthesis (AftD) in the model organism Corynebacterium glutamicum. We show that this interaction is important for proper surface biogenesis and may therefore function to coordinate mycomembrane assembly with AG synthesis. This and other potential regulatory connections controlling envelope biogenesis represent attractive targets for future antibiotic development.

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

A potential role for acyl-phosphate in the coordination of phospholipid and lipopolysaccharide synthesis in Escherichia coli

The envelope of Gram-negative bacteria like Escherichia coli is multilayered with two membranes sandwiching a peptidoglycan cell wall. The inner membrane is a typical phospholipid bilayer whereas the outer membrane is asymmetric with phospholipids in the inner leaflet and lipopolysaccharide (LPS) in the outer leaflet. We recently discovered that inactivation of the conserved peptidoglycan synthesis machinery responsible for cell elongation causes defects in both peptidoglycan and LPS synthesis in E. coli. This finding suggests that the isolation of suppressors that rescue the growth phenotype caused by an impaired cell elongation system is an attractive means of identifying factors involved in coordinating the biogenesis of different envelope layers. Here, we report the results of a global, transposon sequencing-based screen for such suppressors. The inactivation of a number of factors including the phospholipid synthesis enzyme PlsX was found to partially suppress the growth defects of a cell elongation mutant. Deletion of plsX also conferred increased resistance to CHIR-090, an inhibitor of the committed step of LPS synthesis catalyzed by LpxC, suggesting that loss of PlsX function stimulates LPS synthesis. Evidence is presented that increased CHIR-090 resistance is not mediated by changes in the activity of the proteolytic system (YejM-LapB-FtsH) controlling LpxC turnover. Rather, our results are consistent with a model in which the phospholipid precursor acyl-phosphate produced by PlsX serves as an inhibitor of LpxC to lower the rate of LPS synthesis when phospholipid synthesis capacity is reduced. IMPORTANCEOver the last several decades, most proteins essential for Gram-negative cell surface assembly have been characterized. However, relatively little is known about how the synthesis of different envelope layers is coordinated to promote uniform surface growth. Here, we report the results of a transposon sequencing-based genetic screen for mutants that suppress defects in the conserved peptidoglycan synthesis machinery responsible for cell elongation. Inactivation of the plsX gene encoding a phospholipid synthesis enzyme was found to both suppress the growth defect of a cell elongation mutant and to confer elevated resistance to an inhibitor of lipopolysaccharide synthesis. Our results suggest the attractive possibility that the product of PlsX, acyl-phosphate, may play a regulatory role in coordinating the phospholipid and lipopolysaccharide synthesis pathways.

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↗

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

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

Strain-level and phenotypic stability contrasts with plasmid and phage variability in water kefir communities

Microbial communities can change in response to top-down factors, such as phages, and bottom-up factors, such as nutrient availability. Previous studies have successfully investigated bacterial species-level dynamics, but diversity and interactions beyond the species-level is usually lacking. Traditional fermented foods, such as water kefir, provide ideal systems to study ecological and evolutionary dynamics beyond the species-level, as they are simple and trackable systems that are cultivated in non-sterile, nutrient-rich environments which foster microbial growth and invasion. Despite the central role of only a few lactic acid bacteria for fermentation, little is known about the genomic diversity and dynamics of these community members over time. Within the framework of a graduate course, 35 students propagated water kefir across several generations under different nutrient conditions and in different households to study microbial responses over time. We found that water kefir communities were generally stable at the species-level, with only rare bacterial species replaced over long timescales (more than 2 years). While we observed little strain-level diversity with few strain replacements over long timescales, closely related strains exhibited variation in accessory gene content, often encoded on plasmids, particularly those involved in ecologically meaningful functions such as sugar utilization pathways and phage defense systems. We hypothesise that these genomic variations could reflect the adaptations of strains to different sugars and phages. Consistent with this, we observed a diverse array of phages, many likely originating from the unique household environments. By documenting the genomic landscape of microbial species, strains, plasmids, and phages, this study advances our understanding of the diversity and dynamics of microbial communities in fermented foods. Furthermore, our course material is publicly available and offers a blueprint for bridging the gap between teaching and research, inspiring the next generation of scientists to unravel the complexities of microbial ecosystems. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=137 SRC="FIGDIR/small/640646v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@56db6borg.highwire.dtl.DTLVardef@5f6dc6org.highwire.dtl.DTLVardef@11fff14org.highwire.dtl.DTLVardef@1a37a9f_HPS_FORMAT_FIGEXP M_FIG C_FIG

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

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↗

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

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↗

bioRxiv2022

Deep divergence and genomic diversification of gut symbionts of neotropical stingless bees

Social bees harbor conserved gut microbiota that may have been acquired in a common ancestor of social bees and subsequently co-diversified with their hosts. However, most of this knowledge is based on studies on the gut microbiota of honey bees and bumble bees. Much less is known about the gut microbiota of the third and most diverse group of social bees, the stingless bees. Specifically, the absence of genomic data from their microbiota presents an important knowledge gap in understanding the evolution and functional diversity of the social bee microbiota. Here we combined community profiling with culturing and genome sequencing of gut bacteria from six neotropical stingless bee species from Brazil. Phylogenomic analyses show that most stingless bee gut isolates form deep-branching sister clades of core members of the honey bee and bumble bee gut microbiota with conserved functional capabilities, confirming the common ancestry and ecology of their microbiota. However, our bacterial phylogenies were not congruent with those of the host indicating that the evolution of the social bee gut microbiota was not driven by strict co-diversification, but included host switches and independent symbiont gain and losses. Finally, as reported for the honey bee and bumble bee microbiota, we find substantial genomic divergence among strains of stingless bee gut bacteria suggesting adaptation to different host species and glycan niches. Our study offers first insights into the genomic diversity of the stingless bee microbiota, and highlights the need for broader samplings to understand the evolution of the social bee gut microbiota. ImportanceStingless bees are the most diverse group of the corbiculate bees and represent important pollinator species throughout the tropics and subtropics. They harbor specialized microbial communities in their gut that are related to those found in honey bees and bumble bees and that are likely important for bee health. Few bacteria have been cultured from the gut of stingless bees which has prevented characterization of their genomic diversity and functional potential. Here, we established cultures of major community members of the gut microbiota of six stingless bee species and sequenced their genomes. We find that most stingless bee isolates belong to novel bacterial species distantly related to those found in honey bees and bumble bees and encoding similar functional capabilities. Our study offers a new perspective on the evolution of the social bee gut microbiota and presents the basis to characterize the symbiotic relationships between gut bacteria and stingless bees.

microbiology↗

bioRxiv2021

Amoxicillin-resistant Streptococcus pneumoniae can be resensitized by targeting the mevalonate pathway as identified by sCRilecs-seq

Antibiotic resistance in the important opportunistic human pathogen Streptococcus pneumoniae is on the rise. This is particularly problematic in the case of the {beta}-lactam antibiotic amoxicillin, which is the first-line therapy. It is therefore crucial to uncover targets that would kill or resensitize amoxicillin-resistant pneumococci. To do so, we developed a genome-wide, single-cell based, gene silencing screen using CRISPR interference called sCRilecs-seq (subsets of CRISPR interference libraries extracted by fluorescence activated cell sorting coupled to next generation sequencing). Since amoxicillin affects growth and division, sCRilecs-seq was used to identify targets that are responsible for maintaining proper cell size. Our screen revealed that downregulation of the mevalonate pathway leads to extensive cell elongation. Further investigation into this phenotype indicates that it is caused by insufficient transport of cell wall precursors across the cell membrane due to a limitation in the production of undecaprenyl phosphate (Und-P), the lipid carrier responsible for this process. The data suggest that whereas peptidoglycan synthesis continues even with reduced Und-P levels, cell constriction is specifically halted. We successfully exploited this knowledge to create a combination treatment strategy where the FDA-approved drug clomiphene, an inhibitor of Und-P synthesis, is paired up with amoxicillin. Our results show that clomiphene potentiates the antimicrobial activity of amoxicillin and that combination therapy resensitizes amoxicillin-resistant S. pneumoniae. These findings could provide a starting point to develop a solution for the increasing amount of hard-to-treat amoxicillin-resistant pneumococcal infections.

microbiology↗

bioRxiv2020

Exploration of bacterial bottlenecks and Streptococcus pneumoniae pathogenesis by CRISPRi-seq

Streptococcus pneumoniae is a commensal bacterium of the human nasopharynx, but can cause harmful infections if it spreads to other parts of the body, such as pneumonia, sepsis or meningitis. To facilitate pathogenesis studies, we constructed a doxycycline-inducible pooled CRISPR interference (CRISPRi) library targeting all operons in protypical S. pneumoniae strain D39V. Our library design allows fitness within the pool to be assessed by a one-step PCR reaction directly followed by Illumina sequencing (CRISPRi-seq). The doxycycline-inducible CRISPRi system is tightly controllable and suitable for both bottleneck exploration and evaluation of gene fitness in vitro and in vivo. Here, we applied CRISPRi-seq to identify genetic factors important for causing pneumococcal pneumonia. Mice were infected intratracheally with our CRISPRi library and bacteria collected at 24 h (from lung) and 48 h (from both lung and blood) post-infection. CRISPRi-seq showed a critical bottleneck at 48 h after intratracheal infection, with only a few bacteria surviving the brunt of the innate immune response to cause systemic infection. However, earlier at 24 h post-infection, many significant differences in gene fitness cost between in vitro and in vivo conditions were identified, including genes encoding known and putative novel virulence factors, genes essential only in vivo, and genes essential only in vitro. A key advantage of CRISPRi-seq over traditional transposon-based genetic screens is that all genes, including essential genes, can be tested for their role in virulence and pathogenicity. The approaches developed here should be generally applicable to study infection bottlenecks and in vivo fitness for other important human and animal pathogens.

microbiology↗