Biology subjects

Vollmer, D.

Publications and source records attributed to Vollmer, D..

6 recordsLinked to original sources

bioRxiv2026

Trichomonas vaginalis targets Lactobacillus jensenii via pseudopodia-independent phagocytosis and secreted lysozyme TvGH25

A low abundance or absence of protective lactobacilli during acute trichomoniasis is a well-known phenomenon that is associated with T. vaginalis (TV) infection. However, a crucial question that remains unanswered is whether alterations in the lactobacilli population precede TV infection or whether the parasite plays an active role in lactobacilli disappearance. Our findings showed that TV efficiently phagocytosed one of the dominant Lactobacillus species L. jensenii (LJ). Phagocytosis proceeds via a pseudopodia-independent mechanism reminiscent of sinking with a preference for viable cells. The presence of viable LJ leads to an increase in secretion of 27 TV proteins, including TvGH25 lysozyme. This enzyme cleaves peptidoglycan, a major component of the bacterial cell wall. TV overexpressing TvGH25 effectively lowers the bacterial cell count, evidencing the enzymes antimicrobial potential. These data support the notion that TV cells can suppress the Lactobacillus population through a combination of targeted secretory response and phagocytic activity, revealing novel potential targets for developing alternative therapeutic strategies against trichomoniasis. Significance StatementTrichomonas vaginalis (TV) is a sexually transmitted parasite that causes trichomoniasis and is connected to the disruption of the healthy vaginal microbiome, dominated by Lactobacillus species. However, the nature of the interactions between TV and Lactobacillus is poorly understood. In this study, we show that TV uses an unusual form of pseudopodia-independent phagocytosis to engulf L. jensenii alongside a targeted secretory response to the bacterial encounter, involving TvGH25 lysozyme. We found that TV acquired this enzyme by lateral gene transfer from bacteria and repurposed it against bacteria to degrade the major bacterial cell wall component peptidoglycan. TvGH25 thus represents an effective component of TVs antibacterial arsenal. Our findings provide new insights into the mechanistic disruption of the protective Lactobacillus microbiota.

microbiology↗

bioRxiv2026

Vi-TIS: a transposon insertion sequencing framework for quantitative discrimination of viable and non viable mutants

Transposon-insertion sequencing (TIS) is a high-throughput approach that uses randomly generated transposon mutant libraries to assess bacterial gene essentiality and their contribution to fitness under defined conditions. The method integrates classical transposon mutagenesis with next-generation sequencing. However, conventional TIS workflows generally require at least one active growth step to enrich for replicating or fit mutants. The outcome of these experiments can be biased by the recovery of non-replicating cells or by the loss of slow-growing mutants. To overcome this limitation, we modified the traditional TIS workflow by incorporating propidium monoazide (PMA). PMA is membrane-impermeant and excluded from viable cells but can enter dead cells when the membrane has lost its integrity. PMA covalently crosslinks to DNA upon exposure to UV light. Such binding inhibits PCR amplification, an essential step in TIS library preparation. This property enables selective exclusion of DNA originating from dead cells and reduces the signal to noise ratio in TIS experiments. We refer to this modified approach as Viability-TIS (Vi-TIS). Applying Vi-TIS to our ultra-dense Escherichia coli K-12 BW25113 TIS library enhanced the accuracy of essential gene identification. We further exposed the library to subinhibitory concentrations of carbenicillin and found that Vi-TIS produced results consistent with those obtained using culture-based methodologies. In addition, Vi-TIS revealed previously unrecognized carbenicillin-susceptible mutants. Finally, we demonstrate that the mutations are linked to perturbations in peptidoglycan biosynthesis.

microbiology↗

bioRxiv2026

The phenotypic landscape of the model firmicute Bacillus subtilis

Firmicutes are gram-positive bacteria with important roles in human health, disease, and industry. However, more than a quarter of genes in the model firmicute Bacillus subtilis remain completely uncharacterized, including numerous core phylum-specific genes. Here, we design a compact pooled CRISPRi library targeting all protein-coding genes in B. subtilis and test its growth in [~]150 stress conditions. Using data from this screen as a hypothesis generator, we perform targeted experiments, revealing that the conserved essential firmicute protein YneF is part of the SRP co-translational protein secretion pathway. We also demonstrate that ECF-transporters play a previously unknown but broadly conserved role in cell wall homeostasis, perform an unbiased analysis of amino acid crossfeeding, and make additional discoveries about bacterial competition. In addition to these major contributions to our understanding of B. subtilis biology (and gram-positive firmicutes in general), this work provides a rich dataset that will nucleate future studies of uncharacterized genes and presents a framework for accessible full-genome functional genomic screens in other bacteria. SIGNIFICANCELarge-scale chemical genomics screens facilitate the characterization of genes by generating phenotypic data across a library of gene mutants. Here, using CRISPRi, we designed a compact pooled library targeting all genes in B. subtilis and screened growth of the library in [~]150 conditions. Importantly, we used this dataset to expand B. subtilis biology on levels ranging from molecular pathways to bacterial communities. We discovered a role for an essential firmicute gene in protein secretion, implicated two new players in cell wall homeostasis, and unraveled fundamental factors driving competition and cooperation in the soil. This rich dataset will serve as a hypothesis generator, expanding our set of bacterial phenotypic data and driving future experiments in B. subtilis and other gram-positive firmicutes.

microbiology↗

bioRxiv2026

PBP1A and LdtJ support cell envelope homeostasis and impact selection of Colistin-resistance in Acinetobacter baumannii

The multilayered cell envelope of Acinetobacter baumannii is an essential structure that maintains cellular integrity and protects the bacterial cell against external stresses and antibiotics. It consists of an inner membrane, a thin peptidoglycan (PG) layer and an asymmetric outer membrane (OM) enriched in lipooligosaccharide (LOS), whose lipid A moiety is the target of colistin, a last-resource antibiotic. Although lipid A is essential in most Gram-negatives, A. baumannii can survive without LOS through envelope remodeling, particularly in strains producing low levels of the bifunctional penicillin-binding protein PBP1A (encoded by mrcA) or in {Delta}mrcA mutants. Here, we identify a functional interplay between the LD-transpeptidase LdtJ, which generates 3-3 cross-links, and PBP1A, which catalyzes 4-3 transpeptidation during PG synthesis. We show that simultaneous inactivation of both enzymes severely affected growth, viability, morphology, and OM homeostasis. PG analyses revealed that the {Delta}ldtJ {Delta}mrcA mutants displays reduced overall cross-linkage and shorter glycan chains, producing a weakened sacculus. Co-immunoprecipitation demonstrated that PBP1A associates with LdtJ, supporting their coordinated activity at sites of PG synthesis. Notably, {Delta}ldtJ {Delta}mrcA mutants exhibited the highest recovery frequency of colistin-resistant, LOS-deficient variants compared with wild type or single mutants. Together, our findings demonstrate that coupling between 4-3 and 3-3 transpeptidation is critical for envelope stability in A. baumannii and highlight how disrupting this coordination favors the emergence of colistin resistance. This work identifies a conserved PG remodeling vulnerability that directly links PG integrity to the evolution of antibiotic resistance, offering a new conceptual framework for destabilizing the A. baumannii envelope. ImportanceThe global rise of multidrug-resistant Acinetobacter baumannii represents an urgent clinical threat, largely driven by its extraordinary capacity to withstand cell envelope damages and escape last-resort antibiotics such as colistin. Although peptidoglycan synthesis and remodeling are known to influence outer membrane stability, how these processes are coordinated in A. baumannii has remained unclear. Here, we identify a crucial interplay between two critical envelope biogenesis and remodeling activities, 4-3 and 3-3 transpeptidations, mediated by the bifunctional PBP1A and the LD-transpeptidase LdtJ, respectively. Disrupting this coordination weakens the peptidoglycan, destabilizes the outer membrane, and increases the emergence frequency of colistin-resistant, LOS-deficient variants. These findings highlight a previously unrecognized vulnerability in the envelope homeostasis of A. baumannii, suggesting that simultaneously targeting DD and LD-transpeptidation could potentiate therapeutic strategies aimed at limiting antibiotic resistance development.

microbiology↗

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↗

bioRxiv2023

Repair oligodendrocytes demyelinating and disintegrating damaged axons after injury

After a spinal cord injury, axons fail to regrow, which results in permanent loss of function1. This is in contrast with peripheral axons that can regrow efficiently after injury2. These differences are partly due to the different plasticity of myelinating cells, Schwann cells and oligodendrocytes, in these two systems3. The molecular mechanisms underlying this different plasticity remain however poorly understood. Here, we show that the phosphatase Dusp64 is a master inhibitor of oligodendrocyte plasticity after spinal cord injury. Dusp6 is rapidly downregulated in Schwann cells and upregulated in oligodendrocytes after axon injury. Simultaneously, the MAP kinases ERK1/2 are activated and the transcription factor c-Jun is upregulated in Schwann cells5,6, but not in oligodendrocytes. Ablation or inactivation of Dusp6 induces rapid ERK1/2 phosphorylation, c-Jun upregulation and filopodia formation in oligodendrocytes, leading to mechanically-induced, fast disintegration of distal ends of injured axons, myelin clearance and axonal regrowth. Together, our findings provide understanding of the mechanisms underlying the different plasticity of Schwann cells and oligodendrocytes after injury and a method to convert mature oligodendrocytes exhibiting inhibitory cues for axonal regrowth into repair oligodendrocytes reminiscent of repair Schwann cells. We show that repair oligodendrocytes successfully increase the compatibility of the spinal cord environment with axonal regrowth after injury, suggesting a potential use of repair oligodendrocytes as future therapeutic approach to treat spinal cord injuries.

neuroscience↗