Biology subjects

Bumann, D.

Publications and source records attributed to Bumann, D..

14 recordsLinked to original sources

bioRxiv2026

Codon usage determines tRNA-modification requirements for bacterial virulence

Bacterial virulence genes exhibit distinctive codon usage that may impose tRNA-modification requirements for efficient translation, but a comprehensive understanding is lacking. Here, a curated codon-tRNA modification map for Salmonella predicted six modifications that could preferentially support virulence gene translation. Mutant phenotypes in infected mice confirmed requirements for iA37 and C5 modification of U34, whereas essential k2C34 could not be tested. Q34, Cm32/Um32, and Cm34 had limited impact consistent with weak decoding effects in other systems. Non-predicted tRNA modifications were not required. Synonymous reporter variants, proteomics, and targeted recoding demonstrated that virulence-associated codons directly conferred modification dependence. Additionally, four Leu-TTA codons in ssrB, the master regulator of Salmonella pathogenicity island 2 (SPI-2), contributed to MiaA-dependent SPI-2 gene expression. Extending our analysis to other pathogens predicted shared and divergent virulence dependencies consistent with published mutant phenotypes. Thus, codon usage explains tRNA-modification requirements for bacterial virulence.

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

Persistent Staphylococcus aureus in human musculoskeletal tissues despite targeted antibiotic therapy

Staphylococcus aureus is a leading cause of musculoskeletal infection with frequent recurrence despite surgery and antibiotic therapy. Its persistence in human tissues under antibiotic pressure remains unclear. We performed longitudinal in situ analyses of matched tissue samples before and during therapy from patients undergoing repeated surgery using advanced imaging and tissue-based metagenomics. S. aureus remained detectable in all samples--including culture-negative specimens--and whole-genome sequencing confirmed persistence of the infecting strain. Antibiotic initiation reduced bacterial burden by 1.0Slog10 bacteria/mm3 but residual loads stabilized thereafter. Quantitative imaging identified a culture detection threshold ([~]103bacteria/mm3), indicating that culture negativity reflects diagnostic sensitivity limits rather than true bacterial clearance. Persistence under antibiotic therapy was associated with high baseline burden and intracellular sequestration, whereas long-term persistence was characterized by predominantly extracellular bacteria capable of driving relapse. These findings reveal sustained tissue persistence despite apparent microbiological clearance and guide precision strategies to target resilient bacterial reservoirs.

microbiology↗

bioRxiv2026

tRNA modifications enable codon usage signatures to coordinate bacterial virulence in Salmonella

Virulence genes in bacterial pathogens are often A/T-rich and horizontally acquired, yet they must be translated efficiently under stress and nutrient limitation encountered during infection. Here we show that synonymous codon usage contributes directly to this problem. Using CodonPipe, a genome-scale framework for synonymous codon-usage analysis, we find that bacterial genomes contain functionally organized codon-usage landscapes that extend well beyond previous knowledge. In Salmonella enterica, virulence genes form a codon signature that is distinct from ribosomal genes and other mobile elements. This signature favors wobble decoding, increased use of rare tRNA isoacceptors, and enrichment of codons previously shown to better preserve translation during amino-acid limitation. Using codon-recoded fluorescent reporters, we show that virulence codon usage outperforms ribosomal codon usage selectively during nutrient starvation and, more strongly, in host-mimicking conditions. In mouse infections, overexpression of virulence-recoded reporters imposes a strong selective cost. Comparative analyses further indicate that related decoding signatures are conserved across diverse Enterobacteriaceae pathogens. These findings support stress-adapted codon usage as a mechanism that promotes virulence-gene expression during infection. More broadly, our work contributes to explain the long-observed A/T-rich codon bias of virulence genes and identifies tRNA charging and modifications as promising broad anti-virulence targets.

microbiology↗

bioRxiv2026

Opposing range-dependent interactions create complex spatial patterns of antibiotic tolerance in multispecies biofilms

Many microbial communities form multispecies biofilms where cells interact through diffusible molecules. In these biofilms, multiple interactions, often with opposing effects, occur simultaneously, yet we lack quantitative frameworks to predict how they combine to shape community functions. Here, we hypothesized that complex spatial patterns can emerge when opposing interactions have distinct spatial ranges. To test this, we studied how two Pseudomonas aeruginosa exoproducts, HQNO and rhamnolipids, jointly modulate Staphylococcus aureus antibiotic tolerance by respectively increasing and decreasing it. Using microfluidics-based imaging, we quantified spatial-tolerance patterns at single-cell resolution and found that tolerance indeed shows a complex spatial pattern: S. aureus cells survived treatment only at intermediate distances from P. aeruginosa, while cells closer or farther away did not. Combining experiments and modelling, we showed that this remarkable pattern emerges because rhamnolipids have a stronger but short-ranged effect, while HQNO has a weaker but longer-ranged effect. We found that spatial arrangement affects overall tolerance by shifting the balance between the two opposing interactions. Finally, using bioprinting, we confirmed that HQNO and rhamnolipids modulate tolerance in highly mixed biofilms. In more segregated biofilms, spatial arrangement still strongly modulated tolerance, but independently of these compounds, suggesting additional interactions. Together, our results show that spatial-tolerance patterns emerge from the combined effect of opposing range-dependent interactions and cannot be predicted from either alone. By predicting how opposing interactions jointly determine community properties, our framework provides a foundation for understanding and ultimately engineering microbiome functions.

systems biology↗

bioRxiv2025

Long-term control of Salmonella after transient T3SS-2 inhibition

Anti-virulence approaches are promising alternatives for traditional antibiotics to control bacterial infections. Several inhibitors show impressive activities in animal infection models, but the relative contribution of specific virulence inhibition vs off-target effects on both the bacteria and host remain unclear. Here, we developed Salmonella with switchable virulence by putting the type 3 secretion system-2 (T3SS-2) which is essential for systemic virulence, under the control of doxycycline. In infected mice given low-dose doxycycline in drinking water, the strain showed normal fitness and virulence. Doxycycline withdrawal shut down T3SS-2, arrested Salmonella replication and resolved disease symptoms. After ten days of T3SS-2 inhibition, reintroducing doxycycline restored replication, but bacterial loads remained stable, indicating strengthened host immunity. These effects were comparable to treatment with fluoroquinolone antibiotics, a highly effective therapy for human systemic salmonellosis. Thus, selective T3SS-2 inhibition may offer a suitable alternative for controlling invasive Salmonella infections.

microbiology↗

bioRxiv2025

Microgeography of staphyloccoci in human tissue explains antibiotic failure

Summary paragraphBacterial infections remain a major health threat, yet pathogen biology in human tissues is poorly understood. Using AI-guided imaging, we mapped [~]15,500 Staphylococcus aureus cells in biopsies from 33 patients undergoing surgery for musculoskeletal infections. Despite substantial interindividual variability, consistent patterns emerged. Most bacteria resided within non-classical monocytes/macrophages, challenging models of primarily extracellular pathogenesis. Both intra- and extracellular bacteria were predominantly isolated single cells or doublets with low rRNA content, suggesting limited replication. Complementary proteomics implicated inflammation-associated hypoxia and host glucose-to-lactate metabolism as growth constraints. Preoperative antibiotic therapy failed to clear bacteria across microenvironments and cluster sizes, challenging assumptions that antibiotic tolerance is confined to intracellular niches or biofilms and underscoring the clinical need for debridement. In vitro models replicating diverse tissues conditions impaired antibiotic activity, indicating multifactorial resilience. Together, these findings redefine S. aureus infection biology in musculoskeletal infections and establish a framework for mechanism-based prevention and therapy.

microbiology↗

bioRxiv2023

Heterogeneous dual-metal control of Salmonella infection

Iron controls bacterial infections through diverse pathogen and host mechanisms that remain challenging to disentangle. Here, we determined how individual Salmonella cells access iron in infected mice. Our results showed that the iron transporter SLC11A1 restricted iron availability. However, many Salmonella bypassed this restriction by targeting macrophage endosomes that contained remnants of iron-rich red blood cells. These iron-replete bacteria dominated overall Salmonella growth and masked the relieve of iron-starved bacteria under iron overload. These data, combined with our previous discovery of magnesium deprivation as a primary mechanism for controlling Salmonella, reveal a heterogeneous dual-metal mechanism of nutritional immunity, and highlight the power of single-cell analyses under physiological in-vivo conditions to unravel complex anti-bacterial host mechanisms. One sentence summaryIron and magnesium limitations control distinct Salmonella subsets during infection.

cell biology↗

bioRxiv2023

A Far-Red Fluorescent Probe to Visualize Staphylococcus aureus in Patient Samples

Staphylococcus aureus (S. aureus) is the leading bacterial cause of death in high-income countries and can cause invasive infections at various body sites. These infections are associated with prolonged hospital stays, a large economic burden, considerable treatment failure, and mortality rates. So far, there is only limited knowledge about the specific locations where S. aureus resides in the human body during various infections. Hence, the visualization of S. aureus holds significant importance in microbiological research. Herein, we report the development and validation of a far-red-fluorescent probe to detect S. aureus in human biopsies from deep-seated infections. This probe displays strong fluorescence and low background in human tissues, outperforming current tools for S. aureus detection. Several applications are demonstrated, including fixed- and live-cell imaging, flow cytometry, and super-resolution bacterial imaging. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=124 SRC="FIGDIR/small/556223v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@1bcf4e4org.highwire.dtl.DTLVardef@71bf45org.highwire.dtl.DTLVardef@1c468fdorg.highwire.dtl.DTLVardef@1c8baf3_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2023

Ectopical expression of bacterial collagen-like protein supports its role as adhesin in host-parasite coevolution

For a profound understanding of the mechanisms of antagonistic coevolution, it is necessary to identify the coevolving genes. The spore-forming bacterium Pasteuria ramosa and its host, the microcrustacean Daphnia, are a well-characterized paradigm for co-evolution, but the underlying genes remain largely unknown. A genome-wide association study identified a polymorphic carboxy-terminal globular domain of Pasteuria collagen-like protein 7 (Pcl7) as a candidate mediating parasite attachment and driving its coevolution with the host. Since P. ramosa cannot currently be genetically manipulated, we used Bacillus thuringiensis as a surrogate parasite to express a fusion protein of a Pcl7 carboxy-terminus from P. ramosa and the amino-terminal domain of a B. thuringiensis collagen-like protein. Mutant B. thuringiensis (Pcl7-Bt) spores but not wild-type B. thuringiensis (WT-Bt) spores, attached to the same site of susceptible hosts as P. ramosa. Furthermore, Pcl7-Bt spores attached readily to host genotypes that were susceptible to the P. ramosa clone that was the origin of the Pcl7 C-terminus, but only slightly to resistant host genotypes. These findings indicated that the fusion protein was properly expressed and folded and demonstrated that indeed the C-terminus of Pcl7 mediates attachment in a host genotype-specific manner. These results provide strong evidence for the involvement of a CLP in the coevolution of Daphnia and P. ramosa and opens new avenues for genetic epidemiological studies of host-parasite interactions. 150-word "Importance" paragraphDuring host-parasite coevolution, hosts evolve to evade the damaging effect of the parasite, while parasites evolve to maximize their benefits by exploiting the host. The genes underlying this coevolution remain largely unknown. For the prime model-system for coevolutionary research, the crustacean Daphnia and the parasite Pasteuria ramosa, collagen-like proteins (CLPs) in Pasteuria were suggested to play a crucial role for host-parasite interactions. Here we report that transferring part of a CLP coding gene from the unculturable P. ramosa to Bacillus thuringiensis (Bt), confirmed the function of this protein as a genotype-specific adhesin to the hosts cuticle. Our finding highlights the importance of a CLP in host-parasite interactions and will enable us to explore the population genetic dynamics of coevolution in this system.

evolutionary biology↗

bioRxiv2020

Acquisition of ionic copper by a bacterial outer membrane protein

Copper, while toxic in excess, is an essential micronutrient in all kingdoms of life due to its essential role in the structure and function of many proteins. Proteins mediating ionic copper import have been characterised in detail for eukaryotes, but much less so for prokaryotes. In particular, it is still unclear whether and how Gram-negative bacteria acquire ionic copper. Here we show that Pseudomonas aeruginosa OprC is an outer membrane, TonB-dependent transporter that is conserved in many Proteobacteria and which mediates acquisition of both reduced and oxidised ionic copper via an unprecedented CxxxM-HxM metal binding site. Crystal structures of wild type and mutant OprC variants with silver and copper suggest that acquisition of Cu(I) occurs via a surface-exposed "methionine track" leading towards the principal metal binding site. Together with whole-cell copper quantitation and quantitative proteomics in a murine lung infection model, our data identify OprC as an abundant component of bacterial copper biology that may enable copper acquisition under a wide range of conditions. SignificanceCopper is an essential metal in biology due to its role in the structure and function of many proteins. Despite this, it is not very clear how bacteria acquire copper, especially for Gram-negative organisms. In this study we show that the outer membrane protein OprC has an unusual metal binding site that allows OprC to bind both reduced and oxidised ionic copper near-irreversibly. Given the versatility of OprC, its presence in many Proteobacteria and its abundance during lung infection in mice, our study shows that OprC is an important component of prokaryote copper biology that warrants further study to uncover its regulation and to assess its role in bacterial virulence.

microbiology↗

bioRxiv2020

Efficient Dual-Negative Selection for Bacterial Genome Editing

We describe a versatile method for chromosomal gene editing based on classical consecutive single-crossovers. The method exploits rapid plasmid construction using Gibson assembly, a convenient E. coli donor strain, and efficient dual-negative selection for improved suicide vector resolution. We used this method to generate in frame deletions, insertions and point mutations in Salmonella enterica with limited hands-on time. Similar strategies allowed efficient gene editing also in Pseudomonas aeruginosa and multi-drug-resistant (MDR) Escherichia coli clinical isolates.

microbiology↗

bioRxiv2019

Type-3 Secretion System-induced pyroptosis protects Pseudomonas against cell-autonomous immunity

Inflammasome-induced pyroptosis comprises a key cell-autonomous immune process against intracellular bacteria, namely the generation of dying cell structures. These so-called pore-induced intracellular traps (PITs) entrap and weaken intracellular microbes. However, the immune importance of pyroptosis against extracellular pathogens remains unclear. Here, we report that Type-3 secretion system (T3SS)-expressing Pseudomonas aeruginosa (P. aeruginosa) escaped PIT immunity by inducing a NLRC4 inflammasome-dependent macrophage pyroptosis response in the extracellular environment. To the contrary, phagocytosis of Salmonella Typhimurium promoted NLRC4-dependent PIT formation and the subsequent bacterial caging. Remarkably, T3SS-deficient Pseudomonas were efficiently sequestered within PIT-dependent caging, which favored exposure to neutrophils. Conversely, both NLRC4 and caspase-11 deficient mice presented increased susceptibility to T3SS-deficient P. aeruginosa challenge, but not to T3SS-expressing P. aeruginosa. Overall, our results uncovered that P. aeruginosa uses its T3SS to overcome inflammasome-triggered pyroptosis, which is primarily effective against intracellular invaders.\n\nImportanceAlthough innate immune components confer host protection against infections, the opportunistic bacterial pathogen Pseudomonas aeruginosa (P. aeruginosa) exploits the inflammatory reaction to thrive. Specifically the NLRC4 inflammasome, a crucial immune complex, triggers an Interleukin (IL)-1{beta} and -18 deleterious host response to P. aeruginosa. Here, we provide evidence that, in addition to IL-1 cytokines, P. aeruginosa also exploits the NLRC4 inflammasome-induced pro-inflammatory cell death, namely pyroptosis, to avoid efficient uptake and killing by macrophages. Therefore, our study reveals that pyroptosis-driven immune effectiveness mainly depends on P. aeruginosa localization. This paves the way toward our comprehension of the mechanistic requirements for pyroptosis effectiveness upon microbial infections and may initiate targeted approaches in order to ameliorate the innate immune functions to infections.\n\nGraphical abstractMacrophages infected with T3SS-expressing P. aeruginosa die in a NLRC4-dependent manner, which allows bacterial escape from PIT-mediated cell-autonomous immunity and neutrophil efferocytosis. However, T3SS-deficient P. aeruginosa is detected by both NLRC4 and caspase-11 inflammasomes, which promotes bacterial trapping and subsequent efferocytosis of P. aeruginosa-containing-PITs by neutrophils.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=64 SRC=\"FIGDIR/small/650333v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (15K):\norg.highwire.dtl.DTLVardef@17e05c4org.highwire.dtl.DTLVardef@aca55forg.highwire.dtl.DTLVardef@9d2853org.highwire.dtl.DTLVardef@1c275f9_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2018

A multidisciplinary approach towards identification of novel antibiotic scaffolds for Acinetobacter baumannii

Research efforts to discover potential new antibiotics for Gram-negative bacteria suffer from high attrition rates due to the synergistic action of efflux systems and the limited permeability of the outer membrane (OM). One potential strategy to overcome the OM permeability barrier is to identify small molecules that are natural substrates for abundant OM channels, and to use such compounds as scaffolds for the design of efficiently-permeating antibacterials. Here we present a multidisciplinary approach to identify such potential small-molecule scaffolds. Focusing on the pathogenic bacterium Acinetobacter baumannii, we use OM proteomics to identify DcaP as the most abundant channel under various conditions that are relevant for infection. High-resolution X-ray structure determination of DcaP surprisingly reveals a trimeric, porin-like structure and suggests that dicarboxylic acids are potential transport substrates. Electrophysiological experiments and allatom molecular dynamics simulations confirm this notion and provide atomistic information on likely permeation pathways and energy barriers for several small molecules, including a clinically-relevant {beta}-lactamase inhibitor. Our study provides a general blueprint for the identification of molecular scaffolds that will inform the rational design of future antibacterials.

microbiology↗