Biology subjects

Woischnig, A.-K.

Publications and source records attributed to Woischnig, A.-K..

4 recordsLinked to original sources

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↗

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↗

bioRxiv2019

RpiRc regulates RsbU to modulate eDNA-dependent biofilm formation and in vivo virulence of Staphylococcus aureus in a mouse model of catheter infection

Staphylococcus aureus is a major human pathogen. Despite high incidence and morbidity, molecular mechanisms occurring during infection remain largely unknown. Under defined conditions, biofilm formation contributes to the severity of S. aureus related infections. Extracellular DNA (eDNA), a component of biofilm matrix released from apoptotic bacteria, is involved in biofilm structure and stability. In many bacterial biofilms, eDNA originates from cell lysis although eDNA can also be actively secreted or exported by bacterial membrane vesicles. By screening the Nebraska transposon library, we identified rpiRc as a biofilm regulator involved in eDNA regulation. RpiRc is a transcription factor from the pentose phosphate pathway (PPP) whose product is a polysaccharide intercellular adhesin (PIA) precursor. However, rpiRc mutant strain showed neither susceptibility to DispersinB(R) (a commercially available enzyme disrupting PIA biofilms) nor alteration of ica transcription (the operon regulating PIA production). Decreased biofilm formation was linked to Sln, an extracellular compound degrading eDNA in an autolysis independent pathway. Biofilm susceptibility to antibiotics in wt and mutant strains was tested using a similar protocol as the Calgary biofilm device. Involvement of RpiRc in S. aureus virulence was assessed ex vivo by internalization experiments into HEK293 cells and in vivo in a mouse model of subcutaneous catheter infection. While minimum inhibitory concentrations (MICs) of planktonic cells were not affected in the mutant strain, we observed increased biofilm susceptibility to almost all tested antibiotics, regardless of their mode of action. More importantly, the rpiRc mutant showed reduced virulence in both ex vivo and in vivo experiments related to decreased fnbpA-B transcription and eDNA production. RpiRc is an important regulator involved in eDNA degradation inside the matrix of mature PIA independent biofilms. These results illustrate that RpiRc contributes to increased antibiotic tolerance in mature bacterial biofilm and also to S. aureus cell adhesion and virulence during subcutaneous infection.\n\nAuthor summaryBiofilm formation contributes to the severity of Staphylococcus aureus related infections. Biofilm matrix is mainly composed by polysaccharide intercellular adhesion (PIA), proteins and extracellular DNA (eDNA). By screening a mutant library of S. aureus, RpiRc was identified as a new regulator of eDNA dependent biofilm formation. How RpiRc regulates biofilm and its role in S. aureus virulence was studied in four different S. aureus strains. Deletion of RpiRc resulted in a pronounced decreased eDNA dependent biofilm formation, but not PIA dependent biofilm formation. Decreased biofilm formation was not related to increased autolysis, but was linked to extracellular compounds found in the supernatant of mutant biofilms. Sln was identified as one of this compound. RpiRc deletion also decreased biofilm recalcitrance (resistance) to selected antibiotics. Involvement of RpiRc in S. aureus pathogenesis was investigated ex vivo by internalization into HEK293 cells and in vivo in a mouse model of catheter infection. RpiRc deletion resulted in decreased virulence related to decreased expression of surface proteins like the fibronectin binding proteins A and B (FnbpA-B). These results illustrate that RpiRc contributes to increased antibiotic tolerance in mature bacterial biofilm and also to S. aureus cell adhesion and virulence during subcutaneous infection.

microbiology↗