Biology subjects

Bischler, T.

Publications and source records attributed to Bischler, T..

15 recordsLinked to original sources

bioRxiv2026

Host response and cell fates of human U937 monocytes upon infection with vancomycin-resistant E. faecium

In recent years, Enterococcus faecium has become a high-priority human opportunistic pathogen, especially due to its antibiotic and environmental resistance, promoting hospital-acquired infections. However, its interaction with host cells remains poorly understood. In this study, we investigated the interaction of four recently collected, clinical vancomycin-resistant Enterococcus faecium (VREfm) isolates with the human U937 cell line. Early after infection, we observed close bacterial association with U937 monocytes and features suggestive of internalization. We thus investigated host cell fates after washing at 2 hours post infection (hpi) to concentrate on effects induced by internalized bacteria or those with direct host cell contact. Whereas viability was not impaired at 6 hpi, moderate cell death was induced to an isolate-specific extent at 24 hpi, which was abrogated when direct host-pathogen contact as prevented via boyden chamber cell culture inserts, but was enhanced by TNF-R1 deficiency. Host mRNA sequencing revealed a consistent pattern of differentially expressed genes across all VREfm isolates, suggesting a shared innate immune and stress-related response at 6 hpi, with functionally enriched biological processes related to leukocyte activation, cytokine signaling, programmed cell death, and RNA stability. Together, these data show that VREfm triggers an early pro-inflammatory transcriptional response in U937 monocytes without immediate cytotoxicity in U937 monocytes, while late-stage isolate-specific cell death induction depends on direct host cell contact or internalization and involves TNF-R1 mediated regulation. Our findings may assist future efforts to understand how VREfm triggers host cell damage that contributes to severe inflammation and systemic infection such as persistent bacteremia.

microbiology↗

bioRxiv2026

Interdependent mode of RNA recognition by the interacting KH-domain proteins KhpA and KhpB

RNA-binding proteins (RBPs) are central regulators of gene expression. In bacteria, two KH domain-containing proteins, KhpA and KhpB, are increasingly recognized as globally-acting RBPs, including in pathogens that lack well-studied RBPs, such as Hfq and ProQ. While KhpA and KhpB interact with various RNAs in diverse bacteria, their crosstalk and molecular basis of RNA recognition remain poorly understood. Here, we explored the mutual dependence between KhpA and KhpB in the food-borne pathogen Campylobacter jejuni. We demonstrate KhpA and KhpB interact in vivo, and their individual global RNA recognition is largely dependent on the presence of the partner protein. Using RNA co-immunoprecipitation, we reveal that KhpA requires a conserved KH domain motif for RNA binding, whereas KhpB relies on an atypical R3H domain. Moreover, mutating the RNA-binding domain of one protein compromises the RNA interactome of the other. Overall, we demonstrate an interdependent RNA-binding mode of KhpA and KhpB and anticipate that such KH-proteins have co-evolved to elicit joint RNA regulation in bacteria.

microbiology↗

bioRxiv2026

Staphylococcus aureus triggers isolate-specific host transcriptional responses alongside TNF-R1 regulated cell death

BackgroundStaphylococcus aureus (S. aureus) is an increasingly recognized intracellular pathogen, yet infection outcomes vary with bacterial isolate and host cell type. The mechanisms underlying these differences remain poorly understood. This study investigates how distinct intracellular S. aureus isolates influence host signaling programs and infection outcomes by modulating cell death pathways and TNF-R1 dependent regulation of host cell fates across different human cell lines. MethodsFour S. aureus isolates were analyzed for intracellular localization using transmission electron microscopy (TEM), structured illumination microscopy (SIM), serial block-face scanning electron microscopy (SBF-SEM), and imaging flow cytometry. Transcriptional reprogramming of infected U937 monocytes was examined by mRNA sequencing. Infection outcomes were characterized and compared to A549 and SaOS-2 cell lines employing Luminex cytokine assays, flow cytometry and Western blot analysis to characterize host cell death mechanisms in both wild-type and TNF-R1 deficient backgrounds. ResultsAll S. aureus isolates localized to endolysosomal and cytosolic compartments but also peri and putatively intranuclearly, revealing an unexpected intracellular niche. In U937 monocytes, infection induced a conserved stress signature alongside isolatespecific transcriptional programs divergently affecting inflammation, metabolism, and cell fate, which was markedly attenuated in response to the chronicinfection isolate EDCC 5464. Cell death outcomes were likewise isolatedependent, involving intrinsic and extrinsic apoptosis, mitochondrial depolarization, and caspase-1 activation at distinct temporal dynamics. TNFR1 loss initially delayed but exacerbated late, isolate-independent cytotoxicity, identifying TNFR1 as a key regulator of U937 infection outcome. SaOS2 and A549 cell death was far less affected by isolate or TNF-R1 deficiency. ConclusionsThese results highlight the multilayered determinants governing intracellular S. aureus survival, non-canonical intracellular localization, and host cell susceptibility. The TNF/TNF-R1 axis is identified to critically determine regulated host defense during early infection stages in a tissue-specific manner. Together with distinct isolate-driven gene expression profiles, infection risks under TNF-targeted therapies and the contribution of S. aureus heterogeneity should be considered in the design of future host-directed treatment strategies. Plain English summaryThe bacterium Staphylococcus aureus (S. aureus) often lives harmlessly in humans but can cause severe or recurrent infections when the skin barrier is broken or the immune system is weakened. A major reason for its persistence is its ability to hide inside human cells, where it is shielded from immune attacks and antibiotics. To effectively target such bacteria, it is crucial to understand that infections vary depending on both the bacterial strain and the infected cell type. Many reasons behind these differences are still puzzling. We explored how different types of S. aureus (collected from different disease types) change how human cells respond to infection. We focused on how the different strains influence the way immune cells adjust their gene activity during infection, and how a receptor called TNF-R1 is involved in managing cell death responses. Bacteria were found not only in compartments meant to destroy them but also near and even inside the cell nucleus, an unexpected location. All strains triggered a similar stress response but also distinct patterns influencing inflammation, metabolism, and cell survival. A strain linked to chronic infection caused weaker responses, suggesting greater stealth. Cells lacking TNF-R1 initially survived longer but later showed greater damage, indicating this receptors role in infection control. In lung and bone cells, these effects were less pronounced. Concludingly, S. aureus occupies unexpected niches inside human cells and uses varying survival strategies. TNF-R1 is a key regulator of host infection responses in the analyzed immune cells, highlighting that both bacterial diversity and host factors must be considered when developing targeted treatments. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=199 SRC="FIGDIR/small/723175v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@1b4214org.highwire.dtl.DTLVardef@18f4ee6org.highwire.dtl.DTLVardef@1851742org.highwire.dtl.DTLVardef@ba0359_HPS_FORMAT_FIGEXP M_FIG Peri- and intranuclear localization early after S. aureus uptake across host cell lines, with isolate-specific modulation of host fates and a critical role for TNF-R1 to mediate regulated death responses of U937 cells. At 2 hpi, intracellular S. aureus not only localizes in (LAMP-1 decorated) membrane-enclosed compartments or directly in the cytosol, but within invaginations of the nuclear surface and intranuclearly with or without being surrounded by a vesicular membrane in U937wt, SaOS-2wt, and A549wt cells. At 4 hpi, S. aureus triggers differential gene expression in (A) U937wt cells to an isolate-specific extent, with both unique and shared transcriptomic signatures across the four isolates, that is muted for the chronic infection isolate EDCC 5464. Apoptotic cell death is induced to an isolate-dependent extent involving extrinsic initiator caspase-8, intrinsic initiator caspase-9 (EDCC 5055 only), and variable effector caspase-3/-7 activity in the earlier stages of infection (6 hpi), which then barely increases (24 hpi) in U937wt cells. S. aureus-induced cell death and caspase activation is abolished in (B) U937{Delta}TNF-R1 at 6 hpi, but is significantly reinforced at 24 hpi with diminished isolate-specificity. Correspondingly, mitochondrial trans-membrane potential ({Delta}{Psi}m) is disrupted for all isolates upon TNF-R1 knockout, as well as caspase-1 activity, suggesting pyroptotic pathway activation at later stages of infection. (C) SaOS-2 wt cells show moderate caspase-3/-7 and -1 activation, while infection induces detachment of (D) A549wt cells with minimal caspase activation. Infection induces an isolate- and cell line-dependent cytokine release. Coloured arrows indicate the mean proportion of effector-positive cells ({uparrow} [~]20-40%, {uparrow} {uparrow} 40-60%, {uparrow} {uparrow} {uparrow} >60%) representing each S. aureus isolate. Grayed signaling arrows indicate the hypothesis by which TNF-R1 activation and internalization is required to kill lysosomal S. aureus via activation of anti-microbial enzymes and downstream regulated death pathway activation. Created with BioRender.com. C_FIG

microbiology↗

bioRxiv2025

Sex differences define the molecular and cellular phenotypes of pain resolution in dorsal root ganglia

The dorsal root ganglion (DRG), a key site for the initiation and maintenance of neuropathic pain, was examined for sex-dependent phenotypes in sensory neurons, satellite glial cells (SGCs), and local macrophages following traumatic nerve injury and during natural pain resolution. Systematic analysis of 7,495 DRG immunofluorescence images and 62 transcriptomes revealed pronounced sex-specific, multicellular DRG phenotypes, especially during pain resolution. System parameters, including tissue size and neuron density also showed sex-dependent differences. Neuropathic pain resolved without tissue or sensory neuron loss. After injury, macrophages invaded the space between sensory neurons and satellite glial cells (SGCs); this was partially reversed during pain resolution, particularly in males. In females, immune-related gene expression and macrophage phenotypes persisted longer, while SGC activation and contact to sensory neurons was more persistent in males. During resolution, synaptic and excitability-related processes were pronounced in both sexes. However, while injury responses were largely shared between sexes, the resolution phase displayed distinctly sex-specific molecular and cellular signatures. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=197 SRC="FIGDIR/small/691610v1_ufig1.gif" ALT="Figure 1"> View larger version (76K): org.highwire.dtl.DTLVardef@4063c7org.highwire.dtl.DTLVardef@156c0d2org.highwire.dtl.DTLVardef@8d4be4org.highwire.dtl.DTLVardef@7182bd_HPS_FORMAT_FIGEXP M_FIG C_FIG In briefAnalysis of [~]7,500 bioimages and 62 transcriptomes reveals pronounced sex differences in rat dorsal root ganglia during pain resolution after peripheral nerve injury. HighlightsO_LIIn both female and male rats, peripheral nerve injury and subsequent pain resolution occur in the dorsal root ganglia (DRG) without neuronal or tissue loss. C_LIO_LISex influences DRG tissue size, neuron density, immune and glial phenotypes, and molecular-cellular responses to nerve injury and pain resolution. C_LIO_LIFollowing injury, macrophages infiltrate the space between sensory neurons and satellite glial cells (SGCs); this process reverses during pain resolution, particularly in males. C_LIO_LIIn females, immune phenotypes remain more stable throughout pain resolution, while SGC contact is reduced. C_LIO_LIPain resolution involves not only the reversal of injury-induced cell changes but also the activation of resolution-specific gene programs related to synaptic signaling, neuronal excitation, and cell-cell communication. C_LIO_LISex differences on the molecular-cellular level are less prevalent after nerve injury but become prominent during pain resolution. C_LI

neuroscience↗

bioRxiv2025

Condensation of the RNA chaperone Hfq is coupled to inhibition of carbon assimilation and contributes to the stabilisation of regulatory RNAs in nitrogen starved Escherichia coli

Ribonucleoprotein-condensates are membraneless compartments that concentrate RNA-binding proteins and RNA and play key roles in cellular adaptation across both eukaryotes and bacteria. While the biological roles of ribonucleoprotein-condensates are better understood in eukaryotic systems, the knowledge of metabolic processes that govern their formation and their contribution to stress adaptation remains at a nascent stage in bacterial RNA biology. Hfq is an RNA-chaperone conserved in many bacteria that undergoes condensation in response to diverse stresses. Using nitrogen (N) starvation in Escherichia coli as a model stress condition, we show that Hfq condensation occurs independently of any extracellular cues, cytoplasmic shrinkage that cells undergo during N starvation or the canonical NtrBC-dependent adaptive response to N starvation. However, we demonstrate that Hfq condensation is coupled to the inhibition of carbon assimilation in N-starved E. coli. Further, by comparing the transcriptomes of wild-type bacteria and bacteria unable to form Hfq-condensates, we reveal that Hfq-condensates contribute to the stabilisation of Hfq-associated non-coding regulatory RNAs. We propose that coordination of carbon and N metabolism during N starvation, critical for metabolic adaptation, is accompanied by preservation of non-coding regulatory RNAs via Hfq condensation.

microbiology↗

bioRxiv2025

Intramacrophage RIL-seq uncovers an RNA antagonist of the Salmonella virulence-associated small RNA PinT

Salmonella virulence chiefly relies upon two major pathogenicity islands, SPI-1 and SPI-2, which enable host cell invasion and intracellular survival, respectively. There has been increasing evidence for post-transcriptional control of SPI gene expression by Hfq-dependent small RNAs (sRNAs) such as PinT. This 80-nucleotide sRNA is highly expressed after Salmonella enters host cells and modulates the transition from the SPI-1 to SPI-2 program by targeting different virulence factor mRNAs. It has been elusive, however, how PinT activity could be counteracted when virulence gene suppression were to be relieved. To identify putative inhibitors of PinT, we have mapped the RNA interactome of Salmonella recovered from infected macrophages, using an optimized version of the RIL-seq method. Next to offering an unprecedented view of Hfq-mediated RNA interactions during Salmonellas intracellular infection stage, RIL-seq uncovered the 3 end-derived sRNA InvS as a direct negative regulator of PinT. Biochemical and genetic experiments suggest a decoy mechanism whereby InvS lifts the PinT-mediated repression of virulence factors. Additionally, InvS acts as an mRNA repressor of the host cell adhesion protein, MipA, and PinT interaction with InvS relieves mipA repression. Together, our work identifies a unique pair of antagonistic sRNAs in a growing post-transcriptional network of virulence gene regulation.

microbiology↗

bioRxiv2025

Functional genomics of Campylobacter-host interactions in an intestinal tissue model reveals a small lipoprotein essential for flagellar assembly

Campylobacter jejuni is the leading cause of bacterial food-poisoning, with motility being an essential virulence factor. While many aspects of flagella biogenesis have been studied, a complete picture of the components and regulators of these multi-protein machines is still missing. To identify genes crucial for C. jejuni pathogenesis and motility, we applied transposon sequencing in a humanized tissue model. This revealed three largely uncharacterized genes (pflC , pflD , pflE ) as essential for motility. While PflC/D turned out to be components of the flagellar motor disk structures, PflE is a small protein of only 57 aa. PflE strikingly affects motor biogenesis, with complete loss of motor structures upon its deletion. We demonstrate PflE is a lipoprotein and supports outer-membrane localization of the main basal-disk protein FlgP. With motility as a critical Campylobacter virulence factor, our work demonstrates that deletion of a small protein can bring a bacterial pathogen to a halt.

microbiology↗

bioRxiv2025

Resting calcium ion fluxes protect cells from fast mitochondrial fragmentation, cell stress responses, and immediate transcriptional reprogramming

Homeostatic calcium ion (Ca2+) fluxes between the endoplasmic reticulum, cytosol, and extracellular space occur not only in response to cell stimulation but also in unstimulated cells. Using murine astrocytes as a model, we asked whether there is a signaling function of these resting Ca2+-fluxes. The data showed that endoplasmic reticulum (ER) Ca{superscript 2} depletion, induced by sarcoplasmic/endoplasmic reticulum Ca{superscript 2}-ATPase (SERCA) inhibition, resulted to prolonged Ca{superscript 2} influx and mitochondrial fragmentation within 10 to 30 minutes. This mitochondrial fragmentation could be prevented in Ca2+- free medium or by inhibiting store-operated Ca2+ entry (SOCE). Similarly, attenuation of STIM proteins, which are vital ER Ca2+ sensors, protected mitochondrial morphology. On the molecular level, ER Ca2+ depletion, achieved either by removing extracellular Ca2+ or through acute SERCA inhibition, led to changes in gene expression of about 13% and 41% of the transcriptome within an hour, respectively. Transcriptome changes were associated with universal biological processes such as transcription, differentiation, or cell stress. Strong increase in expression was observed for the transcription factor ATF4, which is under control of the kinase PERK (EIF2AK3), a key protein involved in ER stress. Corroborating these findings, PERK was rapidly phosphorylated in Ca2+-free medium or after acute pharmacological inhibition of SOCE. In summary, resting, homeostatic Ca2+ fluxes prevent immediate- early cell stress and transcriptional reprogramming.

physiology↗

bioRxiv2024

Provirus deletion from Haloferax volcanii affects motility, stress resistance and CRISPR RNA expression

Haloferax volcanii harbours four putative proviruses: Halfvol1, Halfvol2, Halfvol3 and Halfvol4. In this study we successfully deleted all four provirus genomes, demonstrating, that they are not essential. Transcriptome comparison between this strain ({Delta}Halfvol1-4) and a wild type strain reveals an increase in archaella and chemotaxis gene expression, resulting in higher swarming motility in {Delta}Halfvol1-4. Furthermore, {Delta}Halfvol1-4 cells show an elongated cell shape and a higher resistance to H2O2 stress compared to the wild type. RNA-seq also revealed down-regulation of CRISPR arrays in the provirus-free strain. Circularised genomes of Halfvol1, Halfvol2 and Halfvol3 were found in the culture supernatant. This confirms excision of the proviruses from the chromosome, which seems to happen more efficiently at low temperature (30{degrees}C). Electron microscopy revealed potential viral particles in the supernatant, and mass spectrometry analysis confirmed the presence of structural viral proteins of Halfvol1 and Halfvol3 in the isolated virus sample. These observations suggest that these proviruses are active and cause a chronic infection in Hfx. volcanii.

molecular biology↗

bioRxiv2024

Internal in-frame translation generates Cas11b, which is important for effective interference in an archaeal CRISPR-Cas system

CRISPR-Cas is a sophisticated defence system used by bacteria and archaea to fend off invaders. CRISPR-Cas systems vary in their Cas protein composition and have therefore been divided into different classes and types. Type I systems of bacteria have been shown to contain the small Cas11 protein as part of the interference complex. Here we show for the first time that an archaeal CRISPR-Cas type I system also contains a Cas11 protein. In addition, we show for the first time an internal in-frame translation of an archaeal protein. The Cas11b protein from the Haloferax volcanii type I-B system is encoded in the cas8b gene. Translation initiation at an internal methionine of the cas8b open reading frame results in synthesis of Cas11b. Cas11b is required for an effective interference reaction and without Cas11b fewer Cascade complexes form. Comparison of transcriptomes from wild type and a Cas11b less strain show that the depletion of Cas11b results in differential regulation of many genes. Taken together Cas11b is important for the defence reaction of the type I-B CRISPR-Cas system and seems to play an additional cellular role.

molecular biology↗

bioRxiv2024

Transcriptome-scale analysis uncovers conserved residues in the hydrophobic core of the bacterial RNA chaperone Hfq required for small regulatory RNA stability

The RNA-chaperone Hfq plays crucial roles in bacterial gene expression and is a major facilitator of small regulatory RNA (sRNA) action. The toroidal molecular architecture of the Hfq hexamer contains three well characterised surfaces which allow it to bind sRNAs to stabilise them and engage target transcripts. Hfq-interacting sRNAs are categorised into two classes based on the surfaces they use to bind Hfq. By characterising a systematic alanine mutant library of Hfq to identify amino acid residues that impact survival of Escherichia coli experiencing nitrogen starvation, we corroborated the important role of the three RNA binding surfaces for Hfq function. Surprisingly, we uncovered two conserved residues, V22 and G34, in the hydrophobic core of Hfq, to have a profound impact on Hfqs RNA binding activity in vivo. Transcriptome-scale analysis revealed that V22A and G34A Hfq mutants cause widespread destabilisation of both sRNA classes. However, the alanine substitutions at these residues had no measurable impact on protein stability, structure or equilibrium binding to target sRNAs in vitro. We propose that V22 and G34 are key to the cooperative function among the RNA-binding surfaces of Hfq, a mechanism especially critical under cellular conditions when there is an increased demand for Hfq.

microbiology↗

bioRxiv2024

Neuronal toxicity and recovery from early bortezomib-induced neuropathy: targeting the blood nerve barrier but not the dorsal root ganglion

The use of the first in class proteasome inhibitor Bortezomib (BTZ) is highly effective in the treatment of multiple myeloma. However, its long-term use is limited by the fact, that most treated patients develop dose limiting painful polyneuropathy. In some of the treated patients, pain resolves after variable timeframes, in others it persists, despite the discontinuation of treatment, with the underlying mechanisms poorly understood. One condition of neural toxicity is the ability to penetrate the blood nerve barrier. Here we present pathways involved in early bortezomib-induced polyneuropathy (BIPN) development and its resolution, in rats and in myeloma patients. One cycle of BTZ elicited transient mechanical hyperalgesia and cold allodynia in rats. Transcriptomic signature and network analysis revealed regulation of circadian, extracellular matrix, and immune genes within the nerve and modest changes in the dorsal root ganglia. Recovery processes resealed the small molecule leakiness of the perineurial barrier, reversed axonal swelling, and normalized small fiber density in the skin. Expression of the microtubule-associated cytoskeletal protein cortactin matched this process in the perineurium. Netrin-1 (Ntn1) as a known barrier sealer was also upregulated in pain resolution in nerve and skin. In patients with painful BIPN skin NTN1 was independent of axonal damage. In summary, our data demonstrate that early BTZ toxicity targets mainly the nerve and indicates that pain resolution could be supported by protective growth factors like Ntn1 for remodeling of the extracellular matrix and neuronal barriers. SummaryBortezomib leads to dose-limiting painful polyneuropathy. Already in the first cycle, BTZ toxicity weakens the blood nerve barrier which reseals upon upregulation of netrin-1.

neuroscience↗

bioRxiv2024

Satellite glial cells from adult DRG dedifferentiate in vitro and can be reprogrammed into nociceptor-like neurons

In dorsal root ganglia (DRG), neuronal loss has been reported in patients with neuropathic pain, raising the question of whether the DRG, as part of the peripheral nervous system (PNS), harbor an endogenous cell source for neural repair. We found that adult mouse DRG harbor glial cells that dedifferentiate in vitro into Sox2/Sox10-positive glial progenitor-like cells. Coexpression of the developmental transcription factors Neurog1 and Neurog2 was sufficient to induce both neuronal and glial phenotypes. Nerve growth factor supported the maturation of a subset of neurons into nociceptor-like cells expressing functional TrpA1, TrpV1, and TTX-resistant NaV channels. We report the limitation that we miss factors allowing consistent maturation to the sensory neuron profile. In summary, in the PNS, adult DRG-derived glial cells can acquire neural progenitor-like properties, show bipotent reprogramming competence, and may serve as an intrinsic cell source for sensory circuit regeneration.

neuroscience↗

bioRxiv2023

Global RNA interactome of nitrogen starved Escherichia coli uncovers a conserved post-transcriptional regulatory axis required for optimal growth recovery

The RNA binding protein Hfq has a central role in the post-transcription control of gene expression in many bacteria. Numerous studies have mapped the transcriptome-wide Hfq-mediated RNA-RNA interactions in growing bacteria or bacteria that have entered short-term growth-arrest. To what extent post-transcriptional regulation underpins gene expression in growth-arrested bacteria remains unknown. Here, we used nitrogen (N) starvation as a model to study the Hfq-mediated RNA interactome as Escherichia coli enter, experience, and exit long-term growth arrest. We observe that the Hfq-mediated RNA interactome undergoes extensive changes during N starvation, with the conserved SdsR sRNA making the most interactions with different mRNA targets exclusively in long-term N-starved E. coli. Taking a proteomics approach, we reveal that in growth-arrested cells SdsR influences gene expression far beyond its direct mRNA targets. We demonstrate that the absence of SdsR significantly compromises the ability of the mutant bacteria to recover growth competitively from the long-term N-starved state and uncover a conserved post-transcriptional regulatory axis which underpins this process.

microbiology↗

bioRxiv2020

MAPS integrates overlooked regulation of actin-targeting effector SteC into the virulence control network of Salmonella small RNA PinT

A full understanding of the contribution of small RNAs (sRNAs) to bacterial virulence demands knowledge of their target suites under infection-relevant conditions. Here, we take an integrative approach to capturing targets of the Hfq-associated sRNA PinT, a known post-transcriptional timer of the two major virulence programs of Salmonella enterica. Using MS2 affinity purification and RNA-sequencing (MAPS), we identify PinT ligands in bacteria under in-vitro conditions mimicking specific stages of the infection cycle, and in bacteria growing inside macrophages. This reveals PinT-mediated translational inhibition of the secreted effector kinase SteC, which had gone unnoticed in previous target searches. Using genetic, biochemical, and microscopic assays, we provide evidence for PinT-mediated repression of steC mRNA, eventually preventing actin rearrangements in infected host cells. Our findings support the role of PinT as a central post-transcriptional regulator in Salmonella virulence and illustrate the need for complementary methods to reveal the full target suites of sRNAs.

microbiology↗