microLife

1.In 2024, the World Health Organisation (WHO) classified Klebsiella pneumoniae as a maximum priority pathogen for the development of new alternatives to antibiotics. In this context, understanding the regulation of key virulence mechanisms is essential. Here, we investigated the role of the orphan quorum-sensing receptor SdiA in modulating virulence-associated processes during macrophage infection. Deletion of sdiA ({Delta}sdiA) significantly increased susceptibility to phagocytosis, as demonstrated using an amoeba predation model in which mutant strains formed larger clearance zones compared to wild-type bacteria. This phenotype was also observed in murine macrophages, where {Delta}sdiA strains exhibited increased adhesion (1.5 to 2.5-fold) and phagocytic uptake. Reduced uronic acid levels were also quantified in mutant strains, indirectly indicating a diminished capsule production, likely contributing to this enhanced phagocytosis. Despite enhanced uptake, {Delta}sdiA strains showed increased intracellular survival and replication rates within macrophages, leading to reduced host cell viability. This effect occurred despite loss of interbacterial killing capacity against E. coli, suggesting that enhanced intracellular fitness is not driven by classical antibacterial offensive mechanisms. Notably, mutant-infected macrophages displayed increased generation of reactive oxygen species (ROS), NF-{kappa}B expression, and pro-inflammatory cytokines (mCXCL10 and mTNF) production, indicating that macrophage defence mechanisms are not impaired during mutant infection. Overall, bacterial survival of {Delta}sdiA could result from overwhelming, rather than actively suppressing, host defences. Together, these findings identify SdiA as a negative regulator of phagocytosis and intracellular survival in K. pneumoniae and highlight a context-dependent role in virulence. This work provides new insights into the regulatory networks governing host-pathogen interactions and bacterial adaptation to the intracellular environment. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/725935v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@1d45bfdorg.highwire.dtl.DTLVardef@e3547forg.highwire.dtl.DTLVardef@c078f9org.highwire.dtl.DTLVardef@46408a_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO Loss of sdiA strongly affects phagocytosis, as mutant strains showed increasing adhesion (1.5 to 2.5-fold) and phagocytic uptake. Diminished capsule production could be contributing to this enhanced phagocytosis, as reduced uronic acid levels were also quantified in mutant strains. Despite being internalized at higher rates, mutants exhibited enhanced intracellular survival and replication, reducing macrophage viability. This fitness advantage occurred independently of classical offensive mechanisms, as evidenced by a lost ability to kill E. coli. Notably, mutant-infected macrophages mounted a stronger immune response, marked by elevated ROS, NF-{kappa}B expression, and pro-inflammatory cytokines production (mCXCL10 and mTNF). Together, these findings suggest that strains survive by overwhelming, rather than suppressing, host immune defences. Created with Biorender (https://www.biorender.com/). C_FIG HighlightsO_LISdiA deletion in K. pneumoniae increases susceptibility to phagocytosis. C_LIO_LIThe mutant strains exhibit reduced uronic acid levels, indicative of capsule production. C_LIO_LISdiA mutants show enhanced intracellular survival and higher macrophage death. C_LIO_LIMutant infected macrophages have higher NF-{kappa}B, TNF, and CXCL10 responses. C_LIO_LISdiA-deficient strains lose predatory capacity against E. coli. C_LI

Clustered regularly interspaced short palindromic repeat (CRISPR) loci and their associated (cas) genes provide adaptive immunity to bacteria and archaea. CRISPR-Cas systems acquire short DNA fragments from the genomes of infecting plasmids and viruses, which are inserted into the CRISPR locus as a "spacer" sequence in between repeats. Spacers constitute a memory of infection that is used to recognize and attack invading genetic elements in future infections. Despite the evolutionarily divergent genetic backgrounds of bacteria and archaea, the same CRISPR-Cas systems are functional in both of these prokaryotic domains. In bacteria, efficient spacer acquisition requires the DNA repair nucleases RecBCD/AddAB. These nucleases, however, are not present in archaea. Here we investigated the importance of the DNA repair systems in the Haloferax volcanii Type I-B CRISPR-Cas response. We found that elimination of the DNA repair nuclease Mre11-Rad50, but not Fen1, substantially reduces spacer acquisition. CRISPR immunity against H. volcanii pleomorphic virus 1 (HFPV-1), on the other hand, was not affected by these deletions. Our results describe how CRISPR-Cas systems have adapted to provide anti-viral defense to hosts from different domains of life.

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

Intestinal inflammation increases the abundance of Enterobacteriaceae in the gastrointestinal tract by several orders of magnitude. These population expansions, or blooms, are associated with disease progression and have been suggested to exacerbate intestinal pathologies in some settings. Murine studies have shown that during the early stages of Escherichia coli colonization, i.e., during engraftment, inflammation enhances fitness through processes that depend on Moco, an enzyme cofactor found in a variety of oxidoreductases that consists of molybdenum coordinated by a pterin molecule. Using a murine commensal E. coli isolate and a DSS-induced colitis model in mice, we investigated whether Moco is also important for blooms of E. coli that are part of the resident microbiota, that is, for E. coli that have engrafted well before the onset of inflammation. We show that resident wild-type and Moco- E. coli exhibit comparable expansions in response to inflammation, indicating that, in this context, Moco-dependent processes such as nitrate respiration or formate oxidation were not important for inflammation-induced blooms. We find that Moco is important, however, for E. coli colonization in the absence of inflammation, suggesting that alternative respiratory pathways or other Moco-dependent processes are necessary for E. coli colonization of a healthy murine gut. Our findings demonstrate that the mechanisms underlying inflammation-induced blooms can depend on the temporal relationship between engraftment and inflammation, and also highlight the importance of considering colonization stage in identifying and interpreting the factors that affect the fitness of microbes colonizing the intestine.

Bacterial pathogens must adapt to dynamic host tissue environments to proliferate. Accordingly, elegant regulatory systems evolved to overcome challenges presented by the host and satisfy nutritional requirements. Sulfur is an essential macronutrient and Gram-positive bacteria such as Staphylococcus aureus balance this nutritional requirement by employing the transcriptional repressor, CymR. Previous investigations defined the S. aureus CymR regulon by comparing transcripts generated in a cymR mutant cultured in cystine replete, rich medium to wild type cells. This study defines the S. aureus CymR-dependent and -independent sulfur-starvation response in chemically defined growth conditions. Results demonstrate that the sulfur starvation and sulfur replete CymR regulons exhibit considerable overlap, including previously noted connections between iron acquisition, oxidative stress, and sulfur metabolism. The link between iron acquisition, oxidative stress, and sulfur metabolism is validated further by the finding that sulfur-containing glutathione (GSH) mitigates heme and peroxide toxicity. In addition to GSH, Cys and thiosulfate fulfill the S. aureus sulfur requirement. Transcriptional responses to organic (cysteine, cystine, reduced and oxidized GSH) or inorganic thiosulfate were quantified, revealing sulfur source-specific expression patterns. Thiosulfate induced the largest number of differentially expressed genes. Consequently, the thiosulfate transporter (SAUSA300_RS10985) has been confirmed as essential for S. aureus growth when thiosulfate is the sulfur source. Furthermore, we demonstrate that a hypothetical protein operonic with SAUSA300_RS10985, SAUSA300_RS10980, supports maximal growth on thiosulfate. Collectively, a resourceful transcriptomics framework is provided which underscores the dynamic nature of S. aureus sulfur metabolism.

Flagella are large transenvelope nanomachines but how they transit the peptidoglycan in Gram positive bacteria is poorly understood. A recent model suggested that flagellar basal bodies diffuse in the membrane and become captured at locations in the peptidoglycan with a pore diameter that could accommodate the axle-like flagellar rod. Mutation of penicillin binding protein 1 (PBP1/PonA), a cell wall repair protein thought to decrease peptidoglycan pore frequency and/or size, resulted in a severe growth defect and cell lysis in the ancestral strain of Bacillus subtilis that was dependent on flagellar synthesis. Genetic analysis indicated that toxicity was due to completion of the flagellar hook, which activated the flagellar sigma factor SigD. SigD, in turn, activated a suite of peptidoglycan hydrolases that caused cellular lysis when PBP1 was absent. In addition, mutations that resulted in high levels of the stress response factor Spx could lessen the toxicity, while PBPX, a putative teichoic acid D-alanylase, was required for autolysis. In sum our results indicate that flagellar synthesis, not normally associated with cell viability, causes cell wall stress and under some conditions, cell death. Moreover, our work indicates that cost of envelope integrity by flagellar synthesis may be underappreciated due to strain domestication, and suggests that specialized systems may compensate for the cost of assembly of transenvelope machines in general. SIGNIFICANCEBacteria assemble nanomachines through the cell envelope but how the machines transit the peptidoglycan is poorly understood. Here we find that assembly of trans-envelope flagella results in cell lysis of Bacillus subtilis when the peptidoglycan repair protein PBP1 is absent. Lysis was due to multiple peptidoglycan lyases expressed as a consequence of flagellar assembly, and lytic activity required another PBP homolog, PBPX. Our work indicates that flagella, not normally thought to impact cell viability, can be lethal at the level of cell envelope integrity.

Salmonella enterica serovar Infantis (SIN) has rapidly become the dominant serovar in poultry worldwide, a success largely linked to the acquisition of the 285 kb megaplasmid pESI. While pESI-encoded antibiotic-resistance and iron-uptake systems are well characterized, pESI-mediated adhesion mechanisms remain poorly understood. Here we identify a novel pESI-encoded monomeric autotransporter adhesin, designated PeaP (pESI-encoded autotransporter protein), and demonstrate its pivotal role in atypical biofilm formation, interference with motility, and colonization of the chicken host. Biofilm assays revealed that pESI-harboring strain SIN 119944 forms robust biofilms at 37 {degrees}C and 42 {degrees}C, temperatures at which CsgD-dependent biofilm formation is negligible. Deletion of csgD did not impair this phenotype, whereas deletion of peaP abolished high-temperature biofilm development and restored motility to wild-type levels. Proteomic profiling of sessile versus planktonic cells highlighted PeaP as the most abundant pESI-derived protein in the biofilm fraction. AlphaFold-based modelling and negative-stain transmission electron microscopy showed that PeaP comprises a C-terminal {beta}-barrel and a 1,500 aa passenger domain with three tandem repeats, projecting filamentous appendages [~]37 nm from the outer membrane. Antibody blockade of PeaP reduced surface adhesion >6-fold, confirming its adhesive function. In an infection model of 2 day-old chicken, the peaP mutant displayed significantly lower colonization, indicating PeaP-mediated adhesion in vivo. Collectively, pESI-positive SIN deploys PeaP for CsgD-independent, temperature-tolerant biofilm formation and enhanced gastrointestinal colonization, providing a mechanistic basis for the epidemic spread of this multidrug-resistant pathogen in poultry.

Listeria monocytogenes can grow as a saprophyte on decaying plant material, but can also switch to a pathogenic lifestyle. This switch is mediated by the virulence regulator PrfA, which activates the expression of most virulence genes. PrfA activity is tightly regulated by several mechanisms to ensure that virulence genes are only expressed within the host. One of these regulatory mechanisms is the sugar-dependent repression. In the presence of readily metabolizable sugars, which are imported via phosphotransferase systems (PTS) such as cellobiose, PrfA is repressed; however, the precise mechanism is still unknown. Using a sugar screen, trehalose was identified as the first PTS-dependent sugar that supports growth of L. monocytogenes, but does not seem to impact PrfA activity. We demonstrated that the PTS permease TreB is the sole trehalose importer. After import, trehalose-6-phosphate is cleaved by the phosphotrehalase TreA; however, loss of TreA does not fully abolish growth on trehalose suggesting that L. monocytogenes encodes an additional phosphotrehalase. 13C-Labeling experiments revealed that trehalose metabolism is repressed in the presence of glucose, while it can be metabolized in the presence of glycerol. Additionally, these experiments provided evidence that trehalose and cellobiose are metabolized via identical pathways, including glycolysis and the incomplete TCA cycle, although trehalose has a slower uptake and/or metabolization rate. We therefore hypothesize that sugar-dependent PrfA repression correlates with sugar transport and/or consumption rates, potentially due to varying availability of phosphoenolpyruvate (PEP), which serves as both a metabolic intermediate and phosphate donor for PTS-dependent transport.

Mycobacterium tuberculosis (Mtb) cultured in minimal medium at acidic pH arrests its growth when provided specific single carbon sources, including glycerol, propionate, and lactate, a phenomenon we refer to as acid growth arrest. To define mechanisms of acid growth arrest on lactate, transposon mutants that suppress growth arrest were selected. Four mutants had insertions in phoT and one had an insertion in pstC2, both components of a phosphate ABC transporter. Mtb grows in minimal media supplemented with lactate at acidic pH when phosphate is depleted, showing that Mtb growth arrest on lactate is dependent on phosphate. The combination of lactate and phosphate at acidic pH causes cytoplasmic acidification below pH 6.7 in wild type Mtb, but a phoT::Tn mutant maintains a cytoplasmic pH of >7.2. Membrane potential in wild type Mtb is slightly decreased by lactate in a dose-dependent manner but is higher in the phoT::Tn mutant. Thus, acidic pH, phosphate, and lactate act together to dissipate proton motive force (PMF), a stress that is associated with acid growth arrest. Transcriptional profiling further supports that lactate causes PMF stress including induction of electron transport chain genes. The phoT::Tn mutant grown in lactate at acidic pH upregulates the senX3/regX3 regulon and using a regX3 mutant, we demonstrate that growth on lactate at low phosphate requires regX3. We propose a model where 1) the combined impact of acidic pH, lactate, and phosphate drives cytoplasmic pH acidification and decreased PMF, thus promoting acid growth arrest, and 2) low phosphate or a mutated phosphate transporter causes upregulation of senX3-regX3, which may induce ESX-5 and PPE/PE-based import mechanisms, thereby altering the mycomembrane or nutrient uptake in a manner that promotes growth on lactate at acidic pH. ImportanceMycobacterium tuberculosis (Mtb) grows well on lactate as a sole carbon source at neutral pH, but not at acidic pH. This study sought to understand why there is a pH-dependent growth restriction on lactate. A genetic selection for mutants that can grow on lactate at acidic pH identified mutants defective in phosphate transport. We found that limiting phosphate through depleting extracellular availability or inactivating a phosphate transporter promotes growth on lactate at acidic pH, and that this growth is dependent on the phosphate responsive two-component regulatory system SenX3-RegX3. Furthermore, we show that lactate, phosphate, and acidic pH combine to cause cytoplasmic pH acidification, a metabolic stress that is associated with acid growth arrest on lactate.

As key drivers of nitrification, ammonia-oxidizing archaea (AOA) play a central role in the global nitrogen cycle and contribute significantly to the emissions of the potent greenhouse gas nitrous oxide (N2O). However, the ecological implications of AOA growth as biofilms, remain poorly understood. Since nitrite production can be used to follow cellular activities directly we were able to compare biofilms with planktonic cells of the terrestrial model AOA Nitrososphaera viennensis at ecologically and agriculturally relevant conditions. Biofilms were more resistant across nearly all tested conditions and remained active at lower temperatures, acidic pH, and high ammonium concentrations. Collectively, activities in biofilm help reconcile discrepancies between earlier laboratory and environmental observations of soil AOA. Additionally, biofilms showed a high general resilience and lowered sensitivities to nitrification inhibitors. Although in situ biofilms grown in microrespiratory chambers exhibited activity and ammonia affinity similar to planktonic cells, biofilm cultures produced only half as much N2O. The enhanced fitness of biofilms across all tested conditions vastly expands the potential ecophysiological niche of AOA and supports the hypothesis that biofilm growth represents the in situ phenotype of AOA in soil environments.

Many environmental bacteria do not readily grow under laboratory conditions and population establishment often occurs stochastically. Although the scout hypothesis has been proposed to explain stochastic population establishment in environmental bacteria, how stochastic population establishment is shaped by individual cell growth behaviors in environmental isolates remains unclear. In the present study, we focused on the ammonia-oxidizing bacterium Nitrosomonas sp. PY1 and showed that environmentally responsive individual cell growth behavior, incorporating time-dependent stochastic growth initiation, shapes both deterministic and stochastic population establishment dynamics. Using single-cell observation, we revealed that PY1 altered cell growth behavior in response to surrounding biomass production ({Delta}Vt). These {Delta}Vt-dependent changes in growth behavior were suppressed by the addition of its own cell-free supernatant (CFS), indicating the presence of a growth regulation mechanism via cell-cell communication. Replicate cultures under the same conditions showed that the population establishment of PY1 was stochastic, whereas the model strain Nitrosomonas europaea exhibited synchronized population establishment, consistent with previous reports. This stochasticity in PY1 was also eliminated by the addition of CFS. Finally, a simulation model based on {Delta}Vt-dependent cell growth behavior of PY1 successfully reproduced synchronized population establishment in the presence of CFS. By contrast, the stochastic population establishment observed in the absence of CFS was successfully reproduced by a model incorporating {Delta}Vt-independent growth initiation following a Weibull distribution. Such environmentally responsive changes in population establishment dynamics may contribute to the low isolation success of environmental bacteria and sudden blooms of the rare biosphere.

Patients with a dominant mutation in the Rho GTPase RAC2, RAC2E62K, which hyperactivates the protein, suffer from a combined immunodeficiency characterized by recurrent bacterial and fungal infections and severe T cell lymphopenia. Patient neutrophils have elevated F-actin and superoxide production yet fail to control growth of S. aureus, and the mechanism underlying this killing defect is unknown. Here we report that hyperactive Rac2 primes neutrophils for primary granule degranulation, potentially depleting myeloperoxidase (MPO) needed for intraphagosomal microbial killing. Using a Rac2+/E62K mouse model, we show that mature bone marrow neutrophils have decreased side scatter, elevated surface CD63, and reduced intracellular MPO. Interestingly, bone marrow architecture and neutrophil development in the mice are normal. Rac2+/E62K neutrophils are hyperactivated, with increased CD11b expression, cell spreading, and bioparticle phagocytosis. In the spleen, Rac2+/E62K mice display extramedullary granulopoiesis and an accumulation of degranulating neutrophils. Splenic T cells, but not B cells, show elevated surface phosphatidylserine, an "eat me" signal that sensitizes them to phagocytic clearance and provides a candidate mechanism for the selective T cell lymphopenia. Together these findings suggest that hyperactive Rac2 compromises antimicrobial neutrophil function and drives selective T cell clearance in the spleen.

Stressful environments can pose a threat to microbial populations, but resistant individuals can emerge and avoid extinction. Adaptation to stress is classically studied in isolated microbial species, ignoring ecological interactions, a key component of natural ecosystems. A growing body of experimental work has shown that community context can affect resistance evolution due to a large variety of mechanisms. Here we set out to identify the minimal components needed to predict the likelihood of acquiring resistance in a focal species embedded within a simple community. To achieve this, we developed a mathematical model based on evolutionary rescue theory and validated it with two experimental systems: Escherichia coli evolving on exposure to the antibiotic nitrofurantoin alone or with one of 14 bacterial isolates from urinary tract infections, and Microbacterium liquefaciens evolving in ampicillin alone or with ampicillin-degrading Comamonas testosteroni. One key factor that emerged from our analyses - the relative strength of competition versus protection - could explain whether a focal species is more or less likely to evolve resistance in the presence of a partner species. While competition always hinders the emergence of resistance, protection can rescue the focal species in two ways: (i) ecological rescue, when the partner species completely removes the antibiotic and favors the survival of the susceptible population, or (ii) evolutionary rescue, when the partner only lowers antibiotic concentrations and favors the emergence of resistant variants, a previously overlooked evolutionary consequence of detoxification. Overall, by integrating theory and experiments, we propose a framework that clarifies how ecological interactions favor or hinder the evolution of resistance to antibiotics or potentially other stressors. SignificanceBacteria can rapidly adapt to resist stressors, such as antibiotics. While resistance evolution in single populations or species is well understood, it remains unclear how ecological interactions with other species influence this process. We develop a mathematical framework to predict what interactions should favor resistance evolution and validate it with two sets of experiments where bacteria adapt to antibiotics in small communities. Our work demonstrates that interactions with other species shape the probability of evolving resistance in a predictable way, determined by the balance between competition and protection against the stressor. By identifying the key factors that drive these dynamics, our work helps explain how bacteria adapt to environmental challenges within species-rich ecosystems.

The evolution of antimicrobial resistance (AMR) in chronic biofilms is often viewed as a unidirectional path toward higher fitness, yet the metabolic constraints governing these trajectories remain poorly understood. We performed a four-passage evolution experiment using a murine lung biofilm model to assess the impact of prolonged ciprofloxacin (CIP) exposure on resistance and host response. This approach integrated population-level adaptive dynamics, whole-genome sequencing (WGS), and NMR-based metabolomics, alongside histopathology and cytokine analysis. Prolonged CIP treatment accelerated resistance, with isolates reaching MICs of 8-12 mg/L (a 32- to 48-fold increase) by the fourth passage. WGS revealed distinct evolutionary trajectories: control isolates accumulated metabolic and regulatory mutations without susceptibility changes, while CIP-treated isolates exhibited a stepwise progression from metabolic adaptation to high-level resistance, marked by early nfxB and late gyrA mutations. Metabolomic profiling revealed progressive divergence, with PCA identifying the nfxB genotype as the primary driver of variation (49.1% of variance). This resistant metabolic state was characterized by the depletion of central carbon metabolites, including glucose and tyrosine, alongside the accumulation of essential amino acids. Importantly, these changes were accompanied by a distinct trade-off; high-level CIP resistance triggered collateral sensitivity to tobramycin and aztreonam. While CIP treatment ultimately reduced neutrophilic inflammation (p = 0.011) and mucin production (p = 0.0496), early-passage lungs exhibited transient elevations in pro-inflammatory cytokines (CXCL2, MMP2, TNF-). In conclusion, the adaptive trajectory to CIP resistance involves metabolic rewiring and collateral sensitivity, offering a framework to exploit the evolutionary costs of resistance in chronic biofilm infections.

Mycobacterium abscessus (Mab) is an opportunistic pathogen that can cause chronic, debilitating lung disease. Mab is intrinsically resistant to most antibiotics, making Mab infections challenging to manage and frequently incurable. During infection, Mab adapts to survive various stresses, including hypoxia and nutrient starvation. In vitro, these conditions drive Mab into a drug-tolerant, non-replicating state. Changes in the Mab proteome that result from entering a non-replicating state have been minimally described despite the clinical importance of this physiological state. Using Mab reference strain ATCC 19977, we collected proteomic data comparing replicating to non-replicating states using a carbon starvation (CS) model of persistence. We identified 2251 proteins overall (46% proteome coverage), and 17% of these proteins were found in only one of the two conditions. A third of identified proteins were significantly changed in abundance, indicating an extensive proteomic response to CS. The response regulator DosR and many DosRS responsive proteins were significantly more abundant under CS, suggesting that the DosRS stress response regulator plays a key role in CS-induced Mab persistence. Many aspects of cell wall biosynthesis were changed, including changes in glycolipid abundance under CS. Proteins involved in other key cellular processes such as secretion, oxidative phosphorylation, and nutrient metabolism were altered under CS. The proteomic analysis presented provides new insights and clarity into how the Mab proteome is regulated during non-replicating persistence, a key consideration for understanding Mab pathophysiology.

Conflicting roles have been proposed for the E. coli protein RatA. Initially described as a ribosome targeting toxin, a later report pronounced it the bacterial homologue to the inner mitochondrial membrane protein Coq10. Coq10 proteins are conserved from prokaryotes to human and implicated to serve a lipid chaperone role in the biosynthesis of ubiquinone, a crucial electron carrier during aerobic respiration. We recently identified that the contradictory results published for RatA can be attributed to a mis-annotation of the gene in the reference genome. Here, we further elucidate the molecular function of RatA. We clarify that RatA is not a toxin but serves as a lipid shuttle for ubiquinone from its cytosolic biosynthesis complex to the inner membrane. Furthermore, we show that the loss of RatA results in an impaired, but not abolished electron transport chain and demonstrate broad metabolic adaptations of the cells as a consequence. Therefore, we propose to rename RatA to UbiM to reflect its function and to be in accordance with the naming convention of other ubiquinone biosynthesis proteins.

Coxiella burnetii is the only member of the order Legionellales known to primarily infect vertebrates. The Q fever pathogen is also unusual in that it replicates within an acidified phagolysosome-like vacuole. The evolutionary origins of the virulence determinants underlying this lifestyle remain unclear. More broadly, little is known about how virulence-related traits arise in specialized intracellular lineages, where access to foreign-origin DNA may be more episodic. To address this question, we used Legionellales-wide comparative phylogenomics to reconstruct the gain and loss of traits affecting host interaction, immune evasion, intracellular survival, and metabolism. We found that many virulence-associated traits in C. burnetii predate the modern pathogen and were assembled stepwise in ancestors that likely occupied niches distinct from the acidified vacuolar niche of modern C. burnetii. The common ancestor shared with soft-tick Coxiella endosymbionts likely encoded most C. burnetii type IVB secretion system effectors, indicating that much of the host-manipulation repertoire in C. burnetii was already present before the emergence of the modern pathogen. Distinctive lipopolysaccharide features associated with immune evasion also appear to have accumulated progressively within the Coxiella lineage, including genes implicated in synthesis of virenose, a unique O-antigen sugar critical for C. burnetii virulence. Traits likely to support replication in the acidic Coxiella-containing vacuole likewise accumulated gradually, with generalized stress-tolerance functions predating acquisition of an Mrp cation/proton antiporter that may have further supported pH homeostasis. Additional changes in sugar transport and catabolism, glycolytic control, and respiratory metabolism likely enhanced metabolic flexibility and access to diverse substrates in this nutrient-rich niche. Together, these findings support a model in which vertebrate pathogenicity in C. burnetii emerged through stepwise remodeling of an ancestral host-associated lineage and provide a framework for understanding how virulence-related traits evolve in specialized intracellular pathogens. AUTHOR SUMMARYCoxiella burnetii is the bacterium that causes Q fever, a disease that can spread from animals to humans. Unlike its close relatives, C. burnetii primarily infects vertebrates and grows inside an acidic compartment within host cells. New bacterial pathogens often evolve by gaining genes from other bacteria, but how virulence evolves in lineages that grow only inside host cells, where opportunities to gain new genes may be infrequent, remains unclear. We wanted to understand how C. burnetii evolved the traits needed for its distinctive intracellular lifestyle. By comparing its genome to those of related bacteria across the order Legionellales, we found that features involved in host manipulation, immune evasion, acid tolerance, and nutrient use appeared at different times in its ancestry rather than being acquired all at once by the modern pathogen. Our findings suggest that specialized intracellular pathogens can emerge through gradual changes in ancestral host-associated lineages, including gene acquisition, gene loss, retention of older traits, and repurposing of existing functions.

1.The secondary messenger cyclic di-GMP is a ubiquitous bacterial signal that regulates the switch from a free-swimming to a sessile biofilm-forming lifestyle. Many biofilm-forming Pseudomonas species possess numerous c-di-GMP-binding proteins (CDGs) which regulate gene expression, protein activity, and protein complexes. However, the mechanisms by which numerous CDG effectors form a coherent signaling network to coordinate lifestyle changes remain poorly understood. We addressed this knowledge gap by focusing on ten CDG proteins involved in biofilm development in P. fluorescens SBW25. We used an integrated approach combining a protein interaction network from genome-wide yeast two-hybrid (Y2H) screens with large-scale biofilm and motility phenotype analyses via CRISPR interference (CRISPRi). Our network associated c-di-GMP signaling with processes such as signal transduction, solute transport, secretion, virulence, transcriptional regulation, DNA repair, and cell division. We discovered unknown functions of two CDG proteins in DNA repair and cell division, supporting the significance of our network. Notably, the phosphodiesterase DipA interacts with numerous CDG proteins through GGDEF domains. Phenotypic analyses revealed that CDG partners were highly correlated or strongly anticorrelated with DipA. These findings suggest that DipA is a central hub for CDG interactions that integrates opposing modules. These findings support the hub-based model of c-di-GMP signaling, which is crucial for localized control and rapid adaptation to environmental changes.

Enterococcus faecalis is a Gram-positive intestinal commensal and opportunistic pathogen capable of causing serious infections, including urinary tract infections, endocarditis, and wound infections. A major contributor to its persistence during infection is the ability to form biofilms on host tissues and medical devices. Biofilm cells have higher phenotypic tolerance to antimicrobial treatment than planktonic bacteria. While mechanisms governing biofilm assembly in E. faecalis have been widely studied, the processes that regulate biofilm dispersion, the final stage of the biofilm life cycle, remain poorly understood. In this study, we found that dispersion is triggered by a tenfold step-change increase in nutrient availability and by cell free supernatant (CFS) of E. faecalis OG1RF cultures. Cells released from biofilms regain sensitivity to antibiotics similar to planktonic cells but maintain a high potential for adherence. We characterized the glycosyltransferase epaOX, which contributes to the structure of the enterococcal polysaccharide antigen as necessary for nutrient step-change induced dispersion, CFS induced dispersion, and adhesion of dispersed cells. Supplementation of epaOX mutant CFS with galactose and N-acetylgalactosamine was sufficient to restore CFS induced dispersion. Together these data suggest that dispersion in OG1RF occurs with fast kinetics, affects antibiotic sensitivity and is regulated in part by known virulence factors. ImportanceE. faecalis causes difficult to treat infections at numerous body sites in human patients. E. faecalis biofilms are adherent populations that require high levels of antibiotics for treatment. Biofilms undergo a disassembly process named dispersion that allows individual cells to leave the biofilm and colonize new locations. Dispersed cells in other species are killed by lower amounts of antibiotics than biofilm cells. Here we showed that dispersion occurs in E. faecalis and lowers the level of antibiotics needed to kill dispersed cells. Dispersion triggers could be used in the future to design treatments that increase the effectiveness of antibiotics.

Per- and polyfluoroalkyl substances (PFAS) are new pollutants in the environment whose effects on bacterias physiology is not well understood. In this study, we show that exposure to PFAS causes membrane depolarization in Salmonella enterica serovar Typhi. This works as a metabolic uncoupler that breaks down proton motive force without immediately killing the cells. This disturbance results in a significant elevation of intracellular NADH and NAD levels while preserving redox equilibrium, signifying an augmented metabolic flux. At the same time, we see that {beta}-oxidation pathways are turned on, which suggests that the cells are shifting toward breaking down fats to make up for the lack of energy. Even though there are more reducing equivalents, ATP levels go down, which is what happens when respiration is uncoupled. This puts the cells in a state of "pseudo-starvation." This metabolic stress triggers the SpoT-dependent stringent response, leading to the accumulation of (p)ppGpp. Genetic analysis employing {Delta}relA and {Delta}relA{Delta}spoT mutants confirm that SpoT is necessary for this adaptive response. Functionally, PFAS-treated populations show an enhanced proportion of persister-like cells, which connects exposure to environmental pollutant in the environment to antibiotic tolerance. Our findings reveal a previously unidentified mechanism by which PFAS alters bacterial metabolism and stress responses, facilitating persistence through membrane depolarization, metabolic reconfiguration, and stringent response activation. This study underscores the potential influence of environmental pollutants on bacterial survival mechanisms and antibiotic resistance.