mBio

Lipoteichoic acid is an essential cell envelope polymer in Staphylococcus aureus and contributes to beta-lactam resistance in methicillin-resistant S. aureus. Mutations that alter lipoteichoic acid synthesis sensitize MRSA to beta-lactam antibiotics, but the mechanism connecting envelope polymer defects to antibiotic susceptibility has remained unclear. Here, we used suppressor genetics, genome sequencing, transcriptomics, inducible gene expression, antibiotic susceptibility assays, and measurements of intracellular potassium, cyclic di-AMP, and penicillin-binding protein production to define this connection. We found that lipoteichoic acid synthesis defects increased expression of a ParB-like protein and the mechanosensitive channel MscS. Repression of this locus in RNA polymerase suppressor mutants restored beta-lactam resistance, whereas induced expression of both genes reduced resistance. Increased ParBL-MscS expression was associated with decreased intracellular potassium and reduced cyclic di-AMP, linking altered membrane-envelope physiology to second messenger signaling. Lipoteichoic acid synthesis mutants also showed reduced production of penicillin-binding protein 4, an important determinant of beta-lactam resistance. Modest restoration of penicillin-binding protein 4 improved resistance in the mutant with reduced lipoteichoic acid abundance, whereas the mutant producing elongated lipoteichoic acid showed a distinct response, indicating that different lipoteichoic acid defects impose different envelope stresses. Together, these findings identify a potassium-dependent pathway connecting lipoteichoic acid synthesis, mechanosensitive channel activity, cyclic di-AMP signaling, penicillin-binding protein 4 production, and beta-lactam resistance. This work reveals how cell envelope perturbations are converted into cytoplasmic regulatory responses that control antibiotic susceptibility and suggests that ion homeostasis and cyclic di-AMP signaling may be exploitable pathways for restoring beta-lactam efficacy against MRSA. Author SummaryAntibiotic-resistant Staphylococcus aureus, including methicillin-resistant S. aureus, is difficult to treat because it can withstand many beta-lactam antibiotics, a widely used class of drugs. Previous work showed that changes in lipoteichoic acid, an important molecule in the bacterial cell envelope, make these bacteria more sensitive to beta-lactams. However, it was not clear how a change at the cell surface affects antibiotic resistance inside the cell. In this study, we found that defects in lipoteichoic acid production activate a pathway involving a predicted membrane channel. This pathway changes the level of potassium inside the bacterial cell and affects a small signaling molecule that helps coordinate cell wall maintenance. These changes also reduce the amount of an enzyme involved in building the cell wall, making the bacteria more vulnerable to beta-lactam antibiotics. Our findings suggest that antibiotic resistance depends not only on the direct targets of antibiotics, but also on how bacteria maintain the balance between their cell envelope, ion levels, and internal signaling. Understanding this connection may help identify new ways to make resistant bacteria more sensitive to existing antibiotics.

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.

Pseudomonas aeruginosa is a globally recognized pathogen causing pulmonary, skin, and severe corneal infections (keratitis), with the potential to induce irreversible blindness if untreated. Spatial transcriptomic analysis of P. aeruginosa infected corneas identified elevated expression of the outer membrane proteins OprF and OprL and PA1414, which encodes the small RNA SicX in the corneal stroma compared with corneal epithelium. Comparative spatial transcriptomics analysis of corneas infected with an oprF transposon (TN) mutant showed reduced expression of the type III effector protein ExoT, which was absent in an oprF deficient mutant ({Delta}oprF) and in contrast to PA14, did not inhibit reactive oxygen species (ROS) production by neutrophils. Corneal infection with the {Delta}oprF mutant resulted in reduced corneal virulence and lower CFU compared to the parental PA14 strain. Collectively, our findings demonstrate a coordinated virulence program connecting OprF functionality with the release of ExoT and its ability to block ROS production and survive in infected corneas.

Shigella OspB, a conserved type 3 effector, is a cysteine protease and peptide recombinase. Developing a protease activity-based screen, we defined and validated an OspB consensus substrate recognition motif. We found that the P1 position is aspartic acid, although cysteine is tolerated, and the P6 position an uncharged nonpolar hydrophobic residue. We demonstrate their predicted proximity to OspB active site residues within a binding groove. These findings will facilitate identification of physiological substrates of OspB and its homologs.

Staphylococcus aureus encounters diverse environmental conditions during colonization and infection, including fluctuations in nutrient availability, oxidative stress, and oxygen limitation. Adaptation to these environments requires regulatory systems that coordinate stress responses with metabolic remodeling. The extracytoplasmic function sigma factor SigS contributes to stress adaptation and virulence in S. aureus and directly activates expression of the sroAB operon, which encodes the small proteins SroA and SroB. While previous work demonstrated that SroA participates in feedback regulation of sigS expression, the broader physiological role of SroA has remained unclear. To define the regulatory functions of SroA, we performed RNA sequencing following inducible overexpression of sroA in S. aureus. Transcriptome analysis revealed extensive remodeling of gene expression, with approximately 200 transcripts significantly altered. Transcriptome analysis revealed coordinated repression of metabolic pathways (including nitrate respiration and nucleotide biosynthesis) alongside activation of stress-response and nutrient acquisition genes. Northern blot and quantitative RT-PCR analysis confirmed repression of narG and narJ transcripts following SroA overexpression. Consistent with these transcriptional changes, nitrate reduction assays demonstrated that SroA overexpression reduces nitrate respiration activity. In addition to repression of nitrate respiration genes, SroA overexpression broadly suppressed genes involved in de novo purine and pyrimidine biosynthesis. In contrast, transcripts associated with stress responses and nutrient acquisition, including the SOS-associated gene sosA and the phosphate transport gene pstS, were upregulated. Together, these findings identify SroA as a regulator that links stress-responsive signaling to metabolic remodeling in S. aureus, particularly through modulation of nitrate respiration pathways. ImportanceStaphylococcus aureus must rapidly adapt its metabolism to survive the diverse environments encountered during colonization and infection, including conditions where oxygen availability is limited. In this study, we identify a previously uncharacterized role for the small protein SroA in regulating metabolic adaptation in S. aureus. Transcriptome analysis revealed that SroA strongly represses genes involved in nitrate respiration, a pathway that enables bacteria to maintain energy production when oxygen is scarce. Consistent with these transcriptional changes, SroA overexpression reduced nitrate respiration activity. These findings reveal a regulatory link between stress-responsive signaling pathways and respiratory metabolism, expanding our understanding of how S. aureus adapts to oxygen-limited environments encountered during infection.

Maintaining the efficacy of {beta}-lactam antibiotics against Staphylococcus aureus is a clinical priority given the prevalence of methicillin-resistant S. aureus (MRSA). We previously showed that the pyrimidine analogues 5-fluorouracil (5-FU) and 5-fluorouridine (5-FUrd) synergize with {beta}-lactams. Here, we extended this by evaluating additional nucleotide metabolism-targeting agents. Gemcitabine (Gem) and mitomycin C (Mito), like 5-FU and 5-FUrd, exhibited intrinsic anti-MRSA activity and potentiated {beta}-lactams, whereas the purine analogue 6-thioguanine (6-TG) showed distinct, often antagonistic effects. Transcriptomic analysis revealed that pyrimidine-targeting agents repress lysine and glutamate biosynthesis, while 6-TG induced these pathways, implicating amino acid metabolism in {beta}-lactam potentiation. Consistent with this, pyrimidine analogues also suppressed GlmS expression, potentially limiting UDP-GlcNAc production required for cell wall synthesis, and synergized with fosfomycin. Fluorescence microscopy confirmed that the potentiation of oxacillin activity by pyrimidine-targeting agents, but not 6-TG, was accompanied by impaired peptidoglycan synthesis. Additionally, glutathione-mediated attenuation of killing implicated reactive oxygen species in the bactericidal activity of cloxacillin combinations. Finally, these agents displayed strong anti-biofilm activity, further enhanced in combination with daptomycin and rifampicin. Together, these findings highlight the potential of pyrimidine analogues to potentiate cell wall-targeting antibiotics and identify an important role for modulation of cell wall precursor pathways in this anti-MRSA activity. ImportanceDrug interactions can complicate the treatment of antimicrobial resistant infections in patients undergoing treatment for cancer highlighting the importance of understanding the effects of anti-cancer drugs on pathogens like MRSA. Here, we investigated several drugs that target nucleotide metabolism and are used to treat cancer, fungal, and viral infections, both alone and in combination with commonly used penicillin-type antibiotics. We found that pyrimidine analogue drugs enhanced the activity of these antibiotics against MRSA, whereas the purine analogue 6-thioguanine reduced antibiotic effectiveness. These drugs altered the bacterial cell wall and other metabolic pathways linked to antibiotic susceptibility. Our findings reveal the potential to repurpose certain anticancer drugs to improve treatment of MRSA infections, while also cautioning that some drug combinations may interfere with antibiotic therapy.

Nocardiosis is a human infectious disease caused by several species of Nocardia and primarily affecting the skin, lungs and central nervous system. The first line treatment is based on cotrimoxazole, combining trimethoprim and sulfamethoxazole. These two drugs target respectively the dihydrofolate synthase (DHFR) and the dihydropteroate synthase (DHPS) involved in the essential folate synthesis pathway. The occurrence of drug resistance to these two drugs is however frequent. While the molecular mechanisms of trimethoprim resistance are well documented in other bacteria, they remain poorly explored and documented in Nocardia. This is partly because few biochemical structural or genetic studies have been conducted on DHFR from this genus. In this study, we report the biochemical and structural characterization of DHFR from Nocardia asteroides (DHFRNad). We show that overexpression of DHFRNad in N. asteroides confers strong resistance to trimethoprim. We recombinantly expressed and purified active DHFRNad and determined its inhibition constant for trimethoprim. We solved the crystal structure of DHFRNad bound to trimethoprim at high resolution. Further, biochemical studies of mutant DHFR variants pinpointed the role of important residues for trimethoprim binding and drug-resistance. HighlightsFirst biochemical and structural characterization of Nocardia asteroides DHFR. Overexpression of DHFRNad induces high-level trimethoprim resistance in N. asteroides. Crystal structure of DHFRNad reveals key residues for trimethoprim binding. Mutagenesis confirms residues critical for trimethoprim susceptibility. IC50 data confirm strong DHFRNad inhibition by trimethoprim and methotrexate

During bloodstream infection, most bacterial pathogens maintain homeostatic levels of heme, which serves as an essential biochemical cofactor and iron source, but becomes toxic at high intracellular concentrations. Well-characterized, surface exposed heme binding and acquisition systems exist in several blood-borne bacterial species. However, some gram-positive bacteria that invade the bloodstream do not encode surface displayed heme acquisition systems, despite showing clear evidence of heme utilization in blood. An example is Streptococcus agalactiae (group B Streptococcus; GBS), which is a major cause of infection in neonatal and immunocompromised populations. Here we show that GBS uses its cell membrane as a dynamic heme reservoir, which functions as the primary site of environmental heme capture, sensing, and transmembrane flux. Using positive and negative genetic selection screens, targeted mutagenesis, membrane fractionation, and spectroscopic heme detection and binding assays, we demonstrate that heme is partitioned into the GBS cell membrane, where it is sensed by the histidine kinase HssS and extracted for intracellular use by the CydDC transporter. Genetically disrupting the function of either HssS heme sensing or CydDC membrane heme extraction attenuates bacterial survival in human whole blood and in a mouse model of bacteremia. These results suggest that cell membrane-localized heme homeostasis is a determinant of fitness during blood survival. This work expands the current models of bacterial heme physiology and provides evidence that membrane localized, homeostatic heme reservoirs may represent an underrecognized strategy for blood-borne pathogens that lack canonical heme acquisition systems.

Gram-negative bacteria must coordinate remodeling of the peptidoglycan cell wall with invagination of the outer membrane to preserve envelope integrity during growth and division. The conserved Tol-Pal system has been implicated in coordinating these processes, yet its physiological contribution to envelope organization remains unclear and may depend on environmental context. Here, we examined the role of Tol-Pal in coordinating envelope remodeling in Acinetobacter baumannii across distinct growth environments. Loss of Tol-Pal did not cause a major population growth defect, and septal peptidoglycan incorporation remained largely preserved under standard laboratory growth conditions. In contrast, under specific environmental conditions--including nutrient-rich media, altered osmotic conditions, and host-like environments--Tol-Pal deficiency disrupted the spatial organization of cell division and cell morphology. Tol-Pal mutants also exhibited modest but reproducible reductions in outer membrane barrier robustness and decreased fitness in environmental and host-associated contexts. Together, these findings demonstrate that Tol-Pal is not an essential component of the core division machinery but instead contributes to the coordinated organization of the Gram-negative envelope under conditions that impose additional physiological demands. More broadly, our results highlight how environmental context can reveal conditional roles for conserved envelope systems that are not apparent during standard laboratory growth. ImportanceThe Gram-negative envelope is a complex, multilayered structure that must remain intact as cells grow and divide across diverse and often challenging environments. Coordination between peptidoglycan remodeling and outer membrane invagination is therefore critical for maintaining envelope organization and cellular fitness. Here, we show that the conserved Tol-Pal system in Acinetobacter baumannii contributes to the spatial organization of cell division and outer membrane robustness under specific environmental conditions. Although Tol-Pal deficiency permits sustained population growth under standard laboratory conditions, its absence disrupts envelope organization and compromises bacterial fitness in environmental and host-associated contexts. These findings demonstrate how environmental conditions can expose conditional roles for conserved envelope systems and highlight the importance of physiological context in shaping bacterial cell envelope organization.

Infection with obligate intracellular Chlamydia trachomatis (Ct) has been associated with cervical and ovarian carcinoma in humans. The possible cause of the tumor-promoting effect of the infection is thought to be a manipulation of host signaling pathways, which are essential for the growth of the bacteria, and which at the same time can support tumor growth. The PI3K and MAPK signaling cascades are outstanding candidates for this concept, as they are persistently activated during infection and required for the growth of Chlamydia and many tumors. The mechanism by which Chlamydia activates these pre-transforming survival signals in the cells is not well understood. Here we show that Ct infection up-regulates the oncomiR, miR21 by activating the host transcription factor AP-1. This depletes the target gene of miR-21, the tumor suppressor PTEN that ensures the persistent activation of the PI3K pathway. Blocking miR21 adversely affects the growth and development of the pathogen. We show here that miR21 KO mice are less susceptible to infection with Ct compared to control mice. Our data thus provides direct in vivo evidence of the induction and dependency of this obligate human pathogen on tumor-promoting miR-21-induced signaling. ImportanceChlamydia trachomatis is an obligate intracellular pathogen that relies on host metabolism for survival, yet the mechanisms by which it sustains nutrient access remain incompletely understood. Here, we identify a conserved host regulatory axis in which infection induces miR-21 to deplete the tumor suppressor PTEN, thereby enabling persistent activation of PI3K signaling. This pathway promotes a tumor-like metabolic state that supports bacterial growth while protecting infected cells from apoptosis. We further demonstrate that the AP-1-miR-21-PTEN circuit is required for efficient chlamydial replication in vitro and in vivo, and that disruption of miR-21 significantly impairs bacterial propagation in epithelial tissues. These findings reveal how C. trachomatis hijacks a central oncogenic signaling network to remodel host cell physiology and highlight a mechanistic link between infection and cancer-associated pathways. Targeting this host-driven signaling axis may provide new strategies for controlling infection independent of traditional antibiotics.

BackgroundGenomic stability is maintained through the coordinated regulation of DNA repair, dNTP pool balance, and histone dynamics--the three pillars of the DNA damage response. Because histones constitute the fundamental 3D physical scaffold of the genome, their precise regulation is essential for the spatial organization that dictates environmental fitness. In the radiotolerant pathogen Cryptococcus neoformans, the Rad53-Bdr1 pathway is a central DDR mediator; however, the mechanisms linking this checkpoint to histone dynamics remain poorly understood. Because conventional one-dimensional analyses cannot capture how spatial chromatin folding shapes transcriptional reprogramming, we integrated high-throughput chromosome conformation capture (Hi-C) with transcriptomic profiling to address this gap. ResultsWe demonstrate that HTA1 and HTB1, encoding H2A and H2B, are essential for viability, whereas H3 and H4 paralogs exhibit functional redundancy. Although most core histones are regulated by Rad53, HHT1 and variant HTZ1 are expressed independently of the Rad53. Notably, loss of the H3 paralog HHT2 induces growth defects under diverse stress conditions. Integrated RNA sequencing and Hi-C analyses reveal that HHT2 deletion drives transcriptional reprogramming of stress-responsive genes, coinciding with large-scale chromatin rearrangements such as A/B compartment switching and topologically associating domain boundary shifts. Furthermore, HHT2 loss impairs virulence factor formation and attenuates virulence. ConclusionOur findings identify core histones as essential architects of the 3D genome in C. neoformans. By establishing a causal link between chromatin structural collapse and transcriptional reprogramming, this study highlights 3D genome architecture as a decisive physical switch linking nucleosome-level dynamics to global transcriptional programs required for environmental survival.

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

Rhizobia are an agronomically valuable group of bacteria capable of entering into endosymbiotic relationships with leguminous plants, during which they fix atmospheric nitrogen using energy derived from the metabolism of plant-provided dicarboxylic acids. It is generally assumed that the gluconeogenic catabolism of dicarboxylic acids proceeds via the Embden-Meyerhoff-Parnas pathway in rhizobia. However, rhizobia are classically thought to lack the phosphofructokinase enzyme required for conversion of fructose-1,6-bisphosphate to fructore-6-phosphate as part of this pathway. Here, we demonstrate that a model rhizobium, Sinorhizobium meliloti, encodes a phosphofructokinase, completing the Embden-Meyerhoff-Parnas pathway of this organism. Biochemical characterization of the S. meliloti phosphofructokinase demonstrates that it can catalyze the reversible phosphorylation of fructose-6-phosphate under in vitro conditions in a pyrophosphate-dependent, rather than ATP-dependent, manner. We further show that S. meliloti also encodes a distinct fructose-1,6-bisphosphatase that can phenotypically complement the loss of the phosphofructokinase enzyme. Loss of both enzymes results in a block of the gluconeogenic pathway in S. meliloti and results in S. meliloti being unable to fix nitrogen in symbiosis with alfalfa (Medicago sativa). Phylogenetic analyses and complementation studies demonstrate that PPi-dependent phosphofructokinases are broadly distributed across the phylum Pseudomonadota (syn. Proteobacteria), including most rhizobial species of the class Alphaproteobacteria, suggesting both that PPi-dependent phosphofructokinases are likely more broadly distributed than is generally recognized, and that the catabolism of dicarboxylic acids in most rhizobia proceeds via a PPi-dependent phosphofructokinase. SIGNIFIGANCECentral carbon metabolism is an important biochemical network that bridges the gap between substrate catabolism and biosynthetic reactions in all living organisms. However, much of what we know about metabolism comes from the study of a few model organisms such as the bacterium Escherichia coli. Here, we identified the enzyme catalyzing a key step of central carbon metabolism in rhizobia (nitrogen-fixing bacterial symbionts of legumes), which until now had remained undetected. We show that this enzyme is dependent on pyrophosphate, which is different than the situation in E. coli, helping to explain why previous studies failed to identify this enzyme in rhizobia and highlighting the limitations associated with generalizing our understanding of metabolism from a limited subset of organisms.

C. glabrata (syn. Nakaseomyces glabratus) is a major fungal pathogen, typically isolated as a haploid but occasionally found in diploid form. We isolated a spontaneous diploid variant of the type strain CBS138 and performed experimental evolution under fluconazole. At physiologically relevant concentrations of fluconazole, we found that haploids and diploids both acquired PDR1 mutations, as is commonly observed in C. glabrata. Diploids additionally acquired heterozygous ERG11 and ERG25 mutations, and were more likely to acquire aneuploidies. Despite an ancestral fitness advantage for haploids, after [~]200 generations the highest-fitness clone, as measured in competitive assays with and without fluconazole, was a diploid PDR1 V329F/+ ERG11 K152E/+ double heterozygote. Diploid clones also had higher MICs. In a follow-up experiment in which we rapidly increased the fluconazole concentration to [~]1 mg/mL, haploids and diploids adapted via entirely different paths: haploids via co-mutation in ERG3 and CgOSH3, a previously unreported path to fluconazole resistance, and diploids via heterozygous mutation in ERG25 coupled with trisomies of chrF, chrG, and chrI (in all clones) and chrC (in most).

The nucleotide diadenosine tetraphosphate (Ap4A) accumulates during stress across organisms and cell types and is widely hypothesized to be an alarmone or second messenger. While Gram-negative bacteria use ApaH-family hydrolases to degrade Ap4A and other dinucleoside tetraphosphates (Ap4Ns), Gram-positive bacteria, including Staphylococcus aureus, use YqeK. Inactivation of Ap4A hydrolases and corresponding Ap4A accumulation cause diverse phenotypic effects in both Gram-negative and Gram-positive bacteria, ranging from increased sensitivity to antimicrobials to reduced virulence. However, the physiological role of YqeK in S. aureus remains uncharacterized. Here, we constructed an isogenic yqeK mutant in S. aureus and showed that {Delta}yqeK was sensitive to nitrosative and organic acid stress. We used a luminescence-based assay to show that {Delta}yqeK had [~]1000-fold higher relative Ap4N levels than wild-type even during unstressed growth, and all phenotypes were restored by complementation. Transcriptomics revealed that {Delta}yqeK exhibited stress-specific dysregulation of translation, nucleotide metabolism, central metabolism, iron acquisition, and stress response genes. In contrast, {Delta}yqeK had few transcriptional differences relative to wild-type during unstressed growth despite the large Ap4N accumulation, suggesting that the effects of Ap4Ns are contingent on the cellular stress state. Unexpectedly, we also found that the entire agr quorum sensing operon and numerous additional virulence genes, including hemolytic toxins, had reduced expression in {Delta}yqeK, correlating with reduced hemolytic activity in the mutant even in the absence of stress. Our data reveal YqeK to be a critical metabolic determinant of S. aureus stress resistance and virulence and position this hydrolase as a promising candidate for anti-virulence drug development. ImportanceS. aureus is a leading cause of antibiotic-resistant bacterial infections worldwide and is resistant to many components of the host immune response. Here, we discovered that deletion of YqeK, an enzyme that degrades a stress-associated nucleotide signaling molecule called Ap4A, rendered S. aureus more susceptible to infection-relevant stress conditions but had little impact on normal growth. Ap4Ns accumulated in the yqeK mutant and caused major stress-specific changes in gene expression, including reduced expression of key virulence genes. This correlated with a reduction in the destruction of red blood cells, a measure of bacterial toxicity toward host cells. Our data suggest that YqeK represents a promising target for new drugs aimed at reducing the virulence of S. aureus.

Shigella sonnei is a leading cause of bacterial dysentery and a high priority WHO pathogen because of the spread of multidrug resistant strains. Understanding microbiome-Shigella-host interactions during colonization of the gastrointestinal tract, and the development of vaccines have been hampered by the lack of small animal models of shigellosis. Here, we developed a murine model of intestinal colonization with S. sonnei. Pre-treatment of mice with antibiotics disturbed the intestinal microbiome and rendered mice susceptible to high level, gastrointestinal colonization with S. sonnei for over one week. Infection with S. sonnei CS14 harbouring a stable virulence plasmid induced an initial inflammatory response in wild type mice, with weight loss and elevated levels of fecal lipocalin 2; the S. sonnei Type III Secretion System was responsible for this inflammatory response. Expression of O-antigen and Group IV capsule by S. sonnei promoted sustained intestinal colonization, with infected mice developing mucosal and systemic antibody responses predominantly directed at these glycans. Finally, infection with S. sonnei induced a degree of protection against subsequent re-challenge. Overall, this murine model successfully mimics aspects of S. sonnei colonization and should be helpful in understanding how S. sonnei successfully survives within the gastrointestinal tract and competes with the microbiota as well as the evaluation of vaccine candidates.

Klebsiella pneumoniae is a leading cause of pneumonia and bacteremia and is especially dangerous in healthcare settings. Despite massive clinical significance, the mechanisms used by macrophages to kill K. pneumoniae are not well defined. Macrophages are critical for controlling K. pneumoniae as mice lacking monocyte-derived or alveolar macrophages have higher bacterial tissue burdens and mortality. Two prominent mechanisms used by macrophages to kill bacteria are the production of reactive oxygen species (ROS) via the NADPH oxidase NOX2 and reactive nitrogen species (RNS) via the inducible nitric oxide synthase iNOS. Previously, we found that K. pneumoniae uses similar genetic factors to survive during bacteremia and within macrophages. The ability of these factors to enhance intracellular fitness was significantly correlated with resistance against RNS, not ROS. Here, we aimed to define whether macrophage ROS and RNS contribute to intracellular K. pneumoniae clearance. Using wild-type, Cybb-/-, and Nos2-/- cells, we measured K. pneumoniae survival within macrophages lacking such defenses. NOX2 was dispensable for K. pneumoniae clearance, and ROS was undetectable in K. pneumoniae-infected macrophages. We confirmed that ROS was undetectable within alveolar-like macrophages, indicating a conserved ROS evasion phenotype across macrophage subsets. Instead, iNOS significantly contributed to macrophage clearance of K. pneumoniae and enhanced cytokine production. iNOS likely enhances K. pneumoniae clearance through coordination of immunity and RNS. Activation of pathways upstream of iNOS may be the most relevant to supporting effective macrophage control of K. pneumoniae. This study defines unexpected differential roles for ROS and RNS in macrophage clearance of K. pneumoniae.

Reduced vancomycin susceptibility phenotypes in Staphylococcus aureus contribute to treatment failure, yet the genetic determinants of survival under inhibitory vancomycin exposure remain incompletely defined. We performed transposon directed insertion-site sequencing (TraDIS) on a methicillin resistant S. aureus (MRSA) ST398 mutant library following exposure to vancomycin at its minimum inhibitory concentration, identifying 52 genes whose disruption was associated with loss of population survival at inhibitory drug concentrations. Prophage associated loci were the largest functional group, spanning predicted structural and regulatory genes as well as multiple conserved hypothetical proteins. Targeted testing of defined transposon mutants in a USA300 background confirmed that disruption of selected loci impaired growth under vancomycin exposure. Our results highlight the contribution of diverse physiological processes, including metabolism, stress responses, and a prominent role for prophage-associated functions, rather than discrete resistance pathways. Together, these findings indicate that vancomycin tolerance is shaped by the general physiological state of the bacterial cell, including metabolic capacity and stress adaptation. ImportanceTreatment failure in Staphylococcus aureus infections often occurs in the absence of known antibiotic resistance determinants, suggesting that additional survival mechanisms influence therapeutic outcomes. In this study, we identify genetic determinants required for survival during inhibitory vancomycin exposure, revealing a broad role for metabolic functions, stress adaptation, and prophage-associated loci. The prominence of these diverse processes highlights that survival reflects global physiological adaptation rather than discrete resistance pathways. This insight underscores the need to consider cellular physiology and stress responses when developing strategies to prevent antibiotic tolerance and improve treatment efficacy.

Enterohemorrhagic Escherichia coli (EHEC) is a foodborne pathogen that causes bloody diarrhea and hemolytic uremic syndrome by disrupting the intestinal brush border. During infection, EHEC injects the transmembrane virulence factor Tir into enterocytes; upon insertion into the apical membrane, this factor mediates bacterial attachment and drives formation of actin-rich pedestals needed for colonization. How Tir is inserted into the host plasma membrane remains unclear. Here, we investigated the role of brush border resident IRTKS, a Tir- and membrane-binding protein, in this process. Using multiple IRTKS gain- and loss-of-function models, we analyzed pedestal organization and component localization. Whereas canonical models position IRTKS downstream of Tir as a scaffolding link to F-actin, we found that perturbing IRTKS disrupted the distribution and abundance of Tir. Moreover, ectopic IRTKS expression enhanced Tir membrane insertion in the absence of other virulence factors. We conclude that IRTKS functions early in pedestal formation to promote Tir accumulation in the plasma membrane and in turn, facilitate bacterial attachment. SUMMARYEHEC attaches to intestinal epithelial cells using injected virulence factor Tir, which forms actin pedestals and promotes bacterial colonization. We found that host protein IRTKS promotes Tir accumulation in the plasma membrane, to facilitate intimate bacterial attachment and pedestal formation.

Cyclic diguanosine monophosphate (c-di-GMP) is a ubiquitous bacterial second messenger that regulates diverse cellular processes, including colony morphology, motility, biofilm formation, and virulence. It is synthesized by diguanylate cyclases (DGCs) containing the GGDEF domain and degraded by phosphodiesterases (PDEs) containing the EAL domain. However, studies on the genetic and physiological characteristics of c-di-GMP metabolism in Pantoea ananatis are lacking. In this study, we identified 26 predicted c-di-GMP metabolism-related genes in the P. ananatis PA13 genome: 9 encode GGDEF-only domain proteins, 5 encode dual GGDEF/EAL domain proteins, and 12 encode EAL-only domain proteins. We constructed overexpression strains and mutants of 26 DGC- and PDE-encoding genes, and then assessed their Congo Red binding, mucoid and rugose phenotypes, pellicle formation, and swimming motility. We identified 14 of 26 DGC and PDE proteins that affect phenotype changes. Among the 26 DGC- and PDE-overexpressing strains, 13 exhibited the phenotypic changes described above, with some showing alterations in multiple phenotypes simultaneously. Notably, overexpression of dgcM induced changes across all phenotypes. Among the 26 DGC and PDE mutants, the pdeC mutant increased pellicle formation and Congo red binding, the pdeM mutant reduced the mucoid phenotype, and the pdeS mutant, which shows high similarity to ydiV, an anti-FlhD factor, increased swimming motility. Overexpression strains and mutants of 14 DGC and PDE proteins that exhibited phenotypic changes had higher intracellular c-di-GMP levels than the wild type. This study provides important insight into the role of the c-di-GMP network in the plant pathogen P. ananatis. IMPORTANCEPantoea ananatis is a versatile bacterium that causes significant diseases in various economically important plants. To survive and infect hosts, bacteria use a key signaling molecule called c-di-GMP to switch between swimming freely and forming protective communities known as biofilms. Despite its importance, the specific genes governing this signaling network in P. ananatis remained unknown. In this study, we systematically identified and characterized 26 genes responsible for regulating c-di-GMP levels in P. ananatis PA13. By analyzing mutants and overexpressing these genes, we pinpointed 14 critical factors that control essential behaviors such as motility, pellicle formation, and colony appearance. Notably, we discovered specific genes, such as dgcM and pdeS, that act as master regulators of these traits. This comprehensive functional map of the c-di-GMP network provides essential insights into how this pathogen adapts to its environment, offering potential targets to control plant infections.