Leptospira interrogans, the agent of leptospirosis, employs complex virulence mechanisms that are not fully understood at a systems level. To elucidate the regulatory landscape of its pathogenicity, we used Weighted Gene Co-expression Network Analysis (WGCNA) on a comprehensive transcriptomic dataset to map the architecture of its virulence programs. Our analysis revealed that the L. interrogans transcriptome is organized into distinct, functionally coherent modules. We discovered that known virulence factors are significantly concentrated in two key modules: a "lightgrey" module that orchestrates host colonization and immune evasion, containing genes for surface adhesion (loa22, ompL1) and defense (lipL21); and a "black" module that functions as an arsenal for tissue invasion (colA), stress adaptation (clpB), and cytotoxicity (sph2). Furthermore, by contextualizing genes within this network, our approach implicated numerous uncharacterized genes (e.g., from the PF07598 family) in pathogenesis due to their strong co-expression with established virulence factors. These findings provide a systems-level blueprint of the regulatory networks driving leptospirosis, offering a rich resource of functionally validated gene modules and novel targets for the development of next-generation vaccines and therapeutics. These findings not only deepen the understanding of L. interrogans virulence regulation but also provide a conceptual framework for integrating transcriptomic data into systems-level models of bacterial pathogenesis, paving the way for translational applications in diagnostics and vaccine design.
A marked difference in virulence was observed between two subpopulations of the Edwardsiella piscicida FSW910410 strain, originally isolated from olive flounder (Paralichthys olivaceus). While the strain maintained under routine laboratory culture conditions retained high virulence, the same strain stored long-term at -80 °C lost its ability to induce disease in olive flounder. Comparative in vivo assays demonstrated complete avirulence in the frozen-stored subpopulation, contrasting with high mortality rates induced by the virulent counterpart. Despite over 99 % genomic sequence identity and identical virulence gene repertoires, the two subpopulations differed significantly in their ability to evade host immune responses. Whole-genome methylation profiling revealed comparable levels of N6-methyladenine (m6A) but notably reduced levels of cytosine methylation (m4C/m5C) in the avirulent subpopulation, particularly in genes associated with type III and type VI secretion systems (T3SS and T6SS). Quantitative RT-PCR analyses confirmed substantially lower expression of T3SS and T6SS genes in the avirulent strain. These results suggest that epigenetic modifications, especially cytosine methylation, may contribute to the observed differences in virulence, although additional regulatory mechanisms cannot be excluded. Collectively, the data highlight the complex interplay of genomic and epigenetic factors that may influence bacterial pathogenicity during prolonged storage or passage.
Klebsiella pneumoniae is a Gram-negative bacterium and an important pathogen implicated in both hospital- and community-acquired pneumonia. K. pneumoniae strains are either classical, hypervirulent (hvKp), or antibiotic-resistant. There are several virulence factors of K. pneumoniae that helps immune evasion and survivability within the host. This review emphasizes the host immune interactions of K. pneumoniae. Evasion strategies of the pathogen and complex risk factors of the infection have been explained. Different diagnostic approaches, such as computed tomography, PCR, and Lateral Flow Immunoassay (LF), have been used for the diagnosis of K. pneumoniae. Multidrug-resistant Klebsiella pneumoniae (MDRKP) has emerged as a significant global public health concern, with higher cases of carbapenem-resistant K. pneumoniae (CRKP). CRKP caused more than 7000 deaths annually in Europe. Although K.pneumoniae has several mechanisms of antibiotic resistance, resistance to β-lactams, specifically carbapenems, causes a notable difference. Newer β-lactam/β-lactamase inhibitor combinations, such as ceftazidime-avibactam and cefiderocol are preferred for KPC-producing infections. This review emphasizes the ongoing challenges in translating therapeutic advancements into successful clinical outcomes, as well as the persisting complications in K. pneumoniae research, including the unsolved differences between hypervirulent and classical strains. Alternative methods, including bacteriophage therapy, antimicrobial peptides, and immunotherapy, are being studied for tackling CRKP. This review addresses recent advances in understanding Klebsiella pneumoniae-induced pneumonia, emphasizing how virulence factors interact with immune defences. Additionally, we highlight key challenges in antimicrobial resistance, current therapeutic strategies, K. pneumoniae's implications for infection control and antibiotic stewardship. Overall, this review aims to contribute to a deeper understanding of K. pneumoniae and to guide future interventions for effective prevention, control, and treatment strategies.
Pseudomonas aeruginosa can exploit its metabolic flexibility during cystic fibrosis lung infections to reduce antibiotic sensitivity and offset resistance costs, traits that influence its evolutionary trajectory. Although both traits are linked to nutrient conditions, their role in resistance evolution remains poorly defined. We examined how single-nutrient conditions influence resistance evolution in P. aeruginosa through phenotypic and genotypic adaptations after adaptive laboratory evolution with different antibiotics in single-nutrient media. Antibiotic susceptibility testing showed limited MIC differences for ceftazidime and imipenem, but stronger effects for ciprofloxacin, colistin, and tobramycin. Ciprofloxacin evolution in glutamate medium yielded the highest MIC increase, with at least a 4-fold rise, whereas tobramycin evolution in glucose resulted in up to a 4-fold MIC reduction compared to lineages evolved under all other nutrient conditions for the same antibiotic. Whole-genome sequencing showed nutrient-specific mutation in wbpL after tobramycin evolution in glucose, and fusA and pmrB across conditions. Ciprofloxacin resistance in glutamate-lineages involved yicC, whereas nfxB mutations were absent in glucose- and arginine-evolved lineages. No distinct nutrient-specific differences were seen for colistin. These findings underscore the significant role of nutrient conditions in shaping resistance and highlight the need to consider physiologically relevant media when studying antibiotic resistance evolution.
PURPOSE:This study aimed to evaluate the role of membrane microdomains (MMd) in virulence of Bacillus anthracis by assessing the effects of known antifungal compounds, referred to as raft-associated lipid biosynthesis inhibitors (RALBIs), on its pathophysiology. MATERIALS AND METHODS:FDA-approved antifungal compounds representing three distinct classes-azoles (ketoconazole), allylamines (terbinafine), and polyenes (nystatin), were tested against B. anthracis. These compounds target different enzymes involved in MMd-associated lipid biosynthesis. Their impact on sporulation, toxin secretion, macrophage interaction, bio-signaling, cell envelope fluidity, and biofilm formation was examined. Additionally, their synergistic potential with conventional antibiotics was evaluated. RESULTS:RALBIs markedly attenuated the pathogenic traits of B. anthracis, including reduced macrophage association, diminished toxin secretion, and impaired sporulation. Treatment also altered growth kinetics, morphology, biofilm development and cell envelope fluidity. Importantly, the combination of RALBIs with erythromycin significantly reduced its minimum inhibitory concentration (MIC) against B. anthracis. CONCLUSION:These findings highlight membrane microdomains as crucial regulators of virulence in B. anthracis and identify RALBIs as promising adjunctive agents. By targeting MMd, sterol inhibitors disrupt multiple pathogenic pathways and enhance antibiotic efficacy, underscoring their potential as novel antibacterial strategies.
Little is known about how subinhibitory concentrations of antibiotics to which bacteria are resistant affect bacterial virulence. In this study, the effect of subinhibitory concentrations of ampicillin on the virulence of E. coli O104:H4 was analyzed. Bacteria were pre-exposed to 0.1, 0.3, or 0.5 mg/mL of ampicillin in LB media and incubated for 4 h at 37 degrees C. Transformation capacity (using plasmids and PCR-amplified DNA sequences), swarming motility, biofilm production, curli formation, and virulence gene expression were determined. Ampicillin increased the transformation of E. coli O104:H4, with the highest number of transformants (>10(4) CFU/ng DNA; p <= 0.05) detected after exposure to DNA sequences of spectinomycin. In addition, bacteria pretreated with 0.5 mg/mL of ampicillin exhibited higher swarming motility (7.6 cm, vs 6.0 cm for control; p <= 0.05) and biofilm production (up to 1.9-fold; p <= 0.05) when subsequently exposed to 0.1 and 0.3 mg/mL of antibiotic compared with the control. Also, significant overexpression of the virulence-related genes flhC (<= 16.1fold), fliA (<= 22.1-fold), csgA (<= 3.6-fold), csgD (<= 9.1-fold), stx2a (<= 32.2-fold), and the antibiotic resistance gene blaTEM-1 (<= 5.5-fold) was observed. In conclusion, ampicillin-resistant E. coli O104:H4 increased the expression of its virulence factors when exposed to most subinhibitory concentrations of ampicillin analyzed in this study.
Biofilm formation is governed by quorum sensing (QS) and intracellular signaling, with cyclic di-GMP (c-di-GMP) acting as a key regulator that modulates biofilm stability in response to environmental cues. The present study aims to explore the regulatory network between QS, c-di-GMP signaling, and amyloid production in the marine biofilm-forming bacterium Pseudomonas aeruginosa PFL-P1 under various physicochemical stressors. P. aeruginosa PFL-P1 demonstrated adaptability to diverse conditions typical of marine habitats, attributed to the activity of diguanylate cyclase (dgc86) and phosphodiesterase (pde94) genes regulating c-di-GMP turnover. Gene expression analysis revealed a coordinated regulatory network during biofilm development, with significant upregulation of dgc86, pde94, fapC (functional amyloid synthesis), lasI, rhlI (QS), and nahAc [polycyclic aromatic hydrocarbon (PAH) degradation] at 48 h, indicating a mature biofilm. Under acidic condition (pH 4), all genes except pde94 exhibited an adaptive response (p<0.0001). Salinity ≤1 % enhanced gene expression, whereas salinity ≥5 % suppressed it due to osmotic stress (p<0.0001). At 40 °C, dgc86 (p=0.0457) and fapC (p=0.0444) were upregulated, promoting biofilm stability. Pyrene exposure induced significant upregulation of dgc86, lasI, rhlI, and nahAc (p<0.05), enhancing biofilm formation and PAH degradation while downregulating pde94. Supplementation with C4-HSL and 3OC12-HSL upregulated these genes, reinforcing the role of QS in biofilm regulation. Terrein, a QS and c-di-GMP inhibitor downregulated fapC and nahAc, disrupting biofilm formation and PAH degradation. The strong correlation between c-di-GMP levels, amyloid production, and its high binding affinity to FapC (-11.8 kcal/mol) suggests a dual role for c-di-GMP as a signaling molecule and molecular chaperone in amyloid assembly.
Over the years, anthrax has become endemic in the state of Andhra Pradesh, India, posing risk for both animals and humans and spreading to the bordering state of Karnataka. The incidence is on the rise and the spread covering the entire state. The article is aimed at projecting spatio-temporal distribution of anthrax in the state of Andhra Pradesh highlighting possible reasons influencing the transmission dynamics in animals and resultant risk to humans. A select sub-sample of these were analyzed by Whole Genome Sequencing. From 2009 to 2024, 122 animal anthrax outbreaks were recorded, whereas 21 human anthrax outbreaks were reported from 2004 to 2024 in the state of Andhra Pradesh. Animal anthrax outbreaks were reported throughout the year with short peaks during March to October, covering high summer and rainy season. Most affected species were sheep (84 %), followed by Bovines (9 %) and Goats (4 %). Spill over was detected in Pig and Hog deer, and a probable transmission in humans. The recent spread of anthrax in livestock across the state, particularly to adjoining pasture lands of Odisha, Karnataka and Tamil Nadu due to migration for grazing might have led to the recent hot spots around state boundaries and forest fringes, calls for effective intervention to stop the spread.
Capsule is a key virulence factor for Bacillus anthracis. In this study, we examined the onset of capsule expression and the ultrastructure of capsule expressed in vitro and in infected hosts. We determined by immunofluorescence and immunoelectron microscopy that capsule was produced as early as 15 min after onset of germination. Further analysis revealed bacilli were often fully encapsulated when they emerged from the exosporium. Using conventional fixative to prepare samples for electron microscopy resulted in loss of capsular material, but using a fixative containing ruthenium red and lysine acetate preserved it. Transmission electron microscopy using enhanced fixation revealed that B. anthracis grown in vitro were surrounded by a thick capsule layer comprised of globular structures and a slime layer. When examined using the enhanced fixation, extracellular bacilli in tissues from moribund animals were surrounded by a thick capsule layer, and had long branching strands like those observed on live but not fixed bacilli by fluorescence microscopy. Scanning electron microscopy using enhanced fixation similarly revealed B. anthracis within spleen sinusoids were covered with long branching strands that appeared to tether them to host cells. This is the first report describing the presence of long capsule strands during infection in vivo.
BACKGROUND:Anthrax outbreaks continue to be reported from several districts of Karnataka in India. Many outbreaks in livestock and humans have reported, the soil and climatic conditions would have supported long-term spore persistence. Hence, the state of Karnataka in India was chosen as an initial pilot site to study, isolate, and characterize Bacillus anthracis. In this study, we integrate molecular typing and soil analysis across endemic and non-endemic areas, an approach that has been scarcely explored and could be a novel contribution. By comparing both endemic and non-endemic regions, it would be an opportunity to examine soil-related ecological factors that could have been linked to the anthrax outbreaks. A total of 45 suspected animal anthrax cases were investigated using culture and PCR-based methods. Colonies with Medusa head-like appearance on blood agar plates were subjected to PCR assays targeting protective antigen (pXO1), capsular gene (pXO2), and the chromosomal rpoB genes. Additionally, soil samples from 12 different sites (six endemic and six non-endemic) were processed using the Ground Anthrax Bacillus Refined Isolation (GABRI) method, which was used to enhance the detection of B. anthracis spores by reducing environmental contaminants that may inhibit spore germination and growth. RESULTS:Out of 45 suspected animal anthrax cases, 09 isolates were confirmed as Bacillus anthracis with colony characteristics of greyish-white, frosted-glass on blood agar, further verified through PCR analysis. Phylogenetic analysis based on the rpoB gene demonstrated a close genetic relationship among these isolates, suggesting that the transmission of spores was localized and likely facilitated by animal movement. The soil analysis showed that the endemic sites had an alkaline pH of 7.81-8.9, higher organic carbon 0.45-4.36 %, elevated phosphorus 10.32 to 123.7 kg/ha, and greater clay content up to 45 % in Bellary, contributing to higher survivability and retention of spores in endemic regions. In contrast, non-endemic soil exhibited neutral to slightly acidic pH (6.1-6.85), lower phosphorus levels, and sandy clay loam texture that may limit long-term spore retention. CONCLUSION:The study offers significant insights into the environmental and genetic factors contributing to the persistence of Bacillus anthracis in both endemic and non-endemic regions. The finding shows that soil plays a crucial role in the survival and transmission of anthrax spores, with higher retention observed in endemic areas. The close genetic relationship among isolates further suggests localized transmission, likely influenced by animal movement. These results underline the need for continued surveillance and prevention strategies, especially in regions with a history of anthrax outbreaks.
Bacillus anthracis, the causative agent of anthrax, poses a persistent threat in endemic regions. In this study, eight archived B. anthracis isolates from the year 2018-2019 were successfully revived, highlighting the resilience of spores and the effectiveness of long-term storage in maintaining virulence. Genomic DNA was extracted using three methods: Qiagen DNeasy kit, Zymo bead-based method, and phenol-chloroform extraction. The inclusion of ampicillin during initial incubation in the phenol-chloroform method enhanced DNA yield by selectively eliminating vegetative cells without inducing sporulation. The highest DNA concentration of 3710 ng/μL was obtained using this method. PCR analysis confirmed the presence of pXO1 and pXO2 virulence plasmids, along with the chromosomal rpoB gene, using OIE-recommended primers. The rpoB marker was chosen over 16S rRNA for its superior resolution among closely related strains. Phylogenetic analysis with bootstrap replicates revealed conserved sequences in plasmid genes, while rpoB exhibited notable diversity, suggesting chromosomal variation among the isolates. These findings contribute to ongoing anthrax surveillance and molecular epidemiology efforts in Karnataka, India, and underscore the potential of combining plasmid and chromosomal markers in understanding strain-level variation. Further whole genome sequencing (WGS) will provide deeper insights into the genomic diversity and evolutionary dynamics of B. anthracis.
Listeria monocytogenes is primarily a saprophytic bacterium, but it can transition into a pathogenic form when ingested by humans, vertebrates, or invertebrates, thereby initiating infection and proliferating within the host. This bacterium serves as a model for studying the molecular and cellular mechanisms by which environmental microorganisms adapt to mammalian and other host systems, thereby promoting the successful colonization and survival of these microorganisms. Environmental contamination is a significant factor in the transmission of listeriosis and other foodborne diseases. This review provides a comprehensive examination of the microbiological characteristics of L. monocytogenes. It systematically evaluates host-pathogen interactions, focusing on a range of clinically relevant isolates and their pathogenic mechanisms. Furthermore, it evaluates in vitro models employed to investigate the virulence factors of the pathogen while also considering the role of animal models, including Drosophila melanogaster and other insect species, in elucidating infection dynamics. In addition, it discusses the host's innate immune response, specifically highlighting the molecular and cellular pathways activated upon infection, thereby providing a comprehensive understanding of the pathogen's interactions with the host's immune system.
Bacillus anthracis, a rod-shaped, spore-forming bacterium, is the causative agent of anthrax. The life cycle of B. anthracis involves sporulation and germination processes that are precisely regulated by distinct sigma factors and associated proteins. Its pathogenicity is primarily attributed to a tripartite toxin consisting of lethal factor (LF), edema factor (EF) and protective antigen (PA), as well as an antiphagocytic capsule of Poly-γ-D-Glutamate. The virulence of B. anthracis is further regulated by various post-translational modifications (PTMs), including protein phosphorylation, acetylation, glycosylation, hydroxylation, and lipidation. These modifications play a key role in modulating bacterial virulence by influencing enzymatic activity and protein expression. This review summarizes the role of PTMs in the regulation of B. anthracis virulence. A deeper understanding of how these PTMs contribute to B. anthracis pathogenesis may offer new insights into novel enzyme targets, strategies to disrupt toxin production and the development of therapeutic approaches to combat anthrax infections.
Phages are bacterial viruses considered as therapeutics for treatment of serious infections with antibiotic-resistant pathogens. In Staphylococcus aureus, resistance to cell wall targeting antibiotics is common in the methicillin resistant (MRSA) or vancomycin-intermediate susceptible (VISA) strains. Furthermore, the cell wall anchors the primary phage receptor, the wall teichoic acid (WTA) glycopolymers. Here we demonstrate that mutations resulting in VISA development affect phage susceptibility of clinically and laboratory evolved strains. For clinical strains we observed both increased and decreased susceptibility compared to the ancestral vancomycin susceptible strains when infected with the therapeutically relevant myoviruses, ΦIPLA-RODI, Stab20, Stab21 and ΦK. For laboratory strains adapted to vancomycin from the MRSA strain, JE2, we observed variable resistance development to the phages ΦIPLA-RODI, Stab21 and ΦK with one strain becoming completely phage resistant. In contrast, half of the VISA strains became susceptible to Stab20 to which JE2 is resistant. These changes in part correlated with altered WTA glycosylation patterns as shown by WTA-specific antibodies and for the resistant strain resulted in compromised phage therapy as shown in a Galleria mellonella infection model. This study highlights the need for understanding antibiotic-driven alterations in bacterial physiology when developing phage-based therapies using combination treatments with antibiotics and phages.
PURPOSE:Cronobacter sakazakii is a foodborne pathogen notable for both virulence and desiccation tolerance. This study investigates the functional roles of the pESA3 plasmid in mediating these two traits and explores the potential trade-off between them. METHODS:Proteomic profiling identified pESA3-encoded proteins, followed by construction of targeted deletion mutants (hcp, ESA_pESA3p05542) and a plasmid-cured strain. Phenotypic assays evaluated growth, desiccation survival, surface hydrophilicity, and virulence in a neonatal rat model. RESULTS:Deletion of ESA_pESA3p05542 impaired desiccation tolerance and reduced surface hydrophilicity, while hcp deletion attenuated virulence but enhanced desiccation survival. Notably, complete loss of pESA3 abolished virulence without affecting desiccation resistance, suggesting functional antagonism between plasmid-encoded virulence and stress-response genes. CONCLUSIONS:pESA3 enables C. sakazakii to balance host pathogenicity and environmental persistence. Its gene modules promote either virulence or desiccation resistance, but not both, reflecting an evolutionary trade-off that supports survival across diverse niches.
The asexual reproduction cycle of the cheese-ripening fungus Penicillium camemberti is typical for a filamentous ascomycete fungus. It involves the production of conidia by successive mitotic divisions from specialized cells called conidiophores. Conidia are used for inoculating the fungus onto French soft cheeses and are produced industrially by submerged fermentation. However, the impact of mycelium macromorphology on conidial production for this type of fermentation has been little investigated. By studying the physiological effect of different sources and concentrations of carbohydrates at the laboratory scale, it was observed that the fungus takes on different morphologies associated with varying ability to produce conidia. Especially, a dispersed morphology was not always associated with production of conidia. Through RNA-seq transcriptomic analyses, we followed the expression profiles of P. camemberti genes during the time course of liquid cultures made in the presence of two concentrations of glucose, one of sucrose and one with a mix of glucose and fructose, which are conditions for which P. camemberti presents varying morphologies and efficiencies to produce conidia. This led to confirm that, like other ascomycetes, P. camemberti likely uses the conidiation pathway first described in the model fungus Aspergillus nidulans. It also enabled the identification of a potential conidiation-inhibiting transcription factor, specific to Eurotiales and only upregulated in conditions without conidia production. Functional studies of its ortholog in A. nidulans, for which the conidiation pathway has been extensively studied, should allow to verify whether this factor indeed plays a role in the asexual cycle.
Cyclic di-guanosine monophosphate (c-di-GMP), a bacterial second messenger, regulates diverse key processes including virulence, biofilm formation, and motility. Its synthesis involves diguanylate cyclases (DGCs) with GGDEF domains, while degradation is mediated by phosphodiesterases (PDEs) containing EAL or HD-GYP domains. This study examines the role of GefC (VP0486), a GGDEF domain protein in Vibrio parahaemolyticus, in c-di-GMP signaling, biofilm dynamics, motility, and virulence. Deletion of gefC markedly reduced cellular c-di-GMP, impaired biofilm formation, and reduced matrix components (exopolysaccharides, extracellular proteins, and extracellular DNA). Conversely, the ΔgefC mutant exhibited enhanced swimming motility and increased cytotoxicity and hemolytic activity, while zebrafish infection assays revealed attenuated lethality. Transcriptional analysis showed GefC differentially regulates EPS-related gene (cpsA), polar flagellar gene (flgM), type III secretion system 1 (vopN and vp1687), type VI secretion system 2 (hcp2, vpa1043, and vpa1044), and the TDH-encoding tdh2. Genetic evidence confirmed vp0485 and gefC form an operon. These findings establish GefC as a critical regulator of c-di-GMP-dependent biofilm development, motility, and virulence in V. parahaemolyticus, highlighting its multifaceted role in pathogenicity.
Pseudomonas aeruginosa can exploit its metabolic flexibility during cystic fibrosis lung infections to reduce antibiotic sensitivity and offset resistance costs, traits that influence its evolutionary trajectory. Although both traits are linked to nutrient conditions, their role in resistance evolution remains poorly defined. We examined how single-nutrient conditions influence resistance evolution in P. aeruginosa through phenotypic and genotypic adaptations after adaptive laboratory evolution with different antibiotics in single-nutrient media. Antibiotic susceptibility testing showed limited MIC differences for ceftazidime and imipenem, but stronger effects for ciprofloxacin, colistin, and tobramycin. Ciprofloxacin evolution in glutamate medium yielded the highest MIC increase, with at least a 4-fold rise, whereas tobramycin evolution in glucose resulted in up to a 4-fold MIC reduction compared to lineages evolved under all other nutrient conditions for the same antibiotic. Whole-genome sequencing showed nutrient-specific mutation in wbpL after tobramycin evolution in glucose, and fusA and pmrB across conditions. Ciprofloxacin resistance in glutamate-lineages involved yicC, whereas nfxB mutations were absent in glucose- and arginine-evolved lineages. No distinct nutrient-specific differences were seen for colistin. These findings underscore the significant role of nutrient conditions in shaping resistance and highlight the need to consider physiologically relevant media when studying antibiotic resistance evolution.
Candida albicans, a prevalent opportunistic fungal pathogen, employs dimorphic transition (yeast-to-hypha) as a central strategy for host tissue invasion and immune evasion. Although prior studies have linked ADH1 deletion to attenuated virulence phenotypes such as impaired hyphal formation, the molecular mechanism underlying this phenomenon remains elusive. Here, we report that ADH1 knockout strains exhibit a striking hyperelongation of hyphae, deviating from the characteristic branched architecture observed in wild-type strains. Transcriptomic profiling identified arginine metabolism as the most significantly activated pathway in adh1Δ/Δ mutants, with marked upregulation of CAR1, encoding a key arginase. Crucially, pharmacological or genetic inhibition of Car1 activity fully restores wild-type hyphal morphology in ADH1 knockout strains, unequivocally establishing that ADH1 governs hyphal development through repression of CAR1 expression. Our findings delineate the Adh1-Car1 metabolic axis as a master regulator of dimorphic switching in C. albicans: while Adh1 constrains Car1 to maintain balanced hyphal branching, its deletion triggers arginine metabolic flux dyshomeostasis, driving uncontrolled hyphal hyperelongation. This work redefines the functional paradigm of Adh1 beyond its canonical role in ethanol metabolism, positions fungal metabolic rewiring as a direct driver of morphogenic plasticity, and nominates the Adh1-Car1 axis as a high-value target for antifungal interventions.
Klebsiella pneumoniae is a major cause of endogenous endophthalmitis, a rapidly progressing intraocular infection associated with severe inflammation and vision loss. The vitreous body presents a hypoxic and iron-restricted environment, yet the bacterial metabolic adaptations that enable persistence in this niche remain largely unknown. Here, we show that K. pneumoniae undergoes metabolic reprogramming to facilitate intraocular survival, characterized by enhanced glycolysis and siderophore-mediated iron acquisition. Proteomic profiling under vitreous-mimicking conditions revealed significant upregulation of PfkA, PykF, and EntB. Targeted deletion of these genes impaired bacterial growth under hypoxia and iron limitation, and significantly reduced intraocular colonization, proinflammatory cytokine production, and visual impairment in a murine model. Double mutants lacking both glycolytic and iron acquisition pathways were nearly avirulent. Correspondingly, infected eyes exhibited lower levels of lactate and iron, reflecting reduced bacterial metabolic activity. These findings establish glycolysis and iron acquisition as critical determinants of K. pneumoniae virulence in the eye and provide insight into the metabolic strategies underpinning bacterial persistence in nutrient-limited host environments.
