Biology subjects

Vidal, J. E.

Publications and source records attributed to Vidal, J. E..

12 recordsLinked to original sources

bioRxiv2026

Lactate Oxidase (LctO) Acts as a Metabolic Checkpoint Restraining Streptococcus pneumoniae Invasion of Respiratory Epithelial Barriers

Streptococcus pneumoniae rapidly translocates across polarized human bronchial epithelial barriers, with viable bacteria recovered from the basolateral compartment within 1 h post-infection. Disruption of the pyruvate node through combined deletion of pyruvate oxidase (spxB) and lactate oxidase (lctO) markedly enhanced transmigration of S. pneumoniae across polarized Calu-3 monolayers without causing early cytotoxicity or loss of monolayer integrity. This hyper-invasive phenotype was conserved in the TIGR4 and EF3030 background and under air-liquid interface conditions. Importantly, single {Delta}lctO mutants exhibited significantly greater translocation than {Delta}spxB mutants or wild-type strains across bronchial (Calu-3), alveolar (A549), and pharyngeal (Detroit 562) epithelial models. Enhanced translocation correlated with increased bacterial adherence but was independent of capsule expression, extracellular H2O2 production, pneumolysin, or tight junction disruption, as evidenced by stable transepithelial electrical resistance (TEER), lack of caspase-3/7 activation, and minimal IL-18 release at early time points. High-resolution confocal microscopy revealed intracellular {Delta}lctO pneumococci localized within N-acetylglucosamine/sialic acid (GN/SA)-containing compartments as early as 1 h post-infection. In murine macrophages, {Delta}lctO mutants were phagocytosed at rates similar to wild-type bacteria but induced greater pneumolysin-dependent cytotoxicity at 24 h. These findings demonstrate that LctO functions as a metabolic checkpoint that restrains pneumococcal invasion of respiratory epithelia, revealing a previously unrecognized role for lactate oxidase in controlling the transition from colonization to invasive disease. ImportanceThis study identifies lactate oxidase (LctO) as a critical metabolic checkpoint that restrains Streptococcus pneumoniae invasion of respiratory epithelial barriers. By linking pyruvate node metabolism to the control of transmigration, these findings reveal a novel mechanism by which central carbon metabolism regulates pneumococcal virulence and the transition from colonization to invasive disease.

microbiology↗

bioRxiv2026

A pan-serotype human monoclonal antibody protects against pneumococcal infection by targeting multiple choline binding domain proteins

Streptococcus pneumoniae remains a global health threat, particularly to young children, the elderly, and immunocompromised individuals. Pneumococcal vaccines targeting the bacterial capsule polysaccharide do not protect against all 100+ pneumococcal serotypes, contributing to non-vaccine serotype infections and antibiotic resistance. To address these limitations, we isolated human monoclonal antibodies (mAbs) targeting pneumococcal surface proteins and identified a first-in-class mAb, derived from a patient with prior pneumococcal infection, namely mAb 5995-40. mAb 5995-40 bound multiple pneumococcal proteins, including PcpA and PspA, through a conserved choline-binding domain shared across serotypes. Functionally, mAb 5995-40 provided complete protection in lethal pneumococcal challenge models and improved survival in influenza A, influenza B, and respiratory syncytial virus-associated bacterial coinfection models. Mechanistic studies showed enhanced opsonophagocytic killing, reduced bacterial dissemination, and blocked epithelial translocation. Cryo-electron microscopy identified a repeating motif within the choline-binding domain targeted by mAb 5995-40, highlighting its potential as a broadly protective pneumococcal therapeutic.

immunology↗

bioRxiv2026

Metabolic Rewiring at the Pyruvate Node Drives Severe Pneumonia and T-Cell Suppression in Serotype 3 Streptococcus pneumoniae Infection

BackgroundSerotype 3 (ST3) Streptococcus pneumoniae remains a major cause of invasive pneumococcal disease and pneumonia despite PCV13 introduction, in part due to potent immune evasion properties. The contribution of the pyruvate metabolic node (SpxB/LctO pathways) to ST3 pathogenesis is poorly defined. MethodsWe investigated oxygen-dependent fitness, virulence, and lung pathology in the ST3 strain WU2 and isogenic {Delta}spxB, {Delta}lctO, and {Delta}spxB{Delta}lctO mutants. In vitro growth was assessed under nasopharyngeal (21% O2) and alveolar (14% O2) conditions. Murine pneumonia models evaluated survival, bacterial burdens, histopathology (H&E, confocal microscopy), and lung transcriptomics (RNA-seq). ResultsST3-strain exhibited a unique oxygen-sensitive growth defect at 21% O2, alleviated by spxB deletion, indicating metabolic burden from pyruvate flux. In mice, wild-type WU2 caused high mortality with severe suppurative bronchopneumonia, alveolar consolidation, hemorrhage, and perivascular inflammation. The {Delta}spxB mutant accelerated lethality with enhanced distal lung damage, uncontrolled dissemination, and amplified inflammation. Wild-type infection uniquely induced targeted reorganization of bronchial epithelial membranes, forming prominent bacterium-laden blebs--host-derived membrane protrusions encapsulating intact pneumococci. These novel structures facilitated organized bacterial translocation into tissue without overt cytotoxicity and were largely absent in spxB-deficient mutants despite comparable lung burdens. RNA-seq analysis revealed SpxB-dependent suppression of T cell activation (e.g., Rag1 and Themis) and acute inflammatory pathways, consistent with immune sequestration via blebs. ConclusionsThe SpxB-dependent pathway orchestrates ST3 virulence by enabling metabolic adaptation and driving bleb-mediated epithelial invasion and immune evasion in the lung. These bacterium-laden blebs represent a novel hallmark mechanism in pneumococcal pathogenesis, offering new insights and potential therapeutic targets.

microbiology↗

bioRxiv2025

Pneumococcal H2O2 Reshapes Mitochondrial Function and Reprograms Host Cell Metabolism

Streptococcus pneumoniae (Spn), a primary cause of pneumonia, induces acute lung parenchymal damage through a unique metabolic pathway generating hydrogen peroxide (H2O2) as a byproduct. This study demonstrates that Spn-derived H2O2, primarily produced by pyruvate oxidase (SpxB), inhibits key tricarboxylic acid (TCA) cycle enzymes (aconitase, glutamate dehydrogenase, and -ketoglutarate dehydrogenase) in lung epithelial cells, leading to citrate accumulation and diminished NADH production for oxidative phosphorylation. RNA sequencing reveals SpxB-dependent upregulation of glycolytic genes (HIF1A, IER3, HK2, PFKP), restricting pyruvate entry into the TCA cycle and increasing glucose consumption and lactate/acetate production, indicative of a Warburg-like metabolic shift that may enhance bacterial survival. Notably, mitochondrial membrane potential remains largely preserved, with minimal apoptosis despite Spn-induced stress. These findings uncover a novel mechanism of Spn-driven host metabolic reprogramming, highlighting potential therapeutic targets for pneumococcal diseases. ImportanceStreptococcus pneumoniae remains a leading cause of community-acquired pneumonia worldwide, yet the mechanisms by which it manipulates host metabolism to promote its survival and pathogenesis are not fully understood. This study reveals a novel metabolic strategy whereby pneumococcus-derived hydrogen peroxide, generated by pyruvate oxidase (SpxB), disrupts the host TCA cycle and drives a Warburg-like metabolic shift in lung epithelial cells. By inhibiting key TCA cycle enzymes and rewiring glycolytic gene expression, S. pneumoniae effectively reprograms host cell metabolism to favor its persistence, while minimizing host cell apoptosis and maintaining mitochondrial function. These insights expand our understanding of host-pathogen metabolic interactions and identify potential metabolic vulnerabilities that could be targeted to mitigate tissue damage and improve treatment outcomes in pneumococcal pneumonia.

microbiology↗

bioRxiv2023

Ultrastructural, metabolic and genetic determinants of the acquisition of macrolide resistance by Streptococcus pneumoniae

AimStreptococcus pneumoniae (Spn) acquires genes for macrolide resistance, MEGA or ermB, in the human host. These genes are carried either in the chromosome, or on integrative conjugative elements (ICEs). Here, we investigated molecular determinants of the acquisition of macrolide resistance. Methods and ResultsWhole genome analysis was conducted for 128 macrolide-resistant pneumococcal isolates to identify the presence of MEGA (44.5%, 57/128) or ermB (100%), and recombination events in Tn916-related elements or in the locus comCDE encoding competence genes. Confocal and electron microscopy studies demonstrated that, during the acquisition of macrolide resistance, pneumococcal strains formed clusters of varying size, with the largest aggregates having a median size of [~]1600 {micro}m2. Remarkably, these pneumococcal aggregates comprise both encapsulated and nonencapsulated pneumococci, exhibited physical interaction, and spanned extracellular and intracellular compartments. We assessed the recombination frequency (rF) for the acquisition of macrolide resistance by a recipient D39 strain, from pneumococcal strains carrying MEGA ([~]5.4 kb) in the chromone, or in large ICEs (>23 kb). Notably, the rF for the acquisition of MEGA, whether in the chromosome or carried on an ICE was similar. However, the rF adjusted to the acquisition of the full-length ICE ([~]52 kb), compared to that of the capsule locus ([~]23 kb) that is acquired by transformation, was three orders of magnitude higher. Finally, metabolomics studies revealed a link between the acquisition of ICE and the metabolic pathways involving nicotinic acid and sucrose. ConclusionsExtracellular and intracellular pneumococcal clusters facilitate the acquisition of full-length ICE at a rF higher than that of typical transformation events, involving distinct metabolic changes that present potential targets for interventions.

microbiology↗

bioRxiv2023

Oxidation of hemoglobin in the lung parenchyma facilitates the differentiation of pneumococci into encapsulated bacteria

Streptococcus pneumoniae (the pneumococcus) causes cytotoxicity and encapsulates within the lung parenchyma, leading to pneumococcal pneumonia. However, the underlying mechanisms remain unclear and likely involve multiple bacterial and host factors. We investigated the selection process of encapsulated pneumococci, a critical factor in lung damage during pneumococcal pneumonia. Our study revealed that pneumococci initially lack capsules but re-encapsulate upon reaching the alveoli. This process is driven by S. pneumoniae-derived hydrogen peroxide (Spn-H2O2), which oxidizes lung hemoglobin, leading to heme release and polymerized hemoglobin formation. Physiologically relevant levels of heme were found to promote the selection of encapsulated bacteria. Furthermore, encapsulation protects bacteria from intracellular heme toxicity, a defense absent in non-encapsulated strains. Ultrastructural analysis demonstrated interactions between hemoglobin and both encapsulated and non-encapsulated pneumococci in human sputum. These findings reveal a critical connection between oxidative stress-mediated lung damage and the selection of encapsulated pneumococci, suggesting potential therapeutic avenues by targeting these oxidative processes.

microbiology↗

bioRxiv2023

Oxidation of hemoproteins by Streptococcus pneumoniae collapses the cell cytoskeleton and disrupts mitochondrial respiration leading to cytotoxicity of human lung cells

Streptococcus pneumoniae (Spn) causes pneumonia that kills millions through acute toxicity and invasion of the lung parenchyma. During aerobic respiration, Spn releases hydrogen peroxide (Spn-H2O2), as a by-product of enzymes SpxB and LctO, and causes cell death with signs of both apoptosis and pyroptosis by oxidizing unknown cell targets. Hemoproteins are molecules essential for life and prone to oxidation by H2O2. We recently demonstrated that during infection-mimicking conditions, Spn-H2O2 oxidizes the hemoprotein hemoglobin (Hb), releasing toxic heme. In this study, we investigated details of the molecular mechanism(s) by which the oxidation of hemoproteins by Spn-H2O2 causes human lung cell death. Spn strains, but not H2O2-deficient Spn{Delta}spxB{Delta}lctO strains caused time-dependent cell cytotoxicity characterized by the rearrangement of the actin, the loss of the microtubule cytoskeleton and nuclear contraction. Disruption of the cell cytoskeleton correlated with the presence of invasive pneumococci and an increase of intracellular reactive oxygen species. In cell culture, the oxidation of Hb or cytochrome c (Cytc) caused DNA degradation and mitochondrial dysfunction from inhibition of complex I-driven respiration, which was cytotoxic to human alveolar cells. Oxidation of hemoproteins resulted in the creation of a radical, which was identified as a protein derived side chain tyrosyl radical by using electron paramagnetic resonance (EPR). Thus, we demonstrate that Spn invades lung cells, releasing H2O2 that oxidizes hemoproteins, including Cytc, catalyzing the formation of a tyrosyl side chain radical on Hb and causing mitochondrial disruption, that ultimately leads to the collapse of the cell cytoskeleton.

microbiology↗

bioRxiv2022

Oxidative reactions catalyzed by hydrogen peroxide produced by Streptococcus pneumoniae and other Streptococci Cause the Release and Degradation of Heme from Hemoglobin

Streptococcus pneumoniae (Spn) strains cause pneumonia that kills millions every year worldwide. Spn produces Ply, a hemolysin that lyses erythrocytes releasing hemoglobin and also produces the pro-oxidant hydrogen peroxide (Spn-H2O2) during growth. The hallmark of the pathophysiology of hemolytic diseases is the oxidation of hemoglobin but oxidative reactions catalyzed by Spn-H2O2 has been poorly studied. We characterized the oxidation of hemoglobin by Spn-H2O2. We prepared a series of single ({Delta}spxB, or {Delta}lctO), double mutant ({Delta}spxB{Delta}lctO) and complemented strains in TIGR4, D39 and EF3030. We then utilized an in vitro model with oxy-hemoglobin to demonstrate that oxy-hemoglobin was oxidized rapidly, within 30 min of incubation, by Spn-H2O2 to met-hemoglobin and that the main source of Spn-H2O2 was pyruvate oxidase (SpxB). Moreover, extended incubation caused the release and the degradation of heme. We then assessed oxidation of hemoglobin and heme degradation by other bacteria inhabitants of the respiratory tract. All hydrogen peroxide-producing streptococci tested caused the oxidation of hemoglobin and heme degradation whereas those bacterial species that produce <1 M H2O2, neither oxidized hemoglobin nor degraded heme. An ex vivo bacteremia model confirmed that oxidation of hemoglobin and heme degradation occurred concurrently with hemoglobin that was released from erythrocytes by Ply. Finally, gene expression studies demonstrated that heme, but not red blood cells or hemoglobin induced an upregulated transcription of the spxB gene. Oxidation of hemoglobin may be important for pathogenesis and for the symbiosis of hydrogen peroxide-producing bacteria with other species by providing nutrients such as iron.

microbiology↗

bioRxiv2022

Synergistic protection against secondary pneumococcal infection by human monoclonal antibodies targeting distinct epitopes

Streptococcus pneumoniae persists as a leading cause of bacterial pneumonia despite the widespread use of polysaccharide-based vaccines. The limited serotype coverage of current vaccines has led to increased incidence of non-vaccine serotypes, as well as an increase in antibiotic resistance among these serotypes. Pneumococcal infection often follows a primary viral infection such as influenza virus, which hinders host defense and results in bacterial spread to the lungs. We previously isolated human monoclonal antibodies (mAbs) against the conserved surface antigen pneumococcal histidine triad protein D (PhtD), and we demonstrated that mAbs to this antigen are protective against lethal pneumococcal challenge prophylactically and therapeutically. In this study, we elucidated the mechanism of protection of a protective anti- pneumococcal human mAb, PhtD3, which is mediated by the presence of complement and macrophages in a mouse model of pneumococcal infection. Treatment with mAb PhtD3 reduced blood and lung bacterial burden in mice, and mAb PhtD3 is able to bind to bacteria in the presence of the capsular polysaccharide, indicating exposure of surface PhtD on encapsulated bacteria. In a mouse model of secondary pneumococcal infection, protection mediated by mAb PhtD3 and another mAb targeting a different epitope, PhtD7, was reduced, however, robust protection was restored by combining mAb PhtD3 with mAb PhtD7, indicating a synergistic effect. Overall, these studies provide new insights into anti-pneumococcal mAb protection and demonstrate for the first time that mAbs to pneumococcal surface proteins can protect against secondary pneumococcal infection in the mouse model. Author SummaryThe persistence of Streptococcus pneumoniae as a leading cause of bacterial pneumonia despite numerous approved pneumococcal vaccines is a serious threat to public health globally. Currently, prophylactic and therapeutic options for Streptococcus pneumoniae are constrained by the limited serotype coverage of vaccines and the emergence of antibiotic resistant strains. An additional hurdle to overcome is the incidence of secondary pneumococcal infection following a viral infection, which leads to increased mortality. Here, we determined the mechanism of action of a monoclonal antibody (mAb) that targets Streptococcus pneumoniae. We found that mAb PhtD3 operates through macrophage and complement mediated functions. mAb PhtD3 was also discovered to reduce bacterial titers in the lungs and blood and bind to a related antigen PhtE. We also tested additional mAbs and discovered that two unique mAbs to the antigen PhtD conferred protection in a pneumococcal-influenza virus co-infection model. Our study provides new insights into the mechanisms and therapeutic potential of mAbs targeting conserved proteins of Streptococcus pneumoniae.

immunology↗

bioRxiv2021

Prophylactic inhibition of colonization by Streptococcus pneumoniae with the secondary bile acid metabolite deoxycholic acid

Streptococcus pneumoniae (Spn) colonizes the nasopharynx of children and the elderly but also kills millions worldwide yearly. The secondary bile acid metabolite, deoxycholic acid (DoC), affects the viability of human pathogens but also plays multiple roles in host physiology. We assessed in vitro the antimicrobial activity of DoC and investigated its potential to eradicate Spn colonization using an ex vivo model of human nasopharyngeal colonization and an in vivo mouse model of colonization. At a physiological concentration DoC (0.5 mg/ml; 1.27 mM) killed all tested Spn strains (N=48) two h post-inoculation. The ex-vivo model of nasopharyngeal colonization showed that DoC eradicated colonization by Spn strains as soon as 10 min post-exposure. The mechanism of action did not involve activation of autolysis since the autolysis-defective double mutants {Delta}lytA{Delta}lytC and{Delta} spxB{Delta}lctO were as susceptible to DoC as was the wild-type (WT). Oral streptococcal species (N=20), however, were not susceptible to DoC (0.5 mg/ml). Unlike trimethoprim, whose spontaneous resistance frequency (srF) for TIGR4 or EF3030 was [≥]1x10-9, no spontaneous resistance was observed with DoC (srF[≥]1x10-12). Finally, the efficacy of DoC to eradicate Spn colonization was assessed in vivo using a topical route via intranasal (i.n.) administration and as a prophylactic treatment. Mice challenged with Spn EF3030 carried a median of 4.05x105 cfu/ml four days post-inoculation compared to 6.67x104 cfu/ml for mice treated with DoC. Mice in the prophylactic group had a [~]99% reduction of the pneumococcal density (median, 2.61 x103 cfu/ml). Thus, DoC, an endogenous human bile salt, has therapeutic potential against Spn.

microbiology↗

bioRxiv2020

Hydrogen peroxide production by Streptococcus pneumoniae results in alpha-hemolysis by oxidation of oxy-hemoglobin to met-hemoglobin

Streptococcus pneumoniae (Spn) and other streptococci produce a greenish halo on blood agar plates referred to as -hemolysis. This phenotype is utilized by clinical microbiology laboratories to report culture findings of -hemolytic streptococci, including Spn, and other bacteria. The -hemolysis halo on blood agar plates has been related to the hemolytic activity of pneumococcal pneumolysin (Ply), or to a lesser extent, to lysis of erythrocytes by Spn-produced hydrogen peroxide. We investigated the molecular basis of the -hemolysis halo produced by Spn. Wild-type strains TIGR4, D39, R6, and EF3030, and isogenic derivative {Delta}ply mutants, produced a similar -hemolytic halo on blood agar plates while cultures of hydrogen peroxide knockout {Delta}spxB/{Delta}lctO mutants lacked this characteristic halo. Spectroscopic studies demonstrated that culture supernatants of TIGR4 released hemoglobin-bound heme (heme-hemoglobin) from erythrocytes and oxidized oxy-hemoglobin to met-hemoglobin within 30 min of incubation. As expected, given Ply hemolytic activity, and that hydrogen peroxide contributes to the release of Ply, TIGR4 isogenic mutants {Delta}ply and {Delta}spxB/{Delta}lctO had a significantly decreased release of heme-hemoglobin from erythrocytes. However, TIGR4{Delta}ply that produces hydrogen peroxide oxidized oxy-hemoglobin to met-hemoglobin, whereas TIGR4{Delta}spxB/{Delta}lctO failed to produce oxidation of oxy-hemoglobin. We demonstrated that the so-called -hemolysis halo is caused by the oxidation oxy-hemoglobin (Fe+2) to a non-oxygen binding met-hemoglobin (Fe+3) by Spn-produced hydrogen peroxide. Since Spn colonizes the human lung, oxidation of oxy-hemoglobin might have important implications for pathogenesis. ImportanceThere is a misconception that -hemolysis observed on blood agar plates cultures of Streptococcus pneumoniae (Spn), and other -hemolytic streptococci is produced by a hemolysin, or alternatively, by lysis of erythrocytes caused by hydrogen peroxide. We noticed in the course of our investigations that wild-type Spn strains and hemolysin (e.g., pneumolysin) knockout mutants, produced the -hemolytic halo on blood agar plates. In contrast, hydrogen peroxide defective mutants prepared in four different strains lacked the characteristic -hemolysis halo. We also demonstrated that wild-type strains and pneumolysin mutants oxidized oxy-hemoglobin to met-hemoglobin. Hydrogen peroxide knockout mutants, however, failed to oxidize oxy-hemoglobin. Therefore, the greenish halo formed on cultures of Spn and other so-called -hemolytic streptococci is caused by the oxidation of oxy-hemoglobin produced by hydrogen peroxide. Oxidation of oxy-hemoglobin to the non-binding oxygen form, met-hemoglobin, might occur in the lungs during pneumococcal pneumonia.

microbiology↗

bioRxiv2019

Captive green iguana is a reservoir of diarrheogenic Escherichia coli pathotypes

The green iguana appears to be a reservoir for bacteria causing gastrointestinal infections in humans. The presence of diarrheogenic E. coli (DEC) pathotypes, however, has not been studied in this reptile. The aim of the current work was to investigate the prevalence of DEC in the intestines of 240 captive green iguanas, their phylogenetic groups, and the antibiotic susceptibility profile. E. coli strains were isolated from 41.7% of the intestinal content of green iguanas. DEC strains was identified in 25.9% of the screened population and were detected in the majority (62%, p=0.009] of those reptiles carrying E. coli strains. Among DEC strains, STEC strains carrying the stx1 gene were the most prevalent pathotype isolated (38.7%), followed by EAEC and ETEC (27.4% each). Genetic markers of DEC strains belonging to the EHEC pathotype were not detected. More than a half of DEC strains were classified into the Clade I-II phylogroup (64.5%), followed by the phylogroup A (14.5%). The antibiotic susceptibility method demonstrated that a high proportion of DEC strains were resistance, or non-susceptible, to carbenicillin, amikacin, and ampicillin (85, 74, and 66%, respectively). We conclude that the green iguana kept in captivity is a reservoir of DEC strains bearing resistance to first-line antibiotics, including penicillins. Given the increase presence of the green iguana in Latin American households, these reptiles represent a potential source of transmission to susceptible humans and therefore a potential source of gastrointestinal disease.\n\nImportanceLatin-American countries present a high burden of diarrheal disease. In this part of the world, besides common pathogens, diarrheal diseases are also caused by pathogenic types of E. coli referred as \"diarrheagenic E. coli\" (DEC). While inhabitants of Latin American countries suffer of self-limiting diarrhea when infected with DEC, a main target of these strains are tourists from developed countries who are not exposed to DEC strains. Efforts are in place to decrease the burden of DEC-associated diarrheal disease. As such, this study investigated a potential reservoir of DEC strains that had been underestimated, the green iguana. These reptiles are very common in Latin American households and are found virtually everywhere in Mexico, Central and South America. We found that 25% of green iguanas carried DEC strains in their intestines. We also demonstrated a high prevalence of antibiotic resistance in these strains, posing a potential risk to humans.

microbiology↗