Biology subjects

Valvano, M. A.

Publications and source records attributed to Valvano, M. A..

8 recordsLinked to original sources

bioRxiv2025

A single-domain response regulator activates exopolysaccharide synthesis by interacting with the initiating phosphoglycosyl transferase

Exported polysaccharides have crucial functions in bacteria. Polysaccharide biosynthesis in the ubiquitous Wzx/Wzy- and ABC-transporter-dependent pathways starts with the transfer of a sugar-1-phosphate from a nucleotide-sugar donor to undecaprenyl phosphate, a reaction catalyzed by a phosphoglycosyl transferase (PGT). Both reaction substrates are limited and shared among multiple glycoconjugate pathways, raising the question of how bacteria regulate these pathways. In Myxococcus xanthus, EpsZ, which belongs to the family of large monotopic PGTs (monoPGTs), starts the Wzx/Wzy-dependent exopolysaccharide (EPS) biosynthesis. The Dif chemosensory system regulates EPS biosynthesis by an unknown mechanism via the phosphorylated single-domain response regulator EpsW (EpsW[~]P). Here, we show that EpsW[~]P stimulates EPS biosynthesis at the post-translational level. Moreover, MiniTurbo-based proximity labeling experiments suggest that EpsW[~]P interacts directly with EpsZ. Additionally, heterologous expression of these two proteins in Salmonella enterica demonstrates that EpsW stimulates EpsZ enzymatic activity. S. enterica WbaP, the prototype large monoPGT, forms a functional homodimer, with dimerization involving a distinct cytoplasmic {beta}-hairpin. However, AlphaFold-based structural modeling shows that EpsZ lacks this {beta}-hairpin, suggesting an alternative mechanism for dimerization. Our structural modeling of a EpsZ-EpsW heterocomplex suggests that EpsW[~]P, by direct interaction, promotes the formation of the stable EpsZ dimer. These findings uncover a new model for the regulation of polysaccharide biosynthesis in which EpsW[~]P allosterically facilitates the formation of the active, dimeric conformation of EpsZ, thereby activating EPS biosynthesis at its initial step. Genomics and structural modeling suggest that the regulation of large monoPGTs by a single-domain response regulator is widespread in myxobacteria and potentially beyond. SignificanceBacteria produce various polysaccharides with important biological functions and biotechnological applications. Polysaccharide synthesis is energy-costly and requires substrates that are in limited supply raising the question of how bacteria regulate these pathways. Here, we explored the regulation of exopolysaccharide biosynthesis in Myxococcus xanthus. We demonstrate that the phosphorylated single-domain response regulator EpsW activates exopolysaccharide biosynthesis at the post-translational level by stimulating the activity of the phosphoglycosyl transferase EpsZ. By directly interacting with EpsZ, phosphorylated EpsW facilitates the formation of the active, dimeric conformation of EpsZ, thereby activating exopolysaccharide biosynthesis at its initial step. We propose that this previously unrecognized regulatory mechanism is broadly conserved, not only in myxobacteria but also beyond.

microbiology↗

bioRxiv2023

Glyceroglycolipids are essential for Burkholderia cenocepacia intracellular survival by preventing phagolysosome acidification

Burkholderia cenocepacia is a problematic pathogen that infects people with cystic fibrosis and often causes fatal "cepacia syndrome". B. cenocepacia infection is difficult to treat due to the high intrinsic resistance of the bacterium to antimicrobials and its ability to survive in macrophages. In this study, we uncover a hitherto unknown aspect of B. cenocepacias pathogenesis related to the formation of new glyceroglycolipids, which is required for intracellular survival. Using lipidomics, we observed that B. cenocepacia can produce three glyceroglycolipid species in phosphate deplete conditions using a PlcP-mediated lipid remodelling pathway originally discovered in soil and ocean-dwelling bacteria. While lipid remodelling as an adaptation strategy for environmental microbes to cope with the scarcity of phosphorus is known, its role in intracellular bacterial survival was not investigated. Using mammalian macrophages and Galleria mellonella larvae as infection models, we showed that the mutant unable to perform membrane lipid remodelling ({Delta}plcP) could not establish infection. Unlike the wild type bacterium, the {Delta}plcP mutant did not replicate within macrophages and failed to prevent phagosome acidification. Comparative genomics analyses showed that this PlcP pathway is conserved in all pathogenic Burkholderia that infect a variety of mammalian and plant hosts. Overall, our results indicate that membrane lipid remodelling plays an essential, yet previously overlooked, role in subverting host immunity.

microbiology↗

bioRxiv2023

The Achromobacter Type 3 secretion system drives pyroptosis and immunopathology via independent activation of NLRC4 and NLRP3 inflammasomes

Achromobacter species are newly recognized opportunistic, pro-inflammatory Gram-negative pathogens in immunocompromised individuals, but how they interact with the innate immune system to drive inflammation is poorly understood. We created sctV (Type 3 Secretion System baseplate) mutants in three Achromobacter clinical isolates from two species and showed that all three required the T3SS to induce cell death in human macrophages. Mutating other critical T3SS components also abolished cell death, which was restored by genetic complementation. Cell death of Achromobacter-infected macrophages was contact-dependent, enhanced by bacterial internalisation, and caused by inflammasome-dependent pyroptosis (typified by Gasdermin-D cleavage and IL-1{beta} secretion). Macrophages deficient in the inflammasome sensors NLRC4 or NLRP3 underwent pyroptosis upon bacterial internalization but those deficient in both NLRC4 and NLRP3 did not, suggesting either sensor can mediate pyroptosis induction in a T3SS-dependent manner. Detailed analysis of the intracellular trafficking of one isolate indicated that the intracellular bacteria reside in an acidic LAMP-1/dextran-positive membrane compartment. Using an intranasal mouse infection model, we observed that Achromobacter damages lung structure and causes severe illness, contingent on a functional T3SS. Together, we demonstrate that Achromobacter species can survive phagocytosis by macrophages and promote macrophage cell death and inflammation by redundant mechanisms of pyroptosis induction.

microbiology↗

bioRxiv2020

A glycoengineered antigen exploiting a conserved protein O-glycosylation pathway in the Burkholderia genus for diagnosis of glanders infections

We recently described a protein O-glycosylation pathway conserved in all species of the Burkholderia genus that results in synthesis and incorporation of a trisaccharide glycan to membrane-exported proteins. Here, we exploited this system to construct and evaluate a diagnostic tool for glanders. Burkholderia mallei, the causative agent of glanders, is a highly infectious and fatal zoonotic pathogen that mainly infects horses, mules, donkeys and occasionally humans. A highly sensitive and specific diagnostic tool is crucial for the control, elimination and eradication of B. mallei infections. We constructed plasmids carrying synthetic genes encoding a modified, previously unannotated Burkholderia glycoprotein containing three glycosylation sequons fused to the cholera toxin B-subunit. The resulting proteins were glycosylated in the B. cenocepacia K56-2 parental strain, but not in glycosylation-deficient mutants, as determined by SDS-PAGE and fluorescent lectin blots. One of these glycoproteins was used as an antigen in ELISA and western blots to screen a panel of serum samples collected from glanders-infected and healthy horses previously investigated by complement fixation test and indirect ELISA based on a semi-purified fraction of B. mallei. We show that ELISA and western blot assays based on our glycoprotein antigen provide 100 % specificity, with a sensitivity greater than 88%. The glycoprotein antigen was recognized by serum samples collected from patients infected with B. pseudomallei, B. mallei, B. multivorans and B. cenocepacia. Our results indicate that protein O-glycosylation in Burkholderia can be exploited as a biomarker for diagnosis of Burkholderia-associated infections. IMPORTANCEGlanders is a severe zoonotic disease caused by the Gram-negative bacterium Burkholderia mallei, which affects horses, mules and donkeys, as well as humans. B. mallei is also considered a category B biothreat agent. Due to insufficient pathognomonic symptoms in the early stages of glanders, diagnosis can be difficult. Complement fixation is the most accurate and reliable serological test prescribed by the World Organization for Animal Health; however, this test has a considerable number of false-positive results. We have recently described a conserved protein O-glycosylation pathway present in all species of the Burkholderia genus; we also demonstrated that Burkholderia-infected humans develop anti-glycan antibodies. Here, we exploited this system to construct and evaluate a synthetic glycoengineered protein antigen as a diagnostic tool for glanders. Our results show 100 % specificity in the detection of antibodies from infected horses, indicating that protein O-glycosylation in Burkholderia can be exploited as a biomarker for diagnosis of Burkholderia-associated infections.

microbiology↗

bioRxiv2020

The DNA mimic protein BCAS0292 is involved in the regulation of virulence of Burkholderia cenocepacia

Adaptation of opportunistic pathogens to their host environment requires reprogramming of a vast array of genes to facilitate survival in the host. Burkholderia cenocepacia, a Gram-negative bacterium that colonizes environmental niches, is exquisitely adaptable to the hypoxic environment of the cystic fibrosis lung and survives in macrophages. B. cenocepacia possesses a large genome encoding multiple virulence systems, stress response proteins and a large locus that responds to low oxygen. We previously identified BCAS0292, an acidic protein encoded on replicon 3. Deletion of the BCAS0292 gene resulted in altered abundance of >1000 proteins; 46 proteins became undetectable while 556 proteins showed >1.5-fold reduced abundance, suggesting BCAS0292 is a global regulator. Moreover, the {triangleup}BCAS0292 mutant showed a range of pleiotropic effects: virulence, host-cell attachment and motility were reduced, antibiotic susceptibility was altered and biofilm formation enhanced. Its growth and survival were impaired in 6% oxygen. Structural analysis revealed BCAS0292 presents a dimeric {beta}-structure with a negative electrostatic surface. Further, the {Delta}BCAS0292 mutant displayed altered DNA supercoiling, implicated in global regulation of gene expression. We propose that BCAS0292 acts as a DNA-mimic, altering DNA topology and regulating the expression of multiple genes, thereby enabling the adaptation of B. cenocepacia to highly diverse environments.

microbiology↗

bioRxiv2020

Characterization of the exopolysaccharide biosynthesis pathway in Myxococcus xanthus

Myxococcus xanthus arranges into two morphologically distinct biofilms depending on its nutritional status, i.e. coordinately spreading colonies in the presence of nutrients and spore-filled fruiting bodies in the absence of nutrients. A secreted polysaccharide referred to as exopolysaccharide (EPS) is a structural component of both biofilms and is also important for type IV pili-dependent motility and fruiting body formation. Here, we characterize the biosynthetic machinery responsible for EPS biosynthesis using bioinformatics, genetics, heterologous expression, and biochemical experiments. We show that this machinery constitutes a Wzx/Wzy-dependent pathway dedicated to EPS biosynthesis. Our data support that EpsZ (MXAN_7415) is the polyisoprenyl-phosphate hexose-1-phosphate transferase responsible for initiation of the repeat unit synthesis. Heterologous expression experiments support that EpsZ has galactose-1-P transferase activity. Moreover, MXAN_7416, renamed WzxEPS, and MXAN_7442, renamed WzyEPS, are the Wzx flippase and Wzy polymerase responsible for translocation and polymerization of the EPS repeat unit, respectively. Also, in this pathway, EpsV (MXAN_7421) is the polysaccharide co-polymerase and EpsY (MXAN_7417) the outer membrane polysaccharide export (OPX) protein. Mutants with single in-frame deletions in the five corresponding genes had defects in type IV pili-dependent motility and a conditional defect in fruiting body formation. Furthermore, all five mutants were deficient in type IV pili formation and genetic analyses suggest that EPS and/or the EPS biosynthetic machinery stimulates type IV pili extension. Additionally, we identify a polysaccharide biosynthesis gene cluster, which together with an orphan gene encoding an OPX protein make up a complete Wzx/Wzy-dependent pathway for synthesis of an unknown polysaccharide. ImportanceThe secreted polysaccharide referred to as exopolysaccharide (EPS) has important functions in the social life cycle of M. xanthus; however, little is known about how EPS is synthesized. Here, we characterized the EPS biosynthetic machinery and show that it makes up a Wzx/Wzy-dependent pathway for polysaccharide biosynthesis. Mutants lacking a component of this pathway had reduced type IV pili-dependent motility and a conditional defect in development. Also, these analysis suggest that EPS and/or the EPS biosynthetic machinery is important for type IV pili formation.

microbiology↗

bioRxiv2019

Loss of O-linked protein glycosylation in Burkholderia cenocepacia impairs biofilm formation and siderophore production via alteration of quorum sensing regulation

O-linked protein glycosylation is a conserved feature of the Burkholderia genus. For Burkholderia cenocepacia, the addition of the trisaccharide {beta}-Gal-(1,3)--GalNAc-(1,3)-{beta}-GalNAc to membrane exported proteins is required for virulence and resistance to environmental stress. However, the underlying causes of the defects observed in the absence of glycosylation are unclear. This study demonstrates that the global B. cenocepacia proteome undergoes dramatic changes consistent with alterations in global transcriptional regulation in the absence of glycosylation. Using luciferase reporter assays and DNA cross-linking analysis, we confirm the repression of the master quorum sensing regulon CepR/I in response to the loss of glycosylation, which leads to the abolition of biofilm formation, defects in siderophore production, and reduced virulence. The abundance of most of the known glycosylated proteins did not significantly change in the glycosylation-defective mutants except for BCAL1086 and BCAL2974, which were found in reduced amount, suggesting they could be degraded. However, the loss of these two proteins was not responsible for driving the proteomic alterations, as well as for reduced virulence and siderophore production. Together, our results show that loss of glycosylation in B. cenocepacia results in a global cell reprogramming via alteration of the CepR/I regulon, which cannot be explained by the abundance changes in known B. cenocepacia glycoproteins.\n\nIMPORTANCEProtein glycosylation is increasingly recognised as a common protein modification in bacterial species. Despite this commonality our understanding of the role of most glycosylation systems in bacterial physiology and pathogenesis is incomplete. In this work, we investigated the effect of the disruption of O-linked glycosylation in the opportunistic pathogen Burkholderia cenocepacia using a combination of proteomic, molecular and phenotypic assays. We find that in contrast to recent findings on the N-linked glycosylation systems of Campylobacter jejuni, O-linked glycosylation does not appear to play a role in proteome stabilization of most glycoproteins. Our results reveal that virulence attenuation observed within glycosylation-null B. cenocepacia strains are consistent with alteration of the master virulence regulator CepR. The repression of CepR transcription and its associated phenotypes support a model in which the virulence defects observed in glycosylation-null strains are at least in part due to transcriptional alteration and not the direct result of the loss of glycosylation per-se. This research unravels the pleotropic effects of O-linked glycosylation in B. cenocepacia, demonstrating that its loss does not simply affect the stability of the glycoproteome, but also interferes with transcription and the broader proteome.

microbiology↗

bioRxiv2018

Modulation of antibiotic sensitivity and biofilm formation in Pseudomonas aeruginosa by interspecies diffusible signal factor analogues

The opportunistic pathogen Pseudomonas aeruginosa can participate in inter-species communication through signaling by cis-2-unsaturated fatty acids of the diffusible signal factor (DSF) family. Sensing these signals involves the histidine kinase PA1396 and leads to altered biofilm formation and increased tolerance to various antibiotics. Here, we show that the membrane-associated sensory input domain of PA1396 has five trans-membrane helices, two of which are required for DSF sensing. DSF binding is associated with enhanced auto-phosphorylation of PA1396 incorporated into liposomes. Further, we examined the ability of synthetic DSF analogues to modulate or inhibit PA1396 activity. Several of these analogues block the ability of DSF to trigger auto-phosphorylation and gene expression, whereas others act as inverse agonists reducing biofilm formation and antibiotic tolerance, both in vitro and in murine infection models. These analogues may thus represent lead compounds for novel adjuvants to improve the efficacy of existing antibiotics.

microbiology↗