scholarly journals Enrichment and Proteomic Characterization of the Cyst Wall from In Vitro Toxoplasma gondii Cysts

mBio ◽  
2019 ◽  
Vol 10 (2) ◽  
Author(s):  
Vincent Tu ◽  
Joshua Mayoral ◽  
Tatsuki Sugi ◽  
Tadakimi Tomita ◽  
Bing Han ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Cyst Wall ◽  
Chronic Infection ◽  
Latent Infection ◽  
Gene Knockout ◽  
Life Stage ◽  
Critical Role ◽  
Data Set ◽  
Content Type ◽  
And Function

ABSTRACT The tissue cyst of Toxoplasma gondii, found in latent infection, serves a critical role in both transmission and reactivation of this organism. Within infected cells, slowly replicating parasites (bradyzoites) are surrounded by a cyst matrix, cyst wall, and cyst membrane. The cyst wall is clearly delineated by ultrastructural analysis; however, the composition and function of this layer in host-parasite interactions are not fully understood. In order to understand the composition of the cyst wall, a proteomic analysis of purified cyst wall fragments, that were enriched with Percoll gradients and subsequently immunoprecipitated with CST1 antibody, was performed. Known cyst wall proteins, such as CST1, BPK1, MCP4, MAG1, GRA2, GRA3, and GRA5, were identified in this preparation by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In addition, dense granule proteins (GRAs) not previously shown to associate with the cyst wall, as well as uncharacterized hypothetical proteins, were identified in this cyst wall preparation. Several of these hypothetical cyst wall (CST) proteins were epitope tagged, and immunofluorescence assays confirmed their localization as novel cyst matrix and cyst wall proteins. Expression of two of these newly identified cyst wall proteins was eliminated by gene knockout (CST2-KO and CST3-KO). CST2-KO parasites were highly attenuated in virulence and did not establish detectable cyst burdens. This targeted proteomic approach allowed the identification of new components of the cyst wall that probably have roles in the parasite/host interface. IMPORTANCE Toxoplasma gondii is a highly prevalent parasite worldwide that presents life-threatening risks to immunocompromised and pregnant individuals. Whereas the life stage responsible for acute infection can be treated, the life stage responsible for chronic infection is refractory to currently available therapeutics. Little is known about the protein composition of the cyst wall, an amorphous structure formed by parasites that is suspected to facilitate persistence within muscle and nervous tissue during chronic (latent) infection. By implementing a refined approach to selectively purify cyst wall fragments, we identified several known and novel cyst wall proteins from our sample preparations. We confirmed the localizations of several proteins from this data set and identified one that is involved in parasite virulence. These data will propel further studies on cyst wall structure and function, leading to therapeutic strategies that can eliminate the chronic infection stage.

scholarly journals GABARAPL2 Is Critical for Growth Restriction of Toxoplasma gondii in HeLa Cells Treated with Gamma Interferon

2020 ◽  
Vol 88 (5) ◽  
Author(s):  
Zhaoxia Zhang ◽  
Haorong Gu ◽  
Qi Li ◽  
Jun Zheng ◽  
Shinuo Cao ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Hela Cells ◽  
Critical Role ◽  
Gamma Interferon ◽  
Growth Restriction ◽  
Membrane Structures ◽  
Content Type ◽  
Receptor Associated Protein ◽  
Ifn Γ ◽  
And Function

ABSTRACT Gamma interferon (IFN-γ)-induced innate immune responses play important roles in the inhibition of Toxoplasma gondii infection. It has been reported that IFN-γ stimulates non-acidification-dependent growth restriction of T. gondii in HeLa cells, but the mechanism remains unclear. Here, we found that γ-aminobutyric acid (GABA) receptor-associated protein-like 2 (GABARAPL2) plays a critical role in parasite restriction in IFN-γ-treated HeLa cells. GABARAPL2 is recruited to membrane structures surrounding parasitophorous vacuoles (PV). Autophagy adaptors are required for the proper localization and function of GABARAPL2 in the IFN-γ -induced immune response. These findings provide further understanding of a noncanonical autophagy pathway responsible for IFN-γ-dependent inhibition of T. gondii growth in human HeLa cells and demonstrate the critical role of GABARAPL2 in this response.

scholarly journals In vitro characterization of protein effector export in the bradyzoite stage of Toxoplasma gondii

2020 ◽  
Author(s):  
Joshua Mayoral ◽  
Peter Shamamian ◽  
Louis M. Weiss
Keyword(s):  
Toxoplasma Gondii ◽  
Host Cell ◽  
Cyst Wall ◽  
Persistent Infection ◽  
Chronic Infection ◽  
Life Stage ◽  
Effector Proteins ◽  
Early Stages ◽  
Bradyzoite Differentiation

ABSTRACTThe ubiquitous parasite Toxoplasma gondii exhibits an impressive ability to maintain a chronic infection of its host for prolonged periods. Despite this, little is known regarding if and how T. gondii bradyzoites, a quasi-dormant life-stage residing within intracellular cysts, manipulate the host cell so as to maintain a persistent infection. A previous proteomic study of the cyst wall, an amorphous layer of proteins that forms underneath the cyst membrane, identified MYR1 as a putative cyst wall protein in vitro. As MYR1 is known to be involved in the translocation of parasite derived effector proteins into the host cell, we sought to determine whether parasites transitioning toward the bradyzoite life stage retain the capacity to translocate proteins via this pathway. By epitope tagging the endogenous loci of four known effectors that translocate from the parasitophorous vacuole into the host cell nucleus, we show by immunofluorescence that most effectors accumulate in the host nucleus at early but not late timepoints post-infection during the tachyzoite to bradyzoite transition and when parasites farther along the bradyzoite differentiation continuum invade a new host cell. We demonstrate that the suppression of interferon-gamma (IFN-γ) signaling, previously shown to be mediated by the effector TgIST, also occurs in the context of prolonged infection with bradyzoites, and that TgIST export is a process that occurs beyond the early stages of host cell infection. These findings have important implications as to how this highly successful parasite maintains a persistent infection of its host.IMPORTANCEToxoplasma bradyzoites persist within tissue cysts and are refractory to current treatments, serving as a reservoir for acute complications in settings of compromised immunity. Much remains to be understood regarding how this life-stage successfully establishes and maintains a persistent infection. In this study, we investigated whether the export of parasite effector proteins into the host cell occurs during the development of in vitro tissue cysts. We quantified the presence of four previously described effectors in host cell nuclei at different timepoints post-bradyzoite differentiation and found that they accumulate largely during the early stages of infection. Despite a decline in nuclear accumulation, we found that one of these effectors still mediates its function after prolonged infection with bradyzoites and provide evidence that this effector is exported beyond early infection stages. These findings suggest that effector export from within developing tissue cysts provides one potential mechanism by which this parasite achieves chronic infection.

scholarly journals A Family of Toxoplasma gondii Genes Related to GRA12 Regulate Cyst Burdens and Cyst Reactivation

mSphere ◽  
2021 ◽  
Vol 6 (2) ◽  
Author(s):  
Rebekah B. Guevara ◽  
Barbara A. Fox ◽  
David J. Bzik
Keyword(s):  
Toxoplasma Gondii ◽  
Cyst Wall ◽  
Chronic Infection ◽  
Acute Infection ◽  
Dense Granule ◽  
Toxoplasmic Encephalitis ◽  
Content Type ◽  
The Central Nervous System ◽  
Related Proteins ◽  
Cyst Development

ABSTRACT Toxoplasma gondii causes a chronic infection that renders the immunocompromised human host susceptible to toxoplasmic encephalitis triggered by cyst reactivation in the central nervous system. The dense granule protein GRA12 is a major parasite virulence factor required for parasite survival during acute infection. Here, we characterized the role of four GRA12-related genes in acute and chronic stages of infection. While GRA12A, GRA12B, and GRA12D were highly expressed in asexual stage tachyzoites and bradyzoites, expression of GRA12C appeared to be restricted to the sexual stages. In contrast to deletion of GRA12 (Δgra12), no major defects in acute virulence were observed in Δgra12A, Δgra12B, or Δgra12D parasites, though Δgra12B parasites exhibited an increased tachyzoite replication rate. Bradyzoites secreted GRA12A, GRA12B, and GRA12D and incorporated these molecules into the developing cyst wall, as well as the cyst matrix in distinct patterns. Similar to GRA12, GRA12A, GRA12B, and GRA12D colocalized with the dense granules in extracellular tachyzoites, with GRA2 and the intravacuolar network in the tachyzoite stage parasitophorous vacuole and with GRA2 in the cyst matrix and cyst wall. Chronic stage cyst burdens were decreased in mice infected with Δgra12A parasites and were increased in mice infected with Δgra12B parasites. However, Δgra12B cysts were not efficiently maintained in vivo. Δgra12A, Δgra12B, and Δgra12D in vitro cysts displayed a reduced reactivation efficiency, and reactivation of Δgra12A cysts was delayed. Collectively, our results suggest that a family of genes related to GRA12 play significant roles in the formation, maintenance, and reactivation of chronic stage cysts. IMPORTANCE If host immunity weakens, Toxoplasma gondii cysts recrudesce in the central nervous system and cause a severe toxoplasmic encephalitis. Current therapies target acute stage infection but do not eliminate chronic cysts. Parasite molecules that mediate the development and persistence of chronic infection are poorly characterized. Dense granule (GRA) proteins such as GRA12 are key virulence factors during acute infection. Here, we investigated four GRA12-related genes. GRA12-related genes were not major virulence factors during acute infection. Instead, GRA12-related proteins localized at the cyst wall and cyst matrix and played significant roles in cyst development, persistence, and reactivation during chronic infection. Similar to GRA12, the GRA12-related proteins selectively associated with the intravacuolar network of membranes inside the vacuole. Collectively, our results support the hypothesis that GRA12 proteins associated with the intravacuolar membrane system support parasite virulence during acute infection and cyst development, persistence, and reactivation during chronic infection.

scholarly journals In Vitro Characterization of Protein Effector Export in the Bradyzoite Stage of Toxoplasma gondii

mBio ◽  
2020 ◽  
Vol 11 (2) ◽  
Author(s):  
Joshua Mayoral ◽  
Peter Shamamian ◽  
Louis M. Weiss
Keyword(s):  
Toxoplasma Gondii ◽  
Host Cell ◽  
Persistent Infection ◽  
Chronic Infection ◽  
Life Stage ◽  
Effector Proteins ◽  
Content Type ◽  
Time Points ◽  
Bradyzoite Differentiation

ABSTRACT The ubiquitous parasite Toxoplasma gondii exhibits an impressive ability to maintain chronic infection of its host for prolonged periods. Despite this, little is known regarding whether and how T. gondii bradyzoites, a quasi-dormant life stage residing within intracellular cysts, manipulate the host cell to maintain persistent infection. A previous proteomic study of the cyst wall, an amorphous layer of proteins that forms underneath the cyst membrane, identified MYR1 as a putative cyst wall protein in vitro. Because MYR1 is known to be involved in the translocation of parasite-derived effector proteins into the host cell, we sought to determine whether parasites transitioning toward the bradyzoite life stage retain the capacity to translocate proteins via this pathway. By epitope tagging the endogenous loci of four known effectors that translocate from the parasitophorous vacuole into the host cell nucleus, we show, by immunofluorescence assays, that most effectors accumulate in the host nucleus at early but not late time points after infection, during the tachyzoite-to-bradyzoite transition and when parasites further along the bradyzoite differentiation continuum invade a new host cell. We demonstrate that the suppression of interferon gamma signaling, which was previously shown to be mediated by the effector TgIST, also occurs in the context of prolonged infection with bradyzoites and that TgIST export is a process that occurs beyond the early stages of host cell infection. These findings have important implications regarding how this highly successful parasite maintains persistent infection of its host. IMPORTANCE Toxoplasma bradyzoites persist within tissue cysts and are refractory to current treatments, serving as a reservoir for acute complications in settings of compromised immunity. Much remains to be understood regarding how this life stage successfully establishes and maintains persistent infection. In this study, we investigated whether the export of parasite effector proteins into the host cell occurs during the development of in vitro tissue cysts. We quantified the presence of four previously described effectors in host cell nuclei at different time points after bradyzoite differentiation and found that they accumulated largely during the early stages of infection. Despite a decline in nuclear accumulation, we found that one of these effectors still mediated its function after prolonged infection with bradyzoites, and we provide evidence that this effector is exported beyond early infection stages. These findings suggest that effector export from within developing tissue cysts provides one potential mechanism by which this parasite achieves chronic infection.

scholarly journals Metabolic Switching of Mycobacterium tuberculosis during Hypoxia Is Controlled by the Virulence Regulator PhoP

2020 ◽  
Vol 202 (7) ◽  
Author(s):  
Prabhat Ranjan Singh ◽  
Anil Kumar Vijjamarri ◽  
Dibyendu Sarkar
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Nitrogen Metabolism ◽  
Electron Acceptor ◽  
Latent Infection ◽  
Critical Role ◽  
Hypoxic Stress ◽  
Content Type ◽  
Metabolic Switching ◽  
Virulence Regulator

ABSTRACT Mycobacterium tuberculosis retains the ability to establish an asymptomatic latent infection. A fundamental question in mycobacterial physiology is to understand the mechanisms involved in hypoxic stress, a critical player in persistence. Here, we show that the virulence regulator PhoP responds to hypoxia, the dormancy signal, and effectively integrates hypoxia with nitrogen metabolism. We also provide evidence to demonstrate that both under nitrogen limiting conditions and during hypoxia, phoP locus controls key genes involved in nitrogen metabolism. Consistently, under hypoxia a ΔphoP strain shows growth attenuation even with surplus nitrogen, the alternate electron acceptor, and complementation of the mutant restores bacterial growth. Together, our observations provide new biological insights into the role of PhoP in integrating nitrogen metabolism with hypoxia by the assistance of the hypoxia regulator DosR. The results have significant implications on the mechanism of intracellular survival and growth of the tubercle bacilli under a hypoxic environment within the phagosome. IMPORTANCE M. tuberculosis retains the unique ability to establish an asymptomatic latent infection. To understand the mechanisms involved in hypoxic stress which play a critical role in persistence, we show that the virulence regulator PhoP is linked to hypoxia, the dormancy signal. In keeping with this, phoP was shown to play a major role in M. tuberculosis growth under hypoxia even in the presence of surplus nitrogen, the alternate electron acceptor. Our results showing regulation of hypoxia-responsive genes provide new biological insights into role of the virulence regulator in metabolic switching by sensing hypoxia and integrating nitrogen metabolism with hypoxia by the assistance of the hypoxia regulator DosR.

scholarly journals Metapopulation Structure of CRISPR-Cas Immunity inPseudomonas aeruginosaand Its Viruses

mSystems ◽  
2018 ◽  
Vol 3 (5) ◽  
Author(s):  
Whitney E. England ◽  
Ted Kim ◽  
Rachel J. Whitaker
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Critical Role ◽  
Opportunistic Pathogen ◽  
Microbial Populations ◽  
Bacterial Strains ◽  
Viral Control ◽  
Data Set ◽  
Content Type ◽  
Global Population

ABSTRACTViruses that infect the widespread opportunistic pathogenPseudomonas aeruginosahave been shown to influence physiology and critical clinical outcomes in cystic fibrosis (CF) patients. To understand how CRISPR-Cas immune interactions may contribute to the distribution and coevolution ofP. aeruginosaand its viruses, we reconstructed CRISPR arrays from a highly sampled longitudinal data set from CF patients attending the Copenhagen Cystic Fibrosis Clinic in Copenhagen, Denmark (R. L. Marvig, L. M. Sommer, S. Molin, and H. K. Johansen, Nat Genet 47:57–64, 2015,https://doi.org/10.1038/ng.3148). We show that new spacers are not added to or deleted from CRISPR arrays over time within a single patient but do vary among patients in this data set. We compared assembled CRISPR arrays from this data set to CRISPR arrays extracted from 726 additional publicly availableP. aeruginosasequences to show that local diversity in this population encompasses global diversity and that there is no evidence for population structure associated with location or environment sampled. We compare over 3,000 spacers from our global data set to 98 lytic and temperate viruses and proviruses and find a subset of related temperate virus clusters frequently targeted by CRISPR spacers. Highly targeted viruses are matched by different spacers in different arrays, resulting in a pattern of distributed immunity within the global population. Understanding the multiple immune contexts thatP. aeruginosaviruses face can be applied to study ofP. aeruginosagene transfer, the spread of epidemic strains in cystic fibrosis patients, and viral control ofP. aeruginosainfection.IMPORTANCEPseudomonas aeruginosais a widespread opportunistic pathogen and a major cause of morbidity and mortality in cystic fibrosis patients. Microbe-virus interactions play a critical role in shaping microbial populations, as viral infections can kill microbial populations or contribute to gene flow among microbes. Investigating howP. aeruginosauses its CRISPR immune system to evade viral infection aids our understanding of how this organism spreads and evolves alongside its viruses in humans and the environment. Here, we identify patterns of CRISPR targeting and immunity that indicateP. aeruginosaand its viruses evolve in both a broad global population and in isolated human “islands.” These data set the stage for exploring metapopulation dynamics occurring within and between isolated “island” populations associated with CF patients, an essential step to inform future work predicting the specificity and efficacy of virus therapy and the spread of invasive viral elements and pathogenic epidemic bacterial strains.

scholarly journals Role of Toxoplasma gondii Chloroquine Resistance Transporter in Bradyzoite Viability and Digestive Vacuole Maintenance

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
Geetha Kannan ◽  
Manlio Di Cristina ◽  
Aric J. Schultz ◽  
My-Hang Huynh ◽  
Fengrong Wang ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Chronic Infection ◽  
Chloroquine Resistance ◽  
Congenital Defects ◽  
Functional Link ◽  
Content Type ◽  
Chloroquine Resistance Transporter

ABSTRACT Toxoplasma gondii is a ubiquitous pathogen that can cause encephalitis, congenital defects, and ocular disease. T. gondii has also been implicated as a risk factor for mental illness in humans. The parasite persists in the brain as slow-growing bradyzoites contained within intracellular cysts. No treatments exist to eliminate this form of parasite. Although proteolytic degradation within the parasite lysosome-like vacuolar compartment (VAC) is critical for bradyzoite viability, whether other aspects of the VAC are important for parasite persistence remains unknown. An ortholog of Plasmodium falciparum chloroquine resistance transporter (CRT), TgCRT, has previously been identified in T. gondii. To interrogate the function of TgCRT in chronic-stage bradyzoites and its role in persistence, we knocked out TgCRT in a cystogenic strain and assessed VAC size, VAC digestion of host-derived proteins and parasite autophagosomes, and the viability of in vitro and in vivo bradyzoites. We found that whereas parasites deficient in TgCRT exhibit normal digestion within the VAC, they display a markedly distended VAC and their viability is compromised both in vitro and in vivo. Interestingly, impairing VAC proteolysis in TgCRT-deficient bradyzoites restored VAC size, consistent with a role for TgCRT as a transporter of products of digestion from the VAC. In conjunction with earlier studies, our current findings suggest a functional link between TgCRT and VAC proteolysis. This study provides further evidence of a crucial role for the VAC in bradyzoite persistence and a new potential VAC target to abate chronic Toxoplasma infection. IMPORTANCE Individuals chronically infected with the intracellular parasite Toxoplasma gondii are at risk of experiencing reactivated disease that can result in progressive loss of vision. No effective treatments exist for chronic toxoplasmosis due in part to a poor understanding of the biology underlying chronic infection and a lack of well-validated potential targets. We show here that a T. gondii transporter is functionally linked to protein digestion within the parasite lysosome-like organelle and that this transporter is necessary to sustain chronic infection in culture and in experimentally infected mice. Ablating the transporter results in severe bloating of the lysosome-like organelle. Together with earlier work, this study suggests the parasite’s lysosome-like organelle is vital for parasite survival, thus rendering it a potential target for diminishing infection and reducing the risk of reactivated disease.

scholarly journals Z-DNA Binding Protein Mediates Host Control of Toxoplasma gondii Infection

2016 ◽  
Vol 84 (10) ◽  
pp. 3063-3070 ◽  
Author(s):  
Kelly J. Pittman ◽  
Patrick W. Cervantes ◽  
Laura J. Knoll
Keyword(s):  
Immune Response ◽  
Toxoplasma Gondii ◽  
Dna Binding ◽  
Chronic Infection ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Host Immune Response ◽  
Activated Macrophages ◽  
Content Type ◽  
Z Dna

Intrinsic toToxoplasma gondiiinfection is the parasite-induced modulation of the host immune response, which ensures establishment of a chronic lifelong infection. This manipulation of the host immune response allowsT. gondiito not only dampen the ability of the host to eliminate the parasite but also trigger parasite differentiation to the slow-growing, encysted bradyzoite form. We previously used RNA sequencing (RNA-seq) to profile the transcriptomes of mice andT. gondiiduring acute and chronic stages of infection. One of the most abundant host transcripts during acute and chronic infection was Z-DNA binding protein 1 (ZBP1). In this study, we determined that ZBP1 functions to controlT. gondiigrowth. In activated macrophages isolated from ZBP1 deletion (ZBP1−/−) mice,T. gondiihas an increased rate of replication and a decreased rate of degradation. We also identified a novel function for ZBP1 as a regulator of nitric oxide (NO) production in activated macrophages, even in the absence ofT. gondiiinfection. Upon stimulation,T. gondii-infected ZBP1−/−macrophages display increased proinflammatory cytokines compared to wild-type macrophages under the same conditions. Thesein vitrophenotypes were recapitulatedin vivo, with ZBP1−/−mice having increased susceptibility to oral challenge, higher cyst burdens during chronic infection, and elevated inflammatory cytokine responses. Taken together, these results highlight a role for ZBP1 in assisting host control ofT. gondiiinfection.

scholarly journals The Toxoplasma gondii Cyst Wall Interactome

mBio ◽  
2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Vincent Tu ◽  
Tadakimi Tomita ◽  
Tatsuki Sugi ◽  
Joshua Mayoral ◽  
Bing Han ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Protein Interactions ◽  
Cyst Wall ◽  
Affinity Purification ◽  
Dense Granule ◽  
Hypothetical Proteins ◽  
Content Type

ABSTRACT A characteristic of the latent cyst stage of Toxoplasma gondii is a thick cyst wall that forms underneath the membrane of the bradyzoite vacuole. Previously, our laboratory group published a proteomic analysis of purified in vitro cyst wall fragments that identified an inventory of cyst wall components. To further refine our understanding of the composition of the cyst wall, several cyst wall proteins were tagged with a promiscuous biotin ligase (BirA*), and their interacting partners were screened by streptavidin affinity purification. Within the cyst wall pulldowns, previously described cyst wall proteins, dense granule proteins, and uncharacterized hypothetical proteins were identified. Several of the newly identified hypothetical proteins were validated to be novel components of the cyst wall and tagged with BirA* to expand the model of the cyst wall interactome. Community detection of the cyst wall interactome model revealed three distinct clusters: a dense granule, a cyst matrix, and a cyst wall cluster. Characterization of several of the identified cyst wall proteins using genetic strategies revealed that MCP3 affects in vivo cyst sizes. This study provides a model of the potential protein interactions within the cyst wall and the groundwork to understand cyst wall formation. IMPORTANCE A model of the cyst wall interactome was constructed using proteins identified through BioID. The proteins within this cyst wall interactome model encompass several proteins identified in a prior characterization of the cyst wall proteome. This model provides a more comprehensive understanding of the composition of the cyst wall and may lead to insights on how the cyst wall is formed.

scholarly journals Analysis of the Topology and Active-Site Residues of WbbF, a Putative O-Polysaccharide Synthase from Salmonella enterica Serovar Borreze

2019 ◽  
Vol 202 (5) ◽  
Author(s):  
Samantha S. Wear ◽  
Brittany A. Hunt ◽  
Bradley R. Clarke ◽  
Chris Whitfield
Keyword(s):  
Salmonella Enterica ◽  
Lipid A ◽  
Critical Role ◽  
Cellulose Synthase ◽  
Site Directed Mutagenesis ◽  
Chain Extension ◽  
Active Site Residues ◽  
Content Type ◽  
Membrane Spanning ◽  
And Function

ABSTRACT Bacterial lipopolysaccharides are major components and contributors to the integrity of Gram-negative outer membranes. The more conserved lipid A-core part of this complex glycolipid is synthesized separately from the hypervariable O-antigenic polysaccharide (OPS) part, and they are joined in the periplasm prior to translocation to the outer membrane. Three different biosynthesis strategies are recognized for OPS biosynthesis, and one, the synthase-dependent pathway, is currently confined to a single example: the O:54 antigen from Salmonella enterica serovar Borreze. Synthases are complex enzymes that have the capacity to both polymerize and export bacterial polysaccharides. Although synthases like cellulose synthase are widespread, they typically polymerize a glycan without employing a lipid-linked intermediate, unlike the O:54 synthase (WbbF), which produces an undecaprenol diphosphate-linked product. This raises questions about the overall similarity between WbbF and conventional synthases. In this study, we examine the topology of WbbF, revealing four membrane-spanning helices, compared to the eight in cellulose synthase. Molecular modeling of the glycosyltransferase domain of WbbF indicates a similar architecture, and site-directed mutagenesis confirmed that residues important for catalysis and processivity in cellulose synthase are conserved in WbbF and required for its activity. These findings indicate that the glycosyltransferase mechanism of WbbF and classic synthases are likely conserved despite the use of a lipid acceptor for chain extension by WbbF. IMPORTANCE Glycosyltransferases play a critical role in the synthesis of a wide variety of bacterial polysaccharides. These include O-antigenic polysaccharides, which form the distal component of lipopolysaccharides and provide a protective barrier important for survival and host-pathogen interactions. Synthases are a subset of glycosyltransferases capable of coupled synthesis and export of glycans. Currently, the O:54 antigen of Salmonella enterica serovar Borreze involves the only example of an O-polysaccharide synthase, and its generation of a lipid-linked product differentiates it from classical synthases. Here, we explore features conserved in the O:54 enzyme and classical synthases to shed light on the structure and function of the unusual O:54 enzyme.

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