Pantothenic Acid Biosynthesis in the Parasite Toxoplasma gondii: a Target for Chemotherapy

ABSTRACTToxoplasma gondiiis a major food pathogen and neglected parasitic infection that causes eye disease, birth defects, and fetal abortion and plays a role as an opportunistic infection in AIDS. In this study, we investigated pantothenic acid (vitamin B5) biosynthesis inT. gondii. Genes encoding the full repertoire of enzymes for pantothenate synthesis and subsequent metabolism to coenzyme A were identified and are expressed inT. gondii. A panel of inhibitors developed to targetMycobacterium tuberculosispantothenate synthetase were tested and found to exhibit a range of values for inhibition ofT. gondiigrowth. Two inhibitors exhibited lower effective concentrations than the currently used toxoplasmosis drug pyrimethamine. The inhibition was specific for the pantothenate pathway, as the effect of the pantothenate synthetase inhibitors was abrogated by supplementation with pantothenate. Hence,T. gondiiencodes and expresses the enzymes for pantothenate synthesis, and this pathway is essential for parasite growth. These promising findings increase our understanding of growth and metabolism in this important parasite and highlight pantothenate synthetase as a new drug target.

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Serine/Threonine Protein Kinase Stk Is Required for Virulence, Stress Response, and Penicillin Tolerance in Streptococcus pyogenes

Infection and Immunity ◽

10.1128/iai.05360-11 ◽

2011 ◽

Vol 79 (10) ◽

pp. 4201-4209 ◽

Cited By ~ 32

Author(s):

Julia Bugrysheva ◽

Barbara J. Froehlich ◽

Jeffrey A. Freiberg ◽

June R. Scott

Keyword(s):

Streptococcus Pyogenes ◽

Fatty Acid Biosynthesis ◽

Group A Streptococcus ◽

Acid Biosynthesis ◽

Regulation Of Expression ◽

Content Type ◽

Reverse Transcription Pcr ◽

Genes Encoding ◽

Group A

ABSTRACTGenes encoding one or more Ser/Thr protein kinases have been identified recently in many bacteria, including one (stk) in the human pathogenStreptococcus pyogenes(group A streptococcus [GAS]). We report that in GAS,stkis required to produce disease in a murine myositis model of infection. Using microarray and quantitative reverse transcription-PCR (qRT-PCR) studies, we found that Stk activates genes for virulence factors, osmoregulation, metabolism of α-glucans, and fatty acid biosynthesis, as well as genes affecting cell wall synthesis. Confirming these transcription studies, we determined that thestkdeletion mutant is more sensitive to osmotic stress and to penicillin than the wild type. We discuss several possible Stk phosphorylation targets that might explain Stk regulation of expression of specific operons and the possible role of Stk in resuscitation from quiescence.

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TgPRELID, a Mitochondrial Protein Linked to Multidrug Resistance in the Parasite Toxoplasma gondii

mSphere ◽

10.1128/msphere.00229-16 ◽

2017 ◽

Vol 2 (1) ◽

Cited By ~ 13

Author(s):

Victoria Jeffers ◽

Edwin T. Kamau ◽

Ananth R. Srinivasan ◽

Jonathan Harper ◽

Preethi Sankaran ◽

...

Keyword(s):

Drug Resistance ◽

Multidrug Resistance ◽

Toxoplasma Gondii ◽

Mitochondrial Protein ◽

Parasitic Infection ◽

Common Mechanism ◽

Parasitic Diseases ◽

Content Type ◽

New Therapies ◽

Resistant Mutants

ABSTRACT We report the discovery of TgPRELID, a previously uncharacterized mitochondrial protein linked to multidrug resistance in the parasite Toxoplasma gondii. Drug resistance remains a major problem in the battle against parasitic infection, and understanding how TgPRELID mutations augment resistance to multiple, distinct compounds will reveal needed insights into the development of new therapies for toxoplasmosis and other related parasitic diseases. New drugs to control infection with the protozoan parasite Toxoplasma gondii are needed as current treatments exert toxic side effects on patients. Approaches to develop novel compounds for drug development include screening of compound libraries and targeted inhibition of essential cellular pathways. We identified two distinct compounds that display inhibitory activity against the parasite’s replicative stage: F3215-0002, which we previously identified during a compound library screen, and I-BET151, an inhibitor of bromodomains, the “reader” module of acetylated lysines. In independent studies, we sought to determine the targets of these two compounds using forward genetics, generating resistant mutants and identifying the determinants of resistance with comparative genome sequencing. Despite the dissimilarity of the two compounds, we recovered resistant mutants with nonsynonymous mutations in the same domain of the same gene, TGGT1_254250, which we found encodes a protein that localizes to the parasite mitochondrion (designated TgPRELID after the name of said domain). We found that mutants selected with one compound were cross resistant to the other compound, suggesting a common mechanism of resistance. To further support our hypothesis that TgPRELID mutations facilitate resistance to both I-BET151 and F3215-0002, CRISPR (clustered regularly interspaced short palindromic repeat)/CAS9-mediated mutation of TgPRELID directly led to increased F3215-0002 resistance. Finally, all resistance mutations clustered in the same subdomain of TgPRELID. These findings suggest that TgPRELID may encode a multidrug resistance factor or that I-BET151 and F3215-0002 have the same target(s) despite their distinct chemical structures. IMPORTANCE We report the discovery of TgPRELID, a previously uncharacterized mitochondrial protein linked to multidrug resistance in the parasite Toxoplasma gondii. Drug resistance remains a major problem in the battle against parasitic infection, and understanding how TgPRELID mutations augment resistance to multiple, distinct compounds will reveal needed insights into the development of new therapies for toxoplasmosis and other related parasitic diseases.

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TgATAT-Mediated α-Tubulin Acetylation Is Required for Division of the Protozoan Parasite Toxoplasma gondii

mSphere ◽

10.1128/msphere.00088-15 ◽

2016 ◽

Vol 1 (1) ◽

Cited By ~ 4

Author(s):

Joseph M. Varberg ◽

Leah R. Padgett ◽

Gustavo Arrizabalaga ◽

William J. Sullivan

Keyword(s):

Toxoplasma Gondii ◽

Drug Target ◽

Drug Targets ◽

Protozoan Parasite ◽

World Population ◽

Content Type ◽

Tubulin Acetylation ◽

Parasite Replication ◽

New Treatments

ABSTRACT Toxoplasma gondii is an opportunistic parasite that infects at least one-third of the world population. New treatments for the disease (toxoplasmosis) are needed since current drugs are toxic to patients. Microtubules are essential cellular structures built from tubulin that show promise as antimicrobial drug targets. Microtubules can be regulated by chemical modification, such as acetylation on lysine 40 (K40). To determine the role of K40 acetylation in Toxoplasma and whether it is a liability to the parasite, we performed mutational analyses of the α-tubulin gene. Our results indicate that parasites cannot survive without K40 acetylation unless microtubules are stabilized with a secondary mutation. Additionally, we identified the parasite enzyme that acetylates α-tubulin (TgATAT). Genetic disruption of TgATAT caused severe defects in parasite replication, further highlighting the importance of α-tubulin K40 acetylation in Toxoplasma and its promise as a potential new drug target. Toxoplasma gondii is a widespread protozoan parasite that causes potentially life-threatening opportunistic disease. New inhibitors of parasite replication are urgently needed, as the current antifolate treatment is also toxic to patients. Microtubules are essential cytoskeletal components that have been selectively targeted in microbial pathogens; further study of tubulin in Toxoplasma may reveal novel therapeutic opportunities. It has been noted that α-tubulin acetylation at lysine 40 (K40) is enriched during daughter parasite formation, but the impact of this modification on Toxoplasma division and the enzyme mediating its delivery have not been identified. We performed mutational analyses to provide evidence that K40 acetylation stabilizes Toxoplasma microtubules and is required for parasite replication. We also show that an unusual Toxoplasma homologue of α-tubulin acetyltransferase (TgATAT) is expressed in a cell cycle-regulated manner and that its expression peaks during division. Disruption of TgATAT with CRISPR/Cas9 ablates K40 acetylation and induces replication defects; parasites appear to initiate mitosis yet exhibit incomplete or improper nuclear division. Together, these findings establish the importance of tubulin acetylation, exposing a new vulnerability in Toxoplasma that could be pharmacologically targeted. IMPORTANCE Toxoplasma gondii is an opportunistic parasite that infects at least one-third of the world population. New treatments for the disease (toxoplasmosis) are needed since current drugs are toxic to patients. Microtubules are essential cellular structures built from tubulin that show promise as antimicrobial drug targets. Microtubules can be regulated by chemical modification, such as acetylation on lysine 40 (K40). To determine the role of K40 acetylation in Toxoplasma and whether it is a liability to the parasite, we performed mutational analyses of the α-tubulin gene. Our results indicate that parasites cannot survive without K40 acetylation unless microtubules are stabilized with a secondary mutation. Additionally, we identified the parasite enzyme that acetylates α-tubulin (TgATAT). Genetic disruption of TgATAT caused severe defects in parasite replication, further highlighting the importance of α-tubulin K40 acetylation in Toxoplasma and its promise as a potential new drug target.

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Evolution of the Sterol Biosynthetic Pathway of Pythium insidiosum and Related Oomycetes Contributes to Antifungal Drug Resistance

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02352-16 ◽

2017 ◽

Vol 61 (4) ◽

Cited By ~ 17

Author(s):

Tassanee Lerksuthirat ◽

Areeporn Sangcakul ◽

Tassanee Lohnoo ◽

Wanta Yingyong ◽

Thidarat Rujirawat ◽

...

Keyword(s):

Drug Target ◽

Biosynthetic Pathway ◽

Subcutaneous Tissue ◽

Antifungal Drugs ◽

Biosynthetic Enzymes ◽

Pythium Insidiosum ◽

Content Type ◽

Clinical Resistance ◽

Genes Encoding ◽

Sterol Biosynthetic Pathway

ABSTRACT Pythiosis is a life-threatening infectious disease caused by the oomycete Pythium insidiosum. Direct exposure to Py. insidiosum zoospores can initiate infections of the eye, limb, gastrointestinal tract, or skin/subcutaneous tissue. Treatments for pythiosis have mostly relied on surgery. Antifungal drugs are generally ineffective against Py. insidiosum. However, one patient with an invasive Py. insidiosum infection recovered completely following treatment with terbinafine and itraconazole. Additionally, the drug target sterol biosynthetic enzymes have been identified in the oomycete Aphanomyces euteiches. It remains an open question whether Py. insidiosum is susceptible to the antifungal drugs and harbors any of the known drug target enzymes. Here, we determined the in vitro susceptibilities of terbinafine and itraconazole against 30 isolates of Py. insidiosum. We also analyzed endogenous sterols and searched for genes encoding the sterol biosynthetic enzymes in the genomes of Py. insidiosum and related oomycetes. The susceptibility assay showed that the growth of each of the Py. insidiosum isolates was inhibited by the antifungal agents, but only at difficult-to-achieve concentrations, which explains the clinical resistance of the drugs in the treatment of pythiosis patients. Genome searches of Py. insidiosum and related oomycetes demonstrated that these organisms contained an incomplete set of sterol biosynthetic enzymes. Gas chromatographic mass spectrometry did not detect any sterol end products in Py. insidiosum. In conclusion, Py. insidiosum possesses an incomplete sterol biosynthetic pathway. Resistance to antifungal drugs targeting enzymes in the ergosterol biosynthetic pathway in Py. insidiosum was due to modifications or losses of some of the genes encoding the drug target enzymes.

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Biological Diagnosis of Ocular Toxoplasmosis: a Nine-Year Retrospective Observational Study

mSphere ◽

10.1128/msphere.00636-19 ◽

2019 ◽

Vol 4 (5) ◽

Cited By ~ 1

Author(s):

Valentin Greigert ◽

Alexander W. Pfaff ◽

Arnaud Sauer ◽

Denis Filisetti ◽

Ermanno Candolfi ◽

...

Keyword(s):

Toxoplasma Gondii ◽

Parasitic Infection ◽

Posterior Uveitis ◽

Ocular Toxoplasmosis ◽

Antibody Detection ◽

Diagnostic Tools ◽

Content Type ◽

Study Results ◽

Biological Tests ◽

Single Positive

ABSTRACT Ocular toxoplasmosis (OT), i.e., the ocular manifestation of Toxoplasma gondii infection, is one of the leading causes of posterior uveitis. While ocular lesions are often typical, atypical forms often require biological confirmation of the diagnosis. Our study sought to review the biological OT diagnoses made in our laboratory to further assess the role of each test in the diagnostic procedure. All ocular samples sent to our laboratory over the last 9 years for OT diagnosis were included. These samples were analyzed using T. gondii PCR and antibody detection by means of immunoblotting and Candolfi coefficient (CC) determinations, either alone or in combination. Since serum analysis is required to interpret both the CC and immunoblotting, blood serology for T. gondii was also performed in most cases. Of the 249 samples analyzed, 80 (32.1%; 95% confidence interval [95%CI], 26.3 to 37.9) were positive for OT. Of these 80 cases, 52/80 (65.0%; 54.6 to 74.5) displayed a positive PCR, 15/80 (18.8%; 10.2 to 27.3) a positive CC, and 33/80 (41.3%; 95%CI, 30.5 to 52.0) a positive immunoblot result. Overall, 63 of the 80 OT diagnoses (78.8%; 95%CI, 69.8 to 87.7) were made on the basis of a single positive test result. Our study results remind us that current biological diagnostic tools for OT must be employed in combination to obtain an optimal diagnosis based on the precious ocular fluids sampled by ophthalmologists. Clinicobiological studies that are focused on correlating the performances of the different tests with clinical features are critically needed to improve our understanding of the pathophysiology and diagnosis of OT. IMPORTANCE Ocular toxoplasmosis (OT), a parasitic infection of the eye, is considered to be the most important infectious cause of posterior uveitis worldwide. Its prevalence is particularly high in South America, where aggressive Toxoplasma gondii strains are responsible for more-severe presentations. The particular pathophysiology of this infection leads, from recurrence to recurrence, to potentially severe vision impairment. The diagnosis of this infection is usually exclusively based on the clinical examination. However, the symptoms may be misleading and are not always sufficient to confirm a diagnosis of OT. In such cases, biological tests performed by means of several techniques on blood and ocular samples may facilitate the diagnosis. In this study, we analyzed the tests that were performed in our laboratory over a 9-year period every time OT was suspected. Our report highlights that the quality of ocular sampling by ophthalmologists and combinations of several techniques are critical for a reliable biological OT diagnosis.

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Disrupted Synthesis of a Di-N-acetylated Sugar Perturbs Mature Glycoform Structure and Microheterogeneity in theO-Linked Protein Glycosylation System ofNeisseria elongatasubsp.glycolytica

Journal of Bacteriology ◽

10.1128/jb.00522-18 ◽

2018 ◽

Vol 201 (1) ◽

Cited By ~ 1

Author(s):

Nelson Wang ◽

Jan Haug Anonsen ◽

Raimonda Viburiene ◽

Joseph S. Lam ◽

Åshild Vik ◽

...

Keyword(s):

Broad Spectrum ◽

Significant Degree ◽

Protein Glycosylation ◽

Acid Biosynthesis ◽

Content Type ◽

Hexuronic Acid ◽

Major Species ◽

Genes Encoding ◽

Health And Disease

ABSTRACTThe genusNeisseriaincludes three major species of importance to human health and disease (Neisseria gonorrhoeae,Neisseria meningitidis, andNeisseria lactamica) that express broad-spectrumO-linked protein glycosylation (Pgl) systems. The potential for related Pgl systems in other species in the genus, however, remains to be determined. Using a strain ofNeisseria elongatasubsp.glycolytica, a unique tetrasaccharide glycoform consisting of di-N-acetylbacillosamine and glucose as the first two sugars followed by a rare sugar whose mass spectrometric fragmentation profile was most consistent with di-N-acetyl hexuronic acid and aN-acetylhexosamine at the nonreducing end has been identified. Based on established mechanisms for UDP-di-N-acetyl hexuronic acid biosynthesis found in other microbes, we searched for genes encoding related pathway components in theN. elongatasubsp.glycolyticagenome. Here, we detail the identification of such genes and the ensuing glycosylation phenotypes engendered by their inactivation. While the findings extend the conservative nature of microbial UDP-di-N-acetyl hexuronic acid biosynthesis, mutant glycosylation phenotypes reveal unique, relaxed specificities of the glycosyltransferases and oligosaccharyltransferases to incorporate pathway intermediate UDP-sugars into mature glycoforms.IMPORTANCEBroad-spectrum protein glycosylation (Pgl) systems are well recognized in bacteria and archaea. Knowledge of how these systems relate structurally, biochemically, and evolutionarily to one another and to others associated with microbial surface glycoconjugate expression is still incomplete. Here, we detail reverse genetic efforts toward characterization of protein glycosylation mutants ofN. elongatasubsp.glycolyticathat define the biosynthesis of a conserved but relatively rare UDP-sugar precursor. The results show both a significant degree of intra- and transkingdom conservation in the utilization of UDP-di-N-acetyl-glucuronic acid and singular properties related to the relaxed specificities of theN. elongatasubsp.glycolyticasystem

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Pyrimidine Pathway-Dependent and -Independent Functions of the Toxoplasma gondii Mitochondrial Dihydroorotate Dehydrogenase

Infection and Immunity ◽

10.1128/iai.00187-16 ◽

2016 ◽

Vol 84 (10) ◽

pp. 2974-2981 ◽

Cited By ~ 8

Author(s):

Miryam Andrea Hortua Triana ◽

Daniela Cajiao Herrera ◽

Barbara H. Zimmermann ◽

Barbara A. Fox ◽

David J. Bzik

Keyword(s):

Toxoplasma Gondii ◽

Drug Target ◽

De Novo ◽

Mutant Gene ◽

Pyrimidine Biosynthesis ◽

Dihydroorotate Dehydrogenase ◽

Content Type ◽

Independent Functions ◽

Fourth Step ◽

Essential Function

Dihydroorotate dehydrogenase (DHODH) mediates the fourth step ofde novopyrimidine biosynthesis and is a proven drug target for inducing immunosuppression in therapy of human disease as well as a rapidly emerging drug target for treatment of malaria. InToxoplasma gondii, disruption of the first, fifth, or sixth step ofde novopyrimidine biosynthesis induced uracil auxotrophy. However, previous attempts to generate uracil auxotrophy by genetically deleting the mitochondrion-associated DHODH ofT. gondii(TgDHODH) failed. To further address the essentiality ofTgDHODH, mutant gene alleles deficient inTgDHODH activity were designed to ablate the enzyme activity. Replacement of the endogenousDHODHgene with catalytically deficientDHODHgene alleles induced uracil auxotrophy. Catalytically deficientTgDHODH localized to the mitochondria, and parasites retained mitochondrial membrane potential. These results show thatTgDHODH is essential for the synthesis of pyrimidines and suggest thatTgDHODH is required for a second essential function independent of its role in pyrimidine biosynthesis.

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Loss of the Conserved Alveolate Kinase MAPK2 Decouples Toxoplasma Cell Growth from Cell Division

mBio ◽

10.1128/mbio.02517-20 ◽

2020 ◽

Vol 11 (6) ◽

Author(s):

Xiaoyu Hu ◽

William J. O’Shaughnessy ◽

Tsebaot G. Beraki ◽

Michael L. Reese

Keyword(s):

Toxoplasma Gondii ◽

Protein Kinases ◽

Daughter Cell ◽

Protozoan Parasite ◽

Productive Infection ◽

Centrosome Duplication ◽

Loss Of Function ◽

Content Type ◽

Mitogen Activated Protein ◽

Parasite Replication

ABSTRACT Mitogen-activated protein kinases (MAPKs) are a conserved family of protein kinases that regulate signal transduction, proliferation, and development throughout eukaryotes. The apicomplexan parasite Toxoplasma gondii expresses three MAPKs. Two of these, extracellular signal-regulated kinase 7 (ERK7) and MAPKL1, have been implicated in the regulation of conoid biogenesis and centrosome duplication, respectively. The third kinase, MAPK2, is specific to and conserved throughout the Alveolata, although its function is unknown. We used the auxin-inducible degron system to determine phenotypes associated with MAPK2 loss of function in Toxoplasma. We observed that parasites lacking MAPK2 failed to duplicate their centrosomes and therefore did not initiate daughter cell budding, which ultimately led to parasite death. MAPK2-deficient parasites initiated but did not complete DNA replication and arrested prior to mitosis. Surprisingly, the parasites continued to grow and replicate their Golgi apparatus, mitochondria, and apicoplasts. We found that the failure in centrosome duplication is distinct from the phenotype caused by the depletion of MAPKL1. As we did not observe MAPK2 localization at the centrosome at any point in the cell cycle, our data suggest that MAPK2 regulates a process at a distal site that is required for the completion of centrosome duplication and the initiation of parasite mitosis. IMPORTANCE Toxoplasma gondii is a ubiquitous intracellular protozoan parasite that can cause severe and fatal disease in immunocompromised patients and the developing fetus. Rapid parasite replication is critical for establishing a productive infection. Here, we demonstrate that a Toxoplasma protein kinase called MAPK2 is conserved throughout the Alveolata and essential for parasite replication. We found that parasites lacking MAPK2 protein were defective in the initiation of daughter cell budding and were rendered inviable. Specifically, T. gondii MAPK2 (TgMAPK2) appears to be required for centrosome replication at the basal end of the nucleus, and its loss causes arrest early in parasite division. MAPK2 is unique to the Alveolata and not found in metazoa and likely is a critical component of an essential parasite-specific signaling network.

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Autophagy-Related Protein ATG8 Has a Noncanonical Function for Apicoplast Inheritance in Toxoplasma gondii

mBio ◽

10.1128/mbio.01446-15 ◽

2015 ◽

Vol 6 (6) ◽

Cited By ~ 43

Author(s):

Maude F. Lévêque ◽

Laurence Berry ◽

Michael J. Cipriano ◽

Hoa-Mai Nguyen ◽

Boris Striepen ◽

...

Keyword(s):

Toxoplasma Gondii ◽

Growth Conditions ◽

Model Organisms ◽

Secondary Endosymbiosis ◽

Related Protein ◽

Content Type ◽

Superresolution Microscopy ◽

Animal Pathogens ◽

Cellular Components ◽

Catabolic Process

ABSTRACT Autophagy is a catabolic process widely conserved among eukaryotes that permits the rapid degradation of unwanted proteins and organelles through the lysosomal pathway. This mechanism involves the formation of a double-membrane structure called the autophagosome that sequesters cellular components to be degraded. To orchestrate this process, yeasts and animals rely on a conserved set of autophagy-related proteins (ATGs). Key among these factors is ATG8, a cytoplasmic protein that is recruited to nascent autophagosomal membranes upon the induction of autophagy. Toxoplasma gondii is a potentially harmful human pathogen in which only a subset of ATGs appears to be present. Although this eukaryotic parasite seems able to generate autophagosomes upon stresses such as nutrient starvation, the full functionality and biological relevance of a canonical autophagy pathway are as yet unclear. Intriguingly, in T. gondii, ATG8 localizes to the apicoplast under normal intracellular growth conditions. The apicoplast is a nonphotosynthetic plastid enclosed by four membranes resulting from a secondary endosymbiosis. Using superresolution microscopy and biochemical techniques, we show that TgATG8 localizes to the outermost membrane of this organelle. We investigated the unusual function of TgATG8 at the apicoplast by generating a conditional knockdown mutant. Depletion of TgATG8 led to rapid loss of the organelle and subsequent intracellular replication defects, indicating that the protein is essential for maintaining apicoplast homeostasis and thus for survival of the tachyzoite stage. More precisely, loss of TgATG8 led to abnormal segregation of the apicoplast into the progeny because of a loss of physical interactions of the organelle with the centrosomes. IMPORTANCE By definition, autophagy is a catabolic process that leads to the digestion and recycling of eukaryotic cellular components. The molecular machinery of autophagy was identified mainly in model organisms such as yeasts but remains poorly characterized in phylogenetically distant apicomplexan parasites. We have uncovered an unusual function for autophagy-related protein ATG8 in Toxoplasma gondii: TgATG8 is crucial for normal replication of the parasite inside its host cell. Seemingly unrelated to the catabolic autophagy process, TgATG8 associates with the outer membrane of the nonphotosynthetic plastid harbored by the parasite called the apicoplast, and there it plays an important role in the centrosome-driven inheritance of the organelle during cell division. This not only reveals an unexpected function for an autophagy-related protein but also sheds new light on the division process of an organelle that is vital to a group of important human and animal pathogens.

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Genes Required for Aerial Growth, Cell Division, and Chromosome Segregation Are Targets of WhiA before Sporulation in Streptomyces venezuelae

mBio ◽

10.1128/mbio.00684-13 ◽

2013 ◽

Vol 4 (5) ◽

Cited By ~ 61

Author(s):

Matthew J. Bush ◽

Maureen J. Bibb ◽

Govind Chandra ◽

Kim C. Findlay ◽

Mark J. Buttner

Keyword(s):

Cell Division ◽

Chromosome Segregation ◽

Regulatory Networks ◽

Liquid Culture ◽

Biological Processes ◽

Streptomyces Venezuelae ◽

Content Type ◽

Master Regulators ◽

Aerial Growth ◽

Genes Encoding

ABSTRACTWhiA is a highly unusual transcriptional regulator related to a family of eukaryotic homing endonucleases. WhiA is required for sporulation in the filamentous bacteriumStreptomyces, but WhiA homologues of unknown function are also found throughout the Gram-positive bacteria. To better understand the role of WhiA inStreptomycesdevelopment and its function as a transcription factor, we identified the WhiA regulon through a combination of chromatin immunoprecipitation-sequencing (ChIP-seq) and microarray transcriptional profiling, exploiting a new model organism for the genus,Streptomyces venezuelae, which sporulates in liquid culture. The regulon encompasses ~240 transcription units, and WhiA appears to function almost equally as an activator and as a repressor. Bioinformatic analysis of the upstream regions of the complete regulon, combined with DNase I footprinting, identified a short but highly conserved asymmetric sequence, GACAC, associated with the majority of WhiA targets. Construction of a null mutant showed thatwhiAis required for the initiation of sporulation septation and chromosome segregation inS. venezuelae, and several genes encoding key proteins of theStreptomycescell division machinery, such asftsZ,ftsW, andftsK, were found to be directly activated by WhiA during development. Several other genes encoding proteins with important roles in development were also identified as WhiA targets, including the sporulation-specific sigma factor σWhiGand the diguanylate cyclase CdgB. Cell division is tightly coordinated with the orderly arrest of apical growth in the sporogenic cell, andfilP, encoding a key component of the polarisome that directs apical growth, is a direct target for WhiA-mediated repression during sporulation.IMPORTANCESince the initial identification of the genetic loci required forStreptomycesdevelopment, all of thebldandwhidevelopmental master regulators have been cloned and characterized, and significant progress has been made toward understanding the cell biological processes that drive morphogenesis. A major challenge now is to connect the cell biological processes and the developmental master regulators by dissecting the regulatory networks that link the two. Studies of these regulatory networks have been greatly facilitated by the recent introduction ofStreptomyces venezuelaeas a new model system for the genus, a species that sporulates in liquid culture. Taking advantage ofS. venezuelae, we have characterized the regulon of genes directly under the control of one of these master regulators, WhiA. Our results implicate WhiA in the direct regulation of key steps in sporulation, including the cessation of aerial growth, the initiation of cell division, and chromosome segregation.

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