High-Resolution Analysis of the Peptidoglycan Composition inStreptomyces coelicolor

ABSTRACTThe bacterial cell wall maintains cell shape and protects against bursting by turgor. A major constituent of the cell wall is peptidoglycan (PG), which is continuously modified to enable cell growth and differentiation through the concerted activity of biosynthetic and hydrolytic enzymes. Streptomycetes are Gram-positive bacteria with a complex multicellular life style alternating between mycelial growth and the formation of reproductive spores. This involves cell wall remodeling at apical sites of the hyphae during cell elongation and autolytic degradation of the vegetative mycelium during the onset of development and antibiotic production. Here, we show that there are distinct differences in the cross-linking and maturation of the PGs between exponentially growing vegetative hyphae and the aerial hyphae that undergo sporulation. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis identified over 80 different muropeptides, revealing that major PG hydrolysis takes place over the course of mycelial growth. Half of the dimers lacked one of the disaccharide units in transition-phase cells, most likely due to autolytic activity. The deacetylation of MurNAc to MurN was particularly pronounced in spores and strongly reduced in sporulation mutants with a deletion ofbldDorwhiG, suggesting that MurN is developmentally regulated. Altogether, our work highlights the dynamic and growth phase-dependent changes in the composition of the PG inStreptomyces.IMPORTANCEStreptomycetes are bacteria with a complex lifestyle and are model organisms for bacterial multicellularity. From a single spore, a large multigenomic multicellular mycelium is formed, which differentiates to form spores. Programmed cell death is an important event during the onset of morphological differentiation. In this work, we provide new insights into the changes in the peptidoglycan composition and over time, highlighting changes over the course of development and between growing mycelia and spores. This revealed dynamic changes in the peptidoglycan when the mycelia aged, with extensive peptidoglycan hydrolysis and, in particular, an increase in the proportion of 3-3 cross-links. Additionally, we identified a muropeptide that accumulates predominantly in the spores and may provide clues toward spore development.

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High-resolution analysis of the peptidoglycan composition inStreptomyces coelicolor

10.1101/319178 ◽

2018 ◽

Cited By ~ 1

Author(s):

Lizah T. van der Aart ◽

Gerwin K. Spijksma ◽

Amy Harms ◽

Waldemar Vollmer ◽

Thomas Hankemeier ◽

...

Keyword(s):

Cell Wall ◽

Mycelial Growth ◽

Antibiotic Production ◽

Hydrolytic Enzymes ◽

Morphological Differentiation ◽

Model Organisms ◽

Major Constituent ◽

Single Spore ◽

High Resolution Analysis ◽

Cell Growth And Differentiation

ABSTRACTThe bacterial cell wall maintains cell shape and protects against bursting by the turgor. A major constituent of the cell wall is peptidoglycan (PG), which is continuously modified to allow cell growth and differentiation through the concerted activity of biosynthetic and hydrolytic enzymes. Streptomycetes are Gram-positive bacteria with a complex multicellular life style alternating between mycelial growth and the formation of reproductive spores. This involves cell-wall remodeling at apical sites of the hyphae during cell elongation and autolytic degradation of the vegetative mycelium during the onset of development and antibiotic production. Here, we show that there are distinct differences in the cross-linking and maturation of the PG between exponentially growing vegetative hyphae and the aerial hyphae that undergo sporulation. LC-MS/MS analysis identified over 80 different muropeptides, revealing that major PG hydrolysis takes place over the course of mycelial growth. Half of the dimers lack one of the disaccharide units in transition-phase cells, most likely due to autolytic activity. De-acetylation of MurNAc to MurN was particularly pronounced in spores, suggesting that MurN plays a role in spore development. Taken together, our work highlights dynamic and growth phase-dependent construction and remodeling of PG inStreptomyces.IMPORTANCEStreptomycetes are bacteria with a complex lifestyle, which are model organisms for bacterial multicellularity. From a single spore a large multigenomic, multicellular mycelium is formed, which differentiates to form spores. Programmed cell death is an important event during the onset of morphological differentiation. In this work we provide new insights into the changes in the peptidoglycan architecture over time, highlighting changes over the course of development and between growing mycelia and spores. This revealed dynamic changes in the peptidoglycan when the mycelia age, showing extensive PG hydrolysis and in particular an increase in the proportion of 3-3-cross-links. Additionally, we identified a muropeptide that is highly abundant specifically in spores, which may relate to dormancy and germination.

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The RNA Polymerase Omega Factor RpoZ Is Regulated by PhoP and Has an Important Role in Antibiotic Biosynthesis and Morphological Differentiation in Streptomyces coelicolor

Applied and Environmental Microbiology ◽

10.1128/aem.00465-11 ◽

2011 ◽

Vol 77 (21) ◽

pp. 7586-7594 ◽

Cited By ~ 27

Author(s):

Fernando Santos-Beneit ◽

Mónica Barriuso-Iglesias ◽

Lorena T. Fernández-Martínez ◽

Miriam Martínez-Castro ◽

Alberto Sola-Landa ◽

...

Keyword(s):

Rna Polymerase ◽

Response Regulator ◽

Antibiotic Production ◽

Streptomyces Coelicolor ◽

Morphological Differentiation ◽

Luciferase Reporter ◽

Antibiotic Biosynthesis ◽

Binding Assays ◽

Content Type ◽

Phosphate Depletion

ABSTRACTThe RNA polymerase (RNAP) omega factor (ω) forms a complex with the α2ββ′ core of this enzyme in bacteria. We have characterized therpoZgene ofStreptomyces coelicolor, which encodes a small protein (90 amino acids) identified as the omega factor. Deletion of therpoZgene resulted in strains with a slightly reduced growth rate, although they were still able to sporulate. The biosynthesis of actinorhodin and, particularly, that of undecylprodigiosin were drastically reduced in the ΔrpoZstrain, suggesting that expression of these secondary metabolite biosynthetic genes is dependent upon the presence of RpoZ in the RNAP complex. Complementation of the ΔrpoZmutant with the wild-typerpoZallele restored both phenotype and antibiotic production. Interestingly, therpoZgene contains a PHO box in its promoter region. DNA binding assays showed that the phosphate response regulator PhoP binds to such a region. Since luciferase reporter studies showed thatrpoZpromoter activity was increased in a ΔphoPbackground, it can be concluded thatrpoZis controlled negatively by PhoP, thus connecting phosphate depletion regulation with antibiotic production and morphological differentiation inStreptomyces.

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Coxiella burnetiiRpoS Regulates Genes Involved in Morphological Differentiation and Intracellular Growth

Journal of Bacteriology ◽

10.1128/jb.00009-19 ◽

2019 ◽

Vol 201 (8) ◽

Cited By ~ 4

Author(s):

Derek E. Moormeier ◽

Kelsi M. Sandoz ◽

Paul A. Beare ◽

Daniel E. Sturdevant ◽

Vinod Nair ◽

...

Keyword(s):

Cell Wall ◽

Stationary Phase ◽

Stress Responses ◽

Q Fever ◽

Morphological Differentiation ◽

Developmental Cycle ◽

Intracellular Growth ◽

Content Type ◽

Small Cell Variant ◽

Cell Variant

ABSTRACTCoxiella burnetii, the etiological agent of Q fever, undergoes a unique biphasic developmental cycle where bacteria transition from a replicating (exponential-phase) large cell variant (LCV) form to a nonreplicating (stationary-phase) small cell variant (SCV) form. The alternative sigma factor RpoS is an essential regulator of stress responses and stationary-phase physiology in several bacterial species, includingLegionella pneumophila, which has a developmental cycle superficially similar to that ofC. burnetii. Here, we used aC. burnetiiΔrpoSmutant to define the role of RpoS in intracellular growth and SCV development. Growth yields following infection of Vero epithelial cells or THP-1 macrophage-like cells with therpoSmutant in the SCV form, but not the LCV form, were significantly lower than that of wild-type bacteria. RNA sequencing and whole-cell mass spectrometry of theC. burnetiiΔrpoSmutant revealed that a substantial portion of theC. burnetiigenome is regulated by RpoS during SCV development. Regulated genes include those involved in stress responses, arginine transport, peptidoglycan remodeling, and synthesis of the SCV-specific protein ScvA. Genes comprising thedot/icmlocus, responsible for production of the Dot/Icm type 4B secretion system, were also dysregulated in therpoSmutant. These data were corroborated with independent assays demonstrating that theC. burnetiiΔrpoSstrain has increased sensitivity to hydrogen peroxide and carbenicillin and a thinner cell wall/outer membrane complex. Collectively, these results demonstrate that RpoS is an important regulator of genes involved inC. burnetiiSCV development and intracellular growth.IMPORTANCEThe Q fever bacteriumCoxiella burnetiihas spore-like environmental stability, a characteristic that contributes to its designation as a potential bioweapon. Stability is likely conferred by a highly resistant, small cell variant (SCV) stationary-phase form that arises during a biphasic developmental cycle. Here, we define the role of the alternative sigma factor RpoS in regulating genes associated with SCV development. Genes involved in stress responses, amino acid transport, cell wall remodeling, and type 4B effector secretion were dysregulated in therpoSmutant. Cellular impairments included defects in intracellular growth, cell wall structure, and resistance to oxidants. These results support RpoS as a central regulator of theCoxielladevelopmental cycle and identify developmentally regulated genes involved in morphological differentiation.

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Polydiglycosylphosphate Transferase PdtA (SCO2578) of Streptomyces coelicolor A3(2) Is Crucial for Proper Sporulation and Apical Tip Extension under Stress Conditions

Applied and Environmental Microbiology ◽

10.1128/aem.01425-16 ◽

2016 ◽

Vol 82 (18) ◽

pp. 5661-5672 ◽

Cited By ~ 8

Author(s):

Steffen Sigle ◽

Nadja Steblau ◽

Wolfgang Wohlleben ◽

Günther Muth

Keyword(s):

Cell Wall ◽

Life Cycle ◽

Vegetative Growth ◽

Cell Envelope ◽

Streptomyces Coelicolor ◽

Morphological Differentiation ◽

Spore Wall ◽

Stress Conditions ◽

Content Type ◽

Tip Extension

ABSTRACTAlthough anionic glycopolymers are crucial components of the Gram-positive cell envelope, the relevance of anionic glycopolymers for vegetative growth and morphological differentiation ofStreptomyces coelicolorA3(2) is unknown. Here, we show that the LytR-CpsA-Psr (LCP) protein PdtA (SCO2578), a TagV-like glycopolymer transferase, has a dual function in theS. coelicolorA3(2) life cycle. Despite the presence of 10 additional LCP homologs, PdtA is crucial for proper sporulation. The integrity of the spore envelope was severely affected in apdtAdeletion mutant, resulting in 34% nonviable spores.pdtAdeletion caused a significant reduction in the polydiglycosylphosphate content of the spore envelope. Beyond that, apical tip extension and normal branching of vegetative mycelium were severely impaired on high-salt medium. This growth defect coincided with the mislocalization of peptidoglycan synthesis. Thus, PdtA itself or the polydiglycosylphosphate attached to the peptidoglycan by the glycopolymer transferase PdtA also has a crucial function in apical tip extension of vegetative hyphae under stress conditions.IMPORTANCEAnionic glycopolymers are underappreciated components of the Gram-positive cell envelope. They provide rigidity to the cell wall and position extracellular enzymes involved in peptidoglycan remodeling. AlthoughStreptomyces coelicolorA3(2), the model organism for bacterial antibiotic production, is known to produce two distinct cell wall-linked glycopolymers, teichulosonic acid and polydiglycosylphosphate, the role of these glycopolymers in theS. coelicolorA3(2) life cycle has not been addressed so far. This study reveals a crucial function of the anionic glycopolymer polydiglycosylphosphate for the growth and morphological differentiation ofS. coelicolorA3(2). Polydiglycosylphosphate is attached to the spore wall by the LytR-CpsA-Psr protein PdtA (SCO2578), a component of theStreptomycesspore wall-synthesizing complex (SSSC), to ensure the integrity of the spore envelope. Surprisingly, PdtA also has a crucial role in vegetative growth under stress conditions and is required for proper peptidoglycan incorporation during apical tip extension.

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The Cell Wall Hydrolytic NlpC/P60 Endopeptidases in Mycobacterial Cytokinesis: A Structural Perspective

Cells ◽

10.3390/cells8060609 ◽

2019 ◽

Vol 8 (6) ◽

pp. 609 ◽

Cited By ~ 3

Author(s):

Flavia Squeglia ◽

Miguel Moreira ◽

Alessia Ruggiero ◽

Rita Berisio

Keyword(s):

Cell Wall ◽

Functional Data ◽

Hydrolytic Enzymes ◽

Diaminopimelic Acid ◽

Major Constituent ◽

Wall Material ◽

Gene Encoding ◽

Daughter Cells ◽

Fine Regulation

In preparation for division, bacteria replicate their DNA and segregate the newly formed chromosomes. A division septum then assembles between the chromosomes, and the mother cell splits into two identical daughters due to septum degradation. A major constituent of bacterial septa and of the whole cell wall is peptidoglycan (PGN), an essential cell wall polymer, formed by glycan chains of β−(1-4)-linked-N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc), cross-linked by short peptide stems. Depending on the amino acid located at the third position of the peptide stem, PGN is classified as either Lys-type or meso-diaminopimelic acid (DAP)-type. Hydrolytic enzymes play a crucial role in the degradation of bacterial septa to split the cell wall material shared by adjacent daughter cells to promote their separation. In mycobacteria, a key PGN hydrolase, belonging to the NlpC/P60 endopeptidase family and denoted as RipA, is responsible for the degradation of septa, as the deletion of the gene encoding for this enzyme generates abnormal bacteria with multiple septa. This review provides an update of structural and functional data highlighting the central role of RipA in mycobacterial cytokinesis and the fine regulation of its catalytic activity, which involves multiple molecular partners.

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Activating Intrinsic Carbohydrate-Active Enzymes of the Smut Fungus Ustilago maydis for the Degradation of Plant Cell Wall Components

Applied and Environmental Microbiology ◽

10.1128/aem.00713-16 ◽

2016 ◽

Vol 82 (17) ◽

pp. 5174-5185 ◽

Cited By ~ 24

Author(s):

Elena Geiser ◽

Michèle Reindl ◽

Lars M. Blank ◽

Michael Feldbrügge ◽

Nick Wierckx ◽

...

Keyword(s):

Cell Wall ◽

Plant Cell ◽

Plant Cell Wall ◽

Hydrolytic Enzymes ◽

Value Added ◽

Ustilago Maydis ◽

Smut Fungus ◽

Content Type ◽

Cell Wall Components ◽

Wall Components

ABSTRACTThe microbial conversion of plant biomass to valuable products in a consolidated bioprocess could greatly increase the ecologic and economic impact of a biorefinery. Current strategies for hydrolyzing plant material mostly rely on the external application of carbohydrate-active enzymes (CAZymes). Alternatively, production organisms can be engineered to secrete CAZymes to reduce the reliance on externally added enzymes. Plant-pathogenic fungi have a vast repertoire of hydrolytic enzymes to sustain their lifestyle, but expression of the corresponding genes is usually highly regulated and restricted to the pathogenic phase. Here, we present a new strategy in using the biotrophic smut fungusUstilago maydisfor the degradation of plant cell wall components by activating its intrinsic enzyme potential during axenic growth. This fungal model organism is fully equipped with hydrolytic enzymes, and moreover, it naturally produces value-added substances, such as organic acids and biosurfactants. To achieve the deregulated expression of hydrolytic enzymes during the industrially relevant yeast-like growth in axenic culture, the native promoters of the respective genes were replaced by constitutively active synthetic promoters. This led to an enhanced conversion of xylan, cellobiose, and carboxymethyl cellulose to fermentable sugars. Moreover, a combination of strains with activated endoglucanase and β-glucanase increased the release of glucose from carboxymethyl cellulose and regenerated amorphous cellulose, suggesting that mixed cultivations could be a means for degrading more complex substrates in the future. In summary, this proof of principle demonstrates the potential applicability of activating the expression of native CAZymes from phytopathogens in a biocatalytic process.IMPORTANCEThis study describes basic experiments that aim at the degradation of plant cell wall components by the smut fungusUstilago maydis. As a plant pathogen, this fungus contains a set of lignocellulose-degrading enzymes that may be suited for biomass degradation. However, its hydrolytic enzymes are specifically expressed only during plant infection. Here, we provide the proof of principle that these intrinsic enzymes can be synthetically activated during the industrially relevant yeast-like growth. The fungus is known to naturally synthesize valuable compounds, such as itaconate or glycolipids. Therefore, it could be suited for use in a consolidated bioprocess in which more complex and natural substrates are simultaneously converted to fermentable sugars and to value-added compounds in the future.

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SIGNIFICANCE OF ANTIBIOTIC PRODUCTION BY CEPHALOSPORIUM GRAMINEUM TO ITS SAPROPHYTIC SURVIVAL

Canadian Journal of Plant Science ◽

10.4141/cjps69-044 ◽

1969 ◽

Vol 49 (3) ◽

pp. 235-246 ◽

Cited By ~ 25

Author(s):

G. W. Bruehl ◽

R. L. Millar ◽

Barry Cunfer

Keyword(s):

Mycelial Growth ◽

Selective Pressure ◽

Wide Spectrum ◽

Antibiotic Production ◽

Selective Advantage ◽

Potato Dextrose Agar ◽

Selective Force ◽

Single Spore ◽

Cephalosporium Gramineum ◽

Parasitic Phase

Of several hundred isolates of Cephalosporium gramineum from nature, all produced a wide-spectrum, antifungal antibiotic. In contrast, certain single-spore isolates from cultures maintained at 6 °C for 2–5 years on potato dextrose agar produced little or no antibiotic. This suggests that in nature a selective force acts to preserve antibiotic producers and to eliminate nonproducers.Selection pressure in favor of antibiotic producers apparently is not exerted in the parasitic phase, because both antibiotic producers and nonproducers are pathogenic. Mycelial growth rates do not give a selective advantage to antibiotic producers as they usually grow more slowly than do nonproducers. The antibiotic itself does not exert selective pressure on nonproducers since nonproducers are not inhibited in culture by producers.Antibiotic producers established in straw reduce the invasion of the straw in soil by other fungi more than do the nonproducers. These observations support the hypothesis that the antibiotic aids the survival of C. gramineum in its saprophytic phases, and that selection pressure in favor of antibiotic producers is probably exerted while the fungus is in the soil.

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Characterization of Conserved and Novel Septal Factors inMycobacterium smegmatis

Journal of Bacteriology ◽

10.1128/jb.00649-17 ◽

2018 ◽

Vol 200 (6) ◽

Cited By ~ 13

Author(s):

Katherine J. Wu ◽

Jenna Zhang ◽

Catherine Baranowski ◽

Vivian Leung ◽

E. Hesper Rego ◽

...

Keyword(s):

Escherichia Coli ◽

Cell Wall ◽

Model Organisms ◽

Wall Structure ◽

Cross Wall ◽

Content Type ◽

A Cell ◽

Mediate Cell ◽

Mycobacterial Cell Wall ◽

Regulatory Functions

ABSTRACTSeptation in bacteria requires coordinated regulation of cell wall biosynthesis and hydrolysis enzymes so that new septal cross-wall can be appropriately constructed without compromising the integrity of the existing cell wall. Bacteria with different modes of growth and different types of cell wall require different regulators to mediate cell growth and division processes. Mycobacteria have both a cell wall structure and a mode of growth that are distinct from well-studied model organisms and use several different regulatory mechanisms. Here, usingMycobacterium smegmatis, we identify and characterize homologs of the conserved cell division regulators FtsL and FtsB, and show that they appear to function similarly to their homologs inEscherichia coli. We identify a number of previously undescribed septally localized factors which could be involved in cell wall regulation. One of these, SepIVA, has a DivIVA domain, is required for mycobacterial septation, and is localized to the septum and the intracellular membrane domain. We propose that SepIVA is a regulator of cell wall precursor enzymes that contribute to construction of the septal cross-wall, similar to the putative elongation function of the other mycobacterial DivIVA homolog, Wag31.IMPORTANCEThe enzymes that build bacterial cell walls are essential for cell survival but can cause cell lysis if misregulated; thus, their regulators are also essential. The number and nature of these regulators is likely to vary in bacteria that grow in different ways. The mycobacteria are a genus that have a cell wall whose composition and construction vary greatly from those of well-studied model organisms. In this work, we identify and characterize some of the proteins that regulate the mycobacterial cell wall. We find that some of these regulators appear to be functionally conserved with their structural homologs in evolutionarily distant species such asEscherichia coli, but other proteins have critical regulatory functions that may be unique to the actinomycetes.

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Ser/Thr Kinase-Dependent Phosphorylation of the Peptidoglycan Hydrolase CwlA Controls Its Export and Modulates Cell Division in Clostridioides difficile

mBio ◽

10.1128/mbio.00519-21 ◽

2021 ◽

Vol 12 (3) ◽

Author(s):

Transito Garcia-Garcia ◽

Sandrine Poncet ◽

Elodie Cuenot ◽

Thibaut Douché ◽

Quentin Giai Gianetto ◽

...

Keyword(s):

Cell Wall ◽

Cell Division ◽

Cell Separation ◽

Hydrolytic Enzymes ◽

Peptidoglycan Hydrolase ◽

Antimicrobial Compounds ◽

Bacterial Cells ◽

Cell Wall Metabolism ◽

Content Type ◽

Clostridioides Difficile

ABSTRACT Cell growth and division require a balance between synthesis and hydrolysis of the peptidoglycan (PG). Inhibition of PG synthesis or uncontrolled PG hydrolysis can be lethal for the cells, making it imperative to control peptidoglycan hydrolase (PGH) activity. The synthesis or activity of several key enzymes along the PG biosynthetic pathway is controlled by the Hanks-type serine/threonine kinases (STKs). In Gram-positive bacteria, inactivation of genes encoding STKs is associated with a range of phenotypes, including cell division defects and changes in cell wall metabolism, but only a few kinase substrates and associated mechanisms have been identified. We previously demonstrated that STK-PrkC plays an important role in cell division, cell wall metabolism, and resistance to antimicrobial compounds in the human enteropathogen Clostridioides difficile. In this work, we characterized a PG hydrolase, CwlA, which belongs to the NlpC/P60 family of endopeptidases and hydrolyses cross-linked PG between daughter cells to allow cell separation. We identified CwlA as the first PrkC substrate in C. difficile. We demonstrated that PrkC-dependent phosphorylation inhibits CwlA export, thereby controlling hydrolytic activity in the cell wall. High levels of CwlA at the cell surface led to cell elongation, whereas low levels caused cell separation defects. Thus, we provided evidence that the STK signaling pathway regulates PGH homeostasis to precisely control PG hydrolysis during cell division. IMPORTANCE Bacterial cells are encased in a PG exoskeleton that helps to maintain cell shape and confers physical protection. To allow bacterial growth and cell separation, PG needs to be continuously remodeled by hydrolytic enzymes that cleave PG at critical sites. How these enzymes are regulated remains poorly understood. We identify a new PG hydrolase involved in cell division, CwlA, in the enteropathogen C. difficile. Lack or accumulation of CwlA at the bacterial surface is responsible for a division defect, while its accumulation in the absence of PrkC also increases susceptibility to antimicrobial compounds targeting the cell wall. CwlA is a substrate of the kinase PrkC in C. difficile. PrkC-dependent phosphorylation controls the export of CwlA, modulating its levels and, consequently, its activity in the cell wall. This work provides a novel regulatory mechanism by STK in tightly controlling protein export.

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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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