Zhicheng Dou

CO2 Fixation Kinetics of Halothiobacillus neapolitanus Mutant Carboxysomes Lacking Carbonic Anhydrase Suggest the Shell Acts as a Diffusional Barrier for CO2

The widely accepted models for the role of carboxysomes in the carbon-concentrating mechanism of autotrophic bacteria predict the carboxysomal carbonic anhydrase to be a crucial component. The enzyme is thought to dehydrate abundant cytosolic bicarbonate and provide ribulose 1.5-bisphosphate carboxylase/oxygenase (RubisCO) sequestered within the carboxysome with sufficiently high concentrations of its substrate, CO2, to permit its efficient fixation onto ribulose 1,5-bisphosphate. In this study, structure and function of carboxysomes purified from wild type Halothiobacillus neapolitanus and from a high CO2-requiring mutant that is devoid of carboxysomal carbonic anhydrase were compared. The kinetic constants for the carbon fixation reaction confirmed the importance of a functional carboxysomal carbonic anhydrase for efficient catalysis by RubisCO. Furthermore, comparisons of the reaction in intact and broken microcompartments and by purified carboxysomal RubisCO implicated the protein shell of the microcompartment as impeding diffusion of CO2 into and out of the carboxysome interior.

Organization, Structure, and Assembly of α-Carboxysomes Determined by Electron Cryotomography of Intact Cells

Carboxysomes are polyhedral inclusion bodies that play a key role in autotrophic metabolism in many bacteria. Using electron cryotomography, we examined carboxysomes in their native states within intact cells of three chemolithoautotrophic bacteria. We found that carboxysomes generally cluster into distinct groups within the cytoplasm, often in the immediate vicinity of polyphosphate granules, and a regular lattice of density frequently connects granules to nearby carboxysomes. Small granular bodies were also seen within carboxysomes. These observations suggest a functional relationship between carboxysomes and polyphosphate granules. Carboxysomes exhibited greater size, shape, and compositional variability in cells than in purified preparations. Finally, we observed carboxysomes in various stages of assembly, as well as filamentous structures that we attribute to misassembled shell protein. Surprisingly, no more than one partial carboxysome was ever observed per cell. Based on these observations, we propose a model for carboxysome assembly in which the shell and the internal RuBisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase) lattice form simultaneously, likely guided by specific interactions between shell proteins and RuBisCOs.

Halothiobacillus neapolitanus Carboxysomes Sequester Heterologous and Chimeric RubisCO Species

Background The carboxysome is a bacterial microcompartment that consists of a polyhedral protein shell filled with ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO), the enzyme that catalyzes the first step of CO2 fixation via the Calvin-Benson-Bassham cycle. Methodology/Principal Findings To analyze the role of RubisCO in carboxysome biogenesis in vivo we have created a series of Halothiobacillus neapolitanus RubisCO mutants. We identified the large subunit of the enzyme as an important determinant for its sequestration into α-carboxysomes and found that the carboxysomes of H. neapolitanus readily incorporate chimeric and heterologous RubisCO species. Intriguingly, a mutant lacking carboxysomal RubisCO assembles empty carboxysome shells of apparently normal shape and composition. Conclusions/Significance These results indicate that carboxysome shell architecture is not determined by the enzyme they normally sequester. Our study provides, for the first time, clear evidence that carboxysome contents can be manipulated and suggests future nanotechnological applications that are based upon engineered protein microcompartments.

Toxoplasma gondii Ingests and Digests Host Cytosolic Proteins

ABSTRACT The protozoan parasite Toxoplasma gondii resides within a nonfusogenic vacuole during intracellular replication. Although the limiting membrane of this vacuole provides a protective barrier to acidification and degradation by lysosomal hydrolases, it also physically segregates the parasite from the host cytosol. Accordingly, it has been suggested that T. gondii acquires material from the host via membrane channels or transporters. The ability of the parasite to internalize macromolecules via endocytosis during intracellular replication has not been tested. Here, we show that Toxoplasma ingests host cytosolic proteins and digests them using cathepsin L and other proteases within its endolysosomal system. Ingestion was reduced in mutant parasites lacking an intravacuolar network of tubular membranes, implicating this apparatus as a possible conduit for trafficking to the parasite. Genetic ablation of proteins involved in the pathway is associated with diminished parasite replication and virulence attenuation. We show that both virulent type I and avirulent type II strain parasites ingest and digest host-derived protein, indicating that the pathway is not restricted to highly virulent strains. The findings provide the first definitive evidence that T. gondii internalizes proteins from the host during intracellular residence and suggest that protein digestion within the endolysosomal system of the parasite contributes to toxoplasmosis. IMPORTANCE Toxoplasma gondii causes significant disease in individuals with weak immune systems. Treatment options for this infection have drawbacks, creating a need to understand how this parasite survives within the cells it infects as a prelude to interrupting its survival strategies. This study reveals that T. gondii internalizes proteins from the cytoplasm of the cells it infects and degrades such proteins within a digestive compartment within the parasite. Disruption of proteins involved in the pathway reduced parasite replication and lessened disease severity. The identification of a novel parasite ingestion pathway opens opportunities to interfere with this process and improve the outcome of infection. Toxoplasma gondii causes significant disease in individuals with weak immune systems. Treatment options for this infection have drawbacks, creating a need to understand how this parasite survives within the cells it infects as a prelude to interrupting its survival strategies. This study reveals that T. gondii internalizes proteins from the cytoplasm of the cells it infects and degrades such proteins within a digestive compartment within the parasite. Disruption of proteins involved in the pathway reduced parasite replication and lessened disease severity. The identification of a novel parasite ingestion pathway opens opportunities to interfere with this process and improve the outcome of infection.

Advances in Understanding Carboxysome Assembly in Prochlorococcus and Synechococcus Implicate CsoS2 as a Critical Component

The marine Synechococcus and Prochlorococcus are the numerically dominant cyanobacteria in the ocean and important in global carbon fixation. They have evolved a CO2-concentrating-mechanism, of which the central component is the carboxysome, a self-assembling proteinaceous organelle. Two types of carboxysome, α and β, encapsulating form IA and form IB d-ribulose-1,5-bisphosphate carboxylase/oxygenase, respectively, differ in gene organization and associated proteins. In contrast to the β-carboxysome, the assembly process of the α-carboxysome is enigmatic. Moreover, an absolutely conserved α-carboxysome protein, CsoS2, is of unknown function and has proven recalcitrant to crystallization. Here, we present studies on the CsoS2 protein in three model organisms and show that CsoS2 is vital for α-carboxysome biogenesis. The primary structure of CsoS2 appears tripartite, composed of an N-terminal, middle (M)-, and C-terminal region. Repetitive motifs can be identified in the N- and M-regions. Multiple lines of evidence suggest CsoS2 is highly flexible, possibly an intrinsically disordered protein. Based on our results from bioinformatic, biophysical, genetic and biochemical approaches, including peptide array scanning for protein-protein interactions, we propose a model for CsoS2 function and its spatial location in the α-carboxysome. Analogies between the pathway for β-carboxysome biogenesis and our model for α-carboxysome assembly are discussed.

Cathepsin Proteases in Toxoplasma gondii

Cysteine proteases are important for the growth and survival of apicomplexan parasites that infect humans. The apicomplexan Toxoplasma gondii expresses five members of the C1 family of cysteine proteases, including one cathepsin L-like (TgCPL), one cathepsin B-like (TgCPB) and three cathepsin C-like (TgCPC1, 2 and 3) proteases. Recent genetic, biochemical and structural studies reveal that cathepsins function in microneme and rhoptry protein maturation, host cell invasion, replication and nutrient acquisition. here, we review the key features and roles of T. gondii cathepsins and discuss the therapeutic potential for specific inhibitor development.

Non-canonical maturation of two papain-family proteases in Toxoplasma gondii

Background: Toxoplasma gondii expresses several cathepsin proteases, but little is known about how they are activated. Results: Toxoplasma cathepsin B protease localizes to lysosome-like compartment, and its maturation requires a parasite cathepsin L protease. Conclusion: Maturation of Toxoplasma cathepsin B is unconventionally nonself-dependent. Significance: The unusual maturation of Toxoplasma cathepsin B suggests a novel mechanism of protease activation within the endolysosomal system. Proteases regulate key events during infection by the pervasive intracellular parasite Toxoplasma gondii. Understanding how parasite proteases mature from an inactive zymogen to an active enzyme is expected to inform new strategies for blocking their actions. Herein, we show that T. gondii cathepsin B protease (TgCPB) does not undergo self-maturation but instead requires the expression of a second papain-family cathepsin protease, TgCPL. Using recombinant enzymes we also show that TgCPL is capable of partially maturing TgCPB in vitro. Consistent with this interrelationship, antibodies with validated specificity detected TgCPB in the lysosome-like vacuolar compartment along with TgCPL. Our findings also establish that TgCPB does not localize to the rhoptries as previously reported. Accordingly, rhoptry morphology and rhoptry protein maturation are normal in TgCPB knock-out parasites. Finally, we show that although maturation of TgCPL is independent of TgCPB, it may involve an additional protease(s) in conjunction with self-maturation.

Toxoplasma depends on lysosomal consumption of autophagosomes for persistent infection.

Globally, nearly 2 billion people are infected with the intracellular protozoan Toxoplasma gondii1. This persistent infection can cause severe disease in immunocompromised people and is epidemiologically linked to major mental illnesses2 and cognitive impairment3. There are currently no options for curing this infection. The lack of effective therapeutics is due partly to a poor understanding of the essential pathways that maintain long-term infection. Although it is known that Toxoplasma replicates slowly within intracellular cysts demarcated with a cyst wall, precisely how it sustains itself and remodels organelles in this niche is unknown. Here, we identify a key role for proteolysis within the parasite lysosomal organelle (the vacuolar compartment or VAC) in turnover of autophagosomes and persistence during neural infection. We found that disrupting a VAC-localized cysteine protease compromised VAC digestive function and markedly reduced chronic infection. Death of parasites lacking the VAC protease was preceded by accumulation of undigested autophagosomes in the parasite cytoplasm. These findings suggest an unanticipated function for parasite lysosomal degradation in chronic infection, and identify an intrinsic role for autophagy in the T. gondii parasite and its close relatives. This work also identifies a key element of Toxoplasma persistence and suggests that VAC proteolysis is a prospective target for pharmacological development.

Microneme protein 5 regulates the activity of Toxoplasma subtilisin 1 by mimicking a subtilisin prodomain.

Background: TgSUB1 is a subtilisin protease that trims invasion proteins on the surface of Toxoplasma gondii. Results: TgMIC5 suppresses TgSUB1 activity and structurally mimics a subtilisin prodomain, suggesting a mechanism for inhibition. Conclusion: The C-terminal region of TgMIC5 is responsible for inhibition of TgSUB1. Significance: We identify a novel subtilisin propeptide mimic. Toxoplasma gondii is the model parasite of the phylum Apicomplexa, which contains obligate intracellular parasites of medical and veterinary importance. Apicomplexans invade host cells by a multistep process involving the secretion of adhesive microneme protein (MIC) complexes. The subtilisin protease TgSUB1 trims several MICs on the parasite surface to activate gliding motility and host invasion. Although a previous study showed that expression of the secretory protein TgMIC5 suppresses TgSUB1 activity, the mechanism was unknown. Here, we solve the three-dimensional structure of TgMIC5 by nuclear magnetic resonance (NMR), revealing that it mimics a subtilisin prodomain including a flexible C-terminal peptide that may insert into the subtilisin active site. We show that TgMIC5 is an almost 50-fold more potent inhibitor of TgSUB1 activity than the small molecule inhibitor N-[N-(N-acetyl-l-leucyl)-l-leucyl]-l-norleucine (ALLN). Moreover, we demonstrate that TgMIC5 is retained on the parasite plasma membrane via its physical interaction with the membrane-anchored TgSUB1.