Repression of Salmonella Host Cell Invasion by Aromatic Small Molecules from the Human Fecal Metabolome

ABSTRACT The human microbiome is a collection of microorganisms that inhabit every surface of the body that is exposed to the environment, generally coexisting peacefully with their host. These microbes have important functions, such as producing vitamins, aiding in maturation of the immune system, and protecting against pathogens. We have previously shown that a small-molecule extract from the human fecal microbiome has a strong repressive effect on Salmonella enterica serovar Typhimurium host cell invasion by modulating the expression of genes involved in this process. Here, we describe the characterization of this biological activity. Using a series of purification methods, we obtained fractions with biological activity and characterized them by mass spectrometry. These experiments revealed an abundance of aromatic compounds in the bioactive fraction. Selected compounds were obtained from commercial sources and tested with respect to their ability to repress the expression of hilA, the gene encoding the master regulator of invasion genes in Salmonella. We found that the aromatic compound 3,4-dimethylbenzoic acid acts as a strong inhibitor of hilA expression and of invasion of cultured host cells by Salmonella. Future studies should reveal the molecular details of this phenomenon, such as the signaling cascades involved in sensing this bioactive molecule. IMPORTANCE Microbes constantly sense and adapt to their environment. Often, this is achieved through the production and sensing of small extracellular molecules. The human body is colonized by complex communities of microbes, and, given their biological and chemical diversity, these ecosystems represent a platform where the production and sensing of molecules occur. In previous work, we showed that small molecules produced by microbes from the human gut can significantly impair the virulence of the enteric pathogen Salmonella enterica. Here, we describe a specific compound from the human gut that produces this same effect. The results from this work not only shed light on an important biological phenomenon occurring in our bodies but also may represent an opportunity to develop drugs that can target these small-molecule interactions to protect us from enteric infections and other diseases.

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Differences in Host Cell Invasion and Salmonella Pathogenicity Island 1 Expression between Salmonella enterica Serovar Paratyphi A and NontyphoidalS. Typhimurium

Infection and Immunity ◽

10.1128/iai.01461-15 ◽

2016 ◽

Vol 84 (4) ◽

pp. 1150-1165 ◽

Cited By ~ 13

Author(s):

Dana Elhadad ◽

Prerak Desai ◽

Guntram A. Grassl ◽

Michael McClelland ◽

Galia Rahav ◽

...

Keyword(s):

Host Cell ◽

Cell Invasion ◽

Salmonella Enterica ◽

Intestinal Inflammation ◽

Pathogenicity Island ◽

Effector Proteins ◽

Host Cells ◽

Host Cell Invasion ◽

Content Type ◽

Microaerobic Conditions

Active invasion into nonphagocytic host cells is central toSalmonella entericapathogenicity and dependent on multiple genes withinSalmonellapathogenicity island 1 (SPI-1). Here, we explored the invasion phenotype and the expression of SPI-1 in the typhoidal serovarS. Paratyphi A compared to that of the nontyphoidal serovarS. Typhimurium. We demonstrate that whileS. Typhimurium is equally invasive under both aerobic and microaerobic conditions,S. Paratyphi A invades only following growth under microaerobic conditions. Transcriptome sequencing (RNA-Seq), reverse transcription-PCR (RT-PCR), Western blot, and secretome analyses established thatS. Paratyphi A expresses much lower levels of SPI-1 genes and secretes lesser amounts of SPI-1 effector proteins thanS. Typhimurium, especially under aerobic growth. Bypassing the native SPI-1 regulation by inducible expression of the SPI-1 activator, HilA, considerably elevated SPI-1 gene expression, host cell invasion, disruption of epithelial integrity, and induction of proinflammatory cytokine secretion byS. Paratyphi A but not byS. Typhimurium, suggesting that SPI-1 expression is naturally downregulated inS. Paratyphi A. Using streptomycin-treated mice, we were able to establish substantial intestinal colonization byS. Paratyphi A and showed moderately higher pathology and intestinal inflammation in mice infected withS. Paratyphi A overexpressinghilA. Collectively, our results reveal unexpected differences in SPI-1 expression betweenS. Paratyphi A andS. Typhimurium, indicate thatS. Paratyphi A host cell invasion is suppressed under aerobic conditions, and suggest that lower invasion in aerobic sites and suppressed expression of immunogenic SPI-1 components contributes to the restrained inflammatory infection elicited byS. Paratyphi A.

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Small-molecule inhibition of a depalmitoylase enhances Toxoplasma host-cell invasion

Nature Chemical Biology ◽

10.1038/nchembio.1315 ◽

2013 ◽

Vol 9 (10) ◽

pp. 651-656 ◽

Cited By ~ 40

Author(s):

Matthew A Child ◽

Carolyn I Hall ◽

Josh R Beck ◽

Leslie O Ofori ◽

Victoria E Albrow ◽

...

Keyword(s):

Host Cell ◽

Small Molecule ◽

Cell Invasion ◽

Host Cell Invasion ◽

Small Molecule Inhibition

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Protein Kinase A Is Essential for Invasion of Plasmodium falciparum into Human Erythrocytes

mBio ◽

10.1128/mbio.01972-19 ◽

2019 ◽

Vol 10 (5) ◽

Cited By ~ 8

Author(s):

Mary-Louise Wilde ◽

Tony Triglia ◽

Danushka Marapana ◽

Jennifer K. Thompson ◽

Alexei A. Kouzmitchev ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Protein Kinase ◽

Protein Kinase A ◽

Host Cell ◽

Cell Invasion ◽

Blood Cells ◽

Host Cell Invasion ◽

Parasite Invasion ◽

Content Type ◽

Kinase A

ABSTRACT Understanding the mechanisms behind host cell invasion by Plasmodium falciparum remains a major hurdle to developing antimalarial therapeutics that target the asexual cycle and the symptomatic stage of malaria. Host cell entry is enabled by a multitude of precisely timed and tightly regulated receptor-ligand interactions. Cyclic nucleotide signaling has been implicated in regulating parasite invasion, and an important downstream effector of the cAMP-signaling pathway is protein kinase A (PKA), a cAMP-dependent protein kinase. There is increasing evidence that P. falciparum PKA (PfPKA) is responsible for phosphorylation of the cytoplasmic domain of P. falciparum apical membrane antigen 1 (PfAMA1) at Ser610, a cAMP-dependent event that is crucial for successful parasite invasion. In the present study, CRISPR-Cas9 and conditional gene deletion (dimerizable cre) technologies were implemented to generate a P. falciparum parasite line in which expression of the catalytic subunit of PfPKA (PfPKAc) is under conditional control, demonstrating highly efficient dimerizable Cre recombinase (DiCre)-mediated gene excision and complete knockdown of protein expression. Parasites lacking PfPKAc show severely reduced growth after one intraerythrocytic growth cycle and are deficient in host cell invasion, as highlighted by live-imaging experiments. Furthermore, PfPKAc-deficient parasites are unable to phosphorylate PfAMA1 at Ser610. This work not only identifies an essential role for PfPKAc in the P. falciparum asexual life cycle but also confirms that PfPKAc is the kinase responsible for phosphorylating PfAMA1 Ser610. IMPORTANCE Malaria continues to present a major global health burden, particularly in low-resource countries. Plasmodium falciparum, the parasite responsible for the most severe form of malaria, causes disease through rapid and repeated rounds of invasion and replication within red blood cells. Invasion into red blood cells is essential for P. falciparum survival, and the molecular events mediating this process have gained much attention as potential therapeutic targets. With no effective vaccine available, and with the emergence of resistance to antimalarials, there is an urgent need for the development of new therapeutics. Our research has used genetic techniques to provide evidence of an essential protein kinase involved in P. falciparum invasion. Our work adds to the current understanding of parasite signaling processes required for invasion, highlighting PKA as a potential drug target to inhibit invasion for the treatment of malaria.

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Dissecting the Gene Expression, Localization, Membrane Topology, and Function of the Plasmodium falciparum STEVOR Protein Family

mBio ◽

10.1128/mbio.01500-19 ◽

2019 ◽

Vol 10 (4) ◽

Cited By ~ 4

Author(s):

J. Stephan Wichers ◽

Judith A. M. Scholz ◽

Jan Strauss ◽

Susanne Witt ◽

Andrés Lill ◽

...

Keyword(s):

Gene Expression ◽

Plasmodium Falciparum ◽

Plasma Membrane ◽

Host Cell ◽

Cell Invasion ◽

Surface Antigens ◽

Membrane Topology ◽

Host Cell Invasion ◽

Content Type ◽

Variant Surface Antigens

ABSTRACT During its intraerythrocytic development, the malaria parasite Plasmodium falciparum exposes variant surface antigens (VSAs) on infected erythrocytes to establish and maintain an infection. One family of small VSAs is the polymorphic STEVOR proteins, which are marked for export to the host cell surface through their PEXEL signal peptide. Interestingly, some STEVORs have also been reported to localize to the parasite plasma membrane and apical organelles, pointing toward a putative function in host cell egress or invasion. Using deep RNA sequencing analysis, we characterized P. falciparum stevor gene expression across the intraerythrocytic development cycle, including free merozoites, in detail and used the resulting stevor expression profiles for hierarchical clustering. We found that most stevor genes show biphasic expression oscillation, with maximum expression during trophozoite stages and a second peak in late schizonts. We selected four STEVOR variants, confirmed the expected export of these proteins to the host cell membrane, and tracked them to a secondary location, either to the parasite plasma membrane or the secretory organelles of merozoites in late schizont stages. We investigated the function of a particular STEVOR that showed rhoptry localization and demonstrated its role at the parasite-host interface during host cell invasion by specific antisera and targeted gene disruption. Experimentally determined membrane topology of this STEVOR revealed a single transmembrane domain exposing the semiconserved as well as variable protein regions to the cell surface. IMPORTANCE Malaria claims about half a million lives each year. Plasmodium falciparum, the causative agent of the most severe form of the disease, uses proteins that are translocated to the surface of infected erythrocytes for immune evasion. To circumvent the detection of these gene products by the immune system, the parasite evolved a complex strategy that includes gene duplications and elaborate sequence polymorphism. STEVORs are one family of these variant surface antigens and are encoded by about 40 genes. Using deep RNA sequencing of blood-stage parasites, including free merozoites, we first established stevor expression of the cultured isolate and compared it with published transcriptomes. We reveal a biphasic expression of most stevor genes and confirm this for individual STEVORs at the protein level. The membrane topology of a rhoptry-associated variant was experimentally elucidated and linked to host cell invasion, underlining the importance of this multifunctional protein family for parasite proliferation.

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Barcoded Consortium Infections Resolve Cell Type-Dependent Salmonella enterica Serovar Typhimurium Entry Mechanisms

mBio ◽

10.1128/mbio.00603-19 ◽

2019 ◽

Vol 10 (3) ◽

Cited By ~ 2

Author(s):

Maria Letizia Di Martino ◽

Viktor Ek ◽

Wolf-Dietrich Hardt ◽

Jens Eriksson ◽

Mikael E. Sellin

Keyword(s):

Host Cell ◽

Salmonella Enterica ◽

Cell Types ◽

Secretion System ◽

Host Cells ◽

Host Cell Invasion ◽

Cell Type ◽

Content Type ◽

Invasion Mechanisms ◽

Serovar Typhimurium

ABSTRACT Bacterial host cell invasion mechanisms depend on the bacterium’s virulence factors and the properties of the target cell. The enteropathogen Salmonella enterica serovar Typhimurium (S.Tm) invades epithelial cell types in the gut mucosa and a variety of immune cell types at later infection stages. The molecular mechanism(s) of host cell entry has, however, been studied predominantly in epithelial cell lines. S.Tm uses a type three secretion system (TTSS-1) to translocate effectors into the host cell cytosol, thereby sparking actin ruffle-dependent entry. The ruffles also fuel cooperative invasion by bystander bacteria. In addition, several TTSS-1-independent entry mechanisms exist, involving alternative S.Tm virulence factors, or the passive uptake of bacteria by phagocytosis. However, it remains ill-defined how S.Tm invasion mechanisms vary between host cells. Here, we developed an internally controlled and scalable method to map S.Tm invasion mechanisms across host cell types and conditions. The method relies on host cell infections with consortia of chromosomally tagged wild-type and mutant S.Tm strains, where the abundance of each strain can be quantified by qPCR or amplicon sequencing. Using this methodology, we quantified cooccurring TTSS-1-dependent, cooperative, and TTSS-1-independent invasion events in epithelial, monocyte, and macrophage cells. We found S.Tm invasion of epithelial cells and monocytes to proceed by a similar MOI-dependent mix of TTSS-1-dependent and cooperative mechanisms. TTSS-1-independent entry was more frequent in macrophages. Still, TTSS-1-dependent invasion dominated during the first minutes of interaction also with this cell type. Finally, the combined action of the SopB/SopE/SopE2 effectors was sufficient to explain TTSS-1-dependent invasion across both epithelial and phagocytic cells. IMPORTANCE Salmonella enterica serovar Typhimurium (S.Tm) is a widespread and broad-host-spectrum enteropathogen with the capacity to invade diverse cell types. Still, the molecular basis for the host cell invasion process has largely been inferred from studies of a few selected cell lines. Our work resolves the mechanisms that Salmonellae employ to invade prototypical host cell types, i.e., human epithelial, monocyte, and macrophage cells, at a previously unattainable level of temporal and quantitative precision. This highlights efficient bacterium-driven entry into innate immune cells and uncovers a type III secretion system effector module that dominates active bacterial invasion of not only epithelial cells but also monocytes and macrophages. The results are derived from a generalizable method, where we combine barcoding of the bacterial chromosome with mixed consortium infections of cultured host cells. The application of this methodology across bacterial species and infection models will provide a scalable means to address host-pathogen interactions in diverse contexts.

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A Member of the Ferlin Calcium Sensor Family Is Essential for Toxoplasma gondii Rhoptry Secretion

mBio ◽

10.1128/mbio.01510-18 ◽

2018 ◽

Vol 9 (5) ◽

Cited By ~ 12

Author(s):

Bradley I. Coleman ◽

Sudeshna Saha ◽

Seiko Sato ◽

Klemens Engelberg ◽

David J. P. Ferguson ◽

...

Keyword(s):

Toxoplasma Gondii ◽

Host Cell ◽

Cell Invasion ◽

Host Cells ◽

Model Organisms ◽

Host Cell Invasion ◽

Content Type ◽

Calcium Dependent ◽

Calcium Sensing ◽

Intracellular Ca

ABSTRACT Invasion of host cells by apicomplexan parasites such as Toxoplasma gondii is critical for their infectivity and pathogenesis. In Toxoplasma, secretion of essential egress, motility, and invasion-related proteins from microneme organelles is regulated by oscillations of intracellular Ca2+. Later stages of invasion are considered Ca2+ independent, including the secretion of proteins required for host cell entry and remodeling from the parasite’s rhoptries. We identified a family of three Toxoplasma proteins with homology to the ferlin family of double C2 domain-containing Ca2+ sensors. In humans and model organisms, such Ca2+ sensors orchestrate Ca2+-dependent exocytic membrane fusion with the plasma membrane. Here we focus on one ferlin that is conserved across the Apicomplexa, T. gondii FER2 (TgFER2). Unexpectedly, conditionally TgFER2-depleted parasites secreted their micronemes normally and were completely motile. However, these parasites were unable to invade host cells and were therefore not viable. Knockdown of TgFER2 prevented rhoptry secretion, and these parasites failed to form the moving junction at the parasite-host interface necessary for host cell invasion. Collectively, these data demonstrate the requirement of TgFER2 for rhoptry secretion in Toxoplasma tachyzoites and suggest a possible Ca2+ dependence of rhoptry secretion. These findings provide the first mechanistic insights into this critical yet poorly understood aspect of apicomplexan host cell invasion. IMPORTANCE Apicomplexan protozoan parasites, such as those causing malaria and toxoplasmosis, must invade the cells of their hosts in order to establish a pathogenic infection. Timely release of proteins from a series of apical organelles is required for invasion. Neither the vesicular fusion events that underlie secretion nor the observed reliance of the various processes on changes in intracellular calcium concentrations is completely understood. We identified a group of three proteins with strong homology to the calcium-sensing ferlin family, which are known to be involved in protein secretion in other organisms. Surprisingly, decreasing the amounts of one of these proteins (TgFER2) did not have any effect on the typically calcium-dependent steps in invasion. Instead, TgFER2 was essential for the release of proteins from organelles called rhoptries. These data provide a tantalizing first look at the mechanisms controlling the very poorly understood process of rhoptry secretion, which is essential for the parasite’s infection cycle.

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Structural Characterization of Acidic M17 Leucine Aminopeptidases from the TriTryps and Evaluation of Their Role in Nutrient Starvation in Trypanosoma brucei

mSphere ◽

10.1128/msphere.00226-17 ◽

2017 ◽

Vol 2 (4) ◽

Cited By ~ 8

Author(s):

Jennifer Timm ◽

Maria Valente ◽

Daniel García-Caballero ◽

Keith S. Wilson ◽

Dolores González-Pacanowska

Keyword(s):

Homologous Recombination ◽

Host Cell ◽

Trypanosoma Brucei ◽

Cell Invasion ◽

Essential Amino Acids ◽

Host Cell Invasion ◽

Content Type ◽

Hydrolysis Of

ABSTRACT Leucine aminopeptidases (LAPs) catalyze the hydrolysis of the N-terminal amino acid of peptides and are considered potential drug targets. They are involved in multiple functions ranging from host cell invasion and provision of essential amino acids to site-specific homologous recombination and transcription regulation. In kinetoplastid parasites, there are at least three distinct LAPs. The availability of the crystal structures provides important information for drug design. Here we report the structure of the acidic LAPs from three kinetoplastids in complex with different inhibitors and explore their role in Trypanosoma brucei survival under various nutrient conditions. Importantly, the acidic LAP is dispensable for growth both in vitro and in vivo, an observation that questions its use as a specific drug target. While LAP-A is not essential, leucine depletion and subcellular localization studies performed under starvation conditions suggest a possible function of LAP-A in the response to nutrient restriction. Leucine aminopeptidase (LAP) is found in all kingdoms of life and catalyzes the metal-dependent hydrolysis of the N-terminal amino acid residue of peptide or amino acyl substrates. LAPs have been shown to participate in the N-terminal processing of certain proteins in mammalian cells and in homologous recombination and transcription regulation in bacteria, while in parasites, they are involved in host cell invasion and provision of essential amino acids for growth. The enzyme is essential for survival in Plasmodium falciparum, where its drug target potential has been suggested. We report here the X-ray structures of three kinetoplastid acidic LAPs (LAP-As from Trypanosoma brucei, Trypanosoma cruzi, and Leishmania major) which were solved in the metal-free and unliganded forms, as well as in a number of ligand complexes, providing insight into ligand binding, metal ion requirements, and oligomeric state. In addition, we analyzed mutant cells defective in LAP-A in Trypanosoma brucei, strongly suggesting that the enzyme is not required for the growth of this parasite either in vitro or in vivo. In procyclic cells, LAP-A was equally distributed throughout the cytoplasm, yet upon starvation, it relocalizes in particles that concentrate in the perinuclear region. Overexpression of the enzyme conferred a growth advantage when parasites were grown in leucine-deficient medium. Overall, the results suggest that in T. brucei, LAP-A may participate in protein degradation associated with nutrient depletion. IMPORTANCE Leucine aminopeptidases (LAPs) catalyze the hydrolysis of the N-terminal amino acid of peptides and are considered potential drug targets. They are involved in multiple functions ranging from host cell invasion and provision of essential amino acids to site-specific homologous recombination and transcription regulation. In kinetoplastid parasites, there are at least three distinct LAPs. The availability of the crystal structures provides important information for drug design. Here we report the structure of the acidic LAPs from three kinetoplastids in complex with different inhibitors and explore their role in Trypanosoma brucei survival under various nutrient conditions. Importantly, the acidic LAP is dispensable for growth both in vitro and in vivo, an observation that questions its use as a specific drug target. While LAP-A is not essential, leucine depletion and subcellular localization studies performed under starvation conditions suggest a possible function of LAP-A in the response to nutrient restriction.

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Flagellin Is Required for Host Cell Invasion and Normal Salmonella Pathogenicity Island 1 Expression by Salmonella enterica Serovar Paratyphi A

Infection and Immunity ◽

10.1128/iai.00468-15 ◽

2015 ◽

Vol 83 (9) ◽

pp. 3355-3368 ◽

Cited By ~ 29

Author(s):

Dana Elhadad ◽

Prerak Desai ◽

Galia Rahav ◽

Michael McClelland ◽

Ohad Gal-Mor

Keyword(s):

Host Cell ◽

Cell Invasion ◽

Salmonella Enterica ◽

Pathogenicity Island ◽

Dependent Manner ◽

Content Type ◽

Type Three Secretion System ◽

Serovar Typhimurium ◽

System 1 ◽

Type Three Secretion

Salmonella entericaserovar Paratyphi A is a human-specific serovar that, together withSalmonella entericaserovar Typhi andSalmonella entericaserovar Sendai, causes enteric fever. Unlike the nontyphoidalSalmonella entericaserovar Typhimurium, the genomes ofS. Typhi andS. Paratyphi A are characterized by inactivation of multiple genes, including in the flagellum-chemotaxis pathway. Here, we explored the motility phenotype ofS. Paratyphi A and the role of flagellin in key virulence-associated phenotypes. Motility studies established that the human-adapted typhoidalS. Typhi,S. Paratyphi A, andS. Sendai are all noticeably less motile thanS. Typhimurium, and comparative transcriptome sequencing (RNA-Seq) showed that inS. Paratyphi A, the entire motility-chemotaxis regulon is expressed at significantly lowers levels than inS. Typhimurium. Nevertheless,S. Paratyphi A, likeS. Typhimurium, requires a functional flagellum for epithelial cell invasion and macrophage uptake, probably in a motility-independent mechanism. In contrast, flagella were found to be dispensable for host cell adhesion. Moreover, we demonstrate that inS. Paratyphi A, but not inS. Typhimurium, the lack of flagellin results in increased transcription of the flagellar and theSalmonellapathogenicity island 1 (SPI-1) regulons in a FliZ-dependent manner and in oversecretion of SPI-1 effectors via type three secretion system 1. Collectively, these results suggest a novel regulatory linkage between flagellin and SPI-1 inS. Paratyphi A that does not occur inS. Typhimurium and demonstrate curious distinctions in motility and the expression of the flagellum-chemotaxis regulon between these clinically relevant pathogens.

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Parasite-Mediated Remodeling of the Host Microfilament Cytoskeleton Enables Rapid Egress of Trypanosoma cruzi following Membrane Rupture

mBio ◽

10.1128/mbio.00988-21 ◽

2021 ◽

Author(s):

Eden R. Ferreira ◽

Alexis Bonfim-Melo ◽

Barbara A. Burleigh ◽

Jaime A. Costales ◽

Kevin M. Tyler ◽

...

Keyword(s):

Trypanosoma Cruzi ◽

Host Cell ◽

Cell Invasion ◽

Lytic Cycle ◽

Host Cells ◽

Host Cell Invasion ◽

High Quality ◽

Content Type ◽

Membrane Rupture

Understanding how Trypanosoma cruzi interacts with host cells has been transformed by high-quality studies that have examined in detail the mechanisms of T. cruzi host cell invasion. In contrast, little is known about the latter stages of the parasite’s lytic cycle: how parasites egress and thereby sustain round after round of infection.

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Functional Analysis of Rhomboid Proteases during Toxoplasma Invasion

mBio ◽

10.1128/mbio.01795-14 ◽

2014 ◽

Vol 5 (5) ◽

Cited By ~ 39

Author(s):

Bang Shen ◽

Jeffrey S. Buguliskis ◽

Tobie D. Lee ◽

L. David Sibley

Keyword(s):

Toxoplasma Gondii ◽

Host Cell ◽

Cell Invasion ◽

Gliding Motility ◽

Surface Proteins ◽

Host Cell Invasion ◽

Content Type ◽

Apicomplexan Parasites ◽

Transmembrane Segments ◽

Rhomboid Proteases

ABSTRACT Host cell invasion by Toxoplasma gondii and other apicomplexan parasites requires transmembrane adhesins that mediate binding to receptors on the substrate and host cell to facilitate motility and invasion. Rhomboid proteases (ROMs) are thought to cleave adhesins within their transmembrane segments, thus allowing the parasite to disengage from receptors and completely enter the host cell. To examine the specific roles of individual ROMs during invasion, we generated single, double, and triple knockouts for the three ROMs expressed in T. gondii tachyzoites. Analysis of these mutants demonstrated that ROM4 is the primary protease involved in adhesin processing and host cell invasion, whereas ROM1 or ROM5 plays negligible roles in these processes. Deletion of ROM4 blocked the shedding of adhesins such as MIC2 (microneme protein 2), causing them to accumulate on the surface of extracellular parasites. Increased surface adhesins led to nonproductive attachment, altered gliding motility, impaired moving junction formation, and reduced invasion efficiency. Despite the importance of ROM4 for efficient invasion, mutants lacking all three ROMs were viable and MIC2 was still efficiently removed from the surface of invaded mutant parasites, implying the existence of ROM-independent mechanisms for adhesin removal during invasion. Collectively, these results suggest that although ROM processing of adhesins is not absolutely essential, it is important for efficient host cell invasion by T. gondii. IMPORTANCE Apicomplexan parasites such as Toxoplasma gondii express surface proteins that bind host cell receptors to aid invasion. Many of these adhesins are subject to cleavage by rhomboid proteases (ROMs) within their transmembrane segments during invasion. Previous studies have demonstrated the importance of adhesin cleavage for parasite invasion and proposed that the ROMs responsible for processing would be essential for parasite survival. In T. gondii, ROM5 was thought to be the critical ROM for adhesin shedding due to its robust protease activity in vitro and posterior localization on the parasite surface. Here, we knocked out all three ROMs in T. gondii tachyzoites and found that ROM4, but not ROM5, was key for adhesin cleavage. However, none of the ROMs individually or in combination was essential for cell entry, further emphasizing that essential pathways such as invasion typically rely on redundant pathways to ensure survival.

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