A Novel Trichomonas vaginalis Surface Protein Modulates Parasite Attachment via Protein:Host Cell Proteoglycan Interaction

ABSTRACT Trichomonas vaginalis is a highly prevalent, sexually transmitted parasite which adheres to mucosal epithelial cells to colonize the human urogenital tract. Despite adherence being crucial for this extracellular parasite to thrive within the host, relatively little is known about the mechanisms or key molecules involved in this process. Here, we have identified and characterized a T. vaginalis hypothetical protein, TVAG_157210 (TvAD1), as a surface protein that plays an integral role in parasite adherence to the host. Quantitative proteomics revealed TvAD1 to be ∼4-fold more abundant in parasites selected for increased adherence (MA parasites) than the isogenic parental (P) parasite line. De novo modeling suggested that TvAD1 binds N-acetylglucosamine (GlcNAc), a sugar comprising host glycosaminoglycans (GAGs). Adherence assays utilizing GAG-deficient cell lines determined that host GAGs, primarily heparan sulfate (HS), mediate adherence of MA parasites to host cells. TvAD1 knockout (KO) parasites, generated using CRISPR-Cas9, were found to be significantly reduced in host cell adherence, a phenotype that is rescued by overexpression of TvAD1 in KO parasites. In contrast, there was no significant difference in parasite adherence to GAG-deficient lines by KO parasites compared with wild-type, which is contrary to that observed for KO parasites overexpressing TvAD1. Isothermal titration calorimetric (ITC) analysis showed that TvAD1 binds to HS, indicating that TvAD1 mediates host cell adherence via HS interaction. In addition to characterizing the role of TvAD1 in parasite adherence, these studies reveal a role for host GAG molecules in T. vaginalis adherence. IMPORTANCE The ability of the sexually transmitted parasite Trichomonas vaginalis to adhere to its human host is critical for establishing and maintaining an infection. Yet how parasites adhere to host cells is poorly understood. In this study, we employed a novel adherence selection method to identify proteins involved in parasite adherence to the host. This method led to the identification of a protein, with no previously known function, that is more abundant in parasites with increased capacity to bind host cells. Bioinformatic modeling and biochemical analyses revealed that this protein binds a common component on the host cell surface proteoglycans. Subsequent creation of parasites that lack this protein directly demonstrated that the protein mediates parasite adherence via an interaction with host cell proteoglycans. These findings both demonstrate a role for this protein in T. vaginalis adherence to the host and shed light on host cell molecules that participate in parasite colonization.

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A Novel Cadherin-like Protein Mediates Adherence to and Killing of Host Cells by the Parasite Trichomonas vaginalis

mBio ◽

10.1128/mbio.00720-19 ◽

2019 ◽

Vol 10 (3) ◽

Cited By ~ 2

Author(s):

Yi-Pei Chen ◽

Angelica M. Riestra ◽

Anand Kumar Rai ◽

Patricia J. Johnson

Keyword(s):

Cell Adhesion ◽

Binding Site ◽

Host Cell ◽

Calcium Binding ◽

Trichomonas Vaginalis ◽

Host Cells ◽

Wild Type ◽

Cell Adherence ◽

Calcium Binding Site ◽

Content Type

ABSTRACT Trichomonas vaginalis, a prevalent sexually transmitted parasite, adheres to and induces cytolysis of human mucosal epithelial cells. We have characterized a hypothetical protein, TVAG_393390, with predicted tertiary structure similar to that of mammalian cadherin proteins involved in cell-cell adherence. TVAG_393390, renamed cadherin-like protein (CLP), contains a calcium-binding site at a position conserved in cadherins. CLP is surface localized, and its mRNA and protein levels are significantly upregulated upon parasite adherence to host cells. To test the roles of CLP and its calcium-binding dependency during host cell adherence, we first demonstrated that wild-type CLP (CLP) binds calcium with a high affinity, whereas the calcium-binding site mutant protein (CLP-mut) does not. CLP and CLP-mut constructs were then used to overexpress these proteins in T. vaginalis. Parasites overexpressing CLP have ∼3.5-fold greater adherence to host cells than wild-type parasites, and this increased adherence is ablated by mutating the calcium-binding site. Additionally, competition with recombinant CLP decreased parasite binding to host cells. We also found that overexpression of CLP induced parasite aggregation which was further enhanced in the presence of calcium, whereas CLP-mut overexpression did not affect aggregation. Lastly, parasites overexpressing wild-type CLP induced killing of host cells ∼2.35-fold, whereas parasites overexpressing CLP-mut did not have this effect. These analyses describe the first parasitic CLP and demonstrate a role for this protein in mediating parasite-parasite and host-parasite interactions. T. vaginalis CLP may represent convergent evolution of a parasite protein that is functionally similar to the mammalian cell adhesion protein cadherin, which contributes to parasite pathogenesis. IMPORTANCE The adherence of pathogens to host cells is critical for colonization of the host and establishing infection. Here we identify a protein with no known function that is more abundant on the surface of parasites that are better at binding host cells. To interrogate a predicted function of this protein, we utilized bioinformatic protein prediction programs which allowed us to uncover the first cadherin-like protein (CLP) found in a parasite. Cadherin proteins are conserved metazoan proteins with central roles in cell-cell adhesion, development, and tissue structure maintenance. Functional characterization of this CLP from the unicellular parasite Trichomonas vaginalis demonstrated that the protein mediates both parasite-parasite and parasite-host adherence, which leads to an enhanced killing of host cells by T. vaginalis. Our findings demonstrate the presence of CLPs in unicellular pathogens and identify a new host cell binding protein family in a human-infective parasite.

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Candida albicans Als3, a Multifunctional Adhesin and Invasin

Eukaryotic Cell ◽

10.1128/ec.00279-10 ◽

2010 ◽

Vol 10 (2) ◽

pp. 168-173 ◽

Cited By ~ 159

Author(s):

Yaoping Liu ◽

Scott G. Filler

Keyword(s):

Candida Albicans ◽

Host Cell ◽

Surface Protein ◽

Host Cells ◽

Content Type ◽

Multifunctional Protein ◽

Multiple Functions ◽

Abiotic Surfaces ◽

Disseminated Disease

ABSTRACTCandida albicansis part of the normal human flora, and it grows on mucosal surfaces in healthy individuals. In susceptible hosts, this organism can cause both mucosal and hematogenously disseminated disease. ForC. albicansto persist in the host and induce disease, it must be able to adhere to biotic and abiotic surfaces, invade host cells, and obtain iron. TheC. albicanshypha-specific surface protein Als3 is a member of the agglutinin-like sequence (Als) family of proteins and is important in all of these processes. Functioning as an adhesin, Als3 mediates attachment to epithelial cells, endothelial cells, and extracellular matrix proteins. It also plays an important role in biofilm formation on prosthetic surfaces, both alone and in mixed infection withStreptococcus gordonii. Als3 is one of two knownC. albicansinvasins. It binds to host cell receptors such as E-cadherin and N-cadherin and thereby induces host cells to endocytose the organism. Als3 also binds to host cell ferritin and enablesC. albicansto utilize this protein as a source of iron. Because of its multiple functions and its high expression levelin vivo, Als3 is a promising target for vaccines that induce protective cell-mediated and antibody responses. This review will summarize the multiple functions of this interesting and multifunctional protein.

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Trichomonas vaginalis Polyamine Metabolism Is Linked to Host Cell Adherence and Cytotoxicity

Infection and Immunity ◽

10.1128/iai.73.5.2602-2610.2005 ◽

2005 ◽

Vol 73 (5) ◽

pp. 2602-2610 ◽

Cited By ~ 32

Author(s):

Ana F. Garcia ◽

M. Benchimol ◽

J. F. Alderete

Keyword(s):

Host Cell ◽

Trichomonas Vaginalis ◽

Cysteine Proteinase ◽

Growth Arrest ◽

Electron Microscopic Examination ◽

Host Cells ◽

Polyamine Metabolism ◽

Cell Adherence ◽

Electron Microscopic ◽

Control Versus

ABSTRACT Trichomonas vaginalis secretes putrescine that is readily detected in vaginal secretions. We wanted to examine the effect of decreased putrescine synthesis by inhibition of ornithine decarboxylase (ODC) on T. vaginalis. One reason is because inhibition of Tritrichomonas foetus ODC results in growth arrest, destruction of hydrogenosomes, and decreased amounts of hydrogenosomal enzymes. Treatment of T. vaginalis T016 with ≥20 mM 1,4-diamino-2-butanone (DAB) to inhibit ODC resulted in growth arrest, which was reversed by addition of exogenous putrescine. No similar reversal of growth arrest was achieved with the polyamines spermine or spermidine or with iron. Electron microscopic examination of control versus DAB-treated trichomonads did not reveal any adverse effects on the number and integrity of hydrogenosomes. Further, the adhesins AP65, AP51, and AP33 mediating binding to immortalized vaginal epithelial cells (VECs) share identity to enzymes of the hydrogenosome organelle, and there was no difference in amounts of adhesins between control versus DAB-treated T. vaginalis parasites. Likewise, similar patterns and extent of fluorescence were evident for the prominent AP65 adhesin. Surprisingly, DAB treatment increased by 4- to 20-fold above untreated trichomonads handled identically the level of adherence mediated by adhesins. Interestingly, the enhanced attachment to VECs was reversed by exogenous putrescine added to DAB-treated trichomonads. Equally noteworthy was that DAB-treated T. vaginalis with enhanced adherence did not possess the previously reported ability to kill host cells in a contact-dependent fashion mediated by cysteine proteinases, and total cysteine proteinase activity patterns were identical between control and DAB-treated trichomonads. Overall, these data suggest that polyamine metabolism and secreted putrescine are linked to host cell adherence and cytotoxicity.

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Microsporidia Interact with Host Cell Mitochondria via Voltage-Dependent Anion Channels Using Sporoplasm Surface Protein 1

mBio ◽

10.1128/mbio.01944-19 ◽

2019 ◽

Vol 10 (4) ◽

Cited By ~ 2

Author(s):

Bing Han ◽

Yanfen Ma ◽

Vincent Tu ◽

Tadakimi Tomita ◽

Joshua Mayoral ◽

...

Keyword(s):

Host Cell ◽

Parasitophorous Vacuole ◽

Surface Protein ◽

Host Cells ◽

Anion Channels ◽

Polar Tube ◽

Content Type ◽

Proteomic Approach ◽

Voltage Dependent ◽

Microsporidian Infection

ABSTRACT Microsporidia are opportunistic intracellular pathogens that can infect a wide variety of hosts ranging from invertebrates to vertebrates. During invasion, the microsporidian polar tube pushes into the host cell, creating a protective microenvironment, the invasion synapse, into which the sporoplasm extrudes. Within the synapse, the sporoplasm then invades the host cell, forming a parasitophorous vacuole (PV). Using a proteomic approach, we identified Encephalitozoon hellem sporoplasm surface protein 1 (EhSSP1), which localized to the surface of extruded sporoplasms. EhSSP1 was also found to interact with polar tube protein 4 (PTP4). Recombinant EhSSP1 (rEhSSP1) bound to human foreskin fibroblasts, and both anti-EhSSP1 and rEhSSP1 caused decreased levels of host cell invasion, suggesting that interaction of SSP1 with the host cell was involved in invasion. Coimmunoprecipitation (Co-IP) followed by proteomic analysis identified host cell voltage-dependent anion channels (VDACs) as EhSSP1 interacting proteins. Yeast two-hybrid assays demonstrated that EhSSP1 was able to interact with VDAC1, VDAC2, and VDAC3. rEhSSP1 colocalized with the host mitochondria which were associated with microsporidian PVs in infected cells. Transmission electron microscopy revealed that the outer mitochondrial membrane interacted with meronts and the PV membrane, mitochondria clustered around meronts, and the VDACs were concentrated at the interface of mitochondria and parasite. Knockdown of VDAC1, VDAC2, and VDAC3 in host cells resulted in significant decreases in the number and size of the PVs and a decrease in mitochondrial PV association. The interaction of EhSSP1 with VDAC probably plays an important part in energy acquisition by microsporidia via its role in the association of mitochondria with the PV. IMPORTANCE Microsporidia are important opportunistic human pathogens in immune-suppressed individuals, such as those with HIV/AIDS and recipients of organ transplants. The sporoplasm is critical for establishing microsporidian infection. Despite the biological importance of this structure for transmission, there is limited information about its structure and composition that could be targeted for therapeutic intervention. Here, we identified a novel E. hellem sporoplasm surface protein, EhSSP1, and demonstrated that it can bind to host cell mitochondria via host VDAC. Our data strongly suggest that the interaction between SSP1 and VDAC is important for the association of mitochondria with the parasitophorous vacuole during microsporidian infection. In addition, binding of SSP1 to the host cell is associated with the final steps of invasion in the invasion synapse.

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A Cell Surface Aggregation-Promoting Factor fromLactobacillus gasseriContributes to Inhibition ofTrichomonas vaginalisAdhesion to Human Vaginal Ectocervical Cells

Infection and Immunity ◽

10.1128/iai.00907-17 ◽

2018 ◽

Vol 86 (8) ◽

Cited By ~ 13

Author(s):

Niha Phukan ◽

Anna E. S. Brooks ◽

Augusto Simoes-Barbosa

Keyword(s):

Trichomonas Vaginalis ◽

Protozoan Parasite ◽

Surface Proteins ◽

Host Cells ◽

Protective Mechanisms ◽

Transmitted Infection ◽

Content Type ◽

Sexually Transmitted ◽

Vaginal Lactobacilli ◽

A Cell

ABSTRACTTrichomoniasis, a prevalent sexually transmitted infection, is commonly symptomatic in women. The causative agent isTrichomonas vaginalis, an extracellular protozoan parasite. The host-protective mechanisms and molecules of vaginal lactobacilli that counteract this pathogen are largely unknown. This study examines the inhibition promoted byLactobacillus gasseriagainst the adhesion ofT. vaginalisto host cells, a critical virulence aspect of this pathogen. We observed that the vaginal strainL. gasseriATCC 9857 is highly inhibitory by various contact-dependent mechanisms and that surface proteins are largely responsible for this inhibitory phenotype. We found that the aggregation-promoting factor APF-2 from these bacteria significantly contributes to inhibition of the adhesion ofT. vaginalisto human vaginal ectocervical cells. Understanding the molecules and mechanisms used by lactobacilli to protect the host againstT. vaginalismight help in the development of novel and specific therapeutic strategies that take advantage of the natural microbiota.

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Differential Expression and Characterization of a Member of the Mucin-Associated Surface Protein Family Secreted by Trypanosoma cruzi

Infection and Immunity ◽

10.1128/iai.05329-11 ◽

2011 ◽

Vol 79 (10) ◽

pp. 3993-4001 ◽

Cited By ~ 25

Author(s):

Luis Miguel De Pablos ◽

Gloria González González ◽

Jennifer Solano Parada ◽

Víctor Seco Hidalgo ◽

Isabel María Díaz Lozano ◽

...

Keyword(s):

Trypanosoma Cruzi ◽

Host Cell ◽

Surface Protein ◽

Surface Proteins ◽

Host Cells ◽

Content Type ◽

The Family ◽

Cell Cytosol ◽

Host Cell Cytosol

ABSTRACTWe describe the characterization, purification, expression, and location of a 52-kDa protein secreted during interaction between the metacyclic form ofTrypanosoma cruziand its target host cell. The protein, which we have named MASP52, belongs to the family of mucin-associated surface proteins (MASPs). The highest levels of expression of both the protein and mRNA occur during the metacyclic and bloodstream trypomastigote stages, the forms that infect the vertebrate host cells. The protein is located in the plasma membrane and in the flagellar pockets of the epimastigote, metacyclic, and trypomastigote forms and is secreted into the medium at the point of contact between the parasite and the cell membrane, as well as into the host-cell cytosol during the amastigote stage. IgG antibodies specific against a synthetic peptide corresponding to the catalytic zone of MASP52 significantly reduce the parasite's capacity to infect the host cells. Furthermore, when the protein is adsorbed onto inert particles of bentonite and incubated with a nonphagocytic cell culture, the particles are able to induce endocytosis in the cells, which seems to demonstrate that MASP52 plays a role in a process whereby the trypomastigote forms of the parasite invade the host cell.

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Initial Characterization of the Two ClpP Paralogs ofChlamydia trachomatisSuggests Unique Functionality for Each

Journal of Bacteriology ◽

10.1128/jb.00635-18 ◽

2018 ◽

Vol 201 (2) ◽

Cited By ~ 5

Author(s):

Nicholas A. Wood ◽

Krystal Y. Chung ◽

Amanda M. Blocker ◽

Nathalia Rodrigues de Almeida ◽

Martin Conda-Sheridan ◽

...

Keyword(s):

Host Cells ◽

Developmental Cycle ◽

Intracellular Bacteria ◽

Clp Protease ◽

Content Type ◽

Sexually Transmitted ◽

Obligate Intracellular ◽

Developmental Form ◽

Obligate Intracellular Bacteria

ABSTRACTMembers ofChlamydiaare obligate intracellular bacteria that differentiate between two distinct functional and morphological forms during their developmental cycle, elementary bodies (EBs) and reticulate bodies (RBs). EBs are nondividing small electron-dense forms that infect host cells. RBs are larger noninfectious replicative forms that develop within a membrane-bound vesicle, termed an inclusion. Given the unique properties of each developmental form of this bacterium, we hypothesized that the Clp protease system plays an integral role in proteomic turnover by degrading specific proteins from one developmental form or the other.Chlamydiaspp. have five uncharacterizedclpgenes,clpX,clpC, twoclpPparalogs, andclpB. In other bacteria, ClpC and ClpX are ATPases that unfold and feed proteins into the ClpP protease to be degraded, and ClpB is a deaggregase. Here, we focused on characterizing the ClpP paralogs. Transcriptional analyses and immunoblotting determined that these genes are expressed midcycle. Bioinformatic analyses of these proteins identified key residues important for activity. Overexpression of inactiveclpPmutants inChlamydiaspp. suggested independent function of each ClpP paralog. To further probe these differences, we determined interactions between the ClpP proteins using bacterial two-hybrid assays and native gel analysis of recombinant proteins. Homotypic interactions of the ClpP proteins, but not heterotypic interactions between the ClpP paralogs, were detected. Interestingly, protease activity of ClpP2, but not ClpP1, was detectedin vitro. This activity was stimulated by antibiotics known to activate ClpP, which also blocked chlamydial growth. Our data suggest the chlamydial ClpP paralogs likely serve distinct and critical roles in this important pathogen.IMPORTANCEChlamydia trachomatisis the leading cause of preventable infectious blindness and of bacterial sexually transmitted infections worldwide. Chlamydiae are developmentally regulated obligate intracellular pathogens that alternate between two functional and morphologic forms, with distinct repertoires of proteins. We hypothesize that protein degradation is a critical aspect to the developmental cycle. A key system involved in protein turnover in bacteria is the Clp protease system. Here, we characterized the two chlamydial ClpP paralogs by examining their expression inChlamydiaspp., their ability to oligomerize, and their proteolytic activity. This work will help understand the evolutionarily diverse Clp proteases in the context of intracellular organisms, which may aid in the study of other clinically relevant intracellular bacteria.

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BspA and Pmp proteins ofTrichomonas vaginalismediate adherence to host cells

10.1101/454884 ◽

2018 ◽

Author(s):

Maria R. Handrich ◽

Sriram G. Garg ◽

Ewen W. Sommerville ◽

Robert P. Hirt ◽

Sven B. Gould

Keyword(s):

Trichomonas Vaginalis ◽

Functional Characterization ◽

Surface Proteins ◽

Host Cells ◽

Protein Families ◽

Sexually Transmitted ◽

Trace Back ◽

Oral Pathogens ◽

Adhesion Performance

AbstractTrichomonas vaginalisis one of the most widespread, sexually transmitted pathogens. The infection involves a morphological switch from a free-swimming pyriform trophozoite to an amoeboid cell upon adhesion to host epithelial cells. While details on how the switch is induced and to what proteins of the host surface the parasite adheres remain poorly characterized, several surface proteins of the parasite itself have been identified as potential candidates. Among those are two expanded protein families that harbor domains that share similarity to functionally investigated surface proteins of prokaryotic oral pathogens; these are the BspA proteins of Bacteroidales and Spirochaetales, and the Pmp proteins of Chlamydiales. We sequenced the transcriptomes of five Trichomonads and screened for the presence of BspA and Pmp domain-containing proteins and tested the ability of individualT. vaginaliscandidates to mediate adhesion. Here we demonstrate that (i) BspA and Pmp domain-containing proteins are specifically expanded inT. vaginalisin comparison to other Trichomonads, and that (ii) individual proteins of both families have the ability to increase adhesion performance in a non-virulentT. vaginalisstrain andTetratrichomonas gallinarum, a parasite usually known to infect birds but not humans. Our results initiate the functional characterization of these two broadly distributed protein families, whose origin we trace back to the origin of Trichomonads themselves.

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The Helicobacter pylori Autotransporter ImaA Tempers the Bacterium's Interaction with α5β1 Integrin

Infection and Immunity ◽

10.1128/iai.00450-16 ◽

2016 ◽

Vol 85 (1) ◽

Cited By ~ 5

Author(s):

William E. Sause ◽

Daniela Keilberg ◽

Soufiane Aboulhouda ◽

Karen M. Ottemann

Keyword(s):

Helicobacter Pylori ◽

Host Cell ◽

Type Iv Secretion ◽

Host Cells ◽

Wild Type ◽

Content Type ◽

Type Iv ◽

H Pylori ◽

Α5β1 Integrin ◽

Cell Interface

ABSTRACT The human pathogen Helicobacter pylori uses the host receptor α5β1 integrin to trigger inflammation in host cells via its cag pathogenicity island (cag PAI) type IV secretion system (T4SS). Here, we report that the H. pylori ImaA protein (HP0289) decreases the action of the cag PAI T4SS via tempering the bacterium's interaction with α5β1 integrin. Previously, imaA-null mutants were found to induce an elevated inflammatory response that was dependent on the cag PAI T4SS; here we extend those findings to show that the elevated response is independent of the CagA effector protein. To understand how ImaA could be affecting cag PAI T4SS activity at the host cell interface, we utilized the Phyre structural threading program and found that ImaA has a region with remote homology to bacterial integrin-binding proteins. This region was required for ImaA function. Unexpectedly, we observed that imaA mutants bound higher levels of α5β1 integrin than wild-type H. pylori, an outcome that required the predicted integrin-binding homology region of ImaA. Lastly, we report that ImaA directly affected the amount of host cell β1 integrin but not other cellular integrins. Our results thus suggest a model in which H. pylori employs ImaA to regulate interactions between integrin and the T4SS and thus alter the host inflammatory strength.

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Toxoplasma gondii Development of Its Replicative Niche: in Its Host Cell and Beyond

Eukaryotic Cell ◽

10.1128/ec.00081-14 ◽

2014 ◽

Vol 13 (8) ◽

pp. 965-976 ◽

Cited By ~ 32

Author(s):

Ira J. Blader ◽

Anita A. Koshy

Keyword(s):

Toxoplasma Gondii ◽

Host Cell ◽

Host Cells ◽

Content Type ◽

Obligate Intracellular ◽

Cellular Processes ◽

High Prevalence ◽

Life Threatening ◽

Cell Processes ◽

Cellular Pathways

ABSTRACTIntracellular pathogens can replicate efficiently only after they manipulate and modify their host cells to create an environment conducive to replication. While diverse cellular pathways are targeted by different pathogens, metabolism, membrane and cytoskeletal architecture formation, and cell death are the three primary cellular processes that are modified by infections.Toxoplasma gondiiis an obligate intracellular protozoan that infects ∼30% of the world's population and causes severe and life-threatening disease in developing fetuses, in immune-comprised patients, and in certain otherwise healthy individuals who are primarily found in South America. The high prevalence ofToxoplasmain humans is in large part a result of its ability to modulate these three host cell processes. Here, we highlight recent work defining the mechanisms by whichToxoplasmainteracts with these processes. In addition, we hypothesize why some processes are modified not only in the infected host cell but also in neighboring uninfected cells.

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