The Guiton Lab
 
 
 
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    Overview of Toxoplasma Gondii

 
Toxoplasma gondii is pathogen of medical and veterinary importance, as it can infect humans and virtually all warm-blooded animals and replicate inside all nucleated cells. The majority T. gondii infection occurs following ingestion of water and vegetables contaminated with oocysts/sporozoites (blue) from environmental reservoirs as well as tissue cysts with bradyzoites (green) in meat from infected animals.

Following ingestion, gastric enzymes and bile degrade the cyst walls of the infective sporozoites or bradyzoites, releasing the parasites into the small intestine where they rapidly invade host cells. Once in the intracellular milieu, the invading parasite resides in a parasitophorous vacuole (PV) witH no to little replication before quickly undergoing a developmental switch to the tachyzoite form (red).

Tachyzoites rapidly replicate within the PV, exit from infected cells, and disseminate to other organs and tissues during the acute phase of infection. In response to unknown stimuli, possibly including the host’s immune response, tachyzoites convert back to the encysted bradyzoite form, establishing a chronic infection. In summary, T. gondii will interconvert between three developmental forms during infection in a single intermediate host: the non-replicating sporozoites, the rapidly growing tachyzoites (red), and the slowly dividing bradyzoites (green).

The life cycle is completed when an infected animal is eaten by a definitive feline host (e.g., a domestic cat preying on an infected mouse), which will shed the oocysts into the environment. Although infection with T. gondii is usually self-limiting in healthy adult humans, severe disease does sometimes occur in immunocompromised individuals, such as the fetus, patients with HIV and transplant patients.

There exists no effective therapy to treat a chronic infection. This presents serious challenges should a chronically infected individual become immunocompromised, as reactivation of tissue cysts can lead to severe complications, especially in HIV-AIDS and transplant patients.

 
 

RESEARCH PROJECTS

 
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Project 1:
Host/Pathogen Interactions

Immunofluorescence image depicting a Toxoplasma protein (green) secreted into the parasitophorous vacuole (left) and cartoon of Toxoplasma infection of a nucleated cell (right).here...

What are the effector proteins Toxoplasma uses when initiating infection in a new host?

Toxoplasma secretes a plethora of proteins into the host cells to modulate host processes, such as host immune responses and cell cycle, in order to ensure its survival and transmission. In this project, we will use a reverse genetic approach to specifically identify genes that encode developmental stage-specific secreted effector proteins. Such proteins are likely to be trafficked into the host cell and operate at the host/pathogen interface. Thus, genetic disruption of such genes using the CRISPR-Cas9 gene editing will be predicted to have detrimental consequences on parasite's infection of IECs. Our hope is to identify novel mediators of Toxoplasma pathogenesis, specifically in the very first interactions of Toxoplasma with its host.

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Project 2:
Developmental Biology

Infectious cycle of Toxoplasma in an intermediate host. After ingestion of oocysts or tissue cysts, the sporozoites (non-dividing form) inside the oocyst or the bradyzoites (slow-dividing form) inside tissue cysts are released into the GI tract where they rapidly infect enterocytes (Initiation of infection). Following processes that are poorly defined, these parasites convert to the tachyzoites (the rapidly dividing form). The tachyzoites will replicate and disseminate to various tissue in the body (Acute phase of the infection). Upon activation of the host immune system, the tachyzoites will convert to the bradyzoites, which will persist in tissue cysts (brain and muscle tissues predominantly) for the lifetime of the infected individual or animal. 

How does Toxoplasma transition between different forms during infection?

Though differentiation is critical to Toxoplasma pathogenesis and transmission, very little is known about the molecular mechanisms underlying this process. In vitro studies have previously shown that bradyzoites and tachyzoites possess distinct energy requirements and metabolic profiles: whereas tachyzoites rely heavily on aerobic glycolysis and oxidative phosphorylation for energy acquisition, bradyzoites obtain their energy from anaerobic glycolysis, presumably via catabolism of amylopectin into lactate. How Toxoplasma alternates between these disparate metabolic states during infection remains an open question and will be the focus of this second project. Because of the technical challenges associated with producing and handling oocysts and the highly infectious nature of sporozoites, the bulk of the experiments proposed in this project will be conducted solely on bradyzoites, the other transmissible form of T. gondii. Studying bradyzoites will shed light on recurrent toxoplasmosis, as this developmental form is also critical for reactivating infection in immunocompromised individuals. Bradyzoites (both wild type and mutant lines) can be readily generated from tachyzoites in vitro using well-established differentiation, molecular biology techniques (CRISPR-cas9 gene editing, PCR, Western blotting), and immunofluorescence microscopy

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PROJECT 3:
Regulation of gene expression and protein trafficking

Cartoon of Toxoplasma depicting the specialized secretory organelles found in Apicomplexans.

How are proteins trafficked to specialized organelles in Toxoplasma?

Toxoplasma belongs to the phylum Apicomplexa, which includes pathogens such as Plasmodium falciparum (causative agent of malaria) and Cryptosporidium parvum (causative agent of cryptosporidiosis). These organisms have specialized secretory organelles, called micronemes, rhoptries, and dense granules. The contents of these organelles are secreted and critical for invasion of the parasite into the host cell, intracellular survival, and modulation of host processes. In this project, we intend to investigate how proteins find their way into these specialized secretory organelles.
 
 
 

PUBLICATIONS


2017

Guiton PS, Sagawa J, Fritz H, JC Boothroyd. An in vitro model of intestinal infection reveals a developmentally regulated transcriptome of Toxoplasma sporozoites and a NF-κB-like signature in infected host cells. PLoS One. 2017 Mar 31;12(3):e0173018.


2013

Guiton PS, Hannan TJ, Ford B, Caparon MG, Hultgren SJ. Enterococcus faecalis overcomes foreign body-mediated inflammation to establish urinary tract infections. Infect. Immun. 2013 81(1):329-39.

Frank KL, Guiton PS, Barnes AM, Manias DA, Chuang-Smith ON, Kohler PL, Spaulding AR, Hultgren SJ, Schlievert, PM, Dunny GM. AhrC and Eep are biofilm infection-associated virulence factors in Enterococcus faecalis. Infect. Immun. 2013 81(5):1696-708.


2012

Guiton PS, Cusumano CK, Kline KA, Dodson KW, Han Z, Janetka JW, Henderson JP, Caparon MG, Hultgren SJ. Combinatorial small-molecular therapy prevents uropathogenic Escherichia coli catheter-associated urinary tract infections in mice. Antimicrob. Agents Chemother. 2012 56(9):4738-45.

Nielsen HV, Guiton PS, Kline KA, Port GC, Pinkner JS, Neiers F, Normark S, Henriques-Normark B, Caparon MG, Hultgren SJ. The metal ion-dependent adhesion site motif of EbpA pilin mediates pilus function in catheter-associated urinary tract infection. Mbio. 2012 3(4):e00177-12.


2010

Guiton PS, Hung CS, Hancock L., Caparon MG, Hultgren SJ. Enterococcal biofilm formation and virulence in an optimized murine model of foreign body-associated urinary tract infection. Infect. Immun. 2010 78(10):4166-4175.


2009

Guiton PS, Hung CS, Kline KA, Roth R, Kau AL, Hayes E, Heuser J, Dodson KW, Caparon MG, Hultgren SJ. Contribution of autolysin and sortase A during Enterococcus faecalis DNA-dependent biofilm development. Infect. Immun. 2009 77(9):3626-38.