Azurin-Like Protein Blocks Invasion ofToxoplasma gondiithrough Potential Interactions with Parasite Surface Antigen SAG1

Abstract

Some pathogenic bacteria produce factors that have evolved a capacity to neutralize competing microbes. The cupredoxin family protein azurin, produced by Pseudomonas aeruginosa, exhibits a remarkable ability to impede invasion of a number of diverse intracellular pathogens, including the human AIDS virus human immunodeficiency virus type 1 and the protozoan parasite Plasmodium falciparum (which causes malaria). Here we report that azurin and an azurin-like protein (Laz) from gonococci/meningococci have activity against Toxoplasma, an apicomplexan parasite that causes opportunistic infection in immunocompromised individuals. We demonstrate that the mechanism of action for Laz involves interfering with the ability of Toxoplasma to adhere to host cells. Computer structural analysis reveals that azurin shares structural features with the predominant surface antigen SAG1, which is known to play an important role in parasite attachment. Interestingly, azurin also has structural similarities to a monoclonal antibody to SAG1. Surface plasmon resonance binding studies validate that SAG1 interacts strongly with Laz and, to lesser extent, azurin. Moreover, Toxoplasma mutants lacking SAG1 are not as susceptible to the growth-inhibitory effects of Laz. Collectively, our data show that Toxoplasma adhesion can be significantly impaired by Laz, and to some extent by azurin, via interactions with SAG1. These observations indicate that Laz can serve as an important tool in the study of host-pathogen interactions and is worthy of further study for development into potential therapeutic agents.

Structural studies on Laz, a promiscuous anticancer Neisserial protein

“…It should also be noted that Paz, Laz and H.8-Paz had not only anticancer activity, but strong anti-viral activity against the AIDScausing HIV-1 virus and strong antiparasitic activity against parasites such as the malarial parasite Plasmodium falciparum 35 and the toxoplasmosis-causing parasite Toxoplasma gondii. 36 The potential clinical importance of such bacterial proteins as Paz and Laz is reflected in the fact that a 28 amino acid peptide fragment from azurin (azurin 50-77), termed p28, has shown very little toxicity in 15 stage IV cancer patients in a phase I clinical trial, but significant beneficial effect including partial and complete regression of the drug-resistant tumors in several patients. 37 Another phase I trial with p28, currently in progress in pediatric brain tumor patients in 11 hospitals in the US (http://clinicaltrials.gov/ct2/show/NCT01975116) for more than a year and 3 months appears to indicate that p28, even lacking the H.8 epitope but with a smaller size than azurin, may be able to enter the brain tumors to allow their regression.…”

Bioengineered bugs, drugs and contentious issues in patenting

“…2 A second unique feature of azurin is its promiscuity in attacking multiple diverse disease agents such as the AIDScausing virus HIV-1, the malarial parasite P. falciparum or the toxoplasmosis-causing parasite T. gondii. 22,23 The ability of azurin to prevent invasion by these attackers of the human body has been shown to be due to azurin's unique structural features similar to the immunoglobulin variable domains. Azurin is a member of a family of copper-containing redox proteins known as cupredoxins.…”

“…2,14,20 With regard to inhibition of parasite invasion of host cells, azurin binds the merozoite surface protein MSP-1 of P. falciparum with a Kd value of 32.2 nM 22 and the surface antigen SAG1 of T. gondii with a Kd value of 12.8 nM. 23 Both MSP-1 and SAG1 are important surface components of the parasites for their invasion of hosts and growth inside such hosts. By binding such important surface components, azurin interferes in their invasion.…”

“…Another interesting aspect of azurin's growth inhibitory action is towards the AIDS-causing virus HIV-1. Both azurin and the azurin-like protein Laz have strong inhibitory action not only towards T. gondii 23 and P. falciparum but also towards HIV-1. 22 Because HIV-1 is highly mutable and acquires resistance to drugs that target and inhibit its essential replication/maturation machinery such as reverse transcriptase or protease, a combination (cocktail) of such anti-retroviral drugs is used to treat AIDS.…”

Comparative evaluation of immunization with recombinant protein and plasmid DNA vaccines of fusion antigen ROP2 and SAG1 from Toxoplasma gondii in mice: cellular and humoral immune responses

“…Among these, we focused our efforts on major surface protein 1 (SAG1) and rhoptry protein 2 (ROP2) of T. gondii. The SAG1 could induce humoral immune responses immediately at the acute phase of infection (Pietkiewicz et al 2004), and had been shown to produce complete protection against lethal T. gondii infection in mice (Haumont et al 2000;Nielsen et al 1999;Naguleswaran et al 2008;Fang et al 2010). Furthermore, the ROP2 was expressed in three stages of the parasite life cycle (tachyzoites, bradyzoites, and sporozoites), and also carried T-cell and B-cell epitopes recognized by a high proportion of the infected human population (Echeverria et al 2006;Beghetto et al 2006;Saavedra et al 1996;Dziadek et al 2009).…”