Protein focus: Don’t blame the cat - the toxoplasmosis effect

By Amaia Sangrador and Alex Mitchell

You may have heard about toxoplasmosis, or read about it in a newspaper or magazine. Toxoplasmosis is a condition caused by the protozoan Toxoplasma gondii, an intracellular parasite that infects a wide variety of warm-blooded animals, including humans. T. gondii has attracted the attention of both the scientific and lay communities, and with good reason. It is one of the most successful parasites, infecting over one third of the human population, with rates varying depending upon geographical location¹.

Acute toxoplasmosis usually only poses a risk for immunocompromised individuals or pregnant women. However, residual parasites persist lifelong after the acute phase. Though this latent form of the infection was thought to be asymptomatic, a growing body of evidence suggests that this is not the case². And the long term effects of infection with T. gondii seem related to the most fascinating aspect of this parasite: its ability to modify host behaviour.

Members of the cat family (Felidae) are the only definitive hosts of T. gondii within which the parasite undergoes sexual reproduction. This culminates with the production of oocysts that are shed in the cat’s faeces. Within intermediate hosts (cat’s natural prey, such as rodents and birds)  and other incidental intermediate hosts (such as humans and domestic livestock), the parasite undergoes asexual reproduction, producing bradyzoites that can encyst in the brain and other tissues, where they remain potentially for the host’s lifetime³. Infection can occur following ingestion of oocysts via contaminated soil or water, or ingestion of tissue cysts through raw/undercooked infected meat.

Fig 1. Life-cycle of the parasite Toxoplasma gondii. Sexual reproduction can only be accomplished in felines, and results in the production of sporozoites-containing oocysts that are shed for a limited period. Within intermediate hosts, the parasite undergoes asexual reproduction, producing rapidly dividing tachyzoites - cleared by the immune system - and slowly dividing bradyzoites that can persist as tissue cysts.

Figure by Sebastien Pesseat  

Given that sexual reproduction of T. gondii can be accomplished only in felines, the parasite needs to secure eventual transmission from its intermediate host reservoir, primarily rodents, to its feline definitive host. T. gondii deals with this by manipulating the intermediate host’s behaviour. Toxoplasma infection draws rats to cat odours, increasing activity in limbic regions related to sexual attraction when exposed to cat urine, turning what should be a fear response into a ‘fatal feline attraction⁴'. The intriguing question is: how does the parasite manage to alter the host behaviour? 

Figure by Sebastien Pesseat

One line of evidence suggests that the parasite alters neurotransmitter signals in the brain through increased dopamine levels, supported by studies showing that parasite-induced behavioural changes can be disrupted with dopamine antagonists^5,6. The genome of T. gondii contains two genes encoding an enzyme capable of producing L-DOPA (3,4-dihydroxy-L-phenylalanine), the precursor to dopamine⁷. One of the genes, TgAaaH1, is constitutively expressed, whilst the other gene, TgAaaH2, is induced during the cyst stages. They encode dual activity amino acid hydrolases, bi-functional enzymes that catabolyse both the amino acids phenylalanine and tyrosine. Thus, they can generate tyrosine from phenylalanine and then use tyrosine to produce L-DOPA. These steps are catalysed in Metazoa by phenylalanine hydroxylase and tyrosine hydroxylase, respectively.

Fig 2. Dopamine biosynthesis pathway

In InterPro, these dual activity amino acid hydrolases are classified as belonging to the aromatic amino acid hydroxylase family (IPR001273). They consist of two domains: an N-terminal ACT domain (IPR002912), and a C-terminal aromatic amino acid hydroxylase domain (IPR019774). The C-terminal domain is responsible for catalysis and the N-terminal domain determines the substrate specificity. You can read more about these proteins and domains on the InterPro website.

Fig 3. InterPro view for aromatic amino acid hydroxylase 1 from T. gondii 
(UniProt protein B2L7T1), the product of gene TgAaaH1.

But if T. gondii can alter the behaviour of cats’ natural prey, what happens when secondary hosts like humans are infected? Altering host behaviour in ‘inappropriate’ hosts seems to be an unnecessary but unavoidable consequence of the parasite’s strategy⁸. Indeed, studies have revealed a range of subtle behavioural alterations associated with T. gondii infection in humans, many of which may be comparable to those observed in infected rodents – such as increased activity and decreased reaction times. Studies indicate that some of these changes can be sex-specific, as infection has been reported to increase testosterone levels in men, but decrease its levels in women. Consequently, infected men have tendency to disregard rules and are more suspicious and jealous. In women, the shift in these two factors is opposite; they are more warm-hearted, extrovert and easy-going⁹. More worrying is the link that may exist between infection and psychiatric conditions, such as schizophrenia, in some individuals. This association is supported by several observations, starting with the prevalence of toxoplasmosis in schizophrenic patients¹⁰. Furthermore, antipsychotic drugs, known to be effective in schizophrenia, also inhibit T. gondii¹¹. Meanwhile, raised or disrupted dopamine levels have been reported in both rodent and human T. gondii infection and within human patients with schizophrenia^12,13. 

And when you think it cannot get more bizarre, well, it does. Some studies show Toxoplasma’s modification of behaviour persists even after all parasites and cysts have been cleared¹⁴. This would contradict the cyst-centric theories, which explain the modification of host behaviour as a result of the parasite’s cysts actively modulating dopamine production or affecting neuronal activity¹⁵.  According to a recent study, an explanation for the persistence of the effects induced by the parasite could reside in its ability to induce epigenetic changes in the host. A change in the  methylation state of the arginine vasopressin promoter has been observed in infected animals, resulting in increased expression of this hormone, and affecting a testosterone-responsive area of the brain known for its role in male sexual behaviour¹⁶.  If T. gondii is capable on inducing epigenetic changes, we should consider whether other parasites and pathogens may use similar strategies.

This brings us to an existential question where philosophy and science meet: what is free will if our behaviour can be manipulated? Perhaps, as the philosopher Jose Ortega y Gasset said, we are us and our circumstances. So don’t worry about what might have been. As a future precaution, remember that most people get infected through ingestion or contact with undercooked meat, so make sure that you wash your hands and utensils after handling raw meat. And don’t blame the cat for your behaviour!

Friend or dinner?
Picture from MorgueFile.com, modified by Hsin-Yu Chang

References

1. Flegr J, Prandota J, Sovičková M, Israili ZH. Toxoplasmosis - a global threat. Correlation of latent for the futuretoxoplasmosis with specific disease burden in a set of 88 countries. PLoS One. 9(3):e90203. 2014. [PMID: 24662942]

2. Bhadra R, Cobb DA, Weiss LM, Khan IA. Psychiatric disorders in toxoplasma seropositive patients--the CD8 connection. Schizophr Bull. 39(3):485-9. 2013. [PMID: 23427221]

3. Webster JP, Kaushik M, Bristow GC, McConkey GA. Toxoplasma gondii infection, from predation to schizophrenia: can animal behaviour help us understand human behaviour? J Exp Biol. 216(Pt 1):99-112. 2013. [PMID: 23225872]

4. House PK, Vyas A, Sapolsky R. Predator cat odors activate sexual arousal pathways in brains of Toxoplasma gondii infected rats. PLoS One. 6(8):e23277. 2011. [PMID: 21858053]

5. Prandovszky E, Gaskell E, Martin H, Dubey JP, Webster JP, McConkey GA. The neurotropic parasite Toxoplasma gondii increases dopamine metabolism. PLoS One. 6(9):e23866. 2011. [PMID: 21957440]

6. Webster JP, Lamberton PH, Donnelly CA, Torrey EF. Parasites as causative agents of human affective disorders? The impact of anti-psychotic, mood-stabilizer and anti-parasite medication on Toxoplasma gondii's ability to alter host behaviour. Proc Biol Sci. 273(1589):1023-30. 2006. [PMID: 16627289]

7. Gaskell EA, Smith JE, Pinney JW, Westhead DR, McConkey GA. A unique dual activity amino acid hydroxylase in Toxoplasma gondii. PLoS One. 4(3):e4801. 2014. [PMID: 19277211]

8. Webster JP, Kaushik M, Bristow GC, McConkey GA. Toxoplasma gondii infection, from predation to schizophrenia: can animal behaviour help us understand human behaviour? J Exp Biol. 216:99-112. 2013. [PMID: 23225872]

9. Flegr J. Influence of latent Toxoplasma infection on human personality, physiology and morphology: pros and cons of the Toxoplasma-human model in studying the manipulation hypothesis. J Exp Biol. 216:127-33. 2013. [PMID: 23225875]

10. Torrey EF, Bartko JJ, Lun ZR, Yolken RH. Antibodies to Toxoplasma gondii in patients with schizophrenia: a meta-analysis. Schizophr Bull. 33(3):729-36. 2007. [PMID: 17085743]

11. Jones-Brando L, Torrey EF, Yolken R. Drugs used in the treatment of schizophrenia and bipolar disorder inhibit the replication of Toxoplasma gondii. Schizophr Res. 62(3):237-44. 2003. [PMID: 12837520]

12. Howes OD, Kapur S. The dopamine hypothesis of schizophrenia: version III--the final common pathway. Schizophr Bull. 35(3):549-62. 2009. [PMID: 19325164]

13. Flegr J. How and why Toxoplasma makes us crazy. Trends Parasitol. 29(4):156-63. 2013. [PMID: 23433494]

14. Ingram WM, Goodrich LM, Robey EA, Eisen MB. Mice infected with low-virulence strains of Toxoplasma gondii lose their innate aversion to cat urine, even after extensive parasite clearance. PLoS One. 8(9):e75246. 2013. [PMID: 24058668]

15. McConkey GA, Martin HL, Bristow GC, Webster JP. Toxoplasma gondii infection and behaviour - location, location, location? J Exp Biol. 216(Pt 1):113-9. 2013. [PMID: 23225873]

16. Hari Dass SA, Vyas A. Toxoplasma gondii infection reduces predator aversion in rats through epigenetic modulation in the host medial amygdala. Mol Ecol. 2014. [PMID: 25142402]

Protein focus: Dionysian mysteries - the aldehyde dehydrogenase (ALDH) family

Do you have friends that cannot handle alcoholic drinks? Just half a pint of beer or a few sips of wine, and their faces turn red, possibly with some hangover symptoms, such as headaches and nausea? You may envy  their cheap night out, but wonder why these people cannot tolerate alcohol as you do. The phenomenon is called ‘alcohol flush reaction’, also known as ‘Asian flush syndrome’, due to its association with the Asian population. It is a condition caused by the accumulation of acetaldehyde, a metabolic byproduct of the catabolic metabolism of alcohol. 

Picture provided by Louise Daugherty

Normally, during the alcohol metabolic process, ethanol is converted to acetaldehyde by an alcohol dehydrogenase enzyme, called ADH1B, and then broken down to acetic acid by an aldehyde dehydrogenase enzyme (ALDH). 

In humans, there are nineteen identified ALDH genes (ALDH1-19). Most Europeans have normal copy of the ALDH2 gene, whilst approximately 30-50% of East Asians carry an allele (ALDH2*2)  that results in the synthesis of a less efficient enzyme ¹.

ALDH2 forms homotetramers.  Each subunit in the tetramer consists of three domains - the catalytic domain, the coenzyme-binding domain and the oligomerisation domain. The low activity of ALDH2*2 is the result of a substitution of lysine for glutamate at position 487 (Glu487) of the 500-amino-acid mature enzyme ². The Glu487 links the coenzyme-binding site to the active site, which creates a stable structural scaffold contributing to catalysis (Figure 1). In the ALDH2*2 apoenzyme, the presence of a lysine at residue 487 disturbs the hydrogen bonds and causes disruptions of the αG helix structure ³. This reduces affinity for the coenzyme and lowers the rate of the metabolic process ³. 

As a result of this mutation, acetaldehyde accumulates whenever alcohol is consumed. Unfortunately, acetaldehyde is a DNA damaging agent that can cause cancer  ⁴, and a higher risk of ALDH2-deficient drinkers developing esophageal cancer has been shown by several studies ^4,5,6. A knock out mouse model also links ethanol consumption with higher risk of acetaldehyde toxicity in ALDH2 deficient individuals ⁷.  But whilst the outlook seems to be dim and gloomy for the ALDH2*2 drinkers, on the bright side, they are less likely to suffer alcohol addiction problems  ⁸. In fact, there is a drug called disulfiram that causes symptoms similar to Asian flush syndrome that is used to treat alcoholism. 

Figure 1. The protein structure of a single subunit of ALDH2. 

Residue 487 is indicated in violet. ALDH2*2 αG helix is shown in red, 

wild type I shown in blue.  Picture modified from Larson et al.  2005. ³

 

Interestingly, some ALDH2*2 individuals have less intense flushing symptoms. This is because they also have a less active form of ADH1B (ADH1B*1/*1). This prevents a steep rise in acetaldehyde after drinking. However, some studies have shown that these individuals may have higher risk of both alcoholism and cancer (Figure 2)  ^{8, 9}.    

Figure 2. Ethanol metabolic process.

It is intriguing that a single mutation in the ALDH2 gene could cause alcohol-related health problems. From an evolutionary point of view, aldehyde dehydrogenases are utilised by different species to detoxify harmful chemical intermediates, and hence play an important role in cell survival. They catalyse the conversion of a wide variety of aldehyde substrates to their respective carboxylic acids, using coenzyme NAD or NADP. The aldehyde dehydrogenase family members contain two conserved sites: a cysteine active site and a glutamic acid active site (Figure 4). These two sites are represented by the InterPro entries IPR016160 and IPR029510, and are conserved across species,  from archaea and bacteria to eukaryotes. 

ALDH in different species

In contrast to humans, budding yeast have only five ALDHs. They are the key enzymes of the pyruvate dehydrogenase (PDH) bypass, which generates additional acetyl-CoA ¹⁰. In the wine producing process, the acetate produced by the PDH bypass accumulates during the alcoholic fermentation of sugars ¹¹.  The level of acetate  has important effects on wine quality - most unspoiled wines have a level of 0.2 to 0.8 g  of acetate per litre ¹².

 

Figure 3. Key enzymes of the PDH bypass pathway. PDH, pyruvate dehydrogenase; PDC, pyruvate decarboxylase; ADH, alcohol dehydrogenase; mtALDH, mitochondria ; cALDH, cytoplasmic ALDH. Modified from Wei et al.  2009. ¹⁶

Plants also have multiple ALDHs, and 14 have been identified in Arabodopsis ¹³. They play an important role in the adaptation of plants to various stresses, such as drought, salinity and extreme temperatures ¹⁴. They may also be involved in different transduction pathways ¹⁵. 

An unsolved mystery

We may not yet understand the reason why the  ALDH2 deficiency is widespread in Asian populations. However, research can help us understand more about the relationship between ALDH2, cancers and alcoholism, as well potentially uncovering the safe number of alcohol units that ALDH2*2  individuals can consume. 

So before you encourage your friends to have another glass of wine or a pint of beer, you may need to check if they have the Asian flush symptoms, or even review their ALDH2 phenotype!

 
By Hsin-Yu Chang and Alex Mitchell

References

1. Helminen A, Väkeväinen S, Salaspuro M. ALDH2 genotype has no effect on salivary acetaldehyde without the presence of ethanol in the systemic circulation. PLoS One. 8(9):e74418. 2013. [PMID: 24058561]

2. Larson HN, Zhou J, Chen Z, Stamler JS, Weiner H, Hurley TD. Structural and functional consequences of coenzyme binding to the inactive asian variant of mitochondrial aldehyde dehydrogenase: roles of residues 475 and 487. 282(17):12940-50. J Biol Chem.  2007. [PMID:17327228]

3. Larson HN, Weiner H, Hurley TD. Disruption of the coenzyme binding site and dimer interface revealed in the crystal structure of mitochondrial aldehyde dehydrogenase "Asian" variant. J Biol Chem. 280(34):30550-6. 2005. [PMID: 15983043]

4. Lewis SJ, Smith GD. Alcohol, ALDH2, and esophageal cancer: a meta-analysis which illustrates the potentials and limitations of a Mendelian randomization approach. Cancer Epidemiol Biomarkers Prev. 14(8):1967-71. 2005. [PMID: 16103445]

5. Yokoyama A, Omori T, Yokoyama T. Alcohol and aldehyde dehydrogenase polymorphisms and a new strategy for prevention and screening for cancer in the upper aerodigestive tract in East Asians. Keio J Med. 59(4):115-30. 2010. [PMID: 21187698]

6. Seitz HK, Meier P. The role of acetaldehyde in upper digestive tract cancer in alcoholics. Transl Res. 149(6):293-7. 2007. [PMID:17543846]

7. Isse T, Oyama T, Matsuno K, Ogawa M, Narai-Suzuki R, Yamaguchi T, Murakami T, Kinaga T, Uchiyama I, Kawamoto T. Paired acute inhalation test reveals that acetaldehyde toxicity is higher in aldehyde dehydrogenase 2 knockout mice than in wild-type mice. J Toxicol Sci. 30(4):329-37. 2005. [PMID: 16404141]

8. Yokoyama A, Omori T, Yokoyama T. Alcohol and aldehyde dehydrogenase polymorphisms and a new strategy for prevention and screening for cancer in the upper aerodigestive tract in East Asians. Keio J Med. 59(4):115-30. 2010. [PMID: 21187698]

9. Lee CH, Lee JM, Wu DC, Goan YG, Chou SH, Wu IC, Kao EL, Chan TF, Huang MC, Chen PS, Lee CY, Huang CT, Huang HL, Hu CY, Hung YH, Wu MT. Carcinogenetic impact of  DH1B and ALDH2 genes on squamous cell carcinoma risk of the esophagus with regard to the consumption of alcohol, tobacco and betel quid. Int J Cancer. 122(6):1347-56. 2008. [PMID:18033686]

10. Boubekeur S, Camougrand N, Bunoust O, Rigoulet M, Guérin B. Participation of acetaldehyde dehydrogenases in ethanol and pyruvate metabolism of the yeast Saccharomyces cerevisiae. Eur J Biochem. 268(19):5057-65. 2001. [PMID: 11589696]

11. Saint-Prix F, Bönquist L, Dequin S. Functional analysis of the ALD gene family of Saccharomyces cerevisiae during anaerobic growth on glucose: the NADP+-dependent Ald6p and Ald5p isoforms play a major role in acetate formation. Microbiology. 150(Pt 7):2209-20. 2004. [PMID: 15256563]

12. Remize F, Roustan JL, Sablayrolles JM, Barre P, Dequin S. Glycerol overproduction by engineered saccharomyces cerevisiae wine yeast strains leads to substantial changes in Byproduct formation and to a stimulation of fermentation rate in stationary phase. Appl Environ
Microbiol. 65(1):143-9. 1999. [PMID: 9872772]

13. Kirch HH, Schlingensiepen S, Kotchoni S, Sunkar R, Bartels D. Detailed expression analysis of selected genes of the aldehyde dehydrogenase (ALDH) gene superfamily in Arabidopsis thaliana. Plant Mol Biol. 57(3):315-32. 2005. [PMID: 15830124]

14. Zhang Y, Mao L, Wang H, Brocker C, Yin X, Vasiliou V, Fei Z, Wang X. Genome-wide identification and analysis of grape aldehyde dehydrogenase (ALDH) gene superfamily. PLoS One. 7(2):e32153. 2012. [PMID: 22355416]

15. Kirch HH, Bartels D, Wei Y, Schnable PS, Wood AJ. The ALDH gene superfamily of Arabidopsis. Trends Plant Sci. 9(8):371-7. 2004. [PMID: 15358267]

16. Wei Y, Lin M, Oliver DJ, Schnable PS. The roles of aldehyde dehydrogenases (ALDHs) in
the PDH bypass of Arabidopsis. BMC Biochem. 10: 7. 2009. [PMID: 19320993]