Citation: Litvintseva AP, Mitchell TG (2012) Population Genetic Analyses Reveal the African Origin and Strain Variation of Cryptococcus neoformans var. grubii. PLoS Pathog8(2): e1002495. https://doi.org/10.1371/journal.ppat.1002495
Editor: Joseph Heitman, Duke University Medical Center, United States of America
Published: February 23, 2012
Copyright: © 2012 Litvintseva, Mitchell. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by US Public Health Service NIH grants 1R01 AI 093257 and 5R01 AI 25783, and by a grant from the Duke University Center for AIDS Research, which is funded by NIH grant P30 AI 64518. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Cryptococcus neoformans is a ubiquitous, saprobic yeast and the cause of life-threatening infections. Humans acquire the infection by inhaling airborne cells from the environment. In the lungs, these cells become encapsulated yeasts and proliferate. In people with healthy immune responses, the infection may resolve or remain latent and subsequently cause disease. However, in immunocompromised people, such as HIV/AIDS patients, and less often in healthy hosts, the yeasts can disseminate to almost any part of the body; however, they are neurotropic, and meningoencephalitis is the most frequent and deadliest clinical manifestation –. An estimated 1 million new infections are acquired each year, and the majority of these cases occur in sub-Saharan Africa, which has the highest prevalence of patients with HIV/AIDS . In this region, C. neoformans is the most common cause of meningitis, and mortality hovers around 50%. Others who succumb to cryptococcosis are apparently immunocompetent and exhibit no evidence of underlying disease. For example, 71% of cryptococcal infections in China occur in people without pre-existing conditions .
There are two varieties, C. neoformans var. grubii (Cng) and C. neoformans var. neoformans (Cnn), which are distinguishable by molecular markers or their capsular serotypes, A or D, respectively. Diploid AD hybrids also occur in the environment and patients –. In addition, a sibling species, Cryptococcus gattii, causes similar infections. However, isolates of both serotype D and AD hybrids, as well as C. gattii, are much less common. At least 90% of human cryptococcal disease and fatalities are caused by Cng (serotype A) –.
Non-African Global Isolates of Cng Are Highly Clonal
Strains of Cng have been isolated from all continents except Antarctica. Molecular epidemiological studies identified significant clonality among global strains, as strains with identical molecular genotypes have been isolated from different geographic areas, continents apart , , . The use of reproducible and robust multilocus sequence typing (MLST) has determined that the overwhelming majority of non-African strains of Cng are represented by only a few genotypes –.
Isolates from southeastern Asia are remarkably homogeneous. For example, all the clinical isolates from a cohort of 120 Chinese patients were infected with the same cosmopolitan MLST genotype, M5 . Similarly, all seven clinical isolates from Japan  and 70 of 75 from South Korea possessed the M5 genotype (designated “VNIc” in the original paper) . In Thailand, 183 clinical and environmental isolates were analyzed, and 96% of the isolates were represented by only three MLST genotypes, one of which was M5 (designated “ST46” in the original paper) .
In the United States, an analysis of over 800 isolates yielded only ten distinct genotypes, and M5 was the most prevalent among both clinical and environmental samples (designated “A5” in the original paper) . In comparison, five genotypes were found in Europe, and nine genotypes among isolates from central and eastern Africa, but only two different MLST genotypes were detected from South American and Australian isolates, although these were small samples . More recently, we found that M5 was the most prevalent genotype among isolates from patients with recurrent cryptococcosis in South Africa (unpublished data), even though, as described below, this region has the highest overall genetic diversity.
Southern African Isolates of Cng Are Highly Diverse
Unlike the rest of the world, southern Africa harbors a geographically restricted, genetically diverse population of Cng. In 2003, isolates from 200 HIV-seropositive patients in Botswana were shown to possess novel genotypes that differed from isolates found anywhere else. Analyses of mating indicated that 12% of these strains possess the MATa mating type, which is exceedingly rare among non-African strains, and population genetic analysis demonstrated evidence for recombination in this population . Subsequent environmental sampling confirmed that two genetically isolated subpopulations are localized in southern Africa: (i) a genetically diverse, endemic population that is restricted to southern African and associated with indigenous African trees, especially the mopane tree (Colophospermum mopane), and (ii) a cosmopolitan population of strains with molecular types that are found worldwide and frequently associated with the excreta of feral pigeons (Columba livia) .
Population genetic analyses of the environmental strains revealed limited genetic interaction between the endemic (arboreal) and cosmopolitan (coprophilic) populations. The arboreal population was characterized by linkage equilibrium among loci and high genetic diversity, which can be explained by recombination, ancestral origin, or both. However, when putative recombinant haplotypes were removed from the analysis, significantly high indices of genetic diversity were still detected in the African population .
Phylogenetic Analyses Indicate That African Strains of Cng Possess Ancestral Haplotypes
Phylogenetic analysis and principal component analysis (PCA) of the MLST profiles indicate that Cng is comprised of three isolated subpopulations, VNI, VNII, and VNB (Figure 1). The global population is markedly clonal and consists of VNI and VNII strains with few unique genotypes. The arboreal, southern African population is geographically confined and comprised of almost all the known VNB strains and a large number of VNI strains that are genetically diverse. Thus, multiple features of the African strains—greatest genetic diversity, prevalence of both mating types, and association with an indigenous reservoir—suggest they represent the ancestral origin of Cng.
Each symbol represents a genotype with a unique eight-digit allelic profile. Red triangles represent genotypes of strains that are endemic to Africa, and blue circles represent genotypes of global strains. Genotypes associated with African trees are enclosed in green circles, and genotypes associated with pigeon excreta are enclosed in brown circles. Genotypes without circles represent clinical strains that to date have not been isolated from the environment. (From reference  and used with permission of the publisher.)
This conclusion was supported by phylogenetic analyses of individual loci using methods of maximum likelihood and parsimony. To illustrate, haplotype networks were analyzed by statistical parsimony to infer any phylogenetic relationships among the haplotypes. The internal nodes of these networks represent ancestral haplotypes from which the distal, derived haplotypes evolved. Numerous haplotypes from the endemic African population occupied both internal (ancestral) and distal (derived) positions on the networks (Figure 2, green circles). Conversely, haplotypes that are unique to the global population were scarce and almost always in distal positions, which suggests they originated more recently. All of these genotypes were obtained from pigeon habitats (Figure 2, brown circles) , . Furthermore, maximum likelihood analyses of three loci (TEF1, CAP59, and PLB1) indicated that the ancestral haplotypes of both the VNI and VNB populations are confined to southern Africa and associated with endemic African trees .
Haplotypes of strains of Cng that have never been found outside Africa are shown in green: filled green circles designate haplotypes of strains that were obtained from trees (most were also found in patients), and empty green circles signify haplotypes that were obtained only from patients. Cosmopolitan haplotypes are shown in brown: filled brown circles designate haplotypes of strains from pigeon excreta (most were also found in patients), and empty brown circles signify haplotypes that were obtained only from patients. Circles that are half green and half brown indicate haplotypes of strains found in trees and pigeon excreta. Haplotypes from the global VNII subpopulation of Cng are included as an outgroup; they are shown in black and lightly encircled. Red dots on the lines connecting the haplotypes represent the most parsimonious number of mutational steps required to generate the allelic polymorphisms. Recombinant haplotypes are excluded. The most common haplotype in Asia and elsewhere, M5, is shown in blue. (From reference  and used with permission of the publisher.)
Evidence That Pigeons Facilitated the Global Dispersion of Southern African Strains
Environmental sampling has demonstrated that African strains of VNI and VNB are associated with native African trees, whereas cosmopolitan strains of VNI are isolated from pigeon droppings. VNI strains with identical MLST genotypes have been isolated from pigeon habitats in North and South America, Europe, Asia, and Africa , , . Coalescent simulations estimated that the cosmopolitan and African populations diverged approximately 5,000 years ago, which is around the postulated time period when C. livia (rock doves or pigeons) were domesticated . Although the exact origin of C. livia is unknown, historical records indicate that pigeons were probably native to the north African Mediterranean region and were spread globally over the last 400 years of European expansion , . Thus, multiple features of the cosmopolitan population of VNI—exceptionally low genetic diversity, dominance of a single mating type, and global distribution in association with pigeon excreta—support the parsimonious conclusion that pigeons facilitated the global exportation and dispersion of African strains.
Phenotypic Diversity among Isolates of Cng
Cryptococcal virulence is complex and polygenic, involving dozens of genes and signal transduction pathways . Several studies have documented variation among strains of Cng in the expression of virulence phenotypes, such as the size, composition, and biological activity of the capsule, susceptibility to antifungal drugs, resistance to phagocytes, and others –. This marked phenotypic variation among wild-type isolates of Cng suggests that some strains are inherently more virulent. As observed in several reports and noted above, patients may survive or succumb to cryptococcosis regardless of their immune status, which suggests that the virulence of an infecting strain may be as important as the host's defenses. Does the genotype of a strain impart any clinical relevance? A recent population genetics investigation used 11 MLST markers to genotype isolates from South African pediatric cases of cryptococcosis . Seventy-one children, nearly all HIV-positive, were infected with 17 different VNI genotypes; most genotypes were equally present in boys and girls, but one genotype was significantly more prevalent in boys . When the relevance of genotype was tested experimentally, clinical and environmental isolates with identical MLST profiles varied dramatically in murine virulence; however, virulence was associated with the clinical or environmental source of a strain rather than its genotype . Clearly, strains with the same genotype exhibit phenotypic variation. Nevertheless, some genotypes are highly prevalent in the environment but rare or absent in patients and vice versa , . The most important phenotype is the production of disease in people. With the advent of next-generation sequencing, it is now possible to use MLST genotypes to select strains differing in genetic diversity and clinical prevalence and to conduct comparative genomic analyses to address the question of why some strains are more pathogenic.
- 1. Lin X, Heitman J (2006) The biology of the Cryptococcus neoformans species Complex. Annu Rev Microbiol 60: 69–105.X. LinJ. Heitman2006The biology of the Cryptococcus neoformans species Complex.Annu Rev Microbiol6069105
- 2. Ma H, May RC (2009) Virulence in Cryptococcus species. Adv Appl Microbiol 67: 131–190.H. MaRC May2009Virulence in Cryptococcus species.Adv Appl Microbiol67131190
- 3. Perfect JR, Dismukes WE, Dromer F, Goldman DL, Graybill JR, et al. (2010) Clinical practice guidelines for the management of cryptococcal disease: 2010 update by the Infectious Diseases Society of America. Clin Infect Dis 50: 291–322.JR PerfectWE DismukesF. DromerDL GoldmanJR Graybill2010Clinical practice guidelines for the management of cryptococcal disease: 2010 update by the Infectious Diseases Society of America.Clin Infect Dis50291322
- 4. Park BJ, Wannemuehler KA, Marston BJ, Govender N, Pappas PG, et al. (2009) Estimation of the current global burden of cryptococcal meningitis among persons living with HIV/AIDS. AIDS 23: 525–530.BJ ParkKA WannemuehlerBJ MarstonN. GovenderPG Pappas2009Estimation of the current global burden of cryptococcal meningitis among persons living with HIV/AIDS.AIDS23525530
- 5. Chen J, Varma A, Diaz MR, Litvintseva AP, Wollenberg KK, et al. (2008) Cryptococcus neoformans strains and infection in apparently immunocompetent patients, China. Emerg Infect Dis 14: 755–762.J. ChenA. VarmaMR DiazAP LitvintsevaKK Wollenberg2008Cryptococcus neoformans strains and infection in apparently immunocompetent patients, China.Emerg Infect Dis14755762
- 6. Lengeler K, Cox G, Heitman J (2001) Serotype AD strains of Cryptococcus neoformans are diploid or aneuploid and heterozygous at the mating-type locus. Infect Immun 69: 115–122.K. LengelerG. CoxJ. Heitman2001Serotype AD strains of Cryptococcus neoformans are diploid or aneuploid and heterozygous at the mating-type locus.Infect Immun69115122
- 7. Litvintseva AP, Lin X, Templeton I, Heitman J, Mitchell TG (2007) Many globally isolated AD hybrid strains of Cryptococcus neoformans originated in Africa. PLoS Pathog 3: e114.AP LitvintsevaX. LinI. TempletonJ. HeitmanTG Mitchell2007Many globally isolated AD hybrid strains of Cryptococcus neoformans originated in Africa.PLoS Pathog3e114
- 8. Xu J, Luo G, Vilgalys RJ, Brandt ME, Mitchell TG (2002) Multiple origins of hybrid strains of Cryptococcus neoformans with serotype AD. Microbiology 148: 203–212.J. XuG. LuoRJ VilgalysME BrandtTG Mitchell2002Multiple origins of hybrid strains of Cryptococcus neoformans with serotype AD.Microbiology148203212
- 9. Govender N, Mitchell TG, litvintseva AP, Miglia KJ (2011) Cryptococcosis in Africa. In: Heitman J, Kozel TR, Kwon-Chung J, Perfect JR, Casadevall A, editors. Cryptococcus: from human pathogen to model yeast. Washington (D.C.): ASM Press. pp. 269–285.N. GovenderTG MitchellAP litvintsevaKJ Miglia2011Cryptococcosis in Africa.J. HeitmanTR KozelJ. Kwon-ChungJR PerfectA. CasadevallCryptococcus: from human pathogen to model yeastWashington (D.C.)ASM Press269285
- 10. Litvintseva AP, Kestenbaum L, Vilgalys R, Mitchell TG (2005) Comparative analysis of environmental and clinical populations of Cryptococcus neoformans. J Clin Microbiol 43: 556–564.AP LitvintsevaL. KestenbaumR. VilgalysTG Mitchell2005Comparative analysis of environmental and clinical populations of Cryptococcus neoformans.J Clin Microbiol43556564
- 11. Xu J, Manosuthi W, Banerjee U, Zhu L-P, Chen J, et al. (2011) Cryptococcosis in Asia. In: Heitman J, Kozel TR, Kwon-Chung J, Perfect JR, Casadevall A, editors. Cryptococcus from human pathogen to model yeast. Washington (D.C.): ASM Press. pp. 287–298.J. XuW. ManosuthiU. BanerjeeL-P ZhuJ. Chen2011Cryptococcosis in Asia.J. HeitmanTR KozelJ. Kwon-ChungJR PerfectA. CasadevallCryptococcus from human pathogen to model yeastWashington (D.C.)ASM Press287298
- 12. Bovers M, Hagen F, Kuramae EE, Boekhout T (2008) Six monophyletic lineages identified within Cryptococcus neoformans and Cryptococcus gattii by multi-locus sequence typing. Fungal Genet Biol 45: 400–421.M. BoversF. HagenEE KuramaeT. Boekhout2008Six monophyletic lineages identified within Cryptococcus neoformans and Cryptococcus gattii by multi-locus sequence typing.Fungal Genet Biol45400421
- 13. Litvintseva AP, Thakur R, Vilgalys R, Mitchell TG (2006) Multilocus sequence typing reveals three genetic subpopulations of Cryptococcus neoformans var. grubii (serotype A), including a unique population in Botswana. Genetics 172: 2223–2238.AP LitvintsevaR. ThakurR. VilgalysTG Mitchell2006Multilocus sequence typing reveals three genetic subpopulations of Cryptococcus neoformans var. grubii (serotype A), including a unique population in Botswana.Genetics17222232238
- 14. Litvintseva AP, Carbone I, Rossouw J, Thakur R, Govender NP, et al. (2011) Evidence that the human pathogenic fungus Cryptococcus neoformans var. grubii may have evolved in Africa. PLoS ONE 6: e19688.AP LitvintsevaI. CarboneJ. RossouwR. ThakurNP Govender2011Evidence that the human pathogenic fungus Cryptococcus neoformans var. grubii may have evolved in Africa.PLoS ONE6e19688
- 15. Simwami SP, Khayhan K, Henk DA, Aanensen DM, Boekhout T, et al. (2011) Low diversity Cryptococcus neoformans variety grubii multilocus sequence types from Thailand are consistent with an ancestral African origin. PLoS Pathog 7: e1001343.SP SimwamiK. KhayhanDA HenkDM AanensenT. Boekhout2011Low diversity Cryptococcus neoformans variety grubii multilocus sequence types from Thailand are consistent with an ancestral African origin.PLoS Pathog7e1001343
- 16. Choi YH, Ngamskulrungroj P, Varma A, Sionov E, Hwang SM, et al. (2010) Prevalence of the VNIc genotype of Cryptococcus neoformans in non-HIV-associated cryptococcosis in the Republic of Korea. FEMS Yeast Res 10: 769–778.YH ChoiP. NgamskulrungrojA. VarmaE. SionovSM Hwang2010Prevalence of the VNIc genotype of Cryptococcus neoformans in non-HIV-associated cryptococcosis in the Republic of Korea.FEMS Yeast Res10769778
- 17. Litvintseva AP, Marra RE, Nielsen K, Heitman J, Vilgalys R, et al. (2003) Evidence of sexual recombination among Cryptococcus neoformans serotype A isolates in sub-Saharan Africa. Eukaryot Cell 2: 1162–1168.AP LitvintsevaRE MarraK. NielsenJ. HeitmanR. Vilgalys2003Evidence of sexual recombination among Cryptococcus neoformans serotype A isolates in sub-Saharan Africa.Eukaryot Cell211621168
- 18. Long JL (1981) Introduced birds of the world: the worldwide history, distribution and influence of birds introduced to new environments. London: David & Charles. pp. 208–211.JL Long1981Introduced birds of the world: the worldwide history, distribution and influence of birds introduced to new environmentsLondonDavid & Charles208211
- 19. Grzimek B, Schlager N, Olendorf D (2004) Pigeons and doves. In: McDade MC, editor. Grzimek's animal life encyclopedia. Volume 9: Birds II. Detroit: Gale. pp. 247–268.B. GrzimekN. SchlagerD. Olendorf2004Pigeons and doves.MC McDadeGrzimek's animal life encyclopedia. Volume 9: Birds IIDetroitGale247268
- 20. Kronstad JW, Attarian R, Cadieux B, Choi J, D'Souza CA, et al. (2011) Expanding fungal pathogenesis: Cryptococcus breaks out of the opportunistic box. Nat Rev Microbiol 9: 193–203.JW KronstadR. AttarianB. CadieuxJ. ChoiCA D'Souza2011Expanding fungal pathogenesis: Cryptococcus breaks out of the opportunistic box.Nat Rev Microbiol9193203
- 21. Kumar P, Yang M, Haynes BC, Skowyra ML, Doering TL (2011) Emerging themes in cryptococcal capsule synthesis. Curr Opin Struct Biol 21: 597–602.P. KumarM. YangBC HaynesML SkowyraTL Doering2011Emerging themes in cryptococcal capsule synthesis.Curr Opin Struct Biol21597602
- 22. Liaw SJ, Wu HC, Hsueh PR (2010) Microbiological characteristics of clinical isolates of Cryptococcus neoformans in Taiwan: serotypes, mating types, molecular types, virulence factors, and antifungal susceptibility. Clin Microbiol Infect 16: 696–703.SJ LiawHC WuPR Hsueh2010Microbiological characteristics of clinical isolates of Cryptococcus neoformans in Taiwan: serotypes, mating types, molecular types, virulence factors, and antifungal susceptibility.Clin Microbiol Infect16696703
- 23. Mitchell TG, Litvintseva AP (2010) Typing species of Cryptococcus and epidemiology of cryptococcosis. In: Ashbee HR, Bignell EM, editors. Pathogenic yeasts. Berlin Heidelberg: Springer-Verlag. pp. 167–190.TG MitchellAP Litvintseva2010Typing species of Cryptococcus and epidemiology of cryptococcosis.HR AshbeeEM BignellPathogenic yeastsBerlin HeidelbergSpringer-Verlag167190
- 24. Pedroso RS, Lavrador MA, Ferreira JC, Candido RC, Maffei CM (2010) Cryptococcus neoformans var. grubii - pathogenicity of environmental isolates correlated to virulence factors, susceptibility to fluconazole and molecular profile. Mem Inst Oswaldo Cruz 105: 993–1000.RS PedrosoMA LavradorJC FerreiraRC CandidoCM Maffei2010Cryptococcus neoformans var. grubii - pathogenicity of environmental isolates correlated to virulence factors, susceptibility to fluconazole and molecular profile.Mem Inst Oswaldo Cruz1059931000
- 25. Alanio A, Desnos-Ollivier M, Dromer F (2011) Dynamics of Cryptococcus neoformans-macrophage interactions reveal that fungal background influences outcome during cryptococcal meningoencephalitis in humans. mBio 2: e00158–11.A. AlanioM. Desnos-OllivierF. Dromer2011Dynamics of Cryptococcus neoformans-macrophage interactions reveal that fungal background influences outcome during cryptococcal meningoencephalitis in humans.mBio2e0015811
- 26. Miglia KJ, Govender NP, Rossouw J, Meiring S, Mitchell TG (2011) Analyses of pediatric isolates of Cryptococcus neoformans from South Africa. J Clin Microbiol 49: 307–314.KJ MigliaNP GovenderJ. RossouwS. MeiringTG Mitchell2011Analyses of pediatric isolates of Cryptococcus neoformans from South Africa.J Clin Microbiol49307314
- 27. Litvintseva AP, Mitchell TG (2009) Most environmental isolates of Cryptococcus neoformans var. grubii (serotype A) are not lethal for mice. Infect Immun 77: 3188–3195.AP LitvintsevaTG Mitchell2009Most environmental isolates of Cryptococcus neoformans var. grubii (serotype A) are not lethal for mice.Infect Immun7731883195
- 28. Illnait-Zaragozi MT, Martinez-Machin GF, Fernandez-Andreu CM, Boekhout T, Meis JF, et al. (2010) Microsatellite typing of clinical and environmental Cryptococcus neoformans var. grubii isolates from Cuba shows multiple genetic lineages. PLoS ONE 5: e9124.MT Illnait-ZaragoziGF Martinez-MachinCM Fernandez-AndreuT. BoekhoutJF Meis2010Microsatellite typing of clinical and environmental Cryptococcus neoformans var. grubii isolates from Cuba shows multiple genetic lineages.PLoS ONE5e9124