Research

Areas: Microbial pathogenesis, protozoan parasites, viruses of protozoan parasites, glycobiology, genetics, genomics, biochemistry, cell biology, host responses/immunology

Much of our work focuses on the protozoan parasite Leishmania, infecting more than 10 million people in tropical regions. We want to understand how the parasite carries out its infectious cycle, which comprises an intracellular phagolysosomal stage within vertebrate macrophages and an extracellular stage within the gut of a sand fly vector.

 

 

 

The development and application of new methods to parasite virulence. Experimentally, Leishmania is EASY to work with. Genetic manipulations are fast and routine, and one can plate them and generate many independent colonies. Parasites can be grown readily in culture under conditions where they differentiate well, and there are excellent mouse models enabling dissection of host defenses.

We have developed and/or applied in parasites powerful methods such as functional genetic rescue, gene knockouts, transposon mutagenesis, expression profiling via microarrays or RNA sequencing, and genome sequencing. We have identified many genes with ‘interesting’ functions, expression patterns and/or database relationships, and now are in the process of assessing their role in virulence using a variety of genetic and biochemical methods.

 

Functional genomics.  One of the challenges of Leishmania is diploidy and an as yet inconvenient sexual cycle in the sand fly vector.  The advent of RNAi tools and more recently CRISPR technology readily overcomes this bottleneck.  CRISPR has many advantages and we are optimizing these tools now for Leishmania.Notably these can be applied not only to the parasite but also to the host genomics, to probe host-parasite interactions from both perspectives. 

 

 

 

A role for the parasite flagellum in virulence.
While the infective amastigote stage lacks an external flagellum, it maintains a ‘stub’ which has largely been ignored.   Recently Gull’s lab proposed this may play a role in parasite virulence, as a conduit for virulence factors as well as a sensing organelle as seen in other eukaryotes.

To test this we sought to knockout the Leishmania flagellum.   Remarkably this proved easy to do through ablation of a gene implicated in intraflagellar transport, IFT140.  In culture as the promastigote stage, these parasites completely lacked a flagellum, yet maintained a fairly normal flagellar pocket, and grew normally (other than unable to swim).   

In contrast, the ift140 mutants were completely avirulent, in a mouse or in a macrophage model.   Current work is now directed towards understand the mechanism by which this occurs.

 

 

 

 

 

 

Leishmania surface glycocalyx.

The Leishmania surface is composed of a dense glycocalyx of glycolipids. One fruitful genetic screen involves a surface glycoconjugate, lipophosphoglycan (LPG), an essential virulence determinant involved in adhesion and survival. LPG genes encode proteins involved in biosynthesis, compartmentalization of LPG intermediates within the eukaryotic secretory pathway, andd regulation.
We are especially interested in understanding the role of the parasite surface interactions in manipulating host cell signal transduction, which is radically altered in Leishmania infections, and in sand fly transmission and co-evolution.

Mitochondrial glycosylation! 

We were surprised to discover that Fucose was an essential metabolite, and even more surprised to find this was because of an essential fucosyl-transferase (FuT1) located in the mitochondrion!   While fut1- mutants were very difficult to obtain, they show strong mitochondrial defects.
Previously mitochondrial glycosylation was not known.  New questions include A) what is the essential fucoconjugate(s)? B) what is its role in mitochondrial function; and C) could FUT1 be a target for anti-parasite chemotherapy? 

 


Parasite Virology!

In collaboration with the group of Nicolas Fasel in Lausanne Switzerland, we showed in 2011 that enigmatic dsRNA Leishmaniavirus (LRV) was associated causally with increased virulence and increased metastasis.

LRV-infected Leishmania induce a hyperimmune response to the parasite responsible for the increased pathogenicity, through a dsRNA and TLR3-dependent pathway.  We are focusing now on the mechanism by which this occurs; preliminary data implicate Type I interferon signaling as a key player


Notably, L. braziliensis and related species of Viannia are responsible for a severe form of leishmaniasis called mucocutaneous leishmaniasis; the LRV represents the first parasite-associated factor implicated in this terrible disease.   Our groups are now working to together to better understand how and where LRV contributes to human disease. We are surveying strains across South America for LRVs in an effort to associate LRV with human disease manifestations, and developing experimental Leishmania LRV ‘virology’ to assess in the laboratory how LRV contributes to pathology. The role of RNAi in LRV biology and LRV-dependent virulence is a subject of considerable interest as well.

A new focus is how general the ‘hypervirulence’ associated with parasite viruses may be; a great many have been described in other parasites but like LRV1, previously overlooked.  We are pursuing known viral elements as carrying out viral discovery efforts, not only in Leishmania (with the discovery of several new viruses) but also in Trichomonas vaginalis (with Pat Johnson at UCLA), Cryptosporidium (with Mark Kuhlenschmidt at the University of Illinois), and Toxoplasma (with David Sibley in the Molecular Microbiology department).  Current data are very encouraging.

 

 

 

 

 

 

 

 

 

 

 

 

 

Genetics/genomics. In 2009 we showed for the first time that genetic crossing is possible in Leishmania. This opens the way to genetic analysis of important traits including tropisms related to different forms of leishmaniasis (cutaneous, mucocutaneous or visceral). We are resequencing several Leishmania genomes to provide SNP markers for this purpose as well as to create the first Leishmania genetic map.
In conjunction with an NHGRI/NIAID sponsored white paper initiative we are working with a consortium of scientists and the Genome Sequencing Center of Washington University to sequence over 50 new trypanosomatid genomes.  Comparative analysis will permit identification of genes associated with phylogenetically with different manifestations of Leishmania pathology and virulence.


RNA interference. In 2010 we showed that while most Leishmania species lack the RNA interference pathway, species belonging to the L. braziliensis group have retained it. This permits the application of RNAi-based methods for functional genomics in this group of species, and towards this end we are developing L. braziliensis (the agent of mucocutaneous leishmaniasis) into a superb experimental system for microbial pathogenesis. Examples include live animal imaging of infection and host response and in vitro systems for differentiation to the pathogenic amastigote stage.

The loss of RNAi in most Leishmania raises the interesting evolutionary question of what forces contribute to the loss of a pathway of such fundamental importance in other organisms. This is being pursued in several ways, through the study of RNAi-deficient L. braziliensis null mutants as well as attempts to restore RNAi to those Leishmania lacking it.

Other interests of the lab include molecular evolution of parasitism and virulence, and the use of genetically modified parasites as vaccines.

 

Selected Publications:


Mandell, M.A. and S. M. Beverley (2017), “Continual renewal and replication of persistent Leishmania major parasites in concomitantly immune hosts”, Proc. Natl. Acad. Sci. USA (in press; on line 1/17/17).   
Kuhlmann, F.M. J.I. Robinson, G. Bluemling, C. Ronet, N. Fasel, and S.M. Beverley (2017), “Anti-viral Screening Identifies Adenosine Analogs Targeting the Endogenous dsRNA Leishmania RNA Virus 1 (LRV1) Pathogenicity Factor”, Proc. Natl. Acad. Sci. USA (in press; on line 1/17/17). 

Brettmann, E.A., J. Shaik, H. Zangger, L-F. Lye, F. M. Kuhlmann, N. S. Akopyants, D. M. Oschwald, K.L. Owens, S. M. Hickerson, C. Ronet, N. Fasel, and S. M. Beverley (2016), “Tilting the balance between RNA interference and replication eradicates Leishmania RNA virus 1 and mitigates the inflammatory response”, Proc. Natl. Acad. Sci. USA 113:11998-12005.  

Favila, M.A, N.S. Geraci, N.S., A. Jayakumar, S. M. Hickerson, J. Mostromi, S.J. Turco, S. M. Beverley and M. A. McDowell, “Differential impact of LPG-and PG-deficient Leishmania major mutants on the immune response of human dendritic cells” (2015), PLoS Neglected tropical Diseases:  9(12):e0004238

Eren, R.O., M. Reverté, M. Rossi, M-A. Hartley, P. Castiglioni, F. Prevel, R. Martin, C. Desponds, L-F. Lye, S. K. Drexler, W. Reith, S. M. Beverley, C. Ronet, and N. Fasel (2016), “Mammalian Innate Immune Response to a Leishmania-Resident RNA Virus Increases Macrophage Survival to Promote Parasite Persistence”, Cell Host Microbe 20:  318-28.

Mandell, M.A., and S.M. Beverley (2016), “Concomitant immunity induced by persistent Leishmania major does not preclude secondary re-infection: implications for genetic exchange, diversity and vaccination”, PLoS Neglected Tropical Diseases 10(6):  e0004811.

Adaui, V., L-F. Lye, N. S. Akopyants, M. Zimic, A. Llanos-Cuentas, L. Garcia, I. Maes, S. De Doncker, D. E. Dobson, J-C. Dujardin, J. Arevalo & S.M. Beverley (2016),  “Association of endobiont dsRNA virus LRV1 with treatment failure of human leishmaniasis caused by Leishmania braziliensis in Peru and Bolivia”, J. Infectious Diseases 213: 112-21.

Ives, A., C. Ronet, F. Prevel, G. Ruzzante, S. Fuertes-Marraco, F. Schutz, H. Zangger, M. Revaz-Breton, L-F. Lye, S.M. Hickerson, S.M. Beverley, H. Acha-Orbea, P. Launois, N. Fasel and S. Masina (2011), "Leishmania RNA Virus controls the severity of LeishmaniasisScience 331: 775-778.

Lye, L-F., K. L. Owens, H. Shi, S.M.F. Murta, A.C. Vieira, S.J. Turco, C. Tschudi, E. Ullu, and S.M. Beverley (2010), “Retention and loss of RNA interference pathways in Trypanosomatid protozoans”, PLoS Pathogens 6:  e1001161.

Akopyants* N, Kimblin* N, SecondinoN, Patrick R, Lawyer P, Dobson DE, Beverley* SM and Sacks* DL. "Demonstration of genetic exchange during cyclical development of Leishmania in the sand fly vector”, Science 2009 324: 265-268. (*1st two authors contributed equally; last two authors contributed equally). (See commentary “Leishmania exploit sex”, Miles MA, Yeo M and Mauricio IL. Science 2009 324: 187-189).

Madeira da Silva L, Owens KL, Murta SFM and Beverley SM. "Regulated expression of the Leishmania major surface virulence factor lipophosphoglycan using conditionally destabilized fusion proteins." Proc. Natl. Acad. Sci. USA 2009 106: 7583-7588.