Goldilocks and the Three Phenotypes: Insights from the Conservation of Mycobacterial Pathogenesis between Zebrafish and Humans

Earlier this month, I had the good fortune to attend a research seminar by Lalita Ramakrishnan, a microbiologist from the University of Washington.  Ramakrishnan and colleagues have been studying the infection of larval zebrafish by Mycobacterium marinum.  M. marinum is a pathogen for a variety of marine and freshwater ectotherms and shares important genomic features with Mycobacterium tuberculosis (Mtb).  The infection of zebrafish larvae by M. marinum recapitulates critical aspects of human infection by Mtb (Tobin and Ramakrishnan, 2008).  In fact, with respect to the hallmark pathological structure of human infection by Mtb, the granuloma, the infection of zebrafish by M. marinum is more similar to human infection by Mtb in some respects than is mouse infection by Mtb (Tobin and Ramakrishnan, 2008; Ramakrishnan, 2012).

In 2010, Tobin et al. (Cell) used a genetic screen of zebrafish to identify a polymorphism at the ortholog of the human leukotriene A₄ hydrolase (lta4h) locus. Variation at this locus controls expression of the corresponding gene product, which is the final enzyme (LTA4H) in the synthesis of leukotriene B₄ (LTB₄) from leukotriene A₄ (LTA₄), a mediator that in zebrafish acts as a macrophage chemoattractant and inducer of increased production and secretion of the inflammation-associated cytokine, tumor necrosis factor alpha, TNFα.  The identified lta4h mutation decreased expression LTA4H, thereby favoring increased production of lipoxin A₄ (LXA₄), instead of LTB₄, from the substrate, LTA_4.  LXA₄  has been associated with decreased inflammation-associated effects, such as reduced neutrophil recruitment.  Increased production of LXA₄ is also associated with decreased TNF production and increased severity of mycobacterial infection in zebrafish.

In human studies in a Vietnamese population, heterozygosity for two lta4h single nucleotide polymorphisms (SNPs) was associated with lower incidence of and greater resistance to Mtb for pulmonary and meningeal tuberculosis than homozygosity for these SNPs (Tobin et al., 2010).  Of special interest was the fact that one homozygous state was associated with low TNF production and the other homozygous state was associated with high TNF production.  The heterozygous state was associated with a level of TNF production that was intermediate, i.e. “just right” for optimizing the clearance of mycobacteria, thereby forcefully illustrating the danger of assuming that if a particular molecule is associated with a desirable phenotype, such as clearance of a pathogen, that more is necessarily better.  For many biological phenomena, the best outcomes are associated with an optimum, as opposed to an extreme (high or low).

In the most recent paper in the series (Cell, 2013), Roca and Ramakrishnan elucidate the cell and molecular biological mechanisms connecting the levels of TNF production with the pathogenetic events that dictate the outcome of mycobacterial infection in zebrafish.  To summarize briefly the very substantial body of work detailed in this report, sub-optimal or supra-optimal production of TNF results in greater death of infected macrophages with increased pathogen replication and spread within the host.  High levels of TNF production lead to increased generation of reactive oxygen species (ROS) that initially facilitates killing of the mycobacteria but can also increase macrophage death leading to extracellular release of surviving mycobacteria and increased net mycobacterial replication if the host cell dies too quickly.

In the context of high TNF production, Roca and Ramakrishnan also delineate two pathways that lead to macrophage death by the process known as programmed necrosis, or necroptosis.  One pathway involves the mitochondrial protein cyclophilin D.  The second involves ceramide production by lysosomal acid sphingomyelinase.  As a bonus, two existing drugs were identified that each inhibit one of these two pathways and synergize to reduce bacterial counts in TNF-high but not wild-type zebrafish and confer increased resistance to mycobacterial infection.

So, the study of the interaction of zebrafish with M. marinum has helped not only to illuminate key aspects of the pathogenesis of Mtb infection in humans, but has also suggested a possible new chemotherapeutic approach to the human infection in the context of high TNF production.  The authors speculate that it could also prove useful to evaluate one or both of these therapeutic agents in the setting of other diseases, such as rheumatoid arthritis, ankylosing spondylitis, and Crohn’s disease, in which excess TNF production plays a central role.

In summary, this research dramatically exemplifies the potential value that can come from exploring the biology of relativey phylogenetically-distant species.  We now also have another instructive example of a balanced polymorphism, such as the one associated with the β-hemoglobin locus, in the human gene pool.  Interest in elucidating the sources of selection on the pertinent lt4ah polymorphism should  prompt future studies as such investigations will likely add important new insights regarding the mechanisms by which evolution shapes human disease susceptibility.

References

Tobin DM, Ramakrishnan L. Comparative pathogenesis of Mycobacterium marinum and Mycobacterium tuberculosis. Cell Microbiol. 2008 May;10(5):1027-39. doi: 10.1111/j.1462-5822.2008.01133.x. Epub 2008 Feb 20. Review. PubMed PMID: 18298637.

Ramakrishnan L. Revisiting the role of the granuloma in tuberculosis. Nat Rev Immunol. 2012 Apr 20;12(5):352-66. doi: 10.1038/nri3211. Review. PubMed PMID: 22517424.

Tobin DM, Vary JC Jr, Ray JP, Walsh GS, Dunstan SJ, Bang ND, Hagge DA, Khadge S, King MC, Hawn TR, Moens CB, Ramakrishnan L. The lta4h locus modulates susceptibility to mycobacterial infection in zebrafish and humans. Cell. 2010 Mar 5;140(5):717-30. doi: 10.1016/j.cell.2010.02.013. PubMed PMID: 20211140; PubMed Central PMCID: PMC2907082.

Roca FJ, Ramakrishnan L. TNF dually mediates resistance and susceptibility to mycobacteria via mitochondrial reactive oxygen species. Cell. 2013 Apr 25;153(3):521-34. doi: 10.1016/j.cell.2013.03.022. Epub 2013 Apr 11. PubMed PMID: 23582643.