Monday, August 31, 2009

Protein census of Leptospira interrogans

A census of proteins in a bacterial cell was conducted for the first time ever. By "census," I don't mean merely identifying all cellular proteins (which can be accomplished by shotgun tandem mass spectrometry). What I mean is counting the number of copies of every protein. The bacterium targeted for the census was the spirochete Leptospira interrogans. Like the census conducted here in the U.S. every ten years, some proteins were missed. The strategy developed by Malmström and colleagues, as described in the August 6 issue of Nature, allowed them to determine the abundance of 1,864 (or 83%) of the 2,221 proteins that were detectable by tandem mass spectrometry (MS) in Leptospira interrogans that had been grown in standard Leptospira culture medium.

The results of the protein census are compiled in the bar graph below. Proteins with related biological functions were grouped together and are color coded. The "Proteome" bar tabulates the number of different proteins in each group.
The next bar, "Copies per cell (control)," gives you an idea of how much of the protein expression machinery in L. interrogans is directed towards the synthesis of proteins in each functional category. The percentage reflects the amounts and size of the proteins in each category. For example, proteins of unknown function (hypothetical proteins) represent only 12.7% (blue) of the total protein synthesis capacity even though they constitute 30% of the identified proteins and over 40% of all genes in L. interrogans. I would surmise that these hypothetical proteins would account for a more sizable fraction of total protein synthesis under some other condition that L. interrogans would encounter during its life cycle (e.g., during infection).

The other observation noted by the authors is that L. interrogans gears 15% of its protein synthesis effort to make a small number of proteins deemed to be components of the "external encapsulating structure" (green), which is a fancy Gene Ontology term encompassing abundant Leptospira proteins that have been demonstrated to be in the inner or outer membrane. Most of the 15% is accounted for by five outer membrane proteins: LipL32, Loa22, LipL41, LipL21, and LipL36, the functions of which are not entirely clear. The five proteins are among the 10 most abundant proteins in L. interrogans.

The last bar shows the effect of the antibiotic ciprofloxacin ("cipro") on global protein levels in L. interrogans. The most striking change is the massive increase in 15 proteins of unknown function (light blue) leading them to constitute ~20% of the total protein content. As ciprofloxacin is an inhibitor of DNA gyrase, transcription of the genes encoding the 15 proteins may be extremely sensitive to DNA topology.

Did the enormous increase in the copy number of the 15 proteins following ciprofloxacin treatment increase the total number of protein molecules in L. interrogans? The authors found little change in the total cellular protein content:
Interestingly, this large redistribution of the proteome did not significantly change the total cellular protein concentration. Therefore, the large increase in the abundance of [the 15 proteins of unknown function] after ciprofloxacin exposure was compensated by a slight reduction of other high abundant protein classes.... This indicates that in L. interrogans, the cells strive to maintain a certain total number of protein components, that is, a constant cellular proteome concentration.
Featured paper

Malmström, J., Beck M., Schmidt, A., Lange, V., Deutsch, E.W., and Aebersold, R. (2009). Proteome-wide cellular protein concentrations of the human pathogen Leptospira interrogans. Nature 460(7256):762-765. DOI: 10.1038/nature08184

Thursday, August 20, 2009

Zebrafish model of leptospirosis: Where's the relevance?

Scrutinized for the past several decades as a model of embryonic development, the zebrafish has recently been promoted as a vertebrate model for investigating the pathogenesis of infectious diseases. Zebrafish embryos are transparent, allowing microbiologists to readily view the course of infections in real time. Another advantage of the zebrafish model is that it's amenable, at least in theory, to large-scale genetic screens for mutated host or microbial genes that affect the infection process.

Davis and colleagues observed the early stages of zebrafish embryo infection by the spirochete Leptospira interrogans, as described in a recent issue of PLoS Neglected Tropical Diseases. They injected 10-100 spirochetes into the hindbrain of zebrafish embryos at 30 hours post-fertilization, when the innate immune response is fully functional. Macrophages rushed to the hindbrain and engulfed the invaders within the first 4 hours following inoculation of the spirochetes. L. interrogans was also rapidly phagocytosed when injected into the caudal vein.

Figure 1a from Lesley and Ramakrishnan, 2008. A zebrafish embryo 30 hours following fertilization. The hindbrain and caudal vein are indicated with the bracket and arrow, respectively.

Macrophages typically kill and destroy their prey following phagocytosis. However, spirochetes were still observed in the macrophages 24 hours following inoculation, suggesting that L. interrogans can survive inside macrophages.

Their most striking observation was the accumulation of infected macrophages near the dorsal aorta in a region known as the aorta-gonad-mesonephros (AGM), where hematopoietic stem cells are born. This was not a general phenomenon of bacterial infections as macrophages harboring Pseudomonas aeruginosa failed to accumlate at the AGM following its injection into zebrafish embryos.

Figures 2C and 2D from Davis et al., 2009. The embryo was infected with fluorescently stained L. interrogans. 24 hours following infection, most of the spirochetes were found near the dorsal aorta (brackets), with a few scattered around the ventral tail. Scale bar, 300 µm in panel C, 100 µm in panel D.

Here's what the authors concluded in the final sentence of the paper:
The strikingly specific delivery of leptospires to [the AGM] by phagocytes provides insights into pathogenesis by suggesting a novel mechanism for targeting of organs during leptospiral dissemination.
In other words, L. interrogans may be capable of steering macrophages towards specific organs. Once the macrophages arrive at their destination, the spirochetes may escape from the macrophage and colonize the organ.

The challenge for the authors in future studies will be to demonstrate the relevance of the zebrafish model to leptospirosis. Hamsters and guinea pigs are appropriate models for leptospirosis because the pathology and lethality of Leptospira infection in these rodents is similar to what's observed in human leptospirosis patients. The fate of Leptospira that macrophages capture in these rodents differs from what is seen in zebrafish embryos. Leptospira that are found inside macrophages in tissue sections from infected rodents often appear to be disintegrating. Nevertheless, it's possible that a few Leptospira survive phagocytosis and subsequently guide the macrophage towards the target organs.

Featured paper

Davis, J.M., Haake, D.A., & Ramakrishnan, L. (2009). Leptospira interrogans stably infects zebrafish embryos, altering phagocyte behavior and homing to specific tissues PLoS Neglected Tropical Diseases, 3 (6) DOI: 10.1371/journal.pntd.0000463

Other references

Lesley, R. and Ramakrishnan, L. (2008). Insights into early mycobacterial pathogenesis from the zebrafish. Current Opinions in Microbiology 11(3):277-283. DOI: 10.1016/j.mib.2008.05.013