Monday, November 21, 2011

Bacteria: nanotech freaks

Nano dimension: graphene cells
Nanotechnology is considered the next "miracle potion" specially if you add the bio term: nanobiotechnology. However, it only refers to the possibility of working at the molecular sclae (100 nanometers). New materials have been created and there are already a lot of promising nano methods that will help to diagnose and treat diseases faster and perhaps even better than before. But, like many other times, we are just re-discovering the wheel. Biological systems are inherently nano in scale. Actually, all living things, including humans, can be considered as nanofactories. Evolution has optimized these nanofactories over millions of years and just now, in the 21st century, we are tapping into this nano-world. 

Who are the experts on this field? The first ones on the ranking are not renowned scientsits but... pathogenic microorganisms. Viruses and bacteria use the most sophisticated nanotools to subvert the machineries of cells to survive. Prominent examples are the systems used by viruses to inject DNA into cells or the systems that bacteria employed to deploy proteins into cells, chiefly the so-called type III secretion systems (T3SS).

T3SS is a complex nanomachine that allows Gram-negative bacteria to deliver proteins, called effectors, across eukaryotic cellular membranes. The system is evolutionary related to the flagelum apparatus, the system that bacteria used for motility. The T3SS system is formed by at least 30 proteins and the hallmark of the system is the needle, also called injectisome, through which proteins are translocated into the cytosol of the host target cell. Quite soon it was perceived that the T3SS was functionally similar to a syringe and, actually, electron microscopy studies demonstrated that, indeed, the system looks like a syringe.

Salmonella T3SS apparatus
Many bacterial pathogens possess a T3SS and the most studied are those from the human pathogens Shigella, Salmonella, Yersinia, Escherichia, Vibrio, Pseudomonas, and from the plant pathogens Erwinia, Ralstonia and Xanthomonas. Most of the research has focused, on the one hand, on the study of the structural components of the T3SS apparatus and, on the other hand, on the identification of the effectors translocated by the system. However, in most cases it is still unknown the signal that triggers the activation of the T3SS and hence the delivery of proteins. In vitro, secretion can be induced by lowering the concetration of calcium (Yersinia and Pseudomonas) or by adding the dye Congo Red (Shigella). Notably, elegant studies from Hans Wolf-Watz laboratory (Science, 1996) demonstrated that bacterial contact with the cell for just 5 minutes is enough to activate the Yersinia T3SS to inject proteins into the cells.

The expression of the T3SSs is tightly regulated such way that the system is expressed only when and where needed. For example, Salmonella activate the so-called SPI-2 T3SS only inside the macrophage whereas the SPI-1 T3SS is only required for invasion of the cells (for a recent review read Microbes and Infection, 2001). Several transcription factors control the expression of the T3SS. There are also feedback mechanisms operating and effectors are only produced if the T3SS apparatus is assemble and functional.

Function of the effectors.
A very active area of research is the characterization of the T3SS effectors. New effectors are found nearly every month. Nevertheless, several general principles can be formulated. Nearly all T3SS deliver effectors that alter the cell cytoskeleton often by targeting the actin filaments. Mechanistically, these effectors modulate the activity of the Rho family GTPases. Interestingly, these proteins play an important role in tumor metastasis. Other effectors are devoted to counteract the activacion of signaling cascades dependent on the transcriptional factor NF-kB and the activation of the MAP kinases. The endpoint of these signaling cascades is the activation of a host defence program against infections. Therefore, bacteria by using T3SS-delivered effectors block the activation of this program. It should be noted that bacteria employ more than one effector to abrogate the activation of these signaling cascades hence demonstrating the critical role of this program for host defense.

Nanobiotechnological applications.
Quite soon it was perceived that T3SS can deliver effectors to a wide range of cells. However, it took a while to manipulate the T3SS in order to inject heterologous proteins. Firstly, it was discovered how effectors were delivered. Secondly, scientists identified the minimal part of the effector necesary for its delivery, the so-called T3SS signal. This knowledge allowed Holger Rüssmann and co-workers to manipulate Salmonella T3SS to inject viral epitopes to cells of the immune system (Science, 1998). Subsequent studies demonstrated that peptides of interest fused to proteins bearing the T3SS signal indeed elicit protective immune responses. Now bacteria-based vectors are being investigated as optimal vehicles for antigen delivery by modified T3SS. But one could think of other applications such as the delivery of drugs to tumor cells or the injection of biologically active molecules such as cytokines. Supporting this, Chamekh and co-workers (Journal Immunolgy, 2008) were able to modify the Shigella T3SS to inject the anti-inflammatory cytokine IL-10 to immune cells. T3SS-delivered IL10 was active in vivo and reduced the inflammatory response in infected animals hence opening new avenues for the use of modified T3SS to deliver proteins for immunomodulation or gene therapy purposes. 

A bright future ahead for nanobactotechnology!!


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