Dr. Francis Nano

 

Research of Dr. Francis E. Nano
PhD Illinois

Nano

Engineering microbial pathogens to temperature sensitivity.

Temperature-sensitivity has been used for decades as a way of attenuating viruses which can then be used as vaccines.  For example the Sabin polio vaccine is a cold-adapted version of the polio virus, and the recently released FluMist® vaccine is a cold-adapted version of the influenza virus. 

In bacterial genetics the generation of temperature sensitive (TS) mutants has long been used as a way of studying essential genes, as the temperature sensitivity can be used as a conditional lethal state.  However, TS mutants of bacteria have only been used as vaccine strains for a limited number of veterinary pathogens.  The growth of the number of sequenced genomes and the analysis of several genomes using saturation transposon mutagenesis has advanced our knowledge of bacterial essential genes so that we can readily identify essential genes in bacteria that have not been extensively analyzed.

The growth of our understanding of essential genes has allowed us to use them as tools in engineering bacterial pathogens.  We have been able to identify essential genes in psychrophilic (cold-loving) bacteria and introduce them into the genomes of pathogenic bacteria, replacing the “native” version of the essential gene.  In many cases the resulting pathogenic bacterium is TS.  Importantly, most of the TS strains that we produce through the introduction of a psychrophilic essential gene are stable, that is, they do not revert to a form that can grow at a higher temperature.  For example, when we introduced the DNA ligase gene from Pseudoalteromonas haloplanktis into the chromosome of Francisella novicida the resulting F. novicida strain did not grow at 37°C, and did not “revert” to a form that can grow at 37°C at a detectable level. 

Although the substitution of a psychrophilic gene into the chromosome of a mesophile (“medium temperature-loving”) can sometimes be simple, more often than not we have to adjust the nature of the foreign gene to make it work properly.  Often we will change the codon usage of the gene to make it function.  We will also measure the strength of the native ribosome binding site (RBS) and engineer the genome to make the in-coming gene have a RBS of the same strength.  We sometimes generate hybrids between the psychrophilic and the mesophilic essential genes, so that they have the proper expression level, but have the TS properties that we desire.

In our initial attempts at making TS bacterial strains we used the mouse pathogen F. novicida, we now seldom use this bacterium as a host for genome engineering.  Currently most of our gene discovery and mutagenesis experiments are done is E. coli, and our target hosts (the pathogens that we want to make TS) are either Salmonella enterica or Mycobacterium tuberculosis

Relevant publications

Nano FE . 2012. Refactoring biological parts, devices and chasses for delivery of therapeutic agents. 2012.  Curr Opin Biotechnol. 23:897-899. doi: 10.1016/

White MD, Bosio CM, Duplantis BN, Nano FE. 2011. Human body temperature and new approaches to constructing temperature-sensitive bacterial vaccines. Cell Mol Life Sci. 68:3019-3031.

Duplantis BN, Bosio CM, Nano FE. 2011. Temperature-sensitive bacterial pathogens generated by the substitution of essential genes from cold-loving bacteria: potential use as live vaccines. J Mol Med (Berl). 2011 89:437-444.

Duplantis BN, Osusky M, Schmerk CL, Ross DR, Bosio CM, Nano FE. 2010. Essential genes from Arctic bacteria used to construct stable, temperature-sensitive bacterial vaccines. Proc Natl Acad Sci U S A. 107:13456-13460.