Monday, January 9, 2012

Illuminating virulence

The advent of molecular methods made necesary to revise the Koch´s postulates, formulated in 1890, as general guidelines that should be followed to identify pathogens causing diseases. As a result, Stanley Falkow established the molecular version of Koch's postulates to guide the identification of microbial genes encoding virulence factors. Falkow established five experimental criteria that a gene must fulfill to be considered a virulence factor. A criterium almost never addressed by scientists is "The gene, which causes virulence, must be expressed during infection." It has been always considered enough to test whether specific inactivation of the gene is associated to a measurable loss of virulence. Actually, the golden standard is to demonstrate in vivo (using suitable animal/plant models) that allelic replacement of the mutated gene leads to restoration of virulence.

However, wouldn´t it be interesting to know exactly when and where the virulence gene is expressed? Certainly this may help to understand the in vivo role of the virulence factor: is it required only for the initial colonization of the tissues? Is it necesary to fight phagocytic cells?, is the expression of this factor coordinated with those of other factors?,...Moreover, are there virulenece factors expressed only in vivo and therefore absolutely dispensable in vitro? These questions led scientists to develop experimental approaches to enlighten virulence gene expression. Conceptually, the methods were originally conceived upon the premise (now considered fact) that most virulence genes are transcriptionally induced at one or more times during infection

DFI work-flow
One of the first experimental strategies was designed by Raphael Valdivia and Stanley Falkow (Science, 1997) taking advantage of the green fluorescent protein. The strategy, differential fluorescence induction (DFI), is elegant and simple. To detect gene expression, they cloned downstream of the gene promoter region a reporter gene, in this case gfp, such way than when the gene is expressed bacterial will become fluorescent. This promoter trap strategy allows isolation of bacteria bearing transcriptionally active gfp promoter fusions directly from infected cells or animal tissues by flow cytometry. As isolation of single bacterial cells from tissues is based on fluorescence, the selection scheme allows for the collection of bacteria bearing promoter elements of various strengths. Above all, as bacteria can be analyzed directly by flow cytometry, gene expression levels can be compared between host and nonhost environments with single cell resolution.

DFI has been succesfully applied to identify genes expressed in hots environments from important pathogens such as Salmonella, Streptococcus pneumoniae, Staphylococcus aureus, Mycobacterium tuberculosis and Brucella. However, DFI is not without its pitfalls mostly derived of the used of gfp as reporter gene. For example, high levels of GFP  should be avoided to prevent nonspecific adverse effects on bacterial fitness. In addition, GFP is notoriously resistant to degradation, turnover of the GFP is largely dependent on signal dilution by bacterial replication. Therefore, in some instances a reduction in replication rates might be misinterpreted as an increase in promoter activity. This latter issue has been largely resolved with the use of de-stabilized versions of GFP.

IVET variations
John Mekalanos laboratory designed a genetic system, termed in vivo expression technology (IVET), that uses an animal as a selective medium to identify genes that pathogenic bacteria specifically express when infecting host tissues (Science, 1993; Proceedings National Academies of Science USA, 1995). IVET is another promoter-trap based strategy using different selectable reporter genes such as a gene mediating antibiotic resistance. This is perhaps the IVET variant that has been most widely used. Salmonella was the first pathogen studied using IVET and now more than 15 pathogens have been analyzed. The disadvantage of the initial IVET system is that it demands that the gene is expressed throughout growth in a particular environment, so this approach would not detect genes that are transiently turned on during adaptation to a new environment.

To circumvent this issue, Andrew Camilli and co-workers designed an IVET variant, called recombination-based IVET (RIVET), based upon a reporter "switch" that results in a permanent inversion (Cell, 1999). In this case, the reporter gene is a resolvase gene such as tnpR from Tnγδ that mediates site-specific DNA recombination. Prior to this step, a gene cassette that serves as the substrate for resolvase is placed at a neutral site in the bacterial genome. Typically, the substrate is an antibiotic resistance gene flanked by resolvase recognition sequences. A gene fused to tnpR results in resolvase production, whose action results in the permanent excision of the antibiotic marker (a reaction termed resolution). This event marks the bacterium by endowing it with an inheritable antibiotic-sensitive phenotype. Resolved strains are then screened for (by replica plating of colonies) after recovery of the bacteria from infected tissues. 

RIVET has been used to analyze the temporal and spatial patterns of virulence gene induction during infection and to dissect the regulatory requirements of in vivo induction with respect to both bacterial regulatory factors and host-inducing environments. Perhaps the main limitation of RIVET to study induction of virulence genes in vivo is that only the initial induction of a gene can be assayed, since resolution is irreversible, and thus expression at later times or within downstream host tissues cannot be detected.

Concluding remarks.
The beauty of DFI, IVET and RIVET is that a live host, with tissue barriers and immune system intact, is used to signal induction of virulence genes. Genetic trickery is then used to identify the in vivo-induced genes. As is true of all genetic screens and selections, these approaches do have limitations. The most significant of these is that the relative level and timing of transcription of an in vivo-induced gene largely dictates whether the gene will be identified in a particular selection or screen. In other words, DFI, IVET and RIVET are not unbiased approaches.

These approaches were designed for the identification of virulence factors and indeed have had tremendous impact on the microbial pathogenesis field. But these methodologies have also led to the discovery of new antigens useful as vaccine components. DFI and RIVET facilitate the isolation of mutations in genes involved in virulence and, therefore, should aid in the construction of live-attenuated vaccines. In addition, the identification of promoters that are expressed optimally in animal tissues provides a means of establishing in vivo-regulated expression of heterologous antigens in live vaccines, an area that it is very problematic. Finally, these methodologies have uncovered many biosynthetic, catabolic, and regulatory genes that are required for growth of microbes only in animal tissues. Evidently, these gene products provide new targets for antimicrobial drug development.

More than 20 years have passed after the advent of these thechnologies and still we have not exploited their full potential and applications. Furthermore, these approaches have so far been used exclusively for investigating pathogen-host interactions, but they should be easily adaptable to the study of other processes such as symbiosis or the impact of antibiotic treatment on the expression of virulence genes in vivo. The light is still on...


  1. This contains some relevant information. But, I wonder why the picture of green lantern seems lost on this post. Anyway, I can say exploiting the full potential and applications of this kind of new technology is really hard to achieve. It takes a lot of time.

    1. Thanks for the comments. Concerning the is a blogger related issue. And coming to the aplications. There are already applivcations around the corner. Some of the genes identified by these approaches are currently used to generate attenuated bacterial strains as carrier vector for vaccines. it takes more or less one year to run these type of screenings so it is not very much time consuming. Nevertheless, I do agree with you that we need to put our efforts to get the most of the information already generated.