Tuberculosis, caused by Mycobacterium tuberculosis, remains an enormous global health problem that has worsened in recent years both with the syndemic with HIV and the emergence of Multi-Drug Resistant (MDR) and Extensively-Drug Resistant (XDR) TB strains. Although M. tuberculosis was the first pathogen for which Robert Koch in 1882 designed the principles to establish a causative relationship between a microbe and a disease, knowledge about much of the basic biology of M. tuberculosis was hampered without the ability to transfer genes in mycobacteria. Using viruses that infect mycobacteria (also called mycobacteriophages), Dr. Jacobs and his lab developed genetic tools that enabled plasmid transformation, transposon mutagenesis, and allelic exchanges in M. tuberculosis. These tools provided the means to elucidate the mechanisms of action of TB drugs, the discovery of the mechanisms of attenuating mutations in BCG and other attenuated M. tuberculosis strains. In addition to answering unknowns of M. tuberculosis biology, genetic tools have allowed for the discovery of numerous new properties associated with the biology of M. tuberculosis infection of host cells.
Developing New Strategies to Kill Persistent M. tuberculosis Cells
One of the greatest challenges to improve TB control strategies is discovering ways to kill persistent M. tuberculosis cells. Persistence is the phenomenon in which a subpopulation of cells is refractory to killing by a bactericidal drug or an immune effector. For M. tuberculosis, when the bactericidal drug Isoniazid (INH) is added to exponentially growing cells we observe a 99 to 99.9% decrease in viable cells in 3 to 4 days. The 0.01 to 0.1% of viable cells cannot be killed over the next 3 or 4 days. Sequence analysis reveals these “perisister” cells are not genetically resistant, but have entered into an adaptive stress response and express tolerance to killing. We have a number of projects aimed at both defining the physiological nature of these cells and finding ways to kill them.
- Transcriptional Analyses of Persister tuberculosis and Development of Persister Reporter Mycobacteriophages: We have performed transcriptional analyses of comparing the persister cells that survive Isoniazid killing to cells that are actively killed by Isoniazid and found a group of stress response genes that are highly upregulated in persister cells. We reasoned that we could utilize the promoters that are up regulated in persister cells by fusing them to the gene encoding the red fluorescent protein and incorporating them into our existing reporter phage Φ2GFP10. We hypothesize that if we use such a phage to infect a population of M. tuberculosis cells they would initially turn the cells green or yellow. After the addition of the drug Isoniazid we reasoned that the green or yellow cells would be killed in the first 4 days and only red cells would be left. We have observed such phenomenon and are currently using this reporter phage to identify persisters in populations of M. tuberculosis cells in vitro and in vivo.
- High Doses of Vitamin C Sterilizes a Culture of tuberculosis: We have discovered that a 4 mM concentration of Vitamin C will sterilize cultures of M. tuberculosis. These high doses are not readily obtainable by oral injection, but this finding is proof of principle that it is possible to kill active tuberculosis as well as persistent M. tuberculosis cells. Active investigations are already underway to elucidate the mechanism by which Vitamin C leads to the killing of persistent M. tuberculosis cells.
- Methionine Starvation Sterilizes Cultures of M. tuberculosis Cells: Previous studies have demonstrated the phenomenon that auxotrophic mutants of tuberculosis fail to grow in mammalian hosts. This phenomenon cam as a surprice for a a leucine auxotroph since many pathogens, such as Legionella or Francisella, that are naturally occurring leucine auxotrophs grow perfectly fine in mammals. We have continued to examine the growth abilities of many auxotrophic mutants of M. tuberculosis. In addition, we have examined the death kinetics of M. tuberculosis when starved for various amino acids or vitamins and found that most starvations are bacteriostatic events that cause the TB to shut down. Dr. Michael Berney has discovered that the methionine starvation of M. tuberculosis leads to the rapid sterilization of an M. tuberculosis culture. We hypothesize enzymes of methionine biosynthetic pathway will be excellent drug targets and are currently exploring this possibility.
- Acid-Fastness of Tuberculosis Correlates With Latent Infection and is Regulated by a Signal Transduction Pathway: The acid-fast stain developed independently by Drs. Ziehl and Neelsen in 1883 and 1884, respectively, has been the defining characteristic for diagnosing Tuberculosis for over a century. We discovered that when the kasB gene is deleted from M. tuberculosis the mutant fails to stain acid-fast, and can cause a truly latent in an infected mouse. Furthermore, we have demonstrated that while this infection with this strain is controlled in an immunocompetent mouse, it is uncontrolled in immunocompromised mice (mice that lack T and B cells). Thus, M. tuberculosis strains that lack the acid-fast quality are models of persistent M. tuberculosis infections in vivo. In collaboration with Dr. Laurent Kremer at Université Montpellier, we have discovered that the KasB activity is regulated by a signal transduction pathway. Current efforts are underway to identify the signals that are sensed by M. tuberculosis in vivo to enter into acid-fast negative state.
Developing a More Efficacious Vaccine
The Jacobs lab continues to use genetic screens to characterize immune and evasion strategies of M. tuberculosis, with the goal that understanding this will lead to a more efficacious TB vaccine. The existing BCG vaccine is attenuated due to the loss of a specialized Type VII secretion system called ESX-1. These Type VII secretion systems are primarily found in mycobacteria and M. tuberculosis has five paralogs including ESX-1. We are systematically deleting each of these and have recently discovered that the ESX-3 system is involved in separable functions including iron acquisition and virulence. We are exploring the potential of this deletion mutant as a vaccine candidate as well as understanding its role in virulence.
In addition to the Type VII secretion systems, the Jacobs lab continues to use the techniques of specialized transduction to systematically delete each of the individual genes of M. tuberculosis in high-throughput strategies. This set of mutants contains barcodes that allow for high-throughput screening methodologies and the Jacobs lab focuses on identifying the genes involved in functions that evade innate or adaptive immune systems of mammals. This knowledge should contribute to a more efficacious TB vaccine.
HSV-2 ΔgD: A Novel Vaccine Mutant for Herpes Infections and a Recombinant Vaccine Vehicle
Herpes simplex virus (HSV) infections are significant health problems that globally and disproportionally impact developing countries. HSV-1 is the leading cause of sporadic infectious encephalitis and corneal blindness and both serotypes are major causes of genital ulcerative disease. Infection with HSV-2 significantly increases the likelihood of acquiring and transmitting HIV, while perinatal transmission of either serotype often leads to severe infant morbidity. The World Health Organization estimated that over 500 million people are infected with HSV-2 worldwide with approximately 20 million new cases annually. The extremely high prevalence of HSV-2 in sub-Saharan Africa (∼70%) may contribute more to the spread of HIV-1 than number of sex partners or other sexually transmitted infections. Moreover, as HSV establishes latency in neurons with frequent subclinical or clinical reactivations, there is a lifelong impact of infection.
Dr. Jacobs has initiated a collaboration with Dr. Betsy Herold at Einstein in which the vaccine candidate they developed for HSV represents a completely new and different paradigm from earlier vaccine efforts and elicits an immune response distinct from that observed with natural infection. We deleted the gene for gD from HSV-2 to generate a novel candidate vaccine. The vaccine strain causes no disease in wild-type or immunodeficient mice (SCID) and could not be detected in dorsal root ganglia (DRG), the site of HSV latency. The vaccine protected multiple strains of mice from skin and vaginal lethal challenge with clinical isolates of both serotypes (HSV-2 and HSV-1) and prevented the establishment of latent infections in DRG. We have further characterized the sterilizing immunity in the skin and found a correlation of antibody infiltration of an isotype known to mediate FcR mediated functions with corresponding innate FcR containing effector cells. We are currently testing this in the guinea pig model of herpes infections as well as using the mouse to elucidate the mechanisms of the robust protection. We also are exploring the possibility of using this vaccine as a vaccine vector platform for generating vaccine for HIV and TB.
Grants from the National Institutes of Health provided support for genetic tool development and isoniazid research.
As of May 3, 2016