About Josephine Clark-Curtiss
Dr. Clark-Curtiss initiated her research career during her Ph.D. studies at the Medical College of Georgia, Augusta, GA. She then joined the research group of Roy Curtiss III at the University of Alabama at Birmingham for postdoctoral studies, initially analyzing the genetic bases for transfer of antibiotic resistance (R) plasmids among enterobacteria. With the inception of recombinant DNA technology, she became part of the Curtiss research team that constructed and characterized the first biologically contained “safe” strains c1776 and DP50 of Escherichia coli that, for a time, were the only approved bacterial strains with which investigators throughout the US could conduct recombinant DNA research in the late 1970s. Dr. Clark-Curtiss subsequently utilized recombinant DNA techniques to conduct research on mycobacteria, beginning in the early 1980s, when she and her colleagues constructed the first recombinant DNA libraries of any mycobacterial strain, thereby initiating molecular genetics research on Mycobacterium leprae and later, on M. tuberculosis and M. avium. Dr. Clark-Curtiss and her research group were among the first to identify specific genes that encoded M. leprae proteins and they were the first to demonstrate the presence of a repeated DNA sequence in the chromosome of M. leprae (now known as Rlep, which is used for the diagnosis of leprosy). Using restriction fragment length polymorphism (RFLP) analyses of genomes from M. leprae isolates from all over the world, Dr. Clark-Curtiss showed that the M. leprae genome is remarkably stable genetically. Dr. Clark-Curtiss’ research group was the first to identify genes encoding antigens of M. leprae that were recognized by antibodies in the sera of leprosy patients.
Dr. Clark-Curtiss has a long-standing interest in understanding the mechanisms whereby pathogenic mycobacteria, particularly M. tuberculosis and M. avium, are able to survive and grow in human macrophages, an attribute essential for the pathogenesis of these bacteria. Thus, she and members of her research group developed methods to identify genes expressed by M. avium and M. tuberculosis at different time points after infection of primary human macrophages in culture. Using these methods (Subtractive RNA Hybridization and Selective Capture of Transcribed Sequences [SCOTS]), the Clark-Curtiss group was the first to identify genes from small numbers of mycobacteria in the macrophages in an unbiased way (i.e., without relying on RT-PCR using primers for specific genes), well before the development of microarray technology. In addition to gene expression per se, Dr. Clark-Curtiss has also endeavored to understand some of the mechanisms whereby M. tuberculosis regulates gene expression. During the past 10 years, the Clark-Curtiss research group has conducted studies analyzing regulation of gene expression in M. tuberculosis by four different two-component regulatory systems (TcrRS, PrrAB, DevRS and NarLS) and other transcriptional regulators (the eukaryotic serine-threonine protein kinase PknK and the M. tuberculosis Rel toxin-antitoxin modules) and deciphering the roles of these regulatory systems in host-pathogen interactions. The genes encoding the PrrAB two-component regulatory system were identified during the SCOTS analyses and were subsequently shown to essential for the viability of M. tuberculosis. The PknK serine-threonine protein kinase was shown to be a global regulator of protein synthesis and to play a significant role in regulation of the growth rate of M. tuberculosis.
In collaboration with Roy Curtiss III, the Clark-Curtiss group is developing a safe, efficacious vaccine to protect humans against infections by M. tuberculosis, using recombinant, attenuated Salmonella vaccine (RASV) strains to deliver M. tuberculosis protective antigens. The RASV strains are engineered to behave like wild type Salmonella as they traverse the mammalian gastrointestinal tract after oral inoculation, but then, using regulated delayed technologies, to begin synthesizing the antigens after the RASVs have colonized internal lymphoid tissues. The RASVs are also engineered to undergo regulated delayed lysis in vivo, to preclude long-term colonization of the immunized host and to release the synthesized antigens into the cytosol of the host cells, to elicit both humoral (antibody) and cell-mediated immune responses. Several of the RASV-M. tuberculosis constructs provide protection in mice against aerosol challenges with virulent M. tuberculosis that is equivalent to or slightly better than that conferred by M. bovis BCG, which is regarded as the “gold standard” for vaccines against M. tuberculosis. The Clark-Curtiss research group is continuing to improve the RASV and is also analyzing the protective capabilities of nine other M. tuberculosis antigens for possible incorporation into candidate RASV-M. tuberculosis constructs.
Tuberculosis has been a scourge to humankind since prehistoric times and remains a serious and significant infectious disease to this day. The World Health Organization estimates that one-third of the world’s population is infected by Mycobacterium tuberculosis, the causative agent of TB, with 9.6 million new cases diagnosed in 2014. Although not all individuals who are infected develop disease, among those who do, more than 1.4 million die each year, making TB the most deadly disease caused by a single bacterial pathogen. Tuberculosis is highly prevalent in those parts of the world where HIV/AIDS occurs and the two infections result in a deadly synergism, with devastating effects on the infected individuals and on the societies in which they live. Morbidity due to chronic respiratory and diarrheal diseases in infancy coupled with malnutrition preclude normal mental development necessary for individuals to achieve self-sufficiency by those who survive to age five.
Tuberculosis can be effectively treated with chemotherapy, but involves a regimen lasting six or more months. Because of the duration of treatment, patient compliance becomes a significant problem. One consequence of non-compliance is the development of drug-resistant strains. In recent years, there has been an increasing number of cases of TB caused by M. tuberculosis strains that are resistant to two or more of the first-line antibiotics used for treatment (multi-drug resistant or MDR strains) and even more alarmingly, strains that are resistant to all of the first-line drugs, plus three or more second-line drugs (extensively drug-resistant or XDR strains). Disease caused by MDR or XDR strains is difficult to impossible to treat, especially in areas of the world with limited access to medical care or antibiotics.
Although there is a vaccine against M. tuberculosis (M. bovis BCG) that is used in many parts of the world to protect infants and young children from serious complications of TB, protection is not long-lasting and by the time individuals reach adolescence, they are fully susceptible to infection. Thus, there is a real need for a better vaccine against M. tuberculosis, preferably one that will confer long-lasting protection against infection. The literature is replete with documentation of benefits that could accrue either by widespread use of existing vaccines against numerous bacterial and viral pathogens or by development of new safe, efficacious vaccines.
The Clark-Curtiss research group currently has two foci of research interests:
(1) Understanding mechanisms of M. tuberculosis pathogenesis through (a) analyses of M. tuberculosis gene expression, (b) identification of operational metabolic pathways during growth in human macrophages and dendritic cells and (c) regulation of gene expression.
To address the first research focus, we identified genes of M. tuberculosis that are expressed at different times after infection of cultured human macrophages, enabling us to identify metabolic pathways that are functional at those times. We have also characterized several classes of regulatory systems that control gene expression, both in vivo and in vitro (two-component regulatory systems, serine-threonine protein kinases and toxin-anti-toxin regulatory modules).
(2) Development of an effective vaccine against M. tuberculosis using recombinant attenuated Salmonella vaccine delivery systems producing M. tuberculosis antigens.
Based on the belief that immunization to protect individuals from infection is superior to the continued development of new antibiotics to combat bacterial pathogens that inevitably acquire resistance to currently available or newly designed antibiotics, we are using recombinant attenuated Salmonella vectors as vaccines (RASVs) to deliver M. tuberculosis antigens to elicit protective immune responses. We use RASVs because (a) Salmonella can elicit mucosal, antibody and cell-mediated immune responses in immunized individuals, (b) much is known about the genetics and physiology of Salmonella, which enables us to rationally design RASVs that are completely attenuated, but can target specific organ, cells and cellular compartments to enhance immune responses and (c) RASVs can be delivered orally, thereby precluding the use of needles or need for refrigeration for transportation of the vaccines to remote geographic locations.
We have constructed RASVs that display regulated delayed lysis and regulated delayed antigen synthesis following immunization. We have introduced plasmids with genes encoding three immunodominant antigens of M. tuberculosis (Early secreted antigenic target 6 kDa [ESAT-6], culture filtrate protein 10 [CFP-10] and Antigen 85A (Ag85A) into the RASVs and used these RASV-M. tuberculosis constructs to orally immunize mice. Mice immunized with RASVs producing these three M. tuberculosis antigens have been protected as well as or slightly better than mice immunized subcutaneously with M. bovis BCG against aerosol infection with virulent M. tuberculosis. We have demonstrated that the RASV- M. tuberculosis vaccine elicits significant antibody and cellular immune responses that contribute to protection against M. tuberculosis infection. We continue to modify our RASV strains to improve their immunogenic potential and we are evaluating nine other M. tuberculosis antigens for possible inclusion to further enhance our RASV-M. tuberculosis vaccine constructs.
- Bacterial pathogenesis
- Host-pathogen interaction
- Mycobacterium Tuberculosis
- Regulation of Gene Expression