|
|
|
|||||
|
|
|
|
|
|
|
|
|
|
|
|
Microbial Pathogenesis and Vaccine Development Microbial pathogens employ a variety of virulence functions to replicate within host tissues and the expression of these virulence determinants changes as the infection proceeds due to host inflammation, tissue breakdown, and immune responses. We are focused on understanding the fundamental mechanisms underlying microbial gene expression and the resultant changes in microbial behavior that are critical to infection. Our applied goal is to utilize this information to develop effective therapies against microbial infections that currently jeopardize human and animal health worldwide. Salmonella Pathogenesis and Gene Regulation Much of our research is focused on the infection process of Salmonella, which causes diseases ranging from food and blood poisoning to typhoid fever and heart disease. The rationale for examining Salmonella as a model system to study microbial pathogenesis is that it provides a well-characterized genetic system to study virulence gene expression and is coupled with an excellent animal model to study infectious disease (typhoid fever). One of the major obstacles in studying Salmonella pathogenesis is that it employs a Trojan Horse strategy: many virulence functions are only expressed during infection ("ON" in vivo) and are not expressed when cells are grown on laboratory medium ("OFF" in vitro). To overcome this problem, we have utilized in vivo Expression Technology (IVET), which is a technique that allows the selection of bacterial virulence genes that are preferentially expressed during infection. We have identified greater than 200 Salmonella in vivo induced (ivi) genes; many encode functions that have been shown to be involved in virulence. We continue to utilize IVET to elucidate the vital changes in virulence gene expression that occur in response to the dynamic environmental and immunological challenges presented in host tissues.
Dam, Gene Expression, and Virulence Alteration in bacterial gene expression in response to the transition from host tissues to the laboratory setting suggested that global regulatory factors controlled the expression of these In vivo Induced (ivi) genes. Therefore, a genetic screen was devised to identify mutations that enabled ivi genes to be expressed when bacteria were grown on laboratory medium (constitutively "ON" in vivo and constitutively "ON" in vitro). It was anticipated that this class of regulatory mutants would abolish the coordinate expression of virulence functions in host tissues exhibited by pathogenic microbes. Consequently, the abrogation of the coordinate regulation in these regulatory mutants might severely attenuate their ability to cause disease. Mutations in DNA adenine methylase (Dam) answered this genetic screen, resulting in the derepression of greater than 35 Salmonella ivi genes when Salmonella was grown in vitro. These results suggested that Dam is a global regulator of Salmonella gene expression. Because Dam affected the expression of many Salmonella genes that were preferentially expressed during infection, we examined the role of dam in bacterial pathogenesis. S. typhimurium strains that lack dam conferred severe virulence defects in several animal models (mouse, chicken, and calf) of typhoid fever. That is, infected animals survived a dam- Salmonella challenge at a dose 10,000 times the dose of the wild-type (dam+) strain that would kill 50% of the animals (LD50). These results indicate that Dam plays an essential role in Salmonella pathogenesis. Dam and Vaccine Development The number of multi-drug resistant strains of bacteria that are known to cause human diseases are increasing at a rapid rate, creating sometimes hopeless situations for patients unfortunate enough to become infected with one of these organisms. One viable approach to meet this challenge is through protective vaccination. It is now more important than ever to focus significant research efforts on the design of efficient and safe vaccines capable of providing strong states of host protection against biowarfare agents and emerging infectious diseases that now threaten the safety of public health worldwide. The mammalian immune system has evolved a sophisticated array of mechanisms specifically in response to, and as a protective measure against, infectious agents which cause diseases. Consequently, attenuated mutants of pathogenic microorganisms, provided to a host via the natural route, provide an effective means to stimulate both the innate and adaptive components of the immune system. One of the more promising examples of an attenuated microorganism serving as an effective vaccine or vaccine vector derives from murine studies employing S. typhimurium, which invade and proliferate within host cells and thus are capable of eliciting strong innate and adaptive immune responses. Therefore, we tested Salmonella dam mutants for their capacity to serve as live vaccines,. Live vaccines are comprised of living organisms that are unable to cause disease, but express many immunogens similar to a natural infection. This interaction elicits a protective response as if the individual had been previously exposed to the pathogen (e.g., smallpox and polio vaccines). Following immunization with dam- Salmonella, animals (mice, chickens, cattle) infected with 10,000 times the LD50 of the wild-type strain were highly protected and exhibited reduced colonization of systemic tissue sites. The elicitation of such protective responses suggests that Salmonella dam mutants are candidates for human and animal live vaccines. Cross-protective Vaccines A major obstacle in vaccine development is that there are often many different isolates of a given pathogen that are proficient in causing disease, and vaccination against one strain may not elicit protection against another strain, or even a variant of the parental strain. This is a principal reason why protective immunity against some microbes may require annual vaccinations with different strains, why vaccine efficacy may depend on the specific pathogenic isolates endemic to a given geographical region, and why mutant variants can cause disease in populations that are immune to infection with the parental strain. Thus, it is desirable to develop bacterial vaccines that can stimulate cross-protective host immune responses to several pathogenic strains. Conceptually, strong and long-lasting cross-protective immunity could be elicited by live vaccines that express multiple antigens that are shared among different pathogenic strains (serotypes). The rationale is that although different strains possess different antigen repertoires, some of the protective antigens may be shared among heterologous serotypes, and expression of these shared antigens may lead to cross-protective immunity. To this end, we have shown that Salmonella dam mutants ectopically express multiple genes that are normally induced during infection. These dam mutants confer cross-protective immunity to heterologous Salmonella serotypes in murine and avian models of typhoid fever. These data provide the foundation for the development of potent cross-protective vaccines against a number of infectious agents. Emergence/ Evolution of Bacterial Pathogens The fundamental principles that distinguish pathogenic strains from non-pathogenic strains are obscure as non-pathogenic strains often contain many of the virulence genes required for a productive infection (e.g., adhesins, invasins, toxins). Thus some of the virulence-, host range-, and disease manifestation- disparities exhibited by bacterial strains may be credited to differential gene regulation, rather than major differences in genomic content. Consistent with this hypothesis, we have recently shown that altered Dam levels differentially affected the expression of several virulence genes in pathogenic strains compared to avirulent laboratory strains. Differential gene regulation may also contribute to the emergence/evolution of pathogenic strains as selective pressures give rise to genetic variants that may have altered virulence properties, e.g., maintaining the ability to cause acute disease in a given natural animal host, while acquiring the ability to cause acute disease or asymptomatic colonization/persistence in a new animal host. Thus, differential gene regulation may contribute to virulence disparities observed among and between strains and, conceivably, to the emergence/evolution of new pathogenic strains, perhaps via epigenetic modifications (DNA methylation) that are not subject to the same evolutionary constraints as are classical genetic mutations. Host Immune Responses to Microbial Infection The host interferon system plays an important role in the host defense against microbial infection. In collaboration with the Samuel laboratory (UCSB), we examined whether Salmonella infection of mice altered the expression of host interferon stimulated genes (ISGs), whose protein products play central roles in promoting immune responses. The results indicated that several ISGs are stimulated after Salmonella infection. These findings are significant since the induction of ISGs have been well-characterized following viral infection, and the fact that they are also induced after bacterial infection suggests a commonality in the host response to a diverse array of microbes. Further investigation into the role of host responses to infection will provide insight into the molecular basis of microbial pathogenesis and the design of therapeutics to either control the infecting microbe or the host immune responses that may contribute to pathogenesis. Model: DAM Affects Gene Expression, Virulence, and Immune Responses The role of dam in virulence and in the elicitation of protective immune responses may rely on its capacity as a global regulator of gene expression. DNA methylation can alter the affinity of regulatory proteins for DNA and, conversely, regulatory proteins can bind to nonmethylated Dam target sites, protecting these sites from methylation. Such competition between Dam and regulatory proteins for Dam target sites in control sequences can affect gene expression. Dysregulation of Dam activity can compromise the ability of a pathogen to cause disease via aberrant virulence gene expression and can contribute to the elicitation of immunity through the production of an expanded repertoire of potential antigens that are delivered to immune cells.
Enhanced immunity conferred by dam mutant immunization may occur via a fundamental difference between the interaction of dam+ vs. dam- cells and lymphoid tissue. That is, many pathogens penetrate the intestinal wall through M cells, which deliver antigens directly to the underlying lymphoid tissue, termed Peyer's patches, which contain dendritic cells and macrophages. Wild-type Salmonella are cytotoxic to these antigen-presenting cells, resulting in restrained immune responses to infection. In contrast, dam- Salmonella proliferate within, but are not cytotoxic to, M cells, dendritic cells, or macrophages. This may allow for enhanced antigen presentation, resulting in heightened immunity in vaccinated hosts.
Future Plans We wish to understand the molecular basis by which dam controls bacterial gene expression, and to characterize those functions that are critical to pathogenesis and to the elicitation of protective immune responses. Additionally, we plan to determine whether Salmonella dam mutant strains can be used as a "carrier platform" for the expression of viral or bacterial antigens to confer heightened immunity against viral or bacterial infection-causing agents. In summary, as we learn more about the mechanisms underlying microbial virulence and the subtle nuances associated with the innate and adaptive immune mechanisms possessed by animal hosts, this information can be translated into effective therapies against microbial infections.
|
|
| | | | | |
|
