Nancy E. Freitag

Bio: Nancy E. Freitag is an academic researcher from University of Illinois at Chicago. The author has contributed to research in topics: Listeria monocytogenes & Virulence. The author has an hindex of 19, co-authored 53 publications receiving 1673 citations.

Listeria monocytogenes - from saprophyte to intracellular pathogen.

Abstract: Bacteria need to adjust as they move between different environments. In this Progress article, Freitag, Port and Miner describe howListeria monocytogenesregulates the transition from saprophyte to human pathogen. Listeria monocytogenes is a bacterium that lives in the soil as a saprophyte but is capable of making the transition into a pathogen following its ingestion by susceptible humans or animals. Recent studies suggest that L. monocytogenes mediates its saprophyte-to-cytosolic-parasite transition through the careful modulation of the activity of a virulence regulatory protein known as PrfA, using a range of environmental cues that include available carbon sources. In this Progress article we describe the regulation of PrfA and its role in the L. monocytogenes transition from the saprophytic stage to the virulent intracellular stage.

The Virulence Regulator PrfA Promotes Biofilm Formation by Listeria monocytogenes

Abstract: Listeria monocytogenes is an opportunistic food-borne pathogen that can be isolated from a variety of environments (5, 14). This Gram-positive bacterium can contaminate foods by persisting on food-processing surfaces, and it has been hypothesized that this persistence is at least partly due to the formation of biofilms, which can be difficult to eradicate (29). Once ingested, L. monocytogenes causes life-threatening infections in fetuses, newborns, and immunocompromised people (9, 23). The high case fatality rate of listeriosis (∼20%) makes L. monocytogenes food contamination, and, thus, biofilms, an important health issue (24). In contrast to its pathogenesis, little is known about L. monocytogenes biofilm development. L. monocytogenes has three distinct life-styles: (i) an intracellular pathogen that utilizes actin-based motility for cytoplasmic movement and cell-to-cell spread; (ii) an extracellular, free-living, flagellum-propelled bacterium in the environment; and (iii) an extracellular bacterial member of a multicellular biofilm community. Biofilm formation and intracellular invasion appear to be mutually exclusive life-styles. The critical virulence transcription factor PrfA regulates the switch from extracellular, flagellum-propelled bacterium to intracellular pathogen (reviewed in references 10, 13, and 39). PrfA binds to a specific DNA sequence (the PrfA box) and positively regulates the expression of the canonical L. monocytogenes virulence genes, including itself. During intracellular invasion, the PrfA-dependent transcription of virulence genes increases first in the phagocytic vacuole. After vacuolar escape into the host cytosol, a subset of PrfA-dependent virulence genes is induced to an even greater extent. PrfA production is regulated at transcriptional, translational, and protein activity levels (13, 39). Three promoters drive prfA transcription (Fig. ​(Fig.1).1). Two of these promoters, PprfAP1 and PprfAP2, control the expression of a monocistronic prfA transcript (11, 12). PprfAP1 is dependent on the housekeeping sigma factor, σA. PprfAP2 can be driven by either σA or the stress response sigma factor, σB (30, 36, 38). In addition, the promoter of the upstream gene plcA, PplcA, contains a canonical PrfA box and is positively autoregulated by PrfA, resulting in the transcription of a bicistronic message containing plcA and prfA (4, 26). The resulting increased level of PrfA expression from PplcA is necessary for wild-type virulence (4, 12). The transcript from PprfAP1 contains a 5′ untranslated region (UTR) that acts as a thermosensor; the translation of this transcript is efficient at the host body temperature but inefficient at 30°C and below (18). In contrast to PplcA, transcription from PprfAP2 is PrfA independent, and in contrast to PprfAP1, the translation of this transcript is temperature independent. It has been postulated that this transcript is important for PrfA production outside mammalian hosts; we provide evidence here to support this hypothesis. FIG. 1. Schematic that shows the three promoters that drive the expression of prfA. PplcA is positively autoregulated by PrfA, which binds to the PrfA box (black box), and produces a bicistronic message. PprfAP1 is σA dependent, and its transcript contains ... PrfA is a member of the cyclic AMP receptor protein-fumarate and nitrate reduction regulator (CRP-FNR) family of transcriptional regulators (20). Small-molecule cofactors regulate the protein activity state of many family members (19, 39). While no such cofactor has been identified yet for PrfA, a variety of point mutations in prfA (prfA* mutants) that result in variant PrfA proteins whose function is locked in the intracellular activity state support the theory that PrfA activity is allosterically regulated (16, 28, 37, 40, 44, 47). These prfA* mutants constitutively express levels of the actin-nucleating protein ActA comparable to those observed when bacteria are in the host cytosol (40). These mutations also result in an increased level of production of the PrfA* protein via the activation of PplcA (43, 47). Another recently isolated mutation in prfA (Y154C) results in a PrfA protein that cannot fully transition from the extracellular activity state to the intracellular activity state (27). The prfA(Y154C) mutant demonstrates a 4-fold increase in actA expression levels in brain heart infusion (BHI) medium compared to the wild type but fails to increase actA expression levels to wild-type levels (100-fold less than the wild type) during intracellular infection (27). It effectively enters the host cytosol but is defective in cell-to-cell spread and is attenuated for virulence (150-fold reduction in a mouse model) (27). Thus, there are PrfA protein variants that are locked in the intracellular activity state (PrfA*) and one [PrfA(Y154C)] that is unable to fully transition to the intracellular activity state. L. monocytogenes has never been observed to form biofilms in host cells; intracellular bacteria are single cells that move through the cytosol using actin-based motility (34). Extracellularly, L. monocytogenes motility is mediated via flagella. Flagellum-mediated motility appears to be important for initial epithelial cell attachment, and flagellum-minus mutants show an early host invasion defect in a competitive oral infection model (32). Similarly, flagellum-mediated motility is critical for initial abiotic surface attachment, and flagellum-minus mutants are defective in biofilm formation (22, 41). This common need for flagellum-mediated motility to transition from an existence as a single, extracellular cell to either environmental biofilm formation or intracellular invasion led us to hypothesize that there might be regulatory cross talk between PrfA and biofilm formation. Indeed, here we report that prfA mutants are defective in biofilm formation at a stage after initial surface attachment. The regulatory connection between PrfA and biofilm formation demonstrates that PrfA has a global role in regulating the life-style of L. monocytogenes.

Identification of Drosophila Mutants Altering Defense of and Endurance to Listeria monocytogenes Infection

Abstract: We extended the use of Drosophila beyond being a model for signaling pathways required for pattern recognition immune signaling and show that the fly can be used to identify genes required for pathogenesis and host–pathogen interactions. We performed a forward genetic screen to identify Drosophila mutations altering sensitivity to the intracellular pathogen Listeria monocytogenes. We recovered 18 mutants with increased susceptibility to infection, none of which were previously shown to function in a Drosophila immune response. Using secondary screens, we divided these mutants into two groups: In the first group, mutants have reduced endurance to infections but show no change in bacterial growth. This is a new fly immunity phenotype that is not commonly studied. In the second group, mutants have a typical defense defect in which bacterial growth is increased and survival is decreased. By further challenging mutant flies with L. monocytogenes mutants, we identified subgroups of fly mutants that affect specific stages of the L. monocytogenes life cycle, exit from the vacuole, or actin-based movement. There is no overlap between our genes and the hundreds of genes identified in Drosophila S2 cells fighting L. monocytogenes infection, using genomewide RNAi screens in vitro. By using a whole-animal model and screening for host survival, we revealed genes involved in physiologies different from those that were found in previous screens, which all had defects in defensive immune signaling.

Constitutive Activation of the PrfA Regulon Enhances the Potency of Vaccines Based on Live-Attenuated and Killed but Metabolically Active Listeria monocytogenes Strains

Abstract: Recombinant vaccines derived from the facultative intracellular bacterium Listeria monocytogenes are presently undergoing early-stage clinical evaluation in oncology treatment settings. This effort has been stimulated in part due to preclinical results that illustrate potent activation of innate and adaptive immune effectors by L. monocytogenes vaccines, combined with efficacy in rigorous animal models of malignant and infectious disease. Here, we evaluated the immunologic potency of a panel of isogenic vaccine strains that varied only in prfA. PrfA is an intracellularly activated transcription factor that induces expression of virulence genes and encoded heterologous antigens (Ags) in appropriately engineered vaccine strains. Mutant strains with PrfA locked into a constitutively active state are known as PrfA* mutants. We assessed the impacts of three PrfA* mutants, G145S, G155S, and Y63C, on the immunologic potencies of live-attenuated and photochemically inactivated nucleotide excision repair mutant (killed but metabolically active [KBMA]) vaccines. While PrfA* substantially increased Ag expression in strains grown in broth culture, Ag expression levels were equivalent in infected macrophage and dendritic cell lines, conditions that more closely parallel those in the immunized host. However, only the prfA(G155S) allele conferred significantly enhanced vaccine potency to KBMA vaccines. In the KBMA vaccine background, we show that PrfA*(G155S) enhanced functional cellular immunity following an intravenous or intramuscular prime-boost immunization regimen. These results form the basis of a rationale for including the prfA(G155S) allele in future live-attenuated or KBMA L. monocytogenes vaccines advanced to the clinical setting.

Disease Tolerance as a Defense Strategy

Abstract: The immune system protects from infections primarily by detecting and eliminating the invading pathogens; however, the host organism can also protect itself from infectious diseases by reducing the negative impact of infections on host fitness. This ability to tolerate a pathogen's presence is a distinct host defense strategy, which has been largely overlooked in animal and human studies. Introduction of the notion of "disease tolerance" into the conceptual tool kit of immunology will expand our understanding of infectious diseases and host pathogen interactions. Analysis of disease tolerance mechanisms should provide new approaches for the treatment of infections and other diseases.

Listeria monocytogenes, a food-borne pathogen

Abstract: Listeria monocytogenes is a Gram positive, aerobic, facultative anaerobic and nonacid fast bacterium, which can cause the disease listeriosis in both human and animals. It is widely distributed thoroughout the environment and has been isolated from various plant and animal food products associated with listeriosis outbreaks. Contaminated ready-to-eat food products such as gravad and cold-smoked salmon and rainbow trout have been associated with human listeriosis in Sweden. The aim of this study was to analyse the occurrence and level of L. monocytogenes in gravad and cold-smoked salmon (Salmo salar) products packed under vacuum or modified atmosphere from retail outlets in Sweden. Isolated strains were characterized by serotyping and the diversity of the strains within and between producers were determined with PFGE (Pulsed-field gel electrophoresis). The characterized fish isolates were compared with previously characterized human strains. L. monocytogenes was isolated from 11 (three manufacturers) of 56 products analysed. This included gravad salmon products from three manufacturers and cold-smoked salmon from one manufacturer. The highest level of L. monocytogenes found was 1500 cfu/g from a cold-smoked salmon product but the level was low (<100 cfu/g) in most of the products. Serovar 1/2a was predominant, followed by 4b. Three products of gravad salmon harboured more than one serovar. PFGE typing of the 56 salmon isolates detected five Asc I types: four types were identical to human clinical strains with Asc I and one was identical and one was closely related to human clinical strains with Apa I. Isolation of identical or closely related L. monocytogenes strains from human clinical cases of listeriosis and gravad and cold-smoked salmon suggested that these kinds of products are possible sources of listeriosis in Sweden. Therefore, these products should be considered risk products for human listeriosis.

Decomposing health: tolerance and resistance to parasites in animals.

Abstract: Plant biologists have long recognized that host defence against parasites and pathogens can be divided into two conceptually different components: the ability to limit parasite burden (resistance) and the ability to limit the harm caused by a given burden (tolerance). Together these two components determine how well a host is protected against the effects of parasitism. This distinction is useful because it recognizes that hosts that are best at controlling parasite burdens are not necessarily the healthiest. Moreover, resistance and tolerance can be expected to have different effects on the epidemiology of infectious diseases and host–parasite coevolution. However, studies of defence in animals have to date focused on resistance, whereas the possibility of tolerance and its implications have been largely overlooked. The aim of our review is to (i) describe the statistical framework for analysis of tolerance developed in plant science and how this can be applied to animals, (ii) review evidence of genetic and environmental variation for tolerance in animals, and studies indicating which mechanisms could contribute to this variation, and (iii) outline avenues for future research on this topic.

Two ways to survive infection: what resistance and tolerance can teach us about treating infectious diseases.

Abstract: A host can evolve two types of defence mechanism to increase its fitness when challenged with a pathogen: resistance and tolerance. Immunology is a well-defined field in which the mechanisms behind resistance to infection are dissected. By contrast, the mechanisms behind the ability to tolerate infections are studied in a less methodical manner. In this Opinion, we provide evidence that animals have specific tolerance mechanisms and discuss their potential clinical impact. It is important to distinguish between these two defence mechanisms because they have different pathological and epidemiological effects. An increased understanding of tolerance to pathogen infection could lead to more efficient treatments for infectious diseases and a better description of host-pathogen interactions.