Evasion of CD8 T CellMediated Protective Immunity
The importance of CD8 T cells during L. monocytogenes infection is currently attributed to the cytosolic niche of the pathogen, allowing it to evade many aspects of immune surveillance. However, L. monocytogenes has also evolved mechanisms to allow it to avoid CD8 T cell-mediated immune responses. The ability of L. monocytogenes to spread intercellularly, from cell to cell, without encountering the extracellular milieu allows the bacteria to avoid perforin-mediated killing. The bacteria are...
Biophysics of ActinBased Movement
Because the process of L. monocytogenes actin-based motility is relatively well understood at a biochemical level, it has captured the attention of biophysicists who are interested in understanding how actin polymerization generates motile force. One key question of interest centers on the magnitude of force generated to power L. monocytogenes motility. Recent measurements suggest a stall force ranging from 7nN to upward of 150 nN, depending on the surface area over which polymerization occurs...
Introduction Evasion and Modulation of the Immune Response by Pathogens
As organisms evolved into multicellular life forms and devoted more time, resources, and energy to life, they needed to insure their investment with a system that could differentiate foreign invaders from self. The immune system allows for recognition of pathogens, in turn leading to an evaluation of the pathogen and then the appropriate response to control or eliminate the pathogen. However, pathogens have also been evolving alongside their hosts. In order to improve their odds for survival,...
The Cell Wall of Listeria monocytogenes and its Role in Pathogenicity
M. Graciela Pucciarelli,1 H l ne Bierne2, and Francisco Garc a-del Portillo1 1 Departamento de Biotecnolog a Microbiana, Centro Nacional de Biotecnolog a-Consejo Superior de Investigaciones Cient ficas CSIC , Darwin 3, 2SG49 Madrid, Spain e-mail mgpuccia cnb.uam.es, fgportillo cnb.uam.es 2Unit des Interactions Bact ries-Cellules, Institut Pasteur, INSERM U G4, INRA USC2G2G, 2S Rue du Docteur Roux, 75724 Paris cedex 15, France e-mail hbierne pasteur.fr Listeria monocytogenes contains a cell wall...
Recognition of Listeria monocytogenes CellWall Components by Eukaryotic
Teichoic acids have been implicated in adhesion of L. monocytogenes to host cells. Thus, purified galactose-containing TA, but not those lacking this modification, adhere to HepG-2 epithelial cells Cowart et al. i990 . Consistently, a gtcA mutant deficient in TA glycosylation see Section 5.2.2 displays a partial defect for adhesion and entry into HepG-2 cells Autret et al. 200i . These observations favor the hypothesis that TA and or LTA could act as bacterial lectins promoting the interaction...
Pathogenesis
Except for vertical transmission from mother to fetus and rare instances of cross-contamination in the delivery suite or neonatal nursery, human-to-human infection has not been documented Farber et al. 1992 Colodner et al. 2003 . Infection most often begins after ingestion of food contaminated with the organism. The oral inoculum required to produce clinical infection is unknown experiments in healthy mammals indicate that gt 109 organisms are required Farber et al. 1991 . Alkalinization of the...
Actin Polymerization Provides the Driving Force for Propulsion
Intracellular movement of L. monocytogenes is always accompanied by the formation of actin comet tails Dabiri et al. 1990 , suggesting that the assembly of actin into the tail is coupled to motility. The direct coupling of actin polymerization and movement was first demonstrated by marking segments of the actin network in comet tails using fluorescent actin derivatives and observing the behavior of the fluorescent marks over time Sanger et al. 1992, Theriot et al. 1992 . If newly polymerizing...
Glucose Catabolism
Listeria monocytogenes growing in complex media e.g., BHI catabolizes first glucose and the other PTS sugars, which are all ultimately converted to glucose-6-phosphate mainly by the glycolytic pathway. The principal glycolysis genes gap, pgk, tpi, pgm, and eno of L. monocytogenes as in most low G C Gp bacteria belong to the predicted highly expressed genes Karlin et al., 2004 . In glucose-containing minimal medium, these genes are, however, down-regulated and genes of the pentose phosphate...
Abbreviations
L. monocytogenes L. innocua L. seeligeri
Programming of the TCell Response
Upon infection with L. monocytogenes, the rapid response of the innate immune system functions to limit infection until the slower adaptive immune response can develop. This coordinated effort typically results in pathogen clearance within a week of infection Harty and Bevan 1995 Badovinac and Harty 2000 . The peak of the MHC Class Ia-restricted CD8 T-cell response is coincident with pathogen clearance followed by rapid contraction of Ag-specific cell numbers. The timing of these events led to...
Contributors
Unit des Interactions Bact ries-Cellules Institut Pasteur, INSERM U604, INRA USC2020 75724 Paris cedex 15, France Cornell University Department of Food Science Ithaca, NY 14853 Unit de G nomique des Microorganisme Pathog nes URACNRS 2171 75724 Paris, France Unit des Interactions Bact ries-Cellules Paris, F-75015 France Seattle Biomedical Research Institute University of Washington Seattle, WA 98109-5219, USA Departamento de Biotecnolog a Microbiana Centro Nacional de Biotecnolog a-Consejo...
Intraspecies and Interspecies Comparisonsthe Listeria Genomes
The initial comparison of the L. monocytogenes EGDe and L. innocua sequences Glaser et al. 2001a revealed several major features of the Listeria genomes a strong conservation of genome organization, a low occurrence of insertion sequences IS elements, and the absence of typical pathogenicity islands, but instead the occurrence of multiple insertions and deletions leading to a general organization of a conserved backbone with multiple interspersed smaller islets. An interesting but still...
LPXTG Proteins and Sortases
5.4.1.1. Species-Specific Role of InlA in Crossing of Intestinal and Placental Epithelia Despite the critical role of InlA as an L. monocytogenes invasion protein, it was for long impossible to associate InlA with virulence in mouse models Gaillard et al. 1996 Gregory et al. 1996 Pron et al. 1998 . This unexpected result was explained by the discovery of a species specificity of InlA interaction with its host receptor, the cell adhesion molecule E-cadherin Ecad . Thus, InlA interacts with human...
Additional Environmental Stimuli That May Influence PrfA Activity
As discussed above, the potential PrfA cofactor or post-translational modification of PrfA remains unknown. Based on the observation that readily metabolized carbon sources repress PrfA-dependent gene expression, Herro et al 2005 recently explored whether components of carbon catabolite repression might influence PrfA activity. Using a heterologous Bacillus subtilis-based expression system, the authors found that the accumulation of the serine phosphory-lated form of the catabolite co-repressor...
Virulence Factors and Surface Proteins
The major virulence factors of L. monocytogenes identified mainly through different genetic approaches include genes encoding the invasion proteins InlA and InlB, genes encoding proteins that allow escape from the phagocytic vacuole Listeriolysin O LLO and a PI-PLC , intracellular actin-based motility ActA , and cell-to-cell spread PlcB . Except inlAB, these genes are clustered together with their common transcription regulator, PrfA, on a 9-kb virulence locus which is absent from the...
Conclusion
For many decades, murine listeriosis has been utilized as a highly reproducible model to safely study both innate and adaptive immune responses. Although T cells are not required for resistance to primary infection with L. monocytogenes, the identification of defined peptide epitopes derived from L. monocytogenes has allowed immunologists to utilize this infection model to dissect many aspects of the antigen-specific T-cell response to intracellular pathogens. The relative ease by which the...
References 1
Arous S, Buchrieser C, Folio P, Glaser P, Namane A, Hebraud M, Hechard Y 2004 Global analysis of gene expression in an rpoN mutant of Listeria monocytogenes. Microbiology 150 1581-90 Aureli P, Fiorucci GC, Caroli D, Marchiaro G, Novara O, Leone L, Salmaso S 2000 An outbreak of febrile gastroenteritis associated with corn contaminated by Listeria monocytogenes. N Engl J Med 342 1236-41 Autret N, Raynaud C, Dubail I, Berche P, Charbit A 2003 Identification of the agr locus of Listeria...
Metabolic Capabilities
It is generally accepted that L. monocytogenes is an aerobically growing microaerophilic carbon dioxydophilic organism. However, L. monocytogenes is able to survive and to colonize the mammalian gut where it encounters anaerobic conditions or to multiply in decaying plants, an environment also devoid of oxygen. This is reflected in the genome sequence as many fermentative pathways are predicted Figure 3.3. . Analysis of the genome sequence identified in L. monocytogenes and in L. innocua a...
InlBMediated Pathway
8.2.2.1. InlB Structure and Function InlB is a 630-amino acids protein from the internalin family encoded in the same operon as InlA Gaillard et al., 1991 . InlB presents an amino-terminal signal peptide followed by the short conserved internalin cap and seven LRRs the IR region that follows the LRR region has a minimal immunoglobulin Ig -like fold that, together with the LRRs and with the cap which presents itself a truncated EF-hand fold structure , constitutes aunique domain with a...
Characteristics of PrfA Target Promoters The PrfA Box
PrfA functions as a transcriptional activator via recognition of a palindromic DNA-binding site located in the -40 region of target promoters Kreft and Vazquez-Boland 2001 Table 7.1 . PrfA recognizes a 14 bp palindromic repeat known as the PrfA box with a consensus sequence of TTAACAnnTGTTAA. DNA footprinting experiments indicate that PrfA binding protects from digestion an approximate 26 bp region, beginning 10 bp upstream and ending 2 bp downstream of the 14 bp PrfA box Dickneite et al. 1998...
Listeria monocytogenesPrevalence and Loads in Foods and FoodAssociated
A relatively small number of human listeriosis cases have been associated with food products contaminated during primary production in agricultural environments, such as raw vegetables, raw milk, and raw milk dairy products, since heating regimes typically applied during food cooking or commercial processing effectively inactivate L. monocytogenes. Instead, most human listeriosis cases are caused by the consumption of RTE meats and dairy food products that contain L. monocytogenes as a...
Ami and Auto GW Proteins Involved in Adhesion and Invasion
Several L. monocytogenes autolysins have been shown to be involved in virulence, including the amidase Ami and Auto. Ami is a 917-amino acids protein with an N-terminal domain that presents similarities to the amidase domain of the Staphylococcus aureus Atl autolysin, while its C-terminal domain is anchored to the bacterial cell wall by eight GW domains Milohanic et al., 2001 . Ami exhibits lytic activity on L. monocytogenes cell walls McLaughlan and Foster, 1998 , but also mediates bacterial...
Transport Systems and Sugar Utilization
The Listeria genomes encode an abundance of transport proteins e.g., 11.6 of all predicted genes of L. monocytogenes EGDe . These comprise, in particular, proteins dedicated to carbohydrate transport conferring Listeria probably in part its ability to colonize a broad range of ecosystems. The overall array of sugar transporters is similar in all Listeria genomes, in particular among the four sequenced L. monocytogenes strains, but also with L. innocua. Listeria are predicted to transport and...
Evasion of AntibodyMediated Killing
Antibodies, produced by B cells, are one of the two main arms of adaptive immunity. Antibodies contribute to the immune response against bacterial pathogens by 1 neutralization of bacteria and their toxins, 2 opsonization of bacteria which promotes uptake by phagocytic cells, and 3 complement activation which enhances opsonization. Humoral responses against many bacterial pathogens are sufficient for protection against disease. For example, vaccines to bacteria such as Bordetella pertussis, the...
Surface Proteins Anchored Covalently
to the Peptidoglycan 5.3.1.1. The LPXTG Protein Family Pioneering work performed with the S. aureus protein A demonstrated that this surface protein is anchored covalently to the peptidoglycan by an enzyme Figure 5.1. Global view of the distinct interactions of the Listeria monocytogenes surface proteins with the cell wall. Listed in the upper part are the proteins identified by proteomics in purified peptidoglycan material in bold in extracts obtained from the cell wall upon incubation of...
Immunity
Resistance to infection with the intracellular bacterium L. monocytogenes is predominantly cell mediated, as evidenced by experiments Mackaness 1962 Mackaness 1971 showing that immunity could be transferred by sensitized lymphocytes but not by antibodies. Further evidence is provided by the overwhelming clinical association between listerial infection and conditions of impaired cellular immunity, including lymphoma, pregnancy, AIDS, and corticosteroid immunosuppression Buchner and Schneierson...
Reconstitution of Motility with Purified Proteins
Ten years of research aimed at understanding the biochemical mechanism of L. monocytogenes actin-based motility culminated in the reconstitution of this process with a system consisting only of purified proteins Loisel et al. 1999 . In a testament to the surprising biochemical simplicity of the system, the basic reconstitution mix consisted of only four factors actin, Arp2 3 complex, capping protein, and ADF cofilin. In the presence of these four core components, motility proceeds slowly 0.5 m...
spDNA Array Analysis
The availability of complete genome sequences allows researchers now to use other techniques, like DNA arrays, to investigate the diversity among a large number of Listeria strains belonging to these different lineages, to different populations or showing different epidemiological characteristics. This approach allows characterization of the genetic differences among and within these lineages, and to investigate whether these differences can be attributed to virulence differences and or...
Clin. Infect. Dis. 40 962-967.
al-Ghazali MR, al-Azawi SK 1988a Storage effects of sewage sludge cake on the survival of Listeria monocytogenes. J Appl Bacteriol 65 209-213 al-Ghazali MR, al-Azawi SK 1988b Effects of sewage treatment on the removal of Listeria monocytogenes. J Appl Bacteriol 65 203-208 Allerberger F, Guggenbichler JP 1989 Listeriosis in Austria report of an outbreak in 1986. Acta Microbiol Hung 36 149-152 Arvanitidou M, Papa A, Constantinidis TC, Danielides V, Katsouyannopoulos V 1997 The occurrence of...
Biochemistry of the Listeria monocytogenes Cell Wall
The first descriptions on the composition and suspected structure of the L. monocytogenes peptidoglycan and associated polymers were made more than 30 years ago Srivastava and Siddique 1973 Ullmann and Cameron 1969 . Despite this long period, the fine structure of its peptidoglycan has not been known until very recently Kloszewska et al. 2006 . Likewise, while the biochemistry of teichoic acids TA and lipoteichoic acids LTA of L. monocytogenes was inferred two decades ago Fiedler 1988 Uchikawa...
Escape from the Primary Vacuole 921 Macrophages
The fate of L. monocytogenes in a macrophage depends upon both the cell lineage and its history. Primary peritoneal macrophages are capable of killing approximately 90 of entering L. monocytogenes Portnoy et al. 1989 , bone marrow-derived macrophages kill approximately half the bacteria, whereas cell lines such as J774 and RAW 264.7 murine macrophage-derived cells are weakly cidal Camilli et al. 1993 De Chastellier 1994 . Treatment of peritoneal macrophages with y interferon IFN-y prior to...
ActA Mediates Actin Polymerization
A bacterial gene that is required for actin polymerization, called actA, was identified in a screen for transposon mutants that failed to spread between cells in a plaque assay Kocks et al. 1992 and also by directed insertional mutagenesis Domann et al. 1992 . Mutations in actA cause a complete failure to assemble actin at the bacterial surface and to undergo intracellular motility. Entry and intracellular growth are not affected, but the motility defect results in the failure to spread from...
Internalins Main Listeria monocytogenes Effectors for Cell Invasion
The L. monocytogenes internalin family of proteins comprises 24 members, characterized by the presence of amino-terminal regions with tandemly arranged leucine-rich repeats LRRs Pizarro-Cerda and Cossart, 2006 . The LRR regions have been found in prokaryotic and eukaryotic proteins, providing a versatile framework for the formation of ligand-binding sites Kobe and Kajava, 2001 . Nineteen members of the internalin family including InlA, the internalin prototype, contain a carboxy-terminal LPXTG...
Surface Proteins with Noncovalent Association to the Cell Wall
5.3.2.1. InlB, a Protein Loosely Associated to the Cell Wall by GW Modules InlB is a surface protein required for L. monocytogenes entry into certain eukaryotic cell types see Section 4.2 . InlB is the only L. monocytogenes surface protein carrying a domain organization consisting of a N-terminal LRR domain and a C-terminal domain of three repetitions of 80 amino-acids, called GW modules Braun et al. 1997 . GW modules are also found in autolysins see below . Domain swapping experiments revealed...
Listeria Phages and Pathogenesis
Although most strains, including clinical isolates of Listeria, contain prophages indeed many are polylysogens Rocourt 1986 containing multiple prophages or cryptic phages no evident phenotype has yet been associated with the presence of prophages under laboratory conditions nor could a pro phage be linked to epidemic strains or outbreaks. However, this situation may reflect our limited understanding of Listeria phage genetics and host cell interactions. What seems clear, however, is that the...
The Third Step in Regulation of the Regulator Posttranslational Control of PrfA
A third and perhaps most critical mechanism exists for control of PrfA activity post-translational regulation. The initial evidence which suggested that PrfA protein activity was regulated on a post-translational level followed the experimental observation that PrfA-dependent gene expression was reduced in the presence of readily metabolized carbon sources, such as glucose and cellobiose, yet there was no significant change in the amount of PrfA protein present in the bacterial cell Renzoni et...
The Peptidoglycan of Listeria monocytogenes
Listeria monocytogenes has a peptidoglycan formed by glycan chains containing alternating units of the disaccharide N-acetylmuramic acid MurNAc - P-1,4 -N-acetyl-D-glucosamine GlcNAc . Bound to the MurNAc residue is a stem peptide that in L. monocytogenes contains L-alanine-7-D-glutamic acid-meso-diaminopimelic acid-D-Ala-D-Ala L-Ala-7-D-Glu-m-Dap-D-Ala-D-Ala Fiedler 1988 Kamisango et al. 1982 . The glycan chains are cross-linked by 4 3 linkages between the D-Ala residue of one lateral peptide...
Vip An LPXTGAnchored Protein Involved in Invasion
Comparison of the genomes of the pathogenic L. monocytogenes and the nonphatogenic L. innocua has led to the identification of several new L. monocy-togenes virulence factors that are absent from the L. innocua genome. One of these new proteins is Vip, an LPXTG-anchored cell wall protein required for the invasion of Caco-2 and L2071 cell lines Cabanes et al., 2005 . The cellular receptor of Vip is Gp96, an endoplasmic reticulum-resident chaperone that can also be present at the plasma membrane...
In1C
Figure 8.2. Schematic representation of different types of internalins involved in invasion of target cells by L. monocytogenes. InlA is an LPXTG-anchored membrane protein that promotes bacterial entry into polarized epithelial cells by interacting with the adhesion molecule E-cadherin through its leucine-rich repeats. InlB is loosely anchored to the bacterial cell wall through its GW domains and can be detached from the L. monocytogenes surface soluble InlB interacts with host cell...
References Vda
Alvarez-Dominguez C, Vazquez-Boland J A, Carrasco-Marin E, Lopez-Mato P, and Leyva-Cobian F 1997 . Host cell heparan sulfate proteoglycans mediate attachment and entry of Listeria monocytogenes, and the listerial surface protein ActA is involved in heparan sulfate receptor recognition. Infect Immun 65, 78-88. Banerjee M, Copp J, Vuga D, Marino M, Chapman T, van der Geer P, and Ghosh P 2004 . GW domains of the Listeria monocytogenes invasion protein InlB are required for potentiation of Met...
The Beginning of the Modern Era A Personal Recollection
I began working on L. monocytogenes in the fall of 1986, shortly after starting my own laboratory at Washington University in St Louis. I should mention that Pascale Cossart also entered the field about the same time Mengaud et al. 1987 and remains a monumental presence contributing to all aspects of L. monocyto-genes pathogenesis. In 1986, basic research on L. monocytogenes was mostly in the realm of immunology while ignored by the bacterial pathogenesis community. I was influenced by a number...
The Actin Cytoskeleton and Intercellular Spread of Listeria monocytogenes
The ability to spread from cell to cell plays a critical role in infection by many intracellular pathogens. L. monocytogenes has evolved the ability to move directly from cell to cell without passing through the intercellular space, enabling it to evade the host immune response. The process of L. monocytogenes' spread was first observed by imaging infections in guinea pig corneal and intestinal epithelia using electron microscopy Racz et al. 1970, 1972 . These early studies documented that,...
References
Ampel NM, Bejarano GC, Saavedra M Jr 1992 Deferoxamine increases the susceptibility of beta-thalassemic, iron-overloaded mice to infection with Listeria monocytogenes. Life Sci 50 1327-1332 Antal E-A, Leberg EM, Bracht P et al 2001 Evidence for intraaxonal spread of Listeria monocytogenes from the periphery to the central nervous system. Brain Pathol 11 432 438 Armstrong RW, Fung PC 1993 Brainstem encephalitis rhombencephalitis due to Listeria monocytogenes Case report and review. Clin Infect...
on PrfADependent Virulence Gene Expression
Previous studies have repeatedly shown that sugars that can be used by L. monocytogenes as carbon source, like glucose, fructose, mannose, and cellobiose, have an inhibitory effect on PrfA activity and hence the PrfA-dependent gene expression Behari and Youngman, 1998b Milenbachs et al., 1997 Milenbachs et al., 2004 . The strongest inhibition is exerted by cellobiose. These sugars are taken up by PTS-mediated transport and result ultimately in the conversion to glucose-6-phosphate and in...
The Listeria monocytogenes CellWall Proteome
Based on genome data, a recent study developed a preliminary proteome reference map of the L. monocytogenes EGD-e strain Ramnath et al. 2003 . Using total cell extracts, 261 spots were differentiated on two-dimensional 2D gels. Of these, only 33 distinct proteins were identified, most of them corresponding to abundant proteins chaperons, translation factors, and enzymes of central metabolism . Noteworthy, no cell-wall-associated protein was identified in this study. A further study, focused on...
Phages and Pathogenicity
The tremendous impact of phages on the pathogenicity of their host has only recently become the focus of detailed research. Phages can introduce new bacterial DNA into their host after infection. Although regulated, the normal packaging of phage DNA into capsids can in certain cases result in incorporation of host DNA into phage particles. This process is called transduction and occurs in two different forms. In specialized transduction, improper excision of phage DNA results in packaging of...
Nitrogen Metabolism
For most bacteria including L. monocytogenes, glutamine Gln is the optimal nitrogen source Merrick and Edwards, 1995 , but in the absence of Gln, L. monocytogenes is capable of utilizing alternative nitrogen sources, such as ammonium Tsai and Hodgson, 2003 , arginine, and even ethanolamine our own unpublished results . These latter N sources might become important, particularly when L. monocytogenes replicates in mammalian host cells where the supply of free Gln is limited and its consumption...
Listeria monocytogenesInduced Apoptosis Modulates the Immune Response
Apoptosis is a process of programmed cell death to eliminate damaged or unneeded cells. Apoptosis of T cells is a natural occurrence during T cell development and during contraction of a T cell response Marsden and Strasser 2003 . In contrast to necrosis, apoptosis is a process that minimizes inflammation. Macrophages express a phosphatidylserine receptor that allows them to recognize, engulf, and degrade apoptotic cells Fadok et al. 1992 . In the spleen, 48 h after L. monocytogenes infection,...
Conclusions 1
Nearly two decades after the discovery that L. monocytogenes exploits the host actin cytoskeleton to undergo intracellular movement, we have reached a reasonable understanding of how this process works at the molecular level. The key molecular components that are involved in motility have been identified, and a core set of proteins has been demonstrated to be sufficient for the reconstitution of the process in vitro. Nevertheless, much remains to be discovered. We do not yet know how actin...
Genome Sequencing and Analysis
The first bacterial genome projects concerned thoroughly analyzed bacteria like the model organisms Escherichia coli Blattner et al. 1997 and Bacillus subtilis Kunst et al. 1997 or the pathogenic Mycobacterium tuberculosis Cole et al. 1998 . Sequencing of these genomes was based on a top-down approach using subcloning of large chromosomal overlapping fragments in lambda phage, plasmids, or cosmids. However, in 1995 the first complete bacterial genome sequence published, that of Haemophilus...