Most eukaryotic and viral mRNAs possess a 5Ј cap that is important for mRNA stability and efficient translation (11). The cap consists of an inverted guanosine, methylated at the N-7 position, linked to the first transcribed RNA nucleotide by a unique 5Ј-5Ј triphosphate bridge (m 7 GpppN; cap 0 structure) (32). The process of RNA capping generally consists of three steps, in which the 5Ј triphosphate end of the nascent RNA transcript is first hydrolyzed to a 5Ј diphosphate by an RNA triphosphatase, then capped with GMP by an RNA guanylyltransferase, and finally methylated at the N-7 position of guanine by an RNA guanine-methyltransferase (N-7 MTase) (13). Additionally, the first and second nucleotides of many cellular and viral mRNAs are further methylated at the ribose 2Ј-OH position by a nucleoside 2Ј-O MTase to form cap 1 (m 7 GpppNm) and cap 2 (m 7 GpppNmNm) structures, respectively (11). Both N-7 and 2Ј-O MTases use S-adenosyl-Lmethionine (AdoMet) as a methyl donor and generate Sadenosyl-L-homocysteine (AdoHcy) as a by-product. The order of the capping and methylation steps is variable among cellular and viral RNAs (11).Many members of the Flavivirus genus are arthropod-borne human pathogens, including West Nile virus (WNV), Yellow fever virus, four serotypes of Dengue virus (DENV), Japanese encephalitis virus, St. Louis encephalitis virus, Murray Valley encephalitis virus, and Tick-borne encephalitis virus (4). The flavivirus genome is a single-stranded, plus-sense RNA of about 11,000 nucleotides that contains a type 1 cap at its 5Ј end (5, 35) and terminates with 5Ј-CU OH -3Ј (35) (see Fig. 1A). 5Ј and 3Ј untranslated regions flank a single open reading frame which encodes a polyprotein that is co-and posttranslationally processed by viral and cellular proteases into three structural proteins (capsid [C], premembrane [prM] or membrane [M], and envelope [E]) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (4). Since flaviviruses replicate in the cytoplasm, they are expected to encode their own capping enzymes, rather than to use the host's capping apparatus located in the nucleus. Alternatively, since all host proteins have to be synthesized in the cytoplasm, it is possible that cellular capping components could be retained in the cytoplasm for viral RNA capping through specific interactions with a viral protein. Of the four enzymes required for flavivirus m 7 GpppAm-cap formation, only the RNA triphosphatase and 2Ј-O MTase have been mapped to NS3 (19,36) and NS5 (8), respectively, whereas the guanylyltransferase and N-7 MTase remain to be identified. The crystal structure of a ternary complex comprising the DENV-2 MTase domain, AdoHcy, and a GTP analogue suggested that, during 2Ј-O methylation, a specific cap-binding site holds the guanine cap to register the ribose 2Ј-OH of the first transcribed adenosine in close proximity to the AdoMet CH 3 donor (2, 8). Structure and sequence alignments of DENV, vaccinia virus VP39, and other 2Ј-O MTases indicate that a conserved K-D-K-E ...

Toll-like receptor 2 (TLR2) deficiency enhances murine susceptibility to infection by Francisella tularensis as indicated by accelerated mortality, higher bacterial burden, and greater histopathology. Analysis of pulmonary cytokine levels revealed that TLR2 deficiency results in significantly lower levels of tumor necrosis factor alpha and interleukin-6 but increased amounts of gamma interferon and monocyte chemoattractant protein 1. This pattern of cytokine production may contribute to the exaggerated pathogenesis seen in TLR2 ؊/؊ mice. Collectively, these findings suggest that TLR2 plays an important role in tempering the host response to pneumonic tularemia.

The intracellular bacterium Francisella tularensis survives in mammals, arthropods, and freshwater amoeba. It was previously established that the conventional media used for in vitro propagation of this microbe do not yield bacteria that mimic those harvested from infected mammals; whether these in vitro-cultivated bacteria resemble arthropod-or amoeba-adapted Francisella is unknown. As a foundation for our goal of identifying F. tularensis outer membrane proteins which are expressed during mammalian infection, we first sought to identify in vitro cultivation conditions that induce the bacterium's infection-derived phenotype. We compared Francisella LVS grown in brain heart infusion broth (BHI; a standard microbiological medium rarely used in Francisella research) to that grown in Mueller-Hinton broth (MHB; the most widely used F. tularensis medium, used here as a negative control) and macrophages (a natural host cell, used here as a positive control). BHIand macrophage-grown F. tularensis cells showed similar expression of MglA-dependent and MglA-independent proteins; expression of the MglA-dependent proteins was repressed by the supraphysiological levels of free amino acids present in MHB. We observed that during macrophage infection, protein expression by intracellular bacteria differed from that by extracellular bacteria; BHI-grown bacteria mirrored the latter, while MHB-grown bacteria resembled neither. Naïve macrophages responding to BHI-and macrophage-grown bacteria produced markedly lower levels of proinflammatory mediators than those in cells exposed to MHBgrown bacteria. In contrast to MHB-grown bacteria, BHI-grown bacteria showed minimal delay during intracellular replication. Cumulatively, our findings provide compelling evidence that growth in BHI yields bacteria which recapitulate the phenotype of Francisella organisms that have emerged from macrophages.

A striking feature of pulmonary infection with the Gram-negative intracellular bacterium Francisella tularensis, a category A biological threat agent, is an intense accumulation of inflammatory cells, particularly neutrophils and macrophages, at sites of bacterial replication. Given the essential role played by host matrix metalloproteinases (MMPs) in modulating leukocyte recruitment and the potentially indiscriminate destructive capacity of these cells, we investigated whether MMP-9, an important member of this protease family released by neutrophils and activated macrophages, plays a role in the pathogenesis of respiratory tularemia. We found that F. tularensis induced expression of MMP-9 in FVB/NJ mice and that the action of this protease is associated with higher bacterial burdens in pulmonary and extrapulmonary tissues, development of more extensive histopathology predominated by neutrophils, and increased morbidity and mortality compared with mice lacking MMP-9 (MMP-9−/−). Moreover, MMP-9−/− mice were able to resolve infection with either the virulence-attenuated type B (live vaccine strain) or the highly virulent type A (SchuS4) strain of F. tularensis. Disease resolution was accompanied by diminished leukocyte recruitment and reductions in both bacterial burden and proinflammatory cytokine production. Notably, neutrophilic infiltrates were significantly reduced in MMP-9−/− mice, owing perhaps to limited release of Pro-Gly-Pro, a potent neutrophil chemotactic tripeptide released from extracellular matrix through the action of MMP-9. Collectively, these results suggest that MMP-9 activity plays a central role in modulating the clinical course and severity of respiratory tularemia and identifies MMPs as novel targets for therapeutic intervention as a means of modulating neutrophil recruitment.

Pathogen-triggered activation of the inflammasome complex leading to caspase-1 activation and IL-1␤ production involves similar sensor proteins between mouse and human. However, the specific sensors used may differ between infectious agents and host species. In mice, Francisella infection leads to seemingly exclusive activation of the Aim2 inflammasome with no apparent role for Nlrp3. Here we examine the IL-1␤ response of human cells to Francisella infection. Francisella strains exhibit differences in IL-1␤ production by influencing induction of IL-1␤ and ASC transcripts. Unexpectedly, our results demonstrate that Francisella activates the NLRP3 inflammasome in human cells. Innate immune responses to pathogens are initiated by recognition of pathogen-associated molecular patterns by both extracellular and intracellular sensors (1-3). Pathogen-associated molecular pattern recognition by members of the Toll-like receptor (TLR) 3 family result in the activation of the NF-B, MAPK, and/or interferon regulatory factor signaling pathways depending on the specific TLR engaged (1, 4 -8). Intracellular receptors of the MAVS/RIG-I family act similarly and are involved in recognition of viral nucleic acids (9, 10). Thus, production of inflammatory cytokines and chemokines such as TNF␣, IL-6, and MCP-1 in response to infection generally follows activation via these receptors. The inflammatory cytokines IL-1␤ and IL-18, however, require processing by caspase-1 to produce their active forms (11). Pathogen-associated molecular patterns and danger-associated molecular patterns activate various members of the nucleotide binding leucine-rich receptor (NLR) family in the cytoplasm, resulting in the assembly of an NLR containing, multiprotein complex (the inflammasome) that recruits and activates caspase-1 leading to IL-1␤ processing (12, 13). Although CARD domain-containing NLRs (e.g. Ipaf) can directly interact with caspase-1, most inflammasomes are assembled by Pyrin domain containing NLRs (NLRPs), which recruit caspase-1 indirectly through the adapter molecule ASC (14).Francisella tularensis is the causative agent of tularemia and a potential bioweapon (15). Pulmonary infection with even a single, virulent F. tularensis bacterium is potentially lethal if untreated (16,17). For humans, the type A strain, SchuS4 (F. tularensis sp. tularensis) results in the highest mortality, whereas neither the attenuated type B live vaccine strain LVS (F. tularensis sp. holarctica) or the U112 strain (Francisella tularensis sp. novicida) are virulent. In mice however, the SchuS4, LVS and U112 strains are lethal (18). Because of its importance in controlling bacterial infection and promoting adaptive immunity, the innate immune response to F. tularensis has been an area of recent interest. In mouse models of tularemia, the macrophage response to F. tularensis LVS is heavily reliant upon TLR2 as TLR2-deficient macrophages fail to produce TNF␣, IL-6, and other NF-B dependent proinflammatory cytokines (19,20). Mouse macrophages infected with U112 ...

Detection of intracellular pathogens is facilitated by members of the nucleotide binding-leucine rich receptor (NLR) family. The role of ASC/Caspase-1 axis for protective innate immunity against Francisella infection is well established, however the NLR(s) involved in initiation of innate immune responses during Francisella infection are yet unknown. Here we demonstrate that human NLRP3 mediates inflammasome dependent IL-1β production following Francisella infection. Notably, both human NLRP3 and mouse Nlrp3 are sufficient to assemble functional inflammasomes in HEK293T cells infected with Francisella. Furthermore, our results also support the existence of more than one Francisella-responsive cytoplasmic inflammasome complex and suggest that intrinsic differences in bacterial strains as well as monocyte-macrophage differentiation status influence inflammasome-dependent innate immune responses to Francisella.