Francisella novicida is a facultative intracellular pathogen capable of growing in macrophages. A spontaneous mutant of F. novicida defective for growth in macrophages was isolated on LB media containing the chromogenic phosphatase substrate 5-bromo-4-chloro-3-indolyl phosphate (X-p) and designated GB2. Using an in cis complementation strategy, four strains were isolated which are restored for growth in macrophages. A locus isolated from one of these strains complements GB2 for the intracellular growth defect, colony morphology on LB (X-p) media, and virulence in mice. The locus consists of an apparent operon of two genes, designated mglAB, for macrophage growth locus. Both mglA and mglB transposon insertion mutants are defective for intracellular growth and have a phenotype similar to GB2 on LB (X-p) media. Sequencing of mglA cloned from GB2 identified a missense mutation, providing evidence that both mglA and mglB are required for the intramacrophage growth of F. novicida. Preliminary studies have also identified a convergently transcribed gene, tentatively designated mglC, immediately downstream of mglB. mglC null mutants are defective for intracellular growth and show the same phenotype on LB (X-p) agar as GB2. mglB expression in GB2 was confirmed using antiserum against recombinant MglB. Western blot analysis revealed the absence of MglA in an mglB null mutant, indicating MglB may influence MglA levels. Analysis of the regulation of mglA expression during growth in broth culture shows a decrease in expression upon entering late log-early stationary phase. mglA is also expressed during culture in macrophages. Cell fractionation studies revealed several differences in the protein profiles of mgl mutants compared with wild-type F. novicida, most notably the absence of a 70 kDa secreted protein. A candidate clone for the gene encoding this 70 kDa protein has been isolated. The deduced amino acid sequences of mglA and mglB show similarity to the SspA and SspB proteins of Escherichia coli and Haemophilus spp. In E. coli, SspA and/or SspB influence the levels of multiple proteins under conditions of nutritional stress, and SspA can associate with the RNA polymerase holoenzyme. Taken together, these observations suggest that in Francisella MglA and MglB may control the expression of genes whose products contribute to survival and growth within macrophages. Roles for the putative MglC and possibly the 70 kDa secreted protein in this activity are also indicated. Acid phosphatases capable of inhibiting the respiratory burst of neutrophils have been identified in certain intracellular pathogens. The gene encoding AcpA, a respiratory burst-inhibiting acid phosphatase of Francisella , was cloned and sequenced. The deduced amino acid sequence of AcpA showed limited similarity to phospholipase C proteins present in Pseudomonas aeruginosa and Mycobacterium tuberculosis. An F. novicida acpA null mutant was found to exhibit wild-type growth kinetics in both cell-line and inflammatory mouse macrophages as well as remaining virulent for mice. These data suggest that AcpA is not essential for the intracellular growth or virulence of F. novicida, and that any role it may play in virulence is subtle. / Graduate

No description available.

Francisella tularensis is a Gram-negative pathogenic organism that causes the disease tularemia. This disease can be potentially fatal without treatment. Francisella tularensis virulent strains can cause disease in humans with an infectious dose as low as 10 organisms. As a result of this low infectious dose, high mortality, and ease to produce an aerosol inoculum, the Centers for Disease Control and Prevention has classified Francisella tularensis as a Tier I select agent, the highest threat level. Much research has been done to determine the cause for the extreme virulence. However, despite these efforts, little is known about the mechanisms by which Francisella goes undetected inside host cells until it is too late for the host to respond. Researchers in the Jones' laboratory utilized a transposon site hybridization (TraSH) screen with human monocyte derived macrophages (MDMs) as the host cell and an enzyme-linked immunosorbent assay (ELISA) screen of pools of transposon mutants searching for virulence determinants and genes responsible for Francisella capsule or LPS. Through the TraSH screen, our group identified a locus of genes, FTT1236, FTT1237, and FTT1238c as being important for survival within human MDMs. From the mutant library screen using ELISA, I identified the same genes, FTT1236 and FTT1238c. In addition, I also identified wzy, wbtA, FTT0846, and hemH as being involved in LPS and or capsule production. A similar ELISA screen was done by researchers in Apicella laboratory using a different monoclonal antibody that identified insertions in, dnaJ, manB and an intergenic region between FTT0673 and FTT0674c that potentially disrupted LPS and capsule biogenesis. Previously, FTT1236, FTT1237, and FTT1238c mutants were observed by our laboratory to be serum sensitive and activate MDMs by an unknown mechanism. I further characterized these mutant strains by analyzing the changes in the LPS core. I identified core truncations for the FTT1236 and FTT1237 mutants, but not FTT1238c. Combining this new data with previously published work and bioinformatical analysis of the FTT1236, FTT1237 and FTT1238c proteins, I hypothesized that these proteins have functions similar to Waa proteins of other organisms, which are involved in LPS core assembly and O-antigen ligation. With functional complementation and mass spectrometry of LPS preparations, I have designated FTT1236, FTT1237, and FTT1238c as WaaY, WaaZ, and WaaL respectively. In addition to this work characterizing the biochemical functions of these gene products, I examined the effect of mutations in these genes on the virulence of Francisella. In contrast to infection with wild type Schu S4, mice infected either intraperitoneally or intranasally displayed significant inflammatory responses to infection and the strains were significantly attenuated by either route of infection. I also observed that waaY and waaL mutant strains disseminated to the liver and spleen after an intranasal infection despite their lack of O-antigen and capsule. At an i.n. dose of 106 CFU these mutant strains still caused lethal murine infection, but death occurred around day 12 post infection; mice infected with <20 CFU of Schu S4 succumb at day 5 post infection. The cause of the death in mice infected with these mutant strains was pulmonary edema, rather than multiple organ failure induced by Schu S4. Of the additional seven mutant strains identified from the ELISA screens, I characterized their physical phenotypes, virulence defects, and their potential as an attenuated live vaccine. All of these strains were determined to be sensitive to human pooled serum to various degrees. Three of these strains, dnaJ::Tn5, hemH::Tn5, and FTT0673p/prsAp::Tn5 did not have identifiable defects in capsule or LPS biosynthesis, nor were they attenuated in mice. The remaining four strains, FTT0846::Tn5, manB::Tn5, wzy::Tn5, and wbtA::Tn5, were found to have LPS O-antigen and capsule defects, and two of these strains had LPS core defects (FTT0846::Tn5 and manB::Tn5). Each of these four strains was attenuated in mice, when compared to WT. I also tested the ability of mice infected with waaY::TrgTn, waaL::TrgTn, and wbt::Tn5 to be protected from lethal challenges of Schu S4. All three strains provided some level of protection against lethal Schu S4 challenges. In addition, I also tested Francisella LPS and capsule to provide protection against lethal challenges of LVS and Schu S4. I determined that LPS and capsule protected against high doses of LVS, but LPS did not provide any protection when immunized mice were challenged with Schu S4. Interestingly, we observed that mice immunized with capsule were partially protected from lethal Schu S4 challenges. In addition, I observed a novel difference between virulent Francisella strains and LVS, in that virulent strains have O-antigen glycosylated and LVS appears to be lacking this characteristic. Collectively, this work adds to the growing data of the importance of LPS and the role of capsule role in immune evasion as well as the significance of capsule and LPS mutant strains to provide protection against Schu S4.

Diss. (sammanfattning) Umeå : Univ., 2004. / Härtill 5 uppsatser.

Thesis (M.S.)--University of Toledo, 2007. / "In partial fulfillment of the requirements for the degree of Master of Science in Biomedical Sciences." Title from title page of PDF document. A role for iglC in the growth and persistence of Francisella tularensis within macrophages. Bibliography: p. 78-85

Die genomische Insel (GI) FhaGI 1 des Stammes Francisella hispaniensis (Fhis) AS02 814 kann sowohl in die tRNAVal integriert als auch als episomale Form vorliegen und kodiert für einen putativen Prophagen. Im Rahmen dieser Arbeit konnte durch Verwendung synthetisch hergestellter, verkürzter Varianten von FhaGI-1 gezeigt werden, dass die GI auf andere Francisella Spezies übertragbar ist. Die ortsspezifische Integration und Exzision der GI sind Integrase-abhängige Prozesse, die durch weitere regulatorische Gene beeinflusst werden. Die Identifizierung der GI FphGI 1 in drei F. philomiragia-Stämmen zeigt, dass die tRNAVal als Integrationsort für GIs in Francisella dient. Die vermutlich nicht funktionale Integrase von FphGI 1 ist wahrscheinlich die Ursache für das Fehlen einer episomalen Form der GI. Das Vorhandensein von GIs in Francisella liefert einen Hinweis darauf, dass horizontaler Gentransfer zwischen verschiedenen Francisella Spezies möglich ist. Auf Grundlage von FhaGI 1 wurden zwei Varianten eines Francisella- Phagenintegrationsvektors (pFIV1-Val und pFIV2 Val) generiert. Der FIV Teil der Vektoren bildet eine zirkuläre, episomale Form, die nach der Transformation in verschiedene Francisella Spezies ortspezifisch in die tRNAVal integriert. Es konnte gezeigt werden, dass die Vektoren für die Expression von Reportergenen sowie die Komplementation von Francisella Deletionsmutanten geeignet sind. Sie sind sowohl in vitro als auch während der Infektion von Wirtszellen ohne Selektionsdruck stabil und zählen zu den low-copy-Vektoren. Damit erweitern die FIV-Vektoren das Repertoire der vorhandenen Werkzeuge zur genetischen Manipulation von Francisellen. Da der Stamm Fhis AS02 814 für Untersuchungen nicht zur Verfügung stand, wurde FhaGI 1 synthetisch in zwei Hälften hergestellt, die jedoch bisher nicht zusammengeführt werden konnten. Damit ist eine Aussage darüber, ob es sich bei FhaGI 1 tatsächlich um einen funktionalen Prophagen handelt, bis jetzt nicht möglich / The genomic island (GI) FhaGI 1 of strain Francisella hispaniensis (Fhis) AS02 814 can exist as a circular episomal form or integrated into the tRNAVal gene and codes for a putative prophage. In this work small-sized variants of FhaGI 1 were used to show that the GI can be transferred to other Francisella species. The site-specific integration and excision of the GI are integrase-dependent processes that are influenced by further regulatory genes. The identification of the GI FphGI 1 in three F. philomiragia strains shows that the tRNAVal gene serves as an integration site for GIs in Francisella. The integrase of FphGI 1 is probably non-functional and hence presumably the reason for the missing episomal form of the GI. The presence of GIs in Francisella might be an indication that horizontal gene transfer between different Francisella species could be possible. Two variants of a Francisella phage integration vector (pFIV1 Val and pFIV2 Val) were successfully constructed based on FhaGI 1. The FIV Val part of the vectors integrates site-specifically into the tRNAVal after transformation into different Francisella species. It was demonstrated that the vectors can be used for the expression of reporter genes as well as for the complementation of Francisella deletion mutants. They remain stable without selective pressure during in vitro growth and during the infection of host cells and fall into the group of low-copy-vectors. The FIV Val vectors expand the repertoire of tools that can be used for the genetic manipulation of Francisella. As strain Fhis AS02 814 could not be obtained for further analysis, FhaGI 1 was synthetically generated in two halves which could not be joined so far. Consequently, it is not possible to state whether FhaGI 1 actually codes for a functional prophage.

Francisella tularensis was a facultative intracellular pathogen and a gram-negative coccobacillus which has been categorized by the CDC as a potential class A select agent due to its highly infectious properties and high mortality rates. Francisella tularensis was also responsible for the zoonotic disease tularemia, which was usually transmitted by arthropod vectors or via contact with infected animals. Francisella tularensis subspecies novicida has been used by many researchers in genetic pathogenesis experiments to try to elucidate genes responsible for virulence factors. One of these virulence factors was a capsular material which has been thought to be involved in either increasing pathogenicity or infectivity of this organism upon engulfment by its principal host cell, the macrophage. There were many potential genetic loci which may be involved in this biosynthetic process of encapsulation. One such locus has excellent homology to the capsule biosynthesis operon of Bacillus anthracis, which, under certain conditions, creates a polyglutamic acid capsule (PGA). A transposon mutation in the amide ligase (capB) in LVS has a reduced virulence in murine infection models. I wished to investigate whether Francisella novicida was capable of producing such a capsule and under which environmental conditions this capsule was made. I have created a site-directed mutant of the capB gene in Francisella novicida U112 using targeted mutagenesis via PCR SOEing and have introduced this mutation via electroporation of a suicide vector. I have tested our mutant against preimmune serum treatments and have shown reduced viability as well as a reduced capacity for replication inside RAW 264.7 murine macrophages. I assayed for production of a PGA capsule via immunodot blot and electron microscopy as well as analysis by mass spectrophotometry of capsular extracts. I also tested various media constituents and different environmental conditions to determine which external stimuli may contribute to PGA capsule biosynthesis as well as regulatory changes in transcript levels of this operon.

Francisella tularensis is the causative agent of the life-threatening disease tularemia. The Centers for Disease Control considers F. tularensis among the most likely agents of biowarfare due to its high mortality rate, ease of aerosol transmission, and low infectious dose. A fundamental aspect of tularemia pathogenesis is the overwhelming accumulation of neutrophils in the lung that are incapable of bacterial clearance and furthermore injurious to the host tissue, as neutrophilia exacerbates disease and blockade of neutrophil influx into the lungs favors host survival. We hypothesized that the pathologic accretion of neutrophils may be the result of decreased neutrophil death and/or decreased clearance by macrophages. Our lab recently demonstrated that F. tularensis delays neutrophil apoptosis by at least 48 hours to preserve its replicative niche, but the mechanism by which this occurs was poorly defined. Here, we investigate alterations in neutrophil apoptosis and survival signaling at the molecular level and find that, in addition to effects on neutrophil transcription, F. tularensis also modulates protein abundance, activity, and subcellular localization. Specifically, we report that F. tularensis preserves mitochondrial integrity by inhibiting the pro-apoptotic proteins Bid and Bax as well as maintaining expression of the pro-survival factors XIAP and calpastatin. Moreover, we found that infection diminishes the ability of R-roscovitine to induce apoptosis, suggesting bacterial modulation of CDK-mediated survival signaling. Following apoptosis, effete neutrophils are rapidly cleared by macrophages in a process termed efferocytosis to avoid neutrophil progression to secondary necrosis and consequent host tissue damage. We demonstrate for the first time that neutrophils laden with F. tularensis are readily consumed by macrophages and release their infectious cargo into the macrophage cytoplasm. The engulfing cell is unable to eradicate the infection and extensive bacterial replication ensues. Intriguingly, we found that unlike other pathogens, covert infection of macrophages by F. tularensis triggers an inflammatory cytokine response that is highly similar to that of directly infected cells, suggesting that efferocytosis is not an essential virulence mechanism for this bacterium. Together, these studies significantly advance our understanding of fundamental F. tularensis virulence mechanisms and disease pathophysiology as well as shed light on other inflammatory disorders characterized by dysregulated neutrophil turnover and clearance.

F. tularensis is an intracellular pathogen, and is the causative agent of tularemia in humans. The ability of F. tularensis to parasitize host cells is largely dependent upon genes within a pathogenicity island (FPI), including those in the iglABCD operon. Specific mechanisms and gene products involved in regulation of the FPI are not well understood. I initiate the study of this regulatory system by creating an efficient Tn5-based mutagenesis system optimized for use in F. tularensis, and utilize this system to construct a lacZ reporter library. I identify genes differentially regulated in response to growth on two different media, including those in the iglABCD and fslABCD operons, and identify iron availability as a factor contributing to the differential regulation. One of these reporter strains, carrying a chromosomal iglB-lacZ fusion, is used as the basis for a secondary transposon mutagenesis to identify mutations that affect iglABCD expression. One such mutation is in FTL_1542 (migR), a hypothetical protein, and reduces expression of the iglABCD approximately 8-fold. The effect of this mutation on igl expression is likely through its effect on another known virulence regulator, fevR, as demonstrated by data from RT-PCR experiments. I compare the phenotypes of LVS fevR and migR mutant strains in primary macrophage and epithelial cell lines and in neutrophils. The mutation in migR effects growth and intracellular trafficking in macrophages but not epithelial cells, and reverses the ability of wild type F. tularensis to block the respiratory burst in neutrophils. When similar mutations were examined in the human virulent F. tularensis strain Schu S4, migR retained its regulatory role, but did not impair replication in macrophages. The migR mutation in Schu S4 did however have an attenuating effect when administered to mice intranasally. Comparison of LVS and Schu S4 in primary human airway epithelial cell infections revealed an inability of LVS to replicate within these cells, which is in contrast to the robust replication of LVS in cultured epithelial cell lines. Together, this work contributes to the understanding of regulatory mechanisms governing virulence gene expression in F. tularensis and highlights differences between LVS and Schu S4 strains.

Francisella tularensis is a facultative intracellular bacterium and the causative agent of tularemia, a severe and potentially fatal disease in humans. This pathogen is extremely infectious by the aerosol route and inhalation of as few as 10 organisms can cause severe pneumonic disease. Consequently, F. tularensis was developed as a bioweapon by several nations and is considered a category A select agent by the Centers for Disease Control and Prevention. The ability of F. tularensis to cause overwhelming infections at low infectious doses suggests this organism has adapted efficient mechanisms to evade containment by the host innate immune system. The goal of this thesis was to better understand the mechanisms by which Francisella modulates innate host defenses, with particular focus on interactions between this pathogen and two important effectors of innate immunity: neutrophils and the complement system. We demonstrate that F. tularensis profoundly modulates neutrophil lifespan during infection, delaying spontaneous apoptosis by inhibiting both the intrinsic and extrinsic apoptotic pathways to maintain an intracellular niche for persistence and proliferation. Furthermore, we show that F. tularensis can override activation of the apoptotic program induced by extracellular apoptotic signals that may drive neutrophil apoptosis at the site of infection. Initial characterization of the molecular mechanisms behind apoptosis inhibition by this pathogen suggests that F. tularensis employs multiple, redundant mechanisms to promote global anti-apoptosis in the cell. Transcriptome analyses of infected PMNs using oligonucleotide microarrays show that 365 unique apoptosis and cell survival genes are differentially regulated between 3-24 hr, several of which directly modulate intrinsic and extrinsic pathway signaling. Moreover, we demonstrate that levels of the potent caspase inhibitor, X-linked inhibitor of apoptosis protein (XIAP), are maintained over the course of infection, which may represent an important mechanism of caspase inhibition by this pathogen. We also confirm reports that F. tularensis can activate complement during incubation in nonimmune serum, and demonstrate for the first time that natural IgM antibodies bind to the bacterial surface and mediate complement opsonization to promote phagocytosis by both human neutrophils and macrophages. Finally, we identify the first neutrophil receptors, CR1 and CR3, involved in the uptake of complement-opsonized F. tularensis. In sum, our data presented here significantly advance our understanding of the host-pathogen relationship between F. tularensis and components of innate immunity, and suggest that this pathogen modulates both neutrophil and complement function to evade innate immune defenses and cause disease.