Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 151-166).

Thesis (Ph. D., Microbiology, Molecular Biology and Biochemistry)--University of Idaho, October 2008. / Major professor: Scott A. Minnich. Includes bibliographical references. Also available online (PDF file) by subscription or by purchasing the individual file.

Francisella tularensis and Francisella novicida are closely related facultative intracellular pathogens capable of survival and growth within macrophages. In this work we present evidence to show that F. tularensis uses phase variation to alter lipopolysaccharide (LPS) antigenicity, macrophage nitric oxide (NO) production, and microbial intramacrophage growth. The LPS and lipid A of F. tularensis LVS fail to stimulate production of significant levels of nitric oxide by rat macrophage monolayers. However, spontaneous variants of F. tularensis expressing an antigenically distinct LPS induce rat macrophages to produce increased levels of NO, thereby suppressing intracellular growth. This new form of LPS produced by F. tularensis is also the predominant form of LPS found normally in F. novicida. Rat macrophages infected with F. novicida produce high levels of NO and exhibit suppression of intracellular growth. LPS and lipid A isolated from F. novicida and variants of F. tularensis stimulate increased levels of NO production. In addition, a reverse phase shift can occur which returns the LPS of the F. tularensis variants to the original antigenic form, resulting in reduced macrophage NO production and restoration of intracellular growth. These results suggest that F. tularensis can modulate macrophage NO production through phase variation of its LPS. It was of interest to initiate a study that would ultimately characterize the molecular mechanism of LPS phase variation in Francisella tularensis . To this end, we used shuttle mutagenesis to create a mutant library of F. novicida. We mutagenized a size- restricted plasmid library of F. novicida with the erythromycin- resistant transposon TnMax2. Putative F. novicida LPS mutants created by shuttle mutagenesis were screened visually for aberrant colony phenotypes on agar plates. Of 10464 mutants screened, 5 unique F. novicida LPS mutants were isolated which exhibit three distinct LPS phenotypes as determined by Western immunoblot. A single mutant from each of the three phenotypic groups was further characterized with respect to DNA sequence analysis, intramacrophage growth, and sensitivity to detergent and serum complement. Furthermore, these three loci were shown to hybridize with a corresponding locus in F. tularensis LVS. However, there was no difference in the restriction pattern of the hybridizing bands between LVS and its LPS phase variants, thus indicating that no major genetic rearrangements or insertion/deletion of a large mobile genetic element occurs in these genes during the phase variation process of F. tularensis. The F. novicida valAB locus has previously been cloned, sequenced, and shown to be functionally homologous to the E. coli genes msbA/lpxK. In order to investigate the hypothesis that valAB is involved in transport of LPS to the cell surface, an E. coli strain harboring an NTG-mutagenized temperature sensitive (t.s.) allele of valAB, a nonfunctional copy of msbA/lpxK, and an IPTG-inducible copy of the gene encoding the Chlamydia trachomatis genus-specific LPS epitope (gseA) was constructed. In this study, DNA sequencing was used to locate the temperature sensitive mutations in the valAB locus. Two C to T transitions were found in the valA coding region which result in a S to F change at amino acid 543 and a T to I change at amino acid 458. The ability of E. coli cells harboring this t.s. copy of valAB to transport the Chlamydia LPS epitope across the inner membrane at the permissive and non-permissive temperatures was determined using sucrose density gradient centrifugation and ELISA. It was determined that there was increased association of the LPS epitope with the inner membrane at the non-permissive temperature, thus suggesting that ValA is required for transport of an LPS precursor across the inner membrane. / Graduate

The innate immune response to pathogens by the host is dependent upon the interplay of both pathogen and host intrinsic factors. Nucleotide-binding domain leucine-rich repeat containing (NLR) and pyrin and HIN200 domain containing (PYHIN) proteins are intracellular sensors of damage-associated and pathogen-associated molecular patterns. The studies presented here focus on the PYHIN molecule, AIM2, and the NLR, NLRP12, and the importance of bacteria- and host-associated proteins in the coordination of the innate immune response to the Gram-negative pathogen Fracisella tularensis. We have found that several genes expressed by F. tularensis encode for proteins that, when disrupted, cause the bacteria to trigger hyper- or hypoactivation of the AIM2 inflammasome. Bacteria with a mutation in FTL_0724, which hyperactivates the AIM2 inflammasome, are highly attenuated in a mouse model of infection, and induce robust caspase-1 processing and secretion of IL-1β by bone marrow derived macrophages (BMDMs). In contrast the hypoactivating mutant of F. tularensis, FTL0699, reduces IL-1β secretion by BMDMs and remains virulent in in vivo models of infection. We have also investigated the role of host-expressed NLRs in F. tularensis infection models. We have found that NLRP12 is important in the coordination of the innate immune response to F. tularensis through the modulation of CXCL1 production. We hypothesized that decreased CXCL1 production by Nlrp12-deficient mice was in turn responsible for the diminished recruitment of neutrophils in to the lungs of Nlrp12-deficient mice following intranasal challenge with F. tularensis. Nlrp12-deficient mice were found to be highly susceptible to infection with F. tularensis, and succumbed to infection at a much higher rate than wild-type mice. Taken together these data demonstrate that a number of pathogen and host factors can play critical roles in the outcome F. tularensis infections.

This thesis is about analyzing genetic differences among isolates of Francisella tularensis – the tularemia-causing bacterium. To elucidate how these bacterial isolates are related, and their geographical and genetic origins, I have developed typing assays for Francisella and used them to study the epidemiology of tularemia. Tularemia is an infectious disease of humans and other mammals found throughout the Northern Hemisphere. The severity of the disease depends on the type of F. tularensis causing the infection. In Sweden, as in other countries of Europe and Eurasia, tularemia is caused by F. tularensis subsp. holarctica, while other varieties of the bacterium occur in Middle Asia and North America. It is important to identify a tularemia infection promptly in order to initiate the correct antibiotic treatment. A rapid identification of the causative F. tularensis variety gives additional clinical information. In recent years, several genomes of various Francisella strains have been sequenced, and in this thesis, I have utilized these genomes to identify genetic markers. In studies reported in the first paper (I) appended to the thesis, we identified and analyzed insertion/deletion mutations (INDELs) inferred to have resulted from a sequence repeat-mediated excision mechanism. We found eight new Regions of Difference (RDs) among Francisella strains. Using RDs together with single nucleotide polymorphisms (SNPs), we were able to predict an evolutionary scenario for F. tularensis in which Francisella novicida was the oldest variety while F. tularensis subsp. holarctica was the youngest. We also found that all virulence-attenuated isolates analyzed had deletions at two specific genetic regions - denoted RD18 and RD19 – suggesting that repeat-mediated excision is a mechanism of attenuation in F. tularensis. In subsequent studies (presented in paper II), we developed a combined analysis of INDELs lacking flanking repeats and variable number of tandem repeats (VNTRs). Both markers could be assayed using the same analytical equipment. The inclusion of INDELs provided increased phylogenetic robustness compared with the use of VNTRs alone, while still maintaining a high level of genetic resolution. In analyses described in the next paper (III), we selected INDELs from paper (II) and discovered novel SNPs by DNA comparisons of multiple Francisella strains. Thirty-four phylogenetically informative genetic markers were included in a hierarchical real-time PCR array for rapid and robust characterization of Francisella. We successfully used the assay to genotype 14 F. tularensis isolates from tularemia patients and DNA in six clinical ulcer specimens. Finally, in paper (IV) we demonstrated a strategy to enhance epidemiological investigations of tularemia by combining GIS-mapping of disease-transmission place collected from patient interviews, with high-resolution genotyping of F. tularensis subsp. holarctica isolates recovered from tularemia patients. We found the geographic distributions of specific F. tularensis subsp. holarctica sub-populations to be highly localized during outbreaks (infections by some genotypes being restricted to areas as small as 2 km2), indicative of a landscape epidemiology of tularemia with distinct point sources of infection. In conclusion, the results acquired during the studies underlying this thesis contribute to our understanding of the genetic genealogy of tularemia at both global and local outbreak scales.

Francisella tularensis is capable of infecting numerous cell types, including professional phagocytes. Upon phagocytosis, F. tularensis resides within the phagosome before escaping into the cytosol to replicate. Phagocytes constitute a hostile environment rich in ROS, which are employed as a means of killing pathogens. ROS interact with and disrupt the function of vital molecules such as DNA, proteins and bacterial structures. Iron potentiates the danger of ROS through the Fenton reaction where ferrous iron reduces H2O2 causing the formation of highly reactive hydroxyl radicals and anions. Low levels of ROS are formed during normal aerobic metabolism and pathogens thus have a need for defense mechanisms to handle the ever present levels of ROS but even more so to combat the onslaught of ROS experienced within a host. This thesis was focused on the investigation of the iron status and oxidative stress response of F. tularensis; thereby identifying key players controlling the bacterial iron content, its adaptation to oxygen-rich environments and defense against ROS. We identified subspecies-specific differences in iron content, where F. tularensis subsp. tularensis was found to contain significantly less iron than strains of subsp. holarctica. The reduced iron content resulted in an increased tolerance to H2O2, despite simultaneously causing a decrease in the activity of catalase - the iron-dependent enzyme responsible for degrading H2O2 in F. tularensis. This strongly suggests that the restricted iron uptake and storage by subsp. tularensis strains is beneficial by rendering the bacteria less susceptible to H2O2, thereby evading the toxic effects of the iron-driven Fenton reaction. This evasion is likely to be an important part of the higher virulence displayed by subsp. tularensis as compared to subsp. holarctica. We further identified that the global regulator, MglA, is important for the adaptation of LVS to oxygen-rich environments. Deletion of mglA from LVS resulted in a mutant, ΔmglA, with impaired defense to oxidative stress, as manifested by an inability to grow to wild-type levels under aerobic conditions, an accumulation of proteins with oxidative damage, a suppressed expression of iron-uptake related genes, an increased catalase activity, and an increased tolerance to H2O2. This phenotype was reversed in a microaerobic environment. We therefore conclude that MglA is an important factor for the defense of LVS to oxidative damage under aerobic conditions and speculate that MglA is of greatest importance in oxygen-rich foci. We also studied the role of OxyR in LVS by creating a ΔoxyR mutant as well as a double mutant, ΔoxyR/ΔkatG. The in vitro response of these mutants, as well as of ΔkatG, to defined ROS was assessed using H2O2, the O2- generating agent paraquat, and the ONOO- generator SIN-1. ΔoxyR was more susceptible to all ROS than LVS as was ΔkatG, with the exception of O2- Strikingly, ΔoxyR/ΔkatG was significantly more susceptible to all ROS tested compared to either single deletion mutant. LVS, ΔoxyR and ΔkatG replicated efficiently in bone marrow-derived macrophages whereas ΔoxyR/ΔkatG showed no replication. In mice, the ΔoxyR mutant displayed impaired replication in liver, but intact replication vs. LVS in spleen. Collectively, our results demonstrate an important role of OxyR in the oxidative stress response and virulence of F. tularensis, and further reveal overlapping roles of OxyR and catalase in the defense against ROS. The results thus shed new light on the complexity of ROS defense in F. tularensis.

The mechanisms by which the facultative intracellular pathogen Francisella tularensis is recognized by the innate immune system and the strategies that F. tularensis uses to avoid this recognition are not well understood. We have identified the basic components of the inflammasome that assemble in response to F. tularensis Live Vaccine Strain (LVS) challenge as containing the cysteine protease caspase-1, the adaptor protein ASC and the PYHIN molecule AIM2. We have also shown here that the nucleotide-binding domain leucine-rich repeat containing receptors (NLRs), NLRC4, NLRP3, NLRP6, NLRP10, and NLRP12 were not necessary for activation of caspase-1 and subsequent IL-1β secretion in response to challenge with F. tularensis LVS in vitro. In vivo, NLRC4, NLRP3, NLRP6, NLRP10, and NLRP12 did not appear to enhance survival. However, caspase-1- and ASC-deficient mice succumbed more rapidly to infection, indicating that the inflammasome played a role in defense against F. tularensis LVS. Additionally, we identified a gene with homology to Escherichia coli mviN, a putative lipid II flippase, that functions as a F. tularensis virulence factor. In vivo infection of mice with a F. tularensis LVS mviN transposon mutant (mviN::Tn5) resulted in improved host survival and decreased bacterial burdens compared to infection with wild-type F. tularensis LVS. Wild-type F. tularensis LVS and the mviN::Tn5 mutant replicated at a similar rate in both macrophages and liquid broth culture. Additionally, the ability to induce the production of TNF-α or IL-6 was also similar between WT F. tularensis and the mviN::Tn5 mutant. In contrast to the similar levels of production of IL-6 and TNF-α, the mviN mutant induced increased AIM2 inflammasome-dependent IL-1β secretion and cytotoxicity in macrophages compared to wild-type F. tularensis. The compromised in vivo virulence associated with the mutation of mviN was dependent upon inflammasome activation, as caspase-1- and ASC-deficient mice did not exhibit preferential survival following infection. These data show that F. tularensis LVS activation of the inflammasome is caspase-1-, ASC-, and AIM2-dependent. These data also identify mviN as a novel F. tularensis virulence factor that enables F. tularensis LVS to evade some AIM2 inflammasome activation.

Francisella tularensis is a highly infective, intracellular bacterium. It is capable of infecting a wide range of mammals and causes the disease tularemia in humans. As a result of its high infectivity there have been a lot of efforts made to create a generally available vaccine against this pathogen. One potential vaccine candidate is the FSC043 strain, a spontaneous mutant that has acquired mutations making it attenuated for replication both in vitro and in the experimental mouse model. However, it was noted that it afforded protection against challenge with a highly virulent F. tularensis strain. The aim of this thesis has been to delineate the mechanisms of its attenuation to better understand F. tularensis pathogenesis and to obtain a better knowledge about the prerequisites of protective immunity against this potent pathogen. Microarray and whole-genome sequencing revealed four mutations in the attenuated FSC043 strain that were not present in the virulent SCHU S4 isolate. One of these mutations has been described earlier as it results in a fusion protein also found in other attenuated strains. Among the other differences, two mutations were identical nonsense mutations in a duplicated gene region known as the Francisella pathogenicity island (FPI). The affected gene, pdpC, is coding for PdpC (pathogenicity determinant protein C). We found that these mutations resulted in a truncated form of PdpC, and also that the downstream gene was severely downregulated due to these mutations. Further, our studies revealed that the intracellular phenotype of the FSC043 strain differed from other tested strains in that a small portion of the intracellular bacteria were able to escape the phagosome and multiply within the host, while the majority of intracellular bacteria stayed confined to the phagosome. We wanted to study the specific function of pdpC and therefore deleted both copies of it in the virulent SCHU S4strain as well as the Live Vaccine Strain, an empirically attenuated strain often used as a model for the virulent strains of F. tularensis. The resulting mutants showed an attenuated phenotype; no intracellular growth in murine cells, and no virulence in mice. When studying the intracellular localization of the LVS Δpdpc mutant, we found that it was uniformly located adjacent to phagosomal membrane-like structures but that the membrane was markedly disrupted. Further, this mutant induced an MOI-dependent cytotoxicity, measured by LDH release, and also the release of IL-1β, an inflammatory cytokine not induced by phagosomally contained mutants. Studies on markers for host cell death revealed that the LVS ΔpdpC mutant induced mitochondrial instability, phosphatidylserine (PS) presentation, and TUNEL-specific DNA fragmentation in infected cells, rather similar to the wild-type strain, despite its lack of replication. This study reveals that the pdpC gene is an important gene required for F. tularensis virulence. We also show that non-replicating intracellular bacteria can induce host cell death, hypothesizing that release of bacterial components in the host cell cytosol is required for this induction. The FSC043 mutant showed a unique phenotype where a small subset of bacteria was able to escape the phagosome and replicate in the host cell. This was also seen in the pdpC deletion mutant of SCHU S4, but not with the LVS ΔpdpC. However, regardless of genetic background, the ΔpdpC mutant had an effect on phagosomal escape; either by affecting the phagosomal membranes in a unique way or by allowing phagosomal escape of a small proportion of the bacteria.

Diss. (sammanfattning) Umeå : University, 2005. / Härtill 5 uppsatser. På omsl. felaktigt: N.S. 954.

Diss. (sammanfattning) Umeå : Univ., 2006. / Härtill 4 uppsatser.