Association of Mycobacterium Proteins with Lipid Droplets.

Mycobacterium tuberculosis is a global pathogen of significant medical importance. A key aspect of its life cycle is the ability to enter into an altered physiological state of nonreplicating persistence during latency and resist elimination by the host immune system. One mechanism by which M. tuberculosis facilitates its survival during latency is by producing and metabolizing intracytoplasmic lipid droplets (LDs). LDs are quasi-organelles consisting of a neutral lipid core such as triacylglycerol surrounded by a phospholipid monolayer and proteins. We previously reported that PspA (phage shock protein A) associates with LDs produced in Mycobacterium In particular, the loss or overproduction of PspA alters LD homeostasis in Mycobacterium smegmatis and attenuates the survival of M. tuberculosis during nonreplicating persistence. Here, M. tuberculosis PspA (PspAMtb) and a ΔpspA M. smegmatis mutant were used as model systems to investigate the mechanism by which PspA associates with LDs and determine if other Mycobacterium proteins associate with LDs using a mechanism similar to that for PspA. Through this work, we established that the amphipathic helix present in the first α-helical domain (H1) of PspA is both necessary and sufficient for the targeting of this protein to LDs. Furthermore, we identified other Mycobacterium proteins that also possess amphipathic helices similar to PspA H1, including a subset that localize to LDs. Altogether, our results indicate that amphipathic helices may be an important mechanism by which proteins target LDs in prokaryotes.IMPORTANCEMycobacterium spp. are one of the few prokaryotes known to produce lipid droplets (LDs), and their production has been linked to aspects of persistent infection by M. tuberculosis Unfortunately, little is known about LD production in these organisms, including how LDs are formed, their function, or the identity of proteins that associate with them. In this study, an established M. tuberculosis LD protein and a surrogate Mycobacterium host were used as model systems to study the interactions between proteins and LDs in bacteria. Through these studies, we identified a commonly occurring protein motif that is able to facilitate the association of proteins to LDs in prokaryotes.

Rv2744c Is a PspA Ortholog That Regulates Lipid Droplet Homeostasis and Nonreplicating Persistence in Mycobacterium tuberculosis.

UNLABELLED: Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), remains a significant cause of morbidity and mortality worldwide, despite the availability of a live attenuated vaccine and anti-TB antibiotics. The vast majority of individuals infected with M. tuberculosis develop an asymptomatic latent infection in which the bacterium survives within host-generated granulomatous lesions in a physiologically altered metabolic state of nonreplicating persistence. The granuloma represents an adverse environment, as M. tuberculosis is exposed to various stressors capable of disrupting the essential constituents of the bacterium. In Gram-negative and Gram-positive bacteria, resistance to cell envelope stressors that perturb the plasma membrane is mediated in part by proteins comprising the phage shock protein (Psp) system. PspA is an important component of the Psp system; in the presence of envelope stress, PspA localizes to the inner face of the plasma membrane, homo-oligomerizes to form a large scaffold-like complex, and helps maintain plasma membrane integrity to prevent a loss of proton motive force. M. tuberculosis and other members of the Mycobacterium genus are thought to encode a minimal functional unit of the Psp system, including an ortholog of PspA. Here, we show that Rv2744c possesses structural and physical characteristics that are consistent with its designation as a PspA family member. However, although Rv2744c is upregulated under conditions of cell envelope stress, loss of Rv2744c does not alter resistance to cell envelope stressors. Furthermore, Rv2744c localizes to the surface of lipid droplets in Mycobacterium spp. and regulates lipid droplet number, size, and M. tuberculosis persistence during anaerobically induced dormancy. Collectively, our results indicate that Rv2744c is a bona fide ortholog of PspA that may function in a novel role to regulate lipid droplet homeostasis and nonreplicating persistence (NRP) in M. tuberculosis IMPORTANCE: Mycobacterium tuberculosis is the causative agent of tuberculosis, a disease associated with significant morbidity and mortality worldwide. M. tuberculosis is capable of establishing lifelong asymptomatic infections in susceptible individuals and reactivating during periods of immune suppression to cause active disease. The determinants that are important for persistent infection of M. tuberculosis or for reactivation of this organism from latency are poorly understood. In this study, we describe our initial characterizations of Rv2744c, an ortholog of phage shock protein A (PspA) that regulates the homeostasis of lipid bodies and nonreplicating persistence in M. tuberculosis This function of PspA in M. tuberculosis is novel and suggests that PspA may represent a unique bacterial target upon which to base therapeutic interventions against this organism.

The MprB extracytoplasmic domain negatively regulates activation of the Mycobacterium tuberculosis MprAB two-component system.

Mycobacterium tuberculosis is an acid-fast pathogen of humans and the etiological agent of tuberculosis (TB). It is estimated that one-third of the world's population is latently (persistently) infected with M. tuberculosis. M. tuberculosis persistence is regulated, in part, by the MprAB two-component signal transduction system, which is activated by and mediates resistance to cell envelope stress. Here we identify MprAB as part of an evolutionarily conserved cell envelope stress response network and demonstrate that MprAB-mediated signal transduction is negatively regulated by the MprB extracytoplasmic domain (ECD). In particular, we report that deregulated production of the MprB sensor kinase, or of derivatives of this protein, negatively impacts M. tuberculosis growth. The observed growth attenuation is dependent on MprAB-mediated signal transduction and is exacerbated in strains of M. tuberculosis producing an MprB variant lacking its ECD. Interestingly, full-length MprB, and the ECD of MprB specifically, immunoprecipitates the Hsp70 chaperone DnaK in vivo, while overexpression of dnaK inhibits MprAB-mediated signal transduction in M. tuberculosis grown in the absence or presence of cell envelope stress. We propose that under nonstress conditions, or under conditions in which proteins present in the extracytoplasmic space are properly folded, signaling through the MprAB system is inhibited by the MprB ECD. Following exposure to cell envelope stress, proteins present in the extracytoplasmic space become unfolded or misfolded, leading to removal of the ECD-mediated negative regulation of MprB and subsequent activation of MprAB.

The tunicamycin derivative TunR2 exhibits potent antibiotic properties with low toxicity in an in vivo Mycobacterium marinum-zebrafish TB infection model.

Tunicamycins (TUN) are well-defined, Streptomyces-derived natural products that inhibit protein N-glycosylation in eukaryotes, and by a conserved mechanism also block bacterial cell wall biosynthesis. TUN inhibits the polyprenylphosphate-N-acetyl-hexosamine-1-phospho-transferases (PNPT), an essential family of enzymes found in both bacteria and eukaryotes. We have previously published the development of chemically modified TUN, called TunR1 and TunR2, that have considerably reduced activity on eukaryotes but that retain the potent antibacterial properties. A mechanism for this reduced toxicity has also been reported. TunR1 and TunR2 have been tested against mammalian cell lines in culture and against live insect cells but, until now, no in vivo evaluation has been undertaken for vertebrates. In the current work, TUN, TunR1, and TunR2 are investigated for their relative toxicity and antimycobacterial activity in zebrafish using a well-established Mycobacterium marinum (M. marinum) infection system, a model for studying human Mycobacterium tuberculosis infections. We also report the relative ability to activate the unfolded protein response (UPR), the known mechanism for the eukaryotic toxicity observed with TUN treatment. Importantly, TunR1 and TunR2 retained their antimicrobial properties, as evidenced by a reduction in M. marinum bacterial burden, compared to DMSO-treated zebrafish. In summary, findings from this study highlight the characteristics of recently developed TUN derivatives, mainly TunR2, and its potential for use as a novel anti-bacterial agent for veterinary and potential medical purposes.

MprA and DosR coregulate a Mycobacterium tuberculosis virulence operon encoding Rv1813c and Rv1812c.

Mycobacterium tuberculosis remains a significant global pathogen, causing extensive morbidity and mortality worldwide. This bacterium persists within granulomatous lesions in a poorly characterized, nonreplicating state. The two-component signal transduction systems MprAB and DosRS-DosT (DevRS-Rv2027c) are responsive to conditions likely to be present within granulomatous lesions and mediate aspects of M. tuberculosis persistence in vitro and in vivo. Here, we describe a previously uncharacterized locus, Rv1813c-Rv1812c, that is coregulated by both MprA and DosR. We demonstrate that MprA and DosR bind to adjacent and overlapping sequences within the promoter region of Rv1813c and direct transcription from an initiation site located several hundred base pairs upstream of the Rv1813 translation start site. We further show that Rv1813c and Rv1812c are cotranscribed, and that the genomic organization of this operon is specific to M. tuberculosis and Mycobacterium bovis. Although Rv1813c is not required for survival of M. tuberculosis in vitro, including under conditions in which MprAB and DosRST signaling are activated, an M. tuberculosis ΔRv1813c mutant is attenuated in the low-dose aerosol model of murine tuberculosis, where it exhibits a lower bacterial burden, delayed time to death, and decreased ability to stimulate proinflammatory cytokines interleukin-1ß (IL-1ß) and IL-12. Interestingly, overcomplementation of these phenotypes is observed in the M. tuberculosis ΔRv1813c mutant expressing both Rv1813c and Rv1812c, but not Rv1813c alone, in trans. Therefore, Rv1813c and Rv1812c may represent general stress-responsive elements that are necessary for aspects of M. tuberculosis virulence and the host immune response to infection.

Components of the Rv0081-Rv0088 locus, which encodes a predicted formate hydrogenlyase complex, are coregulated by Rv0081, MprA, and DosR in Mycobacterium tuberculosis.

Mycobacterium tuberculosis, the etiological agent of tuberculosis, remains a significant cause of morbidity and mortality throughout the world despite a vaccine and cost-effective antibiotics. The success of this organism can be attributed, in part, to its ability to adapt to potentially harmful stress within the host and establish, maintain, and reactivate from long-term persistent infection within granulomatous structures. The DosRS-DosT/DevRS-Rv2027c, and MprAB two-component signal transduction systems have previously been implicated in aspects of persistent infection by M. tuberculosis and are known to be responsive to conditions likely to be found within the granuloma. Here, we describe initial characterization of a locus (Rv0081-Rv0088) encoding components of a predicted formate hydrogenylase enzyme complex that is directly regulated by DosR/DevR and MprA, and the product of the first gene in this operon, Rv0081. In particular, we demonstrate that Rv0081 negatively regulates its own expression and that of downstream genes by binding an inverted repeat element in its upstream region. In contrast, DosR/DevR and MprA positively regulate Rv0081 expression by binding to recognition sequences that either partially or completely overlap that recognized by Rv0081, respectively. Expression of Rv0081 initiates from two promoter elements; one promoter located downstream of the DosR/DevR binding site but overlapping the sequence recognized by both Rv0081 and MprA and another promoter downstream of the DosR/DevR, Rv0081, and MprA binding sites. Interestingly, Rv0081 represses Rv0081 and downstream determinants following activation of DosRS-DosT/DevRS-Rv2027c by nitric oxide, suggesting that expression of this locus is complex and subject to multiple levels of regulation. Based on this and other published information, a model is proposed detailing Rv0081-Rv0088 expression by these transcription factors within particular growth environments.

PepD participates in the mycobacterial stress response mediated through MprAB and SigE.

Currently, one-third of the world's population is believed to be latently infected with Mycobacterium tuberculosis. The mechanisms by which M. tuberculosis establishes latent infection remain largely undefined. mprAB encodes a two-component signal transduction system required by M. tuberculosis for aspects of persistent infection. MprAB regulates a large and diverse group of genetic determinants in response to membrane stress, including the extracytoplasmic function (ECF) sigma factor sigE and the HtrA-like serine protease pepD. Recent studies have demonstrated that PepD functions as both a protease and chaperone in vitro. In addition, inactivation of pepD alters the virulence of M. tuberculosis in a mouse model system of infection. Here, we demonstrate that PepD plays an important role in the stress response network of Mycobacterium mediated through MprAB and SigE. In particular, we demonstrate that the protease activity of PepD requires the PDZ domain, in addition to the catalytic serine at position 317. pepD expression initiates from at least three promoters in M. tuberculosis, including one that is regulated by SigE and is located upstream of the mprA coding sequence. Deletion of pepD or mprAB in Mycobacterium smegmatis and M. tuberculosis alters the stress response phenotypes of these strains, including increasing sensitivity to SDS and cell wall antibiotics and upregulating the expression of stress-responsive determinants, including sigE. Taking these data together, we hypothesize that PepD utilizes its PDZ domain to recognize and process misfolded proteins at the cell membrane, leading to activation of the MprAB and SigE signaling pathways and subsequent establishment of a positive feedback loop that facilitates bacterial adaptation.

Genetics and genetic manipulation in Francisella tularensis.

Francisella tularensis is a gram-negative coccobacillus and the etiological agent of tularemia. The limited knowledge regarding the interaction of F. tularensis with its host is due in part to the previous lack of tools for genetically manipulating the organism. During the past 10 years, the field of F. tularensis genetics has seen a rapid expansion. Plasmids capable of stable or conditional replication in Francisella have been constructed. Methods for the efficient introduction of DNA into Francisella have been developed or optimized. Genetic platforms and procedures for transposon mutagenesis and allelic exchange have been adapted for use in Francisella. Finally, selectable, counterselectable, and screenable genetic markers amenable for use in a variety of F. tularensis species, including highly virulent clinical isolates, have been described. Collectively, these advances have aided in the construction of defined Francisella mutants and helped investigators begin to define the mechanism(s) employed by these organisms to cause disease in the host. In this article, we describe the history of genetic manipulation in Francisella and summarize the current tools and techniques for conducting genetic studies in this organism.

Utilization of a labeled tracking oligonucleotide for visualization and quality control of spotted 70-mer arrays.

BACKGROUND: Spotted 70-mer oligonucleotide arrays offer potentially greater specificity and an alternative to expensive cDNA library maintenance and amplification. Since microarray fabrication is a considerable source of data variance, we previously directly tagged cDNA probes with a third fluorophore for prehybridization quality control. Fluorescently modifying oligonucleotide sets is cost prohibitive, therefore, a co-spotted Staphylococcus aureus-specific fluorescein-labeled "tracking" oligonucleotide is described to monitor fabrication variables of a Mycobacterium tuberculosis oligonucleotide microarray. RESULTS: Significantly (p < 0.01) improved DNA retention was achieved printing in 15% DMSO/1.5 M betaine compared to the vendor recommended buffers. Introduction of tracking oligonucleotide did not effect hybridization efficiency or introduce ratio measurement bias in hybridizations between M. tuberculosis H37Rv and M. tuberculosis mprA. Linearity between the mean log Cy3/Cy5 ratios of genes differentially expressed from arrays either possessing or lacking the tracking oligonucleotide was observed (R2 = 0.90, p < 0.05) and there were no significant differences in Pearson's correlation coefficients of ratio data between replicates possessing (0.72 +/- 0.07), replicates lacking (0.74 +/- 0.10), or replicates with and without (0.70 +/- 0.04) the tracking oligonucleotide. ANOVA analysis confirmed the tracking oligonucleotide introduced no bias. Titrating target-specific oligonucleotide (40 microM to 0.78 microM) in the presence of 0.5 microM tracking oligonucleotide, revealed a fluorescein fluorescence inversely related to target-specific oligonucleotide molarity, making tracking oligonucleotide signal useful for quality control measurements and differentiating false negatives (synthesis failures and mechanical misses) from true negatives (no gene expression). CONCLUSIONS: This novel approach enables prehybridization array visualization for spotted oligonucleotide arrays and sets the stage for more sophisticated slide qualification and data filtering applications.

Reactive nitrogen and oxygen intermediates and bacterial defenses: unusual adaptations in Mycobacterium tuberculosis.

The production of reactive oxygen and reactive nitrogen intermediates is an important host defense mechanism mediated in response to infection by bacterial pathogens. Not surprisingly, intracellular pathogens have evolved numerous defense strategies to protect themselves against the damaging effects of these agents. In enteric bacteria, exposure to oxidative or nitrosative stress induces expression of numerous pathways that allow the bacterium to resist the toxic effects of these compounds during growth in the host. In contrast, members of pathogenic mycobacterial species, including the frank human pathogens Mycobacterium tuberculosis and Mycobacterium leprae, are dysfunctional in aspects of the oxidative and nitrosative stress response, yet they remain able to establish and maintain productive acute and persistent infections in the host. This article reviews the current knowledge regarding reactive oxygen and nitrogen intermediates, and compares the adaptative mechanisms utilized by enteric organisms and mycobacterial species to resist the bactericidal and bacteriostatic effects resulting from exposure to these compounds.

Molecular mechanisms regulating persistent Mycobacterium tuberculosis infection.

Establishing persistent infection and resisting elimination by the host's immune system are key factors contributing to latent infection by Mycobacterium tuberculosis. Recently, bacterial determinants regulating these processes have been identified. Here, we review molecular mechanisms regulating persistent infection and discuss the highly dynamic interaction of M. tuberculosis with the host.

Mechanisms of Francisella tularensis intracellular pathogenesis.

Francisella tularensis is a zoonotic intracellular pathogen and the causative agent of the debilitating febrile illness tularemia. Although natural infections by F. tularensis are sporadic and generally localized, the low infectious dose, with the ability to be transmitted to humans via multiple routes and the potential to cause life-threatening infections, has led to concerns that this bacterium could be used as an agent of bioterror and released intentionally into the environment. Recent studies of F. tularensis and other closely related Francisella species have greatly increased our understanding of mechanisms used by this organism to infect and cause disease within the host. Here, we review the intracellular life cycle of Francisella and highlight key genetic determinants and/or pathways that contribute to the survival and proliferation of this bacterium within host cells.

Adaptation to environmental stimuli within the host: two-component signal transduction systems of Mycobacterium tuberculosis.

Pathogenic microorganisms encounter a variety of environmental stresses following infection of their respective hosts. Mycobacterium tuberculosis, the etiological agent of tuberculosis, is an unusual bacterial pathogen in that it is able to establish lifelong infections in individuals within granulomatous lesions that are formed following a productive immune response. Adaptation to this highly dynamic environment is thought to be mediated primarily through transcriptional reprogramming initiated in response to recognition of stimuli, including low-oxygen tension, nutrient depletion, reactive oxygen and nitrogen species, altered pH, toxic lipid moieties, cell wall/cell membrane-perturbing agents, and other environmental cues. To survive continued exposure to these potentially adverse factors, M. tuberculosis encodes a variety of regulatory factors, including 11 complete two-component signal transduction systems (TCSSs) and several orphaned response regulators (RRs) and sensor kinases (SKs). This report reviews our current knowledge of the TCSSs present in M. tuberculosis. In particular, we discuss the biochemical and functional characteristics of individual RRs and SKs, the environmental stimuli regulating their activation, the regulons controlled by the various TCSSs, and the known or postulated role(s) of individual TCSSs in the context of M. tuberculosis physiology and/or pathogenesis.

The HtrA-like serine protease PepD interacts with and modulates the Mycobacterium tuberculosis 35-kDa antigen outer envelope protein.

Mycobacterium tuberculosis remains a significant global health concern largely due to its ability to persist for extended periods within the granuloma of the host. While residing within the granuloma, the tubercle bacilli are likely to be exposed to stress that can result in formation of aberrant proteins with altered structures. Bacteria encode stress responsive determinants such as proteases and chaperones to deal with misfolded or unfolded proteins. pepD encodes an HtrA-like serine protease and is thought to process proteins altered following exposure of M. tuberculosis to extra-cytoplasmic stress. PepD functions both as a protease and chaperone in vitro, and is required for aspects of M. tuberculosis virulence in vivo. pepD is directly regulated by the stress-responsive two-component signal transduction system MprAB and indirectly by extracytoplasmic function (ECF) sigma factor SigE. Loss of PepD also impacts expression of other stress-responsive determinants in M. tuberculosis. To further understand the role of PepD in stress adaptation by M. tuberculosis, a proteomics approach was taken to identify binding proteins and possible substrates of this protein. Using subcellular fractionation, the cellular localization of wild-type and PepD variants was determined. Purified fractions as well as whole cell lysates from Mycobacterium smegmatis or M. tuberculosis strains expressing a catalytically compromised PepD variant were immunoprecipitated for PepD and subjected to LC-MS/MS analyses. Using this strategy, the 35-kDa antigen encoding a homolog of the PspA phage shock protein was identified as a predominant binding partner and substrate of PepD. We postulate that proteolytic cleavage of the 35-kDa antigen by PepD helps maintain cell wall homeostasis in Mycobacterium and regulates specific stress response pathways during periods of extracytoplasmic stress.

Identification, characterization and immunogenicity of an O-antigen capsular polysaccharide of Francisella tularensis.

Capsular polysaccharides are important factors in bacterial pathogenesis and have been the target of a number of successful vaccines. Francisella tularensis has been considered to express a capsular antigen but none has been isolated or characterized. We have developed a monoclonal antibody, 11B7, which recognizes the capsular polysaccharide of F. tularensis migrating on Western blot as a diffuse band between 100 kDa and 250 kDa. The capsule stains poorly on SDS-PAGE with silver stain but can be visualized using ProQ Emerald glycoprotein stain. The capsule appears to be highly conserved among strains of F. tularensis as antibody 11B7 bound to the capsule of 14 of 14 F. tularensis type A and B strains on Western blot. The capsular material can be isolated essentially free of LPS, is phenol and proteinase K resistant, ethanol precipitable and does not dissociate in sodium dodecyl sulfate. Immunoelectron microscopy with colloidal gold demonstrates 11B7 circumferentially staining the surface of F. tularensis which is typical of a polysaccharide capsule. Mass spectrometry, compositional analysis and NMR indicate that the capsule is composed of a polymer of the tetrasaccharide repeat, 4)-alpha-D-GalNAcAN-(1->4)-alpha-D-GalNAcAN-(1->3)-beta-D-QuiNAc-(1->2)-beta-D-Qui4NFm-(1-, which is identical to the previously described F. tularensis O-antigen subunit. This indicates that the F. tularensis capsule can be classified as an O-antigen capsular polysaccharide. Our studies indicate that F. tularensis O-antigen glycosyltransferase mutants do not make a capsule. An F. tularensis acyltransferase and an O-antigen polymerase mutant had no evidence of an O-antigen but expressed a capsular antigen. Passive immunization of BALB/c mice with 75 microg of 11B7 protected against a 150 fold lethal challenge of F. tularensis LVS. Active immunization of BALB/c mice with 10 microg of capsule showed a similar level of protection. These studies demonstrate that F. tularensis produces an O-antigen capsule that may be the basis of a future vaccine.

Working toward the future: insights into Francisella tularensis pathogenesis and vaccine development.

Francisella tularensis is a facultative intracellular gram-negative pathogen and the etiological agent of the zoonotic disease tularemia. Recent advances in the field of Francisella genetics have led to a rapid increase in both the generation and subsequent characterization of mutant strains exhibiting altered growth and/or virulence characteristics within various model systems of infection. In this review, we summarize the major properties of several Francisella species, including F. tularensis and F. novicida, and provide an up-to-date synopsis of the genes necessary for pathogenesis by these organisms and the determinants that are currently being targeted for vaccine development.

A Francisella tularensis Schu S4 purine auxotroph is highly attenuated in mice but offers limited protection against homologous intranasal challenge.

BACKGROUND: Francisella tularensis is a gram-negative coccobacillus that causes the febrile illness tularemia. Subspecies that are pathogenic for humans include those comprising the type A (subspecies tularensis) or type B (subspecies holarctica) biovars. An attenuated live vaccine strain (LVS) developed from a type B isolate has previously been used to vaccinate at-risk individuals, but offers limited protection against high dose (>1000 CFUs) challenge with type A strains delivered by the respiratory route. Due to differences between type A and type B F. tularensis strains at the genetic level, it has been speculated that utilization of an attenuated type A strain as a live vaccine might offer better protection against homologous respiratory challenge compared with LVS. Here, we report the construction and characterization of an unmarked Delta purMCD mutant in the highly virulent type A strain Schu S4. METHODOLOGY/PRINCIPAL FINDINGS: Growth of Schu S4 Delta purMCD was severely attenuated in primary human peripheral blood monocyte-derived macrophages and in the A549 human lung epithelial cell line. The Schu S4 Delta purMCD mutant was also highly attenuated in mice when delivered via either the intranasal or intradermal infection route. Mice vaccinated intranasally with Schu S4 Delta purMCD were well protected against high dose intradermal challenge with virulent type A or type B strains of F. tularensis. However, intranasal vaccination with Schu S4 Delta purMCD induced tissue damage in the lungs, and conferred only limited protection against high dose Schu S4 challenge delivered by the same route. The level of protection observed was similar to that conferred following vaccination with wild-type LVS or the analogous LVS Delta purMCD mutant. CONCLUSIONS/SIGNIFICANCE: Collectively, these results argue that development of the next generation live attenuated vaccine for Francisella should be based on use of the less pathogenic type B biovar rather than the more reactogenic type A biovar.

Identification of Francisella tularensis Himar1-based transposon mutants defective for replication in macrophages.

Francisella tularensis, the etiologic agent of tularemia in humans, is a potential biological threat due to its low infectious dose and multiple routes of entry. F. tularensis replicates within several cell types, eventually causing cell death by inducing apoptosis. In this study, a modified Himar1 transposon (HimarFT) was used to mutagenize F. tularensis LVS. Approximately 7,000 Km(r) clones were screened using J774A.1 macrophages for reduction in cytopathogenicity based on retention of the cell monolayer. A total of 441 candidates with significant host cell retention compared to the parent were identified following screening in a high-throughput format. Retesting at a defined multiplicity of infection followed by in vitro growth analyses resulted in identification of approximately 70 candidates representing 26 unique loci involved in macrophage replication and/or cytotoxicity. Mutants carrying insertions in seven hypothetical genes were screened in a mouse model of infection, and all strains tested appeared to be attenuated, which validated the initial in vitro results obtained with cultured macrophages. Complementation and reverse transcription-PCR experiments suggested that the expression of genes adjacent to the HimarFT insertion may be affected depending on the orientation of the constitutive groEL promoter region used to ensure transcription of the selective marker in the transposon. A hypothetical gene, FTL_0706, postulated to be important for lipopolysaccharide biosynthesis, was confirmed to be a gene involved in O-antigen expression in F. tularensis LVS and Schu S4. These and other studies demonstrate that therapeutic targets, vaccine candidates, or virulence-related genes may be discovered utilizing classical genetic approaches in Francisella.

Construction and characterization of an attenuated purine auxotroph in a Francisella tularensis live vaccine strain.

Francisella tularensis is a facultative intracellular pathogen and is the etiological agent of tularemia. It is capable of escaping from the phagosome, replicating to high numbers in the cytosol, and inducing apoptosis in macrophages of a variety of hosts. F. tularensis has received significant attention recently due to its potential use as a bioweapon. Currently, there is no licensed vaccine against F. tularensis, although a partially protective live vaccine strain (LVS) that is attenuated in humans but remains fully virulent for mice was previously developed. An F. tularensis LVS mutant deleted in the purMCD purine biosynthetic locus was constructed and partially characterized by using an allelic exchange strategy. The F. tularensis LVS delta purMCD mutant was auxotrophic for purines when grown in defined medium and exhibited significant attenuation in virulence when assayed in murine macrophages in vitro or in BALB/c mice. Growth and virulence defects were complemented by the addition of the purine precursor hypoxanthine or by introduction of purMCDN in trans. The F. tularensis LVS delta purMCD mutant escaped from the phagosome but failed to replicate in the cytosol or induce apoptotic and cytopathic responses in infected cells. Importantly, mice vaccinated with a low dose of the F. tularensis LVS delta purMCD mutant were fully protected against subsequent lethal challenge with the LVS parental strain. Collectively, these results suggest that F. tularensis mutants deleted in the purMCD biosynthetic locus exhibit characteristics that may warrant further investigation of their use as potential live vaccine candidates.