MglA and MglB are required for the intramacrophage growth of Francisella novicida

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 that are restored for growth in macrophages. A locus isolated from one of these strains complements GB2 for both the intracellular growth defect and the colony morphology on LB (X-p) media. 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. mglB expression in GB2 was confirmed using antiserum against recombinant MglB. Cell fractionation studies revealed several differences in the protein profiles of mgl mutants compared with wild-type F. novicida . 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 affect the expression of genes whose products contribute to survival and growth within macrophages.     

Taurine: new implications for an old amino acid

We describe here a technique for allelic exchange in Francisella tularensis subsp. novicida utilizing polymerase chain reaction (PCR) products. Linear PCR fragments containing gene deletions with an erythromycin resistance cassette insertion were transformed into F. tularensis. The subsequent ErmR progeny were found to have undergone allelic exchange at the correct location in the genome; the minimum flanking homology necessary was 500 bp. This technique was used to create mglA, iglC, bla, and tul4 mutants in F. tularensis subsp. novicida strains. The mglA and iglC mutants were defective for intramacrophage growth, and the tul4 mutant lacked detectable Tul4 by Western immunoblot, as expected. Interestingly, the bla mutant maintained resistance to ampicillin, indicating the presence of multiple ampicillin resistance genes in F. tularensis.     

Isolation and characterization of Francisella novicida mutants defective in lipopolysaccharide biosynthesis

In order to identify genes involved in LPS biosynthesis we isolated random mutants generated by transposon insertion in Francisella novicida. The resulting mutant bank yielded mutants with three distinct LPS phenotypes, and three representative mutants were chosen for further study. One mutant that had short O-antigen chains was sensitive to serum; this mutant and one other were more sensitive to killing by deoxycholate than control strains. The third mutant was resistant to deoxycholate killing but slightly sensitive to serum. The three mutants varied in their ability to grow in macrophages. The DNA sequences interrupted by the transposon in two of the three mutants showed similarity to known LPS biosynthetic genes at the deduced amino acid level.     

Expression cloning and periplasmic orientation of the Francisella novicida lipid A 4'-phosphatase LpxF

Francisella tularensis and related intracellular pathogens synthesize lipid A molecules that differ from their Escherichia coli counterparts. Although a functional orthologue of lpxK, the gene encoding the lipid A 4'-kinase, is present in Francisella, no 4'-phosphate moiety is attached to Francisella lipid A. We now demonstrate that a membrane-bound phosphatase present in Francisella novicida U112 selectively removes the 4'-phosphate residue from tetra- and pentaacylated lipid A molecules. A clone that expresses the F. novicida 4'-phosphatase was identified by assaying lysates of E. coli colonies, harboring members of an F. novicida genomic DNA library, for 4'-phosphatase activity. Sequencing of a 2.5-kb F. novicida DNA insert from an active clone located the structural gene for the 4'-phosphatase, designated lpxF. It encodes a protein of 222 amino acid residues with six predicted membrane-spanning segments. Rhizobium leguminosarum and Rhizobium etli contain functional lpxF orthologues, consistent with their lipid A structures. When F. novicida LpxF is expressed in an E. coli LpxM mutant, a strain that synthesizes pentaacylated lipid A, over 90% of the lipid A molecules are dephosphorylated at the 4'-position. Expression of LpxF in wild-type E. coli has no effect, because wild-type hexaacylated lipid A is not a substrate. However, newly synthesized lipid A is not dephosphorylated in LpxM mutants by LpxF when the MsbA flippase is inactivated, indicating that LpxF faces the outer surface of the inner membrane. The availability of the lpxF gene will facilitate re-engineering lipid A structures in diverse bacteria.     

Direct isolation of Francisella spp. from environmental samples

Aims:  To develop a selective medium for isolation of F. tularensis, F. novicida and F. philomiragia from environmental samples.
Methods and Results:  A selective media, cysteine heart agar with 9% chocolatized sheep blood, containing polymyxin B, amphotericin B, cyclohexamide, cefepime and vancomycin (CHAB-PACCV) was developed and evaluated for growth of Francisella spp. No differences were observed in recovered colony forming units (CFUs) for F. tularensis , F. novicida and F. philomiragia on CHAB-PACCV vs nonselective CHAB. Growth of non- Francisella species was inhibited on CHAB-PACCV. When environmental samples were cultured on CHAB and CHAB-PACCV, only CHAB-PACCV allowed isolation of Francisella spp. Three new Francisella strains were isolated directly from seawater and seaweed samples by culture on CHAB-PACCV.
Conclusions:  CHAB-PACCV can be used for direct isolation of Francisella spp from environmental samples.
Significance and Impact of the Study:  Francisella spp. show a close association with environmental sources. Future utilization of CHAB-PACCV for isolation of Francisella spp. directly from environmental samples should prove valuable for investigating outbreaks and human infections attributed to environmental exposure.     

Characterization of the siderophore of Francisella tularensis and role of fslA in siderophore production

We determined that LVS and Schu S4 strains of the human pathogen Francisella tularensis express a siderophore when grown under iron-limiting conditions. We purified this siderophore by conventional column chromatography and high-pressure liquid chromatography and used mass spectrometric analysis to demonstrate that it is structurally similar to the polycarboxylate siderophore rhizoferrin. The siderophore promoted the growth of LVS and Schu S4 strains in iron-limiting media. We identified a potential siderophore biosynthetic gene cluster encoded by fslABCD in the F. tularensis genome. The first gene in the cluster, fslA, encodes a member of the superfamily of nonribosomal peptide synthetase-independent siderophore synthetases (NIS synthetases) characterized by the aerobactin synthetases IucA and IucC. We determined that fslA is transcribed as part of an operon with downstream gene fslB and that the expression of the locus is induced by iron starvation. A targeted in-frame nonpolar deletion of fslA in LVS resulted in the loss of siderophore expression and in a reduced ability of F. tularensis to grow under conditions of iron limitation. Siderophore activity and the ability to grow under iron limitation could be regained by introducing the fslA(+) gene on a complementing plasmid. Our results suggest that the fslA-dependent siderophore is important for survival of F. tularensis in an iron-deficient environment.     

Common ancestry and novel genetic traits of Francisella novicida-like isolates from North America and Australia as revealed by comparative genomic analyses

Francisella novicida is a close relative of Francisella tularensis, the causative agent of tularemia. The genomes of F. novicida-like clinical isolates 3523 (Australian strain) and Fx1 (Texas strain) were sequenced and compared to F. novicida strain U112 and F. tularensis strain Schu S4. The strain 3523 chromosome is 1,945,310 bp and contains 1,854 protein-coding genes. The strain Fx1 chromosome is 1,913,619 bp and contains 1,819 protein-coding genes. NUCmer analyses revealed that the genomes of strains Fx1 and U112 are mostly colinear, whereas the genome of strain 3523 has gaps, translocations, and/or inversions compared to genomes of strains Fx1 and U112. Using the genome sequence data and comparative analyses with other members of the genus Francisella, several strain-specific genes that encode putative proteins involved in RTX toxin production, polysaccharide biosynthesis/modification, thiamine biosynthesis, glucuronate utilization, and polyamine biosynthesis were identified. The RTX toxin synthesis and secretion operon of strain 3523 contains four open reading frames (ORFs) and was named rtxCABD. Based on the alignment of conserved sequences upstream of operons involved in thiamine biosynthesis from various bacteria, a putative THI box was identified in strain 3523. The glucuronate catabolism loci of strains 3523 and Fx1 contain a cluster of nine ORFs oriented in the same direction that appear to constitute an operon. Strains U112 and Schu S4 appeared to have lost the loci for RTX toxin production, thiamine biosynthesis, and glucuronate utilization as a consequence of host adaptation and reductive evolution. In conclusion, comparative analyses provided insights into the common ancestry and novel genetic traits of these strains.     

Genetic dissection of the Francisella novicida restriction barrier

Francisella tularensis is the causative agent of tularemia and is a category A select agent. Francisella novicida, considered by some to be one of four subspecies of F. tularensis, is used as a model in pathogenesis studies because it causes a disease similar to tularemia in rodents but is not harmful to humans. F. novicida exhibits a strong restriction barrier which reduces the transformation frequency of foreign DNA up to 10(6)-fold. To identify the genetic basis of this barrier, we carried out a mutational analysis of restriction genes identified in the F. novicida genome. Strains carrying combinations of insertion mutations in eight candidate loci were created and assayed for reduced restriction of unmodified plasmid DNA introduced by transformation. Restriction was reduced by mutations in four genes, corresponding to two type I, one type II, and one type III restriction system. Restriction was almost fully eliminated in a strain in which all four genes were inactive. The strongest contributor to the restriction barrier, the type II gene, encodes an enzyme which specifically cleaves Dam-methylated DNA. Genome comparisons show that most restriction genes in the F. tularensis subspecies are pseudogenes, explaining the unusually strong restriction barrier in F. novicida and suggesting that restriction was lost during evolution of the human pathogenic subspecies. As part of this study, procedures were developed to introduce unmodified plasmid DNA into F. novicida efficiently, to generate defined multiple mutants, and to produce chromosomal deletions of multiple adjacent genes.     

Paralogous outer membrane proteins mediate uptake of different forms of iron and synergistically govern virulence in Francisella tularensis tularensis

Francisella tularensis subsp. tularensis is a highly infectious bacterium causing acute disease in mammalian hosts. Mechanisms for the acquisition of iron within the iron-limiting host environment are likely to be critical for survival of this intracellular pathogen. FslE (FTT0025) and FupA (FTT0918) are paralogous proteins that are predicted to form β-barrels in the outer membrane of virulent strain Schu S4 and are unique to Francisella species. Previous studies have implicated both FupA, initially identified as a virulence factor and FslE, encoded by the siderophore biosynthetic operon, in iron acquisition. Using single and double mutants, we demonstrated that these paralogs function in concert to promote growth under iron limitation. We used a (55)Fe transport assay to demonstrate that FslE is involved in siderophore-mediated ferric iron uptake, whereas FupA facilitates high affinity ferrous iron uptake. Optimal replication within J774A.1 macrophage-like cells required at least one of these uptake systems to be functional. In a mouse model of tularemia, the ΔfupA mutant was attenuated, but the ΔfslE ΔfupA mutant was significantly more attenuated, implying that the two systems of iron acquisition function synergistically to promote virulence. These studies highlight the importance of specific iron acquisition functions, particularly that of ferrous iron, for virulence of F. tularensis in the mammalian host.     

Macrophage replication screen identifies a novel Francisella hydroperoxide resistance protein involved in virulence

Francisella tularensis is a gram-negative facultative intracellular pathogen and the causative agent of tularemia. Recently, genome-wide screens have identified Francisella genes required for virulence in mice. However, the mechanisms by which most of the corresponding proteins contribute to pathogenesis are still largely unknown. To further elucidate the roles of these virulence determinants in Francisella pathogenesis, we tested whether each gene was required for replication of the model pathogen F. novicida within macrophages, an important virulence trait. Fifty-three of the 224 genes tested were involved in intracellular replication, including many of those within the Francisella pathogenicity island (FPI), validating our results. Interestingly, over one third of the genes identified are annotated as hypothetical, indicating that F. novicida likely utilizes novel virulence factors for intracellular replication. To further characterize these virulence determinants, we selected two hypothetical genes to study in more detail. As predicted by our screen, deletion mutants of FTN_0096 and FTN_1133 were attenuated for replication in macrophages. The mutants displayed differing levels of attenuation in vivo, with the FTN_1133 mutant being the most attenuated. FTN_1133 has sequence similarity to the organic hydroperoxide resistance protein Ohr, an enzyme involved in the bacterial response to oxidative stress. We show that FTN_1133 is required for F. novicida resistance to, and degradation of, organic hydroperoxides as well as resistance to the action of the NADPH oxidase both in macrophages and mice. Furthermore, we demonstrate that F. holarctica LVS, a strain derived from a highly virulent human pathogenic species of Francisella, also requires this protein for organic hydroperoxide resistance as well as replication in macrophages and mice. This study expands our knowledge of Francisella's largely uncharacterized intracellular lifecycle and demonstrates that FTN_1133 is an important novel mediator of oxidative stress resistance.     

Siderophore-mediated iron uptake in Alcaligenes eutrophus CH34 and identification of aleB encoding the ferric iron-alcaligin E receptor.

Siderophore production in response to iron limitation was observed in Alcaligenes eutrophus CH34, and the corresponding siderophore was named alcaligin E. Alcaligin E was characterized as a phenolate-type siderophore containing neither catecholate nor hydroxamate groups. Alcaligin E promoted the growth of siderophore-deficient A. eutrophus mutants under iron-restricted conditions and promoted 59Fe uptake by iron-limited cells. However, the growth of the Sid- mutant AE1152, which was obtained from CH34 by Tn5-Tc mutagenesis, was completely inhibited by the addition of alcaligin E. AE1152 also showed strongly reduced 59Fe uptake in the presence of alcaligin E. This indicates that a gene, designated aleB, which is involved in transport of ferric iron-alcaligin E across the membrane is inactivated. The aleB gene was cloned, and its putative amino acid sequence showed strong similarity to those of ferric iron-siderophore receptor proteins. Both wild-type strain CH34 and aleB mutant AE1152 were able to use the same heterologous siderophores, indicating that AleB is involved only in ferric iron-alcaligin E uptake. Interestingly, no utilization of pyochelin, which is also a phenolate-type siderophore, was observed for A. eutrophus CH34. Genetic studies of different Sid- mutants, obtained after transposon mutagenesis, showed that the genes involved in alcaligin E and ferric iron-alcaligin E receptor biosynthesis are clustered in a 20-kb region on the A. eutrophus CH34 chromosome in the proximity of the cys-232 locus.     

Transport and utilization of rhizoferrin bound iron in Mycobacterium smegmatis

Transport and metabolization of iron bound to the fungal siderophore rhizoferrin was analyzed by transport kinetics, Mössbauer and EPR spectroscopy. Saturation kinetics (v _max=24.4 pmol/(mg min), K _m=64.4M) and energy dependence excluded diffusion and provided evidence for a rhizoferrin transport system in M. smegmatis. Based on the spectroscopic techniques indications for intracellular presence of the ferric rhizoferrin complex were found. This feature could be of practical importance in the search of novel drugs for the treatment of mycobacterial infections. EPR and Mössbauer spectroscopy revealed different ferritin mineral cores depending on the siderophore iron source. This finding was interpreted in terms of different protein shells, i.e. two types of ferritins.     

Akt/Protein kinase B modulates macrophage inflammatory response to Francisella infection and confers a survival advantage in mice

The Gram-negative bacterium Francisella novicida infects primarily monocytes/macrophages and is highly virulent in mice. Macrophages respond by producing inflammatory cytokines that confer immunity against the infection. However, the molecular details of host cell response to Francisella infection are poorly understood. In this study, we demonstrate that F. novicida infection of murine macrophages induces the activation of Akt. Inhibition of Akt significantly decreases proinflammatory cytokine production in infected macrophages, whereas production of the anti-inflammatory cytokine IL-10 is enhanced. Analysis of the mechanism of Akt influence on cytokine response demonstrated that Akt promotes NF-kappaB activation. We have extended these findings to show that Akt activation may be regulated by bacterial genes associated with phagosomal escape. Infection with mglA mutants of F. novicida elicited sustained activation of Akt in comparison to cells infected with wild-type F. novicida. Concomitantly, there was significantly higher proinflammatory cytokine production and lower IL-10 production in cells infected with the mglA mutant. Finally, transgenic animals expressing constitutively active Akt displayed a survival advantage over their wild-type littermates when challenged with lethal doses of F. novicida. Together, these observations indicate that Akt promotes proinflammatory cytokine production by F. novicida-infected macrophages through its influence on NF-kappaB, thereby contributing to immunity against F. novicida infection.     

The Histoplasma capsulatum vacuolar ATPase is required for iron homeostasis, intracellular replication in macrophages and virulence in a murine model of histoplasmosis

Histoplasma capsulatum is a dimorphic fungal pathogen that survives and replicates within macrophages (Mphi). To identify specific genes required for intracellular survival, we utilized Agrobacterium tumefaciens-mediated mutagenesis, and screened for H. capsulatum insertional mutants that were unable to survive in human Mphi. One colony was identified that had an insertion within VMA1, the catalytic subunit A of the vacuolar ATPase (V-ATPase). The vma1 mutant (vma1::HPH) grew normally on iron-replete medium, but not on iron-deficient media. On iron-deficient medium, the growth of the vma1 mutant was restored in the presence of wild-type (WT) H. capsulatum yeasts, or the hydroxamate siderophore, rhodotorulic acid. However, the inability to replicate within Mphi was only partially restored by the addition of exogenous iron. The vma1::HPH mutant also did not grow as a mold at 28 degrees C. Complementation of the mutant (vma/VMA1) restored its ability to replicate in Mphi, grow on iron-poor medium and grow as a mold at 28 degrees C. The vma1::HPH mutant was avirulent in a mouse model of histoplasmosis, whereas the vma1/VMA1 strain was as pathogenic as WT yeasts. These studies demonstrate the importance of V-ATPase function in the pathogenicity of H. capsulatum, in iron homeostasis and in fungal dimorphism.     

Stereochemical characterization of rhizoferrin and identification of its dehydration products

The polycarboxylate siderophore, rhizoferrin, and its dehydration products were separated by preparative HPLC and characterized by¹³C-NMR,¹H-NMR, UV, circular dichroism (CD) and IR spectroscopy, and also by capillary electrophoresis. Assginment of all carbon atoms and protons by NMR spectra confirmed the structure of rhizoferrin and gave evidence for the presence of the cyclized dehydration byproducts, imidorhizoferrin and bis-imidorhizoferrin. The imido forms were also characterized by their mobility during capillary electrophoresis. UV spectra revealed a 1∶1 iron:ligand ratio above pH 3. Based on the absorption maximum of the metal ligand charge transfer hand at 335 nm a molar extinction coefficient of 2300m ^?1 cm^?1 was calculated for ferric rhizoferrin. CD measurements revealed that the quarternary carbon atoms of the two citric acid residues possess anR,R configuration and that the iron complex of rhizoferrin adopts a A configuration around the metal center.     

The Reduced Genome of the Francisella tularensis Live Vaccine Strain (LVS) Encodes Two Iron Acquisition Systems Essential for Optimal Growth and Virulence

Bacterial pathogens require multiple iron-specific acquisition systems for survival within the iron-limiting environment of the host. Francisella tularensis is a virulent intracellular pathogen that can replicate in multiple cell-types. To study the interrelationship of iron acquisition capability and virulence potential of this organism, we generated single and double deletion mutants within the ferrous iron (feo) and ferric-siderophore (fsl) uptake systems of the live vaccine strain (LVS). The Feo system was disrupted by a partial deletion of the feoB gene (ΔfeoB′), which led to a growth defect on iron-limited modified Muller Hinton agar plates. ⁵⁵Fe uptake assays verified that the ΔfeoB′ mutant had lost the capacity for ferrous iron uptake but was still competent for ⁵⁵Fe-siderophore-mediated ferric iron acquisition. Neither the ΔfeoB′ nor the siderophore-deficient ΔfslA mutant was defective for replication within J774A.1 murine macrophage-like cells, thus demonstrating the ability of LVS to survive using either ferrous or ferric sources of intracellular iron. A LVS ΔfslA ΔfeoB′ mutant defective for both ferrous iron uptake and siderophore production was isolated in the presence of exogenous F. tularensis siderophore. In contrast to the single deletion mutants, the ΔfslA ΔfeoB′ mutant was unable to replicate within J774A.1 cells and was attenuated in virulence following intraperitoneal infection of C57BL/6 mice. These studies demonstrate that the siderophore and feoB-mediated ferrous uptake systems are the only significant iron acquisition systems in LVS and that they operate independently. While one system can compensate for loss of the other, both are required for optimal growth and virulence.