Insights into DNA-binding motifs and mechanisms of Francisella tularensis novicida two-component system response regulator proteins QseB, KdpE, and BfpR.

Two component system bacterial response regulators are typically DNA-binding proteins which enable the genetic regulation of many adaptive bacterial behaviors. Despite structural similarity across response regulator families, there is a diverse array of DNA-binding mechanisms. Bacteria usually encode several dozen two-component system response regulators, but Francisella tularensis only encodes three. Due to their simplified response regulatory network, Francisella species are a model for studying the role of response regulator proteins in virulence. Here, we show that Francisella response regulators QseB, KdpE, and BfpR all utilize different DNA-binding mechanisms. Our evidence suggests that QseB follows a simple mechanism whereby it binds a single inverted repeat sequence with a higher affinity upon phosphorylation. This behavior is independent of whether QseB is a positive or negative regulator of the gene as demonstrated by qseB and priM promoter sequences, respectively. Similarly, KdpE binds DNA more tightly upon phosphorylation, but also exhibits a cooperative binding isotherm. While we propose a KdpE binding site, it is possible that KdpE has a complex DNA-binding mechanism potentially involving multiple copies of KdpE being recruited to a promoter region. Finally, we show that BfpR appears to bind a region of its own promoter sequence with a lower affinity upon phosphorylation. Further structural and enzymatic work will need to be performed to deconvolute the KdpE and BfpR binding mechanisms.

Polyaminated, acetylated and stop codon readthrough of recombinant Francisella tularensis universal stress protein in Escherichia coli.

Recombinant Francisella tularensis universal stress protein with a C-terminal histidine-tag (rUsp/His6) was expressed in Escherichia coli. Endogenous F. tularensis Usp has a predicted molecular mass of 30 kDa, but rUsp/His6 had an apparent molecular weight of 33 kDa based on Western blot analyses. To determine the source of the higher molecular weight for rUsp/His6, post translational modifications were examined. Tryptic peptides of purified rUsp/His6 were subjected to liquid chromatography tandem mass spectrometry (LC-MS/MS) and fragmentation spectra were searched for acetylated lysines and polyaminated glutamines. Of the 24 lysines in rUsp/His6, 10 were acetylated (K63, K68, K72, K129, K175, K201, K208, K212, K233, and K238) and three of the four glutamines had putrescine, spermidine and spermine adducts (Q55, Q60 and Q267). The level of post-translational modification was substoichiometric, eliminating the possibility that these modifications were the sole contributor to the 3 kDa extra mass of rUsp/His6. LC-MS/MS revealed that stop codon readthrough had occurred resulting in the unexpected addition of 20 extra amino acids at the C-terminus of rUsp/His6, after the histidine tag. Further, the finding of polyaminated glutamines in rUsp/His6 indicated that E. coli is capable of transglutaminase activity.

Identification of an N-terminal tag (580N) that improves the biosynthesis of fluorescent proteins in Francisella tularensis and other Gram-negative bacteria.

Utilization of fluorescent proteins is widespread for the study of microbial pathogenesis and host-pathogen interactions. Here, we discovered that linkage of the 36 N-terminal amino acids of FTL_0580 (a hypothetical protein of Francisella tularensis) to fluorescent proteins increases the fluorescence emission of bacteria that express these recombinant fusions. This N-terminal peptide will be referred to as 580N. Western blotting revealed that the linkage of 580N to Emerald Green Fluorescent Protein (EmGFP) in F. tularensis markedly improved detection of this protein. We therefore hypothesized that transcripts containing 580N may be translated more efficiently than those lacking the coding sequence for this leader peptide. In support, expression of emGFPFt that had been codon-optimized for F. tularensis, yielded significantly enhanced fluorescence than its non-optimized counterpart. Furthermore, fusing emGFP with coding sequence for a small N-terminal peptide (Serine-Lysine-Isoleucine-Lysine), which had previously been shown to inhibit ribosomal stalling, produced robust fluorescence when expressed in F. tularensis. These findings support the interpretation that 580N enhances the translation efficiency of fluorescent proteins in F. tularensis. Interestingly, expression of non-optimized 580N-emGFP produced greater fluorescence intensity than any other construct. Structural predictions suggested that RNA secondary structure also may be influencing translation efficiency. When expressed in Escherichia coli and Klebsiella pneumoniae bacteria, 580N-emGFP produced increased green fluorescence compared to untagged emGFP (neither allele was codon optimized for these bacteria). In conclusion, fusing the coding sequence for the 580N leader peptide to recombinant genes might serve as an economical alternative to codon optimization for enhancing protein expression in bacteria.

A conserved but structurally divergent loop in acyl protein thioesterase 1 regulates its catalytic activity, ligand binding, and folded stability.

Human acyl protein thioesterases (APTs) catalyze the depalmitoylation of S-acylated proteins attached to the plasma membrane, facilitating reversible cycles of membrane anchoring and detachment. We previously showed that a bacterial APT homologue, FTT258 from the gram-negative pathogen Francisella tularensis, exists in equilibrium between a closed and open state based on the structural dynamics of a flexible loop overlapping its active site. Although the structural dynamics of this loop are not conserved in human APTs, the amino acid sequence of this loop is highly conserved, indicating essential but divergent functions for this loop in human APTs. Herein, we investigated the role of this loop in regulating the catalytic activity, ligand binding, and protein folding of human APT1, a depalmitoylase connected with cancer, immune, and neurological signaling. Using a combination of substitutional analysis with kinetic, structural, and biophysical characterization, we show that even in its divergent structural location in human APT1 that this loop still regulates the catalytic activity of APT1 through contributions to ligand binding and substrate positioning. We confirmed previously known roles for multiple residues (Phe72 and Ile74) in substrate binding and catalysis while adding new roles in substrate selectivity (Pro69), in catalytic stabilization (Asp73 and Ile75), and in transitioning between the membrane binding ß-tongue and substrate-binding loops (Trp71). Even conservative substitution of this tryptophan (Trp71) fulcrum led to complete loss of catalytic activity, a 13°C decrease in total protein stability, and drastic drops in ligand affinity, indicating that the combination of the size, shape, and aromaticity of Trp71 are essential to the proper structure of APT1. Mixing buried hydrophobic surface area with contributions to an exposed secondary surface pocket, Trp71 represents a previously unidentified class of essential tryptophans within α/ß hydrolase structure and a potential allosteric binding site within human APTs.

Functional promiscuity of small multidrug resistance transporters from Staphylococcus aureus, Pseudomonas aeruginosa, and Francisella tularensis.

Small multidrug resistance transporters efflux toxic compounds from bacteria and are a minimal system to understand multidrug transport. Most previous studies have focused on EmrE, the model SMR from Escherichia coli, finding that EmrE has a broader substrate profile than previously thought and that EmrE may perform multiple types of transport, resulting in substrate-dependent resistance or susceptibility. Here, we performed a broad screen to identify potential substrates of three other SMRs: PAsmr from Pseudomonas aeruginosa; FTsmr from Francisella tularensis; and SAsmr from Staphylococcus aureus. This screen tested metabolic differences in E. coli expressing each transporter versus an inactive mutant, for a clean comparison of sequence and substrate-specific differences in transporter function, and identified many substrates for each transporter. In general, resistance compounds were charged, and susceptibility substrates were uncharged, but hydrophobicity was not correlated with phenotype. Two resistance hits and two susceptibility hits were validated via growth assays and IC50 calculations. Susceptibility is proposed to occur via substrate-gated proton leak, and the addition of bicarbonate antagonizes the susceptibility phenotype, consistent with this hypothesis.

Discovery of a glutathione utilization pathway in Francisella that shows functional divergence between environmental and pathogenic species.

Glutathione (GSH) is an abundant metabolite within eukaryotic cells that can act as a signal, a nutrient source, or serve in a redox capacity for intracellular bacterial pathogens. For Francisella, GSH is thought to be a critical in vivo source of cysteine; however, the cellular pathways permitting GSH utilization by Francisella differ between strains and have remained poorly understood. Using genetic screening, we discovered a unique pathway for GSH utilization in Francisella. Whereas prior work suggested GSH catabolism initiates in the periplasm, the pathway we define consists of a major facilitator superfamily (MFS) member that transports intact GSH and a previously unrecognized bacterial cytoplasmic enzyme that catalyzes the first step of GSH degradation. Interestingly, we find that the transporter gene for this pathway is pseudogenized in pathogenic Francisella, explaining phenotypic discrepancies in GSH utilization among Francisella spp. and revealing a critical role for GSH in the environmental niche of these bacteria.

Francisella tularensis disrupts TLR2-MYD88-p38 signaling early during infection to delay apoptosis of macrophages and promote virulence in the host.

Francisella tularensis is a zoonotic pathogen and the causative agent of tularemia. F. tularensis replicates to high levels within the cytosol of macrophages and other host cells while subverting the host response to infection. Critical to the success of F. tularensis is its ability to delay macrophage apoptosis to maintain its intracellular replicative niche. However, the host-signaling pathway(s) modulated by F. tularensis to delay apoptosis are poorly characterized. The outer membrane channel protein TolC is required for F. tularensis virulence and its ability to suppress apoptosis and cytokine expression during infection of macrophages. We took advantage of the F. tularensis ∆tolC mutant phenotype to identify host pathways that are important for activating macrophage apoptosis and that are disrupted by the bacteria. Comparison of macrophages infected with wild-type or ∆tolC F. tularensis revealed that the bacteria interfere with TLR2-MYD88-p38 signaling at early times post infection to delay apoptosis, dampen innate host responses, and preserve the intracellular replicative niche. Experiments using the mouse pneumonic tularemia model confirmed the in vivo relevance of these findings, revealing contributions of TLR2 and MYD88 signaling to the protective host response to F. tularensis, which is modulated by the bacteria to promote virulence. IMPORTANCE Francisella tularensis is a Gram-negative intracellular bacterial pathogen and the causative agent of the zoonotic disease tularemia. F. tularensis, like other intracellular pathogens, modulates host-programmed cell death pathways to ensure its replication and survival. We previously identified the outer membrane channel protein TolC as required for the ability of F. tularensis to delay host cell death. However, the mechanism by which F. tularensis delays cell death pathways during intracellular replication is unclear despite being critical to pathogenesis. In the present study, we address this gap in knowledge by taking advantage of ∆tolC mutants of F. tularensis to uncover signaling pathways governing host apoptotic responses to F. tularensis and which are modulated by the bacteria during infection to promote virulence. These findings reveal mechanisms by which intracellular pathogens subvert host responses and enhance our understanding of the pathogenesis of tularemia.

New structures of Class II Fructose-1,6-Bisphosphatase from Francisella tularensis provide a framework for a novel catalytic mechanism for the entire class.

Class II Fructose-1,6-bisphosphatases (FBPaseII) (EC: 3.1.3.11) are highly conserved essential enzymes in the gluconeogenic pathway of microorganisms. Previous crystallographic studies of FBPasesII provided insights into various inactivated states of the enzyme in different species. Presented here is the first crystal structure of FBPaseII in an active state, solved for the enzyme from Francisella tularensis (FtFBPaseII), containing native metal cofactor Mn2+ and complexed with catalytic product fructose-6-phosphate (F6P). Another crystal structure of the same enzyme complex is presented in the inactivated state due to the structural changes introduced by crystal packing. Analysis of the interatomic distances among the substrate, product, and divalent metal cations in the catalytic centers of the enzyme led to a revision of the catalytic mechanism suggested previously for class II FBPases. We propose that phosphate-1 is cleaved from the substrate fructose-1,6-bisphosphate (F1,6BP) by T89 in a proximal α-helix backbone (G88-T89-T90-I91-T92-S93-K94) in which the substrate transition state is stabilized by the positive dipole of the 〈-helix backbone. Once cleaved a water molecule found in the active site liberates the inorganic phosphate from T89 completing the catalytic mechanism. Additionally, a crystal structure of Mycobacterium tuberculosis FBPaseII (MtFBPaseII) containing a bound F1,6BP is presented to further support the substrate binding and novel catalytic mechanism suggested for this class of enzymes.

Francisella tularensis Glyceraldehyde-3-Phosphate Dehydrogenase Is Relocalized during Intracellular Infection and Reveals Effect on Cytokine Gene Expression and Signaling.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is known for its multifunctionality in several pathogenic bacteria. Our previously reported data suggest that the GAPDH homologue of Francisella tularensis, GapA, might also be involved in other processes beyond metabolism. In the present study, we explored GapA's potential implication in pathogenic processes at the host cell level. Using immunoelectron microscopy, we demonstrated the localization of this bacterial protein inside infected macrophages and its peripheral distribution in bacterial cells increasing with infection time. A quantitative proteomic approach based on stable isotope labeling of amino acids in cell culture (SILAC) combined with pull-down assay enabled the identification of several of GapA's potential interacting partners within the host cell proteome. Two of these partners were further confirmed by alternative methods. We also investigated the impact of gapA deletion on the transcription of selected cytokine genes and the activation of the main signaling pathways. Our results show that ∆gapA-induced transcription of genes encoding several cytokines whose expressions were not affected in cells infected with a fully virulent wild-type strain. That might be caused, at least in part, by the detected differences in ERK/MAPK signaling activation. The experimental observations together demonstrate that the F. tularensis GAPDH homologue is directly implicated in multiple host cellular processes and, thereby, that it participates in several molecular mechanisms of pathogenesis.

Iron-Modified Blood Culture Media Allow for the Rapid Diagnosis and Isolation of the Slow-Growing Pathogen Francisella tularensis.

The life-threatening disease tularemia is caused by Francisella tularensis, an intracellular Gram-negative bacterial pathogen. Due to the high mortality rates of the disease, as well as the low respiratory infectious dose, F. tularensis is categorized as a Tier 1 bioterror agent. The identification and isolation from clinical blood cultures of F. tularensis are complicated by its slow growth. Iron was shown to be one of the limiting nutrients required for F. tularensis metabolism and growth. Bacterial growth was shown to be restricted or enhanced in the absence or addition of iron. In this study, we tested the beneficial effect of enhanced iron concentrations on expediting F. tularensis blood culture diagnostics. Accordingly, bacterial growth rates in blood cultures with or without Fe2+ supplementation were evaluated. Growth quantification by direct CFU counts demonstrated significant improvement of growth rates of up to 6 orders of magnitude in Fe2+-supplemented media compared to the corresponding nonmodified cultures. Fe2+ supplementation significantly shortened incubation periods for successful diagnosis and isolation of F. tularensis by up to 92 h. This was achieved in a variety of blood culture types in spite of a low initial bacterial inoculum representative of low levels of bacteremia. These improvements were demonstrated with culture of either Francisella tularensis subsp. tularensis or subsp. holarctica in all examined commercial blood culture types routinely used in a clinical setup. Finally, essential downstream identification assays, such as matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS), immunofluorescence, or antibiotic susceptibility tests, were not affected in the presence of Fe2+. To conclude, supplementing blood cultures with Fe2+ enables a significant shortening of incubation times for F. tularensis diagnosis, without affecting subsequent identification or isolation assays. IMPORTANCE In this study, we evaluated bacterial growth rates of Francisella tularensis strains in iron (Fe)-enriched blood cultures as a means of improving and accelerating bacterial growth. The shortening of the culturing time should facilitate rapid pathogen detection and isolation, positively impacting clinical diagnosis and enabling prompt onset of efficient therapy.

Atomic Structure of IglD Demonstrates Its Role as a Component of the Baseplate Complex of the Francisella Type VI Secretion System.

Francisella tularensis, a Tier 1 select agent of bioterrorism, contains a type VI secretion system (T6SS) encoded within the Francisella pathogenicity island (FPI), which is critical for its pathogenesis. Among the 18 proteins encoded by FPI is IglD, which is essential to Francisella's intracellular growth and virulence, but neither its location within T6SS nor its functional role has been established. Here, we present the cryoEM structure of IglD from Francisella novicida and show that the Francisella IglD forms a homotrimer that is structurally homologous to the T6SS baseplate protein TssK in Escherichia coli. Each IglD monomer consists of an N-terminal ß-sandwich domain, a 4-helix bundle domain, and a flexible C-terminal domain. While the overall folds of IglD and TssK are similar, the two structures differ in three aspects: the relative orientation between their ß-sandwich and the 4-helix bundle domains; two insertion loops present in TssK's ß-sandwich domain; and, consequently, a lack of subunit-subunit interaction between insertion loops in the IglD trimer. Phylogenetic analysis indicates that IglD is genetically remote from the TssK orthologs in other T6SSs. While the other components of the Francisella baseplate are unknown, we conducted pulldown assays showing IglJ interacts with IglD and IglH, pointing to a model wherein IglD, IglH, and IglJ form the baseplate of the Francisella T6SS. Alanine substitution mutagenesis further established that IglD's hydrophobic pocket in the N-terminal ß-sandwich domain interacts with two loops of IglJ, reminiscent of the TssK-TssG interaction. These results form a framework for understanding the hitherto unexplored Francisella T6SS baseplate. IMPORTANCE Francisella tularensis is a facultatively intracellular Gram-negative bacterium that causes the serious and potentially fatal zoonotic illness, tularemia. Because of its extraordinarily high infectivity and mortality to humans, especially when inhaled, F. tularensis is considered a potential bioterrorism agent and is classified as a Tier 1 select agent. The type VI secretion system (T6SS) encoded within the Francisella pathogenicity island (FPI) is critical to its pathogenesis, but its baseplate components are largely unknown. Here, we report the cryoEM structure of IglD from Francisella novicida and demonstrate its role as a component of the baseplate complex of the Francisella T6SS. We further show that IglD interacts with IglJ and IglH, and propose a model in which these proteins interact to form the Francisella T6SS baseplate. Elucidation of the structure and composition of the Francisella baseplate should facilitate the design of strategies to prevent and treat infections caused by F. tularensis.

ThioredoxinA1 Controls the Oxidative Stress Response of Francisella tularensis Live Vaccine Strain (LVS).

Francisella tularensis is an intracellular, Gram-negative bacterium known for causing a disease known as tularemia in the Northern Hemisphere. F. tularensis is classified as a category A select agent by the CDC based on its possible use as a bioterror agent. F. tularensis overcomes oxidative stress encountered during its growth in the environment or host macrophages by encoding antioxidant enzymes such as superoxide dismutases, catalase, and alkylhydroperoxy reductase. These antioxidant enzymes are regulated by the oxidative stress response regulator, OxyR. In addition to these antioxidant enzymes, F. tularensis also encodes two thioredoxins, TrxA1 (FTL_0611) and TrxA2 (FTL_1224); however, their role in the oxidative stress response of F. tularensis is not known. This study investigated the role of thioredoxins of F. tularensis in the oxidative stress response and intracellular survival. Our results demonstrate that TrxA1 but not TrxA2 plays a major role in the oxidative stress response of F. tularensis. Most importantly, this study elucidates a novel mechanism through which the TrxA1 of F. tularensis controls the oxidative stress response by regulating the expression of the master regulator, oxyR. Further, TrxA1 is required for the intramacrophage survival and growth of Francisella. Overall, this study describes a novel role of thioredoxin, TrxA1, in regulating the oxidative stress response of F. tularensis. IMPORTANCE The role of thioredoxins in the oxidative stress response of F. tularensis is not known. This study demonstrates that of the two thioredoxins, TrxA1 is vital to counter the oxidative stress in F. tularensis live vaccine strain (LVS). Furthermore, this study shows differences in the well-studied thioredoxins of Escherichia coli. First, the expression of TrxA1 of F. tularensis is independent of the oxidative stress response regulator, OxyR. Second and most importantly, TrxA1 regulates the expression of oxyR and, therefore, the OxyR-dependent oxidative stress response of F. tularensis. Overall, this study reports a novel regulatory role of TrxA1 of F. tularensis in the oxidative stress response.

Backbone chemical shift assignment and dynamics of the N-terminal domain of ClpB from Francisella tularensis type VI secretion system.

The Hsp100 family member ClpB is a protein disaggregase which solubilizes and reactivates stress-induced protein aggregates in cooperation with the DnaK/Hsp70 chaperone system. In the pathogenic bacterium Francisella tularensis, ClpB is involved in type VI secretion system (T6SS) disassembly through depolymerization of the IglA-IglB sheath. This leads to recycling and reassembly of T6SS components and this process is essential for the virulence of the bacterium. Here we report the backbone chemical shift assignments and 15N relaxation-based backbone dynamics of the N-terminal substrate-binding domain of ClpB (1-156).

The type IV pili component PilO is a virulence determinant of Francisella novicida.

Francisella tularensis is a highly pathogenic intracellular bacterium that causes the disease tularemia. While its ability to replicate within cells has been studied in much detail, the bacterium also encodes a less characterised type 4 pili (T4P) system. T4Ps are dynamic adhesive organelles identified as major virulence determinants in many human pathogens. In F. tularensis, the T4P is required for adherence to the host cell, as well as for protein secretion. Several components, including pilins, a pili peptidase, a secretin pore and two ATPases, are required to assemble a functional T4P, and these are encoded within distinct clusters on the Francisella chromosome. While some of these components have been functionally characterised, the role of PilO, if any, still is unknown. Here, we examined the role of PilO in the pathogenesis of F. novicida. Our results show that the PilO is essential for pilus assembly on the bacterial surface. In addition, PilO is important for adherence of F. novicida to human monocyte-derived macrophages, secretion of effector proteins and intracellular replication. Importantly, the pilO mutant is attenuated for virulence in BALB/c mice regardless of the route of infection. Following intratracheal and intradermal infection, the mutant caused no histopathology changes, and demonstrated impaired phagosomal escape and replication within lung liver as well as spleen. Thus, PilO is an essential virulence determinant of F. novicida.

Cellular and molecular level host-pathogen interactions in Francisella tularensis: A microbial gene network study.

Due to the high infectivity and fatal effect on human population, Francisella tularensis (F. tularensis) is classified as a potential biological warfare agent. The interaction between host and pathogen behind the successful establishment of F. tularensis infection within the human host is largely unknown. In our present work, we have studied the molecular level interactions between the host cellular components and F. tularensis genes to understand the interplay between the host and pathogen. Interestingly, we have identified the pathways associated with the pathogen offensive strategies that help in invasion of host defensive systems. The F. tularensis genes purL, katG, proS, rpoB and fusA have displayed high number of interactions with the host genes and thus play a crucial role in vital pathogen pathways. The pathways identified were involved in adaptation to different stress conditions within the host and might be crucial for designing new therapeutic interventions against tularemia.

Diverse molecular mechanisms of transcription regulation by the bacterial alarmone ppGpp.

Bacteria must rapidly detect and respond to stressful environmental conditions. Guanosine tetraphosphate (ppGpp) is a universal stress signal that, in most bacteria, drives the reprograming of transcription at a global level. However, recent studies have revealed that the molecular mechanisms utilized by ppGpp to rewire bacterial transcriptomes are unexpectedly diverse. In Proteobacteria, ppGpp regulates the expression of hundreds of genes by directly binding to two sites on RNA polymerase (RNAP), one in combination with the transcription factor, DksA. Conversely, ppGpp indirectly regulates transcription in Firmicutes by controlling GTP levels. In this case, ppGpp inhibits enzymes that salvage and synthesize GTP, which indirectly represses transcription from rRNA and other promoters that use GTP for initiation. More recently, two different mechanisms of transcription regulation involving the direct binding of transcription factors by ppGpp have been described. First, in Francisella tularensis, ppGpp was shown to modulate the formation of a tripartite transcription factor complex that binds RNAP and activates virulence genes. Second, in Firmicutes, ppGpp allosterically regulates the transcription repressor, PurR, which controls purine biosynthesis genes. The diversity in bacterial ppGpp signaling revealed in these studies suggests the likelihood that additional paradigms in ppGpp-mediated transcription regulation await discovery.

An AraC/XylS Family Transcriptional Regulator Modulates the Oxidative Stress Response of Francisella tularensis.

Francisella tularensis is a Gram-negative bacterium that causes a fatal human disease known as tularemia. The Centers for Disease Control and Prevention have classified F. tularensis as a category A tier 1 select agent. The virulence mechanisms of Francisella are not entirely understood. Francisella possesses very few transcription regulators, and most of these regulate the expression of genes involved in intracellular survival and virulence. The F. tularensis genome sequence analysis reveals an AraC (FTL_0689) transcriptional regulator homologous to the AraC/XylS family of transcriptional regulators. In Gram-negative bacteria, AraC activates genes required for l-arabinose utilization and catabolism. The role of the FTL_0689 regulator in F. tularensis is not known. In this study, we characterized the role of FTL_0689 in the gene regulation of F. tularensis and investigated its contribution to intracellular survival and virulence. The results demonstrate that FTL_0689 in Francisella is not required for l-arabinose utilization. Instead, FTL_0689 specifically regulates the expression of the oxidative and global stress response, virulence, metabolism, and other key pathways genes required by Francisella when exposed to oxidative stress. The FTL_0689 mutant is attenuated for intramacrophage growth and virulence in mice. Based on the deletion mutant phenotype, FTL_0689 was termed osrR (oxidative stress response regulator). Altogether, this study elucidates the role of the osrR transcriptional regulator in tularemia pathogenesis. IMPORTANCE The virulence mechanisms of category A select agent Francisella tularensis, the causative agent of a fatal human disease known as tularemia, remain largely undefined. The present study investigated the role of a transcriptional regulator and its overall contribution to the oxidative stress resistance of F. tularensis. The results provide an insight into a novel gene regulatory mechanism, especially when Francisella is exposed to oxidative stress conditions. Understanding such Francisella- specific regulatory mechanisms will help identify potential targets for developing effective therapies and vaccines to prevent tularemia.

A direct spectropolarimetric assay of arabinose 5-phosphate isomerase.

Arabinose 5-phosphate isomerase (API) catalyzes the reversible isomerization of Ribulose 5-phosphate (Ru5P) to Arabinose 5-Phosphate (Ar5P) for the production of 3-deoxy-2-octulosonic acid 8-phosphate (KDO), a component of bacterial lipopolysaccharide (LPS) of gram-negative bacteria. API is an attractive target for therapeutic development against gram-negative bacterial pathogens. The current assay method of API activity utilizes a general reaction for keto sugar determination in a secondary, 3-h color development reaction with 25 N sulfuric acid which poses hazard to both personnel and instrumentation. We therefore aimed to develop a more user friendly assay of the enzyme. Since Ru5P absorbs in the UV region and contains at least 2 chiral centers, it can be expected to display circular dichroism (CD). A wavelength scan revealed indeed Ru5P displays a pronounced negative ellipticity of 30,560 mDeg M-1cm-1 at 279 nm in Tris buffer pH 9.1 but Ar5P does not have any CD. API enzymatic reactions were monitored directly and continuously in real time by following the disappearance of CD from the Ru5P substrate, or by the appearance of CD from Ar5P substrate. The CD signal at this wavelength was not affected by absorption of the enzyme protein or of small molecules, or turbidity of the solution. Common additives in protein and enzyme reaction mixtures such as detergents, metals, and 5% dimethylsulfoxide did not interfere with the CD signal. Assay reactions of 1-3 min consistently yielded reproducible results. Introduction of accessories in a spectropolarimeter will easily adapt this assay to high throughput format for screening thousands of small molecules as inhibitor candidates of API.

A Francisella tularensis L,D-carboxypeptidase plays important roles in cell morphology, envelope integrity, and virulence.

Francisella tularensis is a Gram-negative, intracellular bacterium that causes the zoonotic disease tularemia. Intracellular pathogens, including F. tularensis, have evolved mechanisms to survive in the harsh environment of macrophages and neutrophils, where they are exposed to cell envelope-damaging molecules. The bacterial cell wall, primarily composed of peptidoglycan (PG), maintains cell morphology, structure, and membrane integrity. Intracellular Gram-negative bacteria protect themselves from macrophage and neutrophil killing by recycling and repairing damaged PG--a process that involves over 50 different PG synthesis and recycling enzymes. Here, we identified a PG recycling enzyme, L,D-carboxypeptidase A (LdcA), of F. tularensis that is responsible for converting PG tetrapeptide stems to tripeptide stems. Unlike E. coli LdcA and most other orthologs, F. tularensis LdcA does not localize to the cytoplasm and also exhibits L,D-endopeptidase activity, converting PG pentapeptide stems to tripeptide stems. Loss of F. tularensis LdcA led to altered cell morphology and membrane integrity, as well as attenuation in a mouse pulmonary infection model and in primary and immortalized macrophages. Finally, an F. tularensis ldcA mutant protected mice against virulent Type A F. tularensis SchuS4 pulmonary challenge.

Bacterial Transcription Continues to Surprise: Activation by Alarmone-Mediated σ-Factor Tethering.

Travis et al. (2020) reveal how Francisella tularensis uses stress-induced ppGpp to activate its virulent pathogenesis program by tethering an αCTD-DNA organizer (PigR) to a σ-organizing heterodimer (MglA-SspA), highlighting the remarkable diversity of transcriptional mechanisms in under-studied bacteria.