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.

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.

Open and compressed conformations of Francisella tularensis ClpP.

Caseinolytic proteases are large oligomeric assemblies responsible for maintaining protein homeostasis in bacteria and in so doing influence a wide range of biological processes. The functional assembly involves three chaperones together with the oligomeric caseinolytic protease catalytic subunit P (ClpP). This protease represents a potential target for therapeutic intervention in pathogenic bacteria. Here, we detail an efficient protocol for production of recombinant ClpP from Francisella tularensis, and the structural characterization of three crystal forms which grow under similar conditions. One crystal form reveals a compressed state of the ClpP tetradecamer and two forms an open state. A comparison of the two types of structure infers that differences at the enzyme active site result from a conformational change involving a highly localized disorder-order transition of a ß-strand α-helix combination. This transition occurs at a subunit-subunit interface. Our study may now underpin future efforts in a structure-based approach to target ClpP for inhibitor or activator development. Proteins 2016; 85:188-194. © 2016 Wiley Periodicals, Inc.

Comparative Analysis of Proteome Patterns of Francisella tularensis Isolates from Patients and the Environment.

Francisella tularensis is the causative agent of tularemia. Although major contributors and the main mechanism of the virulence are well known, some of the molecular details are still missing. Proteomics studies regarding F. tularensis have provided snapshot pictures of the organism grown under different culture conditions to understand the mechanism of virulence. In general, such studies were carried out with standard strains e.g., LVS and did not involve comparisons of F. tularensis isolates from either clinical or environmental sources. In this study, we performed two-dimensional gel electrophoresis (2DE)-based proteomic analysis and compared the protein profiles of the F. tularensis subsp. holarctica strains isolated from the clinical and the environmental samples. Regulations were detected in 14 spots when twofold regulation criteria were applied. The regulated protein spots were subjected to MALDI-TOF/TOF analysis and identified. Classification of the identified proteins based on metabolic functions revealed that the translation machinery was the most varying metabolic processes among the isolates. Using normalized protein spot intensities, PCA analysis was also performed. The results indicated that the strain isolated from water source was different then the strains isolated from the patients. Most interestingly, the isolates were strikingly distinguishable from the standard NCTC 10857 strain.

Towards Development of Improved Serodiagnostics for Tularemia by Use of Francisella tularensis Proteome Microarrays.

Tularemia in humans is caused mainly by two subspecies of the Gram-negative facultative anaerobe Francisella tularensis: F. tularensis subsp. tularensis (type A) and F. tularensis subsp. holarctica (type B). The current serological test for tularemia is based on agglutination of whole organisms, and the reactive antigens are not well understood. Previously, we profiled the antibody responses in type A and B tularemia cases in the United States using a proteome microarray of 1,741 different proteins derived from the type A strain Schu S4. Fifteen dominant antigens able to detect antibodies to both types of infection were identified, although these were not validated in a different immunoassay format. Since type A and B subspecies are closely related, we hypothesized that Schu S4 antigens would also have utility for diagnosing type B tularemia caused by strains from other geographic locations. To test this, we probed the Schu S4 array with sera from 241 type B tularemia cases in Spain. Despite there being no type A strains in Spain, we confirmed the responses against some of the same potential serodiagnostic antigens reported previously, as well as determined the responses against additional potential serodiagnostic antigens. Five potential serodiagnostic antigens were evaluated on immunostrips, and two of these (FTT1696/GroEL and FTT0975/conserved hypothetical protein) discriminated between the Spanish tularemia cases and healthy controls. We conclude that antigens from the type A strain Schu S4 are suitable for detection of antibodies from patients with type B F. tularensis infections and that these can be used for the diagnosis of tularemia in a deployable format, such as the immunostrip.

Comparison of radii sets, entropy, QM methods, and sampling on MM-PBSA, MM-GBSA, and QM/MM-GBSA ligand binding energies of F. tularensis enoyl-ACP reductase (FabI).

To validate a method for predicting the binding affinities of FabI inhibitors, three implicit solvent methods, MM-PBSA, MM-GBSA, and QM/MM-GBSA were carefully compared using 16 benzimidazole inhibitors in complex with Francisella tularensis FabI. The data suggests that the prediction results are sensitive to radii sets, GB methods, QM Hamiltonians, sampling protocols, and simulation length, if only one simulation trajectory is used for each ligand. In this case, QM/MM-GBSA using 6 ns MD simulation trajectories together with GB(neck2) , PM3, and the mbondi2 radii set, generate the closest agreement with experimental values (r(2) = 0.88). However, if the three implicit solvent methods are averaged from six 1 ns MD simulations for each ligand (called "multiple independent sampling"), the prediction results are relatively insensitive to all the tested parameters. Moreover, MM/GBSA together with GB(HCT) and mbondi, using 600 frames extracted evenly from six 0.25 ns MD simulations, can also provide accurate prediction to experimental values (r(2) = 0.84). Therefore, the multiple independent sampling method can be more efficient than a single, long simulation method. Since future scaffold expansions may significantly change the benzimidazole's physiochemical properties (charges, etc.) and possibly binding modes, which may affect the sensitivities of various parameters, the relatively insensitive "multiple independent sampling method" may avoid the need of an entirely new validation study. Moreover, due to large fluctuating entropy values, (QM/)MM-P(G)BSA were limited to inhibitors' relative affinity prediction, but not the absolute affinity. The developed protocol will support an ongoing benzimidazole lead optimization program.

Purification and biophysical characterization of the CapA membrane protein FTT0807 from Francisella tularensis.

The capA gene (FTT0807) from Francisella tularensis subsp. tularensis SCHU S4 encodes a 44.4 kDa integral membrane protein composed of 403 amino acid residues that is part of an apparent operon that encodes at least two other membrane proteins, CapB, and CapC, which together play a critical role in the virulence and pathogenesis of this bacterium. The capA gene was overexpressed in Escherichia coli as a C-terminal His6-tagged fusion with a folding reporter green fluorescent protein (frGFP). Purification procedures using several detergents were developed for the fluorescing and membrane-bound product, yielding approximately 30 mg of pure protein per liter of bacterial culture. Dynamic light scattering indicated that CapA-frGFP was highly monodisperse, with a size that was dependent upon both the concentration and choice of detergent. Circular dichroism showed that CapA-frGFP was stable over the range of 3-9 for the pH, with approximately half of the protein having well-defined α-helical and ß-sheet secondary structure. The addition of either sodium chloride or calcium chloride at concentrations producing ionic strengths above 0.1 M resulted in a small increase of the α-helical content and a corresponding decrease in the random-coil content. Secondary-structure predictions on the basis of the analysis of the sequence indicate that the CapA membrane protein has two transmembrane helices with a substantial hydrophilic domain. The hydrophilic domain is predicted to contain a long disordered region of 50-60 residues, suggesting that the increase of α-helical content at high ionic strength could arise because of electrostatic interactions involving the disordered region. CapA is shown to be an inner-membrane protein and is predicted to play a key cellular role in the assembly of polysaccharides.

Adherence to Bürgi-Dunitz stereochemical principles requires significant structural rearrangements in Schiff-base formation: insights from transaldolase complexes.

The Bürgi-Dunitz angle (αBD) describes the trajectory of approach of a nucleophile to an electrophile. The adoption of a stereoelectronically favorable αBD can necessitate significant reactive-group repositioning over the course of bond formation. In the context of enzyme catalysis, interactions with the protein constrain substrate rotation, which could necessitate structural transformations during bond formation. To probe this theoretical framework vis-à-vis biocatalysis, Schiff-base formation was analysed in Francisella tularensis transaldolase (TAL). Crystal structures of wild-type and Lys→Met mutant TAL in covalent and noncovalent complexes with fructose 6-phosphate and sedoheptulose 7-phosphate clarify the mechanism of catalysis and reveal that substrate keto moieties undergo significant conformational changes during Schiff-base formation. Structural changes compelled by the trajectory considerations discussed here bear relevance to bond formation in a variety of constrained enzymic/engineered systems and can inform the design of covalent therapeutics.

IglE is an outer membrane-associated lipoprotein essential for intracellular survival and murine virulence of type A Francisella tularensis.

IglE is a small, hypothetical protein encoded by the duplicated Francisella pathogenicity island (FPI). Inactivation of both copies of iglE rendered Francisella tularensis subsp. tularensis Schu S4 avirulent and incapable of intracellular replication, owing to an inability to escape the phagosome. This defect was fully reversed following single-copy expression of iglE in trans from attTn7 under the control of the Francisella rpsL promoter, thereby establishing that the loss of iglE, and not polar effects on downstream vgrG gene expression, was responsible for the defect. IglE is exported to the Francisella outer membrane as an ∼13.9-kDa lipoprotein, determined on the basis of a combination of selective Triton X-114 solubilization, radiolabeling with [(3)H]palmitic acid, and sucrose density gradient membrane partitioning studies. Lastly, a genetic screen using the iglE-null live vaccine strain resulted in the identification of key regions in the carboxyl terminus of IglE that are required for intracellular replication of Francisella tularensis in J774A.1 macrophages. Thus, IglE is essential for Francisella tularensis virulence. Our data support a model that likely includes protein-protein interactions at or near the bacterial cell surface that are unknown at present.

Comparative proteome profiling of host-pathogen interactions: insights into the adaptation mechanisms of Francisella tularensis in the host cell environment.

The intracellular pathogens have the unique capacity to sense the host cell environment and to respond to it by alteration in gene expression and protein synthesis. Proteomic analysis of bacteria exposed directly to the host cell milieu might thus greatly contribute to the elucidation of processes leading to bacterial adaptation and proliferation inside the host cell. Here we have performed a global proteome analysis of a virulent Francisella tularensis subsp. holarctica strain during its intracellular cycle within the macrophage-like murine cell line J774.2 using the metabolic pulse-labeling of bacterial proteins with (35)S-methionine and (35)S-cysteine in various periods of infection. The two-dimensional gel analysis revealed macrophage-induced bacterial proteome changes in which 64 identified proteins were differentially expressed in comparison to controls grown in tissue culture medium. Nevertheless, activation of macrophages with interferon gamma before in vitro infection decreased the number of detected alterations in protein levels. Thus, these proteomic data indicate the F. tularensis ability to adapt to the intracellular hostile environment that is, however, diminished by prior interferon gamma treatment of host cells.

Francisella tularensis RipA protein topology and identification of functional domains.

Francisella tularensis is a Gram-negative coccobacillus and is the etiological agent of the disease tularemia. Expression of the cytoplasmic membrane protein RipA is required for Francisella replication within macrophages and other cell types; however, the function of this protein remains unknown. RipA is conserved among all sequenced Francisella species, and RipA-like proteins are present in a number of individual strains of a wide variety of species scattered throughout the prokaryotic kingdom. Cross-linking studies revealed that RipA forms homoligomers. Using a panel of RipA-green fluorescent protein and RipA-PhoA fusion constructs, we determined that RipA has a unique topology within the cytoplasmic membrane, with the N and C termini in the cytoplasm and periplasm, respectively. RipA has two significant cytoplasmic domains, one composed roughly of amino acids 1 to 50 and the second flanked by the second and third transmembrane domains and comprising amino acids 104 to 152. RipA functional domains were identified by measuring the effects of deletion mutations, amino acid substitution mutations, and spontaneously arising intragenic suppressor mutations on intracellular replication, induction of interleukin-1ß (IL-1ß) secretion by infected macrophages, and oligomer formation. Results from these experiments demonstrated that each of the cytoplasmic domains and specific amino acids within these domains are required for RipA function.

Chemical synthesis and immunological evaluation of the inner core oligosaccharide of Francisella tularensis.

Francisella tularensis, which is a Gram negative bacterium that causes tularemia, has been classified by the Center for Disease Control and Prevention (CDC) as a category A bioweapon. The development of vaccines, immunotherapeutics, and diagnostics for F. tularensis requires a detailed knowledge of the saccharide structures that can be recognized by protective antibodies. We have synthesized the inner core region of the lipopolysaccharide (LPS) of F. tularensis to probe antigenic responses elicited by a live and subunit vaccine. The successful preparation of the target compound relied on the use of a disaccharide which was modified by the orthogonal protecting groups diethylisopropylsilyl (DEIPS), 2-naphthylmethyl (Nap), allyl ether (All), and levulinoyl (Lev) ester. The ability to remove the protecting groups in different orders made it possible to establish the optimal glycosylations sequence to prepare a highly crowded 1,2,3-cis configured branching point. A variety of different methods were exploited to control anomeric selectivities of the glycosylations. A comparison of the (1)H NMR spectra of isolated material and the synthetic derivative confirmed the reported structural assignment of the inner core oligosaccharide of F. tularensis . The observation that immunizations with LPS lead to antibody responses to the inner core saccharides provides an impetus to further explore this compound as a vaccine candidate.

Immunodetection of inactivated Francisella tularensis bacteria by using a quartz crystal microbalance with dissipation monitoring.

Francisella tularensis are very small, gram-negative bacteria which are capable of infecting a number of mammals. As a highly pathogenic species, it is a potential bioterrorism agent. In this work we demonstrate a fast immunological detection system for whole F. tularensis bacteria. The technique is based on a quartz crystal microbalance with dissipation monitoring (QCMD), which uses sensor chips modified by a specific antibody. This antibody is useful as a capture molecule to capture the lipopolysaccharide structure on the surface of the bacterial cell wall. The QCMD technique is combined with a microfluidic system and allows the label-free online detection of the binding of whole bacteria to the sensor surface in a wide dynamic concentration range. A detection limit of about 4 × 10(3) colony-forming units per milliliter can be obtained. Furthermore, a rather short analysis time and a clear discrimination against other bacteria can be achieved. Additionally, we demonstrate two possibilities for specific and significant signal enhancement by using antibody-functionalized gold nanoparticles or an enzymatic precipitation reaction. These additional steps can be seen as further proof of the specificity and validity.

Iron content differs between Francisella tularensis subspecies tularensis and subspecies holarctica strains and correlates to their susceptibility to H(2)O(2)-induced killing.

Francisella tularensis, the causative agent of tularemia, is one of the most infectious bacterial pathogens known and is classified as a category A select agent and a facultative intracellular bacterium. Why F. tularensis subsp. tularensis causes a more severe form of tularemia than F. tularensis subsp. holarctica does is not known. In this study, we have identified prominent phenotypic differences between the subspecies, since we found that F. tularensis subsp. tularensis strains contained less iron than F. tularensis subsp. holarctica strains. Moreover, strain SCHU S4 of F. tularensis subsp. tularensis was less susceptible than FSC200 and the live vaccine strain (LVS) of F. tularensis subsp. holarctica to H(2)O(2)-induced killing. The activity of the H(2)O(2)-degrading enzyme catalase was similar between the strains, whereas the iron content affected their susceptibility to H(2)O(2), since iron starvation rendered F. tularensis subsp. holarctica strains more resistant to H(2)O(2). Complementing LVS with fupA, which encodes an important virulence factor that regulates iron uptake, reduced its iron content and increased the resistance to H(2)O(2)-mediated killing. By real-time PCR, it was demonstrated that FSC200 and LVS expressed higher levels of gene transcripts related to iron uptake and storage than SCHU S4 did, and this likely explained their high iron content. Together, the results suggest that F. tularensis subsp. tularensis strains have restricted iron uptake and storage, which is beneficial for their resistance to H(2)O(2)-induced killing. This may be an important factor for the higher virulence of this subspecies of F. tularensis, as reactive oxygen species, such as H(2)O(2), are important bactericidal components during tularemia.

Comparative proteomic profiling of culture filtrate proteins of less and highly virulent Francisella tularensis strains.

The facultative intracellular bacterium Francisella tularensis is the causal agent of the serious infectious disease tularemia. Despite the dynamic progress, which has been made in last few years, important questions regarding Francisella pathogenicity still remain to be answered. Generally, secreted proteins play an important role in pathogenicity of intracellular microbes. In this study, we investigated the protein composition of the culture filtrate proteins of highly virulent F. tularensis subsp. tularensis, strain SCHU S4 and attenuated F. tularensis subsp. holarctica, live vaccine strain using a comparative proteomic analysis. The majority of proteins identified in this study have been implicated in virulence mechanisms of other pathogens, and several have been categorized as having moonlighting properties; those that have more than one unrelated function. This profiling study of secreted proteins resulted in the unique detection of acid phosphatase (precursor) A (AcpA), ß-lactamase, and hypothetical protein FTT0484 in the highly virulent strain SCHU S4 secretome. The release of AcpA may be of importance for F. tularensis subsp. tularensis virulence due to the recently described AcpA role in the F. tularensis escape from phagosomes.

Multimethodological approach to identification of glycoproteins from the proteome of Francisella tularensis, an intracellular microorganism.

It appears that most glycoproteins found in pathogenic bacteria are associated with virulence. Despite the recent identification of novel virulence factors, the mechanisms of virulence in Francisella tularensis are poorly understood. In spite of its importance, questions about glycosylation of proteins in this bacterium and its potential connection with bacterial virulence have not been answered yet. In the present study, several putative Francisella tularensis glycoproteins were characterized through the combination of carbohydrate-specific detection and lectin affinity with highly sensitive mass spectrometry utilizing the bottom-up proteomic approach. The protein PilA that was recently found as being possibly glycosylated, as well as other proteins with designation as novel factors of virulence, were among the proteins identified in this study. The reported data compile the list of potential glycoproteins that may serve as a takeoff platform for a further definition of proteins modified by glycans, faciliting a better understanding of the function of protein glycosylation in pathogenicity of Francisella tularensis.

Identification of Francisella tularensis by whole-cell matrix-assisted laser desorption ionization-time of flight mass spectrometry: fast, reliable, robust, and cost-effective differentiation on species and subspecies levels.

Francisella tularensis, the causative agent of tularemia, is a potential agent of bioterrorism. The phenotypic discrimination of closely related, but differently virulent, Francisella tularensis subspecies with phenotyping methods is difficult and time-consuming, often producing ambiguous results. As a fast and simple alternative, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was applied to 50 different strains of the genus Francisella to assess its ability to identify and discriminate between strains according to their designated species and subspecies. Reference spectra from five representative strains of Francisella philomiragia, Francisella tularensis subsp. tularensis, Francisella tularensis subsp. holarctica, Francisella tularensis subsp. mediasiatica, and Francisella tularensis subsp. novicida were established and evaluated for their capability to correctly identify Francisella species and subspecies by matching a collection of spectra from 45 blind-coded Francisella strains against a database containing the five reference spectra and 3,287 spectra from other microorganisms. As a reference method for identification of strains from the genus Francisella, 23S rRNA gene sequencing was used. All strains were correctly identified, with both methods showing perfect agreement at the species level as well as at the subspecies level. The identification of Francisella strains by MALDI-TOF MS and subsequent database matching was reproducible using biological replicates, different culture media, different cultivation times, different serial in vitro passages of the same strain, different preparation protocols, and different mass spectrometers.

Study of matrix additives for sensitive analysis of lipid A by matrix-assisted laser desorption ionization mass spectrometry.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been widely used for structural characterization of bacterial endotoxins (lipid A). However, the mass spectrometric behavior of the lipid A molecule is highly dependent on the matrix. Furthermore, this dependence is strongly linked to phosphorylation patterns. Using lipid A from Escherichia coli O116 as a model system, we have investigated the effects of different matrices and comatrix compounds on the analysis of lipid A. In this paper, we report a highly sensitive matrix system for lipid A analysis, which consists of 5-chloro-2-mercaptobenzothiazole matrix and EDTA ammonium salt comatrix. This matrix system enhances the sensitivity of the analysis of diphosphorylated lipid A species by more than 100-fold and in addition provides tolerance to high concentrations of sodium dodecyl sulfate (SDS) and tolerance to sodium chloride and calcium chloride at 10 muM, 100 muM, and 10 muM concentrations. The method was further evaluated for analysis of lipid A species with different phosphorylation patterns and from different bacteria, including Helicobacter pylori, Salmonella enterica serovar Riogrande, and Francisella novicida.

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of macrophage growth locus A (MglA) protein from Francisella tularensis.

Francisella tularensis, a potential bioweapon, causes a rare infectious disease called tularemia in humans and animals. The macrophage growth locus A (MglA) protein from F. tularensis associates with RNA polymerase to positively regulate the expression of multiple virulence factors that are required for its survival and replication within macrophages. The MglA protein was overproduced in Escherichia coli, purified and crystallized. The crystals diffracted to 7.5 A resolution at the Advanced Photon Source, Argonne National Laboratory and belonged to the hexagonal space group P6(1) or P6(5), with unit-cell parameters a = b = 125, c = 54 A.

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of intracellular growth locus E (IglE) protein from Francisella tularensis subsp. novicida.

Tularaemia is an uncommon but potentially dangerous zoonotic disease caused by the bacterium Francisella tularensis. As few as ten bacterial cells are sufficient to cause disease in a healthy human, making this one of the most infectious disease agents known. The virulence of this organism is dependent upon a genetic locus known as the Francisella pathogenicity island (FPI), which encodes components of a secretion system that is related to the type VI secretion system. Here, the cloning, expression, purification and preliminary X-ray diffraction statistics of the FPI-encoded protein IglE are presented. This putative lipoprotein is required for intra-macrophage growth and is thought to be a constituent of the periplasmic portion of the type VI-like protein complex that is responsible for the secretion of critical virulence factors in Francisella.