STING-Activating Adjuvants Elicit a Th17 Immune Response and Protect against Mycobacterium tuberculosis Infection.

There are a limited number of adjuvants that elicit effective cell-based immunity required for protection against intracellular bacterial pathogens. Here, we report that STING-activating cyclic dinucleotides (CDNs) formulated in a protein subunit vaccine elicit long-lasting protective immunity to Mycobacterium tuberculosis in the mouse model. Subcutaneous administration of this vaccine provides equivalent protection to that of the live attenuated vaccine strain Bacille Calmette-Guérin (BCG). Protection is STING dependent but type I IFN independent and correlates with an increased frequency of a recently described subset of CXCR3-expressing T cells that localize to the lung parenchyma. Intranasal delivery results in superior protection compared with BCG, significantly boosts BCG-based immunity, and elicits both Th1 and Th17 immune responses, the latter of which correlates with enhanced protection. Thus, a CDN-adjuvanted protein subunit vaccine has the capability of eliciting a multi-faceted immune response that results in protection from infection by an intracellular pathogen.

Mycobacterium tuberculosis proteins involved in cell wall lipid biosynthesis improve BCG vaccine efficacy in a murine TB model.

OBJECTIVES: Advances in tuberculosis (TB) vaccine development are urgently required to enhance global disease management. We evaluated the potential of Mycobacterium tuberculosis (M. tb)-derived protein antigens Rv0447c, Rv2957 and Rv2958c to boost BCG vaccine efficacy in the presence or absence of glucopyranosyl lipid adjuvant formulated in a stable emulsion (GLA-SE) adjuvant. METHODS: Mice received the BCG vaccine, followed by Rv0447c, Rv2957 and Rv2958c protein boosting with or without GLA-SE adjuvant 3 and 6 weeks later. Immune responses were examined at given time points. 9 weeks post vaccination, mice were aerosol-challenged with M. tb, and sacrificed at 6 and 12 weeks to assess bacterial burden. RESULTS: Vaccination of mice with BCG and M. tb proteins in the presence of GLA-SE adjuvant triggered strong IFN-γ and IL-2 production by splenocytes; more TNF-α was produced without GLA-SE addition. Antibody responses to all three antigens did not differ, with or without GLA-SE adjuvant. Protein boosting without GLA-SE adjuvant resulted in vaccinated animals having better control of pulmonary M. tb load at 6 and 12 weeks post aerosol infection, while animals receiving the protein boost with GLA-SE adjuvant exhibited more bacteria in the lungs. CONCLUSIONS: Our data provides evidence for developing Rv2958c, Rv2957 and Rv0447c in a heterologous prime-boost vaccination strategy with BCG.

Psychological distress in the workforce: a multilevel and longitudinal analysis of the case of regulated occupations in Canada.

BACKGROUND: This study uses a multidimensional theoretical model to evaluate the role of regulated occupations and working conditions in explaining psychological distress. METHODS: Various multilevel regression analyses were conducted on longitudinal data for which measures repeated over time (n1 = 36,166) were nested in individuals (n2 = 7007). RESULTS: Results showed that when we controlled for working conditions, family situation, the social network outside the workplace, and personal characteristics, the level of psychological distress was significantly lower among professional workers in regulated occupations than among professionals not in regulated occupations. Among the working conditions studied, skill utilisation, psychological demands, and job insecurity were positively associated with psychological distress levels, whereas social support in the workplace was inversely related to distress. Finally, our results suggest that self-esteem reduced the effect of social support in the workplace on psychological distress levels in the workforce. CONCLUSIONS: These results support our hypothesis that working in regulated occupations exerts a direct effect on mental health. These results also make clear the importance of developing new tools for measuring psychological distress among upper-level professional workers. Such tools will be much better suited to the realities characterising today's knowledge-based economies.

A longitudinal and comparative study of psychological distress among professional workers in regulated occupations in Canada.

BACKGROUND: Although several studies are concerned by the phenomenon of psychological distress at work, few studies have looked at the prevalence of psychological distress among professional workers in the regulated occupations and compare this prevalence with other occupations. OBJECTIVES: This study propose to define regulated occupations by laying out the theoretical boundaries that apply to the practice of these occupations and try to understand how regulated occupations contributed to the experience of psychological distress in the Canadian workforce over time. METHOD: Multilevel logistical regression analyses on longitudinal data were performed to compare the odds of experiencing psychological distress over time among professional workers in regulated occupations (n=276) and among other professional workers, classified into 6 categories (n=6731), over a 12-year period. RESULTS: The results show that proportion of distress in the workforce decreases for all occupations between Cycle 1 and Cycle 7 of the NPHS, but this decrease is not linear over time. The results show also that regulated occupations present a lower probability of psychological distress only when compared with white-collar workers. CONCLUSIONS: These results suggest that occupation contributes little toward understanding the prevalence of psychological distress in the Canadian workforce. Further research needs are also discussed.

Induction of cell death after localization to the host cell mitochondria by the Mycobacterium tuberculosis PE_PGRS33 protein.

PE_PGRS33 is the most studied member of the unique PE family of mycobacterial proteins. These proteins are composed of a PE domain (Pro-Glu motif), a linker region and a PGRS domain (polymorphic GC-rich-repetitive sequence). Previous studies have shown that PE_PGRS33 is surface-exposed, constitutively expressed during growth and infection, involved in creating antigenic diversity, and able to induce death in transfected or infected eukaryotic cells. In this study, we showed that PE_PGRS33 co-localizes to the mitochondria of transfected cells, a phenomenon dependent on the linker region and the PGRS domain, but not the PE domain. Using different genetic fusions and chimeras, we also demonstrated a direct correlation between localization to the host mitochondria and the induction of cell death. Finally, although all constructs localizing to the mitochondria did induce apoptosis, only the wild-type PE_PGRS33 with its own PE domain also induced primary necrosis, indicating a potentially important role for the PE domain. Considering the importance of primary necrosis in Mycobacterium tuberculosis dissemination during natural infection, the PE_PGRS33 protein may play a crucial role in the pathogenesis of tuberculosis.

PPE and PE_PGRS proteins of Mycobacterium marinum are transported via the type VII secretion system ESX-5.

ESX-5 is one of the five type VII secretion systems found in mycobacteria. These secretion systems are also known as ESAT-6-like secretion systems. Here, we have determined the secretome of ESX-5 by a proteomic approach in two different strains of Mycobacterium marinum. Comparison of the secretion profile of wild-type strains and their ESX-5 mutants showed that a number of PE_PGRS and PPE-MPTR proteins are dependent on ESX-5 for transport. The PE and PPE protein families are unique to mycobacteria, are highly expanded in several pathogenic species, such as Mycobacterium tuberculosis and M. marinum, and certain family members are cell surface antigens associated with virulence. Using a monoclonal antibody directed against the PGRS domain we showed that nearly all PE_PGRS proteins that are recognized by this antibody are missing in the supernatant of ESX-5 mutants. In addition to PE_PGRS and PPE proteins, the ESX-5 secretion system is responsible for the secretion of a ESAT-6-like proteins. Together, these data show that ESX-5 is probably a major secretion pathway for mycobacteria and that this system is responsible for the secretion of recently evolved PE_PGRS and PPE proteins.

Development of a murine mycobacterial growth inhibition assay for evaluating vaccines against Mycobacterium tuberculosis.

The development and characterization of new tuberculosis (TB) vaccines has been impeded by the lack of reproducible and reliable in vitro assays for measuring vaccine activity. In this study, we developed a murine in vitro mycobacterial growth inhibition assay for evaluating TB vaccines that directly assesses the capacity of immune splenocytes to control the growth of Mycobacterium tuberculosis within infected macrophages. Using this in vitro assay, protective immune responses induced by immunization with five different types of TB vaccine preparations (Mycobacterium bovis BCG, an attenuated M. tuberculosis mutant strain, a DNA vaccine, a modified vaccinia virus strain Ankara [MVA] construct expressing four TB antigens, and a TB fusion protein formulated in adjuvant) can be detected. Importantly, the levels of vaccine-induced mycobacterial growth-inhibitory responses seen in vitro after 1 week of coculture correlated with the protective immune responses detected in vivo at 28 days postchallenge in a mouse model of pulmonary tuberculosis. In addition, similar patterns of cytokine expression were evoked at day 7 of the in vitro culture by immune splenocytes taken from animals immunized with the different TB vaccines. Among the consistently upregulated cytokines detected in the immune cocultures are gamma interferon, growth differentiation factor 15, interleukin-21 (IL-21), IL-27, and tumor necrosis factor alpha. Overall, we have developed an in vitro functional assay that may be useful for screening and comparing new TB vaccine preparations, investigating vaccine-induced protective mechanisms, and assessing manufacturing issues, including product potency and stability.

A comparative study of host response to three Mycobacterium tuberculosis PE_PGRS proteins.

Three Mycobacterium tuberculosis proteins, PE_PGRS 16 (Rv0977), PE_PGRS 26 (Rv1441c) and PE_PGRS 33 (Rv1818c), were expressed in Mycobacterium smegmatis and used to investigate the host response to members of this unique protein family. Following infection of macrophages with the recombinant M. smegmatis (Ms) strains, Ms-PE_PGRS 33 and Ms-PE_PGRS 26 were significantly more persistent (4.4 and 4.2 log c.f.u.) compared with Ms-PE_PGRS 16 (3.4 log c.f.u.) at day 6. Similarly, after infection of mice, Ms-PE_PGRS 33 and Ms-PE_PGRS 26 persisted at significantly higher levels in the spleen (3.5 and 3.2 log c.f.u.) and liver (3 and 2.6 log c.f.u.) compared with Ms-PE_PGRS 16 in the spleen (2 log c.f.u.) and in the liver (1 log c.f.u.) at day 10. Increased persistence of Ms-PE_PGRS 33 and Ms-PE_PGRS 26 was associated with cell death and increased release of lactate dehydrogenase in macrophage cultures as well as increased levels of IL-10 and, in contrast, lower levels of IL-12 and NO both in vitro and in mouse splenocytes. Conversely, poor survival of Ms-PE_PGRS 16 was associated both in macrophage cultures and in vivo with higher levels of NO and IL-12. All three PE_PGRS proteins were found to be cell-surface antigens, but immunization of mice with these PE_PGRS antigens as DNA vaccines showed no protection in a TB aerosol challenge model. In general, the results suggest that variable expression of different PE_PGRS proteins within host cells can affect either the fate of the mycobacterial pathogen or that of the host during infection and point to the importance of studying the expression and function of individual members of the PE_PGRS gene family of M. tuberculosis.

Observations on the calcium dependence and reversibility of cobalamin transport across the outer membrane of Escherichia coli.

The calcium dependence of cobalamin (Cbl) binding to the BtuB protein of Escherichia coli and the reversibility of its function in the transport of Cbl across the outer membrane have been examined. The results show that the two calcium-binding sites in BtuB that were identified previously by others are responsible for the calcium dependence of high affinity Cbl binding. The affinity of the pure BtuB protein for Cbl was approximately 1000-fold higher in the presence of saturating levels of calcium than in its absence. The affinities of BtuB for both Cbl and calcium were decreased by insertion of alanine residues at position 51 of the mature protein and were increased by several mutations and deletions in the TonB box. Experiments on the uptake of Cbl into the periplasmic space showed that this process is reversible and that the exit of Cbl back into the medium does not require the protonmotive force. Our interpretation of these results is that the role of the TonB-ExbB-ExbD complex, potentiated by the protonmotive force, is to reduce the affinity of the Cbl-binding site, thus increasing the rate of Cbl release into the periplasmic space. The evidence also indicates that access of the Cbl-binding site of BtuB to the periplasmic space does not require removal of the hatch domain from the barrel.

A PE protein expressed by Mycobacterium avium is an effective T-cell immunogen.

Infection of mice with Mycobacterium avium or immunization with a novel PE gene expressed by M. avium (MaPE) showed that a dominant T-cell immune response was elicited. Immunization with an MaPE DNA vaccine protected mice against an aerosol challenge with Mycobacterium tuberculosis, suggesting that mycobacteria express PE antigens with cross-protective T-cell epitopes.

Differential substrate-induced signaling through the TonB-dependent transporter BtuB.

The BtuB transporter mediates high-affinity binding and TonB-dependent active transport of vitamin B12 [cyanocobalamin (CNCbl)] across the outer membrane of Escherichia coli. A characteristic feature of TonB-dependent transporters is the Ton box, a conserved sequence near the N terminus and exposed to the periplasm. Crosslinking to TonB and site-directed spin labeling indicated that the Ton box of BtuB undergoes a substantial conformational transition in response to CNCbl binding, but only slight movement was seen in crystal structures. An in vivo method of detecting substrate-induced changes in the Ton box environment measured reaction of a biotin maleimide derivative with cysteine substitutions through the N-terminal region of BtuB between positions 1 and 31. The degree of maleimide labeling of different residues correlated with their accessibility in the crystal structure. Labeling of many positions was increased strongly when CNCbl was present, consistent with the undocking of this region proposed from spin-labeling analyses. The receptor-binding domain of colicin E3, which binds to BtuB competitively with CNCbl, resulted in decreased labeling. Both substrate-induced transitions occur in and beyond the Ton box and were affected by transport-uncoupling substitutions. Thus, two transport substrates that bind competitively to the extracellular face of BtuB stabilize opposite transitions of the Ton box.

Competing ligands stabilize alternate conformations of the energy coupling motif of a TonB-dependent outer membrane transporter.

BtuB is a TonB-dependent outer-membrane transporter for vitamin B12 (or cyanocobalamin, CN-Cbl) in Escherichia coli. The binding of CN-Cbl is believed to promote an unfolding or undocking of the Ton box, the conserved N-terminal energy coupling motif at the periplasmic surface of the transporter. This structural change may facilitate the interaction of BtuB with the inner membrane protein TonB. In this work, the effect of the receptor binding fragment of colicin E3 (E3R) on the conformation of the Ton box was examined with site-directed spin labeling. Addition of E3R reverses the undocking of the Ton box that is promoted by CN-Cbl, consistent with a competitive binding between the substrate and the colicin fragment. EPR spectroscopy indicates that the Ton box is in a two-state equilibrium between docked and undocked conformations. In the absence of substrate, the docked conformation is the predominant state; however, the equilibrium can be shifted to the undocked state by the addition of detergents or site-specific proline substitutions. Even when the undocking is induced by detergents or by certain proline mutations, E3R binding shifts the equilibrium to the docked conformation. Thus, two competitive extracellular ligands, CN-Cbl and ER3, transduce opposite conformations of the N-terminal Ton box. Substrate binding stabilizes an undocked conformation, whereas E3R binding stabilizes a docked conformation of the Ton box.

Substrate-induced conformational changes of the periplasmic N-terminus of an outer-membrane transporter by site-directed spin labeling.

The structure and dynamics of the N-terminal and core regions of BtuB, an outer membrane vitamin B(12) transporter from Escherichia coli, were investigated by site-directed spin labeling. Cysteine mutants were generated by site-directed mutagenesis to place spin labels in the N-terminal region (residues 1-17), the core region (residues 25-30), and double labels into the Ton box (residues 6-12). BtuB mutants were expressed, spin labeled, purified, and reconstituted into phosphatidylcholine. In the presence of substrate (vitamin B(12)), EPR spectroscopy demonstrates that there is a conformational change in the Ton box similar to that seen previously for BtuB in intact outer membranes. The Ton box is positioned within the beta-barrel of BtuB in the absence of substrate (docked configuration) but becomes unfolded and increases its aqueous exposure upon substrate binding (undocked configuration). This conformational change and the similarity in the EPR spectra between reconstituted and native membranes indicate that BtuB is correctly folded and functional in the reconstituted system. The protein segment on the N-terminal side of the Ton box is highly mobile, and it becomes more mobile in the presence of substrate. Side chains in the region C-terminal to the Ton box also show increases in mobility with substrate addition, but position 16 appears to define a hinge point for this conformation change. EPR line shapes and relaxation data indicate that residues 25-30 form a beta-strand structure, which is analogous to the first beta-strand in the cores of the homologous iron transporters. When substrate binds to BtuB, this first beta-strand remains folded. The EPR spectra of double-nitroxide labels within the Ton box are broadened because of dipolar and collisional exchange interactions. The broadening pattern indicates that the Ton box is not helical but is in an extended or beta-strand structure.

Structure and dynamics of the beta-barrel of the membrane transporter BtuB by site-directed spin labeling.

Site-directed spin labeling and EPR spectroscopy were used to map two consecutive beta-strands of the putative transmembrane beta-barrel of BtuB. For these studies, a series of 29 consecutive single cysteine mutants of BtuB were produced covering residues 148-176. The proteins were then expressed, reacted with a sulfhydryl-specific spin label, purified in octyl glucoside (OG), and reconstituted into palmitoyloleoylphosphatidylcholine (POPC) bilayers. The labeled residues spanned from the extracellular region (position 148) to the small periplasmic loop (positions 160-163) and back up to the extracellular side (position 176) of BtuB. Continuous wave power saturation in the presence of oxygen or NiAA yielded an i, i + 2 periodicity for the collision frequencies at these sites and demonstrated the presence of a beta-strand structural motif. For both strands studied, the even-numbered residues were found to be exposed to the hydrophobic phase of the bilayer, whereas the odd-numbered residues pointed toward the interior of the barrel and the core of the protein. In addition, the collision parameters yielded the position of the protein within the bilayer. The phase relationship between the oxygen and metal collision frequencies along with the corresponding membrane depth parameters, Phi, indicates that segments 151-159 and 164-172 are within the bilayer. In POPC bilayers, there is a mobility gradient for spin labels along the barrel indicating enhanced backbone flexibility toward the periplasmic surface of the barrel. In POPC/OG mixed micelles, the even-numbered residues facing the hydrocarbon show an increased mobility compared with the bilayer environment whereas the inward-facing side chains show little change in motion. The data indicate that the protein core remains folded in POPC/OG mixed micelles but that this environment increases the backbone fluctuations of the strands. A model for the beta-barrel of BtuB is presented in part on the basis of these EPR data.

Identification of the periplasmic cobalamin-binding protein BtuF of Escherichia coli.

Cells of Escherichia coli take up vitamin B(12) (cyano-cobalamin [CN-Cbl]) and iron chelates by use of sequential active transport processes. Transport of CN-Cbl across the outer membrane and its accumulation in the periplasm is mediated by the TonB-dependent transporter BtuB. Transport across the cytoplasmic membrane (CM) requires the BtuC and BtuD proteins, which are most related in sequence to the transmembrane and ATP-binding cassette proteins of periplasmic permeases for iron-siderophore transport. Unlike the genetic organization of most periplasmic permeases, a candidate gene for a periplasmic Cbl-binding protein is not linked to the btuCED operon. The open reading frame termed yadT in the E. coli genomic sequence is related in sequence to the periplasmic binding proteins for iron-siderophore complexes and was previously implicated in CN-Cbl uptake in Salmonella. The E. coli yadT product, renamed BtuF, is shown here to participate in CN-Cbl uptake. BtuF protein, expressed with a C-terminal His(6) tag, was shown to be translocated to the periplasm concomitant with removal of a signal sequence. CN-Cbl-binding assays using radiolabeled substrate or isothermal titration calorimetry showed that purified BtuF binds CN-Cbl with a binding constant of around 15 nM. A null mutation in btuF, but not in the flanking genes pfs and yadS, strongly decreased CN-Cbl utilization and transport into the cytoplasm. The growth response to CN-Cbl of the btuF mutant was much stronger than the slight impairment previously described for btuC, btuD, or btuF mutants. Hence, null mutations in btuC and btuD were constructed and revealed that the btuC mutant had a strong impairment similar to that of the btuF mutant, whereas the btuD defect was less pronounced. All mutants with defective transport across the CM gave rise to frequent suppressor variants which were able to respond at lower levels of CN-Cbl but were still defective in transport across the CM. These results finally establish the identity of the periplasmic binding protein for Cbl uptake, which is one of few cases where the components of a periplasmic permease are genetically separated.