Timely detection and repair of envelope damage are paramount for bacterial survival. The Regulator of Capsule Synthesis (Rcs) stress response can transduce the stress signals across the multilayered gram-negative cell envelope to regulate gene expression in the cytoplasm. Previous studies defined the overall pathway, which begins with the sensory lipoprotein RcsF interacting with several outer membrane proteins (OMPs). RcsF can also interact with the periplasmic domain of the negative regulator IgaA, derepressing the downstream RcsCDB phosphorelay. However, how the RcsF/IgaA interaction is regulated at the molecular level to activate the signaling in response to stress remains poorly understood. In this study, we used a site-saturated mutant library of rcsF to carry out several independent genetic screens to interrogate the mechanism of signal transduction from RcsF to IgaA. We analyzed several distinct classes of rcsF signaling mutants, and determined the region of RcsF that is critically important for signal transduction. This region is bifunctional as it is important for RcsF interaction with both IgaA and OMPs. The mutant analysis provides strong evidence for conformational changes in the RcsF/OMP complex mediating signal transduction to IgaA, and the first direct evidence that OMPs play an important regulatory role in Rcs signaling.

The β-barrel assembly machinery (Bam) complex facilitates the assembly of outer membrane proteins (OMPs) in gram-negative bacteria. The Bam complex is conserved and essential for bacterial viability and consists of five subunits, BamA-E. BamA is the transmembrane component, and its β-barrel domain opens laterally to allow folding and insertion of incoming OMPs. The remaining components are regulatory, among which only BamD is essential. Previous studies suggested that BamB regulates BamA directly, while BamE and BamC serve as BamD regulators. However, specific molecular details of their functions remain unknown. Our previous research demonstrated that BamE plays a specialized role in assembling the complex between the lipoprotein RcsF and its OMP partners, required for the Regulator of Capsule Synthesis (Rcs) stress response. Here, we used RcsF/OmpA as a model substrate to investigate BamE function. Our results challenge the current view that BamE only serves as a BamD regulator. We show that BamE also directly interacts with BamA. BamE interaction with both BamA and BamD is important for function. Our genetic and biochemical analysis shows that BamE stabilizes the Bam complex and promotes bidirectional signaling interaction between BamA and BamD. This BamE function becomes essential when direct BamA/BamD communication is impeded.

Spectinamides are a novel series of spectinomycin analogs being developed for the treatment of tuberculosis. The preclinical lead spectinamide 1599 is an antituberculosis drug that possesses robust in vivo efficacy, good pharmacokinetic properties, and excellent safety profiles in rodents. In individuals infected with Mycobacterium tuberculosis or Mycobacterium bovis, causative agents of tuberculosis, the host immune system is capable of restraining these mycobacteria within granulomatous lesions. The harsh microenvironmental conditions of these granuloma lead to phenotypic transformation of mycobacteria. Phenotypically transformed bacteria display suboptimal growth, or complete growth arrest and are frequently associated with drug tolerance. Here we quantified the effect of spectinamide 1599 on log-phase and phenotypically tolerant isoforms of Mycobacterium bovis BCG using various in vitro approaches as a first indicator of spectinamide 1599 activity against various mycobacterial isoforms. We also used the hollow fiber infection model to establish time-kill curves and deployed pharmacokinetic/pharmacodynamic modeling to characterize the activity differences of spectinamide 1599 towards the different phenotypic subpopulations. Our results indicate that spectinamide 1599 is more efficacious against log phase bacteria when compared to its activity against other phenotypically tolerant forms such as acid phase bacteria and hypoxic phase bacteria, a behavior similar to the established antituberculosis drug isoniazid.

In Mycobacterium tuberculosis, proline dehydrogenase (PruB) and ∆1-pyrroline-5-carboxylate (P5C) dehydrogenase (PruA) are monofunctional enzymes that catalyze proline oxidation to glutamate via the intermediates P5C and L-glutamate-γ-semialdehyde. Both enzymes are essential for the replication of pathogenic M. tuberculosis. Highly active enzymes were expressed and purified using a Mycobacterium smegmatis expression system. The purified enzymes were characterized using natural substrates and chemically synthesized analogs. The structural requirements of the quinone electron acceptor were examined. PruB displayed activity with all tested lipoquinone analogs (naphthoquinone or benzoquinone). In PruB assays utilizing analogs of the native naphthoquinone [MK-9 (II-H2)] specificity constants Kcat/Km were an order of magnitude greater for the menaquinone analogs than the benzoquinone analogs. In addition, mycobacterial PruA was enzymatically characterized for the first time using exogenous chemically synthesized P5C. A Km value of 120 ± 0.015 µM was determined for P5C, while the Km value for NAD+ was shown to be 33 ± 4.3 µM. Furthermore, proline competitively inhibited PruA activity and coupled enzyme assays, suggesting that the recombinant purified monofunctional PruB and PruA enzymes of M. tuberculosis channel substrate likely increase metabolic flux and protect the bacterium from methylglyoxal toxicity.

Significance The bacterial cell envelope is the frontline defense against host immune factors and antibiotics. The regulator of capsule synthesis (Rcs) is a complex signaling pathway that maintains the homeostasis of this essential organelle. Several hypotheses for how the sensory component RcsF activates signaling in response to stress were proposed but could not be directly tested because RcsF proper assembly into the complex with outer membrane proteins (OMPs) depends on the essential β-barrel assembly machine (Bam). We used an extensive genetic analysis to tease apart which RcsF interactions are important for the sensing function. We show that RcsF does not monitor the Bam complex function. Instead, the Bam complex is required to assemble the sensory RcsF/OMP complex that monitors membrane integrity.

MenJ, a flavoprotein oxidoreductase, is responsible for the saturation of the β-isoprene unit of mycobacterial menaquinone, resulting in the conversion of menaquinone with nine isoprene units (MK-9) to menaquinone with nine isoprene units where the double bond in the second unit is reduced [MK-9(II-H2)]. The hydrogenation of MK-9 increases the efficiency of the mycobacterial electron transport system, whereas the deletion of MenJ results in decreased survival of the bacteria inside J774A.1 macrophage-like cells but is not required for growth in culture. Thus, it was suggested that MenJ may represent a contextual drug target in M. tuberculosis, that is, a drug target that is valid only in the context of an infected macrophage. However, it was unclear if the conversion of MK-9 to MK-9(II-H2) or the MenJ protein itself was responsible for bacterial survival. In order to resolve this issue, a plasmid expressing folded, full-length, inactive MenJ was engineered. Primary sequence analysis data revealed that MenJ shares conserved FAD binding, NADH binding, and catalytic and C-terminal motifs with archaeal geranylgeranyl reductases. A MenJ mutant deficient in any one of these motifs is devoid of reductase activity. Therefore, point mutations of highly conserved amino acids in the conserved motifs were generated and the recombinant proteins were monitored for conformational changes by circular dichroism and oxidoreductase activity. The mutational analysis indicates that amino acids tryptophan 215 (W215) and cysteine 46 (C46) of M. tuberculosis MenJ, conserved in known archaeal geranylgeranyl reductases and putative menaquinone saturases, are essential to the hydrogenation of MK-9. The mutation of either C46 to serine (C46S) or W215 to leucine (W215L) in MenJ completely abolishes the catalytic activity in vitro, and menJ knockout strains of M. tuberculosis expressing either the C46S or W215L mutant protein are unable to convert MK-9 to MK-9(II-H2) but survive inside the J774A.1 cells. Thus, surprisingly, the survival of M. tuberculosis in J774A.1 cells is dependent on the expression of MenJ rather than its oxidoreductase activity, the conversion of MK-9 to MK-9(II-H2) as previously hypothesized. Overall, the current data suggest that MenJ is a moonlighting protein.

A direct role for IgA either for elimination of malaria parasite or for improvement in tissue pathology has not been investigated in case of Malaria infection while IgG, IgE and IgM were all implicated in the adverse pathology. In this communication, we delineate further that Malaria specific IgA appears to be significant among individuals who had multiple episodes of infection. Interestingly, the IgA elicited by immunization of the homologous peptides derived from Plasmodium berghei ANKA have also resulted in protection of host from adverse lung pathology, while the parasite load is unaffected. The PfrVI immunized mice and mice infected with repeated cycles of ‘infection and recovery’, simulating an endemic like situation, have resulted in development of B cell population that secretes the IgA specific to this region VI. Summarily, our results suggest that the IgA specific to the malarial antigen can confer significant advantage to hosts in protecting the overall tissue pathology.

MenJ, annotated as an oxidoreductase, was recently demonstrated to catalyze the reduction (saturation) of a single double bond in the isoprenyl side-chain of mycobacterial menaquinone. This modification was shown to be essential for bacterial survival in J774A.1 macrophage-like cells, suggesting that MenJ may be a conditional drug target in Mycobacterium tuberculosis and other pathogenic mycobacteria. Recombinant protein was expressed in a heterologous host, and the activity was characterized. Although highly regiospecific in vivo, the activity is not absolutely regiospecific in vitro; in addition, the enzyme is not specific for naphthoquinones vs benzoquinones. Coenzyme Q-1 (a benzoquinone, UQ-1) was used as the lipoquinone substrate, and NADH oxidation was followed spectrophotometrically as the activity readout. NADPH could not be substituted for NADH in the reaction mixture. The enzyme contains a FAD binding site that was 72% occupied in the purified recombinant protein. Enzyme activity was maximal at 37 °C and pH 7.0; addition of divalent cations, EDTA, and reducing agents such as dithiothreitol to the reaction mixture had no effect on activity. The addition of detergents did not stimulate activity, and addition of saturating levels of FAD had relatively little effect on the observed kinetic parameters. These properties allowed the development of a facile assay needed to study this potential drug target, which is also amenable to high throughput screening. The Km values for UQ-1 using recombinant MenJ from Mycobacterium smegmatis or M. tuberculosis without saturating concentrations of FAD were found to be 52 ± 9.6 and 44 ± 4.8 μM, respectively, while the KmNADH values were determined to be 59 ± 14 and 64 ± 15 μM. The Km for MK-1, the menaquinone analogue of UQ-1, using recombinant MenJ from M. tuberculosis without saturating concentrations of FAD but in the presence of 0.5% Tween 80 was shown to be 30 ± 2.9 μM. Thus, this is the first report of a kinetic characterization of a member of the geranylgeranyl reductase family of enzymes.

51V NMR spectroscopy is used to document, using speciation analysis, that one oxometalate is a more potent growth inhibitor of two Mycobacterial strains than other oxovanadates, thus demonstrating selectivity in its interaction with cells. Historically, oxometalates have had many applications in biological and medical studies, including study of the phase-problem in X-ray crystallography of the ribosome. The effect of different vanadate salts on the growth of Mycobacterium smegmatis (M. smeg) and Mycobacterium tuberculosis (M. tb) was investigated, and speciation was found to be critical for the observed growth inhibition. Specifically, the large orange-colored sodium decavanadate (V10O286-) anion was found to be a stronger inhibitor of growth of two mycobacterial species than the colorless oxovanadate prepared from sodium metavanadate. The vanadium(V) speciation in the growth media and conversion among species under growth conditions was monitored using 51V NMR spectroscopy and speciation calculations. The findings presented in this work is particularly important in considering the many applications of polyoxometalates in biological and medical studies, such as the investigation of the phase-problem in X-ray crystallography for the ribosome. The findings presented in this work investigate the interactions of oxometalates with other biological systems.

The biosynthesis of tryptophan in Mycobacterium tuberculosis is initiated by the transformation of chorismate to anthranilate, catalyzed by anthranilate synthase (TrpE/TrpG). Five additional enzymes are required to complete tryptophan biosynthesis. M. tuberculosis strains auxotrophic for tryptophan, an essential amino acid in the human diet, are avirulent. Thus, tryptophan synthesis in M. tuberculosis has been suggested as a potential drug target, and it has been reported that fluorinated anthranilate is lethal to the bacillus. Two mechanisms that could explain the cellular toxicity were tested: (1) the inhibition of tryptophan biosynthesis by a fluorinated intermediate or (2) formation of fluorotryptophan and its subsequent effects. Here, M. tuberculosis mc(2) 6230 cultures were treated with anthranilates fluorinated at positions 4, 5, and 6. These compounds inhibited bacterial growth on tryptophan-free media with 4-fluoroanthranilate being more potent than 5-fluoroanthranilate or 6-fluoroanthranilate. LC-MS based analysis of extracts from bacteria treated with these compounds did not reveal accumulation of any of the expected fluorinated intermediates in tryptophan synthesis. However, in all cases, significant levels of fluorotryptophan were readily observed, suggesting that the enzymes involved in the conversion of fluoroanthranilate to fluorotryptophan were not being inhibited. Inclusion of tryptophan in cultures treated with the fluoroanthranilates obviated the cellular toxicity. Bacterial growth was also inhibited in a dose-dependent manner by exposure to tryptophan substituted with fluorine at positions 5 or 6. Thus, the data suggest that fluorotryptophan rather than fluoroanthranilate or intermediates in the synthesis of fluorotryptophan causes the inhibition of M. tuberculosis growth.

Plasmodium specific IgA has been detected in serum and breast milk among the endemic population but the role it can play in vivo is not clear. In this report, we demonstrate the utility of Malaria specific IgA, elicited by peptide sequences (referred as Mpep3 and Mpep4) of region VI of EBA-175 (PfrVI). Immunization of mice with KLH tagged or untagged peptides of Mpep3, Mpep4 or with PfrVI have resulted in specific IgA response that inhibits the in vitro invasion of Plasmodium falciparum merozoites. Mice having the IgA specific to Mpep4 have exhibited higher tolerance to Plasmodium berghei ANKA parasitemia, exhibited several fold lesser sequestration of infected RBC, lesser damage to microvasculature with no signs of perivascular haemorrhage and lesser lung inflammation in comparison to unimmunized mice. In addition, the immunized mice have B-cell population that secrete the IgA specific to PfrVI. These results suggest that the IgA specific to these malarial antigens can confer significant advantage to hosts and it may also reduce the severity of malaria infection.

The mycobacterial tlyA gene product, Rv1694 (MtbTlyA), has been annotated as "hemolysin" which was re-annotated as 2'-O rRNA methyl transferase. In order to function as a hemolysin, it must reach the extracellular milieu with the help of signal sequence(s) and/or transmembrane segment(s). However, the MtbTlyA neither has classical signals sequences that signify general/Sec/Tat pathways nor transmembrane segments. Interestingly, the tlyA gene appears to be restricted to pathogenic strains such as H37Rv, M. marinum, M. leprae, than M. smegmatis, M. vaccae, M. kansasii etc., which highlights the need for a detailed investigation to understand its functions. In this study, we have provided several evidences which highlight the presence of TlyA on the surface of M. marinum (native host) and upon expression in M. smegmatis (surrogate host) and E. coli (heterologous host). The TlyA was visualized at the bacterial-surface by confocal microscopy and accessible to Proteinase K. In addition, sub-cellular fractionation has revealed the presence of TlyA in the membrane fractions and this sequestration is not dependent on TatA, TatC or SecA2 pathways. As a consequence of expression, the recombinant bacteria exhibit distinct hemolysis. Interestingly, the MtbTlyA was also detected in both membrane vesicles secreted by M. smegmatis and outer membrane vesicles secreted by E. coli. Our experimental evidences unambiguously confirm that the mycobacterial TlyA can reach the extra cellular milieu without any signal sequence. Hence, the localization of TlyA class of proteins at the bacterial surface may highlight the existence of non-classical bacterial secretion mechanisms.

Iron (Fe2+, Fe3+) homeostasis is a tightly regulated process, involving precise control of iron influx and egress from cells. Although the mechanisms of its import into cells by iron carrier molecules are well characterized, iron export remains poorly understood. The current paradigm envisages unique functions associated with specialized macromolecules for its cellular import (transferrin receptors) or export (ferroportin, also known as SLC40A1). Previous studies have revealed that iron-depleted cells recruit glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a multitasking, 'moonlighting' protein, to their surface for internalization of the iron carrier holotransferrin. Here, we report that under the converse condition of intracellular iron excess, cells switch the isoform of GAPDH on their surface to one that now recruits iron-free apotransferrin in close association with ferroportin to facilitate the efflux of iron. Increased expression of surface GAPDH correlated with increased apotransferrin binding and enhanced iron export from cells, a capability lost in GAPDH-knockdown cells. These findings were confirmed in vivo utilizing a rodent model of iron overload. Besides identifying for the first time an apotransferrin receptor, our work uncovers the two-way switching of multifunctional molecules to manage cellular micronutrient requirements.

The pathogenic traits of TlyA proteins of Mycobacterium tuberculosis are not known. Expressions of TlyA in bacteria that do not express endogenous TlyA adhere better to RAW264.7 macrophages and get phagocytosed efficiently. The internalized bacteria avoid acidification to the extent of >65% in the case of both TlyA-expressing E. coli and M. smegmatis. Consistent with this observation, we have observed decreased co-localizaton of Lysosomal Membrane Associated Protein-1 (~35%), Early Endosomal Antigen-1 (~34%), Rab5 (~30%) and Rab7 (~35%) and enhanced co-localizaton of Rab14 (~80%) on both TlyA-expressing bacteria as well as on TlyA-coated latex beads. These results suggest that the mycobacterial TlyA, in general, can modulate phagolysosome maturation pathway immediately after entry into macrophages, while other important molecules may aid the bacterium for long-term, intracellular survival at later point of time.