Iron transport in Mycobacterium smegmatis: occurrence of iron-regulated envelope proteins as potential receptors for iron uptake

Cell-envelope fractions were isolated from the rapidly growing saprophyte Mycobacterium smegmatis following growth in glycerol/asparagine medium under both iron-limited (0.02 microgram Fe ml-1) and iron-sufficient (2.0 to 4.0 micrograms Fe ml-1) conditions. Examination of these preparations by SDS-PAGE demonstrated the production of at least four additional proteins when iron was limiting. These iron-regulated envelope proteins (IREPs) were ascribed apparent molecular masses of 180 kDa (protein I), 84 kDa (protein II), 29 kDa (protein III) and 25 kDa (protein IV). All four proteins were present in both cell-wall and membrane preparations but spheroplast preparations were devoid of the 29 kDa protein. Attempts at labelling the proteins with 55FeCl3 or 55Fe-exochelin, the siderophore for iron uptake, were unsuccessful, though this was attributed to the denatured state of the proteins following electrophoresis. Antibodies were raised to each of the four proteins: the one raised to protein III inhibited exochelin-mediated iron uptake into iron-deficiently grown cells by 70% but was ineffective against iron uptake into iron-sufficiently grown cells. As exochelin is taken up into both types of cells by a similar process, protein III may not be a simple receptor for iron uptake though the results imply some function connected with this process. The role of the other IREPs is less certain.     

Reversible acetylation and inactivation of Mycobacterium tuberculosis acetyl-CoA synthetase is dependent on cAMP

Recent proteomics studies have revealed that protein acetylation is an abundant and evolutionarily conserved post-translational modification from prokaryotes to eukaryotes. Although an astonishing number of acetylated proteins have been identified in those studies, the acetyltransferases that target these proteins remain largely unknown. Here we characterized MSMEG_5458, one of the GCN5-related N-acetyltransferases (GNAT's) in Mycobacterium smegmatis, and show that it is a protein acetyltransferase (MsPat) that specifically acetylates the ε-amino group of a highly conserved lysine residue in acetyl-CoA synthetase (ACS) with a k(cat)/K(m) of nearly 10(4) M(-1) s(-1). This acetylation results in the inactivation of ACS activity. Lysine acetylation by MsPat is dependent on 3',5'-cyclic adenosine monophosphate (cAMP), an important second messenger, indicating that MsPat is a downstream target of the intracellular cAMP signaling pathway. To the best of our knowledge, this is the first protein acetyltransferase in mycobacteria that both is dependent on cAMP and targets a central metabolic enzyme by a specific post-translational modification. Since cAMP is synthesized by adenylate cyclases (AC's) that sense various environmental signals, we hypothesize that the acetylation and inactivation of ACS is important for mycobacteria to adjust to environmental changes. In addition, we show that Rv1151c, a sirtuin-like deacetylase in Mycobacterium tuberculosis, reactivates acetylated ACS through an NAD(+)-dependent deacetylation. Therefore, Pat and the sirtuin-like deacetylase in mycobacteria constitute a reversible acetylation system that regulates the activity of ACS.     

Small-molecule inhibition of siderophore biosynthesis in Mycobacterium tuberculosis and Yersinia pestis

Mycobacterium tuberculosis and Yersinia pestis, the causative agents of tuberculosis and plague, respectively, are pathogens with serious ongoing impact on global public health and potential use as agents of bioterrorism. Both pathogens have iron acquisition systems based on siderophores, secreted iron-chelating compounds with extremely high Fe3+ affinity. Several lines of evidence suggest that siderophores have a critical role in bacterial iron acquisition inside the human host, where the free iron concentration is well below that required for bacterial growth and virulence. Thus, siderophore biosynthesis is an attractive target in the development of new antibiotics to treat tuberculosis and plague. In particular, such drugs, alone or as part of combination therapies, could provide a valuable new line of defense against intractable multiple-drug-resistant infections. Here, we report the design, synthesis and biological evaluation of a mechanism-based inhibitor of domain salicylation enzymes required for siderophore biosynthesis in M. tuberculosis and Y. pestis. This new antibiotic inhibits siderophore biosynthesis and growth of M. tuberculosis and Y. pestis under iron-limiting conditions.     

Iron accumulation in bronchial epithelial cells is dependent on concurrent sodium transport

Airway epithelial cells prevent damaging effects of extracellular iron by taking up the metal and sequestering it within intracellular ferritin. Epithelial iron transport is associated with transcellular movement of other cations including changes in the expression or activity of Na, K-ATPase and epithelial Na(+) channel (ENaC). Given this relationship between iron and Na(+), we hypothesized that iron uptake by airway epithelial cells requires concurrent Na(+) transport. In preliminary studies, we found that Na(+)-free buffer blocked iron uptake by human airway epithelial cell. Na(+) channels inhibitors, including furosemide, bumetanide, and ethylisopropyl amiloride (EIPA) significantly decreased epithelial cell concentrations of non-heme iron suggesting that Na(+)-dependent iron accumulation involves generalized Na(+) flux into the cells rather than participation of one or more specific Na(+) channels. In addition, efflux of K(+) was detected during iron uptake, as was the influx of phosphate to balance the inward movement of cations. Together, these data demonstrate that intracellular iron accumulation by airway epithelium requires concurrent Na(+)/K(+)exchange.     

Protein splicing of SufB is crucial for the functionality of the Mycobacterium tuberculosis SUF machinery

The SufBCD complex is an essential component of the SUF machinery of [Fe-S] cluster biogenesis in many organisms. We show here that in Mycobacterium tuberculosis the formation of this complex is dependent on the protein splicing of SufB, suggesting that this process is a potential new target for antituberculous drugs.     

Surfactant protein D inhibition of human macrophage uptake of Mycobacterium tuberculosis is independent of bacterial agglutination.

The innate immune system in the lung is essential for controlling infections due to inhaled pathogens. Mycobacterium tuberculosis (M.tb) encounters components of the innate immune system when inhaled into the lung, but the consequences of these interactions are poorly understood. Surfactant protein D (SP-D) binds to and agglutinates M.tb bacilli, and reduces the uptake of the bacteria by human macrophages. In the current studies, we utilized a recombinant SP-D variant (CDM) that lacks the collagen domain to further characterize the interaction of SP-D with M.tb, and determine the effects of agglutination on bacterial uptake by human monocyte-derived macrophages. These studies demonstrate that the binding of SP-D and CDM to M.tb is saturable and inhibited by carbohydrate competition and Ca(2+) chelation, implicating the carbohydrate recognition domain in the interaction. Fluorescence microscopy reveals that dodecameric SP-D leads to agglutination of the bacilli, whereas the trimeric CDM does not, demonstrating that the multivalent nature of SP-D is essential for agglutination of M.tb. However, preincubation of M.tb with increasing concentrations of SP-D or CDM leads to a concentration-dependent reduction in the uptake of the bacteria by macrophages, indicating that agglutination does not play a direct role in this observation. Finally, the reduced uptake of M.tb by SP-D is associated with reduced growth of M.tb in monocyte-derived macrophages. These studies provide direct evidence that the inhibition of phagocytosis of M.tb effected by SP-D occurs independently of the aggregation process.     

Iron limitation and its effect on membrane proteins and siderophore transport inNeurospora crassa

Summary Cells of the fungusNeurospora crassa were grown under iron-deficient and iron-sufficient conditions and their plasma membrane proteins were compared. Three strains were studied:N. crassa 74A (wild type), a siderophore-free mutantN. crassa (arg-5 ota aga) as well as a slime variant ofN. crassa which lacks a cell wall. Plasma membranes were purified, solubilized and analyzed by one-dimensional SDS/polyacrylamide gel electrophoresis yielding approximately 50 distinct protein bands with molecular masses in the range 14–160 kDa. Iron-sufficient and iron-deficient growth resulted in nearly identical plasma membrane protein profiles in all strains. Although minor alterations in the proportion of certain proteins could be detected, significant overproduction of certain membrane proteins during iron limitation could not be observed. Transport of⁵⁵ Fe-labeled siderophores seems to be correlated to the degree of iron limitation. For example, transport rates were enhanced five-fold after 16 h of growth in iron-deficient medium compared to growth in iron-sufficient medium. Extraction and HPLC measurement of siderophores from conidiospores yielded approximately 10^–15 mol/spore, indicating that germination tubes and young cells used for transport measurements are not iron-deficient. It is suggested that the putative transport systems for siderophores in fungal plasma membranes are constitutively expressed and enhanced uptake of siderophores during iron limitation is rather the result of cellular transport regulation mechanisms.     

A cell-penetrating peptide derived from mammalian cell uptake protein of Mycobacterium tuberculosis

A Mycobacterium tuberculosis membrane protein called Mycobacterium cell entry protein (Mce1A) was previously shown to mediate the uptake of nonpathogenic Escherichia coli and latex beads by nonphagocytic mammalian cells. Here we characterize further the in vitro invasive activity of Mce1A using colloidal gold nanoparticles and fluorescent latex microspheres. Mce1A-coated colloidal gold particles induced plasma membrane invagination and entered membrane-bound compartments inside HeLa cells. Few of the protein-coated particles were also found in the cytosol compartment. Cytochalasin D and nocodazole inhibited the uptake by HeLa cells, indicating that rearrangement of both microtubules and microfilaments was necessary for the uptake. The functional domain of Mce1A for invasion was narrowed to a highly basic 22-amino acid sequence termed Inv3. A synthetic Inv3 peptide stimulated uptake of colloidal gold particles as well as latex microspheres by HeLa cells. A chimeric protein composed of Inv3 sequence at the N terminus of beta-galactosidase appeared to stain the nuclear membrane, suggesting that it entered the HeLa cell cytoplasm. These observations suggest that the cell uptake activity of Mce1A is confined to a small peptide domain located in the core region of the protein. Inv3 could be used to ferry any protein in fusion with it into mammalian cells and may serve as a potent nonviral delivery system.     

Synergy between the N-terminal and C-terminal domains of Mycobacterium tuberculosis HupB is essential for high-affinity binding, DNA supercoiling and inhibition of RecA-promoted strand exchange

The occurrence of DNA architectural proteins containing two functional domains derived from two different architectural proteins is an interesting emerging research theme in the field of nucleoid structure and function. Mycobacterium tuberculosis HupB, unlike Escherichia coli HU, is a two-domain protein that, in the N-terminal region, shows broad sequence homology with bacterial HU. The long C-terminal extension, on the other hand, contains seven PAKK/KAAK motifs, which are characteristic of the histone H1/H5 family of proteins. In this article, we describe several aspects of HupB function, in comparison with its truncated derivatives lacking either the C-terminus or N-terminus. We found that HupB binds a variety of DNA repair and replication intermediates with K(d) values in the nanomolar range. By contrast, the N-terminal fragment of M. tuberculosis HupB (HupB(MtbN)) showed diminished DNA-binding activity, with K(d) values in the micromolar range, and the C-terminal domain was completely devoid of DNA-binding activity. Unlike HupB(MtbN) , HupB was able to constrain DNA in negative supercoils and introduce negative superhelical turns into relaxed DNA. Similarly, HupB exerted a robust inhibitory effect on DNA strand exchange promoted by cognate and noncognate RecA proteins, whereas HupB(MtbN), even at a 50-fold molar excess, had no inhibitory effect. Considered together, these results suggest that synergy between the N-terminal and C-terminal domains of HupB is essential for its DNA-binding ability, and to modulate the topological features of DNA, which has implications for processes such as DNA compaction, gene regulation, homologous recombination, and DNA repair.     

HupB,a Nucleoid-Associated Protein of Mycobacterium tuberculosis,Is Modified by Serine/Threonine Protein Kinases In Vivo

HU, a widely conserved bacterial histone-like protein, regulates many genes, including those involved in stress response and virulence. Whereas ample data are available on HU-DNA communication, the knowledge on how HU perceives a signal and transmit it to DNA remains limited. In this study, we identify HupB, the HU homolog of the human pathogen Mycobacterium tuberculosis, as a component of serine/threonine protein kinase (STPK) signaling. HupB is extracted in its native state from the exponentially growing cells of M. tuberculosis H₃₇Ra and is shown to be phosphorylated on both serine and threonine residues. The STPKs capable of modifying HupB are determined in vitro and the residues modified by the STPKs are identified for both in vivo and the in vitro proteins through mass spectrometry. Of the identified phosphosites, Thr⁶⁵ and Thr⁷⁴ in the DNA-embracing β-strand of the N-terminal domain of HupB (N-HupB) are shown to be crucial for its interaction with DNA. In addition, Arg⁵⁵ is also identified as an important residue for N-HupB–DNA interaction. N-HupB is shown to have a diminished interaction with DNA after phosphorylation. Furthermore, hupB is shown to be maximally expressed during the stationary phase in M. tuberculosis H₃₇Ra, while HupB kinases were found to be constitutively expressed (PknE and PknF) or most abundant during the exponential phase (PknB). In conclusion, HupB, a DNA-binding protein, with an ability to modulate chromatin structure is proposed to work in a growth-phase-dependent manner through its phosphorylation carried out by the mycobacterial STPKs.     

A genomic view of sugar transport in Mycobacterium smegmatis and Mycobacterium tuberculosis

We present a comprehensive analysis of carbohydrate uptake systems of the soil bacterium Mycobacterium smegmatis and the human pathogen Mycobacterium tuberculosis. Our results show that M. smegmatis has 28 putative carbohydrate transporters. The majority of sugar transport systems (19/28) in M. smegmatis belong to the ATP-binding cassette (ABC) transporter family. In contrast to previous reports, we identified genes encoding all components of the phosphotransferase system (PTS), including permeases for fructose, glucose, and dihydroxyacetone, in M. smegmatis. It is anticipated that the PTS of M. smegmatis plays an important role in the global control of carbon metabolism similar to those of other bacteria. M. smegmatis further possesses one putative glycerol facilitator of the major intrinsic protein family, four sugar permeases of the major facilitator superfamily, one of which was assigned as a glucose transporter, and one galactose permease of the sodium solute superfamily. Our predictions were validated by gene expression, growth, and sugar transport analyses. Strikingly, we detected only five sugar permeases in the slow-growing species M. tuberculosis, two of which occur in M. smegmatis. Genes for a PTS are missing in M. tuberculosis. Our analysis thus brings the diversity of carbohydrate uptake systems of fast- and a slow-growing mycobacteria to light, which reflects the lifestyles of M. smegmatis and M. tuberculosis in their natural habitats, the soil and the human body, respectively.     

A protein secretion pathway critical for Mycobacterium tuberculosis virulence is conserved and functional in Mycobacterium smegmatis

The Snm protein secretion system is a critical determinant of Mycobacterium tuberculosis virulence. However, genes encoding components of this pathway are conserved among all mycobacteria, including the nonpathogenic saprophyte Mycobacterium smegmatis. We show that the Snm system is operational in M. smegmatis and that secretion of its homologous ESAT-6 and CFP-10 substrates is regulated by growth conditions. Importantly, we show that Snm secretion in M. smegmatis requires genes that are homologous to those required for secretion in M. tuberculosis. Using a gene knockout strategy in M. smegmatis, we have also discovered four new gene products that are essential for Snm secretion, including the serine protease mycosin 1. Despite the evolutionary distance between M. smegmatis and M. tuberculosis, the M. smegmatis Snm system can secrete the M. tuberculosis ESAT-6 and CFP-10 proteins, suggesting that substrate recognition is also conserved between the two species. M. smegmatis, therefore, represents a powerful system to study the multicomponent Snm secretory machine and to understand the role of this conserved system in mycobacterial biology.     

The Mycobacterium bovis homologous protein of the Mycobacterium tuberculosis serine/threonine protein kinase Mbk (PknD) is truncated

We previously identified a 70-kDa serine/threonine protein kinase (MbK or PknD) from Mycobacterium tuberculosis Erdman containing a transmembrane domain and bearing a 270-amino acid N-terminal kinase domain. With the use of a polyclonal serum, Mbk has now been identified by Western blotting in protein extracts from M. tuberculosis and confirmed to be localised in the envelope. An identical mbk gene has been found by sequencing different M. tuberculosis and M. africanum strains. Surprisingly, in two virulent M. bovis strains and four different strains of M. bovis BCG, an additional adenine after position 829 of the open reading frame was found that produces a frame shift resulting in a predicted truncated, presumably free cytoplasmic protein, encoding only the N-terminal 30-kDa Mbk kinase domain. This sequence polymorphism has been confirmed by Western blot analysis of M. bovis BCG protein extracts.     

Mycobacterium tuberculosis virulence is mediated by PtpA dephosphorylation of human vacuolar protein sorting 33B

Entry into host macrophages and evasion of intracellular destruction mechanisms, including phagosome-lysosome fusion, are critical elements of Mycobacterium tuberculosis (Mtb) pathogenesis. To achieve this, the Mtb genome encodes several proteins that modify host signaling pathways. PtpA, a low-molecular weight tyrosine phosphatase, is a secreted Mtb protein of unknown function. The lack of tyrosine kinases in the Mtb genome suggests that PtpA may modulate host tyrosine phosphorylated protein(s). We report that a genetic deletion of ptpA attenuates Mtb growth in human macrophages, and expression of PtpA-neutralizing antibodies simulated this effect. We identify VPS33B, a regulator of membrane fusion, as a PtpA substrate. VPS33B and PtpA colocalize in Mtb-infected human macrophages. PtpA secretion combined with active-phosphorylated VPS33B inhibited phagosome-lysosome fusion, a process arrested in Mtb infections. These results demonstrate that PtpA is essential for Mtb intracellular persistence and identify a key host regulatory pathway that is inactivated by Mtb.     

Nuclear transport of parathyroid hormone (PTH)-related protein is dependent on microtubules

PTH-related protein (PTHrP) was first discovered as a circulating factor secreted by certain cancers and is responsible for the syndrome of humoral hypercalcemia of malignancy induced by various tumors. The similarity of its N terminus to that of PTH enables PTHrP to share the signaling properties of PTH, but the rest of the molecule possesses distinct functions, including a role in the nucleus/nucleolus in reducing apoptosis and enhancing cell proliferation. PTHrP nuclear import is mediated by importin beta1. In this study we use the technique of fluorescence recovery after photobleaching to demonstrate the ability of PTHrP to shuttle between cytoplasm and nucleus and to visualize directly the transport of PTHrP into the nucleus in living cells. Endogenous and transfected PTHrP was demonstrated to colocalize with microtubule structures in situ using various high-resolution microscopic approaches, as well as in in vitro binding studies, where importin beta1, but not importin alpha, enhanced the microtubular association of PTHrP with microtubules. Significantly, the dependence of PTHrP nuclear import on microtubules was shown by the inhibitory effect of pretreatment with the microtubule-disrupting agent nocodazole on nuclear-cytoplasmic flux. These results indicate that PTHrP nuclear/nucleolar import is dependent on microtubule integrity and are consistent with a direct role for the cytoskeleton in protein transport to the nucleus.