The cell envelope glycoconjugates of Mycobacterium tuberculosis

Abstract

Tuberculosis (TB) remains the second most common cause of death due to a single infectious agent. The cell envelope of Mycobacterium tuberculosis (Mtb), the causative agent of the disease in humans, is a source of unique glycoconjugates and the most distinctive feature of the biology of this organism. It is the basis of much of Mtb pathogenesis and one of the major causes of its intrinsic resistance to chemotherapeutic agents. At the same time, the unique structures of Mtb cell envelope glycoconjugates, their antigenicity and essentiality for mycobacterial growth provide opportunities for drug, vaccine, diagnostic and biomarker development, as clearly illustrated by recent advances in all of these translational aspects. This review focuses on our current understanding of the structure and biogenesis of Mtb glycoconjugates with particular emphasis on one of the most intriguing and least understood aspect of the physiology of mycobacteria: the translocation of these complex macromolecules across the different layers of the cell envelope. It further reviews the rather impressive progress made in the last 10 years in the discovery and development of novel inhibitors targeting their biogenesis.     

A Trichoderma atroviride stress‐activated MAPK pathway integrates stress and light signals

Cells possess stress‐activated protein kinase (SAPK) signalling pathways, which are activated practically in response to any cellular insult, regulating responses for survival and adaptation to harmful environmental changes. To understand the function of SAPK pathways in T. atroviride, mutants lacking the MAPKK Pbs2 and the MAPK Tmk3 were analysed under several cellular stresses, and in their response to light. All mutants were highly sensitive to cellular insults such as osmotic and oxidative stress, cell wall damage, high temperature, cadmium, and UV irradiation. Under oxidative stress, the Tmk3 pathway showed specific roles during development, which in conidia are essential for tolerance to oxidant agents and appear to play a minor role in mycelia. The function of this pathway was more evident in Δpbs2 and Δtmk3 mutant strains when combining oxidative stress or cell wall damage with light. Light stimulates tolerance to osmotic stress through Tmk3 independently of the photoreceptor Blr1. Strikingly, photoconidiation and expression of blue light regulated genes was severally affected in Δtmk3 and Δpbs2 strains, indicating that this pathway regulates light responses. Furthermore, Tmk3 was rapidly phosphorylated upon light exposure. Thus, our data indicate that Tmk3 signalling cooperates with the Blr photoreceptor complex in the activation of gene expression.     

Hfq reduces envelope stress by controlling expression of envelope‐localized proteins and protein complexes in enteropathogenic Escherichia coli

Gram‐negative bacteria possess several envelope stress responses that detect and respond to damage to this critical cellular compartment. The σ^E envelope stress response senses the misfolding of outer membrane proteins (OMPs), while the Cpx two‐component system is believed to detect the misfolding of periplasmic and inner membrane proteins. Recent studies in several Gram‐negative organisms found that deletion of hfq, encoding a small RNA chaperone protein, activates the σ^E envelope stress response. In this study, we assessed the effects of deleting hfq upon activity of the σ^E and Cpx responses in non‐pathogenic and enteropathogenic (EPEC) strains of Escherichia coli. We found that the σ^E response was activated in Δhfq mutants of all E. coli strains tested, resulting from the misregulation of OMPs. The Cpx response was activated by loss of hfq in EPEC, but not in E. coli K‐12. Cpx pathway activation resulted in part from overexpression of the bundle‐forming pilus (BFP) in EPEC Δhfq. We found that Hfq repressed expression of the BFP via PerA, a master regulator of virulence in EPEC. This study shows that Hfq has a more extensive role in regulating the expression of envelope proteins and horizontally acquired virulence genes in E. coli than previously recognized.     

EspC forms a filamentous structure in the cell envelope of Mycobacterium tuberculosis and impacts ESX‐1 secretion

Pathogenicity of Mycobacterium tuberculosis (M. tb) is mediated by the ESX‐1 secretion system, which exports EsxA and EsxB, the major virulence factors that are co‐secreted with EspA and EspC. Functional information about ESX‐1 components is scarce. Here, it was shown that EspC associates with EspA in the cytoplasm and membrane, then polymerizes during secretion from M. tb. EspC was localized by immuno‐gold electron microscopy in whole cells or cryosections as a surface‐exposed filamentous structure that seems to span the cell envelope. Consistent with these findings, purified EspC homodimerizes via disulphide bond formation, multimerizes and self‐assembles into long filaments in vitro. The C‐terminal domain is required for multimerization as truncation and selected point mutations therein impact EspC filament formation, thus reducing secretion of EsxA and causing attenuation of M. tb. The data are consistent with EspC serving either as a modulator of ESX‐1 function or as a component of the secretion apparatus.     

The functions of OmpATb,a pore-forming protein of Mycobacterium tuberculosis

The functions of OmpATb, the product of the ompATb gene of Mycobacterium tuberculosis and a putative porin, were investigated by studying a mutant with a targeted deletion of the gene, and by observing expression of the gene in wild-type M. tuberculosis H37Rv by real-time polymerase chain reaction (PCR) and immunoblotting. The loss of ompATb had no effect on growth under normal conditions, but caused a major reduction in ability to grow at reduced pH. The gene was substantially upregulated in wild-type bacteria exposed to these conditions. The mutant was impaired in its ability to grow in macrophages and in normal mice, although it was as virulent as the wild type in mice that lack T cells. Deletion of the ompATb gene reduced permeability to several small water-soluble substances. This was particularly evident at pH 5.5; at this pH, uptake of serine was minimal, suggesting that, at this pH, OmpATb might be the only functioning porin. These data indicate that OmpATb has two functions: as a pore-forming protein with properties of a porin, and in enabling M. tuberculosis to respond to reduced environmental pH. It is not known whether this second function is related to the porin-like activity at low pH or involves a completely separate role for OmpATB. The involvement with pH is likely to contribute to the ability of M. tuberculosis to overcome host defence mechanisms and grow in a mammalian host.     

The AhpC and AhpD antioxidant defense system of Mycobacterium tuberculosis

The peroxiredoxin AhpC from Mycobacterium tuberculosis has been expressed, purified, and characterized. It differs from other well characterized AhpC proteins in that it has three rather than one or two cysteine residues. Mutagenesis studies show that all three cysteine residues are important for catalytic activity. Analysis of the M. tuberculosis genome identified a second protein, AhpD, which has no sequence identity with AhpC but is under the control of the same promoter. This protein has also been cloned, expressed, purified, and characterized. AhpD, which has only been identified in the genomes of mycobacteria and Streptomyces viridosporus, is shown here to also be an alkylhydroperoxidase. The endogenous electron donor for catalytic turnover of the two proteins is not known, but both can be turned over with AhpF from Salmonella typhimurium or, particularly in the case of AhpC, with dithiothreitol. AhpC and AhpD reduce alkylhydroperoxides more effectively than H(2)O(2) but do not appear to interact with each other. These two proteins appear to be critical elements of the antioxidant defense system of M. tuberculosis and may be suitable targets for the development of novel anti-tuberculosis strategies.     

EspI regulates the ESX‐1 secretion system in response to ATP levels in Mycobacterium tuberculosis

The function of EspI, a 70 kDa protein in Mycobacterium tuberculosis, has remained unclear. Although EspI is encoded by a gene within the esx‐1 locus, in this study we clarify previous conflicting results and show that EspI is not essential for ESX‐1‐mediated secretion or virulence in M. tuberculosis. We also provide evidence that reduction of cellular ATP levels in wild‐type M. tuberculosis using the drug bedaquiline completely blocks ESX‐1‐mediated secretion. Remarkably, M. tuberculosis lacking EspI fails to exhibit this phenotype. Furthermore, mutagenesis of a highly conserved ATP‐binding motif in EspI renders M. tuberculosis incapable of shutting down ESX‐1‐mediated secretion during ATP depletion. Collectively these results show that M. tuberculosis EspI negatively regulates the ESX‐1 secretion system in response to low cellular ATP levels and this function requires the ATP‐binding motif. In light of our results the potential significance of EspI in ESX‐1 function during latent tuberculosis infection and reactivation is also discussed.     

结核分枝杆菌小RNA及其作用的研究进展

细菌小RNA是一类长度在50~500个核苷酸之间的不具有编码蛋白质功能,但具有转录后调控作用的RNA,在细菌中参与调控细菌多种生理和病理活动,如调节细菌代谢和毒力作用等过程。近年来,在结核分枝杆菌已经鉴定出近200种小RNA,并证明这些小RNA参与结核分枝杆菌的生理和病理过程。本文对结核分枝杆菌小RNA在细菌生长繁殖、毒力因子调控、细菌耐药和巨噬细胞内应激环境的适应等方面的作用进行综述。     

Lipoproteins of Mycobacterium tuberculosis: an abundant and functionally diverse class of cell envelope components

Mycobacterium tuberculosis remains the predominant bacterial scourge of mankind. Understanding of its biology and pathogenicity has been greatly advanced by the determination of whole genome sequences for this organism. Bacterial lipoproteins are a functionally diverse class of membrane-anchored proteins. The signal peptides of these proteins direct their export and post-translational lipid modification. These signal peptides are amenable to bioinformatic analysis, allowing the lipoproteins encoded in whole genomes to be catalogued. This review applies bioinformatic methods to the identification and functional characterisation of the lipoproteins encoded in the M. tuberculosis genomes. Ninety nine putative lipoproteins were identified and so this family of proteins represents ca. 2.5% of the M. tuberculosis predicted proteome. Thus, lipoproteins represent an important class of cell envelope proteins that may contribute to the virulence of this major pathogen.     

The Mycobacterium tuberculosis complex has a pathway for the biosynthesis of 4‐formamido‐4,6‐dideoxy‐d‐glucose

Recent studies have demonstrated that the O‐antigens of some pathogenic bacteria such as Brucella abortus, Francisella tularensis, and Campylobacter jejuni contain quite unusual N‐formylated sugars (3‐formamido‐3,6‐dideoxy‐d ‐glucose or 4‐formamido‐4,6‐dideoxy‐d ‐glucose). Typically, four enzymes are required for the formation of such sugars: a thymidylyltransferase, a 4,6‐dehydratase, a pyridoxal 5'‐phosphate or PLP‐dependent aminotransferase, and an N‐formyltransferase. To date, there have been no published reports of N‐formylated sugars associated with Mycobacterium tuberculosis. A recent investigation from our laboratories, however, has demonstrated that one gene product from M. tuberculosis, Rv3404c, functions as a sugar N‐formyltransferase. Given that M. tuberculosis produces l ‐rhamnose, both a thymidylyltransferase (Rv0334) and a 4,6‐dehydratase (Rv3464) required for its formation have been identified. Thus, there is one remaining enzyme needed for the production of an N‐formylated sugar in M. tuberculosis, namely a PLP‐dependent aminotransferase. Here we demonstrate that the M. tuberculosis rv3402c gene encodes such an enzyme. Our data prove that M. tuberculosis contains all of the enzymatic activities required for the formation of dTDP‐4‐formamido‐4,6‐dideoxy‐d ‐glucose. Indeed, the rv3402c gene product likely contributes to virulence or persistence during infection, though its temporal expression and location remain to be determined.     

Crystal structure of the N‐terminal domain of EccA1 ATPase from the ESX‐1 secretion system of Mycobacterium tuberculosis

EccA₁ is an important component of the type VII secretion system (T7SS) that is responsible for transport of virulence factors in pathogenic mycobacteria. EccA₁ has an N‐terminal domain of unknown function and a C‐terminal AAA+ (ATPases associated with various cellular activities) domain. Here we report the crystal structure of the N‐terminal domain of EccA₁ from Mycobacterium tuberculosis, which shows an arrangement of six tetratricopeptide repeats that may mediate interactions of EccA₁ with secreted substrates. Furthermore, the size and shape of the N‐terminal domain suggest its orientation in the context of a hexamer model of full‐length EccA₁. Proteins 2014; 82:159–163. © 2013 Wiley Periodicals, Inc.     

The use of stress‐70 proteins in physiology: a re‐appraisal

There are few factors more important to the mechanisms of evolution than stress. The stress response has formed as a result of natural selection, improving the capacity of organisms to withstand situations that require action. The ubiquity of the cellular stress response suggests that effective mechanisms to counteract stress emerged early in the history of life, and their commonality proves how vital such mechanisms are to operative evolution. The cellular stress response (CSR) has been identified as a characteristic of cells in all three domains of life and consists of a core 44 proteins that are structurally highly conserved and that have been termed the ‘minimal stress proteome’ (MSP). Within the MSP, the most intensely researched proteins are a family of heat‐shock proteins known as HSP70. Superficially, correlations between the induction of stress and HSP70 differential expression support the use of HSP70 expression as a nonspecific biomarker of stress. However, we argue that too often authors have failed to question exactly what HSP70 differential expression signifies. Herein, we argue that HSP70 up‐regulation in response to stressors has been shown to be far more complex than the commonly accepted quasi‐linear relationship. In addition, in many instances, the uncertain identity and function of heat‐shock proteins and heat‐shock cognates has led to difficulties in interpretation of reports of inducible heat‐shock proteins and constitutive heat‐shock cognates. We caution against the broad application of HSP70 as a biomarker of stress in isolation and conclude that the application of HSP70 as a meaningful index of stress requires a higher degree of validation than the majority of research currently undertakes.     

The HtrA-like serine protease PepD interacts with and modulates the Mycobacterium tuberculosis 35-kDa antigen outer envelope protein

Mycobacterium tuberculosis remains a significant global health concern largely due to its ability to persist for extended periods within the granuloma of the host. While residing within the granuloma, the tubercle bacilli are likely to be exposed to stress that can result in formation of aberrant proteins with altered structures. Bacteria encode stress responsive determinants such as proteases and chaperones to deal with misfolded or unfolded proteins. pepD encodes an HtrA-like serine protease and is thought to process proteins altered following exposure of M. tuberculosis to extra-cytoplasmic stress. PepD functions both as a protease and chaperone in vitro, and is required for aspects of M. tuberculosis virulence in vivo. pepD is directly regulated by the stress-responsive two-component signal transduction system MprAB and indirectly by extracytoplasmic function (ECF) sigma factor SigE. Loss of PepD also impacts expression of other stress-responsive determinants in M. tuberculosis. To further understand the role of PepD in stress adaptation by M. tuberculosis, a proteomics approach was taken to identify binding proteins and possible substrates of this protein. Using subcellular fractionation, the cellular localization of wild-type and PepD variants was determined. Purified fractions as well as whole cell lysates from Mycobacterium smegmatis or M. tuberculosis strains expressing a catalytically compromised PepD variant were immunoprecipitated for PepD and subjected to LC-MS/MS analyses. Using this strategy, the 35-kDa antigen encoding a homolog of the PspA phage shock protein was identified as a predominant binding partner and substrate of PepD. We postulate that proteolytic cleavage of the 35-kDa antigen by PepD helps maintain cell wall homeostasis in Mycobacterium and regulates specific stress response pathways during periods of extracytoplasmic stress.     

The soybean root‐specific protein kinase GmWNK1 regulates stress‐responsive ABA signaling on the root system architecture

In humans, members of the WNK protein kinase family are osmosensitive regulators of cell volume homeostasis and epithelial ion transport, and mutation of these proteins causes a rare inherited form of hypertension due to increased renal NaCl re‐absorption. A related class of kinases was recently discovered in plants, but their functions are largely unknown. We have identified a root‐specific WNK kinase homolog, GmWNK1, in soybean (Glycine max). GmWNK1 expression was detected in the root, specifically in root cells associated with lateral root formation, and was down‐regulated by abscisic acid (ABA), as well as by mannitol, sucrose, polyethylene glycol and NaCl. In vitro and in vivo experiments showed that GmWNK1 interacts with another soybean protein, GmCYP707A1, which is a key ABA 8′‐hydroxylase that functions in ABA catabolism. Furthermore, 35S‐GmWNK1 transgenic soybean plants had reduced lateral root number and length compared with wild‐type, suggesting a role of GmWNK1 in the regulation of root system architecture. We propose that GmWNK1 functions to fine‐tune ABA‐dependent ABA homeostasis, thereby mediating the regulation of the root system architecture by ABA and osmotic signals. The study has revealed a new function of a plant WNK1 gene from the important staple crop soybean, and has identified a new component of a regulatory pathway that is involved not only in ABA signaling, but also in the repression of lateral root formation by an ABA‐dependent mechanism distinct from known ABA signaling pathways.     

ESX‐1‐induced apoptosis is involved in cell‐to‐cell spread of Mycobacterium tuberculosis

Apoptosis modulation is a procedure amply utilized by intracellular pathogens to favour the outcome of the infection. Nevertheless, the role of apoptosis during infection with Mycobacterium tuberculosis, the causative agent of human tuberculosis, is subject of an intense debate and still remains unclear. In this work, we describe that apoptosis induction in host cells is clearly restricted to virulent M. tuberculosis strains, and is associated with the capacity of the mycobacteria to secrete the 6 kDa early secreted antigenic target ESAT‐6 bothunder in vitro and in vivo conditions. Remarkably, only apoptosis‐inducing strains are able to propagate infection into new cells, suggesting that apoptosis is used by M. tuberculosis as a colonization mechanism. Finally, we demonstrate that in vitro modulation of apoptosis affects mycobacterial cell‐to‐cell spread capacity, establishing an unambiguous relationship between apoptosis and propagation of M. tuberculosis. Our data further indicate that BCG and MTBVAC vaccines are inefficient in inducing apoptosis and colonizing new cells, correlating with the strong attenuation profile of these strains previously observed in vitro and in vivo.     

Meropenem inhibits D,D‐carboxypeptidase activity in Mycobacterium tuberculosis

Carbapenems such as meropenem are being investigated for their potential therapeutic utility against highly drug‐resistant tuberculosis. These β‐lactams target the transpeptidases that introduce interpeptide cross‐links into bacterial peptidoglycan thereby controlling rigidity of the bacterial envelope. Treatment of Mycobacterium tuberculosis (Mtb) with the β‐lactamase inhibitor clavulanate together with meropenem resulted in rapid, polar, cell lysis releasing cytoplasmic contents. In Mtb it has been previously demonstrated that 3‐3 cross‐linkages [involving two diaminopimelate (DAP) molecules] predominate over 4‐3 cross‐linkages (involving one DAP and one D‐alanine) in stationary‐phase cells. We purified and analysed peptidoglycan from Mtb and found that 3‐3 cross‐linkages predominate throughout all growth phases and the ratio of 4‐3/3‐3 linkages does not vary significantly under any growth condition. Meropenem treatment was accompanied by a dramatic accumulation of unlinked pentapeptide stems with no change in the tetrapeptide pools, suggesting that meropenem inhibits both a D,D‐carboxypeptidase and an L,D‐transpeptidase. We purified a candidate D,D‐carboxypeptidase DacB2 and showed that meropenem indeed directly inhibits this enzyme by forming a stable adduct at the enzyme active site. These results suggest that the rapid lysis of meropenem‐treated cells is the result of synergistically inhibiting the transpeptidases that introduce 3,3‐cross‐links while simultaneously limiting the pool of available substrates available for cross‐linking.