Mycobacterial lipolytic enzymes: A gold mine for tuberculosis research

Tuberculosis (TB) is one of the deadliest infectious diseases worldwide with a strong impact in developing countries. Mycobacterium tuberculosis, the etiological agent of TB, has a high capacity to evade the host immune system and establish a chronic, asymptomatic and latent infection. In a latent TB infection, persistent bacilli are present in a non-replicating dormant state within host granulomas. During reactivation, bacilli start replicating again leading to an active TB infection that can be highly contagious. Mycobacterial lipids and lipolytic enzymes are thought to play important physiological roles during dormancy and reactivation. The role of lipolytic enzymes in the physiology of M. tuberculosis and physiopathology of the disease will be discussed in this review, with an emphasis on the secreted or cell wall-associated, surface exposed lipolytic enzymes characterized to date. Studies on the localization, enzymatic activity and immunological properties of these enzymes highlighted their possible usefulness as new diagnostic markers in the fight against TB.     

The Mycobacterium tuberculosis shikimate pathway genes: Evolutionary relationship between biosynthetic and catabolic 3-dehydroquinases

Summary The Mycobacterium tuberculosis shikimate pathway genes designated aroB and aroQ encoding 3-dehydroquinate synthase and 3-dehydroquinase, respectively were isolated by molecular cloning and their nucleotide sequences determined. The deduced dehydroquinate synthase amino acid sequence from M. tuberculosis showed high similarity to those of equivalent enzymes from prokaryotes and filamentous fungi. Surprisingly, the deduced M. tuberculosis 3-dehydroquinase amino acid sequence showed no similarity to other characterised prokaryotic biosynthetic 3-dehydroquinases (bDHQases). A high degree of similarity was observed, however, to the fungal catabolic 3-dehydroquinases (cDHQases) which are active in the quinic acid utilisation pathway and are isozymes of the fungal bDHQases. This finding indicates a common ancestral origin for genes encoding the catabolic dehydroquinases of fungi and the biosynthetic dehydroquinases present in some prokaryotes. Deletion of genes encoding shikimate pathway enzymes represents a possible approach to generation of rationally attenuated strains of M. tuberculosis for use as live vaccines.     

Perspectives on mycobacterial vacuole‐to‐cytosol translocation: the importance of cytosolic access

Mycobacterium tuberculosis, the infectious agent of human tuberculosis is a master player in circumventing the defense mechanisms of the host immune system. The host‐pathogen interaction in the case of an infection with M. tuberculosis is highly complex, involving dedicated mycobacterial virulence factors as well as the action of the innate and adapted immune systems, which determine the outcome of infection. Macrophages play a key role in this process through internalizing the bacterium in a phagosomal vacuole. While this action has normally the function of eliminating invading bacteria, M. tuberculosis employs efficient strategies to prevent its extermination. The question on how‐and‐where the bacterium succeeds in doing so has interested generations of scientists and still remains a fascinating and important research subject focused on mycobacterial lipids, secretion systems and other contributing factors. This topic is also central to the longstanding and partially controversial discussion on mycobacterial phagosomal rupture and vacuole‐to‐cytosol translocation, to be reviewed here in more detail.     

Involvement of the autophagy pathway in trafficking of Mycobacterium tuberculosis bacilli through cultured human type II epithelial cells

Interactions between Mycobacterium tuberculosis bacilli and alveolar macrophages have been extensively characterized, while similar analyses in epithelial cells have not been performed. In this study, we microscopically examined endosomal trafficking of M. tuberculosis strain Erdman in A549 cells, a human type II pneumocyte cell line. Immuno‐electron microscopic (IEM) analyses indicate that M. tuberculosis bacilli are internalized to a compartment labelled first with Rab5 and then with Rab7 small GTPase proteins. This suggests that, unlike macrophages, M. tuberculosis bacilli traffic to late endosomes in epithelial cells. However, fusion of lysosomes with the bacteria‐containing compartment appears to be inhibited, as illustrated by IEM studies employing LAMP‐2 and cathepsin‐L antibodies. Examination by transmission electron microscopy and IEM revealed M. tuberculosis‐containing compartments surrounded by double membranes and labelled with antibodies against the autophagy marker Lc3, providing evidence for involvement and intersection of the autophagy and endosomal pathways. Interestingly, inhibition of the autophagy pathway using 3‐methyladenine improved host cell viability and decreased numbers of viable intracellular bacteria recovered after 72 h post infection. Collectively, these datasuggest that trafficking patterns for M. tuberculosis bacilli in alveolar epithelial cells differ from macrophages, and that autophagy is involved this process.     

嗜麦芽窄食单胞菌OUC_Est10全基因组测序及脂类水解酶多样性分析

[目的]本研究的目的是研究嗜麦芽窄食单胞菌OUC_Est10中脂类水解酶的多样性。[方法]使用离子交换层析、全基因组测序和异源表达三种方法研究嗜麦芽窄食单胞菌OUC_Est10中脂类水解酶的多样性。[结果]离子交换层析结果显示嗜麦芽窄食单胞菌OUC_Est10可以分泌多种脂类水解酶。通过全基因组测序,我们给出了该菌的全基因组序列,该基因组大小为4668743 bp,GC含量为66.25%。通过详细的基因组序列分析,我们从该基因组中找到33个可能具有脂类水解酶活性的假定基因。通过异源表达OUC_Est10中的5个假定脂类水解酶基因,来研究其催化特性的多样性,结果显示这些脂类水解酶具有不同的催化特性。[结论]我们证明了嗜麦芽窄食单胞菌OUC_Est10拥有多样的脂类水解酶,这暗示了它在不同领域中的应用潜力。     

<Emphasis Type="Italic">Nramp1</Emphasis> deletion does not confer susceptibility to<Emphasis Type="Italic"> Rhodococcus equi</Emphasis> infection in mice

Rhodococcus equi is an intracellular bacterium that causes pneumonia in immunocompromised people and foals. The Nramp1 gene influences susceptibility to a variety of intracellular bacteria (including mycobacterial species), but not to Mycobacterium tuberculosis. In this study, we demonstrate that mice functionally deleted of the Nramp1 gene were not more susceptible to infection with virulent R. equi (ATCC 33701) than wild-type mice. Susceptibility of mice to infection with the intracellular bacterium R. equi is more similar to that of M. tuberculosis than to other intracellular bacteria, including other mycobacteria.     

Biosynthesis of mycobacterial lipids by polyketide synthases and beyond

Abstract

Over a decade ago, the analysis of the complete sequence of the genome of the human pathogen Mycobacterium tuberculosis revealed an unexpectedly high number of open reading frames encoding proteins with homology to polyketide synthases (PKSs). PKSs form a large family of fascinating multifunctional enzymes best known for their involvement in the biosynthesis of hundreds of polyketide natural products with diverse biological activities. The surprising polyketide biosynthesis capacity of M. tuberculosis has been investigated since its initial inference from genome analysis. This investigation has been based on the genes found in M. tuberculosis or their orthologs found in other Mycobacterium species. Today, the majority of the PKS-encoding genes of M. tuberculosis have been linked to specific biosynthetic pathways required for the production of unique lipids or glycolipid conjugates that are critical for virulence and/or components of the extraordinarily complex mycobacterial cell envelope. This review provides a synopsis of the most relevant studies in the field and an overview of our current understanding of the involvement of PKSs and several other polyketide production pathway-associated proteins in critical biosynthetic pathways of M. tuberculosis and other mycobacteria. In addition, the most relevant studies on PKS-containing biosynthetic pathways leading to production of metabolites from mycobacteria other than M. tuberculosis are reviewed.     

Negative regulation by Ser/Thr phosphorylation of HadAB and HadBC dehydratases from Mycobacterium tuberculosis type II fatty acid synthase system

The type II fatty acid synthase system of mycobacteria is involved in the biosynthesis of major and essential lipids, mycolic acids, key-factors of Mycobacterium tuberculosis pathogenicity. One reason of the remarkable survival ability of M. tuberculosis in infected hosts is partly related to the presence of cell wall-associated mycolic acids. Despite their importance, the mechanisms that modulate synthesis of these lipids in response to environmental changes are unknown. We demonstrate here that HadAB and HadBC dehydratases of this system are phosphorylated by Ser/Thr protein kinases, which negatively affects their enzymatic activity. The phosphorylation of HadAB/BC is growth phase-dependent, suggesting that it represents a mechanism by which mycobacteria might tightly control mycolic acid biosynthesis under non-replicating condition.     

Mammalian heterotrimeric G-protein-like proteins in mycobacteria: implications for cell signalling and survival in eukaryotic host cells

Mammalian heterotrimeric GTP-binding proteins (G proteins) are involved in transmembrane signalling that couples a number of receptors to effectors mediating various physiological processes in mammalian cells. We demonstrate that bacterial proteins such as a Ras-like protein from Pseudomonas aeruginosa or a 65 kDa protein from Mycobacterium smegmatis can form complexes with human or yeast nucleoside diphosphate kinase (Ndk) to modulate their nucleoside triphosphate synthesizing specificity to GTP or UTP. In addition, we demonstrate that bacteria such as M. smegmatis or Mycobacterium tuberculosis harbour proteins that cross react with antibodies against the α-, β- or the γ-subunits of heterotrimeric G proteins. Such antibodies also alter the GTP synthesizing ability of specific membrane fractions isolated from glycerol gradients of such cells, suggesting that a membrane-associated Ndk–G-protein homologue complex is responsible for part of GTP synthesis in these bacteria. Indeed, purified Ndk from human erythrocytes and M. tuberculosis showed extensive complex formation with the purified mammalian α and β G-protein subunits and allowed specific GTP synthesis, suggesting that such complexes may participate in transmembrane signalling in the eukaryotic host. We have purified the α-, β- and γ-subunit homologues from M. tuberculosis and we present their internal amino acid sequences as well as their putative homologies with mammalian subunits and the localization of their genes on the M. tuberculosis genome. Using oligonucleotide probes from the conserved regions of the α- and γ-subunit of M. tuberculosis G-protein homologue, we demonstrate hybridization of these probes with the genomic digest of M. tuberculosis H37Rv but not with that of M. smegmatis, suggesting that M. smegmatis might lack the genes present in M. tuberculosis H37Rv. Interestingly, the avirulent strain H37Ra showed weak hybridization with these two probes, suggesting that these genes might have been deleted in the avirulent strain or are present in limited copy numbers as opposed to those in the virulent strain H37Rv.     

Use of visual loop-mediated isotheral amplification of rimM sequence for rapid detection of Mycobacterium tuberculosis and Mycobacterium bovis

Mycobacterium tuberculosis and Mycobacterium bovis are pathogenic bacterial species in the genus Mycobacterium and the causative agents of most cases of tuberculosis (TB). Detection of M. tuberculosis and M. bovis using conventional culture- and biochemical-based assays is time-consuming and laborious. Therefore, a simple and sensitive method for rapid detection has been anxiously awaited. In the present study, a visual loop-mediated isothermal amplification (LAMP) assay was designed from the rimM (encoding 16S rRNA-processing protein) gene sequence and used to rapidly detect M. tuberculosis and M. bovis from clinical samples in South China. The visual LAMP reaction was performed by adding calcein and manganous ion, allowing the results to be read by simple visual observation of color change in a closed-tube system, and which takes less than 1 h at 65 °C. The assay correctly identified 84 M. tuberculosis isolates, 3 M. bovis strains and 1 M. bovis BCG samples, but did not detect 51 non-tuberculous mycobacteria (NTM) isolates and 8 other bacterial species. Sensitivity of this assay for detection of genomic DNA was 1 pg. Specific amplification was confirmed by the ladder-like pattern of gel electrophoresis and restriction enzyme HhaI digestion. The assay successfully detected M. tuberculosis and M. bovis not only in pure bacterial culture but also in clinical samples of sputum, pleural fluid and blood. The speed, specificity, sensitivity of the rimM LAMP, the lack of a need for expensive equipment, and the visual readout show great potential for clinical detection of M. tuberculosis and M. bovis.     

Structures of citrate synthase and malate dehydrogenase of Mycobacterium tuberculosis

The tricarboxylic acid (TCA) cycle is a central metabolic pathway of all aerobic organisms and is responsible for the synthesis of many important precursors and molecules. TCA cycle plays a key role in the metabolism of Mycobacterium tuberculosis and is involved in the adaptation process of the bacteria to the host immune response. We present here the first crystal structures of M. tuberculosis malate dehydrogenase and citrate synthase, two consecutive enzymes of the TCA, at 2.6 Å and 1.5 Å resolution, respectively. General analogies and local differences with the previously reported homologous protein structures are described. Proteins 2015; 83:389–394. © 2014 Wiley Periodicals, Inc.     

Detection of streptomycin resistance in Mycobacterium tuberculosis clinical isolates from China as determined by denaturing HPLC analysis and DNA sequencing

China is regarded by the World Health Organization as a major hot-spot region for Mycobacterium tuberculosis infection. Streptomycin has been deployed in China for over 50 years and is still widely used for tuberculosis treatment. We have developed a denaturing HPLC (DHPLC) method for detecting various gene mutations conferring drug resistance in M. tuberculosis. The present study focused on rpsL and rrs mutation analysis. Two hundred and fifteen M. tuberculosis clinical isolates (115 proved to be streptomycin-resistant and 100 susceptible by a routine proportional method) from China were tested to determine the streptomycin minimal inhibitory concentration (MIC), and subjected to DHPLC and concurrent DNA sequencing to determine rpsL and rrs mutations. The results showed that 85.2% (98/115) of streptomycin-resistant isolates harbored rpsL or rrs mutation, while rpsL mutation (76.5%, 88/115) dominated. MIC of 98 mutated isolates revealed no close correlation between mutation types and levels of streptomycin resistance. No mutation was found in any of the susceptible isolates. The DHPLC results were completely consistent with those of sequencing. The DHPLC method devised in this study can be regarded as a useful and powerful tool for detection of streptomycin resistance. This is the first report to describe DHPLC analysis of mutations in the rpsL and rrs genes of M. tuberculosis in a large number of clinical isolates.     

Inhibition of Pseudomonas aeruginosa and Mycobacterium tuberculosis disulfide bond forming enzymes

In bacteria, disulfide bonds confer stability on many proteins exported to the cell envelope or beyond, including bacterial virulence factors. Thus, proteins involved in disulfide bond formation represent good targets for the development of inhibitors that can act as antibiotics or anti‐virulence agents, resulting in the simultaneous inactivation of several types of virulence factors. Here, we present evidence that the disulfide bond forming enzymes, DsbB and VKOR, are required for Pseudomonas aeruginosa pathogenicity and Mycobacterium tuberculosis survival respectively. We also report the results of a HTS of 216,767 compounds tested against P. aeruginosa DsbB1 and M. tuberculosis VKOR using Escherichia coli cells. Since both P. aeruginosa DsbB1 and M. tuberculosis VKOR complement an E. coli dsbB knockout, we screened simultaneously for inhibitors of each complemented E. coli strain expressing a disulfide‐bond sensitive β ‐galactosidase reported previously. The properties of several inhibitors obtained from these screens suggest they are a starting point for chemical modifications with potential for future antibacterial development.     

黄翅大白蚁后肠优势菌大白蚁营发酵菌的全基因组序列分析

【目的】营发酵单胞菌属Dysgonomonas是黄翅大白蚁后肠的第二优势微生物。前期研究中,我们从黄翅大白蚁后肠分离出一种命名为大白蚁营发酵菌的新菌。为深入了解大白蚁营发酵菌在宿主白蚁体内发挥的作用和功能,有必要解析大白蚁营发酵菌的基因组序列信息。【方法】使用Illumina Miseq测序平台获取该菌的全基因组序列,将其全基因组序列经过注释的基因蛋白质序列提交COG和KEGG数据库进行BLASTp比对分析,确定该菌潜在的重要酶类和代谢途径,并对个别纤维素酶活进行检测。【结果】大白蚁营发酵菌整个基因组大小为4655756 bp,GC含量为38.54%,DDBJ数据库登录号为BBXL01000001–BBXL01000078。生物信息学分析结果表明菌株大白蚁营发酵菌具有多个木质纤维素降解酶基因,且具备完整的木质纤维素降解和乙酸、乳酸生成通路。此外发现该菌株中存在与氮源代谢和抵御病原体相关的基因。【结论】本研究首次解析大白蚁营发酵菌的全基因组序列,了解其基因组基本特征,初步探讨了该菌降解木质纤维素的过程,为细菌协助宿主白蚁降解木质纤维素提供了理论基础,同时为该菌可能参与宿主白蚁氮源代谢和抵御病原体入侵提供了依据。     

Activation of β- and γ-carbonic anhydrases from pathogenic bacteria with tripeptides

Six tripeptides incorporating acidic amino acid residues were prepared for investigation as activators of β- and γ-carbonic anhydrases (CAs, EC 4.2.1.1) from the pathogenic bacteria Vibrio cholerae, Mycobacterium tuberculosis, and Burkholderia pseudomallei. The primary amino acid residues that are involved in the catalytic mechanisms of these CA classes are poorly understood, although glutamic acid residues near the active site appear to be involved. The tripeptides that contain Glu or Asp residues can effectively activate VchCAβ and VchCAγ (enzymes from V. cholerae), Rv3273 CA (mtCA3, a β-CA from M. tuberculosis) and BpsCAγ (γ-CA from B. pseudomallei) at 0.21–18.1?µM levels. The position of the acidic residues in the peptide sequences can significantly affect bioactivity. For three of the enzymes, tripeptides were identified that are more effective activators than both l-Glu and l-Asp. The tripeptides are also relatively selective because they do not activate prototypical α-CAs (human carbonic anhydrases I and II). Because the role of CA activators in the pathogenicity and life cycles of these infectious bacteria are poorly understood, this study provides new molecular probes to explore such processes.     

Mycobacterium tuberculosis alters the differentiation of monocytes into macrophages in vitro

This paper shows that in vitro infection of human monocytes by Mycobacterium tuberculosis affected monocyte to macrophage differentiation. Despite the low bacterial load used, M. tuberculosis-infected monocytes had fewer granules, displayed a reduced number of cytoplasmic projections and decreased HLA class II, CD68, CD86 and CD36 expression compared to cells differentiated in the absence of mycobacteria. Infected cells produced less IL-12p70, TNF-α, IL-10, IL-6 and high IL-1β in response to lipopolysaccharide and purified protein M. tuberculosis-derived. Reduced T-cell proliferative response and IFN-γ secretion in response to phytohemagglutinin and culture filtrate proteins from M. tuberculosis was also observed in infected cells when compared to non-infected ones. The ability of monocytes differentiated in the presence of M. tuberculosis to control mycobacterial growth in response to IFN-γ stimulation was attenuated, as determined by bacterial plate count; however, they had a similar ability to uptake fluorescent M. tuberculosis and latex beads compared to non-infected cells. Recombinant IL-1β partially altered monocyte differentiation into macrophages; however, treating M. tuberculosis-infected monocytes with IL-1RA did not reverse the effects of infection during differentiation. The results indicated that M. tuberculosis infection altered monocyte differentiation into macrophages and affected their ability to respond to innate stimuli and activate T-cells.     

Substrate delivery by the AAA+ ClpX and ClpC1 unfoldases activates the mycobacterial ClpP1P2 peptidase

Mycobacterial Clp‐family proteases function via collaboration of the heteromeric ClpP1P2 peptidase with a AAA+ partner, ClpX or ClpC1. These enzymes are essential for M. tuberculosis viability and are validated antibacterial drug targets, but the requirements for assembly and regulation of functional proteolytic complexes are poorly understood. Here, we report the reconstitution of protein degradation by mycobacterial Clp proteases in vitro and describe novel features of these enzymes that distinguish them from orthologues in other bacteria. Both ClpX and ClpC1 catalyse ATP‐dependent unfolding and degradation of native protein substrates in conjunction with ClpP1P2, but neither mediates protein degradation with just ClpP1 or ClpP2. ClpP1P2 alone has negligible peptidase activity, but is strongly stimulated by translocation of protein substrates into ClpP1P2 by either AAA+ partner. Interestingly, our results support a model in which both binding of a AAA+ partner and protein‐substrate delivery are required to stabilize active ClpP1P2. Our model has implications for therapeutically targeting ClpP1P2 in dormant M. tuberculosis, and our reconstituted systems should facilitate identification of novel Clp protease inhibitors and activators.     

结核分枝杆菌ESX-1分泌蛋白EspB结构和功能分析

结核分枝杆菌作为肺结核病的病原菌,在人类中致死率远高于其他病原菌.结核分枝杆菌具有特殊的疏水性细胞壁结构,这种致密的细胞壁结构帮助结核分枝杆菌抵御外界环境压力和来自宿主细胞的毒素.同时,它利用特殊的分泌系统将体内的毒力蛋白输出体外,ESX-1分泌系统就是其中之一.结核分枝杆菌ESX-1系统在结核分枝杆菌进入宿主细胞吞噬小体、逃逸至细胞质以及杀死吞噬细胞这些过程中发挥重要作用.研究表明,在结核分枝杆菌内膜上存在一个由多亚基组成、旨在帮助结核分枝杆菌向外输送分泌蛋白的分泌装置.在这个分泌装置的帮助下,结核分枝杆菌重要的毒力蛋白ESAT-6跨内膜向外分泌,EspB也通过这个内膜上的分泌装置被转运至胞外.EspB存在于静置培养的结核分枝杆菌的胶囊层中,也可在振荡培养的结核分枝杆菌的培养液中被检测.通过X射线晶体衍射分析,我们解析了EspB的晶体结构,相比于其他同源结构,发现了EspB的不同构象,即EspB单体能够自组装成为七聚体的规则结构,联系其与毒力因子ESAT-6具有共分泌的特点,七聚体构象的发现为解释EspB在结核分枝杆菌向外分泌蛋白的过程中发挥的作用提供线索,即EspB具有锚定在结核分枝杆菌胶囊层中,作为运输ESAT-6的孔道而存在的可能.     

Enzymatic and antigenic analyses of strains of Mycobacterium bovis,M. bovis BCG,and M. tuberculosis

Strains of Mycobacterium bovis, M. bovis BCG, and M. tuberculosis, including a so-called Canetti strain, were analyzed by means of two-dimensional immunoelectrophoresis (2D-IE), 2D-IE combined with enzyme staining, and multilocus enzyme electrophoresis (MEE). The results demonstrated a close antigentic and enzymatic resemblance among all the strains tested, even though the BCG strains could be divided into two groups based on the presence of one precipitinogen. Eight of the precipitinogens were shown to correspond to enzymes in M. bovis BCG and 10 in M. tuberculosis. Thus, catalase, isocitrate dehydrogenase, malate dehydrogenase, peroxidase, and several others were identified. By means of MEE the strains of M. tuberculosis, M. bovis, and M. bovis BCG could be differentiated. The analyses further indicated that the M. tuberculosis strain Canetti was more closely related to M. bovis than to M. tuberculosis.     

Mycobacterium tuberculosis Rv3034c regulates mTORC1 and PPAR‐γ dependant pexophagy mechanism to control redox levels in macrophages

Mycobacterium tuberculosis survives inside the macrophages by employing several host immune evasion strategies. Here, we reported a novel mechanism in which M. tuberculosis acetyltransferase, encoded by Rv3034c, induces peroxisome homeostasis to regulate host oxidative stress levels to facilitate intracellular mycobacterial infection. Presence of M. tuberculosis Rv3034c induces the expression of peroxisome biogenesis and proliferation factors such as Pex3, Pex5, Pex19, Pex11b, Fis‐1 and DLP‐1; while depletion of Rv3034c decreased the expression of these molecules, thereby selective degradation of peroxisomes via pexophagy. Further studies revealed that M. tuberculosis Rv3034c inhibit induction of pexophagy mechanism by down‐regulating the expression of pexophagy associated proteins (p‐AMPKα, p‐ULK‐1, Atg5, Atg7, Beclin‐1, LC3‐II, TFEB and Keap‐1) and adaptor molecules (NBR1 and p62). Inhibition was found to be dependent on the phosphorylation of mTORC1 and activation of peroxisome proliferator activated receptor‐γ. In order to maintain intracellular homeostasis during oxidative stress, M. tuberculosis Rv3034c was found to induce degradation of dysfunctional and damaged peroxisomes through activation of Pex14 in infected macrophages. In conclusion, this is the first report which demonstrated that M. tuberculosis acetyltransferase regulate peroxisome homeostasis in response to intracellular redox levels to favour mycobacterial infection in macrophage.