On deaf ears,Mycobacterium avium paratuberculosis in pathogenesis Crohn’s and other diseases

The historic suggestion that Mycobacterium avium subsp. paratuberculosis (Map) might be a zoonotic pathogen was based on the apparent similarity of lesions in the intestine of patients with Crohn’s disease (CD) with those present in cattle infected with Map, the etiological agent of Johne’s disease. Reluctance to fully explore this possibility has been attributed to the difficulty in demonstrating the presence of Map in tissues from patients with CD. Advances in technology have resolved this problem and revealed the presence of Map in a significant proportion of patients with CD and other diseases. The seminal finding from recent investigations, however, is the detection of Map in healthy individuals with no clinical signs of disease. The latter observation indicates all humans are susceptible to infection with Map and lends support to the thesis that Map is zoonotic, with a latent stage of infection similar to tuberculosis, where infection leads to the development of an immune response that controls but does not eliminate the pathogen. This clarifies one of the reasons why it has been so difficult to document that Map is zoonotic and associated with the pathogenesis of CD and other diseases. As discussed in the present review, a better understanding of the immune response to Map is needed to determine how infection is usually kept under immune control during the latent stage of infection and elucidate the triggering events that lead to disease progression in the natural host and pathogenesis of CD and immune related diseases in humans.     

E1 of α-ketoglutarate dehydrogenase defends Mycobacterium tuberculosis against glutamate anaplerosis and nitroxidative stress

Enzymes of central carbon metabolism (CCM) in Mycobacterium tuberculosis (Mtb) make an important contribution to the pathogen’s virulence. Evidence is emerging that some of these enzymes are not simply playing the metabolic roles for which they are annotated, but can protect the pathogen via additional functions. Here, we found that deficiency of 2-hydroxy-3-oxoadipate synthase (HOAS), the E1 component of the α-ketoglutarate (α-KG) dehydrogenase complex (KDHC), did not lead to general metabolic perturbation or growth impairment of Mtb, but only to the specific inability to cope with glutamate anaplerosis and nitroxidative stress. In the former role, HOAS acts to prevent accumulation of aldehydes, including growth-inhibitory succinate semialdehyde (SSA). In the latter role, HOAS can participate in an alternative four-component peroxidase system, HOAS/dihydrolipoyl acetyl transferase (DlaT)/alkylhydroperoxide reductase colorless subunit gene (ahpC)-neighboring subunit (AhpD)/AhpC, using α-KG as a previously undescribed source of electrons for reductase action. Thus, instead of a canonical role in CCM, the E1 component of Mtb’s KDHC serves key roles in situational defense that contribute to its requirement for virulence in the host. We also show that pyruvate decarboxylase (AceE), the E1 component of pyruvate dehydrogenase (PDHC), can participate in AceE/DlaT/AhpD/AhpC, using pyruvate as a source of electrons for reductase action. Identification of these systems leads us to suggest that Mtb can recruit components of its CCM for reactive nitrogen defense using central carbon metabolites.The bacterium Mycobacterium tuberculosis (Mtb), which causes tuberculosis, has plagued humanity since antiquity (1), is estimated to infect one-third of the population today, and is the leading cause of death by a bacterium. This success as a pathogen reflects Mtb’s metabolic plasticity and resistance to host immunity (2, 3). Recent evidence suggests that certain enzymes of central carbon metabolism (CCM) can mediate both of these facets of Mtb’s adaptation to the host (4–8). Here, we demonstrate that Rv1248c, recently named 2-hydroxy-3-oxoadipate synthase (HOAS) (9), is one such enzyme.Rv1248c was first annotated as the thiamin diphosphate (ThDP)-dependent E1 component (SucA) of a canonical α-ketoglutarate (α-KG) dehydrogenase complex (KDHC) that produces succinyl CoA (SucCoA) via oxidative decarboxylation of α-KG with concomitant transfer of the resulting succinyl group to CoA (10). Classically, KDHC, composed of three enzymes, joins the oxidative and reductive half-cycles of the TCA cycle (SI Appendix, Fig. S1). The TCA cycle generates high-energy phosphate bonds and biosynthetic precursors of amino acids, nucleotides, and fatty acids (11). However, KDHC activity was not detected in Mtb lysates, and the gene product annotated as the lipoamide-bearing E2 component [Rv2215, dihydrolipoyl succinyl transferase (SucB)] functions as the E2 component dihydrolipoyl acetyl transferase (DlaT) of the pyruvate dehydrogenase complex (PDHC) (5, 12, 13). The Mtb genome does not encode another SucB (12), and KDHC activity could not be demonstrated with purified recombinant Rv1248c plus DlaT and E3 [lipoamide dehydrogenase (Lpd)] in a manner similar to cognate proteins from other actinomycetes (14). Rv1248c by itself produces succinate semialdehyde (SSA) from nonoxidative decarboxylation of α-KG in vitro, and Mtb’s succinate semialdehyde dehydrogenases (SSADHs) can generate succinate from SSA, completing a modified TCA cycle (13). Subsequently, activity-based metabolomic profiling revealed yet another function of Rv1248c that predominated over SSA production: decarboxylation of α-KG, followed by carboligation with glyoxylate to form 2-hydroxy-3-oxoadipate (HOA). Thus, Rv1248c was renamed HOAS (9). Decarboxylation of α-KG is the first step in all three reactions. The Mycobacterium smegmatis α-ketoglutarate decarboxylase (MsKGD) homolog was found to catalyze all three reactions in vitro, with augmentation of catalysis by acetyl CoA (AcCoA)-mediated allosteric modulation (15), whereas Mtb HOAS showed a kinetic preference for the HOAS pathway in vitro (16).HOAS activity is regulated in Mtb by glycogen accumulation regulator A (GarA), whose activity is controlled, in turn, by Ser/Thr kinases PknG and PknB (SI Appendix, Fig. S1B). GarA also regulates glutamate dehydrogenase (GDH) and glutamate synthase/glutamine oxoglutarate aminotransferase (17). Coregulation of these three enzymes by GarA calls attention to the contribution of HOAS to Mtb’s metabolism of glutamate. Glutamate serves as an anaplerotic substrate entering the TCA cycle as α-KG and is also a key intermediate in nitrogen assimilation and metabolism (18, 19).Despite extensive studies, the physiological function of HOAS in Mtb and its contribution to virulence remain unknown. Here, we brought genetics, metabolomics, enzymology, and mouse models of infection to bear on that question, using the hoas deletion mutant in Mtb and the deletion mutant complemented in three ways: with the WT allele, with an allele with a point mutation that abrogates catalysis, or with an allele with a point mutation that is insensitive to allosteric regulation by AcCoA. In standard culture conditions, Δhoas showed no defect in growth or changes in levels of CCM metabolites, arguing against Mtb’s reliance on the conventional function of KDHC. However, situational stresses revealed striking phenotypes in Δhoas, indicative of a defensive role of HOAS against products arising from metabolism of glutamate and against reactive nitrogen intermediates (RNIs). Mechanistic analysis of the latter phenotype revealed a previously undescribed route to antioxidant defense mediated by substrates and enzymes of CCM.     

PCR method is essential for detecting Mycobacterium tuberculosis in oral cavity samples

Tuberculosis is a re-emerging infectious disease, and infection by Mycobacterium tuberculosis has been increasing in immunocompromised hosts, including elderly persons. M. tuberculosis-infected persons may receive dental treatment. To evaluate the risk of M. tuberculosis infection in dental clinics, we examined the detection rates of M. tuberculosis in sample of mixed saliva, dental plaque, extracted teeth, caries lesions, and denture plaque by nested polymerase chain reaction (PCR). The detection rates by PCR in samples from mixed saliva, dental plaque, caries lesions and denture plaque obtained from tuberculosis patients were 98.0%, 92.0%, 89.0%, and 100%, respectively. The detection rates by the culture method were 17.3%, 2.0%, 0%, and 0%, respectively. M. tuberculosis also was detected from the nontuberculous mycobacteria-infected group. Strains of Actinomyces naeslundii, Porphyromonas gingivalis, and Fusobacterium nucleatum inhibited the growth of clinical strains of M. tuberculosis, but strains of Streptococcus mutans, Streptococcus sanguinis, and Actinobacillus actinomycetemcomitans did not. The present study concludes that the PCR method is essential for detecting M. tuberculosis in oral samples.     

On the intrinsic constraint of bacterial growth rate: M. tuberculosis’s view of the protein translation capacity

In nature, the maximal growth rates vary widely among different bacteria species. Fast-growing bacteria species such as Escherichia coli can have a shortest generation time of 20?min. Slow-growing bacteria species are perhaps best known for Mycobacterium tuberculosis, a human pathogen with a generation time being no less than 16?h. Despite of the significant progress made on understanding the pathogenesis of M. tuberculosis, we know little on the origin of its intriguingly slow growth. From a global view, the intrinsic constraint of the maximal growth rate of bacteria remains to be a fundamental question in microbiology. In this review, we analyze and discuss this issue from the angle of protein translation capacity, which is the major demand for cell growth. Based on quantitative analysis, we propose four parameters: rRNA chain elongation rate, abundance of RNA polymerase engaged in rRNA synthesis, polypeptide chain elongation rate, and active ribosome fraction, which potentially limit the maximal growth rate of bacteria. We further discuss the relation of these parameters with the growth rate for M. tuberculosis as well as other bacterial species. We highlight future comprehensive investigation of these parameters for different bacteria species to understand how bacteria set their own specific growth rates.     

The versatility of the CD1 lipid antigen presentation pathway

The family of non‐classical major histocompatibility complex (MHC) class‐I like CD1 molecules has an emerging role in human disease. Group 1 CD1 includes CD1a, CD1b and CD1c, which function to display lipids on the cell surface of antigen‐presenting cells for direct recognition by T‐cells. The recent advent of CD1 tetramers and the identification of novel lipid ligands has contributed towards the increasing number of CD1‐restricted T‐cell clones captured. These advances have helped to identify novel donor unrestricted and semi‐invariant T‐cell populations in humans and new mechanisms of T‐cell recognition. However, although there is an opportunity to design broadly acting lipids and harness the therapeutic potential of conserved T‐cells, knowledge of their role in health and disease is lacking. We briefly summarize the current evidence implicating group 1 CD1 molecules in infection, cancer and autoimmunity and show that although CD1 are not as diverse as MHC, recent discoveries highlight their versatility as they exhibit intricate mechanisms of antigen presentation.     

结核分枝杆菌耐利福平诊断试验的系统评价

目的采用Meta分析的方法评价LiPA和phage-based assays法检测结核分枝杆菌耐利祸平诊断试验的准确性：方法通过检索PubMed、EMbase、CBMWeb、CSJD、CJFD数据库和其他方式广泛收集文献：根据QUADAS质量评价标准评价纳入文献的质量，用Meta-Disc软件对其敏感度、特异度、阳性似然比、阴性似然比等进行合并分析，并进行异质性检验，对无异质性的研究绘制SROC曲线。结果最终纳入42篇文献：（1）LiPA法检测结核分支杆菌耐利福平时，7个研究以BACTEC460法为参考，合并敏感度0.98，合并特异度0.98，SROC（AUC）=0.9924；6个研究以proportion法为参考，合并敏感度0.97，合并特异度1.00，SROC（AUC）=0.9961；3个研究以BACTEC460、Proportion法作为参考，合斤敏感度0.92，合并特异度0.98，SROC（AUC）=0.9842；（2）噬菌体扩增法（商用）检测结核分枝杆菌耐利福平的7个研究，以BACTEC460、Proportion法为参考，合并敏感度0.95，合并特异度0.95，SROC（AUC）=0.9842，（3）噬菌体扩增法（内部的）检测结核分支杆菌耐利福平的7个研究，以BACTEC460、比例法、绝对浓度法和电阻率法为参考，其合并敏感度0.98，合并特异度0.98，SROC（AUC）=0.9949；（4）光素酶噬菌体报告技术检测结核分枝杆菌耐利福平的7个研究，以BACTEC460、比例法、绝对浓度法为参考，其合并敏感度0.98，合并特异度0.98，SROC（AUC）=0.9788。结论现有研究证实：（1）采用分离培养时，噬菌体检测法存检洲结核分枝杆菌耐利福平方面具有较高的敏感度和特异度，在提高耐多药结核病诊断准确性方面具有潜力、（2）LiPA任检测结俊分枝杆菌耐利福平方面也具有较高的敏感度和特异度，但直接应用临床标本检测时敏感度似乎稍有下降一上述结果尚需更多设计严谨、科学的临床试验进一步证实。     

检测结核分枝杆菌rpoB基因突变的研究

目的：建立聚合酶链反应－单链构象多态性（PCR－SSCP）检测结核分枝杆菌rpoB基因突变的方法，并评价共临床应用的价值。方法：依据结核分枝杆菌rpoB基因的耐利福平决定区设计引物，用PCR从临床分离株和直接从痰标本中扩增rpoB基因片段；对扩得的rpoB基因片段做DNA SSCP分析，并随机测定rpoB片段的序列。结果：PCR从所有212株结核分枝杆菌中均扩得230bp片段135份抗酸染色阳性的痰标本中，有113份扩得阳性片段（83．7％），在SSCP分析中，140份经L－J药敏试验检测为利福平耐受的结核分枝杆菌，有130份有rpoB基因突变（符合率为92．9％），72份经L－J药敏试验检测为利福平敏感的菌，有67份的rpoB基因无突变（符合率为93．1％），测序分析发现57份经SSCP检测为突变的rpoB片段均有序列改变，10份经SSCP分析为无突变的有8份无序列改变，SSCP与测序结果的符合率为94．0％，在本研究的菌株中，耐多药结核病（MDR－TB）占76．4％（107／140），有92．5％（99／107）耐多药菌株经SSCP检测有rpoB突变。结论：建立的PCR－SSCP分析方法，是一种较准确和稳定的检测结核分枝杆菌rpoB基因突变的方法，可用于临床快速分析患者结核分枝杆菌对利福平的耐药性，并可作为MDR－TB判断的一个重要指标。     

Synthesis of 4-aminoquinoline analogues and their platinum(II) complexes as new antileishmanial and antitubercular agents

A series of 4-amino-7-chloroquinoline derivatives were synthesized by the reaction of 4,7-dichloro-quinoline with the corresponding diamine and then with propargyl bromide. In addition, platinum(II) complexes were obtained by reacting some of the organic derivatives with K₂PtCl₄. Several of the synthesized compounds displayed antituberculosis activities. Compound 3 was 47.5 times more active than amphotericin B against Leishmania chagasi (IC₅₀ = 0.04 μg/mL). Compounds 5, 6, 7, 9, 10, 11 and 13 presented promising results against Mycobacterium tuberculosis, with MIC values ranging from 12.5 to 15.6 μg/mL, comparable to the “first and second line” drugs used to treat tuberculosis.     

基因芯片技术在结核分支杆菌耐药突变基因检测中的应用

目的通过基因芯片检测系统，快速检测临床样品中结核分支杆菌耐药突变情况。方法根据结核分支杆菌标准株H37Rv序列，设计了覆盖rpoB、katG，inhA基因突变区的系列寡核苷酸探针，制作膜芯片，检测临床样品中结核分支杆菌基因突变情况，以此判断耐药结果。结果在305例临床病例中，共检出阳性病例125例，其中阳性敏感病例64例，阳性突变病例61例，阳性率为40．98％，在125例阳性样品中，共发现有8种突变类型，其中10例531L，占7．94％，19例315M，占阳性样品中总数的15．08％。结论PCR与膜芯片杂交技术可临床检测结核分支杆菌对利福平和异烟肼的耐药性，并具有快速、简便、敏感的特点。