The Effect of Some Antituberculous Drugs upon the Upper Gastrointestinal Tract: an Experiment with Para-Aminosalicylic Acid,Isoniazid, and Streptomycin

The mucosal infrastructure and histology of the upper gastrointestinal tract, intestinal function, and the state of the liver have been followed in 6 patients during antituberculous treatment. In 2 patients a transitory inflammatory cell reaction similar to chronic superficial gastritis developed. The number of eosinophils in the mucosa increased in all cases after 2 weeks’ treatment. This reaction did not correlate with the inflammatory cell reaction. The slight decrease of disaccharidase activities was insufficient to cause impairment of the lactose absorption. The affection of the liver observed in all patients may be toxic rather than allergic. Subjective gastrointestinal symptoms showed no correlation with objective findings of the state of the upper digestive system.     

Tuberculosis in the intensive care unit: A retrospective descriptive cohort study with determination of a predictive fatality score

BACKGROUND:

Despite effective treatments, tuberculosis-related mortality remains high among patients requiring admission to the intensive care unit (ICU).

OBJECTIVE:

To determine prognostic factors of death in tuberculosis patients admitted to the ICU, and to develop a simple predictive scoring system.

METHODS:

A 10-year, retrospective study of 53 patients admitted consecutively to the Hôpitaux de Paris, Hôpital Lariboisière (Paris, France) ICU with confirmed tuberculosis, was conducted. A multivariate analysis was performed to identify risk factors for death. A predictive fatality score was determined.

RESULTS:

Diagnoses included pulmonary tuberculosis (96%) and tuberculous encephalomeningitis (26%). Patients required mechanical ventilation (45%) and vasopressor infusion (28%) on admission. Twenty patients (38%) died, related to direct tuberculosis-induced organ failure (n=5), pulmonary bacterial coinfections (n=14) and pulmonary embolism (n=1). Using a multivariate analysis, three independent factors on ICU admission were predictive of fatality: miliary pulmonary tuberculosis (OR 9.04 [95% CI 1.25 to 65.30]), mechanical ventilation (OR 11.36 [95% CI 1.55 to 83.48]) and vasopressor requirement (OR 8.45 [95% CI 1.29 to 55.18]). A score generated by summing these three independent variables was effective at predicting fatality with an area under the ROC curve of 0.92 (95% CI 0.85 to 0.98).

CONCLUSIONS:

Fatalities remain high in patients admitted to the ICU with tuberculosis. Miliary pulmonary tuberculosis, mechanical ventilation and vasopressor requirement on admission were predictive of death.     

Diagnosis of intestinal tuberculosis using a monoclonal antibody to Mycobacterium tuberculosis

AIM: To investigate the utility of immunohistochemical (IHC) staining with an antibody to Mycobacterium tuberculosis (M. tuberculosis) for the diagnosis of intestinal tuberculosis (TB).METHODS: We retrospectively identified 10 patients (4 males and 6 females; mean age = 65.1 ± 13.6 years) with intestinal TB. Clinical characteristics, including age, gender, underlying disease, and symptoms were obtained. Chest radiograph and laboratory tests, including sputum Ziehl-Neelsen (ZN) staining, M. tuberculosis culture, and sputum polymerase chain reaction (PCR) for tubercle bacilli DNA, as well as Tuberculin skin test (TST) and QuantiFERON-TB gold test (QFT), were examined. Colonoscopic records recorded on the basis of Sato’s classification were also reviewed, in addition to data from intestinal biopsies examined for histopathological findings, including hematoxylin and eosin staining, and ZN staining, as well as M. tuberculosis culture, and PCR for tubercle bacilli DNA. For the present study, archived formalin-fixed paraffin-embedded (FFPE) intestinal tissue samples were immunohistochemically stained using a commercially available species-specific monoclonal antibody to the 38-kDa antigen of the M. tuberculosis complex. These sections were also stained with the pan-macrophage marker CD68 antibody.RESULTS: From the clinical data, we found that no patients were immunocompromised, and that the main symptoms were diarrhea and weight loss. Three patients displayed active pulmonary TB, six patients (60%) had a positive TST, and 4 patients (40%) had a positive QFT. Colonoscopic findings revealed that all patients had type 1 findings (linear ulcers in a circumferential arrangement or linear ulcers arranged circumferentially with mucosa showing multiple nodules), all of which were located in the right hemicolon and/or terminal ileum. Seven patients (70%) had concomitant healed lesions in the ileocecal area. No acid-fast bacilli were detected with ZN staining of the intestinal tissue samples, and both M. tuberculosis culture and PCR for tubercle bacilli DNA were negative in all samples. The histopathological data revealed that tuberculous granulomas were present in 4 cases (40%). IHC staining in archived FFPE samples with anti-M. tuberculosis monoclonal antibody revealed positive findings in 4 patients (40%); the same patients in which granulomas were detected by hematoxylin and eosin staining. M. tuberculosis antigens were found to be mostly intracellular, granular in pattern, and primarily located in the CD68⁺ macrophages of the granulomas.CONCLUSION: IHC staining with a monoclonal antibody to M. tuberculosis may be an efficient and simple diagnostic tool in addition to classic examination methods for the diagnosis of intestinal TB.     

结核病信息管理系统在结核控制中的作用

目的探讨河南省济源市结核病信息管理系统在结核病控制和规范管理中的作用。方法收集1997—2011年运行结核病管理信息系统前后济源市疑似肺结核患者转诊到位情况以及肺结核患者的诊断、治疗、转归等资料,进行对比分析。结果抗结核药品专项管理前分析肺结核患者1805例,专项管理后分析患者3650例;专项管理前与后患者取药延迟率分别为19．28％和12．84％,成功治疗率分别为81．99％和90．99％,涂阳患者治愈率分别为85％的90．33％;总体规则治疗率分别为76．95％和87．68％;两组间差异有统计学意义（P〈0．01）。结论济源市应用结核病信息管理系统后,对结核病患者转诊到位及诊断、治疗、转归都有极大的促进作用。     

铜陵地区结核与非结核分枝杆菌菌型鉴定及耐药性分析

目的通过检测本地区分枝杆菌感染菌株类型以及耐药性,制订化疗方案提供依据。方法采取两种试剂鉴定菌株,绝对浓度法检测药敏耐药性。结果 80株分枝杆菌中人型结核分枝杆菌58例,占72.5%;牛型结核分枝杆菌15例,占18.8%;非结核分枝杆菌7例,占8.7%。结核分枝杆菌复合群耐多药率9.6%。非结核分枝杆菌耐多药率85.7%。牛型结核分枝杆菌对药物敏感性大于人型结核分枝杆菌,人型结核分枝杆菌对药物敏感性大于非结核分枝杆菌。耐药以耐R、H、TH1321、Rtf、PAS多见。结论本地区肺部感染分枝杆菌菌株以结核分枝杆菌复合群为主,耐多药率及非结核分枝杆菌感染率略高于全国水平。     

结核杆菌DNA探针板杂交方法及其应用研究

我们建立了一种在酶标板上进行ＤＮＡ：ＤＮＡ杂交的方法，该方法与ＥＬＩＳＡ类似，简单、易操作、可半定量，便于临床实验室推广应用。对其杂交条件及影响因素进行了详细的探讨。通过该方法，地高辛标记的结核分支杆菌全染色体ＤＮＡ探针和１２种分支杆菌、７种非分支杆菌个染色体ＤＮＡ的杂交百分率有显著差别，除卡介苗、堪萨斯分支杆菌杂交百分率超过３０％，其余受试菌株均低于１０％。结果表明在酶标板上进行核酸杂交是完全可行的，利用已知细菌ＤＮＡ定量包被板，与未知的带标记的细菌ＤＮＡ杂交，可达到菌种鉴定的目的。     

A truncated lipoglycan from mycobacteria with altered immunological properties

Maintenance of cell-wall integrity in Mycobacterium tuberculosis is essential and is the target of several antitubercular drugs. For example, ethambutol targets arabinogalactan and lipoarabinomannan (LAM) biosynthesis through the inhibition of several arabinofuranosyltransferases. Apart from their role in cell-wall integrity, mycobacterial LAMs also exhibit important immunomodulatory activities. Here we report the isolation and detailed structural characterization of a unique LAM molecule derived from Mycobacterium smegmatis deficient in the arabinofuranosyltransferase AftC (AftC-LAM). This mutant LAM expresses a severely truncated arabinan domain completely devoid of 3,5-Araf–branching residues, revealing an intrinsic involvement of AftC in the biosynthesis of LAM. Furthermore, we found that ethambutol efficiently inhibits biosynthesis of the AftC-LAM arabinan core, unambiguously demonstrating the involvement of the arabinofuranosyltransferase EmbC in early stages of LAM-arabinan biosynthesis. Finally, we demonstrate that AftC-LAM exhibits an enhanced proinflammatory activity, which is due to its ability to activate Toll-like receptor 2 (TLR2). Overall, our efforts further describe the mechanism of action of an important antitubercular drug, ethambutol, and demonstrate a role for specific arabinofuranosyltransferases in LAM biosynthesis. In addition, the availability of sufficient amounts of chemically defined wild-type and isogenic truncated LAMs paves the way for further investigations of the structure–function relationship of TLR2 activation by mycobacterial lipoglycans.     

Inhibitors of type II NADH:menaquinone oxidoreductase represent a class of antitubercular drugs

Mycobacterium tuberculosis (Mtb) is an obligate aerobe that is capable of long-term persistence under conditions of low oxygen tension. Analysis of the Mtb genome predicts the existence of a branched aerobic respiratory chain terminating in a cytochrome bd system and a cytochrome aa(3) system. Both chains can be initiated with type II NADH:menaquinone oxidoreductase. We present a detailed biochemical characterization of the aerobic respiratory chains from Mtb and show that phenothiazine analogs specifically inhibit NADH:menaquinone oxidoreductase activity. The emergence of drug-resistant strains of Mtb has prompted a search for antimycobacterial agents. Several phenothiazines analogs are highly tuberculocidal in vitro, suppress Mtb growth in a mouse model of acute infection, and represent lead compounds that may give rise to a class of selective antibiotics.     

Population genomics of Mycobacterium tuberculosis in the Inuit

Nunavik, Québec suffers from epidemic tuberculosis (TB), with an incidence 50-fold higher than the Canadian average. Molecular studies in this region have documented limited bacterial genetic diversity among Mycobacterium tuberculosis isolates, consistent with a founder strain and/or ongoing spread. We have used whole-genome sequencing on 163 M. tuberculosis isolates from 11 geographically isolated villages to provide a high-resolution portrait of bacterial genetic diversity in this setting. All isolates were lineage 4 (Euro-American), with two sublineages present (major, n = 153; minor, n = 10). Among major sublineage isolates, there was a median of 46 pairwise single-nucleotide polymorphisms (SNPs), and the most recent common ancestor (MRCA) was in the early 20th century. Pairs of isolates within a village had significantly fewer SNPs than pairs from different villages (median: 6 vs. 47, P < 0.00005), indicating that most transmission occurs within villages. There was an excess of nonsynonymous SNPs after the diversification of M. tuberculosis within Nunavik: The ratio of nonsynonymous to synonymous substitution rates (dN/dS) was 0.534 before the MRCA but 0.777 subsequently (P = 0.010). Nonsynonymous SNPs were detected across all gene categories, arguing against positive selection and toward genetic drift with relaxation of purifying selection. Supporting the latter possibility, 28 genes were partially or completely deleted since the MRCA, including genes previously reported to be essential for M. tuberculosis growth. Our findings indicate that the epidemiologic success of M. tuberculosis in this region is more likely due to an environment conducive to TB transmission than a particularly well-adapted strain.The tubercule bacillus, Mycobacterium tuberculosis, is a highly successful, medically important human-adapted pathogen. Studies of diverse strain collections reveal a geographic aggregation of the principal M. tuberculosis lineages (1) consistent with a dissemination of this organism around the world with the paleo migration (2). Ancient DNA studies also support the notion that M. tuberculosis has caused disease in humans for thousands of years. Thus, it can be inferred that M. tuberculosis has evolved in step with its human host, successfully responding to changes in the host and its environment that could affect the capacity to cause transmissible disease.In contrast to the global diversity of M. tuberculosis strains (1–3), we have previously observed limited genetic diversity in the Nunavik region of Québec (4). One possible explanation is a founder strain, wherein genetic similarity is due to a single recent introduction of a bacterium and may not necessarily represent ongoing spread between communities. In this scenario, isolates might have indistinguishable genotypes by conventional genotyping modalities (restriction fragment length polymorphism, mycobacterial interspersed repetitive units, spoligotyping) but distinct genotypes when assessed using a higher-resolution method, namely whole-genome sequencing (WGS) (5). An additional explanation is that a single clone of M. tuberculosis is currently spreading both within and between villages; however, the great distances between these communities that are not linked by roads make intervillage spread less likely. These possible explanations need not be mutually exclusive.To evaluate these possibilities, we conducted WGS on M. tuberculosis isolates from Nunavik isolated over 23 y. Estimation of the divergence date of the most recent common ancestor (MRCA) provided evidence that tuberculosis (TB) was introduced into this region in the early 20th century, following which time there has been substantial ongoing transmission, predominantly within villages. This setting provides a unique opportunity to study the genomic characteristics of an epidemiologically successful strain of M. tuberculosis over time.     

Reciprocal seasonal variation in vitamin D status and tuberculosis notifications in Cape Town, South Africa

Vitamin D deficiency is associated with susceptibility to tuberculosis (TB) in HIV-uninfected people in Europe, but it is not known whether such an association exists among HIV-infected people in subtropical Africa. We conducted a cross-sectional study to determine whether vitamin D deficiency was associated with susceptibility to active TB in HIV-uninfected (n = 196) and HIV-infected (n = 174) black Africans in Cape Town, South Africa. We also investigated whether there was evidence of seasonal variation in vitamin D status and TB notifications in this setting over an 8-y period. Vitamin D deficiency (serum 25-hydroxyvitamin D [25(OH)D] <50 nmol/L) was present in 232 (62.7%) of 370 participants and was associated with active TB in both HIV-uninfected (odds ratio = 5.2, 95% confidence interval: 2.8-9.7; P < 0.001) and HIV-infected (odds ratio = 5.6, 95% confidence interval: 2.7-11.6; P < 0.001) people. Vitamin D status varied according to season: The mean serum 25(OH)D concentration was highest in January through March and lowest in July through September (56.8 vs. 30.7 nmol/L, respectively; P < 0.001). Reciprocal seasonal variation in TB notifications was observed: The mean number of TB notifications per quarter for Cape Town in 2003 to 2010 was lowest in April through June and highest in October through December (4,222 vs. 5,080; P < 0.001). Vitamin D deficiency is highly prevalent among black Africans in Cape Town and is associated with susceptibility to active TB both in the presence and absence of HIV infection. Reciprocal seasonal variation in serum 25(OH)D concentration and TB notifications suggests that seasonal variations in vitamin D status and TB incidence in this setting are causally related.     

Incidence and Clinical Outcomes of Clostridium difficile Infection after Treatment with Tuberculosis Medication

Background/Aims

To determine the incidence and clinical characteristics of tuberculosis (TB) medication-associated Clostridium difficile infection.

Methods

This multicenter study included patients from eight tertiary hospitals enrolled from 2008 to 2013. A retrospective analysis was conducted to identify the clinical features of C. difficile infection in patients who received TB medication.

Results

C. difficile infection developed in 54 of the 19,080 patients prescribed TB medication, representing a total incidence of infection of 2.83 cases per 1,000 adults. Fifty-one of the 54 patients (94.4%) were treated with rifampin. The patients were usually treated with oral metronidazole, which produced improvement in 47 of the 54 patients (87%). Twenty-three patients clinically improved with continuous rifampin therapy for C. difficile infection. There were no significant differences in improvement between patients treated continuously (n=21) and patients in whom treatment was discontinued (n=26).

Conclusions

The incidence of C. difficile infection after TB medication was not low considering the relatively low TB medication dosage compared to other antibiotics. It may not be always necessary to discontinue TB medication. Instead, decisions concerning discontinuation of TB medication should be based on TB status.     

Incidence, predictors and prognostic value of cranial nerve involvement in patients with tuberculous meningitis: a retrospective evaluation

Background

Cranial nerve involvement is commonly observed in patients with tuberculous meningitis. The present study evaluated the incidence, predictors and prognostic significance of cranial nerve involvement in tuberculous meningitis.

Materials and method

One hundred-fifty-eight adult patients with tuberculous meningitis were retrospectively evaluated and followed up for 9 months. A detailed clinical evaluation and cranial magnetic resonance imaging were done in every patient.

Result

At inclusion, 60 (38%) patients had cranial neuropathy. Sixteen patients were having involvement of two or more cranial nerves. Abducent nerve was the most frequently (32.3%) affected cranial nerve. Predictors of cranial nerve involvement were age > 25 years, history of vomiting, altered sensorium, hemiparesis, diplopia, papilledema, signs of meningeal irritation, severe functional disability, cerebrospinal fluid protein > 2.5 g/L and cerebrospinal fluid cell count > 100/mm³. The presence of optochiasmatic arachnoiditis and hydrocephalus was also a significant predictor of cranial neuropathy. Presence of cranial neuropathy was significantly associated with poor outcome.

Conclusion

Cranial nerve involvement occurred in more than one third of patients with tuberculous meningitis. The presence of cranial neuropathy was associated with poor outcome.     

Peptidoglycan synthesis in Mycobacterium tuberculosis is organized into networks with varying drug susceptibility

Peptidoglycan (PG), a complex polymer composed of saccharide chains cross-linked by short peptides, is a critical component of the bacterial cell wall. PG synthesis has been extensively studied in model organisms but remains poorly understood in mycobacteria, a genus that includes the important human pathogen Mycobacterium tuberculosis (Mtb). The principle PG synthetic enzymes have similar and, at times, overlapping functions. To determine how these are functionally organized, we carried out whole-genome transposon mutagenesis screens in Mtb strains deleted for ponA1, ponA2, and ldtB, major PG synthetic enzymes. We identified distinct factors required to sustain bacterial growth in the absence of each of these enzymes. We find that even the homologs PonA1 and PonA2 have unique sets of genetic interactions, suggesting there are distinct PG synthesis pathways in Mtb. Either PonA1 or PonA2 is required for growth of Mtb, but both genetically interact with LdtB, which has its own distinct genetic network. We further provide evidence that each interaction network is differentially susceptible to antibiotics. Thus, Mtb uses alternative pathways to produce PG, each with its own biochemical characteristics and vulnerabilities.One of the leading causes of infectious disease deaths worldwide is tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb). One-third of the human population is thought to harbor Mtb and ∼1.5 million individuals died of TB last year (1). Mtb’s success as a pathogen is due in part to its unusual cell wall, which is notorious for its complexity and is implicated in Mtb’s innate resistance to many commonly used antibiotics (2). A critical component of the bacterial cell wall (including Mtb’s) is peptidoglycan (PG), a complex polymer that provides structural support and counteracts turgor pressure (3). PG is essential for cell survival, and its synthesis is targeted by many potent antibiotics (2).PG consists of long glycan chains composed of two different sugars (Fig. 1A) that are cross-linked via short peptide side chains that extend from the glycan chains. Notably, generation of mature PG occurs outside of the cell membrane and is mediated by enzymes that incorporate new PG subunits, which are formed in the cytoplasm, into the PG polymer. PonA1 and PonA2 are the two enzymes in Mtb that can both polymerize glycan strands and cross-link peptides [known as bifunctional penicillin binding proteins (PBPs), Fig. 1A]. The predominant peptide cross-links in mycobacteria join the third amino acids (3–3 link) of adjacent stem peptides (4, 5), which are synthesized by l,d-transpeptidases (Ldts) such as LdtB, one of the major Ldts in Mtb (Fig. 1A). The peptides can also be joined by cross-linking the fourth and third amino acids (4–3 link) (Fig. 1A) through the action of bifunctional or monofunctional (capable of only peptide cross-linking) PBPs. The activity of these distinct factors must be coordinated to ensure proper cell-wall synthesis. One method of coordination is the use of large protein complexes, the elongation complex and divisome, which mediate cell-wall biogenesis during cell elongation or division, respectively (2). The essential activity of these enzymes makes them prime drug targets; indeed, PBPs and Ldts are inhibited by carbapenems and penicillin (6, 7), which remains one of the most clinically important drugs in use.Open in a separate windowFig. 1.Deletion of PG synthases influences growth and morphology of Mtb. (A) Transglycosylation (TG) and transpeptidation (TP) reactions incorporate new PG subunits into the cell wall. PonA1 and PonA2 carry out both TG and 4–3 TP reactions. LdtB only mediates 3–3 TP reactions. M, N-acetylmuramic acid. G, N-acetylglucosamine. (B) Deletion of either ponA2 or ldtB does not greatly affect Mtb growth during log phase, although loss of ldtB reduces population density in stationary phase. Error bars are often too small to see. (C) ponA2 mutant cells (n = 179) have increased width compared with wild-type cells (n = 153) (approximate P value <0.0001 by the Kolmogorov–Smirnov test). (D) ldtB mutant cells (n = 193) have increased width and decreased length compared with wild-type cells (n = 153) (approximate P value <0.0001 by the Kolmogorov–Smirnov test for both length and width).Although the biosynthesis and structure of PG have been investigated for decades, predominantly in organisms such as Escherichia coli or Bacillus subtilis, the mechanisms that coordinate the biochemical activities required to polymerize and modify the cell wall remain incompletely understood. Moreover, much less is known about PG synthesis in many pathogenic organisms, including Mtb (2). However, previous studies in Mtb suggest that PG synthesis in this pathogen does not strictly conform to the E. coli paradigm. For example, E. coli has three bifunctional PBPs [PBP1A, PBP1B, and PBP1C (3)], whereas Mtb has just two [PonA1 and PonA2 (8)]. Additionally, PBP2 (known as PBPA in mycobacteria) is a monofunctional PBP and is required for cell elongation in E. coli, but instead seems to function in cell septation in mycobacteria (3, 9).The structure of PG is also different in Mtb than in E. coli: Mycobacterial PG has an unusual prevalence of 3–3 peptide linkages. The abundance of 3–3 cross-links in mycobacterial PG throughout different growth stages (5) suggests that Ldts are active during normal growth; however, their cellular roles or regulation during growth and PG biogenesis remain largely unknown. Whereas penicillins and cephalosporins target only enzymes that produce 4–3 cross-links, Ldts can be targeted by carbapenems (7). Recent work suggests that these agents might be far more efficacious against both dividing and nondividing bacteria (10). Although little is known about Mtb’s five encoded Ldts (11), one, LdtB, is implicated in antibiotic tolerance (11–13), is required for normal virulence in a mouse model of TB (12), and is important for normal cell shape (13).Previous studies have also revealed that PG biosynthesis differs between Mtb and the related saprophytic Mycobacterium smegmatis (Msm). As opposed to Mtb, Msm has three bifunctional PBPs: PonA1, PonA2, and PonA3 (14). PonA1 is required for Msm but not Mtb growth in culture (15, 16); however, PonA1 is required for robust growth of Mtb during infection (16). In contrast, Mtb and Msm ponA2 mutants do not have growth defects in culture (14, 17). However, Mtb strains with inactivated ponA1 or ponA2 exhibit similar survival defects during growth in a host (16, 18), suggesting that these two similar bifunctional enzymes have nonredundant and important contributions to PG synthesis during infection. Collectively, the differences in PG synthase functionality may imply that different PG synthetic pathways exist across species, which may have consequences for a pathogen’s virulence during infection.Here, we interrogated PG synthesis in Mtb by investigating the genetic interactions of ponA1, ponA2, and ldtB, which encode three PG synthases critical for Mtb’s growth during infection. To identify these interactions, we performed genome-wide transposon mutagenesis screens in Mtb mutant strains that lacked one of these enzymes. Advances in high-throughput sequencing technology coupled with the power of whole-genome studies provide unique insights into key bacterial processes, such as cell-wall biosynthesis. Such studies have been performed to a limited extent in bacteria, and further work would substantially expand our understanding of the organization of prokaryotic metabolic processes. In this study, we identified diverse genetic interaction networks for ponA1, ponA2, and ldtB, suggesting that these synthases are embedded within distinct cellular networks for assembling Mtb’s PG. We found that either ponA1 or ponA2 is required for cell growth, and that ldtB interacts with both ponA1 and ponA2. Moreover, mutants that lack these enzymes have differential susceptibility to agents that interfere with cell-wall biogenesis. Thus, the Mtb cell wall is synthesized using multiple interacting networks that are both overlapping and unique.     

Evasion of peptide, but not lipid antigen presentation, through pathogen-induced dendritic cell maturation

Dendritic cells (DC) present lipid and peptide antigens to T cells on CD1 and MHC Class II (MHCII), respectively. The relative contribution of these systems during the initiation of adaptive immunity after microbial infection is not characterized. MHCII molecules normally acquire antigen and rapidly traffic from phagolysosomes to the plasma membrane as part of DC maturation, whereas CD1 molecules instead continually recycle between these sites before, during, and after DC maturation. We find that in Mycobacterium tuberculosis (Mtb)-infected DCs, CD1 presents antigens quickly. Surprisingly, rapid DC maturation results in early failure and delay in MHCII presentation. Whereas both CD1b and MHCII localize to bacterial phagosomes early after phagocytosis, MHCII traffics from the phagosome to the plasma membrane with a rapid kinetic that can precede antigen availability and loading. Thus, rather than facilitating antigen presentation, a lack of coordination in timing may allow organisms to use DC maturation as a mechanism of immune evasion. In contrast, CD1 antigen presentation occurs in the face of Mtb infection and rapid DC maturation because a pool of CD1 molecules remains available on the phagolysosome membrane that is able to acquire lipid antigens and deliver them to the plasma membrane.     

Human splenic macrophages as a model for in vitro infection with Mycobacterium tuberculosis

Macrophages play an important role during Mycobacterium tuberculosis (MTB) infection. In humans most of the studies on MTB-macrophage interactions have been performed using circulating monocytes and monocyte-derived macrophages. However, little research has been performed on this interaction using tissue macrophages. Herein, we used human splenic macrophages to characterize particular responses to MTB infection. Based on morphological, biochemical, and immunological markers, splenic adherent cells exhibit characteristics of tissue macrophages. They were able to efficiently phagocytose both live and heat-killed (h-k) MTB H37Rv. Upon infection with live, but not h-k MTB, an increase in secreted TNF-alpha was elicited. Splenic macrophages produced high basal levels of IL-10; however, infection with live or h-k MTB resulted in decrease IL-10 secretion. Both IL-12p40 and IL-12p70 basal levels were also decreased upon infection with live or h-k MTB; however, while the reduction for IL-12p40 levels was observed at earlier time points (4h) for both live and h-k MTB, infection with live MTB, but not h-k MTB, resulted in a time-dependent secretion of IL-12p40 at 24 and 48h after infection. IL-12p70 levels were completely reduced upon infection by either live or h-k MTB. These results support that human splenic macrophages may represent a potential useful model to study MTB-macrophage interactions in vitro.     

Inhibition of bacterial disulfide bond formation by the anticoagulant warfarin

Blood coagulation in humans requires the activity of vitamin K epoxide reductase (VKOR), the target of the anticoagulant warfarin (Coumadin). Bacterial homologs of VKOR were recently found to participate in a pathway leading to disulfide bond formation in secreted proteins of many bacteria. Here we show that the VKOR homolog from the bacterium Mycobacterium tuberculosis, the causative agent of human tuberculosis, is inhibited by warfarin and that warfarin-resistant mutations of mycobacterial VKOR appear in similar locations to mutations found in human patients who require higher doses of warfarin. Deletion of VKOR results in a severe growth defect in mycobacteria, and the growth of M. tuberculosis is inhibited by warfarin. The bacterial VKOR homolog may represent a target for antibiotics and a model for genetic studies of human VKOR. We present a simple assay in Escherichia coli, based on a disulfide-sensitive β-galactosidase, which can be used to screen for stronger inhibitors of the M. tuberculosis VKOR homolog.     

EVALUATION OF ANTIMICROBIAL AND CYTOTOXIC ACTIVITIES OF PLANT EXTRACTS FROM SOUTHERN MINAS GERAIS CERRADO

The antimicrobial activity of plant hidroethanolic extracts on bacteriaGram positive, Gram negative, yeasts, Mycobacterium tuberculosis H37and Mycobacterium bovis was evaluated by using the technique of Agardiffusion and microdilution in broth. Among the extracts evaluated by Agar diffusion,the extract of Bidens pilosa leaf presented the most expressiveaverage of haloes of growth inhibition to the microorganisms, followed by the extractof B. pilosa flower, of Eugenia pyriformis'' leafand seed, of Plinia cauliflora leaf which statistically presentedthe same average of haloes inhibitory formation on bacteria Gram positive, Gramnegative and yeasts. The extracts of Heliconia rostrata did notpresent activity. Mycobacterium tuberculosis H37 andMycobacterium bovis (BCG) appeared resistant to all the extracts.The susceptibility profile of Candida albicans andSaccharomyces cerevisiae fungi were compared to one another andto the Gram positive Bacillus subtilis, Enterococcusfaecalis and the Gram negative Salmonella typhimuriumbacteria (p > 0.05). The evaluation of cytotoxicity was carriedout on C6-36 larvae cells of the Aedes albopictus mosquito. Theextracts of stem and flower of Heliconia rostrata, leaf and stem ofPlinia cauliflora, seed of Anonna crassifloraand stem, flower and root of B. pilosa did not present toxicity inthe analyzed concentrations. The highest rates of selectivity appeared in theextracts of stem of A. crassiflora and flower of B.pilosa to Staphylococcus aureus, presenting potentialfor future studies about a new drug development.     

Structural analysis of the dodecameric proteasome activator PafE in Mycobacterium tuberculosis

The human pathogen Mycobacterium tuberculosis (Mtb) requires a proteasome system to cause lethal infections in mice. We recently found that proteasome accessory factor E (PafE, Rv3780) activates proteolysis by the Mtb proteasome independently of adenosine triphosphate (ATP). Moreover, PafE contributes to the heat-shock response and virulence of Mtb. Here, we show that PafE subunits formed four-helix bundles similar to those of the eukaryotic ATP-independent proteasome activator subunits of PA26 and PA28. However, unlike any other known proteasome activator, PafE formed dodecamers with 12-fold symmetry, which required a glycine-XXX-glycine-XXX-glycine motif that is not found in previously described activators. Intriguingly, the truncation of the PafE carboxyl-terminus resulted in the robust binding of PafE rings to native proteasome core particles and substantially increased proteasomal activity, suggesting that the extended carboxyl-terminus of this cofactor confers suboptimal binding to the proteasome core particle. Collectively, our data show that proteasomal activation is not limited to hexameric ATPases in bacteria.Although the ubiquitin proteasome pathway plays essential roles in eukaryotes (reviewed in refs. 1 and 2), most bacterial species do not have proteasome systems and instead degrade proteins using ATP-dependent proteases like ClpP, Lon, and HslUV (reviewed in refs. 3 and 4). However, bacteria of the orders Actinomycetales and Nitrospirales also encode proteasomes that are structurally highly similar to eukaryotic and archaeal proteasomes (reviewed in refs. 5 and 6). Importantly, the human pathogen Mycobacterium tuberculosis (Mtb), an Actinomycete, requires proteasomal function to cause lethal infections in mice (7). Ablation of proteasomal degradation sensitizes bacteria to nitric oxide, an antimicrobial free radical made by macrophages and other cell types, and attenuates bacterial growth in mice (7–9). The potential to target persistent or latent bacteria has made the Mtb proteasome system a prioritized target for the development of antituberculosis drugs (10, 11). Indeed, Mtb-specific proteasome inhibitors have been identified that may provide a promising lead for new drugs to treat tuberculosis (12, 13).There are numerous similarities and differences between eukaryotic and bacterial proteasomes. The 20S proteasome core particle (20S CP), which consists of two seven-membered β-rings between two seven-membered α-rings, is highly conserved structurally between prokaryotes and eukaryotes (14–16). However, the accessory factors that associate with the 20S CPs quickly diverge among the domains of life. Both bacteria and eukaryotes use a covalent small protein modification to mark substrate proteins for degradation; however, the eukaryotic ubiquitin tag is a well-folded protein whereas the Mtb Pup (prokaryotic ubiquitin-like protein) tag is intrinsically disordered (17, 18). Furthermore, degradation of ubiquitylated proteins by eukaryotic 20S CPs largely relies on a complex regulatory particle that caps one or both ends of the 20S CP and includes a heterohexameric ring of adenosine triphosphatases (ATPases) for substrate recognition and unfolding (reviewed in refs. 19 and 20). In contrast, the mycobacterial 20S CP uses a homohexameric ATPase ring called Mpa (mycobacterial proteasome ATPase) for both the recognition and unfolding of pupylated proteins (18, 21, 22).In addition to the ATPase activators, proteolysis by eukaryotic proteasomes can also be stimulated by several ATP-independent factors, such as the 11S activators PA26 and PA28, as well as Blm10 (23–28). We and another group recently discovered that Mtb has an analogous factor encoded by Rv3780 that we call PafE (proteasome accessory factor E; also known as Bpa for bacterial proteasome activator), which stimulates the degradation of small peptides and β-casein in vitro (29, 30). Both studies also showed that a carboxyl (C)-terminal glycine-glutamine-tyrosine-leucine (GQYL) motif is essential for interacting with and activating 20S CPs, and the penultimate tyrosine residue contributes to activation similarly to tyrosines observed in the “HbYX” (hydrophobic-tyrosine-any amino acid) motif in other characterized proteasome activators (reviewed in ref. 28). Our work further showed that PafE promotes the degradation of at least one native Mtb protein substrate, heat-shock protein repressor (HspR), and that an Mtb pafE mutant is sensitive to heat shock and is attenuated for growth in mice (30). Importantly, PafE-mediated degradation does not require pupylation. Thus, there appear to be at least two independent paths for targeting proteins to the mycobacterial proteasome for degradation.Like the eukaryotic 11S proteasome activators, PafE does not require ATP to stimulate proteolysis. However, it was unknown if PafE formed heptameric complexes like PA26 or PA28. In this work, we show that PafE monomers assume a four-helix bundle structure that is similar to that found in 11S activators, but assemble differently into an unprecedented dodecameric ring structure with 12-fold symmetry. We used isothermal titration calorimetry, cryo-electron microscopy (cryo-EM), and X-ray crystallography to analyze interactions between PafE and 20S core particles, and found that PafE binding induces a larger gate-opening change than has been described for other organisms. We also found that PafE has an extended C terminus that limits the ability of PafE to activate proteasomal degradation in vitro and in vivo.