Richard L. Friedman

Mycobacterium tuberculosis Eis Regulates Autophagy, Inflammation, and Cell Death through Redox-dependent Signaling

The “enhanced intracellular survival” (eis) gene of Mycobacterium tuberculosis (Mtb) is involved in the intracellular survival of M. smegmatis. However, its exact effects on host cell function remain elusive. We herein report that Mtb Eis plays essential roles in modulating macrophage autophagy, inflammatory responses, and cell death via a reactive oxygen species (ROS)-dependent pathway. Macrophages infected with an Mtb eis-deletion mutant H37Rv (Mtb-Δeis) displayed markedly increased accumulation of massive autophagic vacuoles and formation of autophagosomes in vitro and in vivo. Infection of macrophages with Mtb-Δeis increased the production of tumor necrosis factor-α and interleukin-6 over the levels produced by infection with wild-type or complemented strains. Elevated ROS generation in macrophages infected with Mtb-Δeis (for which NADPH oxidase and mitochondria were largely responsible) rendered the cells highly sensitive to autophagy activation and cytokine production. Despite considerable activation of autophagy and proinflammatory responses, macrophages infected with Mtb-Δeis underwent caspase-independent cell death. This cell death was significantly inhibited by blockade of autophagy and c-Jun N-terminal kinase-ROS signaling, suggesting that excessive autophagy and oxidative stress are detrimental to cell survival. Finally, artificial over-expression of Eis or pretreatment with recombinant Eis abrogated production of both ROS and proinflammatory cytokines, which depends on the N-acetyltransferase domain of the Eis protein. Collectively, these data indicate that Mtb Eis suppresses host innate immune defenses by modulating autophagy, inflammation, and cell death in a redox-dependent manner.

Mycobacterium tuberculosis produces pili during human infection

Mycobacterium tuberculosis is responsible for nearly 3 million human deaths worldwide every year. Understanding the mechanisms and bacterial factors responsible for the ability of M. tuberculosis to cause disease in humans is critical for the development of improved treatment strategies. Many bacterial pathogens use pili as adherence factors to colonize the host. We discovered that M. tuberculosis produces fine (2- to 3-nm-wide), aggregative, flexible pili that are recognized by IgG antibodies contained in sera obtained from patients with active tuberculosis, indicating that the bacilli produce pili or pili-associated antigen during human infection. Purified M. tuberculosis pili (MTP) are composed of low-molecular-weight protein subunits encoded by the predicted M. tuberculosis H37Rv ORF, designated Rv3312A. MTP bind to the extracellular matrix protein laminin in vitro, suggesting that MTP possess adhesive properties. Isogenic mtp mutants lost the ability to produce Mtp in vitro and demonstrated decreased laminin-binding capabilities. MTP shares morphological, biochemical, and functional properties attributed to bacterial pili, especially with curli amyloid fibers. Thus, we propose that MTP are previously unidentified host-colonization factors of M. tuberculosis.

Identification of a Mycobacterium tuberculosis Gene That Enhances Mycobacterial Survival in Macrophages

Intracellular survival plays a central role in the pathogenesis of Mycobacterium tuberculosis. To identify M. tuberculosis genes required for intracellular survival within macrophages, an M. tuberculosis H37Rv plasmid library was constructed by using the shuttle vector pOLYG. This plasmid library was electroporated into Mycobacterium smegmatis 1-2c, and the transformants were used to infect the human macrophage-like cell line U-937. Because M. smegmatis does not readily survive within macrophages, any increased intracellular survival is likely due to cloned M. tuberculosis H37Rv DNA. After six sequential passages of M. smegmatis transformants through U-937 cells, one clone (p69) was enriched more than 70% as determined by both restriction enzyme and PCR analyses. p69 demonstrated significantly enhanced survival compared to that of the vector control, ranging from 2.4- to 5.3-fold at both 24 and 48 h after infection. DNA sequence analysis revealed three open reading frames (ORFs) in the insert of p69. ORF2 (1.2 kb) was the only one which contained a putative promoter region and a ribosome-binding site. Deletion analysis of the p69 insert DNA showed that disruption of ORF2 resulted in complete loss of the enhanced intracellular survival phenotype. This gene was named the enhanced intracellular survival (eis) gene. By using an internal region of eis as a probe for Southern analysis, eis was found in the genomic DNA of various M. tuberculosis strains and of Mycobacterium bovis BCG but not in that of M. smegmatis or 10 other nonpathogenic mycobacterial species. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis showed that all M. smegmatis eis-containing constructs expressed a unique protein of 42 kDa, the predicted size of Eis. The expression of this 42-kDa protein directly correlated to the enhanced survival of M. smegmatis p69 in U-937 cells. These results suggest a possible role for eis and its protein product in the intracellular survival of M. tuberculosis.

Pertussis: the disease and new diagnostic methods.

Bordetella pertussis, the causative agent of whooping cough, produces an acute and chronic respiratory infection in infants and young children. B. pertussis is still a major health problem of young children throughout the world even though effective immunization against whooping cough is available. While predominantly a childhood disease, it has been reported also to be a cause of persistent cough in adults. This review discusses the numerous bacterial virulence factors that may play roles in the pathogenesis of pertussis and in immunity to infection. The present problems with pertussis diagnosis, recent advances, and future prospects for new and improved rapid diagnostics tests also are explored.

Intracellular survival of virulent Bordetella pertussis in human polymorphonuclear leukocytes.

Little is known regarding the interaction of Bordetella pertussis with polymorphonuclear leukocytes (PMNL) or the role PMNL play as an initial line of defense against B. pertussis infection. An in vitro system was developed to establish conditions for the study of phagocytosis and killing of virulent B. pertussis by human PMNL. Phagocytosis of B. pertussis strains BP504, BP165, and BP338 occurred by opsonization with anti‐B. pertussis antibody, while autologous normal human sera did not induce significant phagocytosis. In PMNL bacterial killing assays virulent B. pertussis strains survived PMNL bactericidal activities while Escherichia coli controls were readily killed. Electron microscopy studies using acid phosphatase as a lysosomal marker strongly suggested that B. pertussis inhibits phagosome‐lysosome fusion in PMNL. These results indicate that virulent B. pertussis strains are capable of surviving intracellularly within PMNL and that such survival may be due to inhibition of phagosome‐lysosome fusion.

Molecular characterization of catalase from Bordetella pertussis: identification of the katA promoter in an upstream insertion sequence

In this report we evaluate the role of catalase in the survival of Bordetella pertussis within human polymorphonuclear leukocytes (PMNs). Crude extracts of B. pertussis exhibited a single catalase activity when subjected to non‐denaturing polyacrylamide gel electrophoresis and assayed for catalase activity. A plasmid containing B. pertussis katA was identified by complementation of UM255, a catalase‐deficient strain of Escherichia coli. The nucleotide sequence of katA predicts a 55kDa protein that shares homology with a class of haem‐containing catalases found in both eubacteria and eukaryotes. Analysis of the nucleotide sequence upstream of katA revealed the presence of a copy of IS481, a B. pertussis‐specific insertion sequence. The start site of transcription of katA was mapped to a T residue in IS481 by primer extension analysis performed with B. pertussis RNA and a katA‐specific primer. A catalase‐deficient strain of B. pertussis, DD900, was constructed by gene replacement. DD900 was more sensitive to killing by 1 and 5 mM H2O2 than the parental strain, BP339. However, there was no difference in the ability of DD900 and BP339 to survive for 2h in human PMNs. This suggests that catalase plays no significant role in the survival of B. pertussis within PMNs

Characterization of the gene encoding Superoxide dismutase of Bordetella pertussis and construction of a SOD-deficient mutant

The Bordetella pertussis gene sodB, encoding superoxide dismutase (SOD), was cloned by complementation of an Escherichia coli sodAsodB double mutant. The nucleotide sequence of sodB predicted a 21-kDa protein with homology to manganese- and iron-containing SODs from other organisms. Examination of SOD activity on gels suggests that B. pertussis extracts have a single SOD containing Fe3+ as a prosthetic group. A SOD-deficient mutant was obtained by insertional inactivation of sodB in B. pertussis, confirming that there is only one SOD in this organism.

Additional Antianginal and Anti-Ischemic Efficacy of Mibefradil in Patients Pretreated With a Beta Blocker for Chronic Stable Angina Pectoris

This study assessed the safety, tolerability, and efficacy of mibefradil when added to beta-blocker monotherapy in patients with chronic stable angina pectoris. Two hundred five patients were randomized to receive double-blind treatment with either placebo (n = 70), mibefradil 25 mg (n = 67), or mibefradil 50 mg (n = 68) for 2 weeks. Exercise tolerance tests (ETTs) were performed at the end of the run-in (baseline) and double-blind treatment periods, and patients maintained an anginal diary. Compared with placebo, treatment with mibefradil 50 mg resulted in significant increases in exercise duration (36 +/- 51 seconds; p = 0.036), time to onset of angina (48 +/- 65 seconds; p = 0.002), and time to persistent 1-mm ST-segment depression (47 +/- 77 seconds; p = 0.004). Greater reductions in heart rate, blood pressure, and the rate-pressure product were more apparent at each stage of the ETT in the 50-mg mibefradil group than in the placebo group. Daily treatment with mibefradil 50 mg was associated with a significant decrease in the number of weekly anginal attacks (-2.1 +/- 4.0, p = 0.020) compared with placebo. The addition of mibefradil to existing beta-blocker therapy was well tolerated. Dizziness was the most frequently reported adverse event in the mibefradil 50-mg dose, and occurred with an incidence of 4.4%. The addition of mibefradil 50 mg, administered once daily, to patients on stable beta-blocker therapy produced additive antianginal and anti-ischemic effects and was well tolerated.

Isolation and characterization of the recA gene of Bordetella pertussis.

This report describes the detection and cloning of the Bordetella pertussis recA gene. Escherichia coli clones having recombinant plasmids containing the B. pertussis recA gene were isolated by complementing an E. coli RecA− mutants inability to survive in the presence of methylmethanesulphonate (MMS). This gene was shown to complement the deficiency of E. coli RecA− strains to tolerate the DNA‐damaging effects of both a chemical agent and ultraviolet light (u.v.). Deletion mapping experiments localized the gene to a 2.5kb Stul‐EcoRI fragment, and expression of the gene in E. coli resulted in the production of a 40kD protein. These data strongly suggest that a region of the B. pertussis chromosome that encodes RecA‐like activity has been isolated and cloned.

Identification of Btr-regulated genes using a titration assay. Search for a role for this transcriptional regulator in the growth and virulence of Bordetella pertussis.

Bordetella pertussis is the causative agent of the respiratory disease pertussis or whopoping cough. Btr, an oxygen-responsive transcriptional regulator of B. pertussis, is homologous to the FNR protein of E. coli. Using a murine respiratory model, we observed in the present study that Btr is important in growth and survival of B. pertussis in vivo. A titration assay was developed that identified genes containing Btr binding sites including B. pertussis sodB and btr, E. coli aspA and a new B. pertussis gene, brg1. The brg1 gene encodes a protein similar to the LysR family of transcriptional regulators and its expression is activated threefold by Btr under anaerobic growth conditions but unaffected by Btr aerobically. The nucleotide sequence flanking brg1 encodes proteins with similarity to various metabolic enzymes. Putative overlapping promoters and a Btr binding site (FNR box) were identified in the DNA sequence between brg1 and the adjacent genes. These intervening sequences may represent sites for regulation by Btr and Brg1.