Eugene L. Opie, 1910

Eugene L. Opie, M.D. Inflammation Lecture delivered February 10th, 1910https://digitalcommons.rockefeller.edu/harvey-lectures/1014/thumbnail.jp

DNA repair systems and the pathogenesis of Mycobacterium tuberculosis: varying activities at different stages of infection

Mycobacteria, including most of all MTB (Mycobacterium tuberculosis), cause pathogenic infections in humans and, during the infectious process, are exposed to a range of environmental insults, including the host's immune response. From the moment MTB is exhaled by infected individuals, through an active and latent phase in the body of the new host, until the time they reach the reactivation stage, MTB is exposed to many types of DNA-damaging agents. Like all cellular organisms, MTB has efficient DNA repair systems, and these are believed to play essential roles in mycobacterial pathogenesis. As different stages of infection have great variation in the conditions in which mycobacteria reside, it is possible that different repair systems are essential for progression to specific phases of infection. MTB possesses homologues of DNA repair systems that are found widely in other species of bacteria, such as nucleotide excision repair, base excision repair and repair by homologous recombination. MTB also possesses a system for non-homologous end-joining of DNA breaks, which appears to be widespread in prokaryotes, although its presence is sporadic within different species within a genus. However, MTB does not possess homologues of the typical mismatch repair system that is found in most bacteria. Recent studies have demonstrated that DNA repair genes are expressed differentially at each stage of infection. In the present review, we focus on different DNA repair systems from mycobacteria and identify questions that remain in our understanding of how these systems have an impact upon the infection processes of these important pathogens

DNA replication fidelity in Mycobacterium tuberculosis is mediated by an ancestral prokaryotic proofreader

The DNA replication machinery is an important target for antibiotic development for increasingly drug resistant bacteria including Mycobacterium tuberculosis1. While blocking DNA replication leads to cell death, disrupting the processes used to ensure replication fidelity can accelerate mutation and the evolution of drug resistance. In E. coli, the proofreading subunit of the replisome, the ε-exonuclease, is essential for high fidelity DNA replication2; however, we find that it is completely dispensable in M. tuberculosis. Rather, the mycobacterial replicative polymerase, DnaE1, encodes a novel editing function that proofreads DNA replication, mediated by an intrinsic 3′-5′ exonuclease activity within its PHP domain. Inactivation of the DnaE1 PHP domain increases the mutation rate by greater than 3,000 fold. Moreover, phylogenetic analysis of DNA replication proofreading in the bacterial kingdom suggests that E. coli is a phylogenetic outlier and that PHP-domain mediated proofreading is widely conserved and indeed may be the ancestral prokaryotic proofreader

Effect of the addition of rocuronium to 2% lignocaine in peribulbar block for cataract surgery

Background and Aims: Peribulbar anesthesia is associated with delayed orbital akinesia compared with retrobulbar anesthesia. To test the hypothesis that rocuronium added to a mixture of local anesthetics (LAs) could improve speed of onset of akinesia in peribulbar block (PB), we designed this study. This study examined the effects of adding rocuronium 5 mg to 2% lignocaine with adrenaline to note orbital and eyelid akinesia in patients undergoing cataract surgery. Material and Methods: In a prospective, randomized, double-blind study, 100 patients were equally randomized to receive a mixture of 0.5 ml normal saline, 6 ml lidocaine 2% with adrenaline and hyaluronidase 50 IU/ml (Group I), a mixture of rocuronium 0.5 ml (5 mg), 6 ml lidocaine 2% with adrenaline and hyaluronidase 50 IU/ml (Group II). Orbital akinesia was assessed on a 0–8 score (0 = no movement, 8 = normal) at 2 min intervals for 10 min. Time to adequate anesthesia was also recorded. Results are presented as mean ± standard deviation. Results: Rocuronium group demonstrated significantly better akinesia scores than control group at 2 min intervals post-PB (significant P value obtained). No significant complications were recorded. Rocuronium added to a mixture of LA improved the quality of akinesia in PB and reduced the need for supplementary injections. Conclusion: The addition of rocuronium 5 mg to a mixture of lidocaine 2% with adrenaline and hyaluronidase 50 IU/ml shortened the onset time of peribulbar anesthesia in patients undergoing cataract surgery without causing adverse effects

A distinct physiological role of MutY in mutation prevention in mycobacteria

Oxidative damage to DNA results in the occurrence of 7,8-dihydro-B-oxoguanine (8-oxoG) in the genome. In eubacteria, repair of such damage is initiated by two major base-excision repair enzymes, MutM and MutY. We generated a MutY-deficient strain of Mycobacterium smegmatis to investigate the role of this enzyme in DNA repair. The MutY deficiency in M. smegmatis did not result in either a noteworthy susceptibility to oxidative stress or an increase in the mutation rate. However, rifampicin resistant isolates of the MutY-deficient strain showed distinct mutations in the rifampicin-resistance-determining region of rpoB. Besides the expected C to A (or G to T) mutations, an increase in A to C (or T to G) mutations was also observed. Biochemical characterization of mycobacterial MutY (M. smegmatis and M. tuberculosis) revealed an expected excision of A opposite 8-oxoG in DNA. Additionally, excision of G and T opposite 8-oxoG was detected. MutY formed complexes with DNA containing 8-oxoG: A, 8-oxoG: G or 8-oxoG: T but not 8-oxoG : C pairs. Primer extension reactions in cell-free extracts of M. smegmatis suggested error-prone incorporation of nucleotides into the DNA. Based on these observations, we discuss the physiological role of MutY in specific mutation prevention in mycobacteria

Novel insertion and deletion mutants of RpoB that render Mycobacterium smegmatis RNA polymerase resistant to rifampicin-mediated inhibition of transcription

The startling increase in the occurrence of rifampicin (Rif) resistance in the clinical isolates of Mycobacterium tuberculosis worldwide is posing a serious concern to tuberculosis management. The majority of Rif resistance in bacteria arises from mutations in the RpoB subunit of the RNA polymerase. We isolated M. smegmatis strains harbouring either an insertion (6 aa) or a deletion (10 aa) in their RpoB proteins. Although these strains showed a compromised fitness for growth in 7H9 Middlebrook medium, their resistance to Rif was remarkably high. The attenuated growth of the strains correlated with decreased specific activities of the RNA polymerases from the mutants. While the RNA polymerases from the parent or a mutant strain (harbouring a frequently occurring mutation, H442Y, in RpoB) were susceptible to Rif-mediated inhibition of transcription from calf thymus DNA, those from the insertion and deletion mutants were essentially refractory to such inhibition. Three-dimensional structure modelling revealed that the RpoB amino acids that interact with Rif are either deleted or unable to interact with Rif due to their unsuitable spatial positioning in these mutants. We discuss possible uses of the RpoB mutants in studying transcriptional regulation in mycobacteria and as potential targets for drug design