Characterization of Mycobacterium leprae diguanylate cyclases

Includes bibliographical references.2016 Summer.Mycobacterium leprae is the causative agent of leprosy, which is still a major health problem in several developing countries. Management of leprosy has been challenging because of the long incubation period of the disease and the development of a spectrum of clinical manifestations. Leprosy treatment is further complicated by the development of drug resistance. Knowledge of infection mechanisms and pathogenesis of leprosy is still limited. These fundamental gaps significantly limit the development of disease management, including treatment and prevention. Although M. leprae is an obligate intracellular pathogen, this bacterium must possess mechanisms to adapt to different host defenses or cell types. The discovery of cyclic diguanylate monophosphate (c-di-GMP) and its potential roles in bacteria as a second messenger to regulate several cellular activities responding to environmental stimuli have stimulated an interest on c-di-GMP studies in Mycobacterium spp., especially M. leprae which has massive gene decay but still harbors several potential proteins functioning as diguanylate cyclases. The hypothesis of this study is that M. leprae has the ability to synthesize c-di-GMP. This study evaluated M. leprae’s potential to synthesize c-di-GMP. Bioinformatics analyses were performed to identify proteins that are involved in c-di-GMP synthesis (diguanylate cyclase, DGC) and turnover (phosphodiesterase, PDE). Bioinformatics revealed that M. leprae harbors a putative DGC-PDE protein (ML1750c) and two putative DGC proteins (ML1419c and ML0397c). Interestingly, homologues of ML1419c and ML0397c are not encoded by Mycobacterium tuberculosis. The M. leprae genes ml1419c, ml0397c, and ml1750c were cloned and expressed in Pseudomonas aeruginosa PAO1 and Escherichia coli BL21(DE3) pLysS. Assays for well-described phenotypes of c-di-GMP production (colony morphology, macromolecule synthesis, and biofilm formation) were performed with the recombinant clones. Direct measurement of c-di-GMP levels was accomplished by LC-MS. RNA was extracted from M. leprae infected mouse footpads, and expression of ml1419c and ml0397c was measured by droplet digital PCR. DGC proteins produced by M. leprae in armadillo tissue were also monitored with protein-specific polyclonal antibodies. Phenotypic studies revealed that recombinant expression of ml1419c in P. aeruginosa altered colony morphology, motility, and biofilm formation, and the recombinant expression of ml0397c increased curli and cellulose production of E. coli. These phenotypes were consistent with increased DGC activity and c-di-GMP production. LC-MS analyses confirmed increased c-di-GMP production by ML1419c and ML0397c. In vivo gene expression studies revealed that ml1419c, ml0397c, and ml1750c are expressed by M. leprae during infection. Additionally, ML1419c and ML1750c proteins were clearly identified in whole cell sonicate of armadillo derived M. leprae. This study demonstrated that M. leprae has significant potential to produce c-di-GMP. ML1419c and ML0397c were confirmed as functional DGCs. This study is significant because it provides evidence that M. leprae has the ability to produce c-di-GMP. Furthermore, these studies will pave the way for future research to characterize the biological roles of c-di-GMP in M. leprae and the pathogenesis of leprosy. Continued studies to elucidate the biological roles and the environmental signals for ML1419c, ML0397c, and ML1750c are being performed. These efforts are directed at defining the function of c-di-GMP in M. leprae. It is anticipated that these future efforts along with the data in this dissertation will shed light on the signaling mechanisms that respond to environmental changes experienced by M. leprae

Performance of RGM Medium for the Isolation of Nontuberculous Mycobacteria from Respiratory Specimens from Non-Cystic Fibrosis Patients

A new selective medium for rapidly growing mycobacteria (RGM medium) was evaluated on respiratory specimens from non-cystic fibrosis patients and compared to the Mycobacterial Growth Indicator Tube (MGIT) system and Middlebrook 7H11 agar for the isolation of all nontuberculous mycobacteria (NTM). A total of 203 mucolyzed respiratory specimens collected from 163 patients were inoculated on RGM medium and incubated at both 30°C (RGM30) and 35°C (RGM35) over a 28-day period. N-acetyl-L-cysteine–sodium hydroxide (NALC-NaOH) decontaminated specimens were inoculated into a MGIT and Middlebrook 7H11 agar and incubated at 35°C for 42 days. NTM were identified by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) or gene sequencing. A total of 133 NTM isolates were recovered overall from 101 (49.8%) specimens collected from 85 (52.1%) patients by a combination of all culture methods. The sensitivity of RGM30 for the recovery of NTM was significantly higher than either MGIT system (76.7% versus 59.4%; p = 0.01) or Middlebrook 7H11 agar (76.7% versus 47.4%; p = 0.0001) alone but it was not significantly different from acid-fast bacilli culture (AFC) which includes both MGIT and Middlebrook 7H11 agar (76.7% versus 63.9%; p = 0.0647). RGM35 had significantly lower sensitivity compared to the MGIT system (49.6% versus 59.4%; p = 0.0367) and AFC (49.6% versus 63.9%; p = 0.0023). RGM medium was highly effective at inhibiting the growth of non-mycobacterial organisms in the respiratory specimens with breakthrough contamination rates of 5.4% and 4.4% for RGM30 and RGM35, respectively

Full-length 16S rDNA sequencing based on Oxford Nanopore Technologies revealed the association between gut-pharyngeal microbiota and tuberculosis in cynomolgus macaques

Abstract Tuberculosis (TB) is an infectious disease caused by the Mycobacterium tuberculosis complex (Mtbc), which develops from asymptomatic latent TB to active stages. The microbiome was purposed as a potential factor affecting TB pathogenesis, but the study was limited. The present study explored the association between gut-pharyngeal microbiome and TB stages in cynomolgus macaques using the full-length 16S rDNA amplicon sequencing based on Oxford Nanopore Technologies. The total of 71 macaques was divided into TB (−) control, TB (+) latent and TB (+) active groups. The differential abundance analysis showed that Haemophilus hemolyticus was decreased, while Prevotella species were increased in the pharyngeal microbiome of TB (+) macaques. In addition, Eubacterium coprostanoligenes in the gut was enriched in TB (+) macaques. Alteration of these bacteria might affect immune regulation and TB severity, but details of mechanisms should be further explored and validated. In summary, microbiota may be associated with host immune regulation and affect TB progression. The findings suggested the potential mechanisms of host-microbes interaction, which may improve the understanding of the role of microbiota and help develop therapeutics for TB in the future