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Nature Communications Mar 2024Antimicrobial resistance is a global health threat that requires the development of new treatment concepts. These should not only overcome existing resistance but be...
Antimicrobial resistance is a global health threat that requires the development of new treatment concepts. These should not only overcome existing resistance but be designed to slow down the emergence of new resistance mechanisms. Targeted protein degradation, whereby a drug redirects cellular proteolytic machinery towards degrading a specific target, is an emerging concept in drug discovery. We are extending this concept by developing proteolysis targeting chimeras active in bacteria (BacPROTACs) that bind to ClpC1, a component of the mycobacterial protein degradation machinery. The anti-Mycobacterium tuberculosis (Mtb) BacPROTACs are derived from cyclomarins which, when dimerized, generate compounds that recruit and degrade ClpC1. The resulting Homo-BacPROTACs reduce levels of endogenous ClpC1 in Mycobacterium smegmatis and display minimum inhibitory concentrations in the low micro- to nanomolar range in mycobacterial strains, including multiple drug-resistant Mtb isolates. The compounds also kill Mtb residing in macrophages. Thus, Homo-BacPROTACs that degrade ClpC1 represent a different strategy for targeting Mtb and overcoming drug resistance.
Topics: Mycobacterium smegmatis; Mycobacterium tuberculosis; Proteolysis; Dimerization; Drug Discovery
PubMed: 38443338
DOI: 10.1038/s41467-024-46218-7 -
Microbiology Spectrum May 2024To address intracellular mycobacterial infections, we developed a cocktail of four enzymes that catalytically attack three layers of the mycobacterial envelope. This...
To address intracellular mycobacterial infections, we developed a cocktail of four enzymes that catalytically attack three layers of the mycobacterial envelope. This cocktail is delivered to macrophages, through a targeted liposome presented here as ENTX_001. Endolytix Cocktail 1 (EC1) leverages mycobacteriophage lysin enzymes LysA and LysB, while also including α-amylase and isoamylase for degradation of the mycobacterial envelope from outside of the cell. The LysA family of proteins from mycobacteriophages has been shown to cleave the peptidoglycan layer, whereas LysB is an esterase that hydrolyzes the linkage between arabinogalactan and mycolic acids of the mycomembrane. The challenge of gaining access to the substrates of LysA and LysB provided exogenously was addressed by adding amylase enzymes that degrade the extracellular capsule shown to be present in . This enzybiotic approach avoids antimicrobial resistance, specific receptor-mediated binding, and intracellular DNA surveillance pathways that limit many bacteriophage applications. We show this cocktail of enzymes is bactericidal against both rapid- and slow-growing nontuberculous mycobacteria (NTM) as well as strains. The EC1 cocktail shows superior killing activity when compared to previously characterized LysB alone. EC1 is also powerfully synergistic with standard-of-care antibiotics. In addition to killing of NTM, ENTX_001 demonstrates the rescue of infected macrophages from necrotic death by and . Here, we demonstrate shredding of mycobacterial cells by EC1 into cellular debris as a mechanism of bactericide.IMPORTANCEThe world needs entirely new forms of antibiotics as resistance to chemical antibiotics is a critical problem facing society. We addressed this need by developing a targeted enzyme therapy for a broad range of species and strains within mycobacteria and highly related genera including nontuberculous mycobacteria such as , , as well as . One advantage of this approach is the ability to drive our lytic enzymes through encapsulation into macrophage-targeted liposomes resulting in attack of mycobacteria in the cells that harbor them where they hide from the adaptive immune system and grow. Furthermore, this approach shreds mycobacteria independent of cell physiology as the drug targets the mycobacterial envelope while sidestepping the host range limitations observed with phage therapy and resistance to chemical antibiotics.
Topics: Mycobacterium tuberculosis; Mycobacteriophages; Macrophages; Humans; Nontuberculous Mycobacteria; Liposomes; Anti-Bacterial Agents; Peptidoglycan; Microbial Sensitivity Tests; Endopeptidases; Galactans
PubMed: 38534149
DOI: 10.1128/spectrum.03534-23 -
Frontiers in Microbiology 2023Animal tuberculosis is a significant infectious disease affecting both livestock and wildlife populations worldwide. Effective disease surveillance and characterization...
Animal tuberculosis is a significant infectious disease affecting both livestock and wildlife populations worldwide. Effective disease surveillance and characterization of strains are essential for understanding transmission dynamics and implementing control measures. Currently, sequencing of genomic information has relied on culture-based methods, which are time-consuming, resource-demanding, and concerning in terms of biosafety. This study explores the use of culture-independent long-read whole-genome sequencing (WGS) for a better understanding of epidemiology in African buffaloes (). By comparing two sequencing approaches, we evaluated the efficacy of Illumina WGS performed on culture extracts and culture-independent Oxford Nanopore adaptive sampling (NAS). Our objective was to assess the potential of NAS to detect genomic variants without sample culture. In addition, culture-independent amplicon sequencing, targeting mycobacterial-specific housekeeping and full-length 16S rRNA genes, was applied to investigate the presence of microorganisms, including nontuberculous mycobacteria. The sequencing quality obtained from DNA extracted directly from tissues using NAS is comparable to the sequencing quality of reads generated from culture-derived DNA using both NAS and Illumina technologies. We present a new approach that provides complete and accurate genome sequence reconstruction, culture independently, and using an economically affordable technique.
PubMed: 38075895
DOI: 10.3389/fmicb.2023.1307440 -
Microbiology Spectrum Jun 2024Four species of non-tuberculous mycobacteria (NTM) rated as biosafety level 1 or 2 (BSL-1/BSL-2) organisms and showing higher genomic similarity with () than previous...
Four species of non-tuberculous mycobacteria (NTM) rated as biosafety level 1 or 2 (BSL-1/BSL-2) organisms and showing higher genomic similarity with () than previous comparator species and were subjected to genomic and phenotypic characterization. These species named , , and might represent "missing links" between low-virulent mycobacterial opportunists and the highly virulent obligate pathogen . We confirmed that is the closest NTM species to currently known and found that it has an optimal growth temperature of 32°C-35°C and not 37°C. showed resistance to rifampicin, isoniazid, and ethambutol, whereas and showed resistance to isoniazid and ethambutol. was sensitive to all three first-line TB drugs, and all four species were sensitive to bedaquiline, a third-generation anti-TB drug. Our results suggest these four NTM may be useful models for the identification and study of new anti-TB molecules, facilitated by their culture under non-BSL-3 conditions as compared to was the most virulent of the four species in cellular and mouse infection models. also multiplied in THP-1 cells at 35°C but was growth impaired at 37°C. Genomic comparisons showed that the locus, essential for the secretion of ESX-1 proteins in , was present only in , which was able to secrete ESAT-6 and CFP-10, whereas secretion of these antigens varied in the other species, making the four species interesting examples for studying ESX-1 secretion mechanisms.IMPORTANCEIn this work, we investigated recently identified opportunistic mycobacterial pathogens that are genomically more closely related to () than previously used comparator species and . We confirmed that is the currently closest known species to the tubercle bacilli, represented by and strains. Surprisingly, the reference strain of (DSM 45176), which was purchased as a biosafety level 1 (BSL-1)-rated organism, was the most virulent of the four species in the tested cellular and mouse infection models, suggesting that a BSL-2 rating might be more appropriate for this strain than the current BSL-1 rating. Our work establishes the four NTM species as interesting study models to obtain new insights into the evolutionary mechanisms and phenotypic particularities of mycobacterial pathogens that likely have also impacted the evolution of the key pathogen .
Topics: Mycobacterium tuberculosis; Antitubercular Agents; Nontuberculous Mycobacteria; Humans; Genome, Bacterial; Genomics; Phenotype; Microbial Sensitivity Tests; Mycobacterium Infections, Nontuberculous; Phylogeny; Animals; Tuberculosis; Drug Resistance, Bacterial; Mice
PubMed: 38700329
DOI: 10.1128/spectrum.04126-23 -
Human Vaccines & Immunotherapeutics Dec 2023The immune escape mutations of SARS-CoV-2 variants emerged frequently, posing a new challenge to weaken the protective efficacy of current vaccines. Thus, the...
The immune escape mutations of SARS-CoV-2 variants emerged frequently, posing a new challenge to weaken the protective efficacy of current vaccines. Thus, the development of novel SARS-CoV-2 vaccines is of great significance for future epidemic prevention and control. We herein reported constructing the attenuated () as a bacterial surface display system to carry the spike (S) and nucleocapsid (N) of SARS-CoV-2. To mimic the native localization on the surface of viral particles, the S or N antigen was fused with truncated PE_PGRS33 protein, which is a transportation component onto the cell wall of (). The sub-cellular fraction analysis demonstrated that S or N protein was exactly expressed onto the surface (cell wall) of the recombinant . After the immunization of the -based COVID-19 vaccine candidate in mice, S or N antigen-specific T cell immune responses were effectively elicited, and the subsets of central memory CD4+ T cells and CD8+ T cells were significantly induced. Further analysis showed that there were some potential cross-reactive CTL epitopes between SARS-CoV-2 and . Overall, our data provided insights that -based bacterial surface display system could be a suitable vector for developing T cell-based vaccines against SARS-CoV-2 and other infectious diseases.
Topics: Mice; Humans; Animals; Mycobacterium smegmatis; COVID-19 Vaccines; COVID-19; SARS-CoV-2; Spike Glycoprotein, Coronavirus
PubMed: 36785935
DOI: 10.1080/21645515.2023.2171233 -
Clinical Infectious Diseases : An... Aug 2023Nontuberculous mycobacteria (NTM) cause pulmonary (PNTM) and extrapulmonary (ENTM) disease. Infections are difficult to diagnose and treat, and exposures occur in...
BACKGROUND
Nontuberculous mycobacteria (NTM) cause pulmonary (PNTM) and extrapulmonary (ENTM) disease. Infections are difficult to diagnose and treat, and exposures occur in healthcare and community settings. In the United States, NTM epidemiology has been described largely through analyses of microbiology data from health departments, electronic health records, and administrative data. We describe findings from a multisite pilot of active, laboratory- and population-based NTM surveillance.
METHODS
The Centers for Disease Control and Prevention's Emerging Infections Program conducted NTM surveillance at 4 sites (Colorado, 5 counties; Minnesota, 2 counties; New York, 2 counties; and Oregon, 3 counties [PNTM] and statewide [ENTM]) from 1 October 2019 through 31 March 2020. PNTM cases were defined using published microbiologic criteria. ENTM cases required NTM isolation from a nonpulmonary specimen, excluding stool and rectal swabs. Patient data were collected via medical record review.
RESULTS
Overall, 299 NTM cases were reported (PNTM: 231, 77%); Mycobacterium avium complex was the most common species group. Annualized prevalence was 7.5/100 000 population (PNTM: 6.1/100 000; ENTM: 1.4/100 000). Most patients had signs or symptoms in the 14 days before positive specimen collection (ENTM: 62, 91.2%; PNTM: 201, 87.0%). Of PNTM cases, 145 (62.8%) were female and 168 (72.7%) had underlying chronic lung disease. Among ENTM cases, 29 (42.6%) were female, 21 (30.9%) did not have documented underlying conditions, and 26 (38.2%) had infection at the site of a medical device or procedure.
CONCLUSIONS
Active, population-based NTM surveillance will provide data for monitoring the burden of disease and characterize affected populations to inform interventions.
Topics: Humans; Female; Male; Mycobacterium Infections, Nontuberculous; Nontuberculous Mycobacteria; Lung; Lung Diseases; Oregon
PubMed: 37083882
DOI: 10.1093/cid/ciad214 -
Tropical Medicine and Health Sep 2023CNS manifestations represent an emerging facet of NTM infection with significant mortality. Due to protean presentation and low index of suspicion, many cases are often... (Review)
Review
BACKGROUND
CNS manifestations represent an emerging facet of NTM infection with significant mortality. Due to protean presentation and low index of suspicion, many cases are often treated erroneously as tubercular meningitis or fungal infections.
OBJECTIVES
Literature on NTM CNS disease is scarce, with most available data on pulmonary disease. This systematic review aimed to evaluate the epidemiology, clinical presentation, diagnostic modalities, and predictors of outcome in CNS NTM infection.
METHODS
The literature search was performed in major electronic databases (PubMed, Google Scholar, and Scopus) using keywords "CNS," "Central nervous system," "brain abscess," "meningitis," "spinal," "Nontuberculous mycobacteria," "NTM". All cases of CNS NTM infection reported between January 1980 and December 2022 were included.
RESULTS
A total of 77 studies (112 cases) were included in the final analysis. The mean age of all patients was 38 years, with most patients male (62.5%). Mycobacterium avium complex (MAC) was the most common aetiology, followed by M. fortuitum and M. abscessus (34.8%, 21.4% and 15.2%, respectively). The disseminated disease was found in 33% of cases. HIV (33.9%) and neurosurgical hardware (22.3%) were the common risk factors. Intracranial abscess (36.6%) and leptomeningeal enhancement (28%) were the most prevalent findings in neuroimaging. The overall case fatality rate was 37.5%. On multivariate analysis, male gender (adjusted OR 2.4, 95% CI 1.2-7.9) and HIV (adjusted OR 3.7, 95% CI 1.8-6.1) were the independent predictors of mortality). M. fortuitum infection was significantly associated with increased survival (adjusted OR 0.18, 95% CI (0.08-0.45), p value 0.012).
CONCLUSIONS
Current evidence shows the emerging role of rapid-grower NTM in CNS disease. Male gender and HIV positivity were associated with significant mortality, while M fortuitum carries favourable outcomes.
PubMed: 37749661
DOI: 10.1186/s41182-023-00546-4 -
BMC Pulmonary Medicine Jul 2023The long-term exercise tolerance changes in patients with nontuberculous mycobacterial pulmonary disease (NTM-PD) are of great interest because of its chronic course.... (Observational Study)
Observational Study
BACKGROUND
The long-term exercise tolerance changes in patients with nontuberculous mycobacterial pulmonary disease (NTM-PD) are of great interest because of its chronic course. This study aimed to characterize the associations between changes over time in six-minute walking test (6MWT) parameters and clinical parameters in patients with NTM-PD.
METHODS
Overall, 188 patients with NTM-PD, visiting outpatient clinics at Keio University Hospital from April 2012 to March 2020 were included in the study. Data were collected using the St. George's Respiratory Questionnaire (SGRQ), pulmonary function test (PFT), blood tests, and the 6MWT at registration and at least once after that. The association of the anchors and clinical indicators with the 6MWT parameters was assessed.
RESULTS
The median age [interquartile range] of the patients was 67 [63-74] years. The median baseline six-minute walk distance (6MWD) and final Borg scale (FBS) were 413 [361-470] m and 1 [0-2], respectively. In the correlation analysis, ΔSGRQ total/year (yr), Δforced vital capacity (FVC, % predicted)/yr, Δforced expiratory volume in 1 s (FEV, % predicted)/yr, and Δdiffusing capacity for carbon monoxide (DL, % predicted)/yr correlated with both Δ6MWD/yr and ΔFBS/yr in the longitudinal analysis (|Rho| > 0.20). When stratified into three quantiles of changes in each anchor, the 6MWT parameters worsened over time in the bottom 25% group by mixed-effects model. Specifically, Δ6MWD was affected by SGRQ activity, SGRQ impacts, PFT (FVC, FEV, and DL), and C-reactive protein (CRP). ΔFBS was affected by all SGRQ components, total score, and PFT. Anchor scores and variables at baseline that worsened Δ6MWD were higher SGRQ scores, lower FVC (% predicted), lower DL (% predicted), higher Krebs von den Lungen-6, old age, and undergoing treatment at registration. Similarly, these clinical parameters and elevated CRP, excluding undergoing treatment at registration, worsened ΔFBS.
CONCLUSIONS
The decreased walking distance and exacerbation of dyspnea on exertion over time in patients with NTM-PD may reflect a deterioration of health-related quality of life and pulmonary function. Thus, the change in 6MWT over time can be used as an indicator to accurately assess the patient's condition and tailor their healthcare environment.
Topics: Aged; Humans; Lung; Lung Diseases; Mycobacterium Infections, Nontuberculous; Pulmonary Disease, Chronic Obstructive; Quality of Life; Walk Test; Walking; Middle Aged
PubMed: 37415094
DOI: 10.1186/s12890-023-02528-y -
Bioorganic & Medicinal Chemistry Letters Aug 2023A series of 2-(tetrazol-5-yl)sulfonylacetamide derivatives were synthesized and evaluated for their in vitro inhibitory activity against Mycobacterium tuberculosis (Mtb)...
A series of 2-(tetrazol-5-yl)sulfonylacetamide derivatives were synthesized and evaluated for their in vitro inhibitory activity against Mycobacterium tuberculosis (Mtb) and Mycobacterium marinum (Mm). The most active compounds exhibited in vitro MIC values of 1.25 μg/mL against Mtb, but they were less effective against Mm (MIC ≥ 10 μg/mL). Despite the most active compounds having favourable physicochemical properties and one of them having a half-life of ∼3 h when incubated with mouse liver microsomes, two representative highly active compounds showed strong chemical reactivity to cysteine derivatives, as surrogate in vivo sulfur-centred nucleophiles, indicating excessive electrophilicity, and therefore, likely indiscriminate chemical reactivity in vivo, representing an unacceptably high risk of general toxicity, and low likelihood of being therapeutically effective.
Topics: Animals; Mice; Mycobacterium tuberculosis; Antitubercular Agents; Mycobacterium marinum; Microbial Sensitivity Tests; Structure-Activity Relationship
PubMed: 37369331
DOI: 10.1016/j.bmcl.2023.129391 -
Frontiers in Medicine 2023Targeted next-generation sequencing (tNGS) has emerged as a rapid diagnostic technology for identifying a wide spectrum of pathogens responsible for pulmonary infections.
BACKGROUND
Targeted next-generation sequencing (tNGS) has emerged as a rapid diagnostic technology for identifying a wide spectrum of pathogens responsible for pulmonary infections.
METHODS
Sputum samples were collected from patients unable or unwilling to undergo bronchoalveolar lavage. These samples underwent tNGS analysis to diagnose pulmonary infections. Retrospective analysis was performed on clinical data, and the clinical efficacy of tNGS was compared to conventional microbiological tests (CMTs).
RESULTS
This study included 209 pediatric and adult patients with confirmed pulmonary infections. tNGS detected 45 potential pathogens, whereas CMTs identified 23 pathogens. The overall microbial detection rate significantly differed between tNGS and CMTs (96.7% vs. 36.8%, < 0.001). Among the 76 patients with concordant positive results from tNGS and CMTs, 86.8% (66/76) exhibited full or partial agreement. For highly pathogenic and rare/noncolonized microorganisms, tNGS, combined with comprehensive clinical review, directly guided pathogenic diagnosis and antibiotic treatment in 21 patients. This included infections caused by complex, certain atypical pathogens, Aspergillus, and nontuberculous Mycobacteria. Among the enrolled population, 38.8% (81/209) of patients adjusted their treatment based on tNGS results. Furthermore, tNGS findings unveiled age-specific heterogeneity in pathogen distribution between children and adults.
CONCLUSION
CMTs often fall short in meeting the diagnostic needs of pulmonary infections. This study highlights how tNGS of sputum samples from patients who cannot or will not undergo bronchoalveolar lavage yield valuable insights into potential pathogens, thereby enhancing the diagnosis of pulmonary infections in specific cases.
PubMed: 38179267
DOI: 10.3389/fmed.2023.1321515