Engineering More Stable, Selectable Marker-Free Autoluminescent Mycobacteria by One Step

<div><p>In our previous study, we demonstrated that the use of the autoluminescent <i>Mycobacterium tuberculosis</i> as a reporter strain had the potential to drastically reduce the time, effort, animals and costs consumed in evaluation of the activities of drugs and vaccines in live mice. However, the strains were relatively unstable and lost reporter with time without selection. The kanamycin selection marker used wasn’t the best choice as it provides resistance to amino glycosides which are an important class of second line drugs used in tuberculosis treatment. In addition, the marker could limit utility of the strains for screening of new potential drugs or evaluating drug combinations for tuberculosis treatment. Limited selection marker genes for mycobacterial genetic manipulation is a major drawback for such a marker-containing strain in many research fields. Therefore, selectable marker-free, more stable autoluminescent mycobacteria are highly needed. After trying several strategies, we created such mycobacterial strains successfully by using an integrative vector and removing both the resistance maker and integrase genes by Xer site-specific recombination in one step. The corresponding plasmid vectors developed in this study could be very convenient in constructing other selectable marker-free, more stable reporter mycobacteria with diverse applications.</p></div

The plasmids constructed in this study for transforming into mycobacteria to create unmarked autoluminescent mycobacteria.

<p><i>oriE</i>, origin region of <i>E</i>. <i>coli</i>; <i>Hsp60</i>, the strong mycobacterial promoter; <i>luxCDABE</i>, the operon for producing autoluminescence; <i>bla</i>, ampicillin resistance gene; <i>Kan</i>, KAN resistance gene; <i>res</i>, the transposonγδ resolvase action site; <i>attP</i>, mycobacteriophage L5 attachment site; <i>int</i>, integrase gene; <i>int’</i>, the remaining part of integrase gene; <i>attB</i>, attachment site from the mycobacterial genome corresponding to <i>attP</i>; <i>oriM</i>, origin region of mycobacteria; <i>Hyg</i>, HYG resistance gene; <i>dif</i>, the recombinases XerCD action site.</p

Plasmids in this study.

<p>AMP: ampicillin; KAN: kanamycin; HYG: hygromycin.</p><p>Plasmids in this study.</p

Photograph of the more stable, selectable marker-free, autoluminescent BCG (UABCG).

<p>Photograph of the more stable, selectable marker-free, autoluminescent BCG (UABCG).</p

Identification of Lysine Acetylation in <i>Mycobacterium abscessus</i> Using LC–MS/MS after Immunoprecipitation

<i>Mycobacterium abscessus</i> (MAB), which manifests in the pulmonary system, is one of the neglected causes of nontuberculous mycobacteria (NTM) infection. Treatment against MAB is difficult, characterized by its intrinsic antibiotic drug resistance. Lysine acetylation can alter the physiochemical property of proteins in living organisms. This study aimed to determine if this protein post-translational modification (PTM) exists in a clinical isolate <i>M. abscessus</i> GZ002. We used the antiacetyl-lysine immunoprecipitation to enrich the low-abundant PTM proteins, followed by the LC–MS/MS analysis. The lysine acetylome of <i>M. abscessus</i> GZ002 was determined. There were 459 lysine acetylation sites found in 289 acetylated proteins. Lysine acetylation occurred in 5.87% of the <i>M. abscessus</i> GZ002 proteome, and at least 25% of them were growth essential. Aerobic respiration and carbohydrate metabolic pathways of <i>M. abscessus</i> GZ002 were enriched with lysine acetylation. Through bioinformatics analysis, we identified four major acetyl motif logos (K^acY, K^acF, K^acH, and DK^ac). Further comparison of the reported <i>M. tuberculosis</i> (MTB) acetylomes and that of MAB GZ002 revealed several common features between these two species. The lysine residues of several antibiotic-resistance, virulence, and persistence-related proteins were acetylated in both MAB GZ002 and MTB. There were 51 identical acetylation sites in 37 proteins found in common between MAB GZ002 and MTB. Overall, we demonstrate a profile of lysine acetylation in MAB GZ002 proteome that shares similarities with MTB. Interventions that target at these conserved sections may be valuable as anti-NTM or anti-TB therapies

DataSheet_1_Comparative genome analysis reveals high-level drug resistance markers in a clinical isolate of Mycobacterium fortuitum subsp. fortuitum MF GZ001.zip

IntroductionInfections caused by non-tuberculosis mycobacteria are significantly worsening across the globe. M. fortuitum complex is a rapidly growing pathogenic species that is of clinical relevance to both humans and animals. This pathogen has the potential to create adverse effects on human healthcare.MethodsThe MF GZ001 clinical strain was collected from the sputum of a 45-year-old male patient with a pulmonary infection. The morphological studies, comparative genomic analysis, and drug resistance profiles along with variants detection were performed in this study. In addition, comparative analysis of virulence genes led us to understand the pathogenicity of this organism.ResultsBacterial growth kinetics and morphology confirmed that MF GZ001 is a rapidly growing species with a rough morphotype. The MF GZ001 contains 6413573 bp genome size with 66.18 % high G+C content. MF GZ001 possesses a larger genome than other related mycobacteria and included 6156 protein-coding genes. Molecular phylogenetic tree, collinearity, and comparative genomic analysis suggested that MF GZ001 is a novel member of the M. fortuitum complex. We carried out the drug resistance profile analysis and found single nucleotide polymorphism (SNP) mutations in key drug resistance genes such as rpoB, katG, AAC(2')-Ib, gyrA, gyrB, embB, pncA, blaF, thyA, embC, embR, and iniA. In addition, the MF GZ001strain contains mutations in iniA, iniC, pncA, and ribD which conferred resistance to isoniazid, ethambutol, pyrazinamide, and para-aminosalicylic acid respectively, which are not frequently observed in rapidly growing mycobacteria. A wide variety of predicted putative potential virulence genes were found in MF GZ001, most of which are shared with well-recognized mycobacterial species with high pathogenic profiles such as M. tuberculosis and M. abscessus.DiscussionOur identified novel features of a pathogenic member of the M. fortuitum complex will provide the foundation for further investigation of mycobacterial pathogenicity and effective treatment.</p

I-309, IL-8 and MIG over-expression is stimulated by Mtb antigens (ESAT-6 and CFP-10) in active TB.

<p>Expression of I-309 (A), IL-8 (B), and MIG (C) induced by PBS or specific TB antigens in whole blood displayed a gradual increase from health controls (n = 80) to individuals with LTBI (n = 45), to those with active TB (n = 80). Mean values ± standard error are shown, *P < 0.05; **P < 0.01 (Student’s <i>t</i>-test).</p

Up-regulation of I-309, IL-8, and MIG in the serum of active TB patients.

<p>(A-C) quantitative humane cytokine array analysis of peripheral serum samples from health controls (n = 20), individuals with LTBI (n = 20) and patients with active TB (n = 20). Blue, I-309; red, IL-8; yellow, MIG. (D-F) ELISA analysis for the confirmation of I-309, IL-8 and MIG expression in peripheral serum samples from health controls, patients with active TB, and individuals with LTBI or other pulmonary diseases (n = 45 in each group). Mean values ± standard error are shown, * P<0.05; ** P < 0.01 (Student’s <i>t</i>-test).</p

Determination of cut-off values for I-309, IL-8 and MIG in the diagnosis of active TB.

<p>Determination of cut-off values for I-309, IL-8 and MIG in the diagnosis of active TB.</p

The arrangement of cytokines and antigens on TB-relate cytokine and TB-specific antigen array.

<p>Mouse anti-humane-IgG IgG was used as positive control and BSA were used as negative control.</p