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Heterologous expression of the M. abscessus CBASS system confers phage TM4 resistance in M. smegmatis

BMC Microbiology volume 25, Article number: 519 (2025) Cite this article

Abstract

Phage therapy for multidrug-resistant Mycobacterium abscessus is constrained by the narrow lytic spectrum of phages due to unknown antiphage mechanisms. Bacteria deploy various defense strategies to prevent phage infection, but few have been comprehensively characterized in mycobacteria. Mycobacteriophage TM4 successfully delivers DNA into M. abscessus but fails to establish infection. Bioinformatic analyses predicted three candidate phage defense systems and multiple individual putative defense proteins in M. abscessus. Among them, Mab_2091, Mab_2092, and Mab_2093, designated CmaABC, are components of a cyclic oligonucleotide-based antiphage signaling system (CBASS) preventing TM4 infection. However, the inability of TM4 to form plaques even in CBASS-deficient M. abscessus implies the involvement of additional resistance mechanisms. Our findings underscore the challenges faced by mycobacteriophages in infecting M. abscessus, and highlight the complex interactions between this pathogen and its viral adversaries.

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Impact importance

Heterologous expression of CmaABC, a CBASS system from M. abscessus, confers resistance against mycobacteriophage TM4 in M. smegmatis. The cmaABC genes reside within a mobile genetic element that is widely distributed across various mycobacteria, suggesting a potential role in phage defense in these organisms. Despite this protective capacity in a surrogate host, deletion of cmaABC in M. abscessus did not confer TM4 sensitivity, suggesting redundant defense mechanisms might be presented. These findings enhance our understanding of phage resistance mechanisms in M. abscessus, and may advance phage therapy as an alternative treatment for its infections.

Introduction

The recent rise in drug-resistant bacterial infections poses significant public health challenges globally. Mycobacterium abscessus grows rapidly and causes opportunistic infections, particularly in individuals with underlying pulmonary conditions such as cystic fibrosis or chronic obstructive pulmonary disease (COPD) [1, 2]. This pathogen is typically resistant to multiple antibiotics, making treatment challenging. Infections caused by M. abscessus are notoriously difficult to eradicate due to its intrinsic resistance mechanisms, often requiring prolonged and aggressive therapeutic regimens [3]. Therefore, alternative therapeutic strategies are urgently needed, with phage therapy emerging as a promising approach for treating M. abscessus infections [4].

Mycobacteriophages, viruses that specifically infect mycobacteria, have been extensively studied for their potential in treating infections caused by drug-resistant mycobacteria. Drug-resistant M. abscessus infections have been successfully treated using mycobacteriophages under compassionate use protocols [5,6,7]. However, M. abscessus exhibits limited susceptibility to mycobacteriophages, with few lytic phages isolated to date [7]. Another challenge is that lytic mycobacteriophages are effective against rough, but not smooth, M. abscessus morphotypes, which limits their eradication potential [8, 9]. Moreover, the presence of phage defense systems in mycobacteria further complicates the effectiveness of mycobacteriophage therapy against M. abscessus. A better understanding of these systems is needed to develop more effective phage therapies, combat drug-resistant infections, and mitigate the global health crisis of antibiotic resistance.

TM4, among the most well-characterized mycobacteriophages, is widely used in research on mycobacteria [10]. TM4 has a broad host range and can infect several mycobacterial species including Mycobacterium smegmatis and Mycobacterium tuberculosis [11]. TM4 can deliver recombinant DNA, such as fluorescent reporters, transposons, and allelic exchange substrates for genetic replacement, into M. abscessus [12,13,14]. However, even at high titers, TM4 is unable to form plaques on M. abscessus, presumably due to specific bacterial defense mechanisms against phage infection [12]. Therefore, elucidating the molecular and genetic bases of M. abscessus resistance to TM4 infection could enhance our understanding of the antiphage mechanisms involved, and facilitate the development of more effective phage therapy strategies, such as engineering TM4 variants capable of evading or inactivating these defense systems.

Bacteria face constant challenges from bacteriophages, the most abundant entities in the biosphere, which drives the evolution of highly specialized immune mechanisms [15, 16]. Among these defense strategies, the cyclic oligonucleotide-based antiphage signaling system (CBASS) is a crucial component in microbial immunity [17]. Identified through structural parallels with mammalian cyclic GMP-AMP synthase (cGAS), the CBASS is an ancient yet sophisticated bacterial immune strategy leveraging cyclic nucleotide second messengers to mount a defense against phage invasion [18]. CBASS operons display remarkable variability across bacterial species, often consisting of minimal constructs with a cGAS/DncV-like cyclic nucleotidyltransferase (CD-NTase) and a CD-NTase-associated protein (Cap) effector, but can include additional regulatory proteins that fine-tune the immune response. These systems are categorized into types I-IV, based on the complexity and regulatory components involved, such as E1/E2 ubiquitin ligase homologs in Type II CBASS that aid in modifying CD-NTase activity [19,20,21,22]. The primary component of this system, the CD-NTase enzyme, is activated upon phage detection and catalyzes the synthesis of diverse cyclic nucleotides [23]. These messengers rapidly diffuse within the bacterial cell, orchestrating an immune response through activation of associated Cap effectors, often resulting in programmed cell death to thwart phage replication [24,25,26]. The mechanisms regulating CD-NTase activation are not yet fully understood. Notably, recent studies suggest interactions with phage-derived RNA or structural proteins as potential triggers [27, 28]. Moreover, the evolutionary arms race with phages has driven CBASS diversification, as evidenced by phage-encoded proteins that either degrade nucleotide signals or inhibit their formation [29, 30]. Despite widespread occurrence across various bacterial species, the CBASS remains poorly characterized in mycobacteria, particularly in M. abscessus.

In this study, we identified Mab_2091, Mab_2092, and Mab_2093, designated cmaABC (CBASS of Mycobacteria abscessus), as components of a CBASS defense system capable of resisting mycobacteriophage TM4 infection when expressed in the surrogate host M. smegmatis. Site-directed mutagenesis of the conserved domains within these proteins eliminated their protective capability. Despite performing escape phage isolation and phage gene expression library screening, we were unable to identify activators of this CBASS. Bioinformatic analyses indicate that this CBASS is ubiquitously present across various mycobacteria. The findings expand our understanding of why mycobacteriophages struggle to infect M. abscessus.

Results

Bioinformatic prediction of candidate phage defense systems in M. abscessus ATCC 19977

To investigate the resistance mechanisms of M. abscessus to mycobacteriophages, we employed DefenseFinder, a prokaryotic antiviral defense system prediction tool, to identify the phage defense systems present in the genome of the standard strain M. abscessus ATCC 19977 [31]. Our analysis predicted three distinct candidate defense systems and multiple putative defense-related genes (Table 1 and Supplementary Table S1). The first candidate defense system predicted was the DNA phosphorothioation (Dnd) defense system, located in the genomic region from Mab_1093c to Mab_1097. This system utilizes DndABCDE to incorporate sulfur into the DNA backbone as a phosphorothioate (PT) modification, while the DndFGH proteins function as restriction enzymes that recognize and degrade non-PT-modified foreign DNA [32]. However, genes encoding DndFGH were not found in the M. abscessus ATCC 19977 genome. Absence of DndFGH suggests an incomplete system likely lacking antiphage function (Fig. 1A). The second candidate phage defense system predicted was the Wadjet defense system, encoded by Mab_1490, Mab_1491, and Mab_1492. Wadjet comprises four genes (JetABCD), predicted to function as a defense system against foreign plasmids [15]. Within this system, JetABC induces bending of plasmid DNA, thereby facilitating DNA kinking and cleavage by JetD [33]. The absence of the JetD protein in the M. abscessus ATCC 19977 genome indicates that JetABC may not function as a phage defense system. Finally, we predicted a Type II CBASS defense system, encompassing cmaABC. The cmaA (Mab_2091) gene may encode a patatin-like phospholipase that serves as an effector protein, while cmaB (Mab_2092) is annotated as a cyclic GMP-AMP synthase DncV-like nucleotidyltransferase. The third, cmaC (Mab_2093) encodes an ancillary protein containing a ubiquitin-conjugating E2-like domain and a ubiquitin-activating E1-like enzymatic domain (Fig. 1A).

Table 1 Defense systems predicted in M. abscessus ATCC_19977
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Fig. 1
figure 1

CmaABC mediates resistance to infection by phage TM4 in the heterologous host M. smegmatis. A Mapping of candidate defense systems within the M. abscessus ATCC_19977 genome. Each gene is represented to scale, with gene positions annotated below the diagram. Transposase genes are colored blue. The diagram was generated using Chiplot (www.chiplot.online). B Defense activity of the predicted phage defense systems. Plaque assays using phages TM4 and D29 were conducted on M. smegmatis mc2155 strains harboring plasmids containing Mab_1093c-1097, Mab_1490-1492, cmaABC, or cmaAB serving as a bacterial lawn. An empty-vector control (pYC601) was showed to represent the baseline phage susceptibility of M. smegmatis. The concentration of ATc used was 10 ng/mL. C Growth curves of M. smegmatis mc2155 with and without induced expression of cmaABC plasmid. OD600 values were measured every 3 h. Results are means ± standard deviation (SD) from three biological replicates. D Growth status of M. smegmatis mc2155 expressing cmaABC plasmid following TM4 treatment with different MOI levels

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To investigate the role of the predicted defense systems, their coding genes were cloned into the replicative plasmid pYC601 and expressed in M. smegmatis mc2155 under the control of the ATc-inducible PmyctetO promoter, which is regulated by the TetR repressor. M. smegmatis mc2155 lacks homologs of these genes and thus serves as a suitable surrogate host for test their roles in anti-phage infection. Double-layer plaque assays revealed that only the CBASS, encoded by cmaABC, provided significant defense against phage TM4 but not D29, resulting in a marked reduction in plaque formation (Fig. 1B). In addition, induced expression of cmaABC had no effect on growth of the M. smegmatis mc2155 strain (Fig. 1C). The CBASS could not sustain host growth when infected with high multiplicity of infection (MOI) phage TM4, while the CBASS effectively resisted low MOI TM4 infection, allowing host bacteria to grow (Fig. 1D). This MOI-dependent protection suggests a potential abortive infection (Abi) phenotype, consistent with documented CBASS-mediated Abi mechanisms in Vibrio cholerae and Escherichia coli [18].

Conserved domains within CBASS components dictate immune function

The cGAS enzyme and Cap effector are critical for the immune functions of Type II CBASS [19]. cGAS catalyzes the synthesis of cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) while the Cap effector and phospholipase domains that cleave membrane lipids are activated by cGAMP [18]. To investigate the role of cGAMP, a conserved amino acid residue (Asp70) within the nucleotidyltransferase domain of CmaB was mutated to alanine (Fig. 2A). This resulted in susceptibility to phage TM4 infection, indicating that CmaB is essential for the phage defense function of the CBASS (Fig. 2B).

Fig. 2
figure 2

Mutating conserved amino acid residues affects the defense function of the CBASS. A Alignment of conserved residues in CmaB. Comparative analysis of M. abscessus CmaB with amino acid sequences of homologs from Kibdelosporangium sp. MJ126-NF4, Kitasatospora sp. NPDC094016, Streptomyces sp. DSM 41602, and Actinomycetota bacterium revealed conserved amino acid sites. B Defense activities of CmaABC carrying point mutations. Plaque assays using phage TM4 were performed on M. smegmatis mc2155 strains expressing wild-type or mutant cmaABC as a bacterial lawn. The concentration of ATc was 10 ng/mL. C Vina scores of CmaA with different cGAMP substrates predicted by CB-DOCK2. D cGAMP-binding pocket of CmaA predicted by CB-DOCK2 highlighted in red. E Alignment of conserved residues in CmaC. Comparative sequence analysis of M. abscessus CmaC and its homologs from Thermobifida fusca, Actinomycetota bacterium, and Streptomyces sp. NPDC005752 highlights conserved amino acid residues within the predicted E1 (down panel) and E2 (up panel) domains. Only the conserved core regions of the E1 and E2 domains are shown. Panels A and E were generated using ENDscript [34]

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To investigate the role of cGAMP in activation of the Cap effector, we employed CB-DOCK2 [35] to investigate the binding pocket for cGAMP in CmaA and found that the binding affinity was strongest for 3’−3’ cGAMP (Fig. 2C). Introduction of a point mutation (G52A) in the binding pocket of CmaA disrupted the defensive capability of the CBASS, based on double-layer plaque assays (Fig. 2B, D; Supplementary Figure S1). Taken together, these results suggested that 3’−3’ cGAMP may be involved in CmaABC-mediated phage defense.

Ancillary proteins may expand the spectrum of phage protection in the Type II CBASS, and may be essential for resistance to some phages [19]. Expression of CmaA and CmaB failed to provide protection against phage TM4 infection (Fig. 1B), indicating an essential role for CmaC. To further investigate the role of CmaC in phage defense, point mutations were introduced at conserved sites within the E1 (residues 341-452, Q385A) and E2 (residues 32-145, L99A) domains (Fig. 2E). Mutation of CmaC in either E1 or E2 abolished its function, highlighting the necessity of both domains for effective phage defense (Fig. 2B).

Identifying the key determinants of immune activation

To explore the activation mechanism of the CBASS, we initially attempted to isolate phage escape variants. However, we were unable to identify any phage TM4 escape mutants after multiple rounds of selection. To further investigate which components of phage TM4 may activate the CBASS, we introduced a previously constructed phage TM4 gene expression plasmid library into M. smegmatis mc2155 expressing cmaABC [36]. Given that activation of this CBASS leads to host cell death, we anticipated that the read counts for TM4 genes capable of activating this defense system would decrease significantly. Unfortunately, sequencing analysis did not reveal any differentially expressed genes (log2FoldChange < 1; Supplementary Table S2). These results suggested that activation of the CBASS may not be triggered by a specific structural protein of TM4, but rather by other unidentified factors.

Disruption of CmaABC does not enhance TM4 susceptibility in M. abscessus

To clarify the contribution of the CBASS system encoded by cmaABC to the resistance of M. abscessus ATCC 19977 to phage TM4 infection, deletion mutants of cmaA and cmaB were constructed using a CRISPR-assisted non-homologous end joining method. However, phage TM4 was unable to form plaques on plates containing these mutants, similar to the wild-type M. abscessus ATCC 19977 strain, even at high phage titers (Fig. 3A; Supplementary Figure S2). This observation suggested that the CBASS is not the only factor mediating the resistance of M. abscessus to TM4 infection and other defense systems might be also involved in this process.

Fig. 3
figure 3

TM4 failed to form plaque on the lawn of M. abscessus CBASS mutants. A Plaque assays employing phage TM4 conducted on bacterial lawns of M. smegmatis mc2155, wild-type M. abscessus, M. abscessus cmaA knockout, and M. abscessus cmaB knockout strains. B Location of CBASS genes within the genome of several isolates of M. abscessus. Genomic locations of CBASS genes in M. abscessus subsp. abscessus strains GD21, GD41, and GD57 are shown alongside those of strains 57626, 57629, and 57596

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Next, we explored the distribution of cmaABC in other M. abscessus strains. Using the cmaABC of M. abscessus ATCC 19977 as the query to perform a BLAST search against the NCBI database M. abscessus genome, we found that only ~ 2% (43 out of 2270) M. abscessus strains contain this system (Fig. 3B, Supplementary Table S3). This finding suggests that the primary role of CmaABC might not be to inhibit TM4 infection directly, but rather to guard against other phage infections in M. abscessus.

CmaABC is widely distributed in Mycobacteria

Defense systems are frequently clustered within genomic regions known as defense islands [15]. Analysis using IslandViewer 4 [37] revealed that the cmaABC genes are situated on a genomic island featuring multiple transposases (Fig. 4). Using the cmaABC sequence to perform a BLAST search against the NCBI genome database, we detected cmaABC in 20 genomes of Mycobacteria sharing > 99% nucleotide sequence identity (Supplementary Table S4). This finding suggests that cmaABC may be part of a mobile genetic element (MGE) and might have been recently transferred among different mycobacterial species.

Fig. 4
figure 4

Comparative analysis and visualization of selected genomes of Mycobacteria. Using the complete genome of M. abscessus strain ATCC 19977 (GenBank accession: CU458896) as a reference, the Proksee webserver [38] was employed for comparative analysis and visualization of selected genomes of Mycobacteria available from GenBank, including M. abscessus strain FDAARGOS_1605 (CP085918), M. chelonae strains Myco3a and MCHE08 (CP050145 and CP058976), M. fortuitum strains W4 and W6 (CP060409 and CP060410), M. intracellulare strains MOTT-02 and S1-32 (CP003323 and CP076380), M. marseillense strains FLAC0026 and JCM 17324 (CP023147 and AP022584), and M. kubicae strains NJH_MKUB1 and NJH_MKUB2 (CP045081 and CP045075)

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To further investigate this system, we selected six pairs of mycobacterial genomes, some containing cmaABC and others lacking it, and conducted a comparative genomic analysis using the Proksee webserver [38]. The cmaABC genes, along with Mab_2089 and Mab_2090, were found within a region flanked by transposase-encoding genes, which were not present in the corresponding genomes lacking cmaABC. This observation indicates that this region may function as part of an MGE that can be transferred between mycobacteria.

Discussion

M. abscessus is notorious for its resistance to conventional antibiotics, making it a significant concern in the treatment of respiratory infections, particularly in immunocompromised patients. Understanding the mechanisms underpinning its phage resistance may guide the design of phage therapy strategies, which could serve as an alternative or adjunct to traditional antibiotic treatments [39]. Exploration of bacterial defense systems, characterized by the formation of genomic defense islands, has led to the identification of various defense mechanism variants [15]. However, these systems have not yet been reported in mycobacteria, including M. abscessus. In the present study, we demonstrate that the cmaABC operon, which encodes a CBASS defense system from M. abscessus, confers resistance to TM4 phage infection when heterologous expressed in the surrogate host M. smegmatis.

In addition to the minimal CD-NTase and Cap effector operon structure that defines Type I CBASS operons, Type II CBASS operons encode an additional Cap2 protein, homologous to E1/E2 ubiquitin ligase [19]. Cap2 forms a thioester bond with conserved glycine or alanine residues at the C-terminus of cGAS, resulting in increased synthesis of nucleotide immune signals [20,21,22]. However, Cap2 is not required for CBASS to counteract certain phage infections, but the reasons and mechanisms are unknown. CmaC, the Cap2 protein of M. abscessus CBASS, is essential for resistance to phage TM4 infection. Consistent with previous findings, mutation of the conserved E1 or E2 sites in CmaC abolishes CBASS protection against phage infection [21]. Similar to other Type II CBASS system, the precise defense mechanism of CmaABC remains poorly understood [20,21,22]. Our results suggest that CmaC may be involved in the activation of CmaB, leading to increased cGAMP synthesis, which subsequently activates the phospholipase activity and ultimately causes abortive infection.

The mechanism by which CmaABC recognizes phage TM4 infection requires further investigation. Unfortunately, we could not identify activators for CmaABC using phage TM4 gene expression [36]. One possibility is that this CBASS is activated not only by signals directly from phage TM4, but also host factors or environmental cues modulating the CBASS response. For instance, previous research showed that Vibrio cholerae can activate the CBASS through quorum sensing and folate depletion [40, 41]. In addition, isolation of escaping TM4 phages that combat the CmaABC system also failed, and escaping phages could not be isolated in previous studies [28, 42, 43]. Failure to isolate escaping phages may be due to the sense mechanism targeting essential phage components, with mutations that allow escape also rendering phages inactive. Additionally, this defense system might require multiple mutations for phage escape, making it unlikely to achieve these combinations in the experimental setup [43]. Future investigations may further elucidate the specificity and adaptability of the CBASS module in conferring defense against other mycobacteriophages.

Our observations suggest that cmaABC might be located in an MGE that is only present in a small portion of M. abscessus strains and some other mycobacteria. The presence of cmaABC might help mycobacteria to avoid infection by some phages. Defense systems were suggested to play a major role in preventing phage TM4 from infecting M. abscessus [12]. However, cmaABC deletion failed to allow plaque formation by TM4 on M. abscessus lawn, indicating that other defense mechanisms are also involved in this process. There are dozens of individual candidate defense genes in M. abscessus (Supplementary Table S1) that may also contribute to avoiding TM4 infection, and their roles remain to be investigated. Future studies should prioritize characterizing these alternative defense systems and their interactions with the CBASS, in order to provide a holistic understanding of bacterial antiphage strategies. This comprehensive understanding may not only advance our knowledge of phage-bacteria interactions, but also inform the development of innovative phage therapy strategies as alternatives or adjuncts to conventional antibiotics, offering renewed hope for managing antibiotic-resistant infections.

Materials and methods

Strains and culture conditions

M. smegmatis mc2155 and M. abscessus ATCC 19977 were cultured at 37 Â°C in Middlebrook 7H9 broth (BD Biosciences), supplemented with 0.2% (w/v) glycerol (Sigma) and 0.05% (w/v) Tween 80 (Sigma), or on Middlebrook 7H10 agar (BD Biosciences) with 0.5% (w/v) glycerol. For antibiotic selection, Middlebrook 7H9 or 7H10 media were supplemented with the following concentrations of antibiotics: 25 Î¼g/mL kanamycin (for M. smegmatis), 100 Î¼g/mL kanamycin (for M. abscessus), 50 Î¼g/mL hygromycin (for M. smegmatis), and 100 Î¼g/mL zeocin (for M. abscessus). In LB broth, the following antibiotics were added: 50 Î¼g/mL kanamycin, 25 Î¼g/mL zeocin, or 100 Î¼g/mL hygromycin. Anhydrotetracycline (ATc) was used at a concentration of 10 ng/mL, unless stated otherwise. For phage infection assays, Tween 80 was replaced with 1 mM CaCl2 in the culture media. The primers utilized in this study are summarized in Supplementary Table S5.

Cloning and site-directed mutagenesis of defense systems

The defense system of M. abscessus ATCC19977 (GenBank accession CU458896) was predicted using DefenseFinder (Version 1.2.2). Three gene clusters were selected for functional characterization: 1) cmaABC, identified by locus tags Mab_2091-Mab_2093, spanning nucleotides 2091174 to 2095061; 2) the five-gene system, Mab_1093c-Mab_1097, spanning nucleotides 1104494 to 1110845; 3) the three-gene system, Mab_1490-Mab_1492, spanning nucleotides 1500576 to 1,506116. All constructs were PCR-amplified from M. abscessus genomic DNA and cloned into plasmid pYC601 [44], a replicative mycobacteria-E. coli shuttle vector. Amplified gene clusters were inserted between the Bam HI and Hind III sites of the pYC601 multiple cloning site using a seamless homologous recombination cloning kit (CU101-02, TransGen Biotech).

Site-directed mutagenesis was performed using overlapping PCR with primers encoding the desired point mutations. Two overlapping DNA fragments were generated and assembled into linearized pYC601 using the same seamless cloning strategy. The G52A mutation in Mab_2091 was constructed in this manner, along with D70A in Mab_2092, and L99A or Q385A in Mab_2093. The resulting recombinant plasmids were introduced into M. smegmatis mc2155 via electroporation (2.5 kV, 1000 Ω, 25 µF) and selected on Middlebrook 7H10 agar supplemented with hygromycin (50 Âµg/mL). Positive colonies were screened by colony PCR, and successful clones were confirmed by sequencing.

Double layer plaque assay

M. smegmatis mc2155 was cultured in 7H9 broth at 37 °C until the optical density at 600 nm (OD600) reached approximately 1.0. Subsequently, 1 mL of the culture was centrifuged at 4000 rpm for 3 min. The harvested cells were washed with 7H9 broth, where Tween 80 was replaced with 1 mM CaCl2. Next, 800 μL of the resuspended culture was combined with 2.2 mL of 7H9 broth (supplemented with 2 mM CaCl2, 100 Î¼g/mL hygromycin, and 20 ng/mL ATc) and 3 mL of top agar (7H9 supplemented with 0.7% agar). This mixture was then plated onto 7H10 agar plates containing 50 Î¼g/mL hygromycin. Phage solutions (TM4 and D29) were serially diluted in tenfold increments. Following dilution, 5 μL from each dilution was spotted onto the surface of the top agar containing the M. smegmatis culture. The plates were incubated at 37 °C for phage D29 and 30 °C for phage TM4 for a duration of 1 to 2 days to allow plaque formation.

CRISPR-NHEJ-mediated genome editing in M. abscessus

M. abscessus cmaAB mutants were generated using a CRISPR-NHEJ based genome editing method as described previously [45]. Briefly, electrocompetent cells harboring pNHEJ-RecXmab were electroporated with 200 ng of the shear plasmid expressing GFP and sgRNA targeting either cmaA or cmaB. Electroporation was performed using a Gene Pulser Xcell (Bio-Rad) at 2.5 kV, 25 μF and 800Ω. Following electroporation, cells were recovered in 1 ml of 7H9 broth supplemented with OADC and incubated at 37 Â°C for 8 h. Cultures were then plated on Middlebrook 7H10 agar containing 50 ng/ml ATc, 100 Î¼g/ml kanamycin, 100 Î¼g/ml zeocin, and OADC supplement. GFP-expressing transformants were selected and screened for successful genome editing by colony PCR and sequencing.

Isolation of phage escaper

For the isolation of escape phages, M. smegmatis mc2155 induced to express CmaABC was used as a bacterial lawn. High-titer TM4 phages were employed to infect this lawn. Following infection, individual plaques were isolated and amplified. To assess the escape capabilities of the isolated phages from the CBASS defense system, we maintained a control condition where CBASS expression was not induced. This process was repeated multiple times; however, we did not observe any definitive evidence of phage escape.

Screening for activator of CmaABC using TM4 gene library

M. smegmatis mc2155 strains containing the pYC601-cmaABC plasmid were inoculated into fresh 7H9 medium supplemented with hygromycin and 10 ng/mL ATc, with an initial OD600 of 0.03. The cultures were grown at 37 Â°C with shaking at 200 rpm until an OD600 of approximately 0.8. The competent cells were prepared and transformed with TM4 gene expression plasmids [36]. After a recovery period of 4 h, the cultures were plated onto 7H10 agar supplemented with hygromycin, zeocin, and 50 ng/mL ATc and incubated at 37 Â°C. A control group was prepared under the same conditions without the addition of ATc to induce defense system expression. The transformants were scraped from the plates, and plasmids were extracted for Next Generation Sequencing (NGS) and comparative analysis.

Data availability

The datasets generated and/or analysed during the current study are available in the Genome Sequence Archive (GSA) repository, CRA027583.

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Acknowledgements

Not applicable.

Funding

This work was supported by funding from the National Key R&D Program of China (2023YFE0113400) and the CAMS Innovation Fund for Medical Sciences (CIFMS2021-I2M-1–043).

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Author notes

  1. Chun-Liang Wang, Ao-Fei Duan and Da-min Pan contributed equally to this work.

Authors and Affiliations

  1. NHC Key Laboratory of Systems Biology of Pathogens, State Key Laboratory of Respiratory Health and Multimorbidity, National Institute of Pathogen Biology and Center for Tuberculosis Research, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 102629, China

    Chun-Liang Wang, Ao-Fei Duan, Da-min Pan, Jian Yang & Yi-Cheng Sun

  2. School of Life Science and Technology, Changchun University of Science and Technology, Changchun, Jilin, 130031, China

    Yi-Ou Zhao

Authors
  1. Chun-Liang Wang
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  2. Ao-Fei Duan
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  3. Da-min Pan
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  4. Yi-Ou Zhao
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  5. Jian Yang
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  6. Yi-Cheng Sun
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Contributions

Experimental design: C-LW, A-FD, D-MP and Y-CS; Experiment carrying out: C-LW, A-FD, D-MP, Y-OZ and JY; Data analysis: C-LW, JY and Y-CS; Article writing: C-LW and Y-CS.

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Correspondence to Jian Yang or Yi-Cheng Sun.

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Supplementary Information

12866_2025_4214_MOESM1_ESM.xlsx

Supplementary Material 1: Table S1. Phage defense genes predicted in M. abscessus ATCC 19977. Table S2. The read counts for TM4 genes of screening for activator of CmaABC. Table S3. M. abscessus strains containing cmaABC. Table S4. Shared >99% nucleotide sequence with cmaABC in 20 mycobacterial genomes. Table S5. The primers and sgRNA utilized in this study.

12866_2025_4214_MOESM2_ESM.docx

Supplementary Material 2: Figure S1. Predicted 3D structure of CmaA in complex with 3′-3′cGAMP. Figure S2. PCR and sequencing confirmation of mab_2091 and mab_2092 deletions in M. abscessus.

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Wang, CL., Duan, AF., Pan, Dm. et al. Heterologous expression of the M. abscessus CBASS system confers phage TM4 resistance in M. smegmatis. BMC Microbiol 25, 519 (2025). https://doi.org/10.1186/s12866-025-04214-x

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Keywords

BMC Microbiology

ISSN: 1471-2180

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