The stress response factor SigH mediates intrinsic resistance to multiple antibiotics in Mycobacterium abscessus

32 Mycobacterium abscessus (Mab) causes pulmonary diseases with limited treatment options due 33 to its high level of intrinsic resistance to available drugs. Mab possesses complex and poorly 34 understood drug resistance mechanisms. Identifying new drug targets and gaining a deeper 35 understanding of drug resistance mechanisms are essential for discovering novel therapeutic 36 alternatives. Here, we investigated the role of a putative sigma factor SigH in intrinsic multi-drug 37 resistance in Mab. Mab SigH shares an 84% peptide sequence identity with Mycobacterium 38 tuberculosis (Mtb) SigH, a well -known stress response protein and global transcriptional 39 regulator. We constructed a sigH gene deletion strain of Mab (Δ sigH) and complemented strains 40 by expressing either Mab sigH (CPMabsigH) or Mtb sigH (CPMtbsigH) in Δ sigH. The Δ sigH 41 strain exhibited hypersensitivity to a broad range of antibiotics, including levofloxacin, 42 moxifloxacin, tigecycline, tetracycline, amikacin, vancomycin, and rifabutin and all 43 complemented strains restored the drug resistance phenotype. Additionally, Δ sigH showed 44 increased sensitivity to oxidative and heat stress compared to the wild -type Mab and 45 complemented strains. Transcriptomic analysis revealed that d eletion of sigH disrupted the 46 balance of gene expression, primarily elevating the expression of genes encoding YrbE and MCE 47 family proteins and downregulating genes expressing ABC -type transporters, sigma and anti -48 sigma factors and other genes associated with antimicrobial resistance. Collectively, our findings 49 indicate that SigH is a key regulator of global gene expression in response to environmental 50 stresses, including antimicrobial treatment, and is crucial for the intrinsic drug resistance of Mab. 51 SigH represents a promising target for the development of novel therapeutic strategies against 52 Mab infections. 53

Non -tuberculous mycobacteria, M. abscessus, SigH, intrinsic resistance, gene 54 expression regulation 55 56 57

58 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 10, 2025. ; https://doi.org/10.1101/2025.03.04.641476doi: bioRxiv preprint 3 Mycobacterium abscessus complex (MABC) includes well -known rapidly growing non -59 tuberculosis mycobacteria (NTM) capable of causing severe acute and chronic lung infections, 60 particularly in patients with underlying lung diseases such as cystic fibrosis and obstructive 61 pulmonary diseases, as well as skin and soft tissue infections (1, 2). Recently, MABC has been 62 associated with a wide range of clinical manifestations due to increasing global morbidity and 63 mortality rates (3). The intrinsic drug resistance mechanisms of MABC remain poorly 64 understood, and the highly resistant phenotype of Mycobacterium abscessus (Mab) presents a 65 significant challenge in its infection therapy. To improve treatment options and combat prevalent 66 Mab infections, novel drugs with new mechanisms of action are urgently needed. 67 MABC accounts for 2.6-13.0% of all NTM-related pulmonary infections (4). Mab demonstrates 68 intrinsic resistance to most therapeutic agents, and cure rates for Mab lung infections are very 69 low (approximately 25 -58%), earning it the moniker "antibiotic nightmare" (5). The primary 70 mechanisms of intrinsic drug resistance include a waxy, impermeable cell wall, drug efflux pump 71 systems, and drug inactivation by hydrolases or modifying enzymes (6, 7, 8). For instance, 72 aminoglycoside phosphotransferases and 2 ′-N-acetyltransferases transfer acetyl or phosphate 73 residues to specific positions within aminoglycoside drugs, rendering them inactive (9). 74 Erythromycin resistance methylase is responsible for macrolide resistance, while 75 MmpL5/MmpS5 confers resistance to bedaquiline and clofazimine (10). The MabTetX, a 76 WhiB7-independent tetracycline-inactivating monooxygenase, increases Mab's resistance to the 77 tetracycline family (11). Additionally, spontaneous mutations in particular genes in response to 78 drugs cause acquired resistance. For example, genetic polymorphism results in resistance to 79 antibiotics like fluoroquinolones (FQs), which are broad -spectrum secondary therapeutic agents 80 for multi-drug-resistant tuberculosis and act by inhibiting DNA gyrase supercoiling activity (12, 81 13). Mab resistance to FQs is due to genetic alterations, especially in quinolone resistance -82 determining regions within DNA gyrase subunits GyrA and GyrB, the primary targets of FQs 83 (14). Tigecycline (TIG) resistance in Mab has been linked to both SigH and RshA (15). 84 Dysregulated environmental stress response sigma factor (SigH) is associated with tigecycline 85 resistance in both Mycobacterium tuberculosis (Mtb) and Mab (16, 17). The limited success in 86 anti-Mab drug discovery primarily stems from high intrinsic resistance and rapidly acquired 87 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 10, 2025. ; https://doi.org/10.1101/2025.03.04.641476doi: bioRxiv preprint 4 resistance to currently available active drugs. However, the mechanisms underlying intrinsic 88 drug resistance in Mab remain not fully elucidated (18). 89 WhiB7 is a redox -sensitive master transcription al regulator crucial for activating intrinsic drug 90 resistance systems in mycobacteria and other bacteria, such as Streptomyces lividans and 91 Rhodococcus jostii (19, 20). The multi -drug resistance WhiB7 transcriptional regulator induces 92 SigH transcription. SigH is an alternative sigma factor involved in the transcriptional regulation 93 of genes responsible for mycobacterial stress responses, including oxidative, heat, and nitrosative 94 stresses (21, 22, 23), and is negatively regulated by RshA (15, 24). The significance of stress 95 responses in etiology and immunity has been extensively studied in Mtb and Mycobacterium 96 smegmatis (Msm) (22, 25). 97 In this study, we investigated the role of SigH (MAB_3543c) in antimicrobial resistance, stress 98 response, and differential gene expression in Mab. Our findings lay the groundwork for further 99 research that could lead to the development of new therapeutics or treatment regimens for Mab 100 infections. 101

102 Bacterial strains and growth conditions 103 The Mab GZ002 (accession number CP034181), exhibiting a smooth phenotype (26 ), was 104 cultivated at 37 °C with shaking at 220 rpm. Growth media included Middlebrook 7H9 (Difco), 105 supplemented with 0.2% glycerol, 0.05% Tween 80, and 10% OADC, or solid 7H10/7H11 agar 106 supplemented with 0.5% glycerol and 10% OADC. Escherichia coli strain DH5α was 107 propagated on solid or in liquid Luria -Bertani medium under identical conditions of 37 °C and 108 220 rpm shaking, with an incubation period of 10 -12 hours, while Mab GZ002 was grown on 109 agar medium for 3 -5 days. Antibiotic usage and their respective concentrations were tailored to 110 the specific experimental demands. 111 CRISPR-Cpf1-assisted knockout of sigH (MAB_3543c) 112 CRISPR-Cpf1-assisted recombineering was employed to knockout the Mab sigH gene via 113 homology-directed repair (27, 28, 29). Specifically, the CRISPR RNA ( crsigH) was designed as 114 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 10, 2025. ; https://doi.org/10.1101/2025.03.04.641476doi: bioRxiv preprint 5 two complementary oligonucleotides, each 24 base pairs in length ( crsigH-F/R), targeting a 115 region adjacent to a protospacer adjacent motif (PAM) characterized by the 5' -YTN-3' 116 trinucleotide sequence. One oligonucleotide was designed with a HindIII overhang, while the 117 other had a BpmI overhang. These oligonucleotides were annealed and cloned into HindIII/BpmI-118 digested pCRZEO to generate pCRZEO-crsigH. 119 The homology -directed repair template ( sigHUD) was constructed by amplifying 640 -bp 120 upstream (U) and 609 -bp downstream (D) regions of the target gene, including flanking 121 sequences from adjacent genes (Figure S2). Both fragments were cloned into pBlueSK to 122 generate pBlueSK -sigHUD. sigHUD was amplified from pBlueSK -sigHUD using the primer 123 pair sigHUD-F/R (Table S3). pCRZEO-crsigH and sigHUD were transformed by electroporation 124 into electrocompetent Mab cells harboring pJV53-Cpf1. The transformants were then plated on a 125 7H11 plate containing zeocin (ZEO, 30 µg/mL), kanamycin (KAN, 100 µg/mL), and 126 anhydrotetracycline (ATc, 100 ng/mL), and incubated at 30 ◦C for 5 days. sigH knockout was 127 verified by PCR and sequencing using the primer pair Id3543c -F/R (Figure S2). To construct the 128 selectable marker -free knockout strain, the knockout strain was grown in 7H9 medium and 129 plated on a drug-free 7H10 plate to obtain single colonies. A colony that grew on drug-free plates 130 but not on KAN - and ZEO -containing plates was selected as the unmarked strain (Δ sigH) to be 131 used in downstream experiments (Figure S3). 132 Complementation and overexpression of sigH 133 Complemented (CP) strains were constructed by integrating sigH into the genome of Δ sigH to 134 express the gene under its native promoter (Np) or hsp60 promoter, or through ectopic expression 135 – i.e. cloning sigH or its Mtb homolog into pMV261 and transforming them into Δ sigH. For 136 overexpression, pMV261 -sigH was transformed into wild -type Mab ( WT ). The transformants 137 were grown on Middlebrook 7H10 agar plates supplemented with KAN at 100 µg/mL for 3 to 5 138 days at 37 °C and verified by PCR and sequencing. Thus, the following strains were used in this 139 study: OEMab sigH (WT overexpressing Mab sigH), Δ sigH (selectable marker -free knockout 140 strain for sigH), CPMabsigH (the CP strain ectopically expressing Mab sigH), CPMtbsigH (the 141 CP strain ectopically expressing Mtb sigH), CPNpMabsigH (the CP strain in which Np-Mab sigH 142 is integrated into Δ sigH genome), and CP hsp60MabsigH (the CP strain in which hsp60-Mab 143 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 10, 2025. ; https://doi.org/10.1101/2025.03.04.641476doi: bioRxiv preprint 6 sigH is integrated into Δ sigH genome). Results of Mab strain verification and all primer 144 sequences are provided in the supplementary material (Figures S4, S5; Table S3). 145 Drug susceptibility testing 146 Mab strains were routinely cultivated on Middlebrook 7H10 agar or in Middlebrook 7H9 broth. 147 Antimicrobial agents including TIG, tetracycline (TET), clarithromycin (CLA), clofazimine 148 (CLF), vancomycin (V AN), amikacin (AMK), levofloxacin (LFX), moxifloxacin (MFX), 149 imipenem (IMP), cefoxitin (CFX), rifabutin (RIB), and linezolid (LZD) were prepared as stock 150 solutions and stored at -20 °C. Broth microdilution assay, adhering to Clinical Laboratory 151 Standards Institute (CLSI) guidelines, was employed to determine drug susceptibility (30, 31). 152 Mycobacterial cultures were standardized to approximately 1 × 10 7 colony-forming units per 153 milliliter (CFU/mL) in 7H9 medium without Tween 80. Bacterial suspensions underwent two -154 fold serial dilutions in the presence of individual drugs within 96- well plates. These plates were 155 incubated at 37 °C for 3 days, with an extended incubation period of 14 days specifically for 156 CLA, before assessing the endpoint. Minimum inhibitory concentrations (MICs) were 157 established according to CLSI criteria, defining them as the minimal drug concentrations capable 158 of visually inhibiting mycobacterial growth. Additionally, spot culture agar methodology was 159 also utilized for drug susceptibility assessments (32). Herein, WT, Δ sigH, and CPMab sigH 160 strains were propagated in 7H9 medium at 37 °C until reaching an optical density (OD 600 nm) of 161 0.6. Subsequently, ten -fold serial dilutions were applied and aliquoted onto plain Middlebrook 162 7H10 agar (Drug -free serving as a control) alongside plates supplemented with varying drug 163 concentrations. Following a 3-day incubation at 37°C, the plates were examined. 164 Thiol-specific oxidative and heat stress assay 165 WT, ΔsigH, and CPMab sigH were grown to the exponential phase and equilibrated at an OD600 166 nm of 0.6 for oxidative and heat stress assays. For oxidative stress assay, a 100 µL bacterial 167 inoculum at OD600 nm 0.6 was spread on agar plates. Whatman paper disks (4.5 mm) were loaded 168 with 10 µL oxidizing agent diamide at concentrations 4 M, 2 M, 1 M, and 0.5 M. These disks 169 were then placed on agar plates and incubated for 3 days at 37 ºC. The susceptibility of bacteria 170 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 10, 2025. ; https://doi.org/10.1101/2025.03.04.641476doi: bioRxiv preprint 7 to diamide was assessed by measuring the zone of inhibition. Different survival curves were 171 observed under heat and oxidative stress conditions. Survival under heat stress was determined 172 based on CFU counts. Bacterial cultures were incubated in a water bath at 45 °C, and at 1 -hour 173 intervals, 100 µL of the bacterial culture was diluted in PBS and plated on 7H10 agar plates to 174 determine the viable cell number ( 33). Additionally, bacterial survival was assessed in the 175 presence of H2O2 and diamide. The bacterial OD 600 nm was adjusted to 0.5-0.6 and cultured with 176 50 mM diamide and 50 mM H 2O2 individually, followed by incubation at 37 °C. At one -hour 177 intervals, bacteria were 10 -fold subjected to serial dilutions and plated to count CFU for 178 determining the survival rates under stress conditions (22, 34). 179 RNA preparation, sequencing, and transcriptomic analysis 180 RNA isolation and library preparation for RNA sequencing were performed on the WT, Δ sigH, 181 and CPMab sigH strains. These bacterial strains were cultured in Middlebrook 7H9 medium 182 supplemented with Tween 80 and incubated at 37°C until reaching the exponential growth phase, 183 characterized by an OD600 nm of approximately 0.6 –0.8. RNA extraction was carried out using 184 the TRIzol method, and sample quality was inspected using a Thermo NanoDrop One and 185 Agilent 4200 Tape Station (35). Approximately, RNA samples were treated with the Epicentre 186 Ribo-Zero rRNA Removal Kit (Illumina) to enrich for mRNA. Sequencing libraries were 187 constructed using the NEBNext Ultra II Directional RNA Library Prep Kit. The constructed 188 libraries underwent a quality inspection before being sequenced on Illumina's high -throughput 189 sequencing platform with PE150 configuration. The resulting reads were trimmed using fastp 190 v0.23.2 (36) and mapped to the Mab reference genome (NCBI accession number CP034181). 191 Quantitative analysis of gene expression levels was conducted to analyze the differentially 192 expressed genes (DEGs) between different samples and to reveal the regulatory mechanisms of 193 these genes by combining sequence function information. Gene expression was quantified using 194 RNA sequencing by expectation maximization (RSEM), and fragments per kilobase of transcript 195 per million mapped reads (FPKM) were calculated using Htseq -count (v0.11.2) (37, 38). 196 Differential gene expression analysis was performed using DESeq2 and edgeR (39, 40), with the 197 default screening conditions set to FDR ≤ 0.05 and |log 2FC (FoldChange)| ≥ 1, applying 198 Benjamini/Hochberg correction for multiple testing. 199 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 10, 2025. ; https://doi.org/10.1101/2025.03.04.641476doi: bioRxiv preprint 8 Functional annotation of the differential gene set was conducted using Gene Ontology (GO) to 200 understand the roles of these genes, metabolic pathways, and other biological processes (41). 201 Additionally, the Kyoto Encyclopedia of Genes and Genomes (KEGG) was used to determine 202 the molecular functional pathways associated with DEGs (42). Functional enrichment analyses, 203 such as GO enrichment and KEGG enrichment, were performed using cluster Profiler (43) . 204 Furthermore, Rockhopper software was employed for small RNA and transcript structure 205 analysis (44). 206 Ethidium bromide accumulation assay 207 The Ethidium bromide (EtBr) accumulation assay was conducted as previously described (45 ) to 208 evaluate the cell envelope permeability of mycobacterial strains. Mycobacterial cultures were 209 grown in Middlebrook 7H9 medium at 37 °C until reaching the mid -log phase. Bacterial 210 suspensions were then normalized to an OD 600 nm of 0.8 in phosphate -buffered saline (PBS) 211 supplemented with 0.8% glucose. EtBr was added to the wells at a final concentration of 2 μ212 g/mL along with 0.4% glucose. Fluorescence measurements were taken using a Flex Station 3 213 Multi-Mode Microplate Reader (Molecular Devices, CA, USA), with excitation and emission 214 wavelengths set at 530 nm and 590 nm, respectively. The fluorescence data from EtBr 215 accumulation were recorded at 60 -second intervals over a period of 60 minutes at 37 °C. Data 216 analysis and plotting were performed using GraphPad Prism version 10.3.1 (GraphPad, San 217 Diego, USA). 218

219 Identification of stresses response factor SigH in Mab 220 Mab is an opportunistic pathogen that causes morbidity in the presence of underlying conditions, 221 including cystic fibrosis (CF). Condition -specific transcriptomics unveiled the molecular factors 222 that drive the persistence and adaptation of Mab in its host. This was demonstrated through Mab 223 exposure to synthetic CF sputum medium, which has been demonstrated to predominantly 224 trigger strong up -regulation in the expression of sigma and anti -sigma factors (46). In addition, 225 the role of these factors in stress response in bacteria has also been demonstrated (25). This 226 therefore demonstrates their potential significance in Mab pathogenesis. These findings inspired 227 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 10, 2025. ; https://doi.org/10.1101/2025.03.04.641476doi: bioRxiv preprint 9 us to investigate whether sigma and anti -sigma factors play a role in resistance to multiple 228 antibiotics in Mab. Thus, we identified two adjacent sigma and anti -sigma factors, sigH 229 (MAB_3543c) and rshA ( MAB_3542c) respectively, for this investigation. We subsequently 230 generated a rshA knockout strain (ΔrhsA) to study the role of rshA gene in drug resistance. The 231 drug susceptibility difference between WT and Δ rhsA was not significant (data not shown). 232 Notably, the rshA gene and sigH lie in the same cluster. Mab SigH shares 84% amino acid 233 identity with Mtb SigH (Figure S1). The stress response factor SigH plays a crucial role in 234 regulating responses to heat and oxidative stress and has been extensively studied in both M tb 235 and Msm (25). Overexpression of sigH in WT (OEMabsigH) resulted in a significant increase i n 236 resistance to TIG and FQs (Table 1). 237 Deletion of sigH increases Mab hypersensitivity to multiple antibiotics 238 To investigate the role of sigH in multiple drug resistance, we constructed an in -frame deletion 239 strain of Mab for sigH (ΔsigH) using CRISPR/Cpf1 -assisted recombineering. The sensitivity of 240 WT, ΔsigH, and its complemented strains was assessed via broth microdilution and spot growth 241 inhibition on agar plates. Drug susceptibility testing revealed that Δ sigH exhibited increased 242 sensitivity to multiple antibiotics, including ribosome -targeting agents such as TIG), TET, and 243 AMK, as well as V AN), rifabutin RIB, and fluoroquinolones LFX and MFX. To further verify 244 the role of sigH in drug resistance in Mab, complemented strains were constructed by 245 reintroducing Mab sigH or its homolog from Mtb (Mtb sigH). The complemented strains 246 restored resistance to the drugs (Figure 1, Table 1). Additionally, complemented strains were 247 constructed by expressing Mab sigH under its native promoter ( Np) (CP NpMabsigH) or the 248 strong mycobacterial hsp60 promoter (CP hsp60MabsigH). Complementation under either 249 promoter led to the restoration of the drug -resistance phenotype (Table 1). These results suggest 250 that sigH plays a significant role in intrinsic multiple drug resistance in Mab. 251 Table 1. MICs of the different Mab strains. 252 Antibioticsa MICb (µg/mL) / Mab strainsc WT ΔsigH CPMabsigH CPMtbsigH OEMabsigH CPhsp60MabsigH CPNpMabsigH TIG 4 0.5 4 2 16 4 8 TET 128 32-64 128 64 ˃128 128 ˃128 CLA 4-64 4-64 4-64 4-64 4-64 4-64 4-64 CLF 4 4 4 4 4 4 4 V AN 128 64 128 128 >128 >128 128 AMK 16 2 16 16 32 16 16 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 10, 2025. ; https://doi.org/10.1101/2025.03.04.641476doi: bioRxiv preprint 10 MFX 8 2 8 8 32 32 32 LFX 16 2 16 16 32-64 32 32 IMP 32 32 32 32 32 32 32 CFX 32 32 32 32 32 32 32 RIB 8 2 8 8 16 8 8 LZD 64 64 64 64 64 64 64 a antimicrobial agents: TIG, tigecycline; TET, tetracycline; CLA, clarithromycin; CLF, clofazimine; V AN vancomycin; AMK, amikacin; LFX, levofloxacin; MFX, moxifloxacin; IMP, imipenem; CFX, cefoxitin; RIB, rifabutin; and LZD, linezolid. b MIC is defined as the lowest concentration of a drug that inhibits visible bacterial growth. c WT, wild type Mab; ΔsigH, sigH deletion strain; CPMabsigH, complemented strain expressing MabsigH; CPMtbsigH, complemented strain expressing MtbsigH; OEMabsigH, wild type Mab overexpressing MabsigH; CPhsp60MabsigH, complemented strain expressing MabsigH under hsp60 promoter; CPNp-MabsigH, complemented strain expressing MabsigH under its Np promoter. The plates were incubated at 37oC for 3 days, except for CLA, which was incubated for 14 days before final reading. 253 254 Figure 1: Susceptibilities of different Mab strains to different antibiotics on 7H10 agar plates. Strains 255 were propagated in 7H9 medium at 37 ºC until reaching an OD 600 nm of 0.6. Subsequently, ten -fold serial 256 dilutions were applied and aliquoted onto plain Middlebrook 7H10 agar (drug -free serving as a control) 257 alongside plates supplemented with varying drug concentrations (µg/mL). Following a 3 -day incubation at 37 258 ºC, the plates were examined. TIG, tigecycline; TET, tetracycline; AMK, amikacin; LFX, levofloxacin; MFX, 259 moxifloxacin and RIB, rifabutin. 260 Deletion of sigH influences stress responses in Mab 261 SigH is a crucial regulator of a large transcriptional network that responds to heat and oxidative 262 stress in Mtb (47). It plays an important role in virulence in animal infection models and in 263 responding to extracellular stresses and intracellular survival (48). To investigate the role of sigH 264 in Mab stress responses, we conducted heat stress assays as well as various oxidative stress 265 assays. The diamide induction assay revealed that the Δ sigH strain is highly sensitive to thiol -266 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 10, 2025. ; https://doi.org/10.1101/2025.03.04.641476doi: bioRxiv preprint 11 specific oxidation by diamide. This sensitivity was evident from a larger inhibition zone in the 267 ΔsigH strain (35 mm) compared to WT (20 mm) and CPMab sigH (26 mm) (Figure 2A; Table 268 S1). After 8 hours, the percentages of survivors for WT, Δ sigH, and CPMab sigH, respectively, 269 under the different conditions were as follows: Diamide (40.38%, 9.71%, and 36.25%), H₂ O₂ 270 (64.99%, 25.74%, and 60.55%), and heat (62.40%, 40.90%, and 53.26%) (Figure 2B-D). 271 272 Figure 2: Sensitivity and survival rates of WT, ΔsigH, and CPMabsigH under stress conditions. 273 Sensitivity of WT, ΔsigH, and CPMabsigH to different concentrations (M) of diamide (A) as well as their 274 survival in the presence of diamide (50 mM) (B), H2O2 (50 mM) (C), and heat (45°C) (D). 275 Identification of d ifferentially expressed genes and analysis of their correlations among 276 WT, ΔsigH, and CPMabsigH 277 The raw reads of transcriptome data were examined to gain insights into gene expression 278 changes in ΔsigH. Several DEGs were found in both Δ sigH and CPMabsigH when compared to 279 WT. Distance heat maps depicting the expression of all genes were utilized to hierarchically 280 cluster the relationships among samples, thereby accurately reflecting inter -sample relationships 281 (Figure 3A). Principal component analysis (PCA) was employed to represent the total variance 282 and correlations among the samples (Figure 3B). This analysis revealed significant differences 283 between Δ sigH and both WT and CPMab sigH. A correlation heat map was plotted to better 284 understand the DEGs and relative expression patterns of shared genes among WT, CPMabsigH, 285 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 10, 2025. ; https://doi.org/10.1101/2025.03.04.641476doi: bioRxiv preprint 12 and ΔsigH. The top 30 DEGs are presented in Figure 3C and supplementary files (XLS 1; XLS 286 2). 287 288 Figure 3: Identification of differentially expressed genes and analysis of their correlations among WT, 289 ΔsigH, and CPMab sigH. Hierarchical clustering based on the expression of all genes can accurately reflect 290 the relationships among samples (A). The first principal component (PC1) and the second principal component 291 (PC2) are plotted in a two -dimensional coordinate graph, with the values in brackets on the axis labels 292 representing the percentage of the total variance explained by each principal component (B). Based on gene 293 expression, we performed hierarchical clustering analysis to explore the relationships between samples and 294 genes. In the figure, each column represents a sample, and each row represents a gene. Different colors 295 indicate the expression levels of genes across various samples. Red indicates higher expression, while blue 296 indicates lower expression. The figure shows the top 30 gene expressions resulting from the clustering analysis 297 (C). Note: WT: WT Mab; KO, ΔsigH; CP, CPMabsigH. 298 299 Deletion of sigH affects global gene expression in Mab 300 To better understand the phenotype of Δ sigH, we identified genes affected by sigH deletion. 301 Whole transcriptome analysis was used to find DEGs in Δ sigH. DEGs were analyzed across 302 three comparison groups: Δ sigH vs. WT, WT vs. CPMab sigH, and Δ sigH vs. CPMab sigH. We 303 identified 863 DEGs in the Δ sigH vs. WT group, with 451 upregulated and 372 downregulated. 304 In the WT vs. CPMab sigH group, 464 DEGs were identified (213 upregulated, 251 305 downregulated). In the Δ sigH vs. CPMab sigH group, 579 DEGs were identified (353 306 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 10, 2025. ; https://doi.org/10.1101/2025.03.04.641476doi: bioRxiv preprint 13 upregulated, 226 downregulated) (Figure 4A). The highest number of DEGs was observed in the 307 ΔsigH vs. WT group. Notably, twelve genes exhibited the lowest expression levels in the Δ sigH 308 vs. WT group compared to the WT vs. CPMabsigH group (Table 2; XLS 3). These genes include 309 MAB_4143c, MAB_1362, MAB_4735, MAB_3016c, MAB_4694c, MAB_2462, MAB_4234c, 310 MAB_4122, MAB_2461, MAB_4843, MAB_2459, and MAB_2460, encoding putative anti -ECF 311 sigma factor, starvation -induced DNA protecting protein/Ferritin and Dps, probable alternative 312 RNA polymerase sigma factor, glycosyltransferase, sulfonate ABC transporter periplasmic 313 protein, reduced flavin mononucleotide (riboflavin 5′ -phosphate) (FMNH2) utilizing oxygenase, 314 acyl-CoA dehydrogenase, and many conserved hypothetical proteins. A high increase in the 315 expression of putative YrbE and MCE family proteins was observed in Δ sigH compared to WT 316 (Table 2), while several putative sigma and anti -sigma factors ( MAB_3028, MAB_3388c, 317 MAB_3016c, MAB_3549c, MAB_3548c, MAB_3546c, MAB_3542c, MAB_3539c, and 318 MAB_3538) were downregulated (Table S2). Other DEGs in the Δ sigH vs. WT group are listed 319 in supplementary materials (XLS 3). The global differential gene expression across different 320 comparison groups is visualized as a heatmap (Figure 4B) and a volcano plot (Figure 4C). These 321

suggest that SigH plays a significant role in shaping global gene expression in Mab. 322 Following the change in global gene expression after sigH deletion, we performed KEGG and 323 GO gene enrichment analyses to determine enriched pathways in Δ sigH. The enriched pathways 324 included those involved in metabolism, cellular processes, human diseases, and the processing of 325 genetic and environmental information (XLS 4; XLS 5). The distribution of KEGG DEGs across 326 the different comparison groups is as follows: Δ sigH vs. WT (45 upregulated, 76 327 downregulated); Δ sigH vs. CPMab sigH (57 upregulated, 10 downregulated); and WT vs. 328 CPMabsigH (45 upregulated, 76 downregulated). The enriched pathways are detected in Δ sigH 329 vs. WT (85 enriched pathways), WT vs. CPMab sigH (64 enriched pathways), and Δ sigH vs. 330 CPMabsigH (72 enriched pathways) groups (Figure S6; XLS 6). For the KEGG enrichment 331 analysis of DEGs, the DEGs in the Δ sigH vs. WT group were mainly enriched in ABC -type 332 transporters. Further, GO enrichment analysis identified changes in DEGs associated with sigma 333 factor, transporter, cofactor, and transmembrane transporter activities (Figure S6; XLS 5). These 334

emphasize that sigH inactivation has a profound impact on transcriptional, sigma, anti -335 sigma, oxidative, and ABC transporter-related gene functions. 336 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 10, 2025. ; https://doi.org/10.1101/2025.03.04.641476doi: bioRxiv preprint 14 337 338 339 Figure 4: DEGs analysis of different comparison groups. The bar graph illustrates the number of 340 upregulated and downregulated DEGs (A). The heat map displays DEGs across different comparison groups; 341 the color intensity indicates the relative expression level of the genes (B). The volcano plot presents 342 differentially expressed genes in the comparison groups, with each point representing a gene. The horizontal 343 axis denotes the log 2 fold change, while the vertical axis represents the negative log 10 of the p-value. Red dots 344 indicate upregulated DEGs, green dots signify downregulated DEGs (down -regulated on the left and up -345 regulated on the right), and gray dots represent non -differentially expressed genes (C). Only genes with 346 |log2FoldChange| > 0 an d p < 0.05 are included in the volcano plot. Note: WT: WT Mab; KO, Δ sigH; CP , 347 CPMabsigH. 348 SigH is involved in drug resistance perhaps by influencing other drug resistance 349 determinants in Mab 350 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 10, 2025. ; https://doi.org/10.1101/2025.03.04.641476doi: bioRxiv preprint 15 SigH is implicated in drug resistance by influencing other drug resistance determinants in Mab. 351 The multi-drug resistance WhiB7 master regulator induces the transcription of sigH. SigH is an 352 alternative sigma factor associated with the transcriptional regulation of genes responsible for 353 mycobacterial stress responses, including oxidative, heat, and nitrosative stress. It is negatively 354 regulated by rshA (15). This sigma factor controls visible physiological alterations and modifies 355 gene expression patterns during antibiotic treatments and diverse environmental stresses, playing 356 a significant role in pathogen drug resistance (49, 50, 51). In this study, several genes were found 357 to be down -regulated in Δ sigH compared to WT and CPMab sigH strains. Examples include 358 MAB_1362, MAB_4143c, MAB_3028, MAB_3388c, MAB_3542c, and MAB_3016c, all 359 previously associated with drug resistance in Mab. For instance, under sigH regulation, 360 MAB_1362 influences intrinsic resistance to AMK, streptomycin (STR), and apramycin (APR) 361 (52). Additionally, the deletion of MAB_3542c increases Mab's sensitivity to TIG (53 ), while a 362 mutation in MAB_3388c (serB2) has been linked to cross-tolerance to CFX and MFX (54). Thus, 363 our results suggest that SigH influences drug resistance in Mab by modulating the expression of 364 other genes directly involved in drug resistance. 365 Deletion of sigH enhanced Mab cell wall permeability 366 The deletion of sigH affects the cell wall permeability of Mab. The hydrophilic fluorescent dye 367 ethidium bromide (EtBr) can intercalate into DNA and RNA and penetrate the cell walls of 368 mycobacteria. Mycobacterial intrinsic resistance to several antimicrobials is thought to be mainly 369 due to decreased cell wall permeability and active efflux mechanisms (55). To investigate 370 whether the sigH deletion affects the cell wall permeability of Mab, we performed an EtBr 371 accumulation assay. We observed that Δ sigH accumulated more EtBr relative to WT, and 372 complementation with CPMab sigH restored the phenotype partially (Figure 5). These findings 373 suggest a possible increase in cell envelope permeability following sigH deletion, highlighting 374 the significance of sigH in maintaining Mab cell wall integrity. 375 376 377 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 10, 2025. ; https://doi.org/10.1101/2025.03.04.641476doi: bioRxiv preprint 16 378 Figure 5: Estimation of cell envelope permeability. Accumulation of EtBr in the cells of WT, Δ sigH, and 379 CPMabsigH strains. 380 0 10 20 30 40 50 60 0 100 200 300 400 500 Time point (min) Relative fluorescence (arbitrary units) WT ΔsigH CPMabsigH ✱✱ ✱✱✱ .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 10, 2025. ; https://doi.org/10.1101/2025.03.04.641476doi: bioRxiv preprint 17 Table 2: The most significant alterations in the transcriptome of DEGs across various Mab groups identified using log2 fold 381 change values 382 Gene Names ΔsigH vs. WT WT vs. CPMabsigH ΔsigH vs. CPMabsigH Description Log2F P value Log2F P value Log2F P value MAB_3543c -12.100632 1.6E-100 -5.51448 1.06E-39 -10.675 2.476 RNA polymerase sigma-H factor MAB_4143c -6.243253 0.0000105 -1.78279 0.013735 -4.7424 5.06E-19 Putative anti-ECF sigma factor MAB_2460 -5.99397 2.80E-53 -5.46303 5.68E-48 -0.53168 0.33503 Conserved hypothetical protein MAB_2459 -5.13153 2.15E-19 -4.76307 1.05E-17 -0.36864 0.68119 Conserved hypothetical protein MAB_4843 -5.0296 0.002647 -4.97085 0.00448 -5.13E-15 1 Hypothetical protein MAB_2461 -4.59441 3.04E-50 -4.62196 9.45E-50 0.028143 1 Putative sulfate ABC transporter, ATP-binding protein MAB_4122 -4.39794 6.07E-14 -3.80961 6.29E-12 -0.58931 0.43792 Putative amino acid permease MAB_4735 -4.3007 2.58E-20 -1.32424 0.000183 -2.98435 4.84E-10 Putative starvation-induced DNA protecting protein/Ferritin and Dps MAB_4234c -4.2974 7.30E-19 -3.88915 5.70E-17 -0.40856 0.595278 Putative reduced flavin mononucleotide (riboflavin 5′- phosphate) (FMNH2)-utilizing oxygenase MAB_4698 4.268768 6.69E-20 1.814478 0.000355 2.454416 5.48E-11 Conserved hypothetical protein MAB_1013 4.265 4.00E-36 1.105148 0.001335 3.160641 3.36E-25 Hypothetical protein MAB_2463 -4.2640 6.39E-25 -4.50564 1.56E-26 0.24279 0.68209 Putative sulfonate ABC transporter, permease MAB_4694c -4.2101 1.21E-17 1.418574 5.91E-05 2.806599 6.84E-18 Glycosyltransferase MAB_1422c -4.2101 1.21E-17 -4.70221 2.94E-20 0.493679 0.372983 Putative acyl-CoA dehydrogenase MAB_1060 -4.20909 6.68E-24 -4.67717 2.85E-26 0.470252 0.378182 Putative alkanesulfonate monooxygenase MAB_2218 -4.19537 1.64E-48 -4.39032 4.67E-51 0.19542 0.561673 Sulfonate ABC transporter periplasmic sulfonate-binding protein SsuA MAB_2462 -4.05529 4.97E-44 -3.8795 6.71E-41 -0.17558 0.653031 Putative sulfonate ABC transporter, periplasmic protein .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 10, 2025. ; https://doi.org/10.1101/2025.03.04.641476doi: bioRxiv preprint 18 MAB_3016c -3.89406 2.23E-17 -0.72556 0.033875 -3.17448 2.67E-12 Conserved hypothetical protein MAB_3574c 5.7915 0.000157 4.651039 0.010345 1.086654 0.169225 3-oxoacyl-[acyl-carrier-protein] synthase II MAB_4123 -4.921 1.56E-40 -5.42211 3.24E-43 0.503349 0.430105 Probable monooxygenase MAB_1011c 4.6210 2.29E-49 1.785083 3.79E-09 2.835979 4.02E-25 Putative YrbE family protein MAB_1012c 4.60967 3.76E-30 2.002296 1.05E-06 2.607384 1.11E-14 Putative YrbE family protein MAB_4908c 4.60967 1.93E-16 0.123578 0.66868 -4.74241 5.06E-19 Putative luciferase-like oxidoreductase MAB_0219 2.74E-72 6.31E-69 -4.0746 4.56E-61 -0.49105 0.035696 Conserved hypothetical protein MAB_4696c 4.477659 3.94E-24 2.729194 1.02E-09 1.748204 5.92E-07 Possible methyltransferase MAB_1005c 4.350183 4.31E-26 2.184348 9.10E-09 2.165824 4.61E-09 Putative MCE family protein MAB_1009c 4.326305 7.32E-30 1.559564 9.28E-06 2.766767 1.89E-15 Putative MCE family protein MAB_1010c 4.207413 1.17E-39 0.919939 0.002153 3.287571 4.01E-28 Putative MCE family protein MAB_1183 -4.17225 6.48E-10 -5.47564 1.64E-12 1.338647 0.3295 Conserved hypothetical protein (rhodanese-like) MAB_4032 4.149218 7.37E-18 2.619616 7.04E-07 1.528882 6.61E-06 Putative Mce family protein MAB_2217 -3.79178 1.06E-36 -3.77927 3.53E-38 -0.01219 1 Sulfonate ABC transporter 2c ATP binding subunit SsuB .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 10, 2025. ; https://doi.org/10.1101/2025.03.04.641476doi: bioRxiv preprint 19

383 The rapidly growing NTM species Mab is an emerging healthcare -associated opportunistic 384 pathogen characterized by its drug -resistant phenotype and high morbidity and mortality rates 385 (56, 57). Mab causes chronic pulmonary diseases that are difficult to manage due to inherent 386 drug resistance, posing a significant public health threat. This underscores the need for 387 investigations to identify novel therapeutic targets and effective treatment options. 388 Bacterial pathogens regulate their gene expression in response to environmental cues during 389 infection, which is crucial for virulence (58). SigH, a sigma factor, plays a critical role in 390 regulating transcription and stress responses in Mtb, Msm, and Mycobacterium avium ssp. 391 paratuberculosis (Mav). SigH controls various genes, including other extracytoplasmic function 392 sigma factors and redox systems (23, 59). It is particularly important for the pathogen's response 393 to heat and oxidative stress, roles extensively studied in Mtb and Msm. Although less well -394 characterized in Mab, SigH has been linked to resistance against TIG and AMK (52, 60). 395 Notably, the peptide sequence of Mab SigH shares 84% similarity with that of Mtb SigH. In this 396 study, we demonstrated that SigH confers resistance not only to TIG and AMK but also to 397 multiple drugs in Mab (Table 1, Figure 1). The increased sensitivity of the ∆ sigH strain to 398 antibiotics such as LFX, MFX, TIG, TET, AMK, V AN, and RIB highlights this role. 399 Complementation of sigH in ∆sigH restored drug resistance, suggesting that SigH's role in drug 400 resistance is conserved between Mab and Mtb (16, 17, 61). Consistently, ∆ sigH showed 401 heightened sensitivity to stressors like diamide, H₂ O₂, and heat, significantly affecting its 402 survival (Figure 2). These findings indicate that SigH's role in stress response is conserved across 403 these mycobacterial species. 404 Sigma factors are global gene regulators and key transcription activators in mycobacterial 405 pathogenesis. They bind to RNA polymerase, enhancing affinity for specific promoters (62). To 406 investigate sigH's influence on global gene expression in Mab, we performed transcriptomic 407 profiling of WT, ∆sigH, and complemented CPMab∆sigH. Significant changes were observed in 408 the gene expression profile of ∆sigH compared to WT, while CPMabsigH closely resembled WT, 409 indicating partial restoration of SigH -associated regulation (Figure 3). A large set of genes was 410 differentially expressed: 863 DEGs (451 upregulated, 372 downregulated) in ∆ sigH vs. WT; 464 411 (213 upregulated, 251 downregulated) in WT vs. CPMab sigH; and 579 (353 upregulated, 226 412 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 10, 2025. ; https://doi.org/10.1101/2025.03.04.641476doi: bioRxiv preprint 20 downregulated) in ∆ sigH vs. CPMab sigH (Figure 4, Table 2). KEGG enrichment analysis 413 revealed that the most downregulated (log 2FC ≤ -3) genes in ∆sigH were ABC-type transporters 414 (22%), potentially explaining the increased antibiotic sensitivity. ABC-type transporters facilitate 415 import/export and drug efflux in mycobacteria (63, 64, 65), so disruption of sigH likely impairs 416 these functions, reducing drug tolerance (Table 1, Figure 1). This is supported by EtBr 417 accumulation in ∆ sigH (Figure 5). Additionally, the downregulation of genes associated with 418 antibiotic resistance was observed, including MAB_1362 (alternative RNA polymerase sigma 419 factor), MAB_3542c (anti -sigma factor), and MAB_3388c (phosphoserine phosphatase serB2) 420 (52, 53, 54). Although we did not test sensitivity to STR and APR, these results align with the 421 observed sensitivity profile. Interestingly, 12 genes were highly upregulated (log 2FC ≥ 2), with 422 five genes (two yrbE and three mce genes) within an mce operon. MCE proteins are associated 423 with virulence and stress responses in mycobacteria (66). Previous studies suggest that oxidative 424 stress, hypoxia, and nutrient deprivation can modify MCE protein expression, implying a role in 425 stress responses (67, 68, 69). Another mce gene ( MAB_4032) was also highly upregulated. We 426 speculate that following sigH disruption and subsequent downregulation of antibiotic resistance 427 genes, Mab attempted to reinforce its cell wall and counter external stressors by upregulating 428 mce genes. However, further experimentation is needed to validate this hypothesis. 429 A synthetic drug -like molecule, SMARt -420 (Small molecule aborting resistance), has been 430 shown to inhibit the DNA binding effect of EthR2 (Rv0078), which is a transcriptional repressor 431 of EthA2 in Mtb. By doing so, SMARt -420 profoundly facilitates the bioactivation and 432 antibacterial effect of ethionamide against Mtb (70). If inhibition of the transcriptional regulator 433 for a single gene would have this profound impact, it would be reasonable to suspect that a 434 protein with genome -wide transcriptional regulatory effect could be an ideal target for drug 435 development. In fact, SigH could be an ideal target for drug development against Mab due to its 436 obvious impact on overall gene expression in this pathogen, as this would have great impact on 437 several pathways that could be involved in drug resistance and stress response. 438 Summary 439 We have identified the role of the mycobacterial sigma factor SigH in stress responses and multi-440 drug resistance in Mab. We hypothesize that disrupting sigH in Mab leads to the downregulation 441 of multiple drug resistance determinants, thereby increasing bacterial sensitivity to drugs. 442 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 10, 2025. ; https://doi.org/10.1101/2025.03.04.641476doi: bioRxiv preprint 21 Additionally, we suggest that as a compensatory mechanism for this downregulation, the bacteria 443 upregulate the expression of certain proteins, such as YrbE and MCE family proteins. Although 444 these findings require further experimental validation, this study lays the foundation for future 445 mechanistic studies to establish the potential of SigH as a drug target in Mab. 446 Author contributions 447 Md Shah Alam : Conceptualization (equal); investigation (lead); methodology (lead); validation 448 (equal); visualization (lead); formal analysis (lead); writing¯¯ original draft (lead); writing¯¯ 449 review and editing (equal); data curation (lead). Mst Sumaia Khatun: Methodology (equal); 450 investigation (equal); formal analysis (equal) and writing¯¯ original draft (supporting). Buhari 451 Yusuf: Original draft (supporting) and writing¯¯ review (lead). Lijie Li : Data curation (equal) 452 and formal analysis (supporting). Aweke Mulu Belachew : Formal analysis (supporting) ; 453 software (supporting). Haftay Abraha Tadesse : Formal analysis (supporting); software 454 (supporting). Jingran Zhang : Original draft (supporting) and formal analysis (supporting). 455 Xirong Tian : Formal analysis (supporting); validation (supporting). Cuiting Fang : Formal 456 analysis (supporting); validation (supporting) . Yamin Gao : Formal analysis (supporting); 457 validation (supporting). Zhiyong Liu: Formal analysis (supporting); validation (supporting). 458 H.M. Adnan Hameed : writing¯¯original draft (supporting) and software (supporting). Jinxing 459 Hu: Resources (supporting), writing¯¯review, and editing (supporting). Xinwen Chen: 460 Resources (supporting), writing¯¯review, and editing (supporting). Nanshan Zhong: Validation 461 (supporting), and writing¯¯review. Shuai Wang : Conceptualization (equal); project 462 administration (lead); funding acquisition (lead); resources (supporting); validation (equal) and 463 writing¯¯review and editing (equal); Tianyu Zhang : Conceptualization (equal); project 464 administration (lead); funding acquisition (lead); resources (supporting), validation (equal) and 465 writing¯¯ review and editing (equal). 466 Acknowledgments 467 This work was supported by the National Key R&D Program of China (2021YFA1300904, 468 2023YFF0713605), the National Natural Science Foundation of China (32300152), partially by 469 Guangdong Provincial Basic and Applied Basic Research Fund (2024A1515012412 ，470 2022A1515110505), the State Key Laboratory of Respiratory Disease, Guangzhou Institute of 471 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 10, 2025. ; https://doi.org/10.1101/2025.03.04.641476doi: bioRxiv preprint 22 Respiratory Diseases, First Affiliated Hospital of Guangzhou Medical University (SKLRD -Z-472 202412, SKLRD-Z-202414, SKLRD-Z-202301). The founders had no role in study design, data 473 collection, and analysis, decision to publish, or preparation of the manuscript. We also 474 acknowledge the group of Yicheng Sun from the Institute of Pathogenic Biology Chinese 475 Academy of Medical Sciences, for kindly sending the pJV53 -Cpfl and pCR -ZEO plasmids as 476 tools for gene deletion. 477 Conflict of interest 478 The authors declare no conflicts of interest. 479

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