Noncanonical mutations in ribosome nascent peptide exit tunnel confer clarithromycin resistance in Mycobacterium abscessus complex.

OBJECTIVES: Mycobacterium abscessus is a non-tuberculous mycobacterial pathogen that causes pulmonary and skin infections globally. Clarithromycin plays a pivotal role in treating M. abscessus infections, with resistance often leading to treatment failure. While canonical mutations in the 23S rRNA residue 2270/2271 are recognized as the primary mechanism for acquired clarithromycin resistance, resistant isolates lacking these mutations have been widely reported. This study aims to identify new mechanisms of clarithromycin resistance in M. abscessus. METHODS: We selected spontaneous resistant mutants derived from two parental strains characterized by erm(41) T28 and C28 sequevars, respectively. Whole-genome sequencing was performed on mutants lacking the 23S rRNA 2270/2271 mutations. Site-directed mutagenesis was used to confirm the resistance phenotypes of newly identified mutations. Bioinformatic analysis of publicly available genomes was conducted to evaluate the presence of these mutations in clinical isolates. The spatial localization of these mutations in the ribosome was analyzed to investigate potential mechanisms of resistance. RESULTS: A total of 135 resistant mutants were selected from the parental strains. Sequencing of the 78 mutants lacking the 23S rRNA 2270/2271 mutations identified mutations within the peptidyl-transferase center and hairpin loops 35, 49, and 74 of the 23S rRNA. These noncanonical mutations were identified in 57 of 1875 genomes of clinical isolates. Thirteen representative mutations were introduced into the bacterial genome, and their contributions to macrolide resistance were confirmed. The newly identified mutations all localized at the entrance of the nascent peptide exit tunnel, potentially contributing to resistance by disrupting the macrolide binding pocket. CONCLUSION: Several noncanonical 23S rRNA mutations conferring clarithromycin resistance were identified. These mutations enhance our understanding of macrolide resistance in M. abscessus and could serve as important markers for diagnosing clarithromycin resistance.

FOXO1 reshapes neutrophils to aggravate acute brain damage and promote late depression after traumatic brain injury.

BACKGROUND: Neutrophils are traditionally viewed as first responders but have a short onset of action in response to traumatic brain injury (TBI). However, the heterogeneity, multifunctionality, and time-dependent modulation of brain damage and outcome mediated by neutrophils after TBI remain poorly understood. METHODS: Using the combined single-cell transcriptomics, metabolomics, and proteomics analysis from TBI patients and the TBI mouse model, we investigate a novel neutrophil phenotype and its associated effects on TBI outcome by neurological deficit scoring and behavioral tests. We also characterized the underlying mechanisms both in vitro and in vivo through molecular simulations, signaling detections, gene expression regulation assessments [including dual-luciferase reporter and chromatin immunoprecipitation (ChIP) assays], primary cultures or co-cultures of neutrophils and oligodendrocytes, intracellular iron, and lipid hydroperoxide concentration measurements, as well as forkhead box protein O1 (FOXO1) conditional knockout mice. RESULTS: We identified that high expression of the FOXO1 protein was induced in neutrophils after TBI both in TBI patients and the TBI mouse model. Infiltration of these FOXO1high neutrophils in the brain was detected not only in the acute phase but also in the chronic phase post-TBI, aggravating acute brain inflammatory damage and promoting late TBI-induced depression. In the acute stage, FOXO1 upregulated cytoplasmic Versican (VCAN) to interact with the apoptosis regulator B-cell lymphoma-2 (BCL-2)-associated X protein (BAX), suppressing the mitochondrial translocation of BAX, which mediated the antiapoptotic effect companied with enhancing interleukin-6 (IL-6) production of FOXO1high neutrophils. In the chronic stage, the "FOXO1-transferrin receptor (TFRC)" mechanism contributes to FOXO1high neutrophil ferroptosis, disturbing the iron homeostasis of oligodendrocytes and inducing a reduction in myelin basic protein, which contributes to the progression of late depression after TBI. CONCLUSIONS: FOXO1high neutrophils represent a novel neutrophil phenotype that emerges in response to acute and chronic TBI, which provides insight into the heterogeneity, reprogramming activity, and versatility of neutrophils in TBI.

Metabotropic glutamate receptor 5 promotes blood-brain barrier recovery after traumatic brain injury.

Blood-brain barrier (BBB) impairment and glutamate release are two pathophysiological features of traumatic brain injury (TBI), contributing to secondary brain damage and neuroinflammation. However, our knowledge of BBB integrity damage and dysfunction are still limited due to the diverse and fluctuating expression of glutamate receptors after trauma. Here, we confirmed the downregulation of metabotropic glutamate receptor 5 (mGluR5) on microvascular endothelial cell within the acute phase of TBI, and the recovered mGluR5 levels on BBB was positively associated with blood perfusion and neurological recovery. In whole body mGluR5-knockout mice, BBB dysfunction and neurological deficiency were exacerbated after TBI compared with wild type mice. In terms of mechanism, the amino acid sequence 201-259 of cytoskeletal protein Alpha-actinin-1 (ACTN1) interacted with mGluR5, facilitating mGluR5 translocation from cytoplasmic compartment to plasma membrane in endothelial cells. Activation of plasma membrane mGluR5 triggers the PLC/PKCµ/c-Jun signaling pathway, leading to increased expression of the tight junction-actin cytoskeleton connecting protein zonula occludens-1 (ZO-1). Our findings uncover a novel mechanism mediated by membrane and cytoplasmic mGluR5 in endothelial cell integrity maintenance and repair, providing the potential therapeutic target for TBI treatment targeting at mGluR5 and mGluR5/ACTN1 complex in BBB.

Mycobacterium tuberculosis PPE7 Enhances Intracellular Survival of Mycobacterium smegmatis and Manipulates Host Cell Cytokine Secretion Through Nuclear Factor Kappa B and Mitogen-Activated Protein Kinase Signaling.

The PE/PPE family proteins of Mycobacterium tuberculosis have been associated with its virulence and interaction with the host immune system. The highly virulent modern lineage of M. tuberculosis possesses a lineage-specific PPE gene (PPE7), which arises from an ancestral mutation and is rarely studied. Here we examined the role of PPE7 in mycobacterial pathogenicity and survival by expressing M. tuberculosis PPE7 in Mycobacterium smegmatis. We show that, PPE7 activates host inflammation by increasing expression of pro-inflammatory cytokines including tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1ß, and IL-6, while suppressing the expression of anti-inflammatory cytokines such as IL-10, possibly through the nuclear factor kappa B, ERK1/2, and p38 mitogen-activated protein kinase pathways. Overexpressing PPE7 in M. smegmatis could enhance bacterial intracellular survival of infected macrophages. Furthermore, higher level of bacterial persistence, higher levels of TNF-α, IL-1ß, and IL-6 cytokines, and more injury in the lung, liver, and spleen tissues of infected mice has been discovered. In conclusion, PPE7 could manipulate host immune response and increase bacterial persistence.

Improvement of the immunogenicity of ESAT-6 via fusion with the dodecameric protein dodecin of Mycobacterium tuberculosis.

Tuberculosis (TB) is a chronic infectious disease that creates a heavy medical burden worldwide. The only approved vaccine, Bacillus Calmette-Guérin (BCG), cannot fully protect adolescents and adults from TB. Therefore, there is an urgent need to develop an effective new vaccine. Previous studies have found that dodecin, a flavin-binding protein of Mycobacterium tuberculosis (Mtb), can form stable dodecamers and has the potential to improve the immunogenicity of Mtb antigens. In this study, we constructed the fusion protein dodecin-ESAT-6 and evaluated the immunogenicity of dodecin, ESAT-6, and dodecin-ESAT-6 separately. Our results showed that dodecin-ESAT-6 is a dodecameric protein that can withstand heat at 95 °C and under SDS-PAGE conditions. Dodecin-ESAT-6 increased the expression of the costimulatory molecules CD80, CD86, and major histocompatibility complex class II (MHC-II) on the surface of RAW264.7 macrophages. Mice immunized with dodecin-ESAT-6 exhibited higher percentages of antigen-specific CD4+ and CD8+ T lymphocytes, higher levels of spleen lymphocyte proliferation and IFN-γ and IL-2 secretion, and a lower level of IL-4 secretion than those immunized with ESAT-6. The IgG, IgG1, and IgG2a titers of the dodecin-ESAT-6 group were significantly higher than those of the ESAT-6 group. Dodecin-ESAT-6 elicited a high IgG2a/IgG1 ratio and tended to produce a predominantly Th1-like response. These results support the conclusion that the dodecin-ESAT-6 dodecameric protein induced strong Th1 immune responses and improved the immunogenicity of ESAT-6, which provides a new strategy for TB vaccine development.

Compensatory effects of M. tuberculosis rpoB mutations outside the rifampicin resistance-determining region.

Mycobacterium tuberculosis has been observed to develop resistance to the frontline anti-tuberculosis drug rifampicin, primarily through mutations in the rifampicin resistance-determining region (RRDR) of rpoB. While these mutations have been determined to confer a fitness cost, compensatory mutations in rpoA and rpoC that may enhance the fitness of resistant strains have been demonstrated. Recent genomic studies identified several rpoB non-RRDR mutations that co-occurred with RRDR mutations in clinical isolates without rpoA/rpoC mutations and may confer fitness compensation. In this study, we identified 33 evolutionarily convergent rpoB non-RRDR mutations through phylogenomic analysis of public genomic data for clinical M. tuberculosis isolates. We found that none of these mutations, except V170F and I491F, can cause rifampin resistance in Mycolicibacterium smegmatis. The compensatory effects of five representative mutations across rpoB were evaluated by an in vitro competition assay, through which we observed that each of these mutations can significantly improve the relative fitness of the initial S450L mutant (0.97-1.08 vs 0.87). Furthermore, we observed that the decreased RNAP transcription efficiency introduced by S450L was significantly alleviated by each of the five mutations. Structural analysis indicated that the fitness compensation observed for the non-RRDR mutations might be achieved by modification of the RpoB active centre or by changes in interactions between RNAP subunits. Our results provide experimental evidence supporting that compensatory effects are exerted by several rpoB non-RRDR mutations, which could be utilized as additional molecular markers for predicting the fitness of clinical rifampin-resistant M. tuberculosis strains.

[Preliminary Study on the Secretory Performance of Mycobacterium tuberculosis Hypoxic Response Protein 1 (Rv2626c)].

OBJECTIVE: To verify the secretory ability of the hypoxic response protein 1 (HRP1) encoded by Mycobacterium tuberculosis (Mtb) Rv2626c. METHODS: The target gene attached with His tag was amplified from the genome of Mtb standard virulence strain H37Rv. The recombinant plasmid contained the above amplified product was constructed and electroporated into Mycobacterium smegmatis (Ms) (MC 2155) to construct a recombinant strain. Protein expression was induced under heat condition, and the expression of protein from the culture filtrates and the bacterial lysates was detected afterward. The 10 kDa culture filtrate antigen (CFP-10) (Ms) and CFP-10 (Mtb) were used as positive controls, and the cytoplasmic protein heat shock protein 65 (GroEL2) (Mtb) was used as negative controls. RESULTS: The HRP1, GroEL2 (Mtb), CFP-10 (Mtb) and CFP-10 (Ms) were successfully amplified by PCR from recombinant plasmid, and sequencing results of the recombinant plasmid is right, confirming the successful construction of the recombinant plasmid. The recombinant Ms was successfully constructed and it could express the proteins GroEL2 (Mtb), HRP1, CFP-10 (Mtb) and CFP-10 (Ms). The target protein HRP1 was detected in both of the lysate and the culture filtrate of the recombinant strain by Western blot, which was consistent with the positive control CFP-10. The negative control GroEL2 (Mtb) was only detected in the bacterial lysate, but not detected in the culture filtrate. CONCLUSION: The protein HRP1 encoded by Mtb Rv2626c can be secreted out of Ms by the secretion system of Ms. It may be a secreted protein and play an important role in the pathogenesis of Mtb.

[Optimizing Mycobacterium Recombineering System (pJV53) to Promote the Screening of the Mycobacterium Mutants].

OBJECTIVE: To establish a way for screening Mycobacterium mutants through adding the screening markers into pJV53. METHODS: The sucrose counter selection gene SacB and mutant hygromycin-resistant gene hygS were inserted into pJV53; The recovery of the hygromycin-resistance indicated the successful homologous recombination in Mycobacterium smegmatis (Ms), which could serve as mutant screening marker; The sucrose counter selection could be used to screen the plasmid-free mutants. RESULTS: The recombinant plasmid pJV53-SacB-hygS were successfully constructed. The rifampin-resistant rpoB D516Y and rpoB H526Q mutants and MSMEG_4487 G188A mutant were efficiently screened out. All mutants had shed the plasmid successfully. CONCLUSION: pJV53-SacB-hygS can efficiently contribute to construct and screen the mutants and to get the mutants shedding the plasmid self, which has high value of extensive application; the D516Y and H526Q mutations in gene rpoB of Mycobacterium tuberculosis contribute to its rifampin-resistance.

[Mycobacterium Tuberculosis Rv3084 Encodes Functional Esterase and Suppresses the Pro-inflammatory Cytokines in vivo].

OBJECTIVE: To explore the biological characteristics of the esterase LipR encoded by Mycobacterium tuberculosis (MTB) Rv3084 and its immunomodulatory function in vivo. METHODS: The LipR gene was amplified from MTB H37Rv strain to construct recombinant expression plasmid. After sequencing, the recombinant plasmid was transformed into E. coli for expression and purification of LipR protein. The expressed protein was confirmed with Western blot assay. The hydrolyzing activity of LipR was detected and the factors affecting LipR enzyme activity were analyzed. Mice were intramuscularly injected with 0.1 mL (containing plasmid DNA 100 µg) recombinant eukaryotic plasmid three times (day 1, 8, and 15); seven days after the last injection, the mice were executed, and the lung and spleen were taken for cytokine detection. RESULTS: The recombinant expression plasmid was successfully constructed and it was found that LipR protein was mainly expressed in the form of inclusion bodies in E. coli with the relative molecular mass of about 33×10 3. LipR was demonstrated as an alkaline eurythermic esterase, due to the preference of hydrolyzing short carbon chain esters with optimal hydrolyzing activity on pNP-acetate (pNPA, C2) and the capability in tolerance of high pH and temperature; in the presence of different detergents or metal ions, the activity of LipR hydrolyzing pNP-butyrate (pNPB, C4) was inhibited to some extent. In the mouse model, it was found that LipR could inhibit the secretion of interferon-γ (IFN- γ) and interleukin-2 (IL-2), but to stimulate the secretion of IL-10. CONCLUSION: The esterase LipR may be one of the esterases help M. tuberculosis withstand harsh environment inside the host in collaboration, and simultaneously act as an immune modulator to inhibit the secretion of pro-inflammatory cytokines and consequently impact the killing effect of host immune system against M. tuberculosis.

PPE11 of Mycobacterium tuberculosis can alter host inflammatory response and trigger cell death.

Tuberculosis (TB), which is caused by Mycobacterium tuberculosis (Mtb), remains a serious global health problem. The PE/PPE family, featuring unique sequences, structures and expression in Mtb, is reported to interfere with the macrophage response to the pathogen and facilitate its infection. PPE11 (Rv0453) existed in pathogenic mycobacteria and was persistently expressed in the infected guinea pig lungs. However, the role it played in the pathogenesis remains unclear. Here, to investigate the interaction and potential mechanism of PPE11 between pathogens and hosts, we heterologously expressed PPE11 in non-pathogenic, rapidly growing Mycobacterium smegmatis strains. We found that the overexpression of the cell wall-associated protein, PPE11, can improve the viability of bacteria in the presence of lysozyme, hydrogen peroxide and acid stress. Expression of PPE11 enhanced the early survival of M. smegmatis in macrophages and sustained a higher bacterial load in mouse tissues that showed exacerbated organ pathology. Macrophages infected with recombinant M. smegmatis produced significantly greater amounts of interleukin (IL)-1ß, IL-6, tumour necrosis factor (TNF)-α and an early decrease in IL-10 along with higher levels of host cell death. Similar cytokines changes were observed in the sera of infected mice. Accordingly, PPE11 protein causes histopathological changes by disrupting the dynamic balance of the inflammatory factors and promoting host-cell death, indicating a potential role in the virulence of Mtb.

The truncated Rv2820c of Mycobacterium tuberculosis Beijing family augments intracellular survival of M. smegmatis by altering cytokine profile and inhibiting NO generation.

Genetic variations among genes of Mycobacterium tuberculosis may be associated with antigenic variation and immune evasion, which complicates the pathogenesis of M. tuberculosis. The hyper-virulent M. tuberculosis Beijing strains harbored several large sequence deletions, among which RD207 attributed to the deletion of CRISPR loci and several Cas genes. RD207 also gave rise to a truncated gene Rv2820c-Bj with 60% deletion in length at the 3'-end and a new 3'-end of five amino acid mutations. It has been reported that Rv2820c-Bj correlated with enhanced intracellular survival of M. smegmatis in macrophages when compared to its full-length counterpart Rv2820c in M. tuberculosis, however, the respective contribution of the truncation and the new 3'-end of Rv2820c-Bj to this enhancement was unclear. Here, by infecting THP-1 macrophages with Ms_Rv2820c-Bj, Ms_Rv2820c and MS_Rv2820c-Tr (expressing the truncated Rv2820c without five amino acid mutations at 3'-end), we found only Ms_Rv2820c-Bj was responsible for the enhancement of survival of M. smegmatis in macrophages. Furthermore, we detected that Ms_Rv2820c-Tr and Ms_Rv2820c-Bj induced similar cytokine profile and NO production after infection of macrophages, which was distinctly different from Ms_Rv2820c. However, Ms_Rv2820c-Bj evoked higher levels of interleukin-10 (IL-10) and lower levels of interleukin- 6 (IL-6), interleukin-1ß (IL-1ß) and interleukin-12 (IL-12) in infected THP-1 macrophages than Ms_Rv2820c-Tr. Accordingly, we concluded that the new 3'-end of Rv2820c-Bj was important to dampen host defense and enhance the intracellular survival of M. smegmatis.