Ajoene: a natural compound with enhanced antimycobacterial and antibiofilm properties mediated by efflux pump modulation and ROS generation against M. Smegmatis.

Tuberculosis (TB) continues to be a primary worldwide health concern due to relatively ineffective treatments. The prolonged duration of conventional antibiotic therapy warrants innovative approaches to shorten treatment courses. In response to challenges, the study explores potential of Ajoene, a naturally occurring garlic extract-derived compound, for potential TB treatment. Mycobacterium smegmatis as a model organism for M. tuberculosis (M. tb) to investigate Ajoene's efficiency. In vitro techniques like antimicrobial susceptibility, antibiofilm, EtBr accumulation assay, and ROS assay evaluate the potency of Ajoene and conventional TB drugs against Mycobacterium smegmatis. An in-silico study also investigated the interaction between Ajoene and quorum-sensing proteins, specifically regX3, MSMEG_5244, and MSMEG_3944, which are involved in biofilm formation and sliding activity. In vitro findings revealed that Ajoene exhibited significant antibacterial activity by inhibiting growth and showing bactericidal effects. It also demonstrated additive interactions with common antibiotics such as Isoniazid and Rifampicin. Furthermore, Ajoene demonstrated a comparative interaction with commonly used antibiotics, such as Isoniazid and Rifampicin, and reduced M. smegmatis motility, both alone and in combination with these antibiotics. In silico analysis shows that Ajoene exhibited a higher binding affinity with regX3, a protein orthologous to the regX3 gene in M.tb. Ajoene also demonstrated consistent antibiofilm effects, particularly when combined synergistically with Isoniazid and Rifampicin. Mechanistic investigations demonstrated Ajoene's potential to inhibit efflux pumps and promote ROS generation in bacteria, suggesting a potential direct killing mechanism. Collectively, the findings emphasize Ajoene's effectiveness as a novel antimycobacterial and antibiofilm molecule for TB treatment.

Point mutation in the stop codon of MAV_RS14660 increases the growth rate of Mycobacterium avium subspecies hominissuis.

Mycobacterium avium subspecies hominissuis (MAH) is a pathogen that causes various non-tuberculous mycobacterial diseases in humans and animals worldwide. Among the genus, MAH is characterized by relatively slow growth. Here, we isolated a rapidly growing variant of the MAH 104 strain. The variant strain (named N104) exhibited an enhanced growth rate and higher motility compared to the parent MAH 104 strain (P104). Whole-genome sequencing analysis of N104 revealed the loss of the stop codon of MAV_RS14660 due to a single nucleotide replacement, resulting in the substitution of the codon for tryptophan. Notably, exclusion of the stop codon ligated the open reading frames and caused the fusion of two adjacent proteins. A revertant parent strain, in which a mutation was introduced to restore the stop codon, revealed that elimination of the stop codon in MAV_RS14660 was responsible for the N104 phenotype. Furthermore, we analysed the phenotypes of the parent and mutated strains by determining the functions of the MAV_RS14660 and MAV_RS14655 coding regions flanking the stop codon. The mutant strains, expected to express a fusion protein, exhibited increased resistance to antimicrobial drugs and exogenous copper toxicity compared to that of the parent strains. These findings suggest that the fusion of the MAV_RS14660- and MAV_RS14655-encoding regions in the mutant N104 strain could be related to the modified functions of these intrinsic proteins.

Synthetic arabinomannan glycolipids impede mycobacterial growth, sliding motility and biofilm structure.

Mycobacterium has evolved distinct cell wall and strategies such as biofilm formation, which helps it to survive in hostile conditions. We have reported previously that arabinofuranoside containing glycolipids exhibit inhibition activities against the above functions of the mycobacterial species M. smegmatis. In search for activities mediated by oligosaccharide glycolipids, we report herein the inhibitory activities of a linear and a branched pentasaccharides having arabinan and mannan moieties. In the presence of the pentasaccharide glycolipids, a significant reduction in mycobacterial growth is observed, concomitant with reductions in sliding motility and colonization through biofilm formation, at the optimal glycolipid concentrations of 50-100 µg mL(-1). Especially the biofilm coat is ruptured by ~80-85 % in the presence of glycolipids. Pentasaccharides alone without the lipidic chain show only a weak effect. The glycolipids are non-toxic, as evaluated through their effect on RBCs. Analysis of the mycolic acid profile of glycolipid treated biofilm shows that α- and epoxy mycolic acids are downregulated significantly, in comparison to glycolipid untreated biofilms. Lipidomics profile analysis through mass spectrometry further reveals profound downregulation of phosphatidylinositol mannosides, acylatedphosphoglycerols and mycolic acid family, namely, keto-, alpha- and methoxymycolic acids.

1,3-Diethylurea-enhanced Mg-ATPase activity of skeletal muscle myosin with a converse effect on the sliding motility.

We investigate the effects of urea and its derivatives on the ATPase activity and on the in vitro motility of chicken skeletal muscle actomyosin. Mg-ATPase rate of myosin subfragment-1 (S1) is increased by 4-fold by 0.3M 1,3-diethylurea (DEU), but it is unaffected by urea, thiourea, and 1,3-dimethylurea at ≤1M concentration. Thus, we further examine the effects of DEU in comparison to those of urea as reference. In in vitro motility assay, we find that in the presence of 0.3M DEU, the sliding speeds of actin filaments driven by myosin and heavy meromyosin (HMM) are significantly decreased to 1/16 and 1/6.6, respectively, compared with the controls. However, the measurement of the actin-activated ATPase activity of HMM shows that the maximal rate, Vmax, is almost unchanged with DEU. Thus, the myosin-driven sliding motility of actin filaments is significantly impeded in the presence of 0.3M DEU, whereas the cyclic interaction of myosin with F-actin occurs during the ATP turnover, the rate of which is close to that without DEU. In contrast to DEU, 0.3M urea exhibits only modest effects on both actin-activated ATPase and sliding motility of actomyosin. Thus, DEU has the effect of uncoupling the sliding motility of actomyosin from its ATP turnover.

Colonization and spreading dynamics of Lactiplantibacillus plantarum spoilage isolates on wet surfaces.

The role of lactic acid bacteria, including Lactiplantibacillus plantarum, in food spoilage is well recognized, while the behavior of these non-motile bacteria on wet surfaces, such as those encountered in food processing environments has gained relatively little attention. Here, we observed a fast colony spreading of non-motile L. plantarum spoilage isolates on wet surfaces via passive sliding using solid BHI agar media as a model. We investigated the effect of physical properties of agar hydrogel substrate on the surface spreading of six L. plantarum food isolates FBR1-6 and a model strain WCFS1, using increasing concentrations of agar from 0.25 up to 1.5% (w/v). Our results revealed that L. plantarum strain FBR2 spreads significantly on low agar concentration plates compared to the other strains studied here (with a factor of 50-60 folds higher surface coverage), due to the formation of very soft biofilms with high water content that can float on the surface. The fast-spreading of FBR2 colonies is accompanied by an increased number of cells, elongated cell morphology, and a higher amount of extracellular components. Our finding highlights colonization dynamics and the spreading capacity of non-motile bacteria on surfaces that are relatively wet, thereby revealing an additional hitherto unnoticed parameter for non-motile bacteria that may contribute to contamination of foods by fast surface spreading of these bacteria in food processing environments.

Effect of biogenic polyamines on sliding motility of mycobacteria in the presence of antibiotics.

Nowadays, sliding is the least investigated mode of bacterial motility. Sliding is a process of passive movement on the surface of semi-liquid mediums which was originally described for mycobacteria and other bacterial species deprived of the organelles specialized for movement. Some mycobacteria are able to colonize surfaces, including tissues of macro-organisms, using glycopeptidolipids localized in the cell envelope for this aim. This is a serious problem for effective therapy of mycobacteriosis caused by nontuberculosis mycobacteria. Furthermore, animal tissues contain biogenic polyamines, which can increase tolerance of microorganisms to stresses, including antibiotics, and modulate cell motility. Therefore, studying mutual effects of biogenic polyamines and antibiotics on the expansion of mycobacteria is important for medicine. Mycobacterial strains, including the parent Mycolicibacterium smegmatis mc2 155 and strains containing single (ΔrelMsm) or double (ΔrelMsmΔrelZ) deletions, were used as the objects of this study. The content of glycopeptidolipids was determined using thin layer chromatography. Sliding motility was assessed by measuring the area of the sliding colony. The effectiveness of antibiotics was measured by comparison of the areas of sliding colonies in the presence of comparable concentrations of antibiotics. The polyamines spermidine and spermine had different effects on the sliding of mycobacteria through an increase or decrease in the colony areas. At the same time, polyamines had neither bactericidal nor bacteriostatic effects. The polyamines contained in the medium decreased the bactericidal effects of the antibiotics streptomycin or isoniazid, but enhanced the effects of DMNP, a synthetic analogue of the natural antibiotic erogorgiaene. Rifampicin was the most effective of all antibiotics investigated here. Moreover, we found that glycopeptidolipids are, apparently, not the only regulators of mycobacterial sliding.

Mab_3083c Is a Homologue of RNase J and Plays a Role in Colony Morphotype, Aggregation, and Sliding Motility of Mycobacterium abscessus.

Mycobacterium abscessus is an opportunistic pathogen causing human diseases, especially in immunocompromised patients. M. abscessus strains with a rough morphotype are more virulent than those with a smooth morphotype. Morphotype switch may occur during a clinical infection. To investigate the genes involved in colony morphotype switching, we performed transposon mutagenesis in a rough clinical strain of M. abscessus. A morphotype switching mutant (smooth) named mab_3083c::Tn was obtained. This mutant was found to have a lower aggregative ability and a higher sliding motility than the wild type strain. However, its glycopeptidolipid (GPL) content remained the same as those of the wild type. Complementation of the mutant with a functional mab_3083c gene reverted its morphotype back to rough, indicating that mab_3083c is associated with colony morphology of M. abscessus. Bioinformatic analyses showed that mab_3083c has a 75.4% identity in amino acid sequence with the well-characterized ribonuclease J (RNase J) of M. smegmatis (RNase JMsmeg). Complementation of the mutant with the RNase J gene of M. smegmatis also switched its colony morphology from smooth back to rough. These results suggest that Mab_3083c is a homologue of RNase J and involved in regulating M. abscessus colony morphotype switching.

Role of the Mycobacterium marinum ESX-1 Secretion System in Sliding Motility and Biofilm Formation.

Mycobacterium marinum is a close relative of Mycobacterium tuberculosis that can cause systemic tuberculosis-like infections in ectotherms and skin infections in humans. Sliding motility correlates with biofilm formation and virulence in most bacteria. In this study, we used a sliding motility assay to screen 2,304 transposon mutants of M. marinum NTUH-M6885 and identified five transposon mutants with decreased sliding motility. Transposons that interrupted the type VII secretion system (T7SS) ESX-1-related genes, espE (mmar_5439), espF (mmar_5440), and eccA1 (mmar_5443), were present in 3 mutants. We performed reverse-transcription polymerase chain reaction to verify genes from mmar_5438 to mmar_5450, which were found to belong to a single transcriptional unit. Deletion mutants of espE, espF, espG (mmar_5441), and espH (mmar_5442) displayed significant attenuation regarding sliding motility and biofilm formation. M. marinum NTUH-M6885 possesses a functional ESX-1 secretion system. However, deletion of espG or espH resulted in slightly decreased secretion of EsxB (which is also known as CFP-10). Thus, the M. marinum ESX-1 secretion system mediates sliding motility and is crucial for biofilm formation. These data provide new insight into M. marinum biofilm formation.

Antibiotic Stimulation of a Bacillus subtilis Migratory Response.

Competitive interactions between bacteria reveal physiological adaptations that benefit fitness. Bacillus subtilis is a Gram-positive species with several adaptive mechanisms for competition and environmental stress. Biofilm formation, sporulation, and motility are the outcomes of widespread changes in a population of B. subtilis. These changes emerge from complex, regulated pathways for adapting to external stresses, including competition from other species. To identify competition-specific functions, we cultured B. subtilis with multiple species of Streptomyces and observed altered patterns of growth for each organism. In particular, when plated on agar medium near Streptomyces venezuelae, B. subtilis initiates a robust and reproducible mobile response. To investigate the mechanistic basis for the interaction, we determined the type of motility used by B. subtilis and isolated inducing metabolites produced by S. venezuelae. Bacillus subtilis has three defined forms of motility: swimming, swarming, and sliding. Streptomyces venezuelae induced sliding motility specifically in our experiments. The inducing agents produced by S. venezuelae were identified as chloramphenicol and a brominated derivative at subinhibitory concentrations. Upon further characterization of the mobile response, our results demonstrated that subinhibitory concentrations of chloramphenicol, erythromycin, tetracycline, and spectinomycin all activate a sliding motility response by B. subtilis. Our data are consistent with sliding motility initiating under conditions of protein translation stress. This report underscores the importance of hormesis as an early warning system for potential bacterial competitors and antibiotic exposure. IMPORTANCE Antibiotic resistance is a major challenge for the effective treatment of infectious diseases. Identifying adaptive mechanisms that bacteria use to survive low levels of antibiotic stress is important for understanding pathways to antibiotic resistance. Furthermore, little is known about the effects of individual bacterial interactions on multispecies communities. This work demonstrates that subinhibitory amounts of some antibiotics produced by streptomycetes induce active motility in B. subtilis, which may alter species interaction dynamics among species-diverse bacterial communities in natural environments. The use of antibiotics at subinhibitory concentrations results in many changes in bacteria, including changes in biofilm formation, small-colony variants, formation of persisters, and motility. Identifying the mechanistic bases of these adaptations is crucial for understanding how bacterial communities are impacted by antibiotics.

Sesamol exhibits potent antimycobacterial activity: Underlying mechanisms and impact on virulence traits.

OBJECTIVES: Novel strategies to overcome multidrug resistance (MDR) in Tuberculosis (TB) still remain a concern. Usage of natural compounds nowadays to surmount the increasing burden of MDR-TB has shown promising results. The aim of this study was to evaluate the antimycobacterial potential of sesamol (Ses) a natural phenolic compound against Mycobacterium smegmatis, a surrogate for MTB and its underlying mechanism of action along with its effect on mycobacterial virulence traits. METHODS: Cell surface phenotypes were estimated microscopically and spectrophotometrically respectively. Membrane parameters were assessed using propidium iodide (PI) uptake, passive diffusion of drug with substrate EtBr and phenotypic susceptibility assay. Changes in lipid profiles were estimated by lipase assay. Oxidative and genotoxic damage were studied using fluorescent probes DCFDA and DAPI. Biofilm formation was studied using crystal violet and calcoflour white staining probes along with biomass measurement. Cell adherence was estimated using buccal epithelial cells. RESULTS: We observed that antimycobacterial activity of Ses was 6mM and it enhances the efficiency of known anti-TB drugs. Ses affects cell surface phenotypes as displayed by altered colony morphology, impaired sliding motility and enhanced cell sedimentation rate. Membrane perturbation was revealed by hypersensitivity against SDS, reduced PI uptake, enhanced passive diffusion and lipase activity. In addition, Ses leads to oxidative and DNA damage along with abrogated iron homeostasis. Furthermore, we uncover phenotypes related to virulence like inhibited biofilm formation and cell adherence to buccal epithelial cells. CONCLUSION: This study for the first time establishes the anti-mycobacterial potential of Ses that may be further exploited for improving the therapeutic strategies and warrants further attention.