Bacterial System Research Articles

MLDSPP: Bacterial Promoter Prediction Tool Using DNA Structural Properties with Machine Learning and Explainable AI.

Bacterial promoters play a crucial role in gene expression by serving as docking sites for the transcription initiation machinery. However, accurately identifying promoter regions in bacterial genomes remains a challenge due to their diverse architecture and variations. In this study, we propose MLDSPP (Machine Learning and Duplex Stability based Promoter prediction in Prokaryotes), a machine learning-based promoter prediction tool, to comprehensively screen bacterial promoter regions in 12 diverse genomes. We leveraged biologically relevant and informative DNA structural properties, such as DNA duplex stability and base stacking, and state-of-the-art machine learning (ML) strategies to gain insights into promoter characteristics. We evaluated several machine learning models, including Support Vector Machines, Random Forests, and XGBoost, and assessed their performance using accuracy, precision, recall, specificity, F1 score, and MCC metrics. Our findings reveal that XGBoost outperformed other models and current state-of-the-art promoter prediction tools, namely Sigma70pred and iPromoter2L, achieving F1-scores >95% in most systems. Significantly, the use of one-hot encoding for representing nucleotide sequences complements these structural features, enhancing our XGBoost model's predictive capabilities. To address the challenge of model interpretability, we incorporated explainable AI techniques using Shapley values. This enhancement allows for a better understanding and interpretation of the predictions of our model. In conclusion, our study presents MLDSPP as a novel, generic tool for predicting promoter regions in bacteria, utilizing original downstream sequences as nonpromoter controls. This tool has the potential to significantly advance the field of bacterial genomics and contribute to our understanding of gene regulation in diverse bacterial systems.

CRISPR-DIPOFF: an interpretable deep learning approach for CRISPR Cas-9 off-target prediction.

CRISPR Cas-9 is a groundbreaking genome-editing tool that harnesses bacterial defense systems to alter DNA sequences accurately. This innovative technology holds vast promise in multiple domains like biotechnology, agriculture and medicine. However, such power does not come without its own peril, and one such issue is the potential for unintended modifications (Off-Target), which highlights the need for accurate prediction and mitigation strategies. Though previous studies have demonstrated improvement in Off-Target prediction capability with the application of deep learning, they often struggle with the precision-recall trade-off, limiting their effectiveness and do not provide proper interpretation of the complex decision-making process of their models. To address these limitations, we have thoroughly explored deep learning networks, particularly the recurrent neural network based models, leveraging their established success in handling sequence data. Furthermore, we have employed genetic algorithm for hyperparameter tuning to optimize these models' performance. The results from our experiments demonstrate significant performance improvement compared with the current state-of-the-art in Off-Target prediction, highlighting the efficacy of our approach. Furthermore, leveraging the power of the integrated gradient method, we make an effort to interpret our models resulting in a detailed analysis and understanding of the underlying factors that contribute to Off-Target predictions, in particular the presence of two sub-regions in the seed region of single guide RNA which extends the established biological hypothesis of Off-Target effects. To the best of our knowledge, our model can be considered as the first model combining high efficacy, interpretability and a desirable balance between precision and recall.

Control of bacterial quorum threshold for metabolic homeostasis and cooperativity.

The mechanisms used by various bacteria to determine whether their density is sufficient to meet the QS threshold, how stringently bacterial cells block QS initiation until the QS threshold is reached, and the impacts of low-density bacterial cells encountering conditions that exceed the QS threshold are longstanding gaps in QS research. We demonstrated that translational control of the QS signaling biosynthetic gene creates a stringent QS threshold to maintain metabolic balance at low cell densities. The emergence of non-cooperative cells underlines the critical role of stringent QS modulation in maintaining the integrity of the bacterial QS system, demonstrating that a lack of such control can serve as a selection pressure. The fate of quorum-calling cells exposed to exceeding the QS threshold clarifies QS bacteria evolution in complex ecosystems.

Synthesis, Bioactivities, and Antibacterial Mechanism of 5-(Thioether)-N-phenyl/benzyl-1,3,4-oxadiazole-2-carboxamide/amine Derivatives.

1,3,4-Oxadiazole thioethers have shown exciting antibacterial activities; however, the current mechanism of action involving such substances against bacteria is limited to proteomics-mediated protein pathways and differentially expressed gene analysis. Herein, we report a series of novel 1,3,4-oxadiazole thioethers containing a carboxamide/amine moiety, most of which show good in vitro and in vivo bacteriostatic activities. Compounds A10 and A18 were screened through CoMFA models as optimums against Xanthomonas oryzae pv. oryzae (Xoo, EC50 values of 5.32 and 4.63 mg/L, respectively) and Xanthomonas oryzae pv. oryzicola (Xoc, EC50 values of 7.58 and 7.65 mg/L, respectively). Compound A10 was implemented in proteomic techniques and activity-based protein profiling (ABPP) analysis to elucidate the antibacterial mechanism and biochemical targets. The results indicate that A10 disrupts the growth and pathogenicity of Xoc by interfering with pathways associated with bacterial virulence, including the two-component regulation system, flagellar assembly, bacterial secretion system, quorum sensing, ABC transporters, and bacterial chemotaxis. Specifically, the translational regulator (CsrA) and the virulence regulator (Xoc3530) are two effective target proteins of A10. Knocking out the CsrA or Xoc3530 gene in Xoc results in a significant reduction in the motility and pathogenicity of the mutant strains. This study contributes available molecular entities, effective targets, and mechanism basis for the management of rice bacterial diseases.

Jatrophihabitans cynanchi sp. nov., isolated from rhizosphere soil of Cynanchum wilfordii.

A novel actinobacterial strain, SB3-54T was isolated from rhizosphere soil of Cynanchum wilfodill, Jaecheon, Chungcheongbuk-do, Republic of Korea. Cells of strain SB3-54T were Gram-stain-positive, aerobic, rod-shaped, and flagellated which formed pale yellow colonies on Reasoner's 2A (R2A) agar. Growth occurred at 15-30°C (optimum 25°C), pH 5-8 (optimum pH 7), and 0-2.5% NaCl (optimum 0%). Phylogenetic and phylogenomic analyses showed that strain SB3-54T formed a separate lineage in the genus Jatrophihabitans with Jatrophihabitans telluris N237T. Strain SB3-54T was positive for catalase activity. Genomic analysis showed that SB3-54T has plant-beneficial function contributing (referred to as PBFC) genes such as root colonization and plant protection from oxidative stress. Furthermore, genome of SB3-54T contained gene clusters related to cytokinin biosynthesis, auxin response, tryptophan biosynthesis, siderophore biosynthesis and bacterial toxin-antitoxin systems. Strain SB3-54T contained iso-C16:0 as the major fatty acid and MK-9(H4) and MK-9(H6) as the predominant quinones. The organism had meso-diaminopimelic acid as the diagnostic diamino acid in the peptidoglycan. The major polar lipids were diphosphatidylglycerol, phosphatidylinositol polymannosides, two unidentified aminoglycophospholipids and three unidentified phospholipids. Based on phylogenetic, physiological and chemotaxonomic characteristics, strain SB3-54T represents a novel species of the genus Jatrophihabitans. The type strain is SB3-54T (= KCTC 49134T = NBRC 114108T).

Role of Type VI secretion system in pathogenic remodeling of host gut microbiota during Aeromonas veronii infection.

Intestinal microbial disturbance is a direct cause of host disease. The bacterial Type VI secretion system (T6SS) often plays a crucial role in the fitness of pathogenic bacteria by delivering toxic effectors into target cells. However, its impact on the gut microbiota and host pathogenesis is poorly understood. To address this question, we characterized a new T6SS in the pathogenic Aeromonas veronii C4. First, we validated the secretion function of the core machinery of A. veronii C4 T6SS. Second, we found that the pathogenesis and colonization of A. veronii C4 is largely dependent on its T6SS. The effector secretion activity of A. veronii C4 T6SS not only provides an advantage in competition among bacteria in vitro, but also contributes to occupation of an ecological niche in the nutritionally deficient and anaerobic environment of the host intestine. Metagenomic analysis showed that the T6SS directly inhibits or eliminates symbiotic strains from the intestine, resulting in dysregulated gut microbiome homeostasis. In addition, we identified three unknown effectors, Tse1, Tse2, and Tse3, in the T6SS, which contribute to T6SS-mediated bacterial competition and pathogenesis by impairing targeted cell integrity. Our findings highlight that T6SS can remodel the host gut microbiota by intricate interplay between T6SS-mediated bacterial competition and altered host immune responses, which synergistically promote pathogenesis of A. veronii C4. Therefore, this newly characterized T6SS could represent a general interaction mechanism between the host and pathogen, and may offer a potential therapeutic target for controlling bacterial pathogens.

Metagenomic insights into jellyfish-associated microbiome dynamics during strobilation.

Host-associated microbiomes can play key roles in the metamorphosis of animals. Most scyphozoan jellyfish undergo strobilation in their life cycles, similar to metamorphosis in classic bilaterians. The exploration of jellyfish microbiomes may elucidate the ancestral mechanisms and evolutionary trajectories of metazoan-microbe associations and interactions during metamorphosis. However, current knowledge of the functional features of jellyfish microbiomes remains limited. Here, we performed a genome-centric analysis of associated microbiota across four successive life stages (polyp, early strobila, advanced strobila, and ephyra) during strobilation in the common jellyfish Aurelia coerulea. We observed shifts in taxonomic and functional diversity of microbiomes across distinct stages and proposed that the low microbial diversity in ephyra stage may be correlated with the high expression of the host-derived antimicrobial peptide aurelin. Furthermore, we recovered 43 high-quality metagenome-assembled genomes and determined the nutritional potential of the dominant Vibrio members. Interestingly, we observed increased abundances of genes related to the biosynthesis of amino acids, vitamins, and cofactors, as well as carbon fixation during the loss of host feeding ability, indicating the functional potential of Aurelia-associated microbiota to support the synthesis of essential nutrients. We also identified several potential mechanisms by which jellyfish-associated microbes establish stage-specific community structures and maintain stable colonization in dynamic host environments, including eukaryotic-like protein production, bacterial secretion systems, restriction-modification systems, and clustered regularly interspaced short palindromic repeats-Cas systems. Our study characterizes unique taxonomic and functional changes in jellyfish microbiomes during strobilation and provides foundations for uncovering the ancestral mechanism of host-microbe interactions during metamorphosis.

Conditions for the spread of CRISPR-Cas immune systems into bacterial populations.

Bacteria contain a wide variety of innate and adaptive immune systems which provide protection to the host against invading genetic material, including bacteriophages (phages). It is becoming increasingly clear that bacterial immune systems are frequently lost and gained through horizontal gene transfer (HGT). However, how and when new immune systems can become established in a bacterial population has remained largely unstudied. We developed a joint epidemiological and evolutionary model that predicts the conditions necessary for the spread of a CRISPR-Cas immune system into a bacterial population lacking this system. We find that whether bacteria carrying CRISPR-Cas will spread (increase in frequency) into a bacterial population depends on the abundance of phages and the difference in the frequency of phage resistance mechanisms between bacteria carrying a CRISPR-Cas immune system, and those not (denoted as ${f}_{\Delta }$). Specifically, the abundance of cells carrying CRISPR-Cas will increase if there is a higher proportion of phage resistance (either via CRISPR-Cas immunity or surface modification) in the CRISPR-Cas possessing population than in the cells lacking CRISPR-Cas. We experimentally validated these predictions using Pseudomonas aeruginosa PA14 and phage DMS3vir as a model. Specifically, by varying the initial ratios of different strains of bacteria that carry alternative forms of phage resistance we confirmed that the spread of cells carrying CRISPR-Cas through a population can be predicted based on phage density and the relative frequency of resistance phenotypes. Understanding which conditions promote the spread of CRISPR-Cas systems helps to predict when and where these defences can establish in bacterial populations after a horizontal gene transfer event, both in ecological and clinical contexts.

Methods optimization for the expression and purification of human calcium calmodulin-dependent protein kinase II alpha.

Calcium/calmodulin-dependent protein kinase II (CaMKII) is a complex multifunctional kinase that is highly expressed in central nervous tissues and plays a key regulatory role in the calcium signaling pathway. Despite over 30 years of recombinant expression and characterization studies, CaMKII continues to be investigated for its impact on signaling cooperativity and its ability to bind multiple substrates through its multimeric hub domain. Here we compare and optimize protocols for the generation of full-length wild-type human calcium/calmodulin-dependent protein kinase II alpha (CaMKIIα). Side-by-side comparison of expression and purification in both insect and bacterial systems shows that the insect expression method provides superior yields of the desired autoinhibited CaMKIIα holoenzymes. Utilizing baculovirus insect expression system tools, our results demonstrate a high yield method to produce homogenous, monodisperse CaMKII in its autoinhibited state suitable for biophysical analysis. Advantages and disadvantages of these two expression systems (baculovirus insect cell versus Escherichia coli expression) are discussed, as well as purification optimizations to maximize the enrichment of full-length CaMKII.

Insights into the inhibition mechanisms of MERS-CoV and SARS-CoV2 papain-like proteases by inhibitors from Crinum distichum: In vitro and in silico analysis

Highly pathogenic coronaviruses, including the Middle East Respiratory Syndrome Coronavirus (Mers-CoV) and Severe Acute Respiratory Syndrome Coronavirus-2 (Sars-CoV2), have recently emerged and represent a serious threat to global health. Accordingly, a search for potent inhibitors against coronavirus diseases is warranted. Therefore, this study aimed at identifying potential inhibitors from Crinum distichum against the papain-like protease (PLpro), an important antiviral target due to its essential role in viral replication. A bacterial expression system was used for the recombinant expression of Mers-CoV-PLpro and Sars-CoV2-PLpro which was used to assess the inhibitory effect of compounds isolated from C. distichum against their proteolytic and deubiquitinating (DUB) activities. Then, the inhibition mechanism of hit compounds was determined through enzyme kinetic studies. Protein-ligand stability was assessed by Differential Scanning Fluorometry and Molecular docking was used to predict the possible molecular interactions. Five compounds, namely (2S)-4´,7-dimethoxyflavan (CDS1), (2S)-4´‑hydroxy-7-methoxyflavan (CDS2), Hippadine (CDS3), (2R)-4´‑hydroxy-5,7-dimethoxyflavan (CDS4) and Hippacine (CDS5) were tested in this study. They markedly inhibited both the proteolytic and deubiquitinating activities of CoV-PLpro. Their IC50 values were in the low micromolar range. The most potent inhibitors were CDS3 and CDS4, with IC50 values of 9.28 and 6.45 µM, and 11.63 and 6.10 µM against the proteolytic and DUB activities of Mers-CoV-PLpro and Sars-CoV2-PLpro, respectively. They exert their inhibitory effect through competitive and non-competitive inhibition mechanisms. These compounds induced the instability of the enzymes by reducing their melting temperature and molecular docking analysis revealed several molecular interactions between the compounds and various amino acids of the enzymes. Overall, our findings suggest C. distichum active constituents to be promising candidates for development into potential inhibitors against coronavirus.

Arbitrium communication controls phage lysogeny through non-lethal modulation of a host toxin-antitoxin defence system.

Temperate Bacillus phages often utilize arbitrium communication to control lysis/lysogeny decisions, but the mechanisms by which this control is exerted remains largely unknown. Here we find that the arbitrium system of Bacillus subtilis phage ϕ3T modulates the host-encoded MazEF toxin-antitoxin system to this aim. Upon infection, the MazF ribonuclease is activated by three phage genes. At low arbitrium signal concentrations, MazF is inactivated by two phage-encoded MazE homologues: the arbitrium-controlled AimX and the later-expressed YosL proteins. At high signal, MazF remains active, promoting lysogeny without harming the bacterial host. MazF cleavage sites are enriched on transcripts of phage lytic genes but absent from the phage repressor in ϕ3T and other Spβ-like phages. Combined with low activation levels of MazF during infections, this pattern explains the phage-specific effect. Our results show how a bacterial toxin-antitoxin system has been co-opted by a phage to control lysis/lysogeny decisions without compromising host viability.

Generation of DNAzyme in Bacterial Cells by a Bacterial Retron System.

DNAzymes are catalytically active single-stranded DNAs in which DNAzyme 10-23 (Dz 10-23) consists of a catalytic core and a substrate-binding arm that reduces gene expression through sequence-specific mRNA cleavage. However, the in vivo application of Dz 10-23 depends on exogenous delivery, which leads to its inability to be synthesized and stabilized in vivo, thus limiting its application. As a unique reverse transcription system, the bacterial retron system can synthesize single-stranded DNA in vivo using ncRNA msr/msd as a template. The objective of this work is to reduce target gene expression using Dz 10-23 generated in vivo by the retron system. In this regard, we successfully generated Dz 10-23 by cloning the Dz 10-23 coding sequence into the retron msd gene and tested its ability to reduce specific gene expression by examining the mRNA levels of cfp encoding cyan fluorescence protein and other functional genes such as mreB and ftsZ. We found that Dz had different repressive effects when targeting different mRNA regions, and in general, the repressive effect was stronger when targeting downstream of mRNAs. Our results also suggested that the reduction effect was due to cleavage of the substrate mRNA by Dz 10-23 rather than the antisense effect of the substrate-binding arm. Therefore, this study not only provided a retron-based method for the intracellular generation of Dz 10-23 but also demonstrated that Dz 10-23 could reduce gene expression by cleaving target mRNAs in cells. We believe that this new strategy would have great potential in the regulation of gene expression.

Aminoacylase efficiently hydrolyses fatty acid amino acid conjugates of Helicoverpa armigera potentially to increase the pool of glutamine

One of the most prevalent bioactive molecules present in the oral secretion (OS) of lepidopteran insects is fatty acid amino acid conjugates (FACs). Insect dietary components have influence on the synthesis and retaining the pool of FACs in the OS. We noted differential and diet-specific accumulation of FACs in the OS of Helicoverpa armigera by using Liquid Chromatography-Quadrupole Time of Flight Mass Spectrometry. Interestingly, we identified FACs hydrolyzing enzyme aminoacylase (HaACY) in the OS of H. armigera through proteomic analysis. Next, we have cloned, expressed, and purified active recombinant HaACY in the bacterial system. Recombinant HaACY hydrolyzes all the six identified FACs in the OS of H. armigera larvae fed on host and non-host plants and releases respective fatty acid and glutamine. In these six FACs, fatty acid moieties vary while amino acid glutamine was common. Glutamine obtained upon hydrolysis of FACs by HaACY might serve as an amino acid pool for insect growth and development. To understand the substrate choices of HaACY, we chemically synthesized, purified, and characterized all the six FACs. Interestingly, rHaACY also shows hydrolysis of synthetic FACs into respective fatty acid and glutamine. Our results underline the importance of diet on accumulation of FACs and role of aminoacylase(s) in regulating the level of FACs and glutamine.

Production of Recombinant Viral Antigens Using the Baculovirus-Insect Cell Expression System.

Insect cell expression has been successfully used for the production of viral antigens as part of commercial vaccine development. As expression host, insect cells offer advantage over bacterial system by presenting the ability of performing post-translational modifications (PTMs) such as glycosylation and phosphorylation thus preserving the native functionality of the proteins especially for viral antigens. Insect cells have limitation in exactly mimicking some proteins which require complex glycosylation pattern. The recent advancement in insect cell engineering strategies could overcome this limitation to some extent. Moreover, cost efficiency, timelines, safety, and process adoptability make insect cells a preferred platform for production of subunit antigens for human and animal vaccines. In this chapter, we describe the method for producing the SARS-CoV2 spike ectodomain subunit antigen for human vaccine development and the virus like particle (VLP), based on capsid protein of porcine circovirus virus 2 (PCV2d) antigen for animal vaccine development using two different insect cell lines, SF9 & Hi5, respectively. This methodology demonstrates the flexibility and broad applicability of insect cell as expression host.

Regulation of Aerobic Succinate Transporter dctA of E. coli by cAMP-CRP, DcuS-DcuR, and EIIAGlc: Succinate as a Carbon Substrate and Signaling Molecule

Introduction: C4-dicarboxylates (C4-DC) have emerged as significant growth substrates and signaling molecules for various Enterobacteriaceae during their colonization of mammalian hosts. Particularly noteworthy is the essential role of fumarate respiration during colonization of pathogenic bacteria. To investigate the regulation of aerobic C4-DC metabolism, the study explored the transcriptional control of the main aerobic C4-DC transporter, dctA, under different carbohydrate conditions. In addition, mutants related to carbon catabolite repression (CCR) and C4-DC regulation (DcuS-DcuR) were examined to better understand the regulatory integration of aerobic C4-DC metabolism into CCR. For initial insight into posttranslational regulation, the interaction between the aerobic C4-DC transporter DctA and EIIAGlc from the glucose-specific phosphotransferase system was investigated. Methods: The expression of dctA was characterized in the presence of various carbohydrates and regulatory mutants affecting CCR. This was accomplished by fusing the dctA promoter (PdctA) to the lacZ reporter gene. Additionally, the interaction between DctA and EIIAGlc of the glucose-specific phosphotransferase system was examined in vivo using a bacterial two-hybrid system. Results: The dctA promoter region contains a class I cAMP-CRP-binding site at position −81.5 and a DcuR-binding site at position −105.5. DcuR, the response regulator of the C4-DC-activated DcuS-DcuR two-component system, and cAMP-CRP stimulate dctA expression. The expression of dctA is subject to the influence of various carbohydrates via cAMP-CRP, which differently modulate cAMP levels. Here we show that EIIAGlc of the glucose-specific phosphotransferase system strongly interacts with DctA, potentially resulting in the exclusion of C4-DCs when preferred carbon substrates, such as sugars, are present. In contrast to the classical inducer exclusion known for lactose permease LacY, inhibition of C4-DC uptake into the cytoplasm affects only its role as a substrate, but not as an inducer since DcuS detects C4-DCs in the periplasmic space (“substrate exclusion”). The work shows an interplay between cAMP-CRP and the DcuS-DcuR regulatory system for the regulation of dctA at both transcriptional and posttranslational levels. Conclusion: The study highlights a hierarchical interplay between global (cAMP-CRP) and specific (DcuS-DcuR) regulation of dctA at the transcriptional and posttranslational levels. The integration of global and specific transcriptional regulation of dctA, along with the influence of EIIAGlc on DctA, fine-tunes C4-DC catabolism in response to the availability of other preferred carbon sources. It attributes DctA a central role in the control of aerobic C4-DC catabolism and suggests a new role to EIIAGlc on transporters (control of substrate uptake by substrate exclusion).

Quantitative Aspects, Engineering and Optimization of Bacterial Sensor Systems

Quantitative Aspects, Engineering and Optimization of Bacterial Sensor Systems

Meningitis a Listeria monocytogenes in a young, immunocompetent male

Listeria Monocytogenes (LM) is a gram-positive intracellular pathogen usually transmitted to humans by ingesting contaminated food. It typically infects immunocompromised individuals, such as pregnant women, newborns, alcoholics, and the elderly. LM is a rare cause of meningitis in immunocompetent, healthy adults. However, its clinical presentation is similar to that of other viral or bacterial central nervous system infections and its course can be rapid and aggressive. Physicians should therefore always consider LM as a possible etiologic agent of meningitis, especially in cases that do not respond to first-line empiric antibiotic therapy. We report a case of Listeria Monocytogenes meningitis in a previously healthy 16-year-old.

Designing and synthesizing peptide-based quorum sensing modulators.

Quorum sensing (QS) is a density-dependent bacterial communication system that uses small molecules as regulatory modulators. Synthetic changes to these molecules can up-or-down-regulate this system, leading to control of phenotypes, like competence and virulence factor production, that have implications in human health. In this chapter, a methodology for library design and screening of synthetic autoinducing peptides (AIPs) to uncover QS SARs is delineated. Additionally, procedures for the synthesis, purification and analysis of linear and cyclic AIPs are detailed. This includes solutions for potential synthetic challenges including diketopiperazine formation when using N-methyl amino acids and cyclization of peptides containing N-terminal cysteine residues. These procedures have and are currently being applied to develop potent QS modulators in Streptococcus pneumoniae, Bacillus cereus, Streptococcus gordonii and Lactiplantibacillus plantarum.

A novel radiolabelled salmochelin derivative for bacteria-specific PET imaging: synthesis, radiolabelling and evaluation.

For specific imaging of bacterial infections we aimed at targeting the exclusive bacterial iron transport system via siderophore-based radiotracers. De novo synthesis and radiolabeling yielded the salmochelin-based PET radiotracer [68Ga]Ga-RMA693, which showed a favourable biodistribution and a bacteria-specific uptake in an animal model of Escherichia coli infection.

New Perspectives on Crosstalks Between Bacterial Regulatory RNAs from Outer Membrane Vesicles and Eukaryotic Cells.

Regulatory small RNAs (sRNAs) help the bacteria to survive harsh environmental conditions by posttranscriptional regulation of genes involved in various biological pathways including stress responses, homeostasis, and virulence. These sRNAs can be found carried by different membrane-bound vesicles like extracellular vesicles (EVs), membrane vesicles (MVs), or outer membrane vesicles (OMVs). OMVs provide myriad functions in bacterial cells including carrying a cargo of proteins, lipids, and nucleic acids including sRNAs. A few interesting studies have shown that these sRNAs can be transported to the host cell by membrane vesicles and can regulate the host immune system. Although there is evidence that sRNAs can be exported to host cells and sometimes can even cross the blood-brain barrier, the exact mechanism is still unknown. In this review, we investigated the new techniques implemented in various studies, to elucidate the crosstalks between bacterial cells and human immune systems by membrane vesicles carrying bacterial regulatory sRNAs.