Auswahl der wissenschaftlichen Literatur zum Thema „Bacterial mechanosensing“

Our understanding of the mechanisms of microgravity perception and response in prokaryotes (Bacteria and Archaea) lag behind those which have been elucidated in eukaryotic organisms. In this hypothesis paper, we: (i) review how eukaryotic cells sense and respond to microgravity using various pathways responsive to unloading of mechanical stress; (ii) we observe that prokaryotic cells possess many structures analogous to mechanosensitive structures in eukaryotes; (iii) we review current evidence indicating that prokaryotes also possess active mechanosensing and mechanotransduction mechanisms; and (iv) we propose a complete mechanotransduction model including mechanisms by which mechanical signals may be transduced to the gene expression apparatus through alterations in bacterial nucleoid architecture, DNA supercoiling, and epigenetic pathways.

After the discovery of Corynebacterium glutamicum from avian feces-contaminated soil, its enigmatic L-glutamate secretion by corynebacterial MscCG-type mechanosensitive channels has been utilized for industrial monosodium glutamate production. Bacterial mechanosensitive channels are activated directly by increased membrane tension upon hypoosmotic downshock; thus; the physiological significance of the corynebacterial L-glutamate secretion has been considered as adjusting turgor pressure by releasing cytoplasmic solutes. In this review, we present information that corynebacterial mechanosensitive channels have been evolutionally specialized as carriers to secrete L-glutamate into the surrounding environment in their habitats rather than osmotic safety valves. The lipid modulation activation of MscCG channels in L-glutamate production can be explained by the “Force-From-Lipids” and “Force-From-Tethers” mechanosensing paradigms and differs significantly from mechanical activation upon hypoosmotic shock. The review also provides information on the search for evidence that C. glutamicum was originally a gut bacterium in the avian host with the aim of understanding the physiological roles of corynebacterial mechanosensing. C. glutamicum is able to secrete L-glutamate by mechanosensitive channels in the gut microbiota and help the host brain function via the microbiota–gut–brain axis.

The bacterial flagellar motor is an ion-powered transmembrane protein complex which drives swimming in many bacterial species. The motor consists of a cytoplasmic ‘rotor’ ring and a number of ‘stator’ units, which are bound to the cell wall of the bacterium. Recently, it has been shown that the number of functional torque-generating stator units in the motor depends on the external load, and suggested that mechanosensing in the flagellar motor is driven via a ‘catch bond’ mechanism in the motor’s stator units. We present a method that allows us to measure—on a single motor—stator unit dynamics across a large range of external loads, including near the zero-torque limit. By attaching superparamagnetic beads to the flagellar hook, we can control the motor’s speed via a rotating magnetic field. We manipulate the motor to four different speed levels in two different ion-motive force (IMF) conditions. This framework allows for a deeper exploration into the mechanism behind load-dependent remodelling by separating out motor properties, such as rotation speed and energy availability in the form of IMF, that affect the motor torque.

Les flagelles ont été décrits comme un facteur de virulence important pour l'attachement initial à l'hôte ou à la surface des équipements hospitaliers. Cependant, la question de savoir comment les bactéries utilisent leurs flagelles pour passer d'un état flottant (planctonique) à un état d'attachement immédiat (sessile) reste ouverte. Cette transition implique la mécanodétection flagellaire. Il a été démontré que les méthylations des lysines dans les flagelles de S. Typhimurium facilitent l'adhésion de la bactérie à la surface ou au récepteur par le biais de l'hydrophobicité. Cependant, l'étude de l'ensemble du méthylome, et plus particulièrement la méthylation des protéines autres que des histones, reste un défi majeur en raison de l'absence de techniques efficaces d’enrichissement des protéines/peptides méthylés. Ici, pour étudier la méthylation des protéines et ses mécanismes chez S. Typhimurium et ses mutants, nous avons appliqué deux stratégies complémentaires pour l'enrichissement des protéines méthylées : l’enrichissement sélectif grâce à la sélection d’aptamères spécifiques, et la séparation de protéines/peptides méthylés par la technologie SCXtips à pH élevé. Des aptamères d'ADN spécifiques ont été obtenus après plusieurs cycles de sélection positive et un cycle de sélection négative à partir d'une bibliothèque d'oligonucléotides aléatoires par le biais de la procédure FluMag-SELEX. Plusieurs oligonucléotides issus du dernier tour de sélection ont été séquencés et des redondances de séquence de 10 %, 11 % et 33 % respectivement contre la MML, DML et TML, ont pu être observées. Une caractérisation plus fine de l’interaction entre l'aptamère sélectionné contre le TML et sa cible a été menée par différentes méthodes (ITC, PCR, liaison sur billes suivie d’une quantification par spectrométrie de masse). La spécificité a été confirmée tandis que l'affinité a été déterminée avec un KD=2,48±0,14 mM. Par la suite, nous avons exploité l’aptamère sélectionné en l’immobilisant à la surface de billes magnétiques afin de disposer d’un outil d’enrichissement des protéines/peptides méthylés sur différents modèles cellulaires. Après avoir validé la démarche sur un contrôle positif (cellules humaines HEK293), nous avons identifié 19 sites de méthylation de la lysine sur 5 protéines de Salmonella. Les résultats préliminaires de l'analyse protéomique ont confirmé que l'aptamère sélectionné était effectivement capable de distinguer et d'enrichir les protéines contenant de la méthyllysine provenant de souches de S. Typhimurium.Parallèlement, la technologie SCXtips à pH élevé a été utilisée comme stratégie de séparation à partir culture de souches bactériennes dans le milieu hM-SILAC. Cette méthode a permis d'identifier 23 sites de méthylation uniques chez la souche ΔmetE, 51 sites de méthylation uniques chez la souche ΔmetEΔmotAB, et enfin 18 sites uniques chez la souche ΔmetEΔfliB sur 19 protéines méthylées. En comparant les différences de méthylation des protéines, nos résultats suggèrent que lorsque les bactéries manquent de motilité, les méthylations de lysine dans les protéines bactériennes semblent être régulées à la hausse. Outre la méthylation par la méthylase FliB, un grand nombre d’événements de méthylation pourraient être régulés par d’autres méthylases
Flagella have been described as an important virulence factor for initial attachment to the host or to the surface of hospital equipment. However, how bacteria use their flagella to switch from a floating (planktonic) state to an immediately attached (sessile) state remains an open question. This transition involves flagellar mechanosensing or surface-sensing. It has been illustrated that lysine methylations in S. Typhimurium flagella facilitate bacterial adhesion to the surface or receptor via hydrophobicity. However, the study of the entire methylome, including non-histone methylation, remains a major challenge because of the lack of efficient methyl protein/peptide enrichment techniques. Here, to investigate protein methylation and its mechanisms in S. Typhimurium and its mutants, we applied two complementary strategies for methyl protein enrichment: aptamer-based enrichment technology and high pH SCXtips separation strategy. The specific DNA aptamers were obtained after performing several rounds of positive selection and one round of negative selection from a random oligonucleotide library through the FluMag-SELEX procedure. The selected ssDNA pools were sequenced and 10%, 11%, and 33% of redundant sequences for MML, DML, and TML, respectively have been observed. The highest redundancy was observed with oligonucleotides directed against TML. The interaction study of this aptamer with its target was then performed by the methods like ITC, PCR, and bead-based binding assays followed by mass spectrometry titration. The specificity was confirmed while the affinity was determined to be KD=2.48±0.14 mM. Then, the selected aptamer has been used as an enrichment tool based on the aptamer-bound beads method, in order to isolate methylated proteins or peptides on various cell lines. After the validation of our approach using a positive control (HEK293 cells), we identified 19 lysine methylation sites on 5 proteins from Salmonella Preliminary results from proteomic analysis confirmed that the selected aptamer was indeed able to distinguish and enrich methyllysine-containing proteins from S. Typhimurium strains and human cell lines. In parallel with this, the high pH SCXtips technology was performed as a complementary separation strategy when cultivating the strains in the hM-SILAC medium. Using this method, 23 unique methylation sites in ΔmetE, 51 unique methylation sites in ΔmetEΔmotAB, and 18 unique sites in ΔmetEΔfliB on 19 methylated proteins were identified. By comparing the differences in protein methylation, our results suggest that when bacteria lack motility, lysine methylations in bacterial proteins seem to be upregulated. Besides being methylated by the methylase FliB, a large number of methylation events could be regulated by other methylases