Disruption of Yarrowia lipolytica biofilms by rhamnolipid biosurfactant

BACKGROUND: Yarrowia lipolytica is an ascomycetous dimorphic fungus that exhibits biofilm mode of growth. Earlier work has shown that biosurfactants such as rhamnolipids are efficient dispersants of bacterial biofilms. However, their effectiveness against fungal biofilms (particularly Y. lipolytica) has not been investigated. The aim of this study was to determine the effect of rhamnolipid on a biofilm forming strain of Y. lipolytica. Two chemical surfactants, cetyl-trimethyl ammonium bromide (CTAB) and sodium dodecyl sulphate (SDS) were used as controls for comparison. RESULTS: The methylene blue dye exclusion assay indicated an increase in fungal cell permeability after rhamnolipid treatment. Microtiter plate assay showed that the surfactant coating decreased Y. lipolytica biofilm formation by 50%. Rhamnolipid treatment disrupted pre-formed biofilms in a more effective manner than the other two surfactants. Confocal laser scanning microscopic studies showed that biofilm formation onto glass surfaces was decreased by 67% after sub-minimum inhibitory concentration (sub-MIC) treatment with rhamnolipids. The disruption of biofilms after rhamnolipid treatment was significant (P<0.05) when compared to SDS and CTAB. CONCLUSION: The results indicate a potential application of the biological surfactant to disrupt Y. lipolytica biofilms

Régulation post-transcriptionnelle de l'expression des porines chez Escherichia coli et impact sur la résistance aux antibiotiques

Antimicrobial resistance (AMR) is a serious and growing health threat as it has been estimated that 700,000 people die every year from drug resistant bacteria. A major factor contributing to AMR is the inability of antibiotics to penetrate the bacterial outer membrane (OM) to reach their requisite target for being effective. In Gram-negative bacteria, such as Escherichia coli, the two classical porins OmpF and OmpC are among the most abundant outer membrane proteins and form water filled channels for the diffusion of small hydrophilic molecules including antibiotics. Porin modifications (in the form of decreased expression or structural modifications) are found in several resistant clinical isolates, limit antibiotic uptake and decrease their intracellular concentration and activity. Given the importance of the OM, it is not surprising that the expression of porins is under complex regulation at multiple levels. Regulation of ompF and ompC at the transcriptional level is well studied, and involves the EnvZ-OmpR two component system in response to external osmolarity. Research has also shown that enterobacteria uses small regulatory RNAs (sRNAs) to fine tune porin expression level at the post-transcriptional level. Among these, MicF and MicC are the two major sRNAs that modulate the expression of OmpF and OmpC, respectively. They suppress porin expression by base pairing with the ribosome binding site of targeted porin mRNA, thereby blocking translation. Interestingly, these two sRNAs are encoded next to porin gene, i.e. micF-ompC and micC-ompN, suggesting a dual regulation. In this work, our goals were: (1) to characterize the regulation of the sRNA MicC and the putative co-regulation of the quiescent porin OmpN in E. coli; (2) to examine the global effect of MicC on the E. coli transcriptome; (3) to analyze the impact of MicC expression on antibiotic susceptibility. First, investigated the factors like external growth conditions and regulatory pathways that lead to increased production of MicC by measuring the β-galactosidase activity of a micC-lacZ transcriptional fusion. For this search, we optimized the reporter gene assay in a 96-wells format and screened collections of compounds provided by the Biolog phenotype MicroarrayTM. Our work shows that the expression of micC was increased in the presence of β-lactam antibiotics (specifically carbapenems and cephalosporins) and in an rpoE depleted mutant. Interestingly, the same conditions enhanced the activity of an ompN–lacZ fusion, suggesting a dual transcriptional regulation of micC and ompN. Increased levels of OmpN in the presence of sub-inhibitory concentrations of chemicals could not be confirmed by Western blot analysis, excepting when the sigma factor σE was depleted. We suggest that the MicC sRNA acts together with the σE envelope stress response pathway to control the OmpC/N levels in response to β-lactam antibiotics. We also performed RNA sequencing to determine the impact of MicC overexpression on E. coli transcriptome. This identified 60 mRNA targets negatively regulated by MicC apart from its original target ompC. Identification of the global target spectra of MicC is of importance to understand its importance on the overall bacterial physiology, and more specifically on AMR. Preliminary results showed that E. coli ΔompF overexpressing MicC exhibit reduced susceptibility to β-lactams, probably due OmpC shutdown. Future studies will aim to investigate to putative connection between β-lactam resistance, loss of OmpC and overexpression of MicC in clinical isolates of Enterobacteriaceae.La résistance aux antibiotiques est une menace sérieuse et grandissante pour la santé publique, causant approximativement 700 000 décès annuels. Chez les bactéries à Gram-négatif, l’imperméabilité de la membrane externe et ainsi l'incapacité des antibiotiques à pénétrer l’enveloppe bactérienne pour atteindre leur cible est un facteur majeur contribuant au développement de la résistance. Chez Escherichia coli, les porines OmpF et OmpC sont des protéines de la membrane externe qui forment des canaux pour la diffusion de petites molécules hydrophiles tels que les antibiotiques. La modification des porines (sous la forme d'une diminution de leur expression ou des modifications structurales) se retrouvent dans de nombreux isolats cliniques résistants, limitent la translocation des antibiotiques, diminuent leur concentration intracellulaire et leur activité. Compte tenu de l'importance de la membrane externe, il n'est pas surprenant que l'expression des porines soit soumise à une régulation complexe à plusieurs niveaux. La régulation de ompF et ompC au niveau transcriptionnel est bien connue et fait intervenir le système à deux composants EnvZ-OmpR en réponse à l'osmolarité du milieu. Plusieurs études ont également montré le rôle des petits ARN non-codants (sRNAs, small RNAs) dans le contrôle de l'expression des porines au niveau post-transcriptionnel. Parmi ceux-ci, MicF et MicC modulent l'expression respective de OmpF et OmpC. Ils fonctionnent par appariement de bases avec le site de liaison du ribosome du messager cible, bloquant ainsi l’initiation de la traduction. De manière intéressante, les gènes codant ces deux sRNAs sont adjacents à deux gènes codant des porines — micF-ompC et micC-ompN — suggérant une co-régulation.Dans ce cadre, et en utilisant E. coli comme bactérie modèle, les objectifs de mon travail de thèse étaient : (1) de caractériser la régulation du sRNA MicC et la co-régulation putative de la porine quiescente OmpN; (2) d’examiner l'effet global de MicC sur le transcriptome; (3) d’analyser l'impact de l'expression de MicC sur la sensibilité aux antibiotiques. Tout d'abord, nous avons étudié le rôle de plusieurs facteurs tels que les conditions de croissance et des voies de régulation connues pouvant conduire à une augmentation de l’expression de MicC. Pour cela, nous avons mesuré l'activité β-galactosidase d'une fusion transcriptionnelle micC-lacZ. Nous avons également optimisé le test du gène rapporteur à un format microplaque afin de cribler plusieurs collections de molécules fournis par la compagnie Biolog. Les résultats obtenus montrent l’induction de MicC en présence d'antibiotiques de la famille des β-lactamines (spécifiquement les carbapénèmes et les céphalosporines) ainsi qu’en déplétant le facteur de transcription sigma spécifique au stress de l’enveloppe, σE. Ces mêmes conditions activent aussi l'activité d'une fusion ompN-lacZ, indiquant une régulation transcriptionnelle commune de micC et ompN. De plus, la production de OmpN a été confirmée par une analyse en immunoblot avec des anticorps spécifiques. Ainsi, MicC pourrait agir conjointement avec σE pour contrôler l’expression de OmpC et OmpN en réponse à la présence de β-lactamines, une famille d’antibiotiques qui cible justement la synthèse du peptidoglycane et l’intégrité de l’enveloppe. Etant donnée la conservation de MicC chez les entérobactéries, nous avons effectué une étude par RNASeq pour déterminer l'impact de la surexpression de MicC sur le transcriptome d’E. coli et identifié 60 ARNm régulés par MicC en plus de sa cible initiale ompC. L'identification des spectres cibles globaux des sRNAs est importante pour comprendre leur importance dans la physiologie bactérienne, ici celui de MicC dans la résistance aux antibiotiques. Les travaux à venir viseront à étudier cet aspect en détail ainsi que le lien putatif entre la résistance aux β-lactamines, la perte d'OmpC et la surexpression de MicC dans des isolats cliniques d’entérobactéries

Dual Regulation of the Small RNA MicC and the Quiescent Porin OmpN in Response to Antibiotic Stress in Escherichia coli

Antibiotic resistant Gram-negative bacteria are a serious threat for public health. The permeation of antibiotics through their outer membrane is largely dependent on porin, changes in which cause reduced drug uptake and efficacy. Escherichia coli produces two major porins, OmpF and OmpC. MicF and MicC are small non-coding RNAs (sRNAs) that modulate the expression of OmpF and OmpC, respectively. In this work, we investigated factors that lead to increased production of MicC. micC promoter region was fused to lacZ, and the reporter plasmid was transformed into E. coli MC4100 and derivative mutants. The response of micC–lacZ to antimicrobials was measured during growth over a 6 h time period. The data showed that the expression of micC was increased in the presence of β-lactam antibiotics and in an rpoE depleted mutant. Interestingly, the same conditions enhanced the activity of an ompN–lacZ fusion, suggesting a dual transcriptional regulation of micC and the quiescent adjacent ompN. Increased levels of OmpN in the presence of sub-inhibitory concentrations of chemicals could not be confirmed by Western blot analysis, except when analyzed in the absence of the sigma factor σE. We suggest that the MicC sRNA acts together with the σE envelope stress response pathway to control the OmpC/N levels in response to β-lactam antibiotics

Stress responses, outer membrane permeability control and antimicrobial resistance in Enterobacteriaceae 2 3

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Tricyclic SpiroLactams Kill Mycobacteria In Vitro and In Vivo by Inhibiting Type II NADH Dehydrogenases

International audienceIt is critical that novel classes of antituberculosis drugs are developed to combat the increasing burden of infections by multidrug-resistant strains. To identify such a novel class of antibiotics, a chemical library of unique 3-D bioinspired molecules was explored revealing a promising, mycobacterium specific Tricyclic SpiroLactam (TriSLa) hit. Chemical optimization of the TriSLa scaffold delivered potent analogues with nanomolar activity against replicating and nonreplicating Mycobacterium tuberculosis. Characterization of isolated TriSLa-resistant mutants, and biochemical studies, found TriSLas to act as allosteric inhibitors of type II NADH dehydrogenases (Ndh-2 of the electron transport chain), resulting in an increase in bacterial NADH/NAD+ ratios and decreased ATP levels. TriSLas are chemically distinct from other inhibitors of Ndh-2 but share a dependence for fatty acids for activity. Finally, in vivo proof-of-concept studies showed TriSLas to protect zebrafish larvae from Mycobacterium marinum infection, suggesting a vulnerability of Ndh-2 inhibition in mycobacterial infections