Abstract:Biofilms are complex communities of microbes that attach to biotic or abiotic surfaces causing chronic infectious diseases. Within a biofilm, microbes are embedded in a self-produced soft extracellular matrix (ECM), which protects them from the host immune system and antibiotics. The nanoscale visualisation of delicate biofilms in liquid is challenging. Here, we develop atmospheric scanning electron microscopy (ASEM) to visualise Gram-positive and -negative bacterial biofilms immersed in aqueous solution. Biof… Show more
“…1). Under the conditions used in this study, curli—but neither type I pili nor flagella—was required for biofilm formation as previously reported 24,26 . In this study, we focused on the conditions that promote curli-dependent biofilm formation.Fig.…”
Biofilms are intricate communities of microorganisms embedded in a self-produced matrix of extracellular polymer, which provides microbes survival advantages in stressful environments and can cause chronic infections in humans. Curli are functional amyloids that assemble on the extracellular surface of enteric bacteria such as Escherichia coli during biofilm development and colonization. The molecular chaperone DnaK, a bacterial Hsp70 homologue, promotes curli biogenesis via unknown mechanism(s). Here we show that DnaK increases the expression of CsgA and CsgB—the major and minor structural components of curli, respectively—via a quantity and quality control of RpoS, a stationary phase-specific alternative sigma factor regulating bacterial transcription, and CsgD, the master transcriptional regulator of curli formation. DnaK also keeps CsgA and CsgB in a translocation-competent state by binding to their signal peptides prone to aggregation. Our findings suggest that DnaK controls the homoeostasis of curli biogenesis at multiple stages to organize the biofilm matrix.
“…1). Under the conditions used in this study, curli—but neither type I pili nor flagella—was required for biofilm formation as previously reported 24,26 . In this study, we focused on the conditions that promote curli-dependent biofilm formation.Fig.…”
Biofilms are intricate communities of microorganisms embedded in a self-produced matrix of extracellular polymer, which provides microbes survival advantages in stressful environments and can cause chronic infections in humans. Curli are functional amyloids that assemble on the extracellular surface of enteric bacteria such as Escherichia coli during biofilm development and colonization. The molecular chaperone DnaK, a bacterial Hsp70 homologue, promotes curli biogenesis via unknown mechanism(s). Here we show that DnaK increases the expression of CsgA and CsgB—the major and minor structural components of curli, respectively—via a quantity and quality control of RpoS, a stationary phase-specific alternative sigma factor regulating bacterial transcription, and CsgD, the master transcriptional regulator of curli formation. DnaK also keeps CsgA and CsgB in a translocation-competent state by binding to their signal peptides prone to aggregation. Our findings suggest that DnaK controls the homoeostasis of curli biogenesis at multiple stages to organize the biofilm matrix.
“…Immunostaining with α-dsDNA combined with CLSM also revealed the existence of long filaments of eDNA in biofilms formed either by Enterococcus faecalis (45) or Haemophilus influenzae ECM (46). Moreover, the presence of eDNA-containing fibrous structures in the ECM of Staphylococcus aureus and Propionibacterium acnes biofilms has also been reported using α-dsDNA and atmospheric scanning electron microscopy (47, 48). The use of different technical approaches reinforces the idea that the fibrous assemblies of eDNA observed in some bacterial biofilms actually exist and also offers a novel perspective of the ECM structure of pneumococcal biofilms.…”
Biofilms are defined as layers of cells of microorganisms 17 adhered to the surface of a substrate and embedded in an extracellular matrix 18 and provide an appropriate environment for increased genetic exchange. 19 Extracellular DNA (eDNA) is an essential component of the extracellular matrix 20 of microbial biofilms, but the pathway(s) responsible for DNA release are largely 21 unknown. Autolysis (either spontaneous or phage-induced) has been proposed 22 the major event leading to the appearance of eDNA. The 'suicidal tendency' of 23 Streptococcus pneumoniae is well-known, with lysis mainly caused by the 24 triggering of LytA, the major autolytic amidase. However, the LytC lysozyme 25 and CbpD (a possible murein hydrolase) have also been shown involved. The 26 present work examines the relationship between eDNA, autolysins, and the 27 formation and maintenance of in vitro pneumococcal biofilms, via fluorescent 28 labelling combined with confocal laser scanning microscopy, plus genetic 29 transformation experiments. Bacterial DNA release mechanisms other than 30 those entailing lytic enzymes were shown to be involved by demonstrating that 31 horizontal gene transfer in biofilms takes place even in the absence of 32 detectable autolytic activity. It had been previously suggested that the quorum 33 sensing systems ComABCDE and LuxS/AI-2 are involved in the production of 34 eDNA as a response to the accumulation of quorum sensing signals, although 35 our immunofluorescence results do not support this hypothesis. Evidence that 36 the release of DNA is somehow linked to the production of extracellular vesicles 37 by S. pneumoniae is provided. 38 IMPORTANCE Most human bacterial infections are caused by 39 microorganisms growing as biofilms. Bacteria in biofilms are less susceptible to 40 antimicrobials and to killing by the host immune system, are very difficult to 41 eliminate and cause recalcitrant and persistent diseases. Extracellular DNA is 42 one of the major components of the bacterial biofilm matrix. In the present 43 study, we provide direct evidence of the existence of biologically active 44 (transforming), extracellular DNA in Streptococcus pneumoniae biofilms. In 45 3previous studies, the involvement of three pneumococcal choline-binding 46 proteins with autolytic activity (LytA, LytC and CbpD) in DNA release had been 47 reported. In contrast, we demonstrate here that pneumococcal in vitro biofilms 48 do contain eDNA, even in the absence of these enzymes. Moreover, our results 49 suggest that DNA release in S. pneumoniae biofilms is connected with the 50 production of extracellular vesicles and that this DNA is associated to the outer 51 part of the vesicles. 52 53 4The human pathogen Streptococcus pneumoniae is a leading cause of 54 pneumonia, meningitis and bloodstream infections in the elderly, and one of the 55 main pathogens responsible for middle ear infections in children. It is carried 56 asymptomatically in the nasopharynx of many healthy adults, and in as many as 57 20-40% of healthy children ...
“…Approaches based on electron microscopy Sugimoto et al . (), which are also often used for imaging biofilms, provide a far superior resolution but cannot be used in our case as they are limited in the volume of the sample.…”
Section: Methodsmentioning
confidence: 99%
“…This imaging modality was chosen for comparison as it is a widespread technique for biofilm observation (Vroom et al, 1999;Choi et al, 2010;Neu & Lawrence, 2015); it also enables imaging large volumes of hundreds of micrometers to a few millimeters. Approaches based on electron microscopy Sugimoto et al (2016), which are also often used for imaging biofilms, provide a far superior resolution but cannot be used in our case as they are limited in the volume of the sample.…”
Optical imaging techniques for biofilm observation, like laser scanning microscopy, are not applicable when investigating biofilm formation in opaque porous media. X-ray micro-tomography (X-ray CMT) might be an alternative but it finds limitations in similarity of X-ray absorption coefficients for the biofilm and aqueous phases. To overcome this difficulty, barium sulphate was used in Davit et al. (2011) to enable high-resolution 3D imaging of biofilm via X-ray CMT. However, this approach lacks comparison with well-established imaging methods, which are known to capture the fine structures of biofilms, as well as uncertainty quantification. Here, we compare two-photon laser scanning microscopy (TPLSM) images of Pseudomonas Aeruginosa biofilm grown in glass capillaries against X-ray CMT using an improved protocol where barium sulphate is combined with low-gelling temperature agarose to avoid sedimentation. Calibrated phantoms consisting of mono-dispersed fluorescent and X-ray absorbent beads were used to evaluate the uncertainty associated with our protocol along with three different segmentation techniques, namely hysteresis, watershed and region growing, to determine the bias relative to image binarization. Metrics such as volume, 3D surface area and thickness were measured and comparison of both imaging modalities shows that X-ray CMT of biofilm using our protocol yields an accuracy that is comparable and even better in certain respects than TPLSM, even in a nonporous system that is largely favourable to TPLSM.
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