Molecular response of Escherichia coli adhering onto nanoscale topography
© Rizzello et al.; licensee Springer. 2012
Received: 18 September 2012
Accepted: 10 October 2012
Published: 18 October 2012
Bacterial adhesion onto abiotic surfaces is an important issue in biology and medicine since understanding the bases of such interaction represents a crucial aspect in the design of safe implant devices with intrinsic antibacterial characteristics. In this framework, we investigated the effects of nanostructured metal substrates on Escherichia coli adhesion and adaptation in order to understand the bio-molecular dynamics ruling the interactions at the interface. In particular, we show how highly controlled nanostructured gold substrates impact the bacterial behavior in terms of morphological changes and lead to modifications in the expression profile of several genes, which are crucially involved in the stress response and fimbrial synthesis. These results mainly demonstrate that E. coli cells are able to sense even slight changes in surface nanotopography and to actively respond by activating stress-related pathways. At the same time, our findings highlight the possibility of designing nanoengineered substrates able to trigger specific bio-molecular effects, thus opening the perspective of smartly tuning bacterial behavior by biomaterial design.
KeywordsBacteria Nanotopography Adhesion Molecular response
Despite the great advancement recently achieved in the development of nanotechnology-based products, as demonstrated by the huge amount of nanomaterials that are present in the market nowadays, a thorough understanding of many biological issues related to these nanotools is still lacking. Among the available nanotech products, nanoengineered biomedical devices are probably one of the most intriguing ones because of their important applications in many research fields, ranging from drug delivery to medical imaging, tissue engineering, and orthopedic implant design. In particular, the fabrication of safe intra-corporeal devices, such as pacemakers, catheters, and bone screws, represents a challenging topic since almost any abiotic surface is prone to contaminations and infections caused by microorganisms that adhere onto the device surface, then colonizing it[2, 3]. Bacteria, in fact, mainly live on surfaces rather than as a suspended swimming community, also producing species- and strain-specific extracellular polymeric substance. This may lead to the formation of a complex combination of polysaccharides, external DNA, and catalytic proteins, usually known as biofilms, which is difficult to eradicate and may result in chronic infections. For this reason, many research efforts have been attempted to investigate the physicochemical bases that regulate the bacterium/abiotic substrate interactions. This is a crucial point because hindering the first step of the adhesion event likely represents the only opportunity to block further biofilm growth and development. In particular, a wide range of substrates presenting different surface chemistries, physical characteristics, and surface topographies has been designed and investigated to date in order to understand which physicochemical cue can avoid bacterial adhesion and persistence[5–9]. In this respect, particular attention has been focused toward the effects of surface micro- and nanostructuration over bacterial attachment, obtaining, however, rather contrasting results. Using multiple linear regression analysis, Bakker et al. showed, for instance, a direct relationship between surface roughness and the number of adherent bacteria on polyurethane-coated glass plates. The importance of the size and morphology of nanoscale features has also been addressed by other works, which confirmed the trend reported by Bakker, showing a general increase in the number of adherent bacteria with increasing surface nanoroughness[11, 12]. On the other side, other studies found out an opposite trend, namely that a decrease in the topographical feature size leads to an increase in the number of attached bacteria. In this respect, many of these studies mainly focused the attention on the theoretical and physicochemical point of view in studying the interaction between abiotic surfaces and bacteria. It should be, however, considered that, since microorganisms are rapidly evolving living systems, they are also able to sense and actively respond to surface cues. Bacteria have, in fact, fine molecular and mechanochemical sensors as well as highly controlled intracellular signaling pathways whose changes in activities with respect to surface nanotopography-related stimuli are nearly completely unknown so far.
In this work, we aimed to investigate the molecular mechanisms underlying the early stage of bacterium/abiotic substrate interaction at the interface. After detecting some important changes in their morphological features, we explored the expression level of several genes in Escherichia coli cells adhering onto flat and nanostructured gold surfaces, detecting the activation of the two-component system stress pathways CpxP/R and the up-regulation of the fimbrial recombinase FimE. These results suggest that nanostructured gold surfaces lead to a general stress condition in adherent bacteria, which down-express and degrade their adhesive organelle type-1 fimbriae and activate their recovery pathway to remove misfolded periplasmatic proteins. These findings highlight how surface nanotopography may play a pivotal role in triggering and guiding specific biological outcomes.
Substrate fabrication and characterization
For substrate preparation, we exploited a method already discussed elsewhere[14, 15]. Briefly, NH2-modified glass slides were coated with 50 nm of Au film by thermal evaporation (0.8 Ǻ/s) in order to obtain a very flat and uniform gold film. Nanorough Au films were achieved by coating 50 nm of Ag film (1.5 Ǻ/s) onto gold pre-coated glass substrates first and then immersing them within a solution of 10−3 M HAuCl4 for 15 min. The surface topography of flat and nanostructured substrates was investigated by scanning electron microscopy (SEM; Nova NanoSEM200, FEI, Hillsboro, OR, USA). Samples were positioned at a working distance of 5 mm and scanned with an 18-KeV e-beam. The substrate line profile was inspected by atomic force microscopy (contact mode in air) using the commercial nanoscope IVMultiMode SPM (Veeco Instruments, Santa Barbara, CA, USA) under ambient conditions (20°C to 25°C, atmospheric pressure, approximately 50% humidity).
Bacterial strain and growth conditions
A loop of glycerol stock of E. coli strain TG1 (K12, lac-pro supE thi hsdD5 (F′traD36 proA + B + lacI q lacZ M15)) was streaked onto a Luria-Bertani medium agar plate and incubated overnight at 37°C. Then, a single colony was picked and grown in Luria-Bertani (LB) liquid medium overnight at 37°C up to an optical density at 600 nm (OD600) of 1.00 ± 0.05 (corresponding to c.a. 8 × 108 cells/mL) in a shaking incubator (240 rpm). The overnight culture was diluted in LB medium to an OD600 of 0.1 and transferred into a six-well plate containing the substrates. The plates were incubated at 37°C for 12 h with shaking (240 rpm). After the incubation, the surfaces were gently rinsed four times with 0.2 M Tris, pH 7.5 to analyze only surface-associated bacteria.
Confocal microscopy analyses
To count the number of adherent bacteria, substrates were immersed in 4% formaldehyde (to fix cells) and then stained with Hoechst 33258 (1 μg/mL final concentration); imaging was performed using a confocal microscopy (Leica TCS-SP5 AOBS, Solms, Germany), and direct counting was carried out on flat and all the nanorough samples. For each replicate (three independent replicates were used), eight scan fields of 400×400 μm2 were analyzed.
Real-time quantitative PCR
Primers used in real-time qPCR analyses
Glyceraldehyde-3-phosphate dehydrogenase A
Outer membrane porinprotein C
Flagellar filament structural protein (flagellin)
Outer membrane lipoprotein
CP4-44 prophage; antigen 43 (Ag43) phase-variable biofilm formation autotransporter
Periplasmic adaptor protein
Oxidoreductase that catalyzes reoxidation of DsbA protein disulfide isomerase I
Serine endoprotease (protease Do), membrane-associated
Sensory histidine kinase/signal sensing protein
Tyrosine recombinase/inversion of on/off regulator of fimA
Results and discussion
Notably, we found a significant over-expression of cpxP and degP genes, which are involved in the bacterial envelope stress response, named as Cpx two-component system. This pathway is activated by the presence of large amounts of misfolded fimbrial protein aggregates, which are associated with the inner membrane. In particular, the periplasmic fimbrial misfolded subunits titrate cpxP and further activate cpxA; the latter then shifts its own phosphatase activity to a kinase and autokinase activity, leading to an accumulation of a phosphorylated transcription factor CpxR in the cytoplasm. This protein activates the expression of envelope folding and degrading factors, including dsbA and degP. However, as indicated by our data, bacteria growing onto nanorough Au surface do not up-regulate the periplasmic protein disulfide isomerase dsbA, which is involved in protein quality control and refolding processes. On the other side, the over-expression of degP suggests that E. coli cells prefer to shift their molecular activity on removing misfolded proteins in the periplasmic space by degrading them, instead of trying to refold them, most probably because of the high presence of extremely unfolded/damaged proteins. Moreover, we found that bacteria growing onto nanostructured gold substrates over-express the fimE gene. fimE encodes for a recombinase protein involved in the on-to-off fimbrial switching (i.e., FimE), leading bacteria to repress the type-1 fimbrial synthesis under particular conditions[24, 25]. These data are in good agreement with the SEM investigation of Figure 2 and better explain also our previous findings.
We also found an up-regulation of luxS gene in the nanorough samples. Such gene is involved in the biosynthesis of a quorum sensing (QS) autoinducer molecule (AI-2), which has been demonstrated as a universal signal that could be used by a variety of bacteria for communication, also among different species. QS molecules are used by microorganisms to coordinate the gene expression also of the surrounding community, thus enabling bacteria to behave like a quasi complex multicellular organism. This phenomenon occurs when bacteria have to overcome some environmental difficulties; in our case, such stress condition is represented by the nanotextured substrates.
On the other hand, the ompC gene, which codifies for the outer membrane porin C, lpxC, which is required for lipid A expression, and murA, which is important for external wall synthesis, are not regulated upon interaction with the nanostructured substrates. Also, the fliC gene that codifies for a flagella subunit, as well as cpxR, which is an effector of the two-component system CpxR-A pathway, is not regulated in the treated samples. In this respect, we can envisage that, although nanostructured Au substrates strongly impact the bacterial adhesion capability, the genes codifying for the biofilm expression seem to be unregulated in the early stage of the adhesion event. Further and more systematic studies are required in order to evaluate any possible influence of nanotopographies on biofilm formation after longer incubation periods. On the other hand, our data suggest that the mechanosensing machinery of E. coli feels the change in surface nanotopography as a physical stress signal. Hence, the bacteria focus their molecular activities on regulating and triggering specific pathways, which are important for recovery from stress conditions.
A detailed understanding of the molecular mechanisms underlying the interactions between nanomaterials and living systems is fundamental for providing more effective products for nanomedicine and drug delivery. The ability to smartly control the response of bacteria by tuning specific physicochemical properties of the nanosurfaces is ultimately the challenging goal. However, in studying nano-biointeractions, it is imperative to take into account the dynamic evolutions of the biosystem/abiotic substrate interaction events. In this context, we have demonstrated that nanostructured gold substrates induce significant changes in the morphological and genetic response of adherent E. coli. Particularly, we found out that nanotopography induces the activation of the stress signaling two-component system Cpx pathways and up-regulation of the fimbrial recombinase FimE. This data suggest that bacteria possess an extra-fine mechanosensing machinery, which is able to detect even nanoscale features in abiotic surface nanotopographies. Finally, this work may pave the way to the design of a new generation of devices which are able to trigger and tune specific biological outcomes.
The authors gratefully acknowledge L. Martiradonna for the SEM analyses, and V. Fiorelli and B. Antonazzo for their expert technical assistance.
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