Diffusion of the MPs through the cell
membrane/envelope
In this section, the diffusional path of the MPs through the cell
membrane/envelope of the bacterial and mammalian cells, as they move
from the bulk to the cytoplasm, is explored. In addition to differing in
shape and size, the boundaries of the cells are constituted by
structures with different layers and composition (Fig. 4). For instance,
animal cells only possess a phospholipidic barrier, the cytoplasmic
membrane. Bacterial cells possess an added barrier, the cell envelope,
which by its turn has several layers, which differ between gram-positive
and gram-negative microorganisms. The gram-negative bacterial cell
envelope is constituted by an outer membrane, followed by the
periplasmic space which comprises a thin peptidoglycan layer, and the
inner cytoplasmic membrane, a phospholipid bilayer (Fig. 4). The
periplasmic space normally presents a higher viscosity compared to the
cytoplasm due to the presence of small molecules and it is often
considered a “crowded” space22. The gram-positive
bacterial cell envelope does not have an outer membrane and the
peptidoglycan layer is thicker than the one present in gram-negative
bacteria, followed by the cytoplasmic membrane (Fig. 4).
Moreover, both bacterial and mammalian cells have proteins channels and
other components in their membranes that are responsible for the
transport of molecules (Fig. 4). It is a complex structural and
functional system, and a comprehensive review about this subject can be
found in Santos et al. 23. The structural and
chemical differences at the membrane level affect the diffusion of the
MPs in the different organisms. Table 1 summarizes the average
dimensions of each barrier for the different cell models here defined.
For bacterial cells the capsule was not considered and the dimensions
displayed for the other barriers are purely reference values, since r
and L depend on the growth phase, temperature and available nutrients,
among other variables24. The thickness of each layer
was set based on the average of available experimental values. In
theory, a lipid bilayer has a thickness of around 4
nm25. However, and most likely due to the extra
constituents of the outer membrane, such as proteins and
lipopolysaccharides (LPS)26, the outer membrane of
gram-negative cells is sometimes considered to be thicker than the
cytoplasmic membrane. In here, the core size of the bilayer was
considered, keeping in mind that the diffusion in this layer may be
slower than in the inner membrane.
To facilitate the interpretation, the different layers were grouped in
terms of composition. The first barrier discussed here is the lipidic
membrane or cytoplasmic membrane, which is the first barrier that the
MPs encounter in animal cells. The outer membrane of gram-negative
bacteria is also included in the lipid membranes section. The next
barriers discussed are the periplasm of gram-negative bacteria followed
by the peptidoglycan layers of both gram-positive and gram-negative
bacteria. The latter is the first physical barrier for the MP in
gram-positive bacteria, and is similar in terms of structure and
chemical composition in both grams, varying mainly in
thickness26.
Molecular diffusion in lipid
membranes
As mentioned above, and in spite of composition differences that might
affect the MP’s diffusion, the cytoplasmic membrane of gram-positive
bacteria, both membranes (outer and inner membrane) of gram-negative
bacteria, and the cytoplasmic membrane of animal cells are approached in
the same manner.
Considering the importance of nucleic acids diffusion in lipid
membranes, there is a remarkable lack of studies for modelling this
process, both in bacterial and animal cells. In one of the few existing
studies regarding this issue, the diffusion time of DNA probes in lipid
membranes has been estimated by tracking the formation of endosomes
after injecting DNA into the outer side of a liposome. For an
oligonucleotide of 21 bp, the process takes around 1
s35. For in vivo hybridization, this value
should be considered the lowest possible diffusion time, because it is
well known that proteins and other molecules can obstruct and bind to
MPs, decreasing the diffusion coefficient36. Although
no exact equation is available to model this process for the probe of 10
and 40 bp, this information can be used as an indication of the
characteristic time a MP takes to go through a lipid membrane.
Molecular diffusion in the periplasm of gram-negative
bacteria
In gram-negative bacteria, the periplasm is located between the outer
and the cytoplasmic membranes and is about 13-25 nm
thick37. In addition to the peptidoglycan layer, the
periplasmic space contains a high concentration of other small molecules
like amino acids and peptides that contribute to the increase of
viscosity in this interface38. As a result, the
periplasm seems to be a gel-like matrix, which makes each molecule
experience an effective viscosity during movement and, as such, the
molecular diffusion in this “crowded” system diverges from a normal
Brownian motion (as described in the bulk). There is limited information
on particle diffusion through the periplasm, especially for nucleic
acids. Most studies report lateral diffusion of proteins in different
membrane layers. Such is the case of the work of Mullineaux et
al. 39, which explores the lateral diffusion of the
globular Green Fluorescence Protein (GFP) in the cytoplasm, periplasm,
and plasma membrane of E. coli . They established that the
diffusion in the periplasm (Dperi) is about three times
smaller than in the cytoplasm (Dcyto), but highlighted
that, in terms of physical properties, the periplasm is a relatively
fluid environment, in comparison to the cytoplasm. For these reasons, it
was assumed that is in the same order of magnitude as . values are
provided in the section concerning the diffusion of the MP in the
cytoplasm. Using the values and Eq. 1, a 10 bp probe takes 3.8
x10-7 s to diffuse through the periplasm, while a 40
bp probe, with a larger radius, takes 1.8 x10-6 s.
Molecular diffusion in the
peptidoglycan
The peptidoglycan layer is structurally similar in gram-negative and
-positive bacteria, mostly varying in thickness. In FISH, the diffusion
of MPs through the peptidoglycan layer most likely represents the
limiting step in both Grams. This is true even for gram-negative
bacteria despite of their severally restrictive outer membrane, because
of the fixation steps prior to the addition of the MPs, which create
pores in the outer membrane, allowing the MPs to directly cross from the
extracellular space to the peptidoglycan layer. Gram-negative bacteria
have a thin peptidoglycan layer, whereas the peptidoglycan of
gram-positive bacteria can be up to 100 nm thick26.
Overcoming this layer is crucial for a successful FISH procedure,
especially in gram-positive bacteria, where longer hybridization times
are required compared to gram-negative bacteria (normally the FISH
procedure takes approximately 120 min for the detection of gram-positive
bacteria and 60 min for gram-negative)16, most likely
due to the differences in thickness. The thickness of the peptidoglycan
makes it particularly important to proceed with the permeabilization of
fixed gram-positive bacteria, or with the enzymatic hydrolysis of the
peptidoglycan prior to the hybridization of large MPs, in order to have
a detectable fluorescence signal40.
Since there is no data in the literature related to the diffusion
coefficient of the MPs in the peptidoglycan barrier, the difference
between the commonly used experimental hybridization times in FISH for
gram-negative and gram-positive bacteria was used to calculate the
diffusion coefficient of the MP in the layer for both grams. For
gram-positive bacteria, a diffusion coefficient of
3.5x10-19 m2/s was obtained, which
corresponds to a characteristic diffusion time of 4524 s (75 min). This
in agreement with the general concept that the peptidoglycan layer of
gram-positive bacteria is one of the most relevant barriers in microbial
FISH experiments40.