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University of Groningen
Cell wall deformation and Staphylococcus aureus surface sensing
Harapanahalli, Akshay
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2015
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Harapanahalli, A. (2015). Cell wall deformation and Staphylococcus aureus surface sensing [Groningen]:
University of Groningen
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Summary
Summary
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Summary
Staphylococcus aureus is one of the major causative bacteria of implant associated
infections. Biomaterial associated infections start with the reversible adhesion of bacteria to
the implant surface, after which adhering bacteria embed themselves in a matrix of
extracellular polymeric substances (EPS) to yield a transition to irreversible adhesion and
biofilm growth commences. The EPS matrix protects biofilm inhabitants against biological,
mechanical and chemical stresses, such as the host immune response, fluid shear and
antibiotic treatment. All these phenomenal changes in S. aureus physiology occurs due to
adhesion and biofilm formation, therefore a sense of touch or mechanical sensitivity towards
surface adhesion is an important characteristic for adaptation and survival. However, very
less is understood about mechanical sensitivity of S. aureus during adhesion to a surface.
Chapter 1. gives an overview of the differences between two major sensory
strategies used by bacteria to sense the external environment, the chemical and
mechanosensing. Bacteria encounter different environmental conditions during the course
of their growth and have developed various mechanisms to sense their environment and
facilitate survival. Bacteria communicate with their environment through sensing of
chemical signals such as pH, ionic strength or sensing of biological molecules, such as
utilized in quorum sensing. However, bacteria do not solely respond to their environment by
means of chemical sensing, but also respond through physical-sensing mechanisms. For
instance, upon adhesion to a surface, bacteria may respond by excretion of EPS through a
mechanism called mechanosensing, allowing them to grow in their preferred, matrix
protected biofilm mode of growth. Therefore, the aim of this thesis was to evaluate the role of
adhesion forces in the response of bacteria to their adhering state. We have used a model
pathogen S. aureus, common in biomaterial associated infections and several of its isogenic
mutants and applied atomic force microscopy (AFM) and surface enhanced fluorescence
(SEF) to quantify adhesion forces and cell wall deformation, respectively. Bacterial response
was evaluated in terms of gene expression on different biomaterials commonly used in
orthopedic implants.
Bacterial adhesion to surfaces is mediated by a combination of different short- and
long-range forces. In Chapter 2, we present a new AFM based method to derive long-range
bacterial adhesion forces from the dependence of bacterial adhesion forces on the loading
force, as applied during the use of AFM. We have used two S. aureus strains, (S. aureus
ATCC12600 and S. aureus NCTC 8325-4) and their isogenic Δpbp4 mutants. The long-range
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adhesion forces of wild-type S. aureus parent strains (0.5 and 0.8 nN) amounted to only one
third of these forces measured for their more deformable isogenic Δpbp4 mutants (2.7 and
1.6 nN) that were deficient in peptidoglycan cross-linking. The measured long-range
Lifshitz-Van der Waals adhesion forces matched those calculated from published Hamaker
constants, provided that a 40% ellipsoidal deformation of the bacterial cell wall was assumed
for the Δpbp4 mutants. Direct imaging of adhering staphylococci using the AFM peak forcequantitative nanomechanical property mapping imaging mode confirmed a height reduction
due to deformation in the Δpbp4 mutants of 100 – 200 nm. Across naturally occurring
bacterial strains, long-range forces do not vary to the extent as observed here for the Δpbp4
mutants. Importantly however, extrapolating from the results of this study it can be
concluded that long-range bacterial adhesion forces are not only determined by the
composition and structure of the bacterial cell surface, but also by a hitherto neglected, small
deformation of the bacterial cell wall, facilitating an increase in contact area and therewith in
adhesion force.
Nanoscale cell wall deformation upon adhesion is difficult to measure, except
for Δpbp4 mutants, deficient in peptidoglycan cross-linking. Chapter 3 discusses a
more advanced technique to quantify cell wall deformation based on surface
enhanced fluorescence in staphylococci adhering on gold surfaces. Adhesion related
fluorescence enhancement depends on the distance of the bacteria from the surface
and the residence-time of the adhering bacteria. In this chapter, a model was
forwarded based on the adhesion related fluorescence enhancement of greenfluorescent microspheres, through which the distance to the surface and cell wall
deformation of adhering bacteria can be calculated from their residence-time
dependent adhesion related fluorescence enhancement. The distances between
adhering bacteria and a surface, including compression of their EPS-layer, decreased
up to 60 min after adhesion, followed by cell wall deformation. Cell wall deformation
is independent on the integrity of the EPS-layer and proceeds fastest for a Δpbp4
strain.
Based on the results from chapter 2 and 3, it can be concluded that cell wall
deformation of both the parent and the Δpbp4 mutant strains occurred upon surface
adhesion. However, what these deformations mean to bacteria in terms of molecular
response in modulating their phenotypes from free floating to surface growing biofilms is
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Summary
unknown. In Chapter 4, we have investigated the influence of staphylococcal adhesion
forces to different biomaterials on icaA (regulating production of EPS matrix components)
and cidA (associated with cell lysis and extracellular DNA release) gene expression in S.
aureus biofilms. Experiments were performed with S. aureus ATCC12600 and its isogenic
mutant S. aureus ATCC12600Δpbp4, deficient in peptidoglycan cross-linking. Deletion of
pbp4 was associated with greater cell-wall deformability, while it did not affect the
planktonic growth rate, biofilm formation, cell surface hydrophobicity or zeta potential of the
strains. The adhesion forces of S. aureus ATCC12600 were strongest on polyethylene (4.9 ±
0.5 nN), intermediate on polymethylmethacrylate (3.1 ± 0.7 nN) and the weakest on
stainless steel (1.3 ± 0.2 nN). The production of poly-N-acetylglucosamine, eDNA presence
and expression of icaA genes decreased with increasing adhesion forces. However, no
relation between adhesion forces and cidA expression was observed. The adhesion forces of
the isogenic mutant S. aureus ATCC12600Δpbp4 were much weaker than those of the parent
strain and did not show any correlation with the production of poly-N-acetylglucosamine,
eDNA presence, or expression of the icaA and cidA genes. This suggests that adhesion forces
modulate the production of matrix molecules poly-N-acetylglucosamine, eDNA presence and
icaA gene expression by inducing nanoscale cell wall deformation, with cross-linked
peptidoglycan layers playing a pivotal role in this adhesion force sensing.
Bacterial adhesion to biomaterial surfaces and associated susceptibility to
antimicrobials is an important threat faced by the medical community. Bacteria not only
form biofilms, but may also gain up to 1000 times more resistance to antibiotics when in a
biofilm than in a planktonic mode of growth. To reveal mechanisms that induce such strong
resistance, in Chapter 5, we investigated the regulation of one of the newly discovered twocomponent
system
nisin-associated-sensitivity-response-regulator
(NsaRS)
and
its
downstream drug transporter NsaAB in S. aureus cells, in presence of chemical stress and
mechanical stress. NsaRS is important for surface adhesion, biofilm formation and bacterial
resistance against chemical stresses in S. aureus. It consists of an intra-membrane located
sensor NasS and a cytoplasmatically located response regulator NsaR, which becomes
activated upon receiving phosphate groups from the NsaS sensor. The intra-membrane
location of the NsaS sensor leads us to hypothesize that the NsaRS system can sense not only
chemical but also mechanical stresses to modulate antibiotic resistance via the NsaAB efflux
pump. To verify this hypothesis, we compared expressions of the NsaS sensor and NsaA
efflux pump in S. aureus SH1000 in their adhering (“mechanical stress”) and planktonic
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state, while the presence of nisin constitutes a chemical stress. NsaS and NsaA gene
expressions by S. aureus SH1000 were higher in a mechanically stressed, adhering state
than in a planktonic one. Chemical stress enhanced NsaS and NsaR gene expressions. Gene
expression became largest, when the organisms experienced a chemical stress in
combination with a strong mechanical stress, in the current study quantitated as the
adhesion force arising from a substratum surface measured using bacterial probe AFM. This
confirms our hypothesis that the NsaRS system can sense both chemical and mechanical
stresses.
In Chapter 6 we have discussed the differences in using AFM and SEM in
quantifying cell wall deformation. Furthermore, we discuss the molecular basis for surface
sensing in S. aureus in comparison with other bacteria and eukaryotic cells. Finally, from the
results obtained in this thesis, we suggested future studies on the role of mechanosensitive
channels in antimicrobial susceptibility.
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