Adhesion force sensing and activation of a membrane-bound sensor to activate nisin efflux pumps in Staphylococcus aureus under mechanical and chemical stresses

Nisin-associated-sensitivity-response-regulator (NsaRS) in Staphylococcus aureus is important for its adhesion to surfaces and resistance against antibiotics, like nisin. NsaRS consists of an intra-membrane-located sensor NsaS and a cytoplasmatically-located response-regulator NsaR, which becomes activated upon receiving phosphate groups from the intratmembrane-located sensor. Hypothesis: The intra-membrane location of the NsaS sensor leads us to hypothesize that the two component NsaRS system not only senses "chemical" (nisin) but also "mechanical" (adhesion) stresses to modulate efflux of antibiotics from the cytoplasm. Experiments: NsaS sensor and NsaAB efflux pump transcript levels in S. aureus SH1000 adhering to surfaces exerting different adhesion forces were compared, in presence and absence of nisin. Adhesion forces were measured using single-bacterial contact probe atomic force microscopy. Findings: Gene expression became largest when staphylococci experienced strong adhesion forces combined with nisin-presence and the two-component NsaRS response to antibiotics was enhanced at a stronger adhesion force. This confirms that the intra-membrane-located sensor NsaS senses both chemical and mechanical stresses to modulate antibiotic clearance through the NsaAB efflux pump. This finding creates better understanding of the antibiotic resistance of bacteria adhering to surfaces and, in the fight against antibiotic-resistant pathogens, may aid development of advanced biomaterials on which bacterial efflux pumps are not activated

Influence of Adhesion Force on icaA and cidA Gene Expression and Production of Matrix Components in Staphylococcus aureus Biofilms

The majority of human infections are caused by biofilms. The biofilm mode of growth enhances the pathogenicity of Staphylococcus spp. considerably, because once they adhere, staphylococci embed themselves in a protective, self-produced matrix of extracellular polymeric substances (EPSs). The aim of this study was to investigate the influence of forces of staphylococcal adhesion to different biomaterials on icaA (which regulates the production of EPS matrix components) and cidA (which is associated with cell lysis and extracellular DNA [eDNA] release) gene expression in Staphylococcus aureus biofilms. Experiments were performed with S. aureus ATCC 12600 and its isogenic mutant, S. aureus ATCC 12600 Delta pbp4, 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 ATCC 12600 were the 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 ATCC 12600 Delta pbp4 (deficient in peptidoglycan cross-linking) 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 the matrix molecule 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

Surface Thermodynamic and Adhesion Force Evaluation of the Role of Chitin-Binding Protein in the Physical Interaction between Pseudomonas aeruginosa and Candida albicans

<p>Candida albicans and Pseudomonas aeruginosa are able to form pathogenic polymicrobial communities. P. aeruginosa colonizes and kills hyphae but is unable to attach to yeast. It is unknown why the interaction of P. aeruginosa is different with yeast than with hyphae. Here we aim to evaluate the role of P. aeruginosa chitin-binding protein (CbpD) in its physical interaction with C. albicans hyphae or yeast, based on surface thermodynamic and atomic force microscopic analyses. A P. aeruginosa mutant lacking CbpD was unable to express strong adhesion forces with hyphae (-2.9 nN) as compared with the parent strain P. aeruginosa PAO1 (-4.8 nN) and showed less adhesion to hyphae. Also blocking of CbpD using N-acetyl-glucosamine yielded a lower adhesion force (-4.3 nN) with hyphae. Strong adhesion forces were restored after complementing the expression of CbpD in P. aeruginosa PA01 Delta cbpD yielding an adhesion force of -5.1 nN. These observations were confirmed with microscopic evaluation of adhesion tests. Regardless of the absence or presence of CbpD on the bacterial cell surfaces, or their blocking, P. aeruginosa experienced favorable thermodynamic conditions for adhesion with hyphae, which were absent with yeast. In addition, adhesion forces with yeast were less than 0.5 nN in all cases. Concluding, CbpD in P. aeruginosa is responsible for strong physical interactions with C. albicans hyphae. The development of this interaction requires time due to the fact that CbpDs have to invade the outermost mannoprotein layer on the hyphal cell surfaces. In order to do this, thermodynamic conditions at the outermost cell surfaces have to be favorable.</p>

Tumor-targeted intracellular delivery of anticancer drugs through the mannose-6-phosphate/insulin-like growth factor II receptor

Tumor-targeting of anticancer drugs is an interesting approach for the treatment of cancer since chemotherapies possess several adverse effects. In the present study, we propose a novel strategy to deliver anticancer drugs to the tumor cells through the mannose-6-phosphate/insulin-like growth factor receptor (M6P/IGF-IIR) which are abundantly expressed in several human tumors. We developed a drug carrier against M6P/IGF-II receptor by modifying human serum albumin (HSA) with M6P moieties. M6P-HSA specifically bound and internalized into M6P/IGF-IIR-expressing B16 melanoma cells as demonstrated with radioactive studies and anti-HSA immunostaining. In vivo, M6P-HSA rapidly accumulated in subcutaneous tumors in tumor and stromal components after an intravenous injection. To demonstrate the application of M6P-HSA as a drug carrier, we coupled doxorubicin to it. Dox-HSA-M6P conjugate could release doxorubicin at lysosomal pH and showed M6P-specific binding and uptake in tumor cells. In vitro, a short exposure with Dox-HSA-M6P induced killing of tumor cells, which could be blocked by excess M6P-HSA. In vivo, Dox-HSA-M6P distributed to tumors and some other organs while free doxorubicin distributed to all organs but slightly to tumors. In B16 tumor-bearing mice, Dox-HSA-M6P significantly inhibited the tumor growth whereas an equimolar dose of free doxorubicin did not show any anti-tumor effect. In addition, targeted doxorubicin did not show any side-effects on liver and kidney function tests, body weight and blood cell counts. In conclusion, M6P-HSA is a suitable carrier for delivery of anticancer drugs to tumors through M6P/IGF-IIR. Improved antitumor effects of the targeted doxorubicin by M6P-HSA suggest that this novel approach may be applied to improve the therapeutic efficacy of anticancer drugs

Bacterial cell wall deformation, mechanosensing, and the measurement of cell wall deformation using surface enhanced fluorescence.

<p><b>A) Left:</b> Intact lipid membrane at equilibrium of an undeformed bacterium, with a closed mechanosensitive channel (MSC). <b>Right:</b> Bacterium adhering to a substratum surface, deformed under the influence of adhesion forces arising from the substratum, yielding hydrophobic mismatch over the thickness of the membrane (water molecules adjacent to hydrophobic lipid tails), and altered lipid bilayer tension in the lipid membrane. Hydrophobic mismatch and pressure profile changes lead to the opening of MSCs. <b>B) Left:</b> A nonactivated stress-sensitive (SS) protein on the bacterial cell surface of an undeformed bacterium and a response regulator protein (RR) suspended freely in the cytoplasm. <b>Right:</b> A SS protein senses cell wall deformation due to adhesion, changes its conformation, and phosphorylates a RR protein which regulates the expression of SS-regulated genes. <b>C) Left:</b> Lifshitz-Van der Waals forces operate between all molecular pairs in a bacterium and a substratum, decreasing with distance between the molecules. <b>Right:</b> Adhering bacterium, deformed due to attractive Lifshitz-Van der Waals forces, with more molecules in the bacterium closer to the substratum, yielding stronger adhesion and more deformation. Deformation stops once the counterforces arising from the deformation of the rigid peptidoglycan layer match those of the adhesion forces. <b>D) Left:</b> Only a small number of fluorophores inside an undeformed bacterium are sufficiently close to a metal substratum surface to experience surface-enhanced fluorescence (brighter dots). <b>Right:</b> In a deformed, adhering bacterium, the volume of the bacterium close to the surface increases and the number of fluorophores subject to surface-enhanced fluorescence becomes higher. Thus, quantitative analysis of fluorescence arising from fluorescent bacteria adhering to a metal surface provides a way to determine cell wall deformation.</p

Residence-time dependent cell wall deformation of different Staphylococcus aureus strains on gold measured using surface-enhanced-fluorescence

Bacterial adhesion to surfaces is accompanied by cell wall deformation that may extend to the lipid membrane with an impact on the antimicrobial susceptibility of the organisms. Nanoscale cell wall deformation upon adhesion is difficult to measure, except for Delta pbp4 mutants, deficient in peptidoglycan cross-linking. This work explores surface enhanced fluorescence to measure the cell wall deformation of 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. A model is forwarded based on the adhesion-related fluorescence enhancement of green-fluorescent 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 extracellular polymeric substance (EPS)-layer, decrease up to 60 min after adhesion, followed by cell wall deformation. Cell wall deformation is independent of the integrity of the EPS-layer and proceeds fastest for a Delta pbp4 strain

Surface Thermodynamic and Adhesion Force Evaluation of the Role of Chitin-Binding Protein in the Physical Interaction between <i>Pseudomonas aeruginosa</i> and <i>Candida albicans</i>

<i>Candida albicans</i> and <i>Pseudomonas aeruginosa</i> are able to form pathogenic polymicrobial communities. <i>P. aeruginosa</i> colonizes and kills hyphae but is unable to attach to yeast. It is unknown why the interaction of <i>P. aeruginosa</i> is different with yeast than with hyphae. Here we aim to evaluate the role of <i>P. aeruginosa</i> chitin-binding protein (CbpD) in its physical interaction with <i>C. albicans</i> hyphae or yeast, based on surface thermodynamic and atomic force microscopic analyses. A <i>P. aeruginosa</i> mutant lacking CbpD was unable to express strong adhesion forces with hyphae (−2.9 nN) as compared with the parent strain <i>P. aeruginosa</i> PAO1 (−4.8 nN) and showed less adhesion to hyphae. Also blocking of CbpD using <i>N</i>-acetyl-glucosamine yielded a lower adhesion force (−4.3 nN) with hyphae. Strong adhesion forces were restored after complementing the expression of CbpD in <i>P. aeruginosa</i> PAO1 ΔcbpD yielding an adhesion force of −5.1 nN. These observations were confirmed with microscopic evaluation of adhesion tests. Regardless of the absence or presence of CbpD on the bacterial cell surfaces, or their blocking, <i>P. aeruginosa</i> experienced favorable thermodynamic conditions for adhesion with hyphae, which were absent with yeast. In addition, adhesion forces with yeast were less than 0.5 nN in all cases. Concluding, CbpD in <i>P. aeruginosa</i> is responsible for strong physical interactions with <i>C. albicans</i> hyphae. The development of this interaction requires time due to the fact that CbpDs have to invade the outermost mannoprotein layer on the hyphal cell surfaces. In order to do this, thermodynamic conditions at the outermost cell surfaces have to be favorable