Development of an antibody fused with an antimicrobial peptide targeting Pseudomonas aeruginosa: A new approach to prevent and treat bacterial infections.

The increase in emerging drug resistant Gram-negative bacterial infections is a global concern. In addition, there is growing recognition that compromising the microbiota through the use of broad-spectrum antibiotics can impact long term patient outcomes. Therefore, there is the need to develop new bactericidal strategies to combat Gram-negative infections that would address these specific issues. In this study, we report and characterize one such approach, an antibody-drug conjugate (ADC) that combines (i) targeting the surface of a specific pathogenic organism through a monoclonal antibody with (ii) the high killing activity of an antimicrobial peptide. We focused on a major pathogenic Gram-negative bacterium associated with antibacterial resistance: Pseudomonas aeruginosa. To target this organism, we designed an ADC by fusing an antimicrobial peptide to the C-terminal end of the VH and/or VL-chain of a monoclonal antibody, VSX, that targets the core of P. aeruginosa lipopolysaccharide. This ADC demonstrates appropriately minimal levels of toxicity against mammalian cells, rapidly kills P. aeruginosa strains, and protects mice from P. aeruginosa lung infection when administered therapeutically. Furthermore, we found that the ADC was synergistic with several classes of antibiotics. This approach described in this study might result in a broadly useful strategy for targeting specific pathogenic microorganisms without further augmenting antibiotic resistance.

Approaching prosthesis infection environment: Development of an innovative in vitro Staphylococcus aureus biofilm model.

The major role and implication of bacterial biofilms in the case of bone and prosthesis infections have been highlighted and often linked to implant colonization. Management strategies of these difficult-to-treat infections consist in surgeries and antibiotic treatment, but the rate of relapse remains high, especially if Staphylococcus aureus, a high-virulent pathogen, is involved. Therapeutic approaches are not adapted to the specific features of biofilm in bone context whereas infectious environment is known to importantly influence biofilm structure. In the present study, we aim to characterize S. aureus SH1000 (methicillin-sensitive strain, MSSA) and USA300 (methicillin-resistant strain, MRSA) biofilm on different surfaces mimicking the periprosthetic environment. As expected, protein adsorption on titanium enhanced the number of adherent bacteria for both strains. On bone explant, USA300 adhered more than SH1000. The simultaneous presence of two different surfaces was also found to change the bacterial behaviour. Thus, proteins adsorption on titanium and bone samples (from bank or directly recovered after an arthroplasty) were found to be key parameters that influence S. aureus biofilm formation: adhesion, matrix production and biofilm-related gene regulation. These results highlighted the need for new biofilm models, more relevant with the infectious environment by using adapted culture medium and presence of surfaces that are representative of in situ conditions to better evaluate therapeutic strategies against biofilm.

Staphylococcus aureus Behavior on Artificial Surfaces Mimicking Bone Environment.

Infections, which interfere with bone regeneration, may be a critical issue to consider during the development of biomimetic material. Calcium phosphate (CaP) and type I collagen substrates, both suitable for bone-regeneration dedicated scaffolds, may favor bacterial adhesion. Staphylococcus aureus possesses adhesins that allow binding to CaP or collagen. After their adhesion, bacteria may develop structures highly tolerant to immune system attacks or antibiotic treatments: the biofilms. Thus, the choice of material used for scaffolds intended for bone sites is essential to provide devices with the ability to prevent bone and joint infections by limiting bacterial adhesion. In this study, we compared the adhesion of three different S. aureus strains (CIP 53.154, SH1000, and USA300) on collagen- and CaP-coating. Our objective was to evaluate the capacity of bacteria to adhere to these different bone-mimicking coated supports to better control the risk of infection. The three strains were able to adhere to CaP and collagen. The visible matrix components were more important on CaP- than on collagen-coating. However, this difference was not reflected in biofilm gene expression for which no change was observed between the two tested surfaces. Another objective was to evaluate these bone-mimicking coatings for the development of an in vitro model. Thus, CaP, collagen-coatings, and the titanium-mimicking prosthesis were simultaneously tested in the same bacterial culture. No significant differences were found compared to adhesion on surfaces independently tested. In conclusion, these coatings used as bone substitutes can easily be colonized by bacteria, especially CaP-coating, and must be used with an addition of antimicrobial molecules or strategies to avoid bacterial biofilm development.

Human Osteoblast-Conditioned Media Can Influence Staphylococcus aureus Biofilm Formation.

Osteoblasts are bone-forming and highly active cells participating in bone homeostasis. In the case of osteomyelitis and more specifically prosthetic joint infections (PJI) for which Staphylococcus aureus (S. aureus) is mainly involved, the interaction between osteoblasts and S. aureus results in impaired bone homeostasis. If, so far, most of the studies of osteoblasts and S. aureus interactions were focused on osteoblast response following direct interactions with co-culture and/or internalization models, less is known about the effect of osteoblast factors on S. aureus biofilm formation. In the present study, we investigated the effect of human osteoblast culture supernatant on methicillin sensitive S. aureus (MSSA) SH1000 and methicillin resistant S. aureus (MRSA) USA300. Firstly, Saos-2 cell line was incubated with either medium containing TNF-α to mimic the inflammatory periprosthetic environment or with regular medium. Biofilm biomass was slightly increased for both strains in the presence of culture supernatant collected from Saos-2 cells, stimulated or not with TNF-α. In such conditions, SH1000 was able to develop microcolonies, suggesting a rearrangement in biofilm organization. However, the biofilm matrix and regulation of genes dedicated to biofilm formation were not substantially changed. Secondly, culture supernatant obtained from primary osteoblast culture induced varied response from SH1000 strain depending on the different donors tested, whereas USA300 was only slightly affected. This suggested that the sensitivity to bone cell secretions is strain dependent. Our results have shown the impact of osteoblast secretions on bacteria and further identification of involved factors will help to manage PJI.

A qnrD-Plasmid Promotes Biofilm Formation and Class 1 Integron Gene Cassette Rearrangements in Escherichia coli.

Bacteria within biofilms may be exposed to sub-minimum inhibitory concentrations (sub-MICs) of antibiotics. Cell-to-cell contact within biofilms facilitates horizontal gene transfers and favors induction of the SOS response. Altogether, it participates in the emergence of antibiotic resistance. Aminoglycosides at sub-MICs can induce the SOS response through NO accumulation in E. coli carrying the small plasmid with the quinolone resistance qnrD gene (pDIJ09-518a). In this study, we show that in E. coli pDIJ09-518a, the SOS response triggered by sub-MICs of aminoglycosides has important consequences, promoting genetic rearrangement in class 1 integrons and biofilm formation. We found that the integrase expression was increased in E. coli carrying pDIJ09-518a in the presence of tobramycin, which was not observed for the WT isogenic strain that did not carry the qnrD-plasmid. Moreover, we showed that biofilm production was significantly increased in E. coli WT/pDIJ09-518a compared to the WT strain. However, such a higher production was decreased when the Hmp-NO detoxification pathway was fully functional by overexpressing Hmp. Our results showing that a qnrD-plasmid can promote biofilm formation in E. coli and potentiate the acquisition and spread of resistance determinants for other antibiotics complicate the attempts to counteract antibiotic resistance and prevention of biofilm development even further. We anticipate that our findings emphasize the complex challenges that will impact the decisions about antibiotic stewardship, and other decisions related to retaining antibiotics as effective drugs and the development of new drugs.

Comparison of Two Cutibacterium acnes Biofilm Models.

The study of biofilms in vitro is complex and often limited by technical problems due to simplified models. Here, we compared C. acnes biofilm formation, from species involved in bone and prosthesis infection, in a static model with a dynamic model. Using similar parameters, the percentage of live bacteria within the biofilm was higher in dynamic than in static approach. In both models, bacterial internalization in osteoblast-like cells, playing the role of stress factor, affected this proportion but in opposite ways: increase of live bacteria proportion in the static model (×2.04 ± 0.53) and of dead bacteria proportion (×3.5 ± 1.03) in the dynamic model. This work highlights the huge importance in the selection of a relevant biofilm model in accordance with the environmental or clinical context to effectively improve the understanding of biofilms and the development of better antibiofilm strategies.

Synergy between Indoloquinolines and Ciprofloxacin: An Antibiofilm Strategy against Pseudomonas aeruginosa.

Antibiotic treatments can participate in the formation of bacterial biofilm in case of under dosage. The interest of indoloquinoline scaffold for drug discovery incited us to study the preparation of new indolo [2,3-b]quinoline derivatives by a domino radical process. We tested the effect of two different "indoloquinoline" molecules (Indol-1 and Indol-2) without antimicrobial activity, in addition to ciprofloxacin, on biofilm formation thanks to crystal violet staining and enumeration of adhered bacteria. This association of ciprofloxacin and Indol-1 or Indol-2 attenuated the formation of biofilm up to almost 80% compared to ciprofloxacin alone, or even prevented the presence of adhered bacteria. In conclusion, these data prove that the association of non-antimicrobial molecules with an antibiotic can be a solution to fight against biofilm and antibiotic resistance emergence.

Staphylococcus aureus Strain-Dependent Biofilm Formation in Bone-Like Environment.

Staphylococcus aureus species is an important threat for hospital healthcare because of frequent colonization of indwelling medical devices such as bone and joint prostheses through biofilm formations, leading to therapeutic failure. Furthermore, bacteria within biofilm are less sensitive to the host immune system responses and to potential antibiotic treatments. We suggested that the periprosthetic bone environment is stressful for bacteria, influencing biofilm development. To provide insights into S. aureus biofilm properties of three strains [including one methicillin-resistant S. aureus (MRSA)] under this specific environment, we assessed several parameters related to bone conditions and expected to affect biofilm characteristics. We reported that the three strains harbored different behaviors in response to the lack of oxygen, casamino acids and glucose starvation, and high concentration of magnesium. Each strain presented different biofilm biomass and live adherent cells proportion, or matrix production and composition. However, the three strains shared common responses in a bone-like environment: a similar production of extracellular DNA and engagement of the SOS response. This study is a step toward a better understanding of periprosthetic joint infections and highlights targets, which could be common among S. aureus strains and for future antibiofilm strategies.

Interaction of implant infection-related commensal bacteria with mesenchymal stem cells: a comparison between Cutibacterium acnes and Staphylococcus aureus.

Staphylococcus aureus and Cutibacterium acnes are involved in several tissue infections and can encounter mesenchymal stem cells (MSCs) during their role in tissue regenerative process. C. acnes and S. aureus internalization by three types of MSCs derived from bone marrow, dental pulp and Wharton's jelly; and bacterial biofilm production were compared. Internalization rates ranged between 1.7-6.3% and 0.8-2.7% for C. acnes and S. aureus, respectively. While C. acnes strains exhibited limited cytotoxic effect on MSCs, S. aureus were more virulent with marked effect starting after only 3 h of interaction. Both bacteria were able to produce biofilms with respectively aggregated and monolayered structures for C. acnes and S. aureus. The increase in C. acnes capacity to develop biofilm following MSCs' internalization was not linked to the significant increase in number of live bacteria, except for bone marrow-MSCs/C. acnes CIP 53.117 with 79% live bacteria compared to the 36% before internalization. On the other hand, internalization of S. aureus had no impact on its ability to form biofilms composed mainly of living bacteria. The present study underlined the complexity of MSCs-bacteria cross-interaction and brought insights into understanding the MSCs behavior in response to bacterial infection in tissue regeneration context.

Antibiotic Tolerance of Staphylococcus aureus Biofilm in Periprosthetic Joint Infections and Antibiofilm Strategies.

The need for bone and joint prostheses is currently growing due to population aging, leading to an increase in prosthetic joint infection cases. Biofilms represent an adaptive and quite common bacterial response to several stress factors which confer an important protection to bacteria. Biofilm formation starts with bacterial adhesion on a surface, such as an orthopedic prosthesis, further reinforced by matrix synthesis. The biofilm formation and structure depend on the immediate environment of the bacteria. In the case of infection, the periprosthetic joint environment represents a particular interface between bacteria, host cells, and the implant, favoring biofilm initiation and maturation. Treating such an infection represents a huge challenge because of the biofilm-specific high tolerance to antibiotics and its ability to evade the immune system. It is crucial to understand these mechanisms in order to find new and adapted strategies to prevent and eradicate implant-associated infections. Therefore, adapted models mimicking the infectious site are of utmost importance to recreate a relevant environment in order to test potential antibiofilm molecules. In periprosthetic joint infections, Staphylococcus aureus is mainly involved because of its high adaptation to the human physiology. The current review deals with the mechanisms involved in the antibiotic resistance and tolerance of Staphylococcus aureus in the particular periprosthetic joint infection context, and exposes different strategies to manage these infections.

Infection of Human Dental Pulp Stromal Cells by Streptococcus mutans: Shedding Light on Bacteria Pathogenicity and Pulp Inflammation.

Cariogenic Streptococcus mutans (S. mutans) is implicated in the dental pulp necrosis but also in cardiovascular tissue infections. Herein, the purpose was to elucidate how human dental pulp derived stromal cells (DPSCs) react toward a direct interaction with S. mutans. DPSCs were challenged with S. mutans. Following 3 h of interaction, DPSCs were able to internalize S. mutans (rate < 1%), and F-actin fibers played a significant role in this process. S. mutans persisted in the DPSCs for 48 h without causing a cytotoxic effect. S. mutans was, however, able to get out of the DPSCs cytoplasm and to proliferate in the extracellular environment. Yet, we noticed several adaptive responses of bacteria to the extracellular environment such as a modification of the kinetic growth, the increase in biofilm formation on type I collagen and polyester fabrics, as well as a tolerance toward amoxicillin. In response to infection, DPSCs adopted a proinflammatory profile by increasing the secretion of IL-8, lL-1ß, and TNF-α, strengthening the establishment of the dental pulp inflammation. Overall, these findings showed a direct impact of S. mutans on DPSCs, providing new insights into the potential role of S. mutans in infective diseases.

Cutibacterium acnes Biofilm Study during Bone Cells Interaction.

Cutibacterium acnes is an opportunistic pathogen involved in Bone and Prosthesis Infections (BPIs). In this study, we observed the behavior of commensal and BPI C. acnes strains in the bone environment through bacterial internalization by osteoblast-like cells and biofilm formation. For the commensal strains, less than 1% of the bacteria were internalized; among them, about 32.7 ± 3.9% persisted intracellularly for up to 48 h. C. acnes infection seems to have no cytotoxic effect on bone cells as detected by LDH assay. Interestingly, commensal C. acnes showed a significant increase in biofilm formation after osteoblast-like internalization for 50% of the strains (2.8-fold increase). This phenomenon is exacerbated on a titanium support, a material used for medical devices. For the BPI clinical strains, we did not notice any increase in biofilm formation after internalization despite a similar internalization rate by the osteoblast-like cells. Furthermore, fluorescent staining revealed more live bacteria within the biofilm after osteoblast-like cell interaction, for all strains (BPIs and commensal). The genomic study did not reveal any link between their clinical origin and phylotype. In conclusion, we have shown for the first time the possible influence of internalization by osteoblast-like cells on commensal C. acnes.

Biopolymers-calcium phosphate antibacterial coating reduces the pathogenicity of internalized bacteria by mesenchymal stromal cells.

A multifunctional material system that kills bacteria and drives bone healing is urgently sought to improve bone prosthesis. Herein, the osteoinductive coating made of calcium phosphate/chitosan/hyaluronic acid, named Hybrid, was proposed as an antibacterial substrate for stromal cell adhesion. This Hybrid coating possesses a contact-killing effect reducing by 90% the viability of Gram-positive Staphylococcus aureus (S. aureus) and Gram-negative Pseudomonas aeruginosa (P. aeruginosa) strains after 48 h of contact. In addition to the production of immunomodulatory mediators, Wharton's jelly (WJ-SCs), dental pulp (DPSCs) and bone marrow (BM-MSCs) derived stromal cells were able to release antibacterial and antibiofilm agents effective against S. aureus and P. aeruginosa strains, respectively. Studying the effect of the Hybrid coating on the internalization of S. aureus by the stromal cells, in acute-mimicking bone infection, highlighted an increase in the bacteria internalization by DPSCs and BM-MSCs when cultured on the Hybrid coating versus uncoated glass. Despite the internalization, Hybrid coating showed a beneficial effect by reducing the pathogenicity of the internalized bacteria. The formation of biofilm was reduced by at least 50% in comparison to internalized bacteria by stromal cells on uncoated glass. This work opens the route for the development of innovative antibacterial coatings by taking into account the internalization of bacteria by stromal cells.

Abundant Extractable Metabolites from Temperate Tree Barks: The Specific Antimicrobial Activity of Prunus Avium Extracts.

Tree barks are mainly considered as wood wastes from forestry activities, but represent valuable resources as they may contain antimicrobial compounds. Here, we aimed to evaluate the possible antimicrobial activities of bark extracts and to characterize the chemical composition of the most active extract. Ten methanol bark extracts were tested in vitro against 17 bacterial strains and 5 yeast strains, through minimum inhibitory concentration (MIC) and minimum bactericidal (or fungicidal) concentration (MBC/MFC) assays. The extract from Prunus avium (E2-4) displayed the largest bactericidal activity against Gram-positive bacteria, with a lethal effect on 6 out of 8 strains. Antibiofilm assays of E2-4 were performed by crystal violet staining and enumeration of adhered bacteria. Assays demonstrated a biofilm inhibitory effect of E2-4 against Staphylococcus aureus CIP 53.154 at concentrations equal to or higher than 250 µg/mL. Chemical profiling of E2-4 by 13C nuclear magnetic resonance (NMR) revealed the presence of dihydrowogonin as a major constituent of the extract. E2-4 was fractionated by centrifugal partition chromatography and the three fractions containing dihydrowogonin were tested for their antibacterial and antibiofilm activities, revealing similar activities to those of E2-4. Dihydrowogonin was positively assessed as an interesting antimicrobial compound, which could be valued from wastes of Prunus avium barks.

Bone Environment Influences Irreversible Adhesion of a Methicillin-Susceptible Staphylococcus aureus Strain.

Prosthesis and joint infections are an important threat in public health, especially due to the development of bacterial biofilms and their high resistance to antimicrobials. Biofilm-associated infections increase mortality and morbidity rates as well as hospitalization costs. Prevention is the best strategy for this serious issue, so there is an urgent need to understand the signals that could induce irreversible bacterial adhesion on a prosthesis. In this context, we investigated the influence of the bone environment on surface adhesion by a methicillin-susceptible Staphylococcus aureus strain. Using static and dynamic biofilm models, we tested various bone environment factors and showed that the presence of Mg2+, lack of oxygen, and starvation each increased bacterial adhesion. It was observed that human osteoblast-like cell culture supernatants, which contain secreted components that would be found in the bone environment, increased bacterial adhesion capacity by 2-fold (p = 0.015) compared to the medium control. Moreover, supernatants from osteoblast-like cells stimulated with TNF-α to mimic inflammatory conditions increased bacterial adhesion by almost 5-fold (p = 0.003) without impacting on the overall biomass. Interestingly, the effect of osteoblast-like cell supernatants on bacterial adhesion could be counteracted by the activity of synthetic antibiofilm peptides. Overall, the results of this study demonstrate that factors within the bone environment and products of osteoblast-like cells directly influence S. aureus adhesion and could contribute to biofilm initiation on bone and/or prosthetics implants.

Spread of Klebsiella pneumoniae ST395 non-susceptible to carbapenems and resistant to fluoroquinolones in North-Eastern France.

OBJECTIVES: Fluoroquinolones (FQs) are a potential treatment for infections caused by extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae that are susceptible to these agents. METHODS: Owing to increasing non-susceptibility to carbapenems among Enterobacteriaceae, in this study FQ resistance mechanisms were characterised in 36 ertapenem-non-susceptible Klebsiella pneumoniae isolated from North-Eastern France in 2012. The population structure was described by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). RESULTS: Among the 36 isolates, 13 (36%) carried a carbapenemase encoding-gene. Decreased expression of the OmpK35-encoding gene might be considered a major resistance determinant that could explain the non-susceptibility to carbapenems. The carbapenemase-producing isolates carried the well-known IncL pOXA-48a plasmid. All 36 K. pneumoniae isolates also harboured a FQ resistance determinant. The aac(6')-Ib-cr gene was the major plasmid-mediated quinolone resistance (PMQR) determinant found in K. pneumoniae (89%; 32/36). MLST identified five sequence types (STs), with the most common being ST395 (69%; 25/36), followed by ST147 (17%; 6/36). ST395 strains showed ertapenem minimum inhibitory concentrations (MICs) ranging from 0.75-32µg/mL. Klebsiella pneumoniae ST395 isolates did not show enhanced biofilm formation or environmental survival but showed higher chlorhexidine MICs compared with ST147 isolates. CONCLUSIONS: These findings showed that (i) K. pneumoniae ST395 carrying pOXA-48a has spread in North-Eastern France, (ii) aac(6')-Ib-cr is predominant in carbapenem-non-susceptible K. pneumoniae, (iii) K. pneumoniae ST395 is resistant to chlorhexidine and (iv) FQs as an alternative to ß-lactams to treat ertapenem-non-susceptible K. pneumoniae are compromised.

Novel roles for two-component regulatory systems in cytotoxicity and virulence-related properties in Pseudomonas aeruginosa.

The rapid adaptation of the opportunistic bacterial pathogen Pseudomonas aeruginosa to various growth modes and environmental conditions is controlled in part through diverse two-component regulatory systems. Some of these systems are well studied, but the majority are poorly characterized, even though it is likely that several of these systems contribute to virulence. Here, we screened all available strain PA14 mutants in 50 sensor kinases, 50 response regulators and 5 hybrid sensor/regulators, for contributions to cytotoxicity against cultured human bronchial epithelial cells, as assessed by the release of cytosolic lactate dehydrogenase. This enabled the identification of 8 response regulators and 3 sensor kinases that caused substantial decreases in cytotoxicity, and 5 response regulators and 8 sensor kinases that significantly increased cytotoxicity by 15-58% or more. These regulators were additionally involved in motility, adherence, type 3 secretion, production of cytotoxins, and the development of biofilms. Here we investigated in more detail the roles of FleSR, PilSR and WspR. Not all cognate pairs contributed to cytotoxicity (e.g. PhoPQ, PilSR) in the same way and some differences could be detected between the same mutants in PAO1 and PA14 strain backgrounds (e.g. FleSR, PhoPQ). This study highlights the potential importance of these regulators and their downstream targets on pathogenesis and demonstrates that cytotoxicity can be regulated by several systems and that their contributions are partly dependent on strain background.

Two Isoforms of Clp Peptidase in Pseudomonas aeruginosa Control Distinct Aspects of Cellular Physiology.

Caseinolytic peptidases (ClpPs) regulate diverse aspects of cellular physiology in bacteria. Some species have multiple ClpPs, including the opportunistic pathogen Pseudomonas aeruginosa, in which there is an archetypical isoform, ClpP1, and a second isoform, ClpP2, about which little is known. Here, we use phenotypic assays to investigate the biological roles of ClpP1 and ClpP2 and biochemical assays to characterize purified ClpP1, ClpP2, ClpX, and ClpA. Interestingly, ClpP1 and ClpP2 have distinct intracellular roles for motility, pigment production, iron scavenging, and biofilm formation. Of particular interest, ClpP2, but not ClpP1, is required for microcolony organization, where multicellular organized structures first form on the pathway to biofilm production. We found that purified ClpP1 with ClpX or ClpA was enzymatically active, yet to our surprise, ClpP2 was inactive and not fully assembled in vitro; attempts to assist ClpP2 assembly and activation by mixing with the other Clp components failed to turn on ClpP2, as did solution conditions that have helped activate other ClpPs in vitro We postulate that the active form of ClpP2 has yet to be discovered, and we present several potential models to explain its activation as well as the unique role ClpP2 plays in the development of the clinically important biofilms in P. aeruginosaIMPORTANCEPseudomonas aeruginosa is responsible for severe infections of immunocompromised patients. Our work demonstrates that two different isoforms of the Clp peptidase, ClpP1 and ClpP2, control distinct aspects of cellular physiology for this organism. In particular, we identify ClpP2 as being necessary for microcolony organization. Pure active forms of ClpP1 and either ClpX or ClpA were characterized as assembled and active, and ClpP2 was incompletely assembled and inactive. By establishing both the unique biological roles of ClpP1 and ClpP2 and their initial biochemical assemblies, we have set the stage for important future work on the structure, function, and biological targets of Clp proteolytic enzymes in this important opportunistic pathogen.

Design of an anti-adhesive surface by a pilicide strategy.

Biofilm formation on surfaces is one of major problems in medical, cosmetic and food industries. Nowadays any efficient treatment is known, as consequence, research of new strategies to inhibit biofilm formation is urgent. Recently, virstatin, which interferes with bacterial type IV pili formation, has demonstrated a capacity to inhibit biofilm formation developed by Acinetobacter baumannii after 24h. In this study, we aim to elaborate anti-adhesive surfaces preventing biofilm development by the covalent immobilization of virstatin on silicon surface. Surfaces were functionalized by self-assembled monolayers of two aminosilanes (11-aminoundecyltrimethoxysilane (AUTMS) and 3-aminopropyltrimethoxysilane (APTMS)). Then, virstatin (2mM) was immobilized on those modified surfaces. We observed an increase in surface hydrophobicity of AUTMS modified substratum leading to an increase of A. baumannii ATCC 17978 adhesion (after 4h). Immobilization of virstatin molecule on APTMS modified surface was efficient to decrease cell attachment by 32.1±5.7% compared to unmodified surface. As virstatin is known to inhibit type IV pili formation in solution, the observed decrease of bacterial adhesion might be due to this pilicide action. We also demonstrated that hydrophobicity of strains plays a role in adhesion according to surface properties. In conclusion, immobilized virstatin succeeded to inhibit bacterial attachment of various Acinetobacter baumannii strains comparing to APTMS modified support.

The Structure of a Type 3 Secretion System (T3SS) Ruler Protein Suggests a Molecular Mechanism for Needle Length Sensing.

The type 3 secretion system (T3SS) and the bacterial flagellum are related pathogenicity-associated appendages found at the surface of many disease-causing bacteria. These appendages consist of long tubular structures that protrude away from the bacterial surface to interact with the host cell and/or promote motility. A proposed "ruler" protein tightly regulates the length of both the T3SS and the flagellum, but the molecular basis for this length control has remained poorly characterized and controversial. Using the Pseudomonas aeruginosa T3SS as a model system, we report the first structure of a T3SS ruler protein, revealing a "ball-and-chain" architecture, with a globular C-terminal domain (the ball) preceded by a long intrinsically disordered N-terminal polypeptide chain. The dimensions and stability of the globular domain do not support its potential passage through the inner lumen of the T3SS needle. We further demonstrate that a conserved motif at the N terminus of the ruler protein interacts with the T3SS autoprotease in the cytosolic side. Collectively, these data suggest a potential mechanism for needle length sensing by ruler proteins, whereby upon T3SS needle assembly, the ruler protein's N-terminal end is anchored on the cytosolic side, with the globular domain located on the extracellular end of the growing needle. Sequence analysis of T3SS and flagellar ruler proteins shows that this mechanism is probably conserved across systems.