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Because of their increasing resistance to antibiotics, bacteria have become one of the top ten threats to global health according to the World Health Organisation [

The phage replication cycle begins with its adsorption to its host and ends with the release of new virions upon bacterial lysis. These steps are important, not only for the proper conduct of the phage life cycle, but also because proteins mediating attachment and lysis have a significant biotechnological potential. The adsorption step involves viral proteins called receptor binding proteins (RBP) and specific receptors at the host surface, while membrane permeabilization and cell wall degradation leading to lysis are achieved by the concerted actions of holins and endolysins. This thesis aimed to better understand the adsorption and lysis processes of phages targeting the Bacillus cereus group. The first part of this work showed that phage Deep-Blue is a promising candidate for controlling emetic strains, although its narrow host range requires its integration in a phage cocktail. In the second part, the viral proteins involved in the adsorption process were investigated. Comparison of the tail proteins of B. cereus siphoviruses revealed diverse genetic organizations and the prevalence of carbohydrate binding modules (CBM). The role in adsorption of these CBM was experimentally demonstrated for phage Deep-Purple. Regarding myoviruses, Deep-Blue and Vp4 RBP were identified, and their tail regions compared to that of the well-known Listeria phage A511 revealing important differences in key proteins. The third part of this thesis showed that different lysis mechanisms are present in phages infecting B. cereus and that endolysins have a broader activity spectrum than their related phages revealing that they could be used in biocontrol and detection., (AGRO - Sciences agronomiques et ingénierie biologique) -- UCL, 2022

Holins are small transmembrane proteins involved in the final stage of the lytic cycle of double-stranded DNA (dsDNA) phages. They cooperate with endolysins to achieve bacterial lysis, thereby releasing the phage progeny into the extracellular environment. Besides their role as membrane permeabilizers, allowing endolysin transfer and/or activation, holins also regulate the lysis timing. In this work, we provide functional characterization of the holins encoded by three phages targeting the Bacillus cereus group. The siphovirus Deep-Purple has a lysis cassette in which holP30 and holP33 encode two proteins displaying holin properties, including a transmembrane domain. The holin genes were expressed in Escherichia coli and induced bacterial lysis, with HolP30 being more toxic than HolP33. In Bacillus thuringiensis, the simultaneous expression of both holins was necessary to observe lysis, suggesting that they may interact to form functional pores. The myoviruses Deep-Blue and Vp4 both encode a single candidate holin (HolB and HolV, respectively) with two transmembrane domains, whose genes are not located near the endolysin genes. Their function as holin proteins was confirmed as their expression in E. coli impaired cell growth and viability. The HolV expression in B. thuringiensis also led to bacterial lysis, which was enhanced by coexpressing the holin with its cognate endolysin. Despite similar organizations and predicted topologies, truncated mutants of the HolB and HolV proteins showed different toxicity levels, suggesting that differences in amino acid composition influence their lysis properties.

pXO16, a large plasmid from Bacillus thuringiensis serovar israelensis, exhibits unique features. Not only is pXO16 able to transfer at high frequencies within few minutes, but it is also able to transfer among virtually all members of the Bacillus cereus group. Among the proteins encoded in the tip transfer locus of pXO16, TipB displays an atypical organization compared to known conjugative cell wall hydrolases with the large central soluble lytic transglycosylase (SLT) domain missing from the protein. We constructed a tipB deletion mutant which led to significant reduction in transfer efficiencies in both liquid and filter mating. The initial transfer frequencies could be restored when complementing tipB in trans thus showing the TipB implication in pXO16 conjugative transfer. Additionally, truncated versions of TipB were expressed in Escherichia coli to assess the protein lytic activity. When applied exogenously, TipB-2TM, in which the two N-terminal TM domains were removed, yielded a decrease of ca. 40% in optical density of B. thuringiensis sv. israelensis, a lytic activity that could partially be explained by the C-terminal CHAP-like domain. These results confirm TipB conjugative hydrolase function and provide new insights into pXO16 unique conjugative apparatus.

Porous silicon (PSi) has been widely used as a biosensor in recent years due to its large surface area and its optical properties. Most PSi biosensors consist in close-ended porous layers, and, because of the diffusion-limited infiltration of the analyte, they lack sensitivity and speed of response. In order to overcome these shortcomings, PSi membranes (PSiMs) have been fabricated using electrochemical etching and standard microfabrication techniques. In this work, PSiMs have been used for the optical detection of Bacillus cereus lysate. Before detection, the bacteria are selectively lysed by PlyB221, an endolysin encoded by the bacteriophage Deep-Blue targeting B. cereus. The detection relies on the infiltration of bacterial lysate inside the membrane, which induces a shift of the effective optical thickness. The biosensor was able to detect a B. cereus bacterial lysate, with an initial bacteria concentration of 105 colony forming units per mL (CFU/mL), in only 1 h. This proof-of-concept also illustrates the specificity of the lysis before detection. Not only does this detection platform enable the fast detection of bacteria, but the same technique can be extended to other bacteria using selective lysis, as demonstrated by the detection of Staphylococcus epidermidis, selectively lysed by lysostaphin.

The rapid detection of hazardous bacteria is important for healthcare situations where such identification can lead to substantial gains for patient treatment and recovery, and a reduced usage of broad-spectrum antibiotics. Potential biosensors must be able to provide a fast, sensitive and selective response with as little sample preparation as possible. Indeed, some of these pathogens such as Staphylococcus aureus can be yet harmful at very low concentrations in the blood stream, e.g. below 10 colony forming units per mL (CFU/mL). These stringent requirements limit the number of candidates, especially for point-of-care applications. Amongst several biosensing techniques, optical sensing using porous silicon (PSi) substrate has been widely suggested in recent years thanks to unique features such as a large surface area, tunable optical characteristics, and above all relatively easy and affordable fabrication techniques. In most configurations, PSi optical biosensors are close-ended porous layers that limits their sensitivity and responsiveness due to diffusion-limited infiltration of the analytes in the porous layer. Also, PSi is a reactive material, its oxidation in buffer solutions results in time-varying shifts. Despite its attractive properties, several challenges must still be overcome in order to reach practical applications. Our work addresses three main improvement points. The first one is the stability over time in saline solutions helped by atomic layer deposition of metal oxides inside the pores. Besides a better stability, our solution is helping with an increase of the optical signal to noise ratio thus reducing the limit of detection. The second one is to perform the lysis of the bacteria prior to its exposure to the sensor, such that the selective detection is based upon the percolation of bacterial residues inside the pores rather than the bacteria themselves. The third one is to remove the bulk silicon below a PSi layer to create a membrane, that al

The phage life cycle is initiated by the interaction between viral proteins and specific receptor at the bacterial surface. This adsorption step is a key determinant of the phage host spectrum and also the main target of resistance mechanisms developed by bacteria to overcome phage infection. Thus, in the light of the increase interest for phage in medical and industrial applications, it is paramount to characterize the molecular mechanisms underlying phage-host primary interactions. In this work, we were interested in identifying and characterizing the phage structural proteins involved in the adsorption of Deep-Purple, a Siphoviridae (i.e. phage possessing a long non contractile tail) infecting members of the B. cereus group [1]. Commonly, the central actors in the adsorption step are structures called Receptor Binding Proteins (RBP) which are responsible for the specific recognition of the bacterial host and subsequent triggering of genome delivery. However, recently, another type of adsorption associated protein, called evolved Distal tail (evoDit) proteins, were identified in Siphoviridae infecting Lactic Acid Bacteria (LAB). These proteins were previously thought to have only a structural role, but it appears that the presence of a carbohydrate-binding module (CBM) allows them to interact with the host surface and thus assist the RBP. Bioinformatic analysis of Deep-Purple tail protein-encoding region allowed to identify gp28 and gp29 as the putative evoDit protein and RBP, respectively. Both proteins contain a CBM, suggesting that these proteins can interact with host associated polysaccharides. When fused to a fluorescent tag, both proteins were able to decorate the bacterial surface but differences in binding spectra were observed. The RBP-gp29 only recognized strains that are sensitive to the phage whereas evoDit-gp28 bound to many strains including some that are insensitive to Deep-Purple, highlighting the fact that the evoDit is not the sole determinant o

Paper substrates have shown a high potential for development of cost-effective and efficient point-of-care biosensors, essential for public healthcare and environmental diagnostics. Most paper-based biosensors rely on qualitative colorimetric detection schemes with high limits of detection. To overcome this limitation, technologies that combine paper-based substrates and electrochemical detection are being developed to allow for quantification and achieve better performances. In this work, we explore the potential of dielectric measurements towards electrical detection of whole-cell bacteria in nitrocellulose membranes, a paper-derivative. Impedance spectroscopy was considered to characterize the membranes with and without Bacillus thuringiensis cells, used as model microorganism. To specifically target this bacterial strain, phage endolysin cell- wall binding domain (CBD) encoded by a bacteriophage targeting B. thuringiensis were prepared and integrated into the membranes as recognition biointerface. The fluid sample containing the bacteria is conducted in the membrane through passive capillarity, and the bacteria are specifically immobilized in the test zone. Resulting changes of the dielectric properties of the membrane are sensed through impedance changes, highlighting the contribution of ions in the bacterial detection mechanism. This experimental proof-of-concept illustrates the electrical detection of 108 CFU/mL bacteria in low-salinity buffers within 5 min.

Antibiotic microbial resistance is, according to the World Health Organization, one of the top 10 threats to global health. One way to prevent and control antibiotic resistance is the development of novel diagnostic tools. In this work, we present a biosensor for the rapid optical detection of bacteria after their selective lysis, based on open-ended Porous Silicon (PSi) membranes. PSi is widely used for biosensing applications, thanks to its large surface area and unique optical properties [1]. In this study, Bacillus cereus was selectively lysed by PlyB221, a recently characterized endolysin encoded by the Deep-Blue bacteriophage targeting B. cereus [2]. The bacterial lysate was then pushed through a PSi membrane, which generated changes in its optical properties. A method called Reflective Interferometric Fourier Transform Spectroscopy (RIFTS) was used to quantify these changes, by measuring the effective optical thickness (EOT) of the PSi membrane overtime. This EOT equals 2nL, with n the refractive index of the porous matrix, and L is thickness. With this detection protocol, we were able to selectively detect 105 colony forming units per mL (CFU/mL) of B. cereus lysate in 1h.

Endolysins are phage-encoded enzymes implicated in the breaching of the bacterial cell wall at the end of the viral cycle. This study focuses on the endolysins of Deep-Blue (PlyB221) and Deep-Purple (PlyP32), two phages preying on the Bacillus cereus group. Both enzymes exhibit a typical modular organization with an enzymatically active domain (EAD) located in the N-terminal and a cell wall binding domain (CBD) in the C-terminal part of the protein. In silico analysis indicated that the EAD domains of PlyB221 and PlyP32 are endowed with peptidase and muramidase activities, respectively, whereas in both proteins SH3 domains are involved in the CBD. To evaluate their antimicrobial properties and binding specificity, both endolysins were expressed and purified. PlyB221 and PlyP32 efficiently recognized and lysed all the tested strains from the B. cereus group. Biochemical characterization showed that PlyB221 activity was stable under a wide range of pHs (5–9), NaCl concentrations (up to 200 mM), and temperature treatments (up to 50 °C). Although PlyP32 activity was less stable than that of PlyB221, the endolysin displayed high activity at pH 6–7, NaCl concentration up to 100 mM and the temperature treatment up to 45 °C. Overall, PlyB221 and PlyP32 display suitable characteristics for the development of biocontrol and detection tools.

Some strains of the Bacillus cereus group can be implicated in two types of foodborne intoxication syndromes, namely diarrheal and emetic. The emetic syndrome results from the ingestion of the cereulide toxin produced by particular strains of B. cereus and Bacillus weihenstephanensis. It represents a major concern since it can lead, in severe cases, to the death of the intoxicated patients. This study aimed to characterize the virulent phage Deep-Blue and investigate its biocontrol potential against psychrotolerant B. weihenstephanensis. This myovirus was isolated from an agricultural soil and is stable for at least 6 months at 4 °C. It is specific to members of the B. cereus group and shows a lytic activity against five of twelve B. weihenstephanensis. Its lytic cycle lasts less than 1 h with a final phage progeny of ca. 300 virions. Regarding its bactericidal potential, Deep-Blue was able to reduce B. weihenstephanensis contamination under laboratory conditions (LB broth) and in milk, and to control bacterial growth in food matrices such as milk rice. Finally, the application of Deep-Blue as preventive treatment was shown to efficiently control the development of B. weihenstephanensis biofilm.

pXO16, the large conjugative plasmid from Bacillus thuringiensis serovar israelensis is able to efficient self-transfer, to mobilize and retro-mobilize non-conjugative plasmids, including "non-mobilizable" plasmids, and to transfer chromosomal loci. It also displays a remarkable aggregation phenotype associated with conjugation under liquid conditions. However, it was recently shown that aggregation boosts pXO16 transfer but is not mandatory. In this paper, we have further explored pXO16 transfers under various mating conditions and with different members of the Bacillus cereus group. The results indicated that colony or filter mating largely compensate the transfer deficit observed when using a pXO16 aggregation-minus mutant. Using filter mating, pXO16 transfer efficiency and host range were both improved. For instance, pXO16 was shown to transfer itself, and to mobilize the small pUB110 plasmid, from B. thuringiensis serovar israelensis to the thermotolerant Bacillus cytotoxicus at frequencies of 3.3 × 10-3 and 5.2 × 10-4 transconjugants per donor (T/D), respectively. All together, these results indicate that pXO16 can potentially "circulate" among members of the Bacillus cereus group. Yet, this is contrasting with pXO16's known natural distribution, which is apparently limited to the israelensis serovar of B. thuringiensis.

Binding of Staphylococcus aureus to the large plasma glycoprotein von Willebrand factor (vWF) is controlled by hydrodynamic flow conditions. Currently, we know little about the molecular details of this shear-stress-dependent interaction. Using single-molecule atomic force microscopy, we demonstrate that vWF binds to the S. aureus surface protein A (SpA) via a previously undescribed force-sensitive mechanism. We identify an extremely strong SpA-vWF interaction, capable of withstanding forces of ∼2 nN, both in laboratory and in clinically relevant methicillin-resistant S. aureus (MRSA) strains. Strong bonds are activated by mechanical stress, consistent with flow experiments revealing that bacteria adhere in larger amounts to vWF surfaces when the shear rate is increased. We suggest that force-enhanced adhesion may involve conformational changes in vWF. Under force, elongation of vWF may lead to the exposure of a high-affinity cryptic SpA-binding site to which bacteria firmly attach. In addition, force-induced structural changes in the SpA domains may also promote strong, high-affinity binding. This force-regulated interaction might be of medical importance as it may play a role in bacterial adherence to platelets and to damaged blood vessels. IMPORTANCE Staphylococcus aureus protein A (SpA) binds to von Willebrand factor (vWF) under flow. While vWF binding to SpA plays a role in S. aureus adherence to platelets and endothelial cells under shear stress, the molecular basis of this stress-dependent interaction has not yet been elucidated. Here we show that the SpA-vWF interaction is regulated by a new force-dependent mechanism. The results suggest that mechanical extension of vWF may lead to the exposure of a high-affinity cryptic SpA-binding site, consistent with the shear force-controlled functions of vWF. Moreover, strong binding may be promoted by force-induced structural changes in the SpA domains. This study highlights the role of mechanoregulation in controllin

Introduction There is a renewed interest in phages and their derived proteins for various applications, including in the agro-food sector. Endolysins are peptidoglycan degrading enzymes synthetized at the end of the lytic cycle. In combination with holins, they are responsible for the lysis of the host cell allowing the release of newly synthetized virions. Endolysins from phages infecting Gram-positive bacteria are organized into two domains: a C-terminal Cell Wall Binding domain (CBD) and an N-terminal Enzymatically Active Domain (EAD) that cleaves the peptidoglycan. This study focuses on endolysins encoded by Deep-Blue [1] and Deep-Purple [2], two phages infecting Bacillus weihenstephanensis and belonging to the Myoviridae and Siphoviridae families, respectively. Materials and Methods Endolysin candidates were selected by bioinformatics and whole genes were cloned into the expression vector pET30a. Similarly, the corresponding CBD were cloned into pET30a with a N-terminal GFP fusion. The constructions were transformed into the strain BL21(DE3) and expression of each recombinant protein was induced by addition of IPTG. The proteins were purified on a Ni-NTA column. The lytic activity of the putative endolysins was assess by CFU reduction assay and reduction in Optical Density. As for the CBD, the interaction between the GFP fused domain and the bacteria was detected by fluorescent microscopy. Results and Discussion In silico analyses revealed that Deep-Blue gp221 and Deep-purple gp032 are good endolysin candidates. Gp221 has a Peptidase_M15_4 conserved domain from the VanY superfamily as EAD, which is also present in the endolysins of phages B4 [3] (LysB4) and Phrodo [4] (PlyP56) infecting members of the Bacillus cereus group. Gp032 possesses a GH25_PlyB-like domain belonging to the GH25_muramidase superfamily. PlyB is an endolysin from phage BcpI that displays activity against Bacillus anthracis [5]. As for the CBD, two SH3 domains were detected at the C terminus