Cross-regulation of Aps-promoters in Lacticaseibacillus paracasei by the PsdR response regulator in response to lantibiotics.

The PsdRSAB and ApsRSAB detoxification modules, together with the antimicrobial peptides (AMPs)-resistance determinants Dlt system and MprF protein, play major roles in the response to AMPs in Lacticaseibacillus paracasei BL23. Sensitivity assays with a collection of mutants showed that the PsdAB ABC transporter and the Dlt system are the main subtilin resistance determinants. Quantification of the transcriptional response to subtilin indicate that this response is exclusively regulated by the two paralogous systems PsdRSAB and ApsRSAB. Remarkably, a cross-regulation of the derAB, mprF and dlt-operon genes-usually under control of ApsR-by PsdR in response to subtilin was unveiled. The high similarity of the predicted structures of both response regulators (RR), and of the RR-binding sites support this possibility, which we experimentally verified by protein-DNA binding studies. ApsR-P shows a preferential binding in the order PderA > Pdlt > PmprF > PpsdA. However, PsdR-P bound with similar apparent affinity constants to the four promoters. This supports the cross-regulation of derAB, mprF and the dlt-operon by PsdR. The possibility of cross-regulation at the level of RR-promoter interaction allows some regulatory overlap with two RRs controlling the expression of systems involved in maintenance of critical cell membrane functions in response to lantibiotics.

The Novel Synthetic Antibiotic BDTL049 Based on a Dendritic System Induces Lipid Domain Formation while Escaping the Cell Envelope Stress Resistance Determinants.

The threat of antimicrobial-resistant bacteria is ever increasing and over the past-decades development of novel therapeutic counter measurements have virtually come to a halt. This circumstance calls for interdisciplinary approaches to design, evaluate and validate the mode of action of novel antibacterial compounds. Hereby, carbosilane dendritic systems that exhibit antimicrobial properties have the potential to serve as synthetic and rationally designed molecules for therapeutic use. The bow-tie type topology of BDTL049 was recently investigated against the Gram-positive model organism Bacillus subtilis, revealing strong bactericidal properties. In this study, we follow up on open questions concerning the usability of BDTL049. For this, we synthesized a fluorescent-labeled version of BDTL049 that maintained all antimicrobial features to unravel the interaction of the compound and bacterial membrane. Subsequently, we highlight the bacterial sensitivity against BDTL049 by performing a mutational study of known resistance determinants. Finally, we address the cytotoxicity of the compound in human cells, unexpectedly revealing a high sensitivity of the eukaryotic cells upon BDTL049 exposure. The insights presented here further elaborate on the unique features of BDTL049 as a promising candidate as an antimicrobial agent while not precluding that further rounds of rational designing are needed to decrease cytotoxicity to ultimately pave the way for synthetic antibiotics toward clinical applicability.

Synthesis and mechanism-of-action of a novel synthetic antibiotic based on a dendritic system with bow-tie topology.

Over the course of the last decades, the continuous exposure of bacteria to antibiotics-at least in parts due to misprescription, misuse, and misdosing-has led to the widespread development of antimicrobial resistances. This development poses a threat to the available medication in losing their effectiveness in treating bacterial infections. On the drug development side, only minor advances have been made to bring forward novel therapeutics. In addition to increasing the efforts and approaches of tapping the natural sources of new antibiotics, synthetic approaches to developing novel antimicrobials are being pursued. In this study, BDTL049 was rationally designed using knowledge based on the properties of natural antibiotics. BDTL049 is a carbosilane dendritic system with bow-tie type topology, which has antimicrobial activity at concentrations comparable to clinically established natural antibiotics. In this report, we describe its mechanism of action on the Gram-positive model organism Bacillus subtilis. Exposure to BDTL049 resulted in a complex transcriptional response, which pointed toward disturbance of the cell envelope homeostasis accompanied by disruption of other central cellular processes of bacterial metabolism as the primary targets of BDTL049 treatment. By applying a combination of whole-cell biosensors, molecular staining, and voltage sensitive dyes, we demonstrate that the mode of action of BDTL049 comprises membrane depolarization concomitant with pore formation. As a result, this new molecule kills Gram-positive bacteria within minutes. Since BDTL049 attacks bacterial cells at different targets simultaneously, this might decrease the chances for the development of bacterial resistances, thereby making it a promising candidate for a future antimicrobial agent.

Streptococcus dentisani is a common inhabitant of the oral microbiota worldwide and is found at higher levels in caries-free individuals.

Streptococcus dentisani has been proposed as a promising probiotic against tooth decay, due to its ability to buffer acidic pH and to inhibit the growth of oral pathogens. However, it is unknown if this bacterial species has a global distribution. The current study aimed to establish the presence of S. dentisani in oral samples from different geographic locations by identifying the sequence of its 16S rRNA gene in available datasets from across the globe. In addition, an analytical and cross-sectional study was carried out to determine if the levels of this probiotic strain are higher in caries-free individuals compared to those with dental caries. Samples from various geographical sources demonstrated that S. dentisani is present in saliva and dental plaque from individuals of different continents. Typical S. dentisani levels in saliva ranged from 104 to 105 cells/ml and a total of 106-107 cells in dental plaque. Using real-time qPCR, S. dentisani was quantified from supragingival dental plaque of 25 caries-free and 29 caries-active individuals from a Mexican children population, where significantly higher proportions of S. dentisani were found in the caries-free group (p = 0.002). Finally, a negative correlation was found between caries levels (as measured by the dmft caries index) and the percentage of S. dentisani (p < 0.001). Thus, the current manuscript indicates that this species has a global distribution, can be found in saliva and dental plaque, and appears to be present in higher numbers in plaque samples from caries-free children.

Amphotericin B Specifically Induces the Two-Component System LnrJK: Development of a Novel Whole-Cell Biosensor for the Detection of Amphotericin-Like Polyenes.

The rise of drug-resistant fungal pathogens urges for the development of new tools for the discovery of novel antifungal compounds. Polyene antibiotics are potent agents against fungal infections in humans and animals. They inhibit the growth of fungal cells by binding to sterols in the cytoplasmic membrane that subsequently causes pore formation and eventually results in cell death. Many polyenes are produced by Streptomycetes and released into the soil environment, where they can then target fungal hyphae. While not antibacterial, these compounds could nevertheless be also perceived by bacteria sharing the same habitat and serve as signaling molecules. We therefore addressed the question of how polyenes such as amphotericin B are perceived by the soil bacterium, Bacillus subtilis. Global transcriptional profiling identified a very narrow and specific response, primarily resulting in strong upregulation of the lnrLMN operon, encoding an ABC transporter previously associated with linearmycin resistance. Its strong and specific induction prompted a detailed analysis of the lnrL promoter element and its regulation. We demonstrate that the amphotericin response strictly depends on the two-component system LnrJK and that the target of LnrK-dependent gene regulation, the lnrLMN operon, negatively affects LnrJK-dependent signal transduction. Based on this knowledge, we developed a novel whole-cell biosensor, based on a P lnrL -lux fusion reporter construct in a lnrLMN deletion mutant background. This highly sensitive and dynamic biosensor is ready to be applied for the discovery or characterization of novel amphotericin-like polyenes, hopefully helping to increase the repertoire of antimycotic and antiparasitic polyenes available to treat human and animal infections.

ABC Transporter DerAB of Lactobacillus casei Mediates Resistance against Insect-Derived Defensins.

Bce-like systems mediate resistance against antimicrobial peptides in Firmicutes bacteria. Lactobacillus casei BL23 encodes an "orphan" ABC transporter that, based on homology to BceAB-like systems, was proposed to contribute to antimicrobial peptide resistance. A mutant lacking the permease subunit was tested for sensitivity against a collection of peptides derived from bacteria, fungi, insects, and humans. Our results show that the transporter specifically conferred resistance against insect-derived cysteine-stabilized αß defensins, and it was therefore renamed DerAB for defensin resistance ABC transporter. Surprisingly, cells lacking DerAB showed a marked increase in resistance against the lantibiotic nisin. This could be explained by significantly increased expression of the antimicrobial peptide resistance determinants regulated by the Bce-like systems PsdRSAB (formerly module 09) and ApsRSAB (formerly module 12). Bacterial two-hybrid studies in Escherichia coli showed that DerB could interact with proteins of the sensory complex in the Psd resistance system. We therefore propose that interaction of DerAB with this complex in the cell creates signaling interference and reduces the cell's potential to mount an effective nisin resistance response. In the absence of DerB, this negative interference is relieved, leading to the observed hyperactivation of the Psd module and thus increased resistance to nisin. Our results unravel the function of a previously uncharacterized Bce-like orphan resistance transporter with pleiotropic biological effects on the cell.IMPORTANCE Antimicrobial peptides (AMPs) play an important role in suppressing the growth of microorganisms. They can be produced by bacteria themselves-to inhibit competitors-but are also widely distributed in higher eukaryotes, including insects and mammals, where they form an important component of innate immunity. In low-GC-content Gram-positive bacteria, BceAB-like transporters play a crucial role in AMP resistance but have so far been primarily associated with interbacterial competition. Here, we show that the orphan transporter DerAB from the lactic acid bacterium Lactobacillus casei is crucial for high-level resistance against insect-derived AMPs. It therefore represents an important mechanism for interkingdom defense. Furthermore, our results support a signaling interference from DerAB on the PsdRSAB module that might prevent the activation of a full nisin response. The Bce modules from L. casei BL23 illustrate a biological paradox in which the intrinsic nisin detoxification potential only arises in the absence of a defensin-specific ABC transporter.

Genetically Engineered Bacterial Biohybrid Microswimmers for Sensing Applications.

Bacterial biohybrid microswimmers aim at exploiting the inherent motion capabilities of bacteria (carriers) to transport objects (cargoes) at the microscale. One of the most desired properties of microswimmers is their ability to communicate with their immediate environment by processing the information and producing a useful response. Indeed, bacteria are naturally equipped with such communication skills. Hereby, two-component systems (TCSs) represent the key signal transducing machinery and enable bacteria to sense and respond to a variety of stimuli. We engineered a natural microswimmer based on the Gram-positive model bacterium Bacillus subtilis for the development of biohybrids with sensing abilities. B. subtilis naturally adhered to silica particles, giving rise to different motile biohybrids systems with variable ratios of carrier(s)-to-cargo(es). Genetically engineered TCS pathways allowed us to couple the binding to the inert particles with signaling the presence of antibiotics in their surroundings. Activation of the antibiotic-induced TCSs resulted in fluorescent bacterial carriers as a response readout. We demonstrate that the genetically engineered TCS-mediated signaling capabilities of B. subtilis allow for the custom design of bacterial hybrid microswimmers able to sense and signal the presence of target molecules in the environment. The generally recognized as safe (GRAS) status of B. subtilis makes it a promising candidate for human-related applications of these novel biohybrids.

Bacterial Biohybrid Microswimmers.

Over millions of years, Nature has optimized the motion of biological systems at the micro and nanoscales. Motor proteins to motile single cells have managed to overcome Brownian motion and solve several challenges that arise at low Reynolds numbers. In this review, we will briefly describe naturally motile systems and their strategies to move, starting with a general introduction that surveys a broad range of developments, followed by an overview about the physical laws and parameters that govern and limit motion at the microscale. We characterize some of the classes of biological microswimmers that have arisen in the course of evolution, as well as the hybrid structures that have been constructed based on these, ranging from Montemagno's ATPase motor to the SpermBot. Thereafter, we maintain our focus on bacteria and their biohybrids. We introduce the inherent properties of bacteria as a natural microswimmer and explain the different principles bacteria use for their motion. We then elucidate different strategies that have been employed for the coupling of a variety of artificial microobjects to the bacterial surface, and evaluate the different effects the coupled objects have on the motion of the "biohybrid." Concluding, we give a short overview and a realistic evaluation of proposed applications in the field.

Physiological Role of Two-Component Signal Transduction Systems in Food-Associated Lactic Acid Bacteria.

Two-component systems (TCSs) are widespread signal transduction pathways mainly found in bacteria where they play a major role in adaptation to changing environmental conditions. TCSs generally consist of sensor histidine kinases that autophosphorylate in response to a specific stimulus and subsequently transfer the phosphate group to their cognate response regulators thus modulating their activity, usually as transcriptional regulators. In this review we present the current knowledge on the physiological role of TCSs in species of the families Lactobacillaceae and Leuconostocaceae of the group of lactic acid bacteria (LAB). LAB are microorganisms of great relevance for health and food production as the group spans from starter organisms to pathogens. Whereas the role of TCSs in pathogenic LAB (most of them belonging to the family Streptococcaceae) has focused the attention, the roles of TCSs in commensal LAB, such as most species of Lactobacillaceae and Leuconostocaceae, have been somewhat neglected. However, evidence available indicates that TCSs are key players in the regulation of the physiology of these bacteria. The first studies in food-associated LAB showed the involvement of some TCSs in quorum sensing and production of bacteriocins, but subsequent studies have shown that TCSs participate in other physiological processes, such as stress response, regulation of nitrogen metabolism, regulation of malate metabolism, and resistance to antimicrobial peptides, among others.

Peptide and amino acid metabolism is controlled by an OmpR-family response regulator in Lactobacillus casei.

A Lactobacillus casei BL23 strain defective in an OmpR-family response regulator encoded by LCABL_18980 (PrcR, RR11), showed enhanced proteolytic activity caused by overexpression of the gene encoding the proteinase PrtP. Transcriptomic analysis revealed that, in addition to prtP expression, PrcR regulates genes encoding peptide and amino acid transporters, intracellular peptidases and amino acid biosynthetic pathways, among others. Binding of PrcR to twelve promoter regions of both upregulated and downregulated genes, including its own promoter, was demonstrated by electrophoretic mobility shift assays showing that PrcR can act as a transcriptional repressor or activator. Phosphorylation of PrcR increased its DNA binding activity and this effect was abolished after replacement of the phosphorylatable residue Asp-52 by alanine. Comparison of the transcript levels in cells grown in the presence or absence of tryptone in the growth medium revealed that PrcR activity responded to the presence of a complex amino acid source in the growth medium. We conclude that the PrcR plays a major role in the control of the peptide and amino acid metabolism in L. casei BL23. Orthologous prcR genes are present in most members of the Lactobacillaceae and Leuconostocaceae families. We hypothesize that they play a similar role in these bacterial groups.

Defence against antimicrobial peptides: different strategies in Firmicutes.

The Firmicutes constitute a phylum of bacteria that can be found in a wide variety of habitats, from soil to the gastrointestinal tract of animals, where they have to thrive in complex communities. Competition in these communities usually involves the production of compounds such as antimicrobial peptides (AMPs) to eliminate competitor organisms. Animals and plants also produce AMPs to control their associated microbiota. In turn, defence mechanisms have evolved to prevent the action of these compounds. The close association of some Firmicutes with humans as prominent pathogens or commensal organisms has driven a considerable research effort on defence mechanisms used by these bacteria against antimicrobial compounds. This review focuses on the most recent advances on two well-characterized defence mechanisms against AMPs: the modification of the cell wall by D-alanylation and the role of peptide antibiotic-specific adenosine triphosphate-binding cassette transporters.

Characterization of a regulatory network of peptide antibiotic detoxification modules in Lactobacillus casei BL23.

Two-component systems (TCS) are major signal transduction pathways that allow bacteria to detect and respond to environmental and intracellular changes. A group of TCS has been shown to be involved in the response against antimicrobial peptides (AMPs). These TCS are characterized by the possession of intramembrane-sensing histidine kinases, and they are usually associated with ABC transporters of the peptide-7 exporter family (Pep7E). Lactobacillus casei BL23 encodes two TCS belonging to this group (TCS09 and TCS12) that are located next to two ABC transporters (ABC09 and ABC12), as well as a third Pep7E ABC transporter not genetically associated with any TCS (orphan ABC). This study addressed the involvement of modules TCS09/ABC09 and TCS12/ABC12 in AMP resistance. Results showed that both systems contribute to L. casei resistance to AMPs, and that each TCS constitutes a functional unit with its corresponding ABC transporter. Analysis of transcriptional levels showed that module 09 is required for the induction of ABC09 expression in response to nisin. In contrast, module 12 controls a wider regulon that encompasses the orphan ABC, the dlt operon (d-alanylation of teichoid acids), and the mprF gene (l-lysinylation of phospholipids), thereby controlling properties of the cell envelope. Furthermore, the characterization of a dltA mutant showed that Dlt plays a major role in AMP resistance in L. casei. This is the first report on the regulation of the response of L. casei to AMPs, giving insight into its ability to adapt to the challenging environments that it encounters as a probiotic microorganism.

NADP+ binding to the regulatory subunit of methionine adenosyltransferase II increases intersubunit binding affinity in the hetero-trimer.

Mammalian methionine adenosyltransferase II (MAT II) is the only hetero-oligomer in this family of enzymes that synthesize S-adenosylmethionine using methionine and ATP as substrates. Binding of regulatory ß subunits and catalytic α2 dimers is known to increase the affinity for methionine, although scarce additional information about this interaction is available. This work reports the use of recombinant α2 and ß subunits to produce oligomers showing kinetic parameters comparable to MAT II purified from several tissues. According to isothermal titration calorimetry data and densitometric scanning of the stained hetero-oligomer bands on denatured gels, the composition of these oligomers is that of a hetero-trimer with α2 dimers associated to single ß subunits. Additionally, the regulatory subunit is able to bind NADP(+) with a 1:1 stoichiometry, the cofactor enhancing ß to α2-dimer binding affinity. Mutants lacking residues involved in NADP(+) binding and N-terminal truncations of the ß subunit were able to oligomerize with α2-dimers, although the kinetic properties appeared altered. These data together suggest a role for both parts of the sequence in the regulatory role exerted by the ß subunit on catalysis. Moreover, preparation of a structural model for the hetero-oligomer, using the available crystal data, allowed prediction of the regions involved in ß to α2-dimer interaction. Finally, the implications that the presence of different N-terminals in the ß subunit could have on MAT II behavior are discussed in light of the recent identification of several splicing forms of this subunit in hepatoma cells.

Influence of two-component signal transduction systems of Lactobacillus casei BL23 on tolerance to stress conditions.

Lactobacillus casei BL23 carries 17 two-component signal transduction systems. Insertional mutations were introduced into each gene encoding the cognate response regulators, and their effects on growth under different conditions were assayed. Inactivation of systems TC01, TC06, and TC12 (LCABL_02080-LCABL_02090, LCABL_12050-LCABL_12060, and LCABL_19600-LCABL_19610, respectively) led to major growth defects under the conditions assayed.