The CRISPR effector Cam1 mediates membrane depolarization for phage defence.

Prokaryotic type III CRISPR-Cas systems provide immunity against viruses and plasmids using CRISPR-associated Rossman fold (CARF) protein effectors1-5. Recognition of transcripts of these invaders with sequences that are complementary to CRISPR RNA guides leads to the production of cyclic oligoadenylate second messengers, which bind CARF domains and trigger the activity of an effector domain6,7. Whereas most effectors degrade host and invader nucleic acids, some are predicted to contain transmembrane helices without an enzymatic function. Whether and how these CARF-transmembrane helix fusion proteins facilitate the type III CRISPR-Cas immune response remains unknown. Here we investigate the role of cyclic oligoadenylate-activated membrane protein 1 (Cam1) during type III CRISPR immunity. Structural and biochemical analyses reveal that the CARF domains of a Cam1 dimer bind cyclic tetra-adenylate second messengers. In vivo, Cam1 localizes to the membrane, is predicted to form a tetrameric transmembrane pore, and provides defence against viral infection through the induction of membrane depolarization and growth arrest. These results reveal that CRISPR immunity does not always operate through the degradation of nucleic acids, but is instead mediated via a wider range of cellular responses.

The cell cycle of Staphylococcus aureus: An updated review.

As bacteria proliferate, DNA replication, chromosome segregation, cell wall synthesis, and cytokinesis occur concomitantly and need to be tightly regulated and coordinated. Although these cell cycle processes have been studied for decades, several mechanisms remain elusive, specifically in coccus-shaped cells such as Staphylococcus aureus. In recent years, major progress has been made in our understanding of how staphylococci divide, including new, fundamental insights into the mechanisms of cell wall synthesis and division site selection. Furthermore, several novel proteins and mechanisms involved in the regulation of replication initiation or progression of the cell cycle have been identified and partially characterized. In this review, we will summarize our current understanding of the cell cycle processes in the spheroid model bacterium S. aureus, with a focus on recent advances in the understanding of how these processes are regulated.

Antibiotic combinations reduce Staphylococcus aureus clearance.

The spread of antibiotic resistance is attracting increased attention to combination-based treatments. Although drug combinations have been studied extensively for their effects on bacterial growth1-11, much less is known about their effects on bacterial long-term clearance, especially at cidal, clinically relevant concentrations12-14. Here, using en masse microplating and automated image analysis, we systematically quantify Staphylococcus aureus survival during prolonged exposure to pairwise and higher-order cidal drug combinations. By quantifying growth inhibition, early killing and longer-term population clearance by all pairs of 14 antibiotics, we find that clearance interactions are qualitatively different, often showing reciprocal suppression whereby the efficacy of the drug mixture is weaker than any of the individual drugs alone. Furthermore, in contrast to growth inhibition6-10 and early killing, clearance efficacy decreases rather than increases as more drugs are added. However, specific drugs targeting non-growing persisters15-17 circumvent these suppressive effects. Competition experiments show that reciprocal suppressive drug combinations select against resistance to any of the individual drugs, even counteracting methicillin-resistant Staphylococcus aureus both in vitro and in a Galleria mellonella larva model. As a consequence, adding a ß-lactamase inhibitor that is commonly used to potentiate treatment against ß-lactam-resistant strains can reduce rather than increase treatment efficacy. Together, these results underscore the importance of systematic mapping the long-term clearance efficacy of drug combinations for designing more-effective, resistance-proof multidrug regimes.

Characterization and validation of an alternative reference bacterium Korean Pharmacopoeia Staphylococcus aureus strain.

The National Culture Collection of Pathogens (NCCP) is a microbial resource bank in Korea that collects pathogen resources causing infectious disease in human and distributes them for research and education. The NCCP bank attempts to discover strains with various characteristics and specific purposes to provide diverse resources to researchers. Staphylococcus aureus American Type Culture Collection (ATCC) 6538P is used as a reference strain in the microbial assay for antibiotics in the Korean and in the United States Pharmacopoeias. We aimed to analyze domestically isolated microbial resources from the NCCP to replace the S. aureus reference strain. Staphylococcus aureus strains were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and the VITEK-2 system and characterized by multilocus sequence typing, 16S rRNA sequencing, and antibiotic susceptibility testing. Several candidate strains had similar characteristics as the reference strain. Among them, the nucleotide sequence of the 16S rRNA region of NCCP 16830 was 100% identical to that of the reference strain; it was sensitive to six types of antibiotics and showed results most similar to the reference strain. A validity evaluation was conducted using the cylinder-plate method. NCCP 16830 presented valid results and had the same performance as ATCC 6538P; therefore, it was selected as an alternative candidate strain.

Sortase A: A chemoenzymatic approach for the labeling of cell surfaces.

Sortase A, a transpeptidase enzyme is present in many Gram-positive bacteria and helps in the recruitment of the cell surface proteins. Over the last two decades, Sortase A has become an attractive tool for performing in vivo and in vitro ligations. Sortase A-mediated ligation has continuously been used for its specificity, robustness, and highly efficient nature. These properties make it a popular choice among protein engineers as well as researchers from different fields. In this review, we give an overview of Sortase A-mediated ligation of various molecules on the cell surfaces, which can have diverse applications in interdisciplinary fields.

Crystallographic analysis of TarI and TarJ, a cytidylyltransferase and reductase pair for CDP-ribitol synthesis in Staphylococcus aureus wall teichoic acid biogenesis.

The cell wall of many pathogenic Gram-positive bacteria contains ribitol-phosphate wall teichoic acid (WTA), a polymer that is linked to virulence and regulation of essential physiological processes including cell division. CDP-ribitol, the activated precursor for ribitol-phosphate polymerization, is synthesized by a cytidylyltransferase and reductase pair known as TarI and TarJ, respectively. In this study, we present crystal structures of Staphylococcus aureus TarI and TarJ in their apo forms and in complex with substrates and products. The TarI structures illustrate the mechanism of CDP-ribitol synthesis from CTP and ribitol-phosphate and reveal structural changes required for substrate binding and catalysis. Insights into the upstream step of ribulose-phosphate reduction to ribitol-phosphate is provided by the structures of TarJ. Furthermore, we propose a general topology of the enzymes in a heterotetrameric form built using restraints from crosslinking mass spectrometry analysis. Together, our data present molecular details of CDP-ribitol production that may aid in the design of inhibitors against WTA biosynthesis.

Plasma medicine and wound management: Evaluation of the antibacterial efficacy of a medically certified cold atmospheric argon plasma jet.

OBJECTIVES: A major problem for wound healing is contamination with bacteria, often resulting in biofilm formation and wound infection, which, in turn, needs immediate intervention such as surgical debridement and through irrigation. A topical treatment with cold atmospheric pressure plasma (CAP) for wound disinfection may present an alternative and less painful approach. METHODS: This study investigated the antibacterial effects of a cold atmospheric pressure argon plasma jet (kINPen® MED) as a CAP source, using the three-dimensional Staphylococcus aureus immunocompetent biofilm system hpBIOM in addition to a standard planktonic test. Furthermore, skin cell compatibility was evaluated using a keratinocyte (HaCat) model. RESULTS: CAP treatment (0-240 s) followed by incubation (15, 120 min) within the CAP-treated media showed slight bactericidal efficacy under planktonic conditions but no effect on biofilms. However, indirect CAP treatment of keratinocytes performed under the same conditions resulted in a significant decrease in metabolic activity. Short CAP treatment and exposure time (30 s; 15 min) induced a slight increase in the metabolic activity; however, longer treatments and/or exposure times led to pronounced reductions up to 100%. These effects could partially be reversed by addition of catalase, indicating a dominant role of CAP-generated hydrogen peroxide. CONCLUSIONS: These results indicate that plasma treatment does not lead to the desired disinfection or significant reduction in the bacterial burden of Staphylococcus aureus in a wet milieu or in biofilms. Thus, treatment with CAP could not be recommended as a single anti-bacterial therapy for wounds but could be used to support standard treatments.

SERS characterization of aggregated and isolated bacteria deposited on silver-based substrates.

Surface-enhanced Raman scattering (SERS), based on the enhancement of the Raman signal of molecules positioned within a few nanometres from a structured metal surface, is ideally suited to provide bacterial-specific molecular fingerprints which can be used for analytical purposes. However, for some complex structures such as bacteria, the generation of reproducible SERS spectra is still a challenging task. Among the various factors influencing the SERS variability (such as the nature of SERS-active substrate, Raman parameters and bacterial specificity), we demonstrate in this study that the environment of Gram-positive and Gram-negative bacteria deposited on ultra-thin silver films also impacts the origin of the SERS spectra. In the case of densely packed bacteria, the obtained SERS signatures were either characteristic of the secretion of adenosine triphosphate for Staphylococcus aureus (S. aureus) or the cell wall and the pili/flagella for Escherichia coli (E. coli), allowing for an easy discrimination between the various strains. In the case of isolated bacteria, SERS mapping together with principal component analysis revealed some variabilities of the spectra as a function of the bacteria environment and the bactericidal effect of the silver. However, the variability does not preclude the SERS signatures of various E. coli strains to be discriminated.

Enhancement of Antibacterial and Mechanical Properties of Photocurable ε-Poly-l-lysine Hydrogels by Tannic Acid Treatment.

In this study, a photocurable hydrogel based on an ε-poly-l-lysine (EPL) composite was fabricated by a grafting reaction using glycidyl methacrylate and then complexed with tannic acid (TA) to improve the mechanical stability and antibacterial performance of the EPL hydrogels. UV-visible spectrophotometry, nuclear magnetic resonance, and Fourier transform infrared spectroscopy were introduced to characterize the chemical construction. The obtained EPLMA hydrogel was immersed into TA solution to induce the forming of the H-bond between EPL and TA, resulting in double networks in the composite hydrogel (EPLMA-TA). Due to the additional hydrogen-bond interaction between TA and EPLMA, the mechanical properties of hydrogels were improved and supported cell growth and proliferation. In addition, the antibacterial properties and antioxidant activities of the EPLMA-TA hydrogels were greatly enhanced due to the addition of TA. All the findings indicate that the EPLMA-TA hydrogels with multiple properties show great potential for biomedicine applications.

Novel luminescent affiprobes for molecular detection of Staphylococcus aureus using flow cytometry.

AIMS: Diagnosis of Staphylococcus aureus is important in various diseases from hospital-acquired infections to foodborne diseases. This work reports two new luminescent affiprobes for specific detection of S. aureus. METHODS AND RESULTS: To develop advanced luminescent affiprobes, enhanced green fluorescent protein (EGFP) was flanked by single and double repeats of ZpA963 affibody using molecular biology studies. The recombinant proteins including fluorescent monomeric affibody (fA1 ) and fluorescent dimeric affibody (fA2 ) were expressed in the bacterial expression system, purified and used to identify the S. aureus. Fluorescence microscope and flow cytometry results demonstrated that the treated samples with fA1 and fA2 had relatively high fluorescent mean intensities in comparison to the untreated S. aureus cells. Moreover, it was revealed that 'fA2 ' affiprobe had lower dissociation constant value (about 25-fold) and was more effective for detection of S. aureus than the 'fA1 ' affiprobe. In addition, the binding of the affiprobes for some other pathogenic bacteria i.e. Escherichia coli, Bacillus cereus, Enterococcus faecalis and Staphylococcus saprophyticus was examined. Expectedly, no cross-reaction was observed for binding the constructed affiprobes to these bacteria, eliminating possibilities for false positive results. CONCLUSIONS: The results show that 'fA1 ' affiprobe and 'fA2 ' affiprobe are two new efficient luminescent affiprobes for detecting S. aureus. SIGNIFICANCE AND IMPACT OF THE STUDY: We developed a new approach for detection of Staphylococcus aureus in a simple one-step process and in low concentrations of probes. In the best of our knowledge, this is the first study to direct detection of bacterial cells by affiprobes and may be used to develop new diagnostic kits.

Size-Dependent Antibacterial Immunity of Staphylococcus aureus Protoplast-Derived Particulate Vaccines.

BACKGROUND: Vaccination provides a viable alternative to antibiotics for the treatment of drug-resistant bacterial infection. Bacterial protoplasts have gained much attention for a new generation vaccine due to depleting toxic outer wall components. PURPOSE: The objective of this study was to reveal the effects of bacterial protoplast-derived nanovesicles (PDNVs) size on antibacterial immunity. METHODS: Herein, we prepared bacterial PDNVs with different sizes by removing the cell wall of Staphylococcus aureus (S. aureus) to generate multi-antigen nanovaccines. Furthermore, we investigated the ability of PDNVs in different sizes to activate dendritic cells (DCs) and trigger humoral and cellular immune responses in vivo. RESULTS: We obtained particles of ∼200 nm, 400 nm, and 700 nm diameters and found that all the PDNVs readily induce efficient maturation of DCs in the draining lymph nodes of the vaccinated mice. Dramatically, the activation of DCs was increased with decreasing particle sizes. In addition, vaccination with PDNVs generated elevated expression levels of specific antibody and the production of INF-γ, especially the smaller ones, indicating the capability of inducing strong humoral immunity and Th1 biased cell responses against the source bacteria. CONCLUSION: These observed results provide evidence for size-dependent orchestration of immune responses of PDNVs and help to rationally design and develop effective antibacterial vaccines.

Antibacterial Action of Nanoparticles by Lethal Stretching of Bacterial Cell Membranes.

It is commonly accepted that nanoparticles (NPs) can kill bacteria; however, the mechanism of antimicrobial action remains obscure for large NPs that cannot translocate the bacterial cell wall. It is demonstrated that the increase in membrane tension caused by the adsorption of NPs is responsible for mechanical deformation, leading to cell rupture and death. A biophysical model of the NP-membrane interactions is presented which suggests that adsorbed NPs cause membrane stretching and squeezing. This general phenomenon is demonstrated experimentally using both model membranes and Pseudomonas aeruginosa and Staphylococcus aureus, representing Gram-positive and Gram-negative bacteria. Hydrophilic and hydrophobic quasi-spherical and star-shaped gold (Au)NPs are synthesized to explore the antibacterial mechanism of non-translocating AuNPs. Direct observation of nanoparticle-induced membrane tension and squeezing is demonstrated using a custom-designed microfluidic device, which relieves contraction of the model membrane surface area and eventual lipid bilayer collapse. Quasi-spherical nanoparticles exhibit a greater bactericidal action due to a higher interactive affinity, resulting in greater membrane stretching and rupturing, corroborating the theoretical model. Electron microscopy techniques are used to characterize the NP-bacterial-membrane interactions. This combination of experimental and theoretical results confirm the proposed mechanism of membrane-tension-induced (mechanical) killing of bacterial cells by non-translocating NPs.

A Novel Surface-Exposed Polypeptide Is Successfully Employed as a Target for Developing a Prototype One-Step Immunochromatographic Strip for Specific and Sensitive Direct Detection of Staphylococcus aureus Causing Neonatal Sepsis.

Neonatal sepsis is a life-threatening condition and Staphylococcus aureus is one of its major causes. However, to date, no rapid and sensitive diagnostic tool has been developed for its direct detection. Bioinformatics analyses identified a surface-exposed 112-amino acid polypeptide of the cell wall protein NWMN_1649, a surface protein involved in cell aggregation and biofilm formation, as being a species-specific and highly conserved moiety. The polypeptide was cloned, purified, and used to immunize mice to raise specific immunoglobulins. The purified antibodies were conjugated to gold nano-particles and used to assemble an immunochromatographic strip (ICS). The developed prototype ICS detected as low as 5 µg purified polypeptide and 102 CFU/mL S. aureus within 15 min. The strip showed superior ability to directly detect S. aureus in neonatal sepsis blood specimens without prior sample processing. Moreover, it showed no cross-reaction in specimens infected with two other major causes of neonatal sepsis; coagulase-negative staphylococci and Klebsiella pneumoniae. The selected NWMN_1649-derived polypeptide demonstrates success as a promising biomolecule upon which a prototype ICS has been developed. This ICS provides a rapid, direct, sensitive, and specific option for the detection of S. aureus causing neonatal sepsis. Such a tool is urgently needed especially in resources-limited countries.

Antibacterial Effects and Mechanism of Mandarin (Citrus reticulata L.) Essential Oil against Staphylococcus aureus.

Staphylococcus aureus (S. aureus) creates an array of challenges for the food industry and causes foodborne diseases in people, largely due to its strong antibiotic resistance. Mandarin (Citrus reticulata L.) essential oil (MEO) is recognized as a natural and safe preservative; however, the antibacterial effects and mechanism of MEO to combat S. aureus are not yet clearly understood. This study will examine the inhibitory effects of MEO against S. aureus and explore the antibacterial mechanism thereof from the perspective of membrane destruction. The antibacterial activity of MEO on planktonic S. aureus was examined to determine the minimal inhibitory concentration (MIC). Scanning electron microscope (SEM) images revealed the direct impacts of MEO treatment on the cell structure of S. aureus. The cell membrane was observed to be depolarized, the determination of extracellular nucleic acids, proteins and intracellular adenosine triphosphate (ATP) confirmed the increased permeability of the cell membrane, its integrity was destroyed and the cellular constituents had leaked. These results, thus, provided conclusive evidence that MEO constrains the growth of planktonic S. aureus by affecting the permeability and integrity of its cell membrane. Our study provides a basis for the further development and utilization of MEO as a natural antibacterial agent in the food industry.

Efficient microbial colony growth dynamics quantification with ColTapp, an automated image analysis application.

Populations of genetically identical bacteria are phenotypically heterogeneous, giving rise to population functionalities that would not be possible in homogeneous populations. For instance, a proportion of non-dividing bacteria could persist through antibiotic challenges and secure population survival. This heterogeneity can be studied in complex environmental or clinical samples by spreading the bacteria on agar plates and monitoring time to growth resumption in order to infer their metabolic state distribution. We present ColTapp, the Colony Time-lapse application for bacterial colony growth quantification. Its intuitive graphical user interface allows users to analyze time-lapse images of agar plates to monitor size, color and morphology of colonies. Additionally, images at isolated timepoints can be used to estimate lag time. Using ColTapp, we analyze a dataset of Staphylococcus aureus time-lapse images including populations with heterogeneous lag time. Colonies on dense plates reach saturation early, leading to overestimation of lag time from isolated images. We show that this bias can be corrected by taking into account the area available to each colony on the plate. We envision that in clinical settings, improved analysis of colony growth dynamics may help treatment decisions oriented towards personalized antibiotic therapies.

Community-acquired infection caused by small-colony variant of Staphylococcus aureus.

Staphylococcus aureus and other Gram negative bacteria produce small colony variants (SCV) which usually emerge after exposure to antimicrobials. They cause repeated infections, treatment failures and often pass unnoticed during cultures due to unusual appearance and incomplete incubation. This infectious disease grand round highlights a similar clinical case with atypical history and appearance of a SCV of S. aureus and why prolonged incubation is necessary for aspirates from patients with recurrent infections like abscesses.

Synthesis, DNA/RNA-interaction and biological activity of benzo[k,l]xanthene lignans.

Interactions of two newly synthesized and six previously reported benzoxanthene lignans (BXLs), analogues of rare natural products, with DNA/RNA, G-quadruplex and HSA were evaluated by a set of spectrophotometric methods. Presence/absence of methoxy and hydroxy groups on the benzoxanthene core and minor modifications at C-1/C-2 side pendants - presence/absence of phenyl ring and presence/absence of methoxy and hydroxy groups on phenyl ring - influenced the fluorescence changes and the binding strength to double-stranded (ds-) and G-quadruplex structures. In general, compounds without phenyl ring showed stronger fluorescence changes upon binding than phenyl-substituted BXLs. On the other hand, BXLs with an unsubstituted phenyl ring showed the best stabilization effects of G-quadruplex. Circular dichroism spectroscopy results suggest mixed binding mode, groove binding and partial intercalation, to ds-DNA/RNA and end-stacking to top or bottom G-tetrads as the main binding modes of BXLs to those targets. All compounds exhibited micromolar binding affinities toward HSA and an increased protein thermal stability. Moderate to strong antiradical scavenging activity was observed for all BXLs with hydroxy groups at C-6, C-9 and C-10 positions of the benzoxanthene core, except for derivative bearing methoxy groups at these positions. BXLs with unsubstituted or low-substituted phenyl ring and one derivative without phenyl ring showed strong growth inhibition of Gram-positive Staphylococcus aureus. All compounds showed moderate to strong tumor cell growth-inhibitory activity and cytotoxicity.

Cellulase-Linked Immunomagnetic Microbial Assay on Electrodes: Specific and Sensitive Detection of a Single Bacterial Cell.

Pathogen-associated infections represent one of the major threats to human health and require reliable methods for immediate and robust identification of pathogenic microorganisms. Here, an inexpensive cellulase-linked immunomagnetic methodology was developed for the specific and ultrasensitive analysis of bacteria at their single-cell levels within a 3 h procedure. Detection of a model bacterium, Escherichia coli, was performed in a sandwich reaction with E. coli-specific either aptamer or antibody (Ab)-modified magnetic beads (MBs) and Ab/aptamer reporter molecules linked to cellulase. The cellulase-labeled immuno-aptamer sandwich applied onto nitrocellulose-film-modified electrodes digested the film and changed its electrical conductivity. Electrode's chronocoulometric responses at 0.3 V, in the absence of any redox indicators, allowed a single E. coli cell detection and from 1 to 4 × 104 CFU mL-1 E. coli quantification. No interference/cross-reactivity from Salmonella enteritidis, Enterobacter agglomerans, Pseudomonas putida, Staphylococcus aureus, and Bacillus subtilis was observed when the assay was performed on Ab-modified MBs, and E. coli could be quantified in tap water and milk. This electrochemically label-free methodology is sufficiently fast, highly specific, and sensitive to be used in direct in-field applications. The assay can be adapted for specific detection of other bacterial strains of either the same or different species and offers new analytical tools for fast, specific, and reliable analysis of bacteria in the clinic, food, and environment.

Reassessment of the distinctive geometry of Staphylococcus aureus cell division.

Staphylococcus aureus is generally thought to divide in three alternating orthogonal planes over three consecutive division cycles. Although this mode of division was proposed over four decades ago, the molecular mechanism that ensures this geometry of division has remained elusive. Here we show, for three different strains, that S. aureus cells do not regularly divide in three alternating perpendicular planes as previously thought. Imaging of the divisome shows that a plane of division is always perpendicular to the previous one, avoiding bisection of the nucleoid, which segregates along an axis parallel to the closing septum. However, one out of the multiple planes perpendicular to the septum which divide the cell in two identical halves can be used in daughter cells, irrespective of its orientation in relation to the penultimate division plane. Therefore, division in three orthogonal planes is not the rule in S. aureus.

Design, Synthesis, Conjugation, and Reactivity of Novel trans,trans-1,5-Cyclooctadiene-Derived Bioorthogonal Linkers.

The tetrazine/trans-cyclooctene (TCO) inverse electron-demand Diels-Alder (IEDDA) reaction is the fastest bioorthogonal "click" ligation process reported to date. In this context, TCO reagents have found widespread applications; however, their availability and structural diversity is still somewhat limited due to challenges connected with their synthesis and structural modification. To address this issue, we developed a novel strategy for the conjugation of TCO derivatives to a biomolecule, which allows for the creation of greater structural diversity from a single precursor molecule, i.e., trans,trans-1,5-cyclooctadiene [(E,E)-COD] 1, whose preparation requires standard laboratory equipment and readily available reagents. This two-step strategy relies on the use of new bifunctional TCO linkers (5a-11a) for IEDDA reactions, which can be synthesized via 1,3-dipolar cycloaddition of (E,E)-COD 1 with different azido spacers (5-11) carrying an electrophilic function (NHS-ester, N-succinimidyl carbonate, p-nitrophenyl-carbonate, maleimide) in the ω-position. Following bioconjugation of these electrophilic linkers to the nucleophilic residue (cysteine or lysine) of a protein (step 1), the resulting TCO-decorated constructs can be subjected to a IEDDA reaction with tetrazines functionalized with fluorescent or near-infrared (NIR) tags (step 2). We successfully used this strategy to label bovine serum albumin with the TCO linker 8a and subsequently reacted it in a cell lysate with the fluorescein-isothiocyanate (FITC)-derived tetrazine 12. The same strategy was then used to label the bacterial wall of Gram-positive Staphylococcus aureus, showing the potential of these linkers for live-cell imaging. Finally, we determined the impact of structural differences of the linkers upon the stability of the bioorthogonal constructs. The compounds for stability studies were prepared by conjugation of TCO linkers 6a, 8a, and 10a to mAbs, such as Rituximab and Obinutuzumab, and subsequent labeling with a reactive Cy3-functionalized tetrazine.