Undecaprenyl phosphate translocases confer conditional microbial fitness.

The microbial cell wall is essential for maintenance of cell shape and resistance to external stressors1. The primary structural component of the cell wall is peptidoglycan, a glycopolymer with peptide crosslinks located outside of the cell membrane1. Peptidoglycan biosynthesis and structure are responsive to shifting environmental conditions such as pH and salinity2-6, but the mechanisms underlying such adaptations are incompletely understood. Precursors of peptidoglycan and other cell surface glycopolymers are synthesized in the cytoplasm and then delivered across the cell membrane bound to the recyclable lipid carrier undecaprenyl phosphate7 (C55-P, also known as UndP). Here we identify the DUF368-containing and DedA transmembrane protein families as candidate C55-P translocases, filling a critical gap in knowledge of the proteins required for the biogenesis of microbial cell surface polymers. Gram-negative and Gram-positive bacteria lacking their cognate DUF368-containing protein exhibited alkaline-dependent cell wall and viability defects, along with increased cell surface C55-P levels. pH-dependent synthetic genetic interactions between DUF368-containing proteins and DedA family members suggest that C55-P transporter usage is dynamic and modulated by environmental inputs. C55-P transporter activity was required by the cholera pathogen for growth and cell shape maintenance in the intestine. We propose that conditional transporter reliance provides resilience in lipid carrier recycling, bolstering microbial fitness both inside and outside the host.

Structural variations and roles of rhamnose-rich cell wall polysaccharides in Gram-positive bacteria.

Rhamnose-rich cell wall polysaccharides (Rha-CWPSs) have emerged as crucial cell wall components of numerous Gram-positive, ovoid-shaped bacteria-including streptococci, enterococci, and lactococci-of which many are of clinical or biotechnological importance. Rha-CWPS are composed of a conserved polyrhamnose backbone with side-chain substituents of variable size and structure. Because these substituents contain phosphate groups, Rha-CWPS can also be classified as polyanionic glycopolymers, similar to wall teichoic acids, of which they appear to be functional homologs. Recent advances have highlighted the critical role of these side-chain substituents in bacterial cell growth and division, as well as in specific interactions between bacteria and infecting bacteriophages or eukaryotic hosts. Here, we review the current state of knowledge on the structure and biosynthesis of Rha-CWPS in several ovoid-shaped bacterial species. We emphasize the role played by multicomponent transmembrane glycosylation systems in the addition of side-chain substituents of various sizes as extracytoplasmic modifications of the polyrhamnose backbone. We provide an overview of the contribution of Rha-CWPS to cell wall architecture and biogenesis and discuss current hypotheses regarding their importance in the cell division process. Finally, we sum up the critical roles that Rha-CWPS can play as bacteriophage receptors or in escaping host defenses, roles that are mediated mainly through their side-chain substituents. From an applied perspective, increased knowledge of Rha-CWPS can lead to advancements in strategies for preventing phage infection of lactococci and streptococci in food fermentation and for combating pathogenic streptococci and enterococci.

Rearrangement of Thiodepsipeptides by S → N Acyl Shift Delivers Homodetic Autoinducing Peptides.

Group behavior in many bacteria relies on chemically induced communication called quorum sensing (QS), which plays important roles in the regulation of colonization, biofilm formation, and virulence. In Gram-positive bacteria, QS is often mediated by cyclic ribosomally synthesized and posttranslationally modified peptides (RiPPs). In staphylococci, for example, most of these so-called autoinducing peptides (AIPs) contain a conserved thiolactone functionality, which has also been predicted to constitute a structural feature of AIPs from other genera. Here, we show that pentameric AIPs from Lactiplantibacillus plantarum, Clostridium perfringens, and Listeria monocytogenes that were previously presumed to be thiolactone-containing structures readily rearrange to become homodetic cyclopeptides. This finding has implications for the developing understanding of cross-species and potential cross-genus communication of bacteria and may help guide the discovery of peptide ligands to perturb their function.

Entropy-driven translocation of disordered proteins through the Gram-positive bacterial cell wall.

In Gram-positive bacteria, a thick cross-linked cell wall separates the membrane from the extracellular space. Some surface-exposed proteins, such as the Listeria monocytogenes actin nucleation-promoting factor ActA, remain associated with the bacterial membrane but somehow thread through tens of nanometres of cell wall to expose their amino terminus to the exterior. Here, we report that entropy enables the translocation of disordered transmembrane proteins through the Gram-positive cell wall. We build a physical model, which predicts that the entropic constraint imposed by a thin periplasm is sufficient to drive the translocation of an intrinsically disordered protein such as ActA across a porous barrier similar to a peptidoglycan cell wall. We experimentally validate our model and show that ActA translocation depends on the cell-envelope dimensions and disordered-protein length, and that translocation is reversible. We also show that disordered regions of eukaryotic proteins can translocate Gram-positive cell walls via entropy. We propose that entropic forces are sufficient to drive the translocation of specific proteins to the outer surface.

Membrane vesicles of Lacticaseibacillus rhamnosus JB-1 contain immunomodulatory lipoteichoic acid and are endocytosed by intestinal epithelial cells.

Intestinal bacteria have diverse and complex influence on their host. Evidence is accumulating that this may be mediated in part by bacterial extracellular membrane vesicles (MV), nanometer-sized particles important for intercellular communication. Little is known about the composition of MV from gram-positive beneficial bacteria nor how they interact with intestinal epithelial cells (IEC). Here we demonstrate that MV from Lacticaseibacillus rhamnosus JB-1 are endocytosed in a likely clathrin-dependent manner by both mouse and human IEC in vitro and by mouse IEC in vivo. We further show that JB-1 MV contain lipoteichoic acid (LTA) that activates Toll-like receptor 2 (TLR2) and induces immunoregulatory interleukin-10 expression by dendritic cells in an internalization-dependent manner. By contrast, neither LTA nor TLR2 appear to be required for JB-1 MV endocytosis by IEC. These results demonstrate a novel mechanism by which bacterial MV can influence host physiology and suggest one potential route for beneficial influence of certain bacteria and probiotics.

Myrindole A, an Antimicrobial Bis-indole from a Marine Sponge Myrmekioderma sp.

Myrindole A, a bis-indole alkaloid, was isolated from the deep-sea sponge Myrmekioderma sp. The high degree of unsaturation of the molecule complicated the assignment of its structure by standard 2D-NMR experiments but was ultimately achieved by a combination of 1H-15N-HMBC and 1,n-ADEQUATE experiments as well as the comparison of measured and calculated CD spectra. Myrindole A showed antimicrobial activity against Gram-positive and Gram-negative bacteria.

Passive Diffusion of Ciprofloxacin and its Metalloantibiotic: A Computational and Experimental study.

Fluoroquinolones (FQ) are antibiotics widely used in clinical practise, but the development of bacterial resistance to these drugs is currently a critical public health problem. In this context, ternary copper complexes of FQ (CuFQPhen) have been studied as a potential alternative. In this study, we compared the passive diffusion across the lipid bilayer of one of the most used FQ, ciprofloxacin (Cpx), and its ternary copper complex, CuCpxPhen, that has shown previous promising results regarding antibacterial activity and membrane partition. A combination of spectroscopic studies and molecular dynamics simulations were used and two different model membranes tested: one composed of anionic phospholipids, and the other composed of zwitterionic phospholipids. The obtained results showed a significantly higher membrane permeabilization activity, larger partition, and a more favourable free energy landscape for the permeation of CuCpxPhen across the membrane, when compared to Cpx. Furthermore, the computational results indicated a more favourable translocation of CuCpxPhen across the anionic membrane, when compared to the zwitterionic one, suggesting a higher specificity towards the former. These findings are important to decipher the influx mechanism of CuFQPhen in bacterial cells, which is crucial for the ultimate use of CuFQPhen complexes as an alternative to FQ to tackle multidrug-resistant bacteria.

Biodiversity, Bioactivity, and Metabolites of High Desert Derived Oregonian Soil Bacteria.

From arid, high desert soil samples collected near Bend, Oregon, 19 unique bacteria were isolated. Each strain was identified by 16S rRNA gene sequencing, and their organic extracts were tested for antibacterial and antiproliferative activities. Noteworthy, six extracts (30 %) exhibited strong inhibition resulting in less than 50 % cell proliferation in more than one cancer cell model, tested at 10 µg/mL. Principal component analysis (PCA) of LC/MS data revealed drastic differences in the metabolic profiles found in the organic extracts of these soil bacteria. In total, fourteen potent antibacterial and/or cytotoxic metabolites were isolated via bioactivity-guided fractionation, including two new natural products: a pyrazinone containing tetrapeptide and 7-methoxy-2,3-dimethyl-4H-chromen-4-one, as well as twelve known compounds: furanonaphthoquinone I, bafilomycin C1 and D, FD-594, oligomycin A, chloramphenicol, MY12-62A, rac-sclerone, isosclerone, tunicamycin VII, tunicamycin VIII, and (6S,16S)-anthrabenzoxocinone 1.264-C.

Preparation of Boronic Acid-Functionalized Cryogels Using Modular and Clickable Building Blocks for Bacterial Separation.

Composite cryogels containing boronic acid ligands are synthesized for effective separation and isolation of bacteria. The large and interconnected pores in cryogels enable fast binding and release of microbial cells. To control bacterial binding, an alkyne-tagged boronic acid ligand is conjugated to azide-functionalized cryogel via the Cu(I)-catalyzed azide-alkyne cycloaddition reaction. The boronic acid-functionalized cryogel binds Gram-positive and Gram-negative bacteria through reversible boronate ester bonds, which can be controlled by pH and simple monosaccharides. To increase the capacity of affinity separation, a new approach is used to couple the alkyne-tagged phenylboronic acid to cryogel via an intermediate polymer layer that provides multiple immobilization sites. The morphology and chemical composition of the composite cryogel are characterized systematically. The capability of the composite cryogel for the separation of Gram-positive and Gram-negative bacteria is investigated. The binding capacities of the composite cryogel for Escherichia coli and Staphylococcus epidermidis are 2.15 × 109 and 3.36 × 109 cfu/g, respectively. The bacterial binding of the composite cryogel can be controlled by adjusting pH. The results suggest that the composite cryogel may be used as affinity medium for rapid separation and isolation of bacteria from complex samples.

Multitask ATPases (NBDs) of bacterial ABC importers type I and their interspecies exchangeability.

ATP-binding cassette (ABC) type I importers are widespread in bacteria and play a crucial role in its survival and pathogenesis. They share the same modular architecture comprising two intracellular nucleotide-binding domains (NBDs), two transmembrane domains (TMDs) and a substrate-binding protein. The NBDs bind and hydrolyze ATP, thereby generating conformational changes that are coupled to the TMDs and lead to substrate translocation. A group of multitask NBDs that are able to serve as the cellular motor for multiple sugar importers was recently discovered. To understand why some ABC importers share energy-coupling components, we used the MsmX ATPase from Bacillus subtilis as a model for biological and structural studies. Here we report the first examples of functional hybrid interspecies ABC type I importers in which the NBDs could be exchanged. Furthermore, the first crystal structure of an assigned multitask NBD provides a framework to understand the molecular basis of the broader specificity of interaction with the TMDs.

A novel electrochemical biosensor based on peptidoglycan and platinum-nickel-copper nano-cube for rapid detection of Gram-positive bacteria.

A novel non-enzyme electrochemical biosensor for the rapid detection of Gram-positive bacteria has been constructed that relys on a stable and efficient combination between the peptidoglycan layer and platinum-nickel-copper nanocubes (Pt-Ni-Cu NCs). Briefly, bacteria were first captured by a specific antibody. Then, the electrochemical signal materials (Pt-Ni-Cu NCs) were bound to the bacteria peptidoglycan layer using specific structural and surface features. The rapid and sensitive bacterial detection was then achieved using intrinsic electrochemical characteristics and superoxidase-like activity of the Pt-Ni-Cu NCs. Moreover, the nature of peptidoglycan covering the whole bacteria provided the premise for signal amplification. Under optimal conditions, the electrochemical signal variation was proportional to the concentration of bacteria ranging from 1.5 × 102 to 1.5 × 108 CFU/mL with a detection limit of 42 CFU/mL using a working potential of - 0.4 V. This electrochemical biosensor has been successfully applied to detect bacteria concentrations in urine samples, and the recoveries range from 90.4 to 107%. The proposed biosensor could be applied for broad-spectrum detection of Gram-positive bacteria since most Gram-positive bacteria possess a thick peptidoglycan layer. The developed electrochemical biosensing strategy might be used as a potential tool for clinical pathogenic bacteria detection and point-of-care testing (POCT).

Targeting Protein Folding: A Novel Approach for the Treatment of Pathogenic Bacteria.

Infectious diseases are a major cause of morbidity and mortality worldwide, exacerbated by increasing antibiotic resistance in many bacterial species. The development of drugs with new modes of action is essential. A leading strategy is antivirulence, with the aim to target bacterial proteins that are important in disease causation and progression but do not affect growth, resulting in reduced selective pressure for resistance. Immunophilins, a superfamily of peptidyl-prolyl cis-trans isomerase (PPIase) enzymes have been shown to be important for virulence in a broad-spectrum of pathogenic bacteria. This Perspective will provide an overview of the recent advances made in understanding the role of each immunophilin family, cyclophilins, FK506 binding proteins (FKBPs), and parvulins in bacteria. Inhibitor design and medicinal chemistry strategies for development of novel drugs against bacterial FKBPs will be discussed. Furthermore, drugs against human cyclophilins and parvulins will be reviewed in their current indication as antiviral and anticancer therapies.

Clinical characteristics of bloodstream infection by Parvimonas micra: retrospective case series and literature review.

BACKGROUND: Gram-positive anaerobic (GPA) bacteria inhabit different parts of the human body as commensals but can also cause bacteremia. In this retrospective observational study, we analyzed GPA bacteremia pathogens before (2013-2015) and after (2016-2018) the introduction of the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). METHOD: We conducted a retrospective observational study by searching the microbiology database to identify all positive GPA blood cultures of patients with GPA bacteremia diagnosed using the new technique, MALDI-TOF MS, between January 1, 2016 and December 31, 2018; and using a conventional phenotypic method between January 1, 2013 and December 31, 2015 at a single tertiary center in Japan. Parvimonas micra (P. micra) (17.5%) was the second most frequently identified GPA (MALDI-TOF MS); we then retrospectively reviewed electronic medical records for 25 P. micra bacteremia cases at our hospital. We also conducted a literature review of published cases in PubMed from January 1, 1980, until December 31, 2019; 27 cases were retrieved. RESULTS: Most cases of P. micra bacteremia were identified after 2015, both, at our institute and from the literature review. They were of mostly elderly patients and had comorbid conditions (malignancies and diabetes). In our cases, laryngeal pharynx (7/25, 28%) and gastrointestinal tract (GIT; 6/25, 24%) were identified as the most likely sources of bacteremia; however, the infection source was not identified in 9 cases (36%). P. micra bacteremia were frequently associated with spondylodiscitis (29.6%), oropharyngeal infection (25.9%), intra-abdominal abscess (14.8%), infective endocarditis (11.1%), septic pulmonary emboli (11.1%), and GIT infection (11.1%) in the literature review. Almost all cases were treated successfully with antibiotics and by abscess drainage. The 30-day mortalities were 4 and 3.7% for our cases and the literature cases, respectively. CONCLUSIONS: Infection sites of P. micra are predominantly associated with GIT, oropharyngeal, vertebral spine, intra-abdominal region, pulmonary, and heart valves. Patients with P. micra bacteremia could have good prognosis following appropriate treatment.

The mannose phosphotransferase system (Man-PTS) - Mannose transporter and receptor for bacteriocins and bacteriophages.

Mannose transporters constitute a superfamily (Man-PTS) of the Phosphoenolpyruvate Carbohydrate Phosphotransferase System (PTS). The membrane complexes are homotrimers of protomers consisting of two subunits, IIC and IID. The two subunits without recognizable sequence similarity assume the same fold, and in the protomer are structurally related by a two fold pseudosymmetry axis parallel to membrane-plane (Liu et al. (2019) Cell Research 29 680). Two reentrant loops and two transmembrane helices of each subunit together form the N-terminal transport domain. Two three-helix bundles, one of each subunit, form the scaffold domain. The protomer is stabilized by a helix swap between these bundles. The two C-terminal helices of IIC mediate the interprotomer contacts. PTS occur in bacteria and archaea but not in eukaryotes. Man-PTS are abundant in Gram-positive bacteria living on carbohydrate rich mucosal surfaces. A subgroup of IICIID complexes serve as receptors for class IIa bacteriocins and as channel for the penetration of bacteriophage lambda DNA across the inner membrane. Some Man-PTS are associated with host-pathogen and -symbiont processes.

Bismuth(III) Flavonolates: The Impact of Structural Diversity on Antibacterial Activity, Mammalian Cell Viability and Cellular Uptake.

A series of homoleptic and heteroleptic bismuth(III) flavonolate complexes derived from six flavonols of varying substitution have been synthesised and structurally characterised. The complexes were evaluated for antibacterial activity towards several problematic Gram-positive (Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), and vancomycin-resistant Enterococcus (VRE)) and Gram-negative (Escherichia coli, Pseudomonas aeruginosa) bacteria. The cell viability of COS-7 (monkey kidney) cells treated with the bismuth flavonolates was also studied to determine the effect of the complexes on mammalian cells. The heteroleptic complexes [BiPh(L)2 ] (in which L=flavonolate) showed good antibacterial activity towards all of the bacteria but reduced COS-7 cell viability in a concentration-dependent manner. The homoleptic complexes [Bi(L)3 ] exhibited activity towards the Gram-positive bacteria and showed low toxicity towards the mammalian cell line. Bismuth uptake studies in VRE and COS-7 cells treated with the bismuth flavonolate complexes indicated that Bi accumulation is influenced by both the substitution of the flavonolate ligands and the degree of substitution at the bismuth centre.

Binding of an antimicrobial peptide to bacterial cells: Interaction with different species, strains and cellular components.

Antimicrobial peptides (AMPs) selectively kill bacteria by disrupting their cell membranes, and are promising compounds to fight drug-resistant microbes. Biophysical studies on model membranes have characterized AMP/membrane interactions and the mechanism of bilayer perturbation, showing that accumulation of cationic peptide molecules in the external leaflet leads to the formation of pores ("carpet" mechanism). However, similar quantitative studies on real cells are extremely limited. Here, we investigated the interaction of the dansylated PMAP23 peptide (DNS-PMAP23) with a Gram-positive bacterium, showing that 107 bound peptide molecules per cell are needed to kill it. This result is consistent with our previous finding for Gram-negative strains, where a similar high threshold for killing was determined, demonstrating the general relevance of the carpet model for real bacteria. However, in the case of the Gram-positive strain, this number of molecules even exceeds the total surface available on the bacterial membrane. The high affinity of DNS-PMAP23 for the anionic teichoic acids of the Gram-positive cell wall, but not for the lipopolysaccharides of Gram-negative bacteria, provides a rationale for this finding. To better define the role of anionic lipids in peptide/cell association, we studied DNS-PMAP23 interaction with E. coli mutant strains lacking phosphatidylglycerol and/or cardiolipin. Surprisingly, these strains showed a peptide affinity similar to that of the wild type. This finding was rationalized by observing that these bacteria have an increased content of other anionic lipids, thus maintaining the total membrane charge essentially constant. Finally, studies of DNS-PMAP23 association to dead bacteria showed an affinity an order of magnitude higher compared to that of live cells, suggesting strong peptide binding to intracellular components that become accessible after membrane perturbation. This effect could play a role in population resistance to AMP action, since dead bacteria could protect the surviving cells by sequestering significant amounts of peptide molecules. Overall, our data indicate that quantitative studies of peptide association to bacteria can lead to a better understanding of the mechanism of action of AMPs.

1-hydroxy-7-oxolavanducyanin and Δ7″,8″-6″-hydroxynaphthomevalin from Streptomyces sp. CPCC 203577.

Lavanducyanin is a bioactive phenazine-containing secondary metabolite, and naphthomevalin is an antibacterial polyketide secondary metabolite. Herein, new analogues of lavanducyanin (2) and of naphthomevalin (4), together with lavanducyanin (1) and naphthomevalin (3), were identified from Streptomyces sp. CPCC 203577, an actinomycete soil isolate. The structures of 2 and 4 were elucidated as 1-hydroxy-7-oxolavanducyanin and Δ7″,8″-6″-hydroxynaphthomevalin, respectively, by 1D and 2D NMR. Antibacterial assays revealed that 2 had significant but reduced anti-Gram-positive bacterial activity compared with 1, and 4 was devoid of anti-Gram-positive bacterial activity. This indicated that the phenazinone nucleus in lavanducyanin and the monoterpene side chain in naphthomevalin might be important for their anti-Gram-positive bacterial activity. Compounds 1-4 were all inactive against Gram-negative bacteria.

Bacterial DNA promotes Tau aggregation.

A hallmark feature of Alzheimer's disease (AD) and other tauopathies is the misfolding, aggregation and cerebral accumulation of tau deposits. Compelling evidence indicates that misfolded tau aggregates are neurotoxic, producing synaptic loss and neuronal damage. Misfolded tau aggregates are able to spread the pathology from cell-to-cell by a prion like seeding mechanism. The factors implicated in the initiation and progression of tau misfolding and aggregation are largely unclear. In this study, we evaluated the effect of DNA extracted from diverse prokaryotic and eukaryotic cells in tau misfolding and aggregation. Our results show that DNA from various, unrelated gram-positive and gram-negative bacteria results in a more pronounced tau misfolding compared to eukaryotic DNA. Interestingly, a higher effect in promoting tau aggregation was observed for DNA extracted from certain bacterial species previously detected in the brain, CSF or oral cavity of patients with AD. Our findings indicate that microbial DNA may play a previously overlooked role in the propagation of tau protein misfolding and AD pathogenesis, providing a new conceptual framework that positions the compromised blood-brain and intestinal barriers as important sources of microbial DNA in the CNS, opening novel opportunities for therapeutic interventions.

π-Extended Donor-Acceptor Porphyrins and Metalloporphyrins for Antimicrobial Photodynamic Inactivation.

Free base, zinc and palladium π-extended porphyrins containing fused naphthalenediamide units were employed as photosensitizers in antimicrobial photodynamic therapy (aPDT). Their efficacy, assessed by photophysical and in vitro photobiological studies on Gram-positive bacteria, was found to depend on metal coordination, showing a dramatic enhancement of photosensitizing activity for the palladium complex.

Effect of AMPs MSI-78 and BP100 on the lipid acyl chains of 2H-labeled intact Gram positive bacteria.

The lipid bilayer disrupting effect of antimicrobial peptides (AMPs) has been widely studied in model-lipid systems by applying biophysical techniques such as 2H NMR spectroscopy. Real bacteria cell envelopes contain non-lipid components, such as peptidoglycan, and thus it is important to assess the effects of such non-lipid components on the lipid-disrupting effects of AMPs. To this end, our group and other have developed methods that promote uptake of deuterium-labeled acyl chains in bacterial cells to produce 2H-membrane-enriched Bacillus subtilis. In this work, we studied changes in the static 2H NMR spectra of B. subtilis induced by the AMPs MSI-78 and BP100. Addition of both AMPs resulted in the increase of lipid acyl chain disorder consistent with disruption of the bacterial membrane. In addition, the peptide to lipid molar ratios (P:L) that give rise to observable effects fall between the P:L molar ratios necessary to generate membrane disruption in model-lipid-only systems and the P:L molar ratios needed to inhibit bacterial cell growth. This observation supports a role for the non-lipid components in modulating the AMP-lipid interactions.