Lethal puncturing of planktonic Gram-positive and Gram-negative bacteria by magnetically-rotated silica hexapods.

Planktonic bacterial presence in many industrial and environmental applications and personal health-care products is generally countered using antimicrobials. However, antimicrobial chemicals present an environmental threat, while emerging resistance reduces their efficacy. Suspended bacteria have no defense against mechanical attack. Therefore, we synthesized silica hexapods on an α-Fe2O3 core that can be magnetically-rotated to inflict lethal cell-wall-damage to planktonic Gram-negative and Gram-positive bacteria. Hexapods possessed 600 nm long nano-spikes, composed of SiO2, as shown by FTIR and XPS. Fluorescence staining revealed cell wall damage caused by rotating hexapods. This damage was accompanied by DNA/protein release and bacterial death that increased with increasing rotational frequency up to 500 rpm. Lethal puncturing was more extensive on Gram-negative bacteria than on Gram-positive bacteria, which have a thicker peptidoglycan layer with a higher Young's modulus. Simulations confirmed that cell-wall-puncturing occurs at lower nano-spike penetration levels in the cell walls of Gram-negative bacteria. This approach offers a new way to kill bacteria in suspension, not based on antimicrobial chemicals.

Biochemical and structural characterization of chlorhexidine as an ATP-assisted inhibitor against type 1 methionyl-tRNA synthetase from Gram-positive bacteria.

Methionyl-tRNA synthetase (MetRS) catalyzes the attachment of l-methionine (l-Met) to tRNAMet to generate methionyl-tRNAMet, an essential substrate for protein translation within ribosome. Owing to its indispensable biological function and the structural discrepancies with human counterpart, bacterial MetRS is considered an ideal target for developing antibacterials. Herein, chlorhexidine (CHX) was identified as a potent binder of Staphylococcus aureus MetRS (SaMetRS) through an ATP-aided affinity screening. The co-crystal structure showed that CHX simultaneously occupies the enlarged l-Met pocket (EMP) and the auxiliary pocket (AP) of SaMetRS with its two chlorophenyl groups, while its central hexyl linker swings upwards to interact with some conserved hydrophobic residues. ATP adopts alternative conformations in the active site cavity, and forms ionic bonds and water-mediated hydrogen bonds with CHX. Consistent with this synergistic binding mode, ATP concentration-dependently enhanced the binding affinity of CHX to SaMetRS from 10.2 µM (no ATP) to 0.45 µM (1 mM ATP). While it selectively inhibited two representative type 1 MetRSs from S. aureus and Enterococcus faecalis, CHX did not show significant interactions with three tested type 2 MetRSs, including human cytoplasmic MetRS, in the enzyme inhibition and biophysical binding assays, probably due to the conformational differences between two types of MetRSs at their EMP and AP. Our findings on CHX may inspire the design of MetRS-directed antimicrobials in future.

Metabolic labeling-mediated visualization, capture, and inactivation of Gram-positive bacteria via biotin-streptavidin interactions.

We introduce a biotinylated D-amino acid probe capable of metabolically incorporating into bacterial PG. Leveraging the robust affinity between biotin and streptavidin, the probe has demonstrated efficacy in imaging, capture, and targeted inactivation of Gram-positive bacteria through synergistic pairings with commercially available streptavidin-modified fluorescent dyes and nanomaterials. The versatility of the probe is underscored by its compatibility with a variety of commercially available streptavidin-modified reagents. This adaptability allows the probe to be applied across diverse scenarios by integrating with these commercial reagents.

Human IgG Fc-engineering for enhanced plasma half-life, mucosal distribution and killing of cancer cells and bacteria.

Monoclonal IgG antibodies constitute the fastest growing class of therapeutics. Thus, there is an intense interest to design more potent antibody formats, where long plasma half-life is a commercially competitive differentiator affecting dosing, frequency of administration and thereby potentially patient compliance. Here, we report on an Fc-engineered variant with three amino acid substitutions Q311R/M428E/N434W (REW), that enhances plasma half-life and mucosal distribution, as well as allows for needle-free delivery across respiratory epithelial barriers in human FcRn transgenic mice. In addition, the Fc-engineered variant improves on-target complement-mediated killing of cancer cells as well as both gram-positive and gram-negative bacteria. Hence, this versatile Fc technology should be broadly applicable in antibody design aiming for long-acting prophylactic or therapeutic interventions.

Unveiling the mechanism of bactericidal activity of a cecropin A-fused endolysin LNT113.

Endolysins are lytic enzymes produced by bacteriophages at the end of their lytic cycle and degrade the peptidoglycan layer of the bacterial cell wall. Thus, they have been extensively explored as a promising antibacterial agent to replace or supplement current antibiotics. Gram-negative bacteria, however, are prone to resist exogenous endolysins owing to their protective outer membrane. We previously engineered endolysin EC340, encoded by the Escherichia coli phage PBEC131, by substituting its seven amino acids and fusing an antimicrobial peptide cecropin A at its N-terminus. The engineered endolysin LNT113 exerted superior activity to its intrinsic form. This study investigated how cecropin A fusion facilitated the bactericidal activity of LNT113 toward Gram-negative bacteria. Cecropin A of LNT113 markedly increased the interaction with lipopolysaccharides, while the E. coli defective in the core oligosaccharide was less susceptible to endolysins, implicating the interaction between the core oligosaccharide and endolysins. In fact, E. coli with compromised lipid A construction was more vulnerable to LNT113 treatment, suggesting that the integrity of the lipid A layer was important to resist the internalization of LNT113 across the outer membrane. Cecropin A fusion further accelerated the inner membrane destabilization, thereby enabling LNT113 to deconstruct it promptly. Owing to the increased membrane permeability, LNT113 could inactivate some Gram-positive bacteria as well. This study demonstrates that cecropin A fusion is a feasible method to improve the membrane permeability of endolysins in both Gram-negative and Gram-positive bacteria.

Serine-rich repeat proteins: well-known yet little-understood bacterial adhesins.

Serine-rich-repeat proteins (SRRPs) are large mucin-like glycoprotein adhesins expressed by a plethora of pathogenic and symbiotic Gram-positive bacteria. SRRPs play major functional roles in bacterial-host interactions, like adhesion, aggregation, biofilm formation, virulence, and pathogenesis. Through their functional roles, SRRPs aid in the development of host microbiomes but also diseases like infective endocarditis, otitis media, meningitis, and pneumonia. SRRPs comprise shared domains across different species, including two or more heavily O-glycosylated long stretches of serine-rich repeat regions. With loci that can be as large as ~40 kb and can encode up to 10 distinct glycosyltransferases that specifically facilitate SRRP glycosylation, the SRRP loci makes up a significant portion of the bacterial genome. The significance of SRRPs and their glycans in host-microbe communications is becoming increasingly evident. Studies are beginning to reveal the glycosylation pathways and mature O-glycans presented by SRRPs. Here we review the glycosylation machinery of SRRPs across species and discuss the functional roles and clinical manifestations of SRRP glycosylation.

A widespread single amino acid mutation in AcrA reduces tigecycline susceptibility in Klebsiella pneumoniae.

IMPORTANCE: Tigecycline, a glycecycline antibiotic with broad-spectrum activity against almost all Gram-positive and Gram-negative bacteria, is a highly concerned "last-resort" antibiotic. In addition to plasmid-hosted mobile tet(X) conferring high-level resistance to tigecycline, there are many reports suggesting increased expression of AcrAB-TolC efflux pump leads to tigecycline non-susceptibility. However, the role of mutations in AcrAB-TolC on tigecycline resistance has not been identified. This study reports a novel T188A mutation of the AcrA subunit of AcrAB-TolC complex in a clinical tigecycline-resistant Klebsiella pneumoniae strain and reveals the role of AcrA mutation on tigecycline resistance in K. pneumoniae. High prevalence of A188 type AcrA in hypervirulent multidrug-resistant K. pneumoniae indicates that mutations of the AcrAB-TolC complex may play a larger role in determining bacterial pathogenesis and antibiotic susceptibility than previously expected.

Models of Hfq interactions with small non-coding RNA in Gram-negative and Gram-positive bacteria.

Hfq is required by many Gram-negative bacteria to chaperone the interaction between small non-coding RNA (sRNA) and mRNA to facilitate annealing. Conversely and despite the presence of Hfq in many Gram-positive bacteria, sRNAs in Gram-positive bacteria bind the mRNA target independent of Hfq. Details provided by the Hfq structures from both Gram-negative and Gram-positive bacteria have demonstrated that despite a conserved global structure of the protein, variations of residues on the binding surfaces of Hfq results in the recognition of different RNA sequences as well as the ability of Hfq to facilitate the annealing of the sRNA to the mRNA target. Additionally, a subset of Gram-negative bacteria has an extended C-terminal Domain (CTD) that has been shown to affect the stability of the Hfq hexamer and increase the rate of release of the annealed sRNA-mRNA product. Here we review the structures of Hfq and biochemical data that have defined the interactions of the Gram-negative and Gram-positive homologues to highlight the similarities and differences in the interactions with RNA. These interactions provided a deeper understanding of the how Hfq functions to facilitate the annealing of sRNA-mRNA, the selectivity of the interactions with RNA, and the role of the CTD of Hfq in the interactions with sRNA.

A marine-derived fatty acid targets the cell membrane of Gram-positive bacteria.

IMPORTANCE: With the lack of new antibiotics in the drug discovery pipeline, coupled with accelerated evolution of antibiotic resistance, new sources of antibiotics that target pathogens of clinical importance are paramount. Here, we use bacterial cytological profiling to identify the mechanism of action of the monounsaturated fatty acid (Z)-13-methyltetra-4-decenoic acid isolated from the marine bacterium Olleya marilimosa with antibacterial effects against Gram-positive bacteria. The fatty acid antibiotic was found to rapidly destabilize the cell membrane by pore formation and membrane aggregation in Bacillus subtilis, suggesting that this fatty acid may be a promising adjuvant used in combination to enhance antibiotic sensitivity.

The DBL-1/TGF-ß signaling pathway tailors behavioral and molecular host responses to a variety of bacteria in Caenorhabditis elegans.

Generating specific, robust protective responses to different bacteria is vital for animal survival. Here, we address the role of transforming growth factor ß (TGF-ß) member DBL-1 in regulating signature host defense responses in Caenorhabditis elegans to human opportunistic Gram-negative and Gram-positive pathogens. Canonical DBL-1 signaling is required to suppress avoidance behavior in response to Gram-negative, but not Gram-positive bacteria. We propose that in the absence of DBL-1, animals perceive some bacteria as more harmful. Animals activate DBL-1 pathway activity in response to Gram-negative bacteria and strongly repress it in response to select Gram-positive bacteria, demonstrating bacteria-responsive regulation of DBL-1 signaling. DBL-1 signaling differentially regulates expression of target innate immunity genes depending on the bacterial exposure. These findings highlight a central role for TGF-ß in tailoring a suite of bacteria-specific host defenses.

Insight into the structure, biosynthesis, isolation method and biological function of teichoic acid in different gram-positive microorganisms: A review.

Teichoic acid (TA) is a weakly anionic polymer present in the cell walls of Gram-positive bacteria. It can be classified into wall teichoic acid (WTA) and lipoteichoic acid (LTA) based on its localization in the cell wall. The structure and biosynthetic pathway of TAs are strain-specific and have a significant role in maintaining cell wall stability. TAs have various beneficial functions, such as immunomodulatory, anticancer and antioxidant activities. However, the purity and yield of TAs are generally not high, and different isolation methods may even affect their structural integrity, which limits the research progress on the probiotic functions of TA. This paper reviews an overview of the structure and biosynthetic pathway of TAs in different strains, as well as the research progress of the isolation and purification methods of TAs. Furthermore, this review also highlights the current research status on the biological functions of TAs. Through a comprehensive understanding of this review, it is expected to pave the way for advancements in isolating and purifying high-quality TAs and, in turn, lay a foundation for contributing to the development of targeted probiotic therapies.

Hydroxychloroquine-Loaded Chitosan Nanoparticles Induce Anticancer Activity in A549 Lung Cancer Cells: Design, BSA Binding, Molecular Docking, Mechanistic, and Biological Evaluation.

The current study describes the encapsulation of hydroxychloroquine, widely used in traditional medicine due to its diverse pharmacological and medicinal uses, in chitosan nanoparticles (CNPs). This work aims to combine the HCQ drug with CS NPs to generate a novel nanocomposite with improved characteristics and bioavailability. HCQ@CS NPs are roughly shaped like roadways and have a smooth surface with an average size of 159.3 ± 7.1 nm, a PDI of 0.224 ± 0.101, and a zeta potential of +46.6 ± 0.8 mV. To aid in the development of pharmaceutical systems for use in cancer therapy, the binding mechanism and affinity of the interaction between HCQ and HCQ@CS NPs and BSA were examined using stopped-flow and other spectroscopic approaches, supplemented by molecular docking analysis. HCQ and HCQ@CS NPs binding with BSA is driven by a ground-state complex formation that may be accompanied by a non-radiative energy transfer process, and binding constants indicate that HCQ@CS NPs-BSA was more stable than HCQ-BSA. The stopped-flow analysis demonstrated that, in addition to increasing BSA affinity, the nanoformulation HCQ@CS NPS changes the binding process and may open new routes for interaction. Docking experiments verified the development of the HCQ-BSA complex, with HCQ binding to site I on the BSA structure, primarily with the amino acids, Thr 578, Gln 579, Gln 525, Tyr 400, and Asn 404. Furthermore, the nanoformulation HCQ@CS NPS not only increased cytotoxicity against the A549 lung cancer cell line (IC50 = 28.57 ± 1.72 µg/mL) compared to HCQ (102.21 ± 0.67 µg/mL), but also exhibited higher antibacterial activity against both Gram-positive and Gram-negative bacteria when compared to HCQ and chloramphenicol, which is in agreement with the binding constants. The nanoformulation developed in this study may offer a viable therapy option for A549 lung cancer.

Gene amplifications cause high-level resistance against albicidin in gram-negative bacteria.

Antibiotic resistance is a continuously increasing concern for public healthcare. Understanding resistance mechanisms and their emergence is crucial for the development of new antibiotics and their effective use. The peptide antibiotic albicidin is such a promising candidate that, as a gyrase poison, shows bactericidal activity against a wide range of gram-positive and gram-negative bacteria. Here, we report the discovery of a gene amplification-based mechanism that imparts an up to 1000-fold increase in resistance levels against albicidin. RNA sequencing and proteomics data show that this novel mechanism protects Salmonella Typhimurium and Escherichia coli by increasing the copy number of STM3175 (YgiV), a transcription regulator with a GyrI-like small molecule binding domain that traps albicidin with high affinity. X-ray crystallography and molecular docking reveal a new conserved motif in the binding groove of the GyrI-like domain that can interact with aromatic building blocks of albicidin. Phylogenetic studies suggest that this resistance mechanism is ubiquitous in gram-negative bacteria, and our experiments confirm that STM3175 homologs can confer resistance in pathogens such as Vibrio vulnificus and Pseudomonas aeruginosa.

Controlling biofilm and virulence properties of Gram-positive bacteria by targeting wall teichoic acid and lipoteichoic acid.

Wall teichoic acid (WTA) and lipoteichoic acid (LTA) are structural components of Gram-positive bacteria's peptidoglycan and cell membrane, which are mostly anionic glycopolymers. WTA confers numerous physiological, virulence, and pathogenic features to bacterial pathogens. It controls cell shape, cell division, and the localisation of autolytic enzymes and ion homeostasis. In the context of virulence and pathogenicity, it aids bacterial cell attachment and colonisation and protects against the host defence system and antibiotics. Having such a broad function in pathogenic bacteria's lifecycle, WTA/LTA become one of the potential targets for antibacterial agents to reduce bacterial infection in the host. The number of reports for targeting the WTA/LTA pathway has risen, mostly by focusing on three distinct targets: antivirulence targets, ß-lactam potentiator targets, and essential targets. The current review looked at the role of WTA/LTA in biofilm development and virulence in a range of Gram-positive pathogenic bacteria. Furthermore, alternate strategies, such as the application of natural and synthetic compounds that target the WTA/LTA pathway, have been thoroughly discussed. Moreover, the application of nanomaterials and a combination of drugs have also been discussed as a viable method for targeting the WTA/LTA in numerous Gram-positive bacteria. In addition, a future perspective for controlling bacterial infection by targeting the WTA/LTA is proposed.

Lysozyme-immobilized bandage contact lens inhibits the growth and biofilm formation of common eye pathogens in vitro.

Bandage contact lenses have an increased affinity to accumulate tear film proteins and bacteria during wear. Among the wide variety of tear film proteins, lysozyme has attracted the most attention for several reasons, including the fact that it is found at a high concentration in the tear film, has exceptional antibacterial and antibiofilm properties, and its significant deposits onto contact lenses. This study aims to evaluate the effect of lysozyme on bacterial biofilm formation on bandage contact lenses. For this purpose, several methods, including microtiter plate test and Colony Forming Unit (CFU) assay have been used to determine antibacterial and antibiofilm characteristics of lysozyme against the two most frequent contact lens-induced bacterial ocular infections, Staphylococcus aureus, and Pseudomonas aeruginosa. The results of these assays demonstrate lysozyme potential to inhibit 57.9% and 80.7% of the growth of S. aureus and P. aeruginosa, respectively. In addition, biofilm formations of P. aeruginosa and S. aureus reduced by 38.3% and 62.7%, respectively due to the antibiofilm effect of lysozyme. SEM and AFM imaging were utilized to visualize lysozyme antibacterial activity and topography changes of the contact lens surface, respectively, in the presence/absence of lysozyme. The results indicated that lysozyme can efficiently attack both gram-positive and gram-negative bacteria and consequently lysozyme-functionalized bandage contact lenses can reduce the risk of ocular infection after eye surgery.

Role of bacterial efflux pump proteins in antibiotic resistance across microbial species.

Efflux proteins are transporter molecules that actively pump out a variety of substrates, including antibiotics, from cells to the environment. They are found in both Gram-positive and Gram-negative bacteria and eukaryotic cells. Based on their protein sequence homology, energy source, and overall structure, efflux proteins can be divided into seven groups. Multidrug efflux pumps are transmembrane proteins produced by microbes to enhance their survival in harsh environments and contribute to antibiotic resistance. These pumps are present in all bacterial genomes studied, indicating their ancestral origins. Many bacterial genes encoding efflux pumps are involved in transport, a significant contributor to antibiotic resistance in microbes. Efflux pumps are widely implicated in the extrusion of clinically relevant antibiotics from cells to the extracellular environment and, as such, represent a significant challenge to antimicrobial therapy. This review aims to provide an overview of the structures and mechanisms of action, substrate profiles, regulation, and possible inhibition of clinically relevant efflux pumps. Additionally, recent advances in research and the pharmacological exploitation of efflux pump inhibitors as a promising intervention for combating drug resistance will be discussed.

Antibacterial and immunoregulatory activity of an antimicrobial peptide hepcidin in loach (Misgurnus anguillicaudatus).

Antimicrobial peptides (AMPs) are members of humoral immunity and particpate in resisting microbial invasion. In this study, an AMP gene hepcidin was obtained from the oriental loach Misgurnus anguillicaudatus and named Ma-Hep. This Ma-Hep encodes a peptide of 90 amino acids, with a predicted active peptide segment (Ma-sHep) of 25 amino acids at C terminus. Stimulation by a bacterial pathogen Aeromonas hydrophila resulted in significant up-regulation of Ma-Hep transcripts in loach midgut, head kidney, and gill. Ma-Hep and Ma-sHep proteins were expressed in Pichia pastoris and their antibacterial activity was examined. Results showed that Ma-sHep possessed stronger antibacterial activity against various Gram-positive and Gram-negative bacteria, compared to Ma-Hep. Scanning electron microscopy showed that Ma-sHep might kill bacteria by destroying bacterial cell membranes. Moreover, we found that Ma-sHep had an inhibitory effect on blood cell apoptosis induced by A. hydrophila and facilitated the bacterial phagocytosis and clearance in loach. Histopathological analysis indicated Ma-sHep could protect liver and gut of loach from bacterial infection. Ma-sHep has high thermal stability and PH stability, which is conducive to further feed addition. Feed supplemented with Ma-sHep expressing yeast improved the intestinal flora of loach by increasing the dominant bacteria and decreasing the harmful bacteria. Feed supplemented with Ma-sHep expressing yeast also regulated the expression of inflammatory related factors in various tissues of loach and reduced the mortality of loach upon bacterial infection. These findings show that the antibacterial peptide Ma-sHep is involved in the antibacterial defense of loach and can be used as a candidate for new antimicrobial agents in aquaculture.

Modulation of the gut microbiome with nisin.

Nisin is a broad spectrum bacteriocin used extensively as a food preservative that was identified in Lactococcus lactis nearly a century ago. We show that orally-ingested nisin survives transit through the porcine gastrointestinal tract intact (as evidenced by activity and molecular weight determination) where it impacts both the composition and functioning of the microbiota. Specifically, nisin treatment caused a reversible decrease in Gram positive bacteria, resulting in a reshaping of the Firmicutes and a corresponding relative increase in Gram negative Proteobacteria. These changes were mirrored by the modification in relative abundance of pathways involved in acetate, butyrate (decreased) and propionate (increased) synthesis which correlated with overall reductions in short chain fatty acid levels in stool. These reversible changes that occur as a result of nisin ingestion demonstrate the potential of bacteriocins like nisin to shape mammalian microbiomes and impact on the functionality of the community.

Molecular Modelling Study and Antibacterial Evaluation of Diphenylmethane Derivatives as Potential FabI Inhibitors.

The need for new antibiotics has become a major worldwide challenge as bacterial strains keep developing resistance to the existing drugs at an alarming rate. Enoyl-acyl carrier protein reductases (FabI) play a crucial role in lipids and fatty acid biosynthesis, which are essential for the integrity of the bacterial cell membrane. Our study aimed to discover small FabI inhibitors in continuation to our previously found hit MN02. The process was initially started by conducting a similarity search to the NCI ligand database using MN02 as a query. Accordingly, ten compounds were chosen for the computational assessment and antimicrobial testing. Most of the compounds showed an antibacterial activity against Gram-positive strains, while RK10 exhibited broad-spectrum activity against both Gram-positive and Gram-negative bacteria. All tested compounds were then docked into the saFabI active site followed by 100 ns MD simulations (Molecular Dynamics) and MM-GBSA (Molecular Mechanics with Generalised Born and Surface Area Solvation) calculations in order to understand their fitting and estimate their binding energies. Interestingly, and in line with the experimental data, RK10 was able to exhibit the best fitting with the target catalytic pocket. To sum up, RK10 is a small compound with leadlike characteristics that can indeed act as a promising candidate for the future development of broad-spectrum antibacterial agents.

Domain shuffling of a highly mutable ligand-binding fold drives adhesin generation across the bacterial kingdom.

Bacterial fibrillar adhesins are specialized extracellular polypeptides that promote the attachment of bacteria to the surfaces of other cells or materials. Adhesin-mediated interactions are critical for the establishment and persistence of stable bacterial populations within diverse environmental niches and are important determinants of virulence. The fibronectin (Fn)-binding fibrillar adhesin CshA, and its paralogue CshB, play important roles in host colonization by the oral commensal and opportunistic pathogen Streptococcus gordonii. As paralogues are often catalysts for functional diversification, we have probed the early stages of structural and functional divergence in Csh proteins by determining the X-ray crystal structure of the CshB adhesive domain NR2 and characterizing its Fn-binding properties in vitro. Despite sharing a common fold, CshB_NR2 displays an ~1.7-fold reduction in Fn-binding affinity relative to CshA_NR2. This correlates with reduced electrostatic charge in the Fn-binding cleft. Complementary bioinformatic studies reveal that homologues of CshA/B_NR2 domains are widely distributed in both Gram-positive and Gram-negative bacteria, where they are found housed within functionally cryptic multi-domain polypeptides. Our findings are consistent with the classification of Csh adhesins and their relatives as members of the recently defined polymer adhesin domain (PAD) family of bacterial proteins.