Bacterial Cells Research Articles

A synthetic antibiotic class with a deeply-optimized design for overcoming bacterial resistance

The lack of new drugs that are effective against antibiotic-resistant bacteria has caused increasing concern in global public health. Based on this study, we report development of a modified antimicrobial drug through structure-based drug design (SBDD) and modular synthesis. The optimal modified compound, F8, was identified, which demonstrated in vitro and in vivo broad-spectrum antibacterial activity against drug-resistant bacteria and effectively mitigated the development of resistance. F8 exhibits significant bactericidal activity against bacteria resistant to antibiotics such as methicillin, polymyxin B, florfenicol (FLO), doxycycline, ampicillin and sulfamethoxazole. In a mouse model of drug-resistant bacteremia, F8 was found to increase survival and significantly reduce bacterial load in infected mice. Multi-omics analysis (transcriptomics, proteomics, and metabolomics) have indicated that ornithine carbamoyl transferase (arcB) is a antimicrobial target of F8. Further molecular docking, Isothermal Titration Calorimetry (ITC), and Differential Scanning Fluorimetry (DSF) studies verified arcB as a effective target for F8. Finally, mechanistic studies suggest that F8 competitively binds to arcB, disrupting the bacterial cell membrane and inducing a certain degree of oxidative damage. Here, we report F8 as a promising candidate drug for the development of antibiotic formulations to combat antibiotic-resistant bacteria-associated infections.

The ability of photochemical decomposition products of the Roundup to induce oxidative stress in bacterial cells

BACKGROUND: A common non-selective systemic herbicide Roundup (Glyphosate, active ingredient N-phosphonomethylglycine, N-PMG) is used to control perennial weeds. It is necessary to assess the hazard of the products of photochemical decomposition of N-FMG formed under the influence of solar UV and ozone. AIM: Using lux-biosensors based on Escherichia coli, studying the ability of N-FMG photochemical degradation products to induce oxidative stress in bacterial cells. MATERIALS AND METHODS: The work used the active substance of the herbicide Roundup N-phosphonomethylglycine (N-PMG), biosensors E. coli (pSoxS-lux), E. coli (pKatG-lux). UV radiation, Mass spectrometry. RESULTS: Using biosensors, it was shown that the products of photochemical decomposition of N-PMG (2-(N-hydroxymethyl-hydroxyamine) ethanoic acid) cause an increase in the concentration of superoxide anion radical and H2O2 in E. coli cells, which induces oxidative stress in the bacterial cell. CONCLUSIONS: The photochemical decomposition product of N-PMG (2-(N-hydroxymethyl-hydroxyamine) ethanoic acid) induces the formation of superoxide anion radical and H2O2 in bacterial cells.

When the Host Encounters the Cell Wall and Vice Versa.

Peptidoglycan (PGN) and associated surface structures such as secondary polymers and capsules have a central role in the physiology of bacteria. The exoskeletal PGN heteropolymer is the major determinant of cell shape and allows bacteria to withstand cytoplasmic turgor pressure. Thus, its assembly, expansion, and remodeling during cell growth and division need to be highly regulated to avoid compromising cell survival. Similarly, regulation of the assembly impacts bacterial cell shape; distinct shapes enhance fitness in different ecological niches, such as the host. Because bacterial cell wall components, in particular PGN, are exposed to the environment and unique to bacteria, these have been coopted during evolution by eukaryotes to detect bacteria. Furthermore, the essential role of the cell wall in bacterial survival has made PGN an important signaling molecule in the dialog between host and microbes and a target of many host responses. Millions of years of coevolution have resulted in a pivotal role for PGN fragments in shaping host physiology and in establishing a long-lasting symbiosis between microbes and the host. Thus, perturbations of this dialog can lead to pathologies such as chronic inflammatory diseases. Similarly, pathogens have devised sophisticated strategies to manipulate the system to enhance their survival and growth.

Deciphering the Relationship between Cell Growth and Cell Cycle in Individual Escherichia coli Cells by Flow Cytometry.

Accurate coordination of chromosome replication and cell division is essential for cellular processes, yet the regulatory mechanisms governing the bacterial cell cycle remain contentious. The lack of quantitative data connecting key cell cycle players at the single-cell level across large samples hinders consensus. Employing high-throughput flow cytometry, we quantitatively correlated the expression levels of key cell cycle proteins (FtsZ, MreB, and DnaA) with DNA content in individual bacteria. Our findings reveal distinct correlations depending on the chromosome number (CN), specifically whether CN ≤2 or ≥4, unveiling a mixed regulatory scenario in populations where CN of 2 or 4 coexist. We observed function-dependent regulations for these key proteins across nonoverlapping division cycles and various nutrient conditions. Notably, a logarithmic relationship between total protein content and replication origin number across nutrient conditions suggests a unified mechanism governing cell cycle progression, confirming the applicability of Schaechter's growth law to cells with CN ≥4. For the first time, we established a proportional relationship between the synthesis rates of key cell cycle proteins and chromosome dynamics in cells with CN ≥4. Drug experiments highlighted CN 2 and 4 as pivotal turning points influencing cellular resource allocation. This high-throughput, single-cell analysis provides interconnected quantitative insights into key molecular events, facilitating a predictive understanding of the relationship between cell growth and cell cycle.

Trojan Horse virus delivering CRISPR-AsCas12f1 controls plant bacterial wilt caused by Ralstonia solanacearum.

Plant bacterial wilt caused by Ralstonia solanacearum results in huge losses. Accordingly, developing an effective control method for this disease is urgently required. Filamentous phages, which do not lyse host bacteria and exert minimal burden, offer a potential biocontrol solution. A filamentous phage RSCq that infects R. solanacearum was isolated in this study through genome mining. We constructed engineered filamentous phages based on RSCq by employing our proposed approach with wide applicability to non-model phages, enabling the exogenous genes delivery into bacterial cells. CRISPR-AsCas12f1 is a miniature class 2 type V-F CRISPR-Cas system. A CRISPR-AsCas12f1-based gene editing system that targets the key virulence regulator gene hrpB was developed, generating the engineered phage RSCqCRISPR-Cas. Similar to the Greek soldiers in the Trojan Horse, our findings demonstrated that the engineered phage-delivered CRISPR-Cas system could disarm the key "weapon," hrpB, of R. solanacearum, in medium and plants. Remarkably, pretreatment with RSCqCRISPR-Cas significantly controlled tobacco bacterial wilt, highlighting the potential of engineered filamentous phages as promising biocontrol agents against plant bacterial diseases.IMPORTANCEBacterial disease, one of the major plant diseases, causes huge food and economic losses. Phage therapy, an environmentally friendly control strategy, has been frequently reported in plant bacterial disease control. However, host specificity, sensitivity to ultraviolet light and certain conditions, and bacterial resistance to phage impede the widespread application of phage therapy in crop production. Filamentous phages, which do not lyse host bacteria and exert minimal burden, offer a potential solution to overcome the limitations of lytic phage biocontrol. This study developed a genetic engineering approach with wide applicability to non-model filamentous phages and proved the application possibility of engineered phage-based gene delivery in plant bacterial disease biocontrol for the first.

Comparative study of the structural, magnetic and electrical properties of cuprous delafossites CuBO2(B = Zn, Mn and Er) prepared using the flash auto-combustion technique

Abstract The flash auto-combustion method was utilized to produce Cu-based delafossites of CuBO2 (B = Zn, Mn, and Er). X-ray diffraction (XRD), high-resolution transmission electron microscopy (HRTEM), and X-ray photoelectron spectroscopy (XPS) were employed to verify the phase formation, surface morphology, and oxidation states of the synthesized delafossite samples. The crystallite sizes were determined to be 43, 16.76, and 21.66 nm for CuZnO2, CuMnO2, and CuErO2 nanoparticles (NPs), respectively. The magnetic characteristics of CuZnO2, CuMnO2, and CuErO2 samples were studied at room temperature, revealing their paramagnetic nature through the hysteresis effect. The Seebeck coefficient (S) for CuZnO2 was found to be positive, while it was negative for CuMnO2 and CuErO2. The thermoelectric power of CuZnO2 NPs was high, indicating their potential as materials for more efficient thermoelectric devices. Additionally, CuZnO2 exhibited an antimicrobial response against four-gram (+ve) bacteria, four-gram (-ve) bacteria, and the fungus Candida albicans (CA). The data obtained demonstrated that CuZnO2 NPs altered bacterial cell morphology, ultimately leading to bacterial cell death.

Influence of riboflavin on the corrosion of X80 pipeline steel by Sulfate reducing bacteria

Abstract The sulfate reducing bacteria(SRB) is commonly attached to the surface of buried pipeline steel, and the electron shuttle in the corrosion medium can promote the release of electrons from iron oxidation through the bacterial cell wall into the cytoplasm to accelerate the corrosion of anode iron. This study investigated the impact of riboflavin (RF) as an endogenous electron shuttle on the corrosion behavior of X80 pipeline steel in SRB system. The findings indicated that while the type of corrosion products remains unchanged in samples under SRB+10mg/L RF system, there was an expansion in both area and depth of corrosion pits on the sample surface, resulting in a corrosion loss rate approximately 3 times higher than that observed in SRB system. Furthermore, the polarization resistance (Rp) value of the sample in SRB system is about 2~5 times that of the sample in SRB+10 mg/L RF system. Additionally, the corrosion current density of X80 pipeline steel samples soaked in SRB and SRB+10 mg/L RF system for 14 days is 9.31×10-6 A·cm-2 and 1.28×10-5 A·cm-2, and the addition of 10 mg/L RF increases the corrosion current density of SRB system by about 37.49%. These results indicated that the reaction resistance of SRB-induced MIC in X80 pipeline steel was significantly reduced due to the presence of RF.

Film-Forming Capability and Antibacterial Activity of Surface-Deacetylated Chitin Nanocrystals: Role of Degree of Deacetylation.

Developing sustainable food-active packaging materials is a major issue in food preservation applications. Chitin nanocrystals (ChNCs) are regarded as unique bioderived nanomaterials due to their inherent nitrogen moiety. By tuning the chemical functionality of this nanomaterial, it is possible to affect its properties, such as film-forming capability and antibacterial activity. In this work, surface-deacetylated chitin nanocrystals (D-ChNCs) with different degrees of deacetylation (DDs) were prepared by partial deacetylation of native chitin and subsequent acid hydrolysis, and their film-forming capability and antibacterial activity were studied systematically. The D-ChNCs showed favorable film-forming ability and antibacterial activity, which are closely related to their DD. With the increase in DD (from 5.7% to 45.4%), the formed transparent films based on ChNCs showed gradually increased elongation at break (from 0.5% to 2.5%) and water contact angle (from 25.5° to 87.0°), but decreased break strength (from 3.13 to 0.89 MPa), Young's modulus (from 0.84 to 0.24 MPa), and water vapor permeability (from 4.7 × 10-10 to 4.1 × 10-10g/m s Pa). Moreover, the antibacterial activity of the D-ChNCs against E. coli and S. aureus also increased with the increase of DD. This study also found that the depolarization and potential dissipation of the bacterial cell membrane induced by the contact between amino-rich D-ChNCs and bacteria through electrostatic attraction are the possible mechanisms causing bacterial cell death. This study provides a basis for understanding the effects of DD on the film-forming capability and antibacterial activity of ChNCs, which is conducive to the design of novel active packaging films based on ChNCs.

Coumarin derivatives ameliorate the intestinal inflammation and pathogenic gut microbiome changes in the model of infectious colitis through antibacterial activity

Coumarin, a phenolic compound, is a secondary metabolite produced by plants such as Tanga and Lime. Coumarin derivatives were prepared via Pechmann condensation. In this study, we performed in vitro and in vivo experiments to determine the antimicrobial and gut immune-regulatory functions of coumarin derivatives. For the in vitro antimicrobial activity assay, coumarin derivatives C1 and C2 were selected based on their pathogen-killing activity against various pathogenic microbes. We further demonstrated that the selected coumarin derivatives disrupted bacterial cell membranes. Next, we examined the regulatory function of the coumarin derivatives in gut inflammation using an infectious colitis model. In an in vivo infectious colitis model, administration of selected C1 coumarin derivatives reduced pathogen loads, the number of inflammatory immune cells (Th1 cells and Th17 cells), and inflammatory cytokine levels (IL-6 and IL-1b) in the intestinal tissue after pathogen infection. In addition, we found that the administration of C1 coumarin derivatives minimized abnormal gut microbiome shift-driven pathogen infection. Potential pathogenic gut microbes, such as Enterobacteriaceae and Staphylococcaceae, were increased by pathogen infection. However, this pathogenic microbial expansion was minimized and beneficial bacteria, such as Ligilactobacillus and Limosilactobacillus, increased with C1 coumarin derivative treatment. Functional gene enrichment assessment revealed that the relative abundance of genes associated with lipid and nucleotide metabolism was reduced by pathogen infection; however, this phenomenon was not observed in C1 coumarin derivative-treated animals. Collectively, our data suggest that C1 coumarin derivative is effective antibacterial agents that minimize pathogen-induced gut inflammation and abnormal gut microbiome modulation through their antibacterial activity.

Determination of bisphosphonate properties in terms of bioavailability, bone affinity, and cytotoxicity.

The study aimed to evaluate the therapeutic potential of fourteen newly synthesized bisphosphonates by assessing their bioavailability, bone affinity, and cytotoxicity. These bisphosphonates included a series of aminomethylenebisphosphonates and standard compounds such as risedronate and tiludronate. Drug permeability was determined using Parallel Artificial Membrane Permeability Assays (PAMPA), while bone affinity was assessed by sorption on hydroxyapatite. Bacterial cell response to the bisphosphonates was also examined using Lactobacillus paracasei cells as a model. Several tested compounds, including BP3 to BP8 and BP11, which feature substituents in the pyridine ring such as methyl groups, iodine, bromine, chlorine, or hydroxyl groups, demonstrated potentially more beneficial therapeutic properties than commercially used bisphosphonates. These compounds showed stronger bone affinity and higher gastrointestinal absorption with comparable or lower cytotoxic effects. Specifically, BP11 exhibited the highest bone affinity, while BP8 and BP11 showed the greatest permeability. The findings suggest that BP3 BP8, and BP11 are promising candidates for further research. These results highlight the importance of comprehensively evaluating bisphosphonates' therapeutic properties to identify effective treatments for osteoporosis and other bone diseases.

Revolutionizing Antibacterial Strategies: Lipid Nanoparticles as Game-Changers in Combatting Multi-Drug-Resistant Infections.

To overcome the limits of traditional antibiotic medications, novel approaches are needed to combat the growing global epidemic of Multidrug-resistant (MDR) infections. As drug-resistant bacteria develop, the importance of innovative antimicrobial methods is underscored by antibiotic abuse and misuse. The global threat of MDR microorganisms is increasing, which calls for a coordinated global response. Lipid Nanoparticles (LNPs) possess several characteristics that make them attractive choices for managing multidrug resistant (MDR) infections, as well as potential delivery systems for antimicrobial agents. Thus, LNPs improve drug solubility, stability, and targeted delivery, thereby mitigating the drawbacks of conventional antibiotic therapy. Several characteristics of LNPs, which stop MDR bacteria from developing resistance mechanisms, serve as guidelines for precision medicine. It presents a powerful approach for combating the growing concern of MDR bacteria by increasing Anti-Microbial Peptides (AMPs) bioavailability and targeting distribution to bacterial cells. LNPs have the potential to redefine antibacterial treatments for MDR illnesses in the context of this study. Further, it discusses LNP use in larger applications, such as fighting Anti-Microbial Resistance (AMR) and MDR. A complete understanding of the unique features, many uses, and importance of collaborative efforts to overcome the global challenge of antibiotic resistance are also conveyed in the study.

Surface-Charge Tuned Polymeric Nanoemulsions for Carvacrol Delivery in Interkingdom Biofilms.

Biofilms, intricate microbial communities entrenched in extracellular polymeric substance (EPS) matrices, pose formidable challenges in infectious disease treatment, especially in the context of interkingdom biofilms prevalent in the oral environment. This study investigates the potential of carvacrol-loaded biodegradable nanoemulsions (NEs) with systematically varied surface charges─cationic guanidinium (GMT-NE) and anionic carboxylate (CMT-NE). Zeta potentials of +25 mV (GMT-NE) and -33 mV (CMT-NE) underscore successful nanoemulsion fabrication (∼250 nm). Fluorescent labeling and dynamic tracking across three dimensions expose GMT-NE's superior diffusion into oral biofilms, yielding a robust antimicrobial effect with 99.99% killing for both streptococcal and Candida species and marked reductions in bacterial cell viability compared to CMT-NE (∼4-log reduction). Oral mucosa tissue cultures affirm the biocompatibility of both NEs with no morphological or structural changes, showcasing their potential for combating intractable biofilm infections in oral environment. This study advances our understanding of NE surface charges and their interactions within interkingdom biofilms, providing insights crucial for addressing complex infections involving bacteria and fungi in the demanding oral context.

Multimodal Affinity-Modulated Efficient Separation of Lysozyme with a Hierarchical MXene@MOF Hybrid Framework.

The development of abiotic protein affinity adsorbents remains challenging for the accurate acquisition and analysis of specific protein species. Inspired by bacterial cell walls, a hierarchical hybrid framework is fabricated through the oriented growth of an Fe-based metal organic framework (MOF) on V2C MXene for the efficient separation of lysozyme (Lys). After directed evolution of adsorptive materials, the MXene@MOF composite rich in hydroxyl groups (termed as MX@MOF-DH) is found exerting exceptional affinity for Lys. Benefiting from hydrogen-bonding, coordination, and electrostatic interaction-mediated multimodal and multivalent affinity, MX@MOF-DH reveals rapid adsorption rate (5 min), superb enrichment factor (83.1), and favorable binding capacity (609.7 mg g-1), which outperforms other latest adsorbents. Moreover, femtomolar sensitivity is achieved even in the presence of high-abundant interfering proteins, as confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometer analysis. This work not only provides an efficient approach for selective enrichment of lysozyme but also paves an avenue to construct the protein affinity reagents for specific biological medicine and analysis applications.

Electric wiring of bacteria using redox polymers and selective measurement of metabolic activity in the presence of surrounding planktonic bacteria

Non-electroactive bacteria (n-EAB), constituting the majority of known bacteria to date, have been underutilized in electrochemical conversion technologies due to their lack of direct electron transfer to electrodes. In this study, we established an electric wiring between n-EAB (gram-positive Bacillus subtilis and gram-negative Escherichia coli) and an extracellular electrode via a ferrocene-polyethyleneimine-based redox polymer (Fc-PEI). Chronoamperometry recordings indicated that Fc-PEI can transfer intracellular electrons to the extracellular electrode regardless of the molecular organization of PEI (linear or branched) and the membrane structure of bacteria (gram-positive or -negative). As fluorescence staining suggested, Fc-PEI improves the permeability of the bacterial cell membrane, enabling electron carriers in the cell to react with Fc. In addition, experiments with Fc-immobilized electrodes without PEI suggested the existence of an alternative electron transfer pathway from B. subtilis to the extracellular Fc adsorbed onto the cell membrane. Furthermore, we proposed for the first time that the bacteria/Fc-linear PEI modified structure enables selective measurement of immobilized bacterial activity by physically blocking contact between the electrode surface and planktonic cells co-existing in the surrounding media. Such electrodes can be a powerful analytical tool for elucidating the metabolic activities of specific bacteria wired to the electrode even within complex bacterial communities.

Cancer Antibody Engineering: Comparison of Mammalian, Yeast, Bacterial, Plants, Cell-free and Hybridoma Expression Systems.

Cancer is a significant issue worldwide. Generally, commercially available treatments, such as surgery, radiotherapy, and chemotherapy, are associated with undesirable complications. Hence, immunotherapy serves as a crucial alternative to those treatment options. This modality is aimed to boost the immune system through the application of engineered antibodies, which can be produced using recombinant DNA technology. The discussion of the technologies leads to an introduction of the single-chain variable fragment (scFv). Thereafter, the advantages, disadvantages, and challenges associated with different expression systems, such as mammalian cells, yeast cells, bacterial cells, plant cells, and phage display were discussed comprehensively. Furthermore, conventional approaches such as hybridoma and modern approaches such as cell-free protein synthesis (CFPS) and simple colony assays are included. In short, this article has compiled evidence relating to each display system and may serve as a reference for those who aim to explore antibody engineering using one of the methods listed in this article.

The Efficiency of Flaxseed Against Cancer and Bacterial Infection

Flaxseed ( Linum usitatissimum) has garnered attention for its potential health benefits , particularly its role in combating colon cancer and bacterial infections . This study aims to elucidate the mechanisms by which flaxseed exerts its effects and evaluate its overall efficacy. A comprehensive review of existing literature was conducted , alongside laboratory involving in vitro models . The anticancer effects were evaluated using human colon cancer cell lines, while antibacterial properties were tested against a spectrum of pathogenic bacteria. Flaxseed exhibited significant antiproliferative effects on colon cancer cells ,primarily through the induction of apoptosis and inhibition of cell cycle progression . Key bioactive compounds, such as lignans , omega-3fatty acids, and fiber, were identified as major contributors to these effects. In antibacterial assays, flaxseed demonstrated broad-spectrum activity, inhibiting the growth of several clinically relevant bacteria strains. The presence of lignans and omega-3fatty acids played a crucial role in disrupting bacterial cell walls and interfering with metabolic processes. The findings suggest that flaxseed holds substantial promise as a natural therapeutic agent against colon cancer and bacterial infections. Its dual action , mediated by a complex interplay of bioactive compounds , highlights its potential for inclusion in dietary strategies and development into functional foods or complementary therapies.

A classic antibiotic reimagined: Rationally designed bacitracin variants exhibit potent activity against vancomycin-resistant pathogens

Bacitracin is a macrocyclic peptide antibiotic that is widely used as a topical treatment for infections caused by gram-positive bacteria. Mechanistically, bacitracin targets bacteria by specifically binding to the phospholipid undecaprenyl pyrophosphate (C55PP), which plays a key role in the bacterial lipid II cycle. Recent crystallographic studies have shown that when bound to C55PP, bacitracin adopts a highly ordered amphipathic conformation. In doing so, all hydrophobic side chains align on one face of the bacitracin-C55PP complex, presumably interacting with the bacterial cell membrane. These insights led us to undertake structure-activity investigations into the individual contribution of the nonpolar amino acids found in bacitracin. To achieve this we designed, synthesized, and evaluated a series of bacitracin analogues, a number of which were found to exhibit significantly enhanced antibacterial activity against clinically relevant, drug-resistant pathogens. As for the natural product, these next-generation bacitracins were found to form stable complexes with C55PP. The structure-activity insights thus obtained serve to inform the design of C55PP-targeting antibiotics, a key and underexploited antibacterial strategy.

Molecular dynamics simulations reveal differences in the conformational stability of FtsZs derived from Staphylococcus aureus and Bacillus subtilis

FtsZ is highly conserved among bacteria and plays an essential role in bacterial cell division. The tense conformation of FtsZ bound to GTP assembles into a straight filament via head-to-tail associations, and then the upper subunit of FtsZ hydrolyzes GTP bound to the lower FtsZ subunit. The subunit with GDP bound disassembles accompanied by a conformational change in the subunit from the tense to relaxed conformation. Although crystal structures of FtsZ derived from several bacterial species have been determined, the conformational change from the relaxed to tense conformation has only been observed in Staphylococcus aureus FtsZ (SaFtsZ). Recent cryo-electron microscopy analyses revealed the three-dimensional reconstruction of the protofilament, in which tense molecules assemble via head-to-tail associations. However, the lower resolution of the protofilament suggested that the flexibility of the FtsZ protomers between the relaxed and tense conformations caused them to form in less-strict alignments. Furthermore, this flexibility may also prevent FtsZs other than SaFtsZ from crystalizing in the tense conformation, suggesting that the flexibility of bacterial FtsZs differs. In this study, molecular dynamics simulations were performed using SaFtsZ and Bacillus subtilis FtsZ in several situations, which suggested that different features of the FtsZs affect their conformational stability.

Initiation of H1-T6SS dueling between Pseudomonas aeruginosa.

The Type VI secretion system (T6SS) is a multicomponent apparatus, present in many Gram-negative bacteria, which can inhibit bacterial prey in various ecological niches. Pseudomonas aeruginosa assembles one of its three T6SS (H1-T6SS) to respond to attacks from adjacent competing bacteria. Surprisingly, repeated assemblies of the H1-T6SS, termed dueling, were described in a monoculture in the absence of an attacker strain; however, the underlying mechanism was unknown. Here, we explored the role of H2-T6SS of P. aeruginosa in triggering H1-T6SS assembly. We show that H2-T6SS inactivation in P. aeruginosa causes a significant reduction in H1-T6SS dueling and that H2-T6SS activity directly triggers retaliation by the H1-T6SS. Intraspecific competition experiments revealed that elimination of H2-T6SS in non-immune prey cells conferred protection from H1-T6SS. Moreover, we show that the H1-T6SS response is triggered independently of the characterized lipase effectors of the H2-T6SS, as well as those of Acinetobacter baylyi and Vibrio cholerae. Our results suggest that H1-T6SS response to H2-T6SS in P. aeruginosa can impact intraspecific competition, particularly when the H1-T6SS effector-immunity pairs differ between strains, and could determine the outcome of multistrain colonization.IMPORTANCEThe opportunistic pathogen Pseudomonas aeruginosa harbors three different Type VI secretion systems (H1, H2, and H3-T6SS), which can translocate toxins that can inhibit bacterial competitors or inflict damage to eukaryotic host cells. Unlike the unregulated T6SS assembly in other Gram-negative bacteria, the H1-T6SS in P. aeruginosa is precisely assembled as a response to various cell damaging attacks from neighboring bacterial cells. Surprisingly, it was observed that neighboring P. aeruginosa cells repeatedly assemble their H1-T6SS toward each other. Mechanisms triggering this "dueling" behavior between sister cells were unknown. In this report, we used a combination of microscopy, genetic and intraspecific competition experiments to show that H2-T6SS initiates H1-T6SS dueling. Our study highlights the interplay between different T6SS clusters in P. aeruginosa, which may influence the outcomes of multistrain competition in various ecological settings such as biofilm formation and colonization of cystic fibrosis lungs.

High throughput platform technology for rapid target identification in personalized phage therapy

As bacteriophages continue to gain regulatory approval for personalized human therapy against antibiotic-resistant infections, there is a need for transformative technologies for rapid target identification through multiple, large, decentralized therapeutic phages biobanks. Here, we design a high throughput phage screening platform comprised of a portable library of individual shelf-stable, ready-to-use phages, in all-inclusive solid tablets. Each tablet encapsulates one phage along with luciferin and luciferase enzyme stabilized in a sugar matrix comprised of pullulan and trehalose capable of directly detecting phage-mediated adenosine triphosphate (ATP) release through ATP bioluminescence reaction upon bacterial cell burst. The tablet composition also enhances desiccation tolerance of all components, which should allow easier and cheaper international transportation of phages and as a result, increased accessibility to therapeutic phages. We demonstrate high throughput screening by identifying target phages for select multidrug-resistant clinical isolates of Pseudomonas aeruginosa, Salmonella enterica, Escherichia coli, and Staphylococcus aureus with targets identified within 30-120 min.