Pseudomonas aeruginosa, a gram-negative bacterium of clinical importance, forms more robust biofilm during anaerobic respiration, a mode of growth presumed to occur in abnormally thickened mucus layer lining the cystic fibrosis (CF) patient airway. However, molecular basis behind this anaerobiosis-triggered robust biofilm formation is not clearly defined yet. Here, we identified a morphological change naturally accompanied by anaerobic respiration in P. aeruginosa and investigated its effect on the biofilm formation in vitro. A standard laboratory strain, PAO1 was highly elongated during anaerobic respiration compared with bacteria grown aerobically. Microscopic analysis demonstrated that cell elongation likely occurred as a consequence of defective cell division. Cell elongation was dependent on the presence of nitrite reductase (NIR) that reduces nitrite (NO2 −) to nitric oxide (NO) and was repressed in PAO1 in the presence of carboxy-PTIO, a NO antagonist, demonstrating that cell elongation involves a process to respond to NO, a spontaneous byproduct of the anaerobic respiration. Importantly, the non-elongated NIR-deficient mutant failed to form biofilm, while a mutant of nitrate reductase (NAR) and wild type PAO1, both of which were highly elongated, formed robust biofilm. Taken together, our data reveal a role of previously undescribed cell biological event in P. aeruginosa biofilm formation and suggest NIR as a key player involved in such process.

Background Staphylococcus epidermidis is one of the most abundant colonizers of healthy human mucosa including that in the respiratory tract. As the respiratory microbiome has been linked to host immune responses, this study sought to determine the role of nasal mucosa-associated S. epidermidis in innate immune responses against the influenza A virus (IAV). S. epidermidis strains were isolated from nasal mucus samples of healthy individuals. The effects of these mucosa-derived commensal strains on interferon (IFN)-dependent innate immunity and IAV infection dynamics were tested in vitro using normal human nasal epithelial (NHNE) cells and human turbinate mucosa. The effects of S. epidermidis on antiviral immunity were also tested in vivo using an acute IAV infection mouse model. Results Exposure of NHNE cells to nasal mucosa-derived S. epidermidis increased IFN-λ mRNA and secreted protein levels in the absence of viral stimulation. In the context of IAV infection, NHNE exposure to S. epidermidis prevented an increase in the viral burden, as revealed by IAV PA mRNA abundance, IAV nucleoprotein levels, and viral titers. S. epidermidis also enhanced transcription of IFN-stimulated genes independently of Toll-like receptor 2 and further induced IFN-λ production in IAV-infected cells by promoting phosphorylation of interferon regulatory factor 7. In a murine infection model, S. epidermidis prevented the spread of IAV to the lungs by stimulating IFN-λ innate immunity and suppressing IAV replication in the nasal mucosa. Conclusion The human nasal commensal S. epidermidis mediates front-line antiviral protection against IAV infection through modulation of IFN-λ-dependent innate immune mechanisms in the nasal mucosa, thereby demonstrating the role of host-bacterial commensalism in shaping human antiviral responses. Electronic supplementary material The online version of this article (10.1186/s40168-019-0691-9) contains supplementary material, which is available to authorized users.

Pseudomonas aeruginosa establishes airway infections in Cystic Fibrosis patients. Here, we investigate the molecular interactions between P. aeruginosa and airway mucus secretions (AMS) derived from the primary cultures of normal human tracheal epithelial (NHTE) cells. PAO1, a prototype strain of P. aeruginosa, was capable of proliferating during incubation with AMS, while all other tested bacterial species perished. A PAO1 mutant lacking PA4834 gene became susceptible to AMS treatment. The ΔPA4834 mutant was grown in AMS supplemented with 100 μM ferric iron, suggesting that the PA4834 gene product is involved in iron metabolism. Consistently, intracellular iron content was decreased in the mutant, but not in PAO1 after the AMS treatment. Importantly, a PAO1 mutant unable to produce both pyoverdine and pyochelin remained viable, suggesting that these two major siderophore molecules are dispensable for maintaining viability during incubation with AMS. The ΔPA4834 mutant was regrown in AMS amended with 100 μM nicotianamine, a phytosiderophore whose production is predicted to be mediated by the PA4836 gene. Infectivity of the ΔPA4834 mutant was also significantly compromised in vivo. Together, our results identify a genetic element encoding a novel iron acquisition system that plays a previously undiscovered role in P. aeruginosa airway infection.

Background: The human intestine, in which Vibrio cholerae exerts its virulence, is an anaerobic environment. Results: When grown anaerobically with trimethylamine N-oxide (TMAO), V. cholerae exhibited enhanced growth and cholera toxin (CT) production was remarkably induced. Conclusion: Anaerobic TMAO respiration may serve as a signal to increase V. cholerae virulence. Significance: A novel growth condition that induces CT production is uncovered.

Highlights d Cleaved inner ear cochlin LCCL secretes to perilymph space post-bacterial infection d LCCL induces bacterial aggregation in the scala tympani d Spatiotemporal innate immune response by LCCL protects the sensory organ of Corti d LCCL rescues Coch À/À mouse post-Pseudomonas inner ear infection hearing loss

ABSTRACTPseudomonas aeruginosaundergoes cell elongation and forms robust biofilms during anaerobic respiratory growth using nitrate (NO3−) as an alternative electron acceptor. Understanding the mechanism of cell shape change induced upon anaerobiosis is crucial to the development of effective treatments againstP. aeruginosabiofilm infection. Here, we uncovered the molecular basis of anaerobiosis-triggered cell elongation and identified vitamin B12to be a molecule that can reinstate defective anaerobic growth ofP. aeruginosa. The ratio of total cellular DNA content to protein content was significantly decreased in the PAO1 strain grown under anaerobic conditions, indicating that DNA replication is impaired during anaerobic growth. Anaerobic growth of PAO1 reached a higher cell density in the presence of vitamin B12, an essential coenzyme of class II ribonucleotide reductase. In addition, cell morphology returned to a normal rod shape and transcription of stress-response genes was downregulated under the same anaerobic growth conditions. These results suggest that vitamin B12, the production of which was suppressed during anaerobic growth, can restore cellular machineries for DNA replication and therefore facilitate better anaerobic growth ofP. aeruginosawith normal cell division. Importantly, biofilm formation was substantially decreased when grown with vitamin B12, further demonstrating that anaerobiosis-induced cell elongation is responsible for robust biofilm formation. Taken together, our data reveal mechanistic details of a morphological change that naturally occurs during anaerobic growth ofP. aeruginosaand illustrates the ability of vitamin B12to modulate the biofilm-forming capacity ofP. aeruginosaunder such condition.

The pathogen is the causative agent of cholera. Emergence of antibiotic-resistant strains is increasing, but the underlying mechanisms remain unclear. Herein, we report that the stringent response regulator and stress alarmone guanosine tetra- and pentaphosphate ((p)ppGpp) significantly contributes to antibiotic tolerance in We found that N16961, a pandemic strain, and its isogenic (p)ppGpp-overexpressing mutant ΔΔ are both more antibiotic-resistant than (p)ppGpp (ΔΔΔ) and Δ mutants, which cannot produce or utilize (p)ppGpp, respectively. We also found that additional disruption of the aconitase B-encoding and tricarboxylic acid (TCA) cycle gene in the (p)ppGpp mutant increases its antibiotic tolerance. Moreover, expression of TCA cycle genes, including , was increased in (p)ppGpp, but not in the antibiotic-resistant ΔΔ mutant, suggesting that (p)ppGpp suppresses TCA cycle activity, thereby entailing antibiotic resistance. Importantly, when grown anaerobically or incubated with an iron chelator, the (p)ppGpp mutant became antibiotic-tolerant, suggesting that reactive oxygen species (ROS) are involved in antibiotic-mediated bacterial killing. Consistent with that hypothesis, tetracycline treatment markedly increased ROS production in the antibiotic-susceptible mutants. Interestingly, expression of the Fe(III) ABC transporter substrate-binding protein FbpA was increased 10-fold in (p)ppGpp, and gene deletion restored viability of tetracycline-exposed (p)ppGpp cells. Of note, FbpA expression was repressed in the (p)ppGpp-accumulating mutant, resulting in a reduction of intracellular free iron, required for the ROS-generating Fenton reaction. Our results indicate that (p)ppGpp-mediated suppression of central metabolism and iron uptake reduces antibiotic-induced oxidative stress in .

Herein, we introduce a simple route to fabricating hydrophilic microfluidic chips with an alternative material, a UV‐cured polyurethane‐related polymer, known as Norland Optical Adhesive (NOA 63). Conventionally, polydimethylsiloxane (PDMS) is widely used to fabricate microfluidic chips as an alternative to glass or SiO2 because PDMS is easily molded and relatively cheap. However, despite these advantages, the hydrophobicity of PDMS entails critical problems when it is used in microfluidic chips because microchannels inside the microfluidic chips, which have extremely low surface tension, are difficult to fill with aqueous solution without an extra pumping system. To overcome these problems, significant efforts have been focused on developing procedures to change the PDMS surface to be hydrophilic. However, the resulting hydrophilicity is generally short‐lived and the modification procedures require cumbersome multi‐steps. In the present study, we demonstrate that microchannel‐molding and microfluidic chip construction are easier using NOA 63 than when using PDMS and that the hydrophilicity of the NOA surface, which is induced by treatment with O2 plasma, lasts longer, for at least one month. Due to the longer lasting hydrophilicity, microchannels in NOA 63 microfluidic chips are spontaneously filled with solution by capillary reaction without any extra pumping over the period. The feasibility of NOA 63‐based microfabrication is verified by demonstrating NOA 63 microfluidic platforms with antibody‐immobilized beads for immunoassays.