Membrane vesicles released by Pseudomonas aeruginosa PA14 can elicit septic acute lung injury due to loss of endothelial barrier integrity and inflammasome activationhttps://bit.ly/3gMnkPu

Bacterial lung infections lead to greater than 4 million deaths per year with antibiotic treatments driving an increase in antibiotic resistance and a need to establish new therapeutic approaches. Recently, we have generated mouse and rat stem cell-derived alveolar-like macrophages (ALMs), which like primary alveolar macrophages (1'AMs), phagocytose bacteria and promote airway repair. Our aim was to further characterize ALMs and determine their bactericidal capabilities. The characterization of ALMs showed that they share known 1'AM cell surface markers, but unlike 1'AMs are highly proliferative in vitro. ALMs effectively phagocytose and kill laboratory strains of P. aeruginosa (P.A.), E. coli (E.C.) and S. aureus, and clinical strains of P.A. In vivo, ALMs remain viable, adapt additional features of native 1'AMs, but proliferation is reduced. Mouse ALMs phagocytose P.A. and E.C. and rat ALMs phagocytose and kill P.A. within the lung 24 h post-instillation. In a pre-clinical model of P.A.-induced lung injury, rat ALM administration mitigated weight loss and resolved lung injury observed seven days post-instillation. Collectively, ALMs attenuate pulmonary bacterial infections and promote airway repair. ALMs could be utilized as an alternative or adjuvant therapy where current treatments are ineffective against antibiotic-resistant bacteria or to enhance routine antibiotic delivery.

β-lactam (BL) antibiotics are the most prescribed antimicrobial class. The efficacy of β-lactams is threatened by the production of β-lactamase enzymes, the predominant resistance mechanism impacting these agents in Gram-negative bacterial pathogens.

The age-old saying, seeing is believing, could not be truer when we think about the value of imaging interactions between epithelial cells and bacterial pathogens. Imaging and culturing techniques have vastly improved over the years, and the breadth and depth of these methods is ever increasing. These technical advances have benefited researchers greatly; however, due to the large number of potential model systems and microscopy techniques to choose from, it can be overwhelming to select the most appropriate tools for your research question. This Review discusses a variety of available epithelial culturingmethods and quality control experiments that can be performed, and outlines various options commonly used to fluorescently label bacterial and mammalian cell components. Both light- and electron-microscopy techniques are reviewed, with descriptions of both technical aspects and common applications. Several examples of imaging bacterial pathogens and their interactions with epithelial cells are discussed to provide researchers with an idea of the types of biological questions that can be successfully answered by using microscopy.

Intracellular bacterial pathogens have evolved sophisticated infection strategies, including the release and secretion of virulence factors to interfere with host cell functions and to perturb immune responses. For Salmonella enterica serovar Typhimurium ( S . Typhimurium), the type III secretion systems encoded on Salmonella pathogenicity islands (SPI) 1 and 2 mediates invasion of the bacterium into innate immune cells and regulates bacterial replication and survival within the hostile environment of the host, respectively. Here, we explore the temporal and strain-specific dual perspective response of both the host and pathogen during cellular infection via quantitative proteomics. We report time- and pathogenicity island-specific expression and secretion of infection-associated proteins (i.e., SL1344_1263, SL1344_3112, SL1344_1563, and YnhG) and regulated immune response proteins in macrophage, including Cd86, Cd40, Casp4, C3, IL-1α, and Cd69). Through intracellular macrophage and in vivo murine models of infection, we reveal a role in virulence for three of the bacterial proteins (SL1344_1263, SL1344_1563, and YnhG), defining their importance as novel T3SS effectors. We characterize the temporal intra- and extracellular production of the effectors and identify their interaction networks in host cells, representing inhibitory and stimulatory pathways mounted by invading bacterial pathogens. Author Summary The relationship between a host and pathogen is intricate, and regulation of the host immune response correlates with the progressive timing of infection and tailored responses to the pathogen. Relying on detection and quantification of protein-level changes using mass spectrometry-based proteomics, we explore the production of known and novel effectors by Salmonella during intracellular survival within macrophage. Our results portray a role for these effectors in bacterial virulence using an in vivo murine model of infection, and we define a dynamic network of interaction between the effectors and host proteins. These interactions reveal opportunity for drug design to disrupt modulation of the host by the invading bacterium as a new strategy for combatting infection. Our approach is dynamic and universal, with the power to alter therapeutic discovery against infectious diseases.

The age-old saying, seeing is believing, could not be truer when we think about the value of imaging interactions between epithelial cells and bacterial pathogens. Imaging and culturing techniques have vastly improved over the years, and the breadth and depth of these methods is ever increasing. These technical advances have benefited researchers greatly; however, due to the large number of potential model systems and microscopy techniques to choose from, it can be overwhelming to select the most appropriate tools for your research question. This Review discusses a variety of available epithelial culturing methods and quality control experiments that can be performed, and outlines various options commonly used to fluorescently label bacterial and mammalian cell components. Both light- and electron-microscopy techniques are reviewed, with descriptions of both technical aspects and common applications. Several examples of imaging bacterial pathogens and their interactions with epithelial cells are discussed to provide researchers with an idea of the types of biological questions that can be successfully answered by using microscopy.

In Escherichia coli, the N-terminal domain of the essential protein FtsK (FtsKN) is proposed to modulate septum formation through the formation of dynamic and essential protein interactions with both the Z-ring and late-stage division machinery. Using genomic mutagenesis, complementation analysis, and in vitro pull-down assays, we aimed to identify protein interaction partners of FtsK essential to its function during division. Here, we identified the cytoplasmic Z-ring membrane anchoring protein FtsA as a direct protein-protein interaction partner of FtsK. Random genomic mutagenesis of an ftsK temperature-sensitive strain of E. coli revealed an FtsA point mutation (G50E) that is able to fully restore normal cell growth and morphology, and further targeted site-directed mutagenesis of FtsA revealed several other point mutations capable of fully suppressing the essential requirement for functional FtsK. Together, this provides insight into a potential novel co-complex formed between these components during division and suggests FtsA may directly impact FtsK function.

Our understanding of the biofilm matrix components utilized by Gram-positive bacteria, and the signalling pathways that regulate their production are largely unknown. In a companion study, we developed a computational pipeline for the unbiased identification of homologous bacterial operons and applied this algorithm to the analysis of synthase-dependent exopolysaccharide biosynthetic systems. Here, we explore the finding that many species of Gram-positive bacteria have operons with similarity to the Pseudomonas aeruginosa pel locus. Our characterization of the pelDEADAFG operon from Bacillus cereus ATCC 10987, presented herein, demonstrates that this locus is required for biofilm formation and produces a polysaccharide structurally similar to Pel. We show that the degenerate GGDEF domain of the B. cereus PelD ortholog binds cyclic-3',5'-dimeric guanosine monophosphate (c-di-GMP), and that this binding is required for biofilm formation. Finally, we identify a diguanylate cyclase, CdgF, and a c-di-GMP phosphodiesterase, CdgE, that reciprocally regulate the production of Pel. The discovery of this novel c-di-GMP regulatory circuit significantly contributes to our limited understanding of c-di-GMP signalling in Gram-positive organisms. Furthermore, conservation of the core pelDEADAFG locus amongst many species of bacilli, clostridia, streptococci, and actinobacteria suggests that Pel may be a common biofilm matrix component in many Gram-positive bacteria.

In Escherichia coli, formation of new cells is mediated by the elongasome and divisome that govern cell elongation and septation, respectively. Proper transition between these events is essential to ensure viable progeny are produced; however, the components of each complex responsible for transmission of the cell signal to shift from elongation to septation are unclear. Recently, a region within the N-terminal domain of the essential divisome protein FtsK (FtsK(N)) was identified that points to a key role for FtsK as a checkpoint of cell envelope remodeling during division. Here, we used site-specific in vivo UV cross-linking to probe the periplasmic loops of FtsK(N) for protein interaction partners critical for FtsK(N) function. Mass spectrometry analysis of five unique FtsK(N) periplasmic cross-links revealed a network of potential FtsK(N) interactors, one of which included the septal peptidoglycan binding protein rare lipoprotein A (RlpA). This protein was further verified as a novel interaction partner of FtsK(N) by an in vitro pull-down assay. Deletion of rlpA from an FtsK temperature-sensitive E. coli strain partially restored cell growth and largely suppressed cellular filamentation compared to the wild-type strain. This suggests that interaction with RlpA may be critical in suppressing septation until proper assembly of the divisome.

ABSTRACT Cystic fibrosis (CF) is caused by mutations in the CFTR gene and is associated with progressive and ultimately fatal infectious lung disease. There can be considerable variability in disease severity among individuals with the same CFTR mutations, and recent genome-wide association studies have identified secondary genetic factors that contribute to this. One of these modifier genes is SLC6A14, which encodes an amino acid transporter. Importantly, variants of this gene have been associated with age at first acquisition of Pseudomonas aeruginosa. In this study, we aimed to determine the function of SLC6A14 in airway epithelia and how it might affect colonization by P. aeruginosa. We show that SLC6A14 is expressed in respiratory epithelial cells and transports l-arginine out of the airway surface liquid (ASL). Exposure of airway epithelia to flagellin from P. aeruginosa led to upregulation of SLC6A14 expression and increased SLC6A14-dependent uptake of l-arginine from the ASL. In support of the hypothesis that l-arginine affects P. aeruginosa attachment, we showed that l-arginine supplementation promoted P. aeruginosa attachment to an abiotic surface in a dose-dependent manner. In a coculture model, we found that inhibition of SLC6A14-dependent l-arginine transport enhanced P. aeruginosa attachment. In Slc6a14−/y (knockout) mice, P. aeruginosa attachment to lung tissue was also significantly enhanced. Together, these findings suggest that SLC6A14 activity plays a role in the modification of the initial stages of airway infection by altering the level of l-arginine in the ASL, which in turn affects the attachment of P. aeruginosa. IMPORTANCE CF patients with shared CFTR gene mutations show significant variability in their clinical presentation of infectious lung disease. Genome-wide association studies have been used to identify secondary genetic factors that may explain the variable susceptibility to infection by opportunistic pathogens, including P. aeruginosa, the leading cause of pathogen-induced lung damage in nonpediatric CF patients. Once identified and characterized, these secondary genetic modifiers may allow for the development of personalized medicine for patients and ultimately the extension of life. In this study, we interrogated the biological role of one of these modifiers, SLC6A14, and showed that it contributes to host defense by depleting extracellular arginine (an attachment-promoting metabolite for P. aeruginosa) from the airway surface liquid.

ABSTRACT Bacterial cell division is an essential and highly coordinated process. It requires the polymerization of the tubulin homologue FtsZ to form a dynamic ring (Z-ring) at midcell. Z-ring formation relies on a group of FtsZ-associated proteins (Zap) for stability throughout the process of division. In Escherichia coli, there are currently five Zap proteins (ZapA through ZapE), of which four (ZapA, ZapB, ZapC, and ZapD) are small soluble proteins that act to bind and bundle FtsZ filaments. In particular, ZapD forms a functional dimer and interacts with the C-terminal tail of FtsZ, but little is known about its structure and mechanism of action. Here, we present the crystal structure of Escherichia coli ZapD and show it forms a symmetrical dimer with centrally located α-helices flanked by β-sheet domains. Based on the structure of ZapD and its chemical cross-linking to FtsZ, we targeted nine charged ZapD residues for modification by site-directed mutagenesis. Using in vitro FtsZ sedimentation assays, we show that residues R56, R221, and R225 are important for bundling FtsZ filaments, while transmission electron microscopy revealed that altering these residues results in different FtsZ bundle morphology compared to those of filaments bundled with wild-type ZapD. ZapD residue R116 also showed altered FtsZ bundle morphology but levels of FtsZ bundling similar to that of wild-type ZapD. Together, these results reveal that ZapD residues R116, R221, and R225 likely participate in forming a positively charged binding pocket that is critical for bundling FtsZ filaments. IMPORTANCE Z-ring assembly underpins the formation of the essential cell division complex known as the divisome and is required for recruitment of downstream cell division proteins. ZapD is one of several proteins in E. coli that associates with the Z-ring to promote FtsZ bundling and aids in the overall fitness of the division process. In the present study, we describe the dimeric structure of E. coli ZapD and identify residues that are critical for FtsZ bundling. Together, these results advance our understanding about the formation and dynamics of the Z-ring prior to bacterial cell division.

FtsZ is an essential cell division protein in Escherichia coli, and its localization, filamentation, and bundling at the mid-cell are required for Z-ring stability. Once assembled, the Z-ring recruits a series of proteins that comprise the bacterial divisome. Zaps (FtsZ-associated proteins) stabilize the Z-ring by increasing lateral interactions between individual filaments, bundling FtsZ to provide a scaffold for divisome assembly. The x-ray crystallographic structure of E. coli ZapA was determined, identifying key structural differences from the existing ZapA structure from Pseudomonas aeruginosa, including a charged α-helix on the globular domains of the ZapA tetramer. Key helix residues in E. coli ZapA were modified using site-directed mutagenesis. These ZapA variants significantly decreased FtsZ bundling in protein sedimentation assays when compared with WT ZapA proteins. Electron micrographs of ZapA-bundled FtsZ filaments showed the modified ZapA variants altered the number of FtsZ filaments per bundle. These in vitro results were corroborated in vivo by expressing the ZapA variants in an E. coli ΔzapA strain. In vivo, ZapA variants that altered FtsZ bundling showed an elongated phenotype, indicating improper cell division. Our findings highlight the importance of key ZapA residues that influence the extent of FtsZ bundling and that ultimately affect Z-ring formation in dividing cells.

Photoconvertible fluorescent proteins (FPs) are recent additions to the biologists’ toolbox for understanding the living cell. Like green fluorescent protein (GFP), monomeric EosFP is bright green in color but is efficiently photoconverted into a red fluorescent form using a mild violet-blue excitation. Here, we report mEosFP-based probes that localize to the cytosol, plasma membrane invaginations, endosomes, prevacuolar vesicles, vacuoles, the endoplasmic reticulum, Golgi bodies, mitochondria, peroxisomes, and the two major cytoskeletal elements, filamentous actin and cortical microtubules. The mEosFP fusion proteins are smaller than GFP/red fluorescent protein-based probes and, as demonstrated here, provide several significant advantages for imaging of living plant cells. These include an ability to differentially color label a single cell or a group of cells in a developing organ, selectively highlight a region of a cell or a subpopulation of organelles and vesicles within a cell for tracking them, and understanding spatiotemporal aspects of interactions between similar as well as different organelles. In addition, mEosFP probes introduce a milder alternative to fluorescence recovery after photobleaching, whereby instead of photobleaching, photoconversion followed by recovery of green fluorescence can be used for estimating subcellular dynamics. Most importantly, the two fluorescent forms of mEosFP furnish bright internal controls during imaging experiments and are fully compatible with cyan fluorescent protein, GFP, yellow fluorescent protein, and red fluorescent protein fluorochromes for use in simultaneous, multicolor labeling schemes. Photoconvertible mEosFP-based subcellular probes promise to usher in a much higher degree of precision to live imaging of plant cells than has been possible so far using single-colored FPs.