Imaging host-pathogen interactions using epithelial and bacterial cell infection models.

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.

Therapeutic stem cell-derived alveolar-like macrophages display bactericidal effects and resolve Pseudomonas aeruginosa-induced lung injury.

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.

Discovery and characterization of a Gram-positive Pel polysaccharide biosynthetic gene cluster.

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.

FtsA G50E mutant suppresses the essential requirement for FtsK during bacterial cell division in Escherichia coli.

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.

Structure and Mutational Analyses of Escherichia coli ZapD Reveal Charged Residues Involved in FtsZ Filament Bundling.

UNLABELLED: 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 ß-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.

Crystal structure and site-directed mutational analysis reveals key residues involved in Escherichia coli ZapA function.

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.

The Next-Generation ß-Lactamase Inhibitor Taniborbactam Restores the Morphological Effects of Cefepime in KPC-Producing Escherichia coli.

Gram-negative bacteria producing carbapenemases are resistant to a variety of ß-lactam antibiotics and pose a significant health risk. Given the dearth of new antibiotics, combinations of new broad-spectrum ß-lactamase inhibitors (BLIs) with approved ß-lactams have provided treatment options for resistant bacterial infections. Taniborbactam (formerly VNRX-5133) is an investigational BLI that is effective against both serine- and metallo-ß-lactamases, including the serine carbapenemase KPC. In the current study, we assessed the effectiveness of taniborbactam to restore antibacterial activity of cefepime against KPC-3-producing Escherichia coli by inhibiting the KPC-3-dependent hydrolysis of cefepime. Time-lapse microscopy revealed that cells treated with greater than 1× MIC of cefepime (128 µg/ml) and cefepime-taniborbactam (4 µg/ml cefepime and 4 µg/ml taniborbactam) exhibited significant elongation, whereas cells treated with taniborbactam alone did not owing to a lack of standalone antibacterial activity of the BLI. The elongated cells also had frequent cellular voids thought to be formed by attempted cell divisions and pinching of the cytoplasmic membrane. Additionally, the effect of taniborbactam continued even after its removal from the growth medium. Pretreatment with 4 µg/ml taniborbactam helped to restore the antibacterial action of cefepime by neutralizing the effect of the KPC-3 ß-lactamase. IMPORTANCE ß-lactam (BL) antibiotics are the most prescribed antimicrobial class. The efficacy of ß-lactams is threatened by the production of ß-lactamase enzymes, the predominant resistance mechanism impacting these agents in Gram-negative bacterial pathogens. This study visualizes the effects of a combination treatment of taniborbactam, a broad spectrum ß-lactamase inhibitor (BLI), and the BL antibiotic cefepime on a carbapenemase-producing E. coli strain. While this treatment has been described in the context of other cephalosporin-resistant bacteria, this is the first description of a microscopic evaluation of a KPC-3-producing strain of E. coli challenged by this BL-BLI combination. Live-cell microscopy analysis of cells treated with taniborbactam and cefepime demonstrated the antimicrobial effects on cellular morphology and highlighted the long-lasting inhibition of ß-lactamases by taniborbactam even after it was removed from the medium. This research speaks to the importance of taniborbactam in fighting BL-mediated antibiotic resistance.

Outer membrane lipoprotein RlpA is a novel periplasmic interaction partner of the cell division protein FtsK in Escherichia coli.

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 (FtsKN) 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 FtsKN for protein interaction partners critical for FtsKN function. Mass spectrometry analysis of five unique FtsKN periplasmic cross-links revealed a network of potential FtsKN 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 FtsKN 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.

SLC6A14 Is a Genetic Modifier of Cystic Fibrosis That Regulates Pseudomonas aeruginosa Attachment to Human Bronchial Epithelial Cells.

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. aeruginosaIMPORTANCE 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.