Genetic and Structural Characterization of Pneumolancidin, a Broad Spectrum Inhibitory Lantibiotic, Produced by Streptococcus pneumoniae.

Streptococcus pneumoniae, a member of the diverse microbial community of the human nasopharynx, is subjected to competitive interactions. Since colonization is a prerequisite for pneumococcal pathogenesis, understanding the dynamics of bacterial competition is important for identifying factors that aid in colonization and carriage. To eliminate competitors, pneumococcus is known to secrete antimicrobial peptides or bacteriocins. Pneumococcal bacteriocins and their role in competition have been well characterized. However, a class of modified bacteriocins named lantibiotics, and their role in promoting pneumococcal competition is less well understood. Though several lantibiotic loci have been identified in pneumococcus, none have been found with antimicrobial activity. Recently, our laboratory identified a clinical isolate of pneumococcus, P174, with a broad spectrum of inhibitory activity attributed to a novel lantibiotic locus, termed pneumolancidin (pld). In addition to encoding genes required for the modification, processing, regulation and immunity to lantibiotic peptides, four open reading frames predicted to encode four highly homologous lantibiotic peptides were also found. This posed the question of whether the Pld peptides were redundant in function or had specialized roles. Lantibiotic peptides are known to function as antimicrobials and autoinducers. To determine the role of each peptide as it relates to inhibition and induction, individual in-frame peptide deletions were constructed. Strains carrying the mutation were assayed for their ability to inhibit the growth of other strains and their ability to upregulate the pld locus. The first three peptides, PldA1-3, were found to be required for signaling while PldA4 was found to be dispensable. Because upregulation of the locus is needed to determine whether specific peptides were involved in inhibition, the ability of the peptide deletion strains to inhibit could not be evaluated. However, a serendipitous mutant, P174act, was discovered that allowed for distinct phenotypes to be observed for each of the Pld peptide deletion strains. Through structural elucidation, it was found that PldA1 and PldA3 are structurally similar yet have specialized roles in signaling and inhibition, respectively. The Pld peptides represent a novel strategy for bacterial competition and provide insight into structure-function relationships of lantibiotics.PHDMicrobiology and ImmunologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/133297/1/natmar_1.pd

Substituted Lactam and Cyclic Azahemiacetals Modulate Pseudomonas aeruginosa Quorum Sensing

Quorum sensing (QS) is a population-dependent signaling process bacteria use to control multiple processes including virulence that is critical for establishing infection. The most common QS signaling molecule used by Gram-negative bacteria are acylhomoserine lactones. The development of non-native acylhomoserine lactone (AHL) ligands has emerged as a promising new strategy to inhibit QS in Gram-negative bacteria. In this work, we have synthesized a set of optically pure γ-lactams and their reduced cyclic azahemiacetal analogues, bearing the additional alkylthiomethyl substituent, and evaluated their effect on the AHL-dependent Pseudomonas aeruginosa las and rhl QS pathways. The concentration of these ligands and the simple structural modification such as the length of the alkylthio substituent has notable effect on activity. The γ-lactam derivatives with nonylthio or dodecylthio chains acted as inhibitors of las signaling with moderate potency. The cyclic azahemiacetal with shorter propylthio or hexylthio substituent was found to strongly inhibit both las and rhl signaling at higher concentrations while the propylthio analogue strongly stimulated the las QS system at lower concentrations

Investigating an Alternative Treatment to Cystic Fibrosis: Testing the Effect of Panax ginseng C.A. Meyer extracts on Pseudomonas aeruginosa

The predominant pathogen found in the lungs of cystic fibrosis (CF) patients is Pseudomonas aeruginosa. The success of the infection is partially due to virulence factor production, which is regulated by quorum sensing (QS) signaling. Currently, antibiotics are used to treat the infection, but resistant forms of P. aeruginosa have evolved, necessitating alternative treatments. Previous animal studies showed that treatment with extracts from the Chinese herb Panax ginseng C.A. Meyer reduced bacterial load resulting in a favorable immune response. It is hypothesized that ginsenosides, the major bioactive compounds in ginseng, is responsible for this effect. This study explores the role of ginseng extracts in attenuating P. aeruginosa virulence. A sequential extraction was performed using hexane, methylene chloride, methanol, and water. High performance liquid chromatography (HPLC) analysis showed the methanol and water ginseng extracts contained the known ginsenosides Rb1, Rb2, Rc, Rd, Re, and Rg1• All extracts were tested on biomonitor strains of Agrobacterium tumefaciens,Chromobacterium violaceum, and P. aeruginosa. Antibacterial and anti-QS activity were assessed using a disc diffusion assay. This was then followed by thin layer chromatography (TLC) bioautographic assay to further separate active compounds. The hexane and dichloromethane extracts, that lacked ginsenosides, displayed antibacterial activity against C. violaceum, whereas methanol and water extracts had anti-QS activity. The results of the bioassay with the pure ginsenoside standards showed that they lack antibacterial or anti-QS activity. Our results indicate that there are bioactive compounds, other than ginsenosides, that are the cause of antibacterial effects and anti-QS in the ginseng extracts

Characterization of a Multipeptide Lantibiotic Locus in Streptococcus pneumoniae

Bacterial communities are established through a combination of cooperative and antagonistic interactions between the inhabitants. Competitive interactions often involve the production of antimicrobial substances, including bacteriocins, which are small antimicrobial peptides that target other community members. Despite the nearly ubiquitous presence of bacteriocin-encoding loci, inhibitory activity has been attributed to only a small fraction of gene clusters. In this study, we characterized a novel locus (the pld locus) in the pathogen Streptococcus pneumoniae that drives the production of a bacteriocin called pneumolancidin, which has broad antimicrobial activity. The locus encodes an unusual tandem array of four inhibitory peptides, three of which are absolutely required for antibacterial activity. The three peptide sequences are similar but appear to play distinct roles in regulation and inhibition. A modification enzyme typically found in loci encoding a class of highly modified bacteriocins called lantibiotics was required for inhibitory activity. The production of pneumolancidin is controlled by a two-component regulatory system that is activated by the accumulation of modified peptides. The locus is located on a mobile element that has been found in many pneumococcal lineages, although not all elements carry the pld genes. Intriguingly, a minimal region containing only the genes required for pneumolancidin immunity was found in several Streptococcus mitis strains. The pneumolancidin-producing strain can inhibit nearly all pneumococci tested to date and provided a competitive advantage in vivo. These peptides not only represent a unique strategy for bacterial competition but also are an important resource to guide the development of new antimicrobials

Zinc coordination is essential for the function and activity of the type II secretion ATPase EpsE

The type II secretion system Eps in Vibrio cholerae promotes the extracellular transport of cholera toxin and several hydrolytic enzymes and is a major virulence system in many Gram‐negative pathogens which is structurally related to the type IV pilus system. The cytoplasmic ATPase EpsE provides the energy for exoprotein secretion through ATP hydrolysis. EpsE contains a unique metal‐binding domain that coordinates zinc through a tetracysteine motif (CXXCX29CXXC), which is also present in type IV pilus assembly but not retraction ATPases. Deletion of the entire domain or substitution of any of the cysteine residues that coordinate zinc completely abrogates secretion in an EpsE‐deficient strain and has a dominant negative effect on secretion in the presence of wild‐type EpsE. Consistent with the in vivo data, chemical depletion of zinc from purified EpsE hexamers results in loss of in vitro ATPase activity. In contrast, exchanging the residues between the two dicysteines with those from the homologous ATPase XcpR from Pseudomonas aeruginosa does not have a significant impact on EpsE. These results indicate that, although the individual residues in the metal‐binding domain are generally interchangeable, zinc coordination is essential for the activity and function of EpsE.Type II secretion ATPases contain a unique zinc‐binding domain which is absent from homologous type IV pilus retraction ATPases and type IV secretion ATPases. Removal of the entire zinc‐binding domain or disruption of zinc coordination in the type II secretion ATPase EpsE abrogates secretion and prevents ATP hydrolysis, indicating that zinc coordination is essential for the function and activity of type II secretion ATPases.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/134427/1/mbo3376_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/134427/2/mbo3376.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/134427/3/mbo3376-sup-0001-FigureS1-S4.pd

Repurposing the Streptococcus mutans CRISPR-Cas9 System to Understand Essential Gene Function.

A recent genome-wide screen identified ~300 essential or growth-supporting genes in the dental caries pathogen Streptococcus mutans. To be able to study these genes, we built a CRISPR interference tool around the Cas9 nuclease (Cas9Smu) encoded in the S. mutans UA159 genome. Using a xylose-inducible dead Cas9Smu with a constitutively active single-guide RNA (sgRNA), we observed titratable repression of GFP fluorescence that compared favorably to that of Streptococcus pyogenes dCas9 (Cas9Spy). We then investigated sgRNA specificity and proto-spacer adjacent motif (PAM) requirements. Interference by sgRNAs did not occur with double or triple base-pair mutations, or if single base-pair mutations were in the 3' end of the sgRNA. Bioinformatic analysis of >450 S. mutans genomes allied with in vivo assays revealed a similar PAM recognition sequence as Cas9Spy. Next, we created a comprehensive library of sgRNA plasmids that were directed at essential and growth-supporting genes. We discovered growth defects for 77% of the CRISPRi strains expressing sgRNAs. Phenotypes of CRISPRi strains, across several biological pathways, were assessed using fluorescence microscopy. A variety of cell structure anomalies were observed, including segregational instability of the chromosome, enlarged cells, and ovococci-to-rod shape transitions. CRISPRi was also employed to observe how silencing of cell wall glycopolysaccharide biosynthesis (rhamnose-glucose polysaccharide, RGP) affected both cell division and pathogenesis in a wax worm model. The CRISPRi tool and sgRNA library are valuable resources for characterizing essential genes in S. mutans, some of which could prove to be promising therapeutic targets

Substituted Lactam and Cyclic Azahemiacetals Modulate Pseudomonas aeruginosa Quorum Sensing

Quorum sensing (QS) is a population-dependent signaling process bacteria use to control multiple processes including virulence that is critical for establishing infection. The most common QS signaling molecule used by Gram-negative bacteria are acylhomoserine lactones. The development of non-native acylhomoserine lactone (AHL) ligands has emerged as a promising new strategy to inhibit QS in Gram-negative bacteria. In this work, we have synthesized a set of optically pure γ-lactams and their reduced cyclic azahemiacetal analogues, bearing the additional alkylthiomethyl substituent, and evaluated their effect on the AHL-dependent Pseudomonas aeruginosa las and rhl QS pathways. The concentration of these ligands and the simple structural modification such as the length of the alkylthio substituent has notable effect on activity. The γ-lactam derivatives with nonylthio or dodecylthio chains acted as inhibitors of las signaling with moderate potency. The cyclic azahemiacetal with shorter propylthio or hexylthio substituent was found to strongly inhibit both las and rhl signaling at higher concentrations while the propylthio analogue strongly stimulated the las QS system at lower concentrations