Novel small molecules that increase the susceptibility of Neisseria gonorrhoeae to cationic antimicrobial peptides by inhibiting lipid A phosphoethanolamine transferase.

OBJECTIVES: Neisseria gonorrhoeae is an exclusively human pathogen that commonly infects the urogenital tract resulting in gonorrhoea. Empirical treatment of gonorrhoea with antibiotics has led to multidrug resistance and the need for new therapeutics. Inactivation of lipooligosaccharide phosphoethanolamine transferase A (EptA), which attaches phosphoethanolamine to lipid A, results in attenuation of the pathogen in infection models. Small molecules that inhibit EptA are predicted to enhance natural clearance of gonococci via the human innate immune response. METHODS: A library of small-fragment compounds was tested for the ability to enhance susceptibility of the reference strain N. gonorrhoeae FA1090 to polymyxin B. The effect of these compounds on lipid A synthesis and viability in models of infection were tested. RESULTS: Three compounds, 135, 136 and 137, enhanced susceptibility of strain FA1090 to polymyxin B by 4-fold. Pre-treatment of bacterial cells with all three compounds resulted in enhanced killing by macrophages. Only lipid A from bacterial cells exposed to compound 137 showed a 17% reduction in the level of decoration of lipid A with phosphoethanolamine by MALDI-TOF MS analysis and reduced stimulation of cytokine responses in THP-1 cells. Binding of 137 occurred with higher affinity to purified EptA than the starting material, as determined by 1D saturation transfer difference NMR. Treatment of eight MDR strains with 137 increased susceptibility to polymyxin B in all cases. CONCLUSIONS: Small molecules have been designed that bind to EptA, inhibit addition of phosphoethanolamine to lipid A and can sensitize N. gonorrhoeae to killing by macrophages.

Contribution of Streptococcus pseudopneumoniae and Streptococcus salivarius to vocal fold mucosal integrity and function.

Structural changes to the vocal fold (VF) epithelium, namely, loosened intercellular junctions, have been reported in VF benign lesions. The potential mechanisms responsible for the disruption of cell junctions do not address the contribution of resident microbial communities to this pathological phenomenon. In this study, we focused on determining the relationship between Streptococcus pseudopneumoniae (SP), a dominant bacterial species associated with benign lesions, and Streptococcus salivarius (SS), a commensal bacterium, with human VF epithelial cells in our three-dimensional model of the human VF mucosa. This experimental system enabled direct deposition of bacteria onto constructs at the air/liquid interface, allowing for the assessment of bacterium-host interactions at the cellular, molecular and ultrastructural levels. Our findings demonstrate that SP disrupts VF epithelial integrity and initiates inflammation via the exported products HtrA1 and pneumolysin. In contrast, SS attaches to the VF epithelium, reduces inflammation and induces Mmp2-mediated apical desquamation of infected cells to mitigate the impact of pathogens. In conclusion, this study highlights the complexity of microbial involvement in VF pathology and potential VF mucosal restoration in the presence of laryngeal commensals.

Localization of TRPV3/4 and PIEZO1/2 sensory receptors in murine and human larynges.

Objective: The primary aim of this study was to identify expression of TRPV3 and TRPV4 chemoreceptors across perinatal and adult stages using a murine model with direct comparisons to human laryngeal mucosa. Our secondary aim was to establish novel cell expression patterns of mechanoreceptors PIEZO1 and PIEZO2 in human tissue samples. Study design: In vivo. Methods: We harvested murine laryngeal tissue to localize and describe TRPV3/4 endogenous protein expression patterns via immunofluorescence analyses across two developmental (E16.5, P0) and adult (6 weeks) timepoints. Additionally, we obtained a 60-year-old female larynx including the proximal trachea and esophagus to investigate TRPV3/4 and PIEZO1/2 protein expression patterns via immunofluorescence analyses for comparison to murine adult tissue. Results: Murine TRPV3/4 expression was noted at E16.5 with epithelial cell colocalization to supraglottic regions of the arytenoids, aryepiglottic folds and epiglottis through to birth (P0), extending to the adult timepoint. Human TRPV3/4 protein expression was most evident to epithelium of the arytenoid region, with additional expression of TRPV3 and TRPV4 to proximal esophageal and tracheal epithelium, respectively. Human PIEZO1 expression was selective to differentiated, stratified squamous epithelia of the true vocal fold and esophagus, while PIEZO2 expression exhibited selectivity for intermediate and respiratory epithelia of the false vocal fold, ventricles, subglottis, arytenoid, and trachea. Conclusion: Results exhibited expression of TRPV3/4 chemoreceptors in utero, suggesting their importance during fetal/neonatal stages. TRPV3/4 and PIEZO1/2 were noted to adult murine and human laryngeal epithelium. Data indicates conservation of chemosensory receptors across species given similar regional expression in both the murine and human larynx.

A method for identification of vocal fold lamina propria fibroblasts in culture.

OBJECTIVE: Vocal fold biology research is emerging as a vital area of study in laryngology. One impediment is the lack of both commercially available vocal fold lamina propria fibroblasts and a constitutively expressed specific marker for fibroblasts. We present an in vitro technique that allows for identification of fibroblasts by ruling out the possibility of the cells belonging to other lineages that are found in vocal fold tissue. STUDY DESIGN: An in vitro study. METHODS: Two primary vocal fold fibroblast cell lines and one immortalized vocal fold fibroblast cell line were cultured. Immunohistologic staining for alpha-actinin, cytokeratin 19, and von Willebrand factor was completed for the three fibroblast lines in addition to skeletal, endothelial, and epithelial cell lines. Cell type was differentiated by positive staining for alpha-actinin, cytokeratin 19, and von Willebrand factor. RESULTS: Fibroblast cultures did not express alpha-actinin, cytokeratin 19, and von Willebrand factor, whereas skeletal muscle, endothelial, and epithelial cultured cells expressed each respectively. CONCLUSIONS: This simple rule-out methodology for fibroblast confirmation is an important step when establishing cell culture, and it establishes sound internal validity particularly in the early stages of this emerging area of study.

Acquisition of the capsule locus by horizontal gene transfer in Neisseria meningitidis is often accompanied by the loss of UDP-GalNAc synthesis.

Pathogenic meningococci have acquired a 24 kb capsule synthesis island (cps) by horizontal gene transfer which consists of a synthetic locus and associated capsule transport genes flanked by repetitive Regions D and D'. Regions D and D' contain an intact gene encoding a UDP-galactose epimerase (galE1) and a truncated remnant (galE2), respectively. In this study, GalE protein alleles were shown to be either mono-functional, synthesising UDP-galactose (UDP-Gal), or bi-functional, synthesising UDP-Gal and UDP-galactosamine (UDP-GalNAc). Meningococci possessing a capsule null locus (cnl) typically possessed a single bi-functional galE. Separation of functionality between galE1 and galE2 alleles in meningococcal isolates was retained for all serogroups except serogroup E which has a synthetic requirement for UDP-GalNAc. The truncated galE2 remnant in Region D' was also phylogenetically related to the bi-functional galE of the cnl locus suggesting common ancestry. A model is proposed in which the illegitimate recombination of the cps island into the galE allele of the cnl locus results in the formation of Region D' containing the truncated galE2 locus and the capture of the cps island en bloc. The retention of the duplicated Regions D and D' enables inversion of the synthetic locus within the cps island during bacterial growth.

Vocal fold myofibroblast profile of scarring.

OBJECTIVES/HYPOTHESIS: Vocal fold fibroblasts (VFF) are responsible for extracellular matrix synthesis supporting lamina propria in normal and diseased conditions. When tissue is injured, VFF become activated and differentiate into myofibroblasts to facilitate wound healing response. We investigated if vocal fold myofibroblasts can be utilized as surrogate cells for scarred VFF. STUDY DESIGN: In vitro. METHODS: Normal VFF cell lines from a 21-year-old male (N21), 59-year-old female (N59), and a scar VFF cell line from a 56-year-old female (S56) were used in this study. 10 ng/mL of transforming growth factor (TGFß1) was applied for 5 days to normal VFF. Myofibroblast differentiation was determined with immunocytochemistry and western blot, measuring alpha smooth muscle actin (α-SMA). Cell growth, proliferation, contractile properties, and gene expression profiles were evaluated. RESULTS: N21, N59, and S56 VFF presented elongated configuration. N21+ and N21- VFF demonstrated significantly greater proliferation compared to N59+, N59-, and S56 VFF at 6 days. α-SMA was expressed in all cells. Fibronectin, alpha smooth actin, connective tissue growth factor, and metallopeptidase inhibitor were the highest genes expression in VFF treated with transforming growth factor ß1 (TGFß1). At 24 hours, S56 VFF showed lower contraction compared to N21+ and N59+ VFF, but at 60 hours S56 VFF had lower collagen contraction compared to all cell groups. Highest collagen contraction matrices were measured with VFF treated with TGFß1 at 24 hours and N59- VFF at 60 hours. CONCLUSION: VFF treated with TGFß1 (myofibroblasts) appear to have similar phenotypic characteristics but different genotypic behavior compared to scar VFF. LEVEL OF EVIDENCE: N/A. Laryngoscope, 126:E110-E117, 2016.

Identification of the polycomb group protein SU(Z)12 as a potential molecular target for human cancer therapy.

We have previously identified SU(Z)12 as an E2F target gene. Because many E2F target genes encode proteins that are critical for the control of cell proliferation, we have further characterized the regulation and expression of SU(Z)12. To understand the molecular mechanisms responsible for expression of SU(Z)12 mRNA, we have analyzed the promoter region. We found that the SU(Z)12 gene is controlled by dual promoters, one of which functions bidirectionally. In addition to the E2F binding site, we have identified two binding sites for T cell factor (TCF)/beta-catenin complexes. Using gel mobility shift assays, we demonstrated that both TCF sites can be bound by TCF4. TCF/beta-catenin complexes have been shown to be a critical regulator of gene expression in tumors of the colon, breast, and liver. Accordingly, we have used chromatin immunoprecipitation assays to confirm that TCF4/beta-catenin complexes are bound to the SU(Z)12 promoter in colon cancer cells but not in HeLa cells. We next adapted the chromatin immunoprecipitation assay for use with primary colon tumor samples, and, using matched pairs of normal and tumor tissue obtained from several different colon cancer patients, we demonstrate that levels of beta-catenin bound to the SU(Z)12 promoter are increased in colon tumors. Finally, we show that the SU(Z)12 mRNA is up-regulated in a number of different human tumors, including tumors of the colon, breast, and liver. Recent studies have found that SU(Z)12 is a component of the Drosophila ESC-E(Z) and the human EED-EZH2 Polycomb chromatin remodeling complexes. Therefore, we suggest that SU(Z)12, which may modulate the tumor phenotype by changing gene expression profiles, may be a logical target for the design of a new antitumor agent

The use of high-throughput DNA sequencing in the investigation of antigenic variation: application to Neisseria species.

Antigenic variation occurs in a broad range of species. This process resembles gene conversion in that variant DNA is unidirectionally transferred from partial gene copies (or silent loci) into an expression locus. Previous studies of antigenic variation have involved the amplification and sequencing of individual genes from hundreds of colonies. Using the pilE gene from Neisseria gonorrhoeae we have demonstrated that it is possible to use PCR amplification, followed by high-throughput DNA sequencing and a novel assembly process, to detect individual antigenic variation events. The ability to detect these events was much greater than has previously been possible. In N. gonorrhoeae most silent loci contain multiple partial gene copies. Here we show that there is a bias towards using the copy at the 3' end of the silent loci (copy 1) as the donor sequence. The pilE gene of N. gonorrhoeae and some strains of Neisseria meningitidis encode class I pilin, but strains of N. meningitidis from clonal complexes 8 and 11 encode a class II pilin. We have confirmed that the class II pili of meningococcal strain FAM18 (clonal complex 11) are non-variable, and this is also true for the class II pili of strain NMB from clonal complex 8. In addition when a gene encoding class I pilin was moved into the meningococcal strain NMB background there was no evidence of antigenic variation. Finally we investigated several members of the opa gene family of N. gonorrhoeae, where it has been suggested that limited variation occurs. Variation was detected in the opaK gene that is located close to pilE, but not at the opaJ gene located elsewhere on the genome. The approach described here promises to dramatically improve studies of the extent and nature of antigenic variation systems in a variety of species.

The role of oxidoreductases in determining the function of the neisserial lipid A phosphoethanolamine transferase required for resistance to polymyxin.

The decoration of the lipid A headgroups of the lipooligosaccharide (LOS) by the LOS phosphoethanolamine (PEA) transferase (LptA) in Neisseria spp. is central for resistance to polymyxin. The structure of the globular domain of LptA shows that the protein has five disulphide bonds, indicating that it is a potential substrate of the protein oxidation pathway in the bacterial periplasm. When neisserial LptA was expressed in Escherichia coli in the presence of the oxidoreductase, EcDsbA, polymyxin resistance increased 30-fold. LptA decorated one position of the E. coli lipid A headgroups with PEA. In the absence of the EcDsbA, LptA was degraded in E. coli. Neisseria spp. express three oxidoreductases, DsbA1, DsbA2 and DsbA3, each of which appear to donate disulphide bonds to different targets. Inactivation of each oxidoreductase in N. meningitidis enhanced sensitivity to polymyxin with combinatorial mutants displaying an additive increase in sensitivity to polymyxin, indicating that the oxidoreductases were required for multiple pathways leading to polymyxin resistance. Correlates were sought between polymyxin sensitivity, LptA stability or activity and the presence of each of the neisserial oxidoreductases. Only meningococcal mutants lacking DsbA3 had a measurable decrease in the amount of PEA decoration on lipid A headgroups implying that LptA stability was supported by the presence of DsbA3 but did not require DsbA1/2 even though these oxidoreductases could oxidise the protein. This is the first indication that DsbA3 acts as an oxidoreductase in vivo and that multiple oxidoreductases may be involved in oxidising the one target in N. meningitidis. In conclusion, LptA is stabilised by disulphide bonds within the protein. This effect was more pronounced when neisserial LptA was expressed in E. coli than in N. meningitidis and may reflect that other factors in the neisserial periplasm have a role in LptA stability.

Attachment and invasion of Neisseria meningitidis to host cells is related to surface hydrophobicity, bacterial cell size and capsule.

We compared exemplar strains from two hypervirulent clonal complexes, strain NMB-CDC from ST-8/11 cc and strain MC58 from ST-32/269 cc, in host cell attachment and invasion. Strain NMB-CDC attached to and invaded host cells at a significantly greater frequency than strain MC58. Type IV pili retained the primary role for initial attachment to host cells for both isolates regardless of pilin class and glycosylation pattern. In strain MC58, the serogroup B capsule was the major inhibitory determinant affecting both bacterial attachment to and invasion of host cells. Removal of terminal sialylation of lipooligosaccharide (LOS) in the presence of capsule did not influence rates of attachment or invasion for strain MC58. However, removal of either serogroup B capsule or LOS sialylation in strain NMB-CDC increased bacterial attachment to host cells to the same extent. Although the level of inhibition of attachment by capsule was different between these strains, the regulation of the capsule synthesis locus by the two-component response regulator MisR, and the level of surface capsule determined by flow cytometry were not significantly different. However, the diplococci of strain NMB-CDC were shown to have a 1.89-fold greater surface area than strain MC58 by flow cytometry. It was proposed that the increase in surface area without changing the amount of anchored glycolipid capsule in the outer membrane would result in a sparser capsule and increase surface hydrophobicity. Strain NMB-CDC was shown to be more hydrophobic than strain MC58 using hydrophobicity interaction chromatography and microbial adhesion-to-solvents assays. In conclusion, improved levels of adherence of strain NMB-CDC to cell lines was associated with increased bacterial cell surface and surface hydrophobicity. This study shows that there is diversity in bacterial cell surface area and surface hydrophobicity within N. meningitidis which influence steps in meningococcal pathogenesis.

The identification of E2F1-specific target genes.

The E2F family of transcriptional regulators consists of six different members. Analysis of E2F-regulated promoters by using cultured cells suggests that E2Fs may have redundant functions. However, animal studies have shown that loss of individual E2Fs can have distinct biological consequences. Such seemingly conflicting results could be due to a difference in E2F-mediated regulation in cell culture vs. animals. Alternatively, there may be genes that are specifically regulated by an individual E2F which have not yet been identified. To investigate this possibility further, we have analyzed gene expression in E2F1 nullizygous mice. We found that loss of E2F1 did not cause changes in expression of known E2F target genes, suggesting that perhaps E2F1-specific promoters are distinct from known E2F target promoters. Therefore, we used oligonucleotide microarrays to identify mRNAs whose expression is altered on loss of E2F1. We demonstrate by chromatin immunoprecipitation that several of the promoters that drive expression of the deregulated mRNAs selectively recruit E2F1, but not other E2Fs, and this recruitment is via an element distinct from a consensus E2F binding site. To our knowledge, these are as yet undocumented examples of promoters being occupied in asynchronously growing cells by a single E2F family member. Interestingly, the E2F1-specific target genes that we identified encode proteins having functions quite different from the function of known E2F target genes. Thus, whereas E2F1 may share redundant functions in cell growth control with other E2F family members, it may also play an important biological role distinct from the other E2Fs.

Silencing of human polycomb target genes is associated with methylation of histone H3 Lys 27.

Polycomb group (PcG) complexes 2 and 3 are involved in transcriptional silencing. These complexes contain a histone lysine methyltransferase (HKMT) activity that targets different lysine residues on histones H1 or H3 in vitro. However, it is not known if these histones are methylation targets in vivo because the human PRC2/3 complexes have not been studied in the context of a natural promoter because of the lack of known target genes. Here we report the use of RNA expression arrays and CpG-island DNA arrays to identify and characterize human PRC2/3 target genes. Using oligonucleotide arrays, we first identified a cohort of genes whose expression changes upon siRNA-mediated removal of Suz12, a core component of PRC2/3, from colon cancer cells. To determine which of the putative target genes are directly bound by Suz12 and to precisely map the binding of Suz12 to those promoters, we combined a high-resolution chromatin immunoprecipitation (ChIP) analysis with custom oligonucleotide promoter arrays. We next identified additional putative Suz12 target genes by using ChIP coupled to CpG-island microarrays. We showed that HKMT-Ezh2 and Eed, two other components of the PRC2/3 complexes, colocalize to the target promoters with Suz12. Importantly, recruitment of Suz12, Ezh2 and Eed to target promoters coincides with methylation of histone H3 on Lys 27.