Maciej Lis

The Stress-Responsive dgk Gene from Streptococcus mutans Encodes a Putative Undecaprenol Kinase Activity

ABSTRACT We analyzed a previously constructed stress-sensitive Streptococcus mutans mutant Tn-1 strain resulting from disruption by transposon Tn916 of a gene encoding a protein exhibiting amino acid sequence similarity to the Escherichia coli diacylglycerol kinase. It was confirmed that the mutation led to significantly reduced lipid kinase activity, while expression of the intact gene on a plasmid restored both kinase activity and the wild-type phenotype. Further analysis revealed that the product of the dgk gene in S. mutans predominantly recognizes a lipid substrate other than diacylglycerol, most likely undecaprenol, as demonstrated by its efficient phosphorylation and the resistance of the product of the reaction to saponification. The physiological role of the product of the dgk gene as a putative undecaprenol kinase was further supported by a significantly higher sensitivity of the mutant to bacitracin compared with that of the parental strain.

Antimicrobial peptide MUC7 12‐mer activates the calcium/calcineurin pathway in Candida albicans

MUC7 12-mer is a cationic antimicrobial peptide derived from the N-terminal region of human low-molecular-weight salivary mucin. In order to gain new insights into the modes of action of the 12-mer against opportunistic fungal pathogen Candida albicans, we examined changes in the gene expression profile of C. albicans upon exposure to this peptide. Cells at an early logarithmic phase were exposed to 6 muM peptide and grown until an OD(600 nm) of approximately 0.4 was reached. Changes in gene expression were determined by microarray analysis and showed that 19 out of the total of 531 genes, whose expression was elevated in response to the peptide, are regulated by the calcium/calcineurin signalling pathway. Inactivation of this pathway by deletions, or by FK506, caused hypersensitivity to the peptide, demonstrating the importance of this pathway to the defense of C. albicans against the MUC7 peptide. Other differentially expressed genes that were detected include those encoding subunits of proteasome, and genes involved in cell stress, iron metabolism, cell wall maintenance and small-molecule transport. The presented results suggest that the calcium/calcineurin signalling pathway plays a role in the adaptation of C. albicans to the MUC7 antimicrobial peptide.

Proteomic and metabolic characterization of a Candida albicans mutant resistant to the antimicrobial peptide MUC7 12-mer.

MUC7 12-mer is a cationic peptide derived from the N-terminal portion of human mucin MUC7, exhibiting potent antibacterial and antifungal properties. To advance our knowledge regarding the mechanisms of action of MUC7 peptide against an opportunistic fungal pathogen Candida albicans, we sought to develop and characterize mutant(s) resistant to this peptide. One of the selected mutants, designated #37, was much less susceptible to the MUC7 12-mer in a killing assay than the parental strain (ED(50)>40 vs. c. 6 microM, respectively). Difference gel electrophoresis (DIGE) analysis of the mutant revealed elevation of several glycolytic enzymes. The mutant also exhibited lowered ATP contents along with a relatively lower rate of oxygen consumption, as well as inability to grow on nonfermentable carbon sources. These properties are likely to be associated with changes in metabolic regulation, rather than lack of functional mitochondria, as determined by rhodamine 123 staining. Analysis of interaction between fluorescently labeled peptide and cells of both strains revealed that resistance of the mutant #37 is associated with changes in the process of transition between surface-bound state of the peptide to its internalization marking cell death.

Exploring the Mode of Action of Antimicrobial Peptide MUC7 12-Mer by Fitness Profiling of Saccharomyces cerevisiae Genomewide Mutant Collection

ABSTRACT The MUC7 12-mer (RKSYKCLHKRCR) is a cationic antimicrobial peptide derived from the human salivary mucin MUC7. To study its effect/mechanism of action on fungi, we performed a fitness screen of a tagged, diploid, homozygous gene deletion mutant pool of the yeast Saccharomyces cerevisiae grown in the presence of the MUC7 peptide. Forty-five strains exhibiting reduced fitness and 13 strains exhibiting increased fitness (sensitivity or resistance, respectively) were identified by hybridization intensities to tag arrays. The strongest fitness defects were observed with deletions in genes encoding elements of the RIM101 signaling pathway (regulating response to alkaline and neutral pH and other environmental conditions) and of the endosomal sorting complex required for transport (ESCRT; functioning mainly in protein sorting for degradation, but also required for activation of the RIM101 pathway). Other deletions identified as conferring fitness defect or gain are in genes associated with a variety of functions, including transcription regulation, protein trafficking, transport, metabolism, and others. The results of the pool fitness screen were validated by a set of mutant strains tested individually in the presence of the MUC7 12-mer. All tested RIM101-related deletion strains showing fitness defects confirmed their sensitivities. Taken together, the results led us to conclude that deletions of genes associated with the RIM101 pathway confer sensitivity to the peptide by preventing activation of this pathway and that this stress response plays a major role in the protection of S. cerevisiae against damage inflicted by the MUC7 12-mer peptide.

Chemical Genomic Screening of a Saccharomyces cerevisiae Genomewide Mutant Collection Reveals Genes Required for Defense against Four Antimicrobial Peptides Derived from Proteins Found in Human Saliva

ABSTRACT To compare the effects of four antimicrobial peptides (MUC7 12-mer, histatin 12-mer, cathelicidin KR20, and a peptide containing lactoferricin amino acids 1 to 11) on the yeast Saccharomyces cerevisiae, we employed a genomewide fitness screen of combined collections of mutants with homozygous deletions of nonessential genes and heterozygous deletions of essential genes. When an arbitrary fitness score cutoffs of 1 (indicating a fitness defect, or hypersensitivity) and −1 (indicating a fitness gain, or resistance) was used, 425 of the 5,902 mutants tested exhibited altered fitness when treated with at least one peptide. Functional analysis of the 425 strains revealed enrichment among the identified deletions in gene groups associated with the Gene Ontology (GO) terms “ribosomal subunit,” “ribosome biogenesis,” “protein glycosylation,” “vacuolar transport,” “Golgi vesicle transport,” “negative regulation of transcription,” and others. Fitness profiles of all four tested peptides were highly similar, particularly among mutant strains exhibiting the greatest fitness defects. The latter group included deletions in several genes involved in induction of the RIM101 signaling pathway, including several components of the ESCRT sorting machinery. The RIM101 signaling regulates response of yeasts to alkaline and neutral pH and high salts, and our data indicate that this pathway also plays a prominent role in regulating protective measures against all four tested peptides. In summary, the results of the chemical genomic screens of S. cerevisiae mutant collection suggest that the four antimicrobial peptides, despite their differences in structure and physical properties, share many interactions with S. cerevisiae cells and consequently a high degree of similarity between their modes of action.

Characterization of a suppressor mutation complementing an acid-sensitive mutation in Streptococcus mutans

We isolated a spontaneous suppressor mutant complementing the acid-sensitive phenotype of Streptococcus mutans strain Tn-1, a mutant previously generated in this laboratory, defective in the activity of the dgk-encoded putative undecaprenol kinase. A relatively simple genetic method was developed to identify the suppressor mutation, based on selection for transformants containing two closely linked markers: a selectable allele of the unknown suppressor gene and an antibiotic resistance gene introduced on a suicide plasmid at random sites into the chromosome via homologous recombination. While we have not actually identified the original suppressor mutation, another mutated gene restoring acid resistance has been isolated, which suggests a possible mechanism of suppression.

Humanized Mouse Model Used to Monitor MUC Gene Expression in Nasal Polyps and to Preclinically Evaluate the Efficacy of Montelukast in Reducing Mucus Production

Objectives: To determine whether MUC gene expression could be down-regulated in nasal polyps by the leukotriene receptor antagonist montelukast, we developed a system in which nondisrupted human nasal polyps could be successfully implanted into severely immunocompromised mice, and in which the histopathology of the original nasal polyp tissue could be preserved for long periods. In addition, the histopathologic changes in the human nasal polyps were carefully examined to determine the origin of the submucosal glands (SMGs) that develop in true nasal polyps found in the anterior third of the nose. Methods: Small, nondisrupted pieces of human nasal polyp tissues were subcutaneously implanted into NOD-scid IL-2rγnull mice. Xenograft-bearing mice were treated with either montelukast or saline solution. Xenografts at 8 to 12 weeks after implantation were examined histologically, and expression of MUC genes 4, 5AC, and 7 was studied in the polyps before implantation and in the 8-week xenograft. Alzet pumps were inserted into the mice, and montelukast (Singulair) was continuously delivered to determine its effect on goblet cell hyperplasia, mucus production, and the enlargement of nasal polyps over an 8-week period. Results: The xenografts were maintained in a viable and functional state for up to 3 months and retained a histopathology similar to that of the original tissue, but with a noticeable increase in goblet cell hyperplasia and marked mucus accumulation in the SMGs. MUC4 and MUC5AC were significantly increased in the xenograft 8 weeks after implantation, but MUC7 was significantly decreased compared to the preimplantation polyps. Inasmuch as MUC7 is found exclusively in serous glands, the findings suggest that serous glands are not found in polyps in the anterior third of the nose. The histopathologic findings confirm the original findings of Tos et al suggesting that the SMGs are derived from pinching-off of the epithelium of the enlarging polyp following inflammatory changes. These SMGs have the same epithelium as surface epithelium and consist of multiple goblet cells that secrete periodic acid Schiff stain-positive mucin into the interior of the SMGs. A progressive increase in the volume of the xenografts was observed, with little or no evidence of mouse cell infiltration into the human leukocyte antigen-positive human tissue. An average twofold increase in polyp volume was found 2 months after engraftment. Montelukast did not decrease the growth of the xenograft in the 8-week NOD-scid mice, nor did it affect MUC gene expression. Conclusions: The use of innate and adaptive immunodeficient NOD-scid mice homozygous for targeted mutations in the IL-2 gamma-chain locus NOD-scid IL-2rγnull for establishing engraftment of nondisrupted pieces of human nasal polyp tissues represents a significant advancement in studying chronic inflammation over a long period of time. In the present study, we utilized this humanized mouse model to confirm our prediction that MUC genes 4 and 5AC are highly expressed and significantly increased over those of preimplanted polyps. The overexpression of these 2 MUC genes correlates with both the goblet cell hyperplasia and the excessive mucus production that are found in nasal polyp xenografts. MUC7, which is primarily associated with the submucosa, as opposed to MUC4 and MUC5AC, which are primarily expressed in the epithelium, was significantly decreased in the nasal polyp xenografts. Montelukast had no significant effect on MUC gene expression in the xenografts. In addition to the MUC gene expression patterns, the histology of the xenografts supports the concept that mucinous glands that are characteristic of true nasal polyps are significantly different from those in the mucosa found in the lateral wall of the nose in patients with chronic sinusitis without nasal polyps. The mucinous glands seen in nasal polyps (which appear to be derived from an invagination of hyperplastic epithelial mucosa containing large numbers of goblet cells) are histologically distinct from the seromucinous glands found in the submucosa of hyperplastic middle turbinates. The data presented here establish a humanized mouse model as a viable approach to study nasal polyp growth, to assess the therapeutic efficacy of various drugs in this chronic inflammatory disease, and to contribute to our understanding of the pathogenesis of this disease.