Antimicrobial Peptides Originating from Expression Libraries of Aurelia aurita and Mnemiopsis leidyi Prevent Biofilm Formation of Opportunistic Pathogens.

The demand for novel antimicrobial compounds is rapidly growing due to the rising appearance of antibiotic resistance in bacteria; accordingly, alternative approaches are urgently needed. Antimicrobial peptides (AMPs) are promising, since they are a naturally occurring part of the innate immune system and display remarkable broad-spectrum activity and high selectivity against various microbes. Marine invertebrates are a primary resource of natural AMPs. Consequently, cDNA expression (EST) libraries from the Cnidarian moon jellyfish Aurelia aurita and the Ctenophore comb jelly Mnemiopsis leidyi were constructed in Escherichia coli. Cell-free size-fractionated cell extracts (<3 kDa) of the two libraries (each with 29,952 clones) were consecutively screened for peptides preventing the biofilm formation of opportunistic pathogens using the crystal violet assay. The 3 kDa fraction of ten individual clones demonstrated promising biofilm-preventing activities against Klebsiella oxytoca and Staphylococcus epidermidis. Sequencing the respective activity-conferring inserts allowed for the identification of small ORFs encoding peptides (10-22 aa), which were subsequently chemically synthesized to validate their inhibitory potential. Although the peptides are likely artificial products from a random translation of EST inserts, the biofilm-preventing effects against K. oxytoca, Pseudomonas aeruginosa, S. epidermidis, and S. aureus were verified for five synthetic peptides in a concentration-dependent manner, with peptide BiP_Aa_5 showing the strongest effects. The impact of BiP_Aa_2, BiP_Aa_5, and BiP_Aa_6 on the dynamic biofilm formation of K. oxytoca was further validated in microfluidic flow cells, demonstrating a significant reduction in biofilm thickness and volume by BiP_Aa_2 and BiP_Aa_5. Overall, the structural characteristics of the marine invertebrate-derived AMPs, their physicochemical properties, and their promising antibiofilm effects highlight them as attractive candidates for discovering new antimicrobials.

Adenine nucleotide metabolism and a role for AMP in modulating flagellar waveforms in mouse sperm.

While most ATP, the main energy source driving sperm motility, is derived from glycolysis and oxidative phosphorylation, the metabolic demands of the cell require the efficient use of power stored in high-energy phosphate bonds. In times of high energy consumption, adenylate kinase (AK) scavenges one ATP molecule by transphosphorylation of two molecules of ADP, simultaneously yielding one molecule of AMP as a by-product. Either ATP or ADP supported motility of detergent-modeled cauda epididymal mouse sperm, indicating that flagellar AKs are functional. However, the ensuing flagellar waveforms fueled by ATP or ADP were qualitatively different. Motility driven by ATP was rapid but restricted to the distal region of the sperm tail, whereas ADP produced slower and more fluid waves that propagated down the full flagellum. Characterization of wave patterns by tracing and superimposing the images of the flagella, quantifying the differences using digital image analysis, and computer-assisted sperm analysis revealed differences in the amplitude, periodicity, and propagation of the waves between detergent-modeled sperm treated with either ATP or ADP. Surprisingly, addition of AMP to the incubation medium containing ATP recapitulated the pattern of sperm motility seen with ADP alone. In addition to AK1 and AK2, which we previously demonstrated are present in outer dense fibers and mitochondrial sheath of the mouse sperm tail, we show that another AK, AK8, is present in a third flagellar compartment, the axoneme. These results extend the known regulators of sperm motility to include AMP, which may be operating through an AMP-activated protein kinase.

BNC1 is required for maintaining mouse spermatogenesis.

Basonuclin (BNC1) is a zinc finger protein expressed primarily in gametogenic cells and proliferative keratinocytes. Our previous work suggested that BNC1 is present in spermatogonia, spermatocytes, and spermatids, but absent in the Sertoli cells. BNC1's role in spermatogenesis is unknown. Here, we show that BNC1 is required for the maintenance of spermatogenesis. Bnc1-null male mice were sub-fertile, losing germ cells progressively with age. The Bnc1-null seminiferous epithelia began to degenerate before 8 weeks of age and eventually became Sertoli cell-only. Sperm count and motility also declined with age. Furthermore, Bnc1 heterozygotes, although fertile, showed a significant drop in sperm count and in testis weight by 24 weeks of age, suggesting a dosage effect of Bnc1 on testis development. In conclusion, our data demonstrate for the first time BNC1's essential role in maintaining mouse spermatogenesis.

Sorbitol can fuel mouse sperm motility and protein tyrosine phosphorylation via sorbitol dehydrogenase.

Energy sources that can be metabolized to yield ATP are essential for normal sperm functions such as motility. Two major monosaccharides, sorbitol and fructose, are present in semen. Furthermore, sorbitol dehydrogenase (SORD) can convert sorbitol to fructose, which can then be metabolized via the glycolytic pathway in sperm to make ATP. Here we characterize Sord mRNA and SORD expression during mouse spermatogenesis and examine the ability of sorbitol to support epididymal sperm motility and tyrosine phosphorylation. Sord mRNA levels increased during the course of spermatogenic differentiation. SORD protein, however, was first detected at the condensing spermatid stage. By indirect immunofluorescence, SORD was present along the length of the flagella of caudal epididymal sperm. Furthermore, immunoelectron microscopy showed that SORD was associated with mitochondria and the plasma membranes of sperm. Sperm incubated with sorbitol maintained motility, indicating that sorbitol was utilized as an energy source. Sorbitol, as well as glucose and fructose, were not essential to induce hyperactive motility. Protein tyrosine phosphorylation increased in a similar manner when sorbitol was substituted for glucose in the incubation medium used for sperm capacitation. These results indicate that sorbitol can serve as an alternative energy source for sperm motility and protein tyrosine phosphorylation.

Adenylate kinases 1 and 2 are part of the accessory structures in the mouse sperm flagellum.

Proper sperm function depends on adequate ATP levels. In the mammalian flagellum, ATP is generated in the midpiece by oxidative respiration and in the principal piece by glycolysis. In locations where ATP is rapidly utilized or produced, adenylate kinases (AKs) maintain a constant adenylate energy charge by interconverting stoichiometric amounts of ATP and AMP with two ADP molecules. We previously identified adenylate kinase 1 and 2 (AK1 and AK2) by mass spectrometry as part of a mouse SDS-insoluble flagellar preparation containing the accessory structures (fibrous sheath, outer dense fibers, and mitochondrial sheath). A germ cell-specific cDNA encoding AK1 was characterized and found to contain a truncated 3' UTR and a different 5' UTR compared to the somatic Ak1 mRNA; however, it encoded an identical protein. Ak1 mRNA was upregulated during late spermiogenesis, a time when the flagellum is being assembled. AK1 was first seen in condensing spermatids and was associated with the outer microtubular doublets and outer dense fibers of sperm. This localization would allow the interconversion of ATP and ADP between the fibrous sheath where ATP is produced by glycolysis and the axonemal dynein ATPases where ATP is consumed. Ak2 mRNA was expressed at relatively low levels throughout spermatogenesis, and the protein was localized to the mitochondrial sheath in the sperm midpiece. AK1 and AK2 in the flagellar accessory structures provide a mechanism to buffer the adenylate energy charge for sperm motility.

Deficiency of SPAG16L causes male infertility associated with impaired sperm motility.

The axonemes of cilia and flagella contain a "9+2" structure of microtubules and associated proteins. Proteins associated with the central doublet pair have been identified in Chlamydomonas that result in motility defects when mutated. The murine orthologue of the Chlamydomonas PF20 gene, sperm-associated antigen 16 (Spag16), encodes two proteins of M(r) approximately 71 x 10(3) (SPAG16L) and M(r) approximately 35 x 10(3) (SPAG16S). In sperm, SPAG16L is found in the central apparatus of the axoneme. To determine the function of SPAG16L, gene targeting was used to generate mice lacking this protein but still expressing SPAG16S. Mutant animals were viable and showed no evidence of hydrocephalus, lateralization defects, sinusitis, bronchial infection, or cystic kidneys-symptoms typically associated with ciliary defects. However, males were infertile with a lower than normal sperm count. The sperm had marked motility defects, even though ultrastructural abnormalities of the axoneme were not evident. In addition, the testes of some nullizygous animals showed a spermatogenetic defect, which consisted of degenerated germ cells in the seminiferous tubules. We conclude that SPAG16L is essential for sperm flagellar function. The sperm defect is consistent with the motility phenotype of the Pf20 mutants of Chlamydomonas, but morphologically different in that the mutant algal axoneme lacks the central apparatus.