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Marsupial and monotreme cathelicidins display antimicrobial activity, including against methicillin-resistant Staphylococcus aureus
Abstract
With the growing demand for new antibiotics to combat increasing multi-drug resistance, a family of antimicrobial peptides known as cathelicidins has emerged as potential candidates. Expansions in cathelicidin-encoding genes in marsupials and monotremes are of specific interest as the peptides they encode have evolved to protect immunologically naive young in the harsh conditions of the pouch and burrow. Our previous work demonstrated that some marsupial and monotreme cathelicidins have broad-spectrum antibacterial activity and kill resistant bacteria, but the activity of many cathelicidins is unknown. To investigate associations between peptide antimicrobial activity and physiochemical properties, we tested 15 cathelicidin mature peptides from tammar wallaby, grey short-tailed opossum, platypus and echidna for antimicrobial activity against a range of bacterial and fungal clinical isolates. One opossum cathelicidin ModoCath4, tammar wallaby MaeuCath7 and echidna Taac-CATH1 had broad-spectrum antibacterial activity and killed methicillin-resistant Staphylococcus aureus. However, antimicrobial activity was reduced in the presence of serum or whole blood, and non-specific toxicity was observed at high concentrations. The active peptides were highly charged, potentially increasing binding to microbial surfaces, and contained amphipathic helical structures, which may facilitate membrane permeabilisation. Peptide sequence homology, net charge, amphipathicity and alpha helical content did not correlate with antimicrobial activity. However active peptides contained a significantly higher percentage of cationic residues than inactive ones, which may be used to predict active peptides in future work. Along with previous studies, our results indicate that marsupial and monotreme cathelicidins show potential for development as novel therapeutics to combat increasing antimicrobial resistance.
... Therefore, such biological characteristics may have contributed to the expansion and diversification of AMPs during evolution in marsupials (4,13). Cathelicidins of Monodelphis domestica have been studied previously; however, the genes were not completely identified and characterized (14). ...
... The antimicrobial activity of a single opossum cathelicidin, ModoCath4, has been previously demonstrated (14). ...
... Determination of the active core sequence for cathelicidin propeptides depends on the predicted enzyme cleavage site, secondary structures, cationic charges, and hydrophobicity (3,28,29). In this study, we slightly revised the core sequence of three opossum cathelicidins previously thought to have no antimicrobial activity after adjusting the neutrophil elastase cleavage site (14). Interestingly, we were able to detect antimicrobial activity against pathogenic bacteria for all three tested peptides, suggesting that the precise determination of the active domain sequence is critical to assess the activity of cathelicidins. ...
This study aimed to characterize cathelicidins from the gray short-tailed opossum in silico and experimentally validate their antimicrobial effects against various pathogenic bacteria and West Nile virus (WNV). Genome-wide in silico analysis against the current genome assembly of the gray short-tailed opossum yielded 56 classical antimicrobial peptides (AMPs) from eight different families, among which 19 cathelicidins, namely ModoCath1 – 19, were analyzed in silico to predict their antimicrobial domains and three of which, ModoCath1, -5, and -6, were further experimentally evaluated for their antimicrobial activity, and were found to exhibit a wide spectrum of antimicroial effects against a panel of gram-positive and gram-negative bacterial strains. In addition, these peptides displayed low-to-moderate cytotoxicity in mammalian cells as well as stability in serum and various salt and pH conditions. Circular dichroism analysis of the spectra resulting from interactions between ModoCaths and lipopolysaccharides (LPS) showed formation of a helical structure, while a dual-dye membrane disruption assay and scanning electron microscopy analysis revealed that ModoCaths exerted bactericidal effects by causing membrane damage. Furthermore, ModoCath5 displayed potent antiviral activity against WNV by inhibiting viral replication, suggesting that opossum cathelicidins may serve as potentially novel antimicrobial endogenous substances of mammalian origin, considering their large number. Moreover, analysis of publicly available RNA-seq data revealed the expression of eight ModoCaths from five different tissues, suggesting that gray short-tailed opossums may be an interesting source of cathelicidins with diverse characteristics.
... Z tego względu torbacze są doskonałym obszarem badań pod kątem nowych, skutecznych AMPs [36]. W genomie diabłów tasmańskich zlokalizowano geny kodujące sześć różnych katelicydyn: Saha--CATH1 -Saha-CATH6 [37]. ...
... Saha-CATH 3 wykazuje specyficzną aktywność przeciwgrzybiczą względem Cryptococcus neoformans, natomiast Saha-CATH 6 wykazuje właściwości bakteriobójcze w stosunku do niektórych gatunków Streptococcus oraz Enterococcus faecalis opornych na wankomycynę. Spośród badanych związków najszersze spektrum bakteriobójcze wykazuje Saha-CATH 5: udowodniono jego skuteczność zarówno wobec wielu gatunków bakterii Gram-dodatnich, jak i Gram-ujemnych, a zwłaszcza szczepów opornych na wankomycynę (VRE) oraz metycylinę (MRSA) [37]. Dalsze analizy wykazały, że w przypadku peptydów Saha-CATH5 i 6 nie zaob-serwowano toksycznego wpływu na komórki eukariotyczne w stężeniach sięgających 250 μg/ml [37]. ...
... Spośród badanych związków najszersze spektrum bakteriobójcze wykazuje Saha-CATH 5: udowodniono jego skuteczność zarówno wobec wielu gatunków bakterii Gram-dodatnich, jak i Gram-ujemnych, a zwłaszcza szczepów opornych na wankomycynę (VRE) oraz metycylinę (MRSA) [37]. Dalsze analizy wykazały, że w przypadku peptydów Saha-CATH5 i 6 nie zaob-serwowano toksycznego wpływu na komórki eukariotyczne w stężeniach sięgających 250 μg/ml [37]. ...
... Monotremes diverged from therian mammals (marsupials and eutherians) about 166 million years ago (Warren et al. 2008). Young, immunologically naïve monotremes develop immunocompetence through AMPs while living in soil burrows and being exposed to several pathogens (Peel et al. 2017). Transcriptomic analysis of echidna milk cells led to the identification of a novel AMP, echidna antimicrobial protein (EchAMP), whose expression is specific to monotremes and showed in vitro antimicrobial activity, including activity against mastitiscausing pathogens, S. aureus, E. coli, B. subtilis and P. aeruginosa (Bisana et al. 2013;Elhadidy and Elsayyad 2013;Halasa et al. 2007;Kumar et al. 2019). ...
... Hence, there is a requirement for a far-reaching range of antimicrobial agents that can target diverse bacterial strains. Lactation-specific AMPs evolved in the monotreme lineage protect both the mother and young ones from microbial contamination, proving them to be effective bacteriolytic molecules (Newman et al. 2018;Peel et al. 2017). Previous study from our laboratory showed that monotreme-specific EchAMP protein has in vitro broad-spectrum antibacterial activity (Bisana et al. 2013). ...
... EchAMP showed the maximum inhibitory action against S. aureus, known to be found in soil, air and skin (Schulz et al. 2012). Previous studies also showed that lactation proteins from monotremes are effective against S. aureus (Enjapoori et al. 2014;Peel et al. 2017), indicating the more bactericidal activity functions to protect the mammary gland from infections through exposed skin in their habitat (Munday et al. 1998). EchAMP did not show bactericidal activity against two gram-negative strains-E. ...
EchAMP, the tenth most abundant transcript expressed in the mammary gland of echidna, has in vitro broad-spectrum antibacterial effects. However, the effects of EchAMP on mastitis, a condition where inflammation is triggered following mammary gland infection, has not been investigated. To investigate the impact of EchAMP against mastitis, EchAMP transgenic mice were generated. In antibacterial assays, the whey fractions of milk from transgenic mice significantly reduced growth of Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa compared with whey fractions from wildtype mice. Furthermore, a mastitis model created by infecting mammary gland with these four bacterial strains displayed a significant reduction in bacterial load in transgenic mice injected with S. aureus and B. subtilis. On further confirmation, histomorphologic analysis showed absence of necrosis and cell infiltration in the mammary glands of transgenic mice. To understand the role of EchAMP against inflammation, we employed an LPS-injected mastitis mouse model. LPS is known to induce phopshorylation of NF-κB and MAPK pathways, which in turn activate downstream proinflammatory signaling mediators, to promote inflammation. In LPS-treated EchAMP transgenic mice, phosphorylation levels of NF-κB, p38 and ERK1/2 were significantly downregulated. Furthermore, in mammary gland of transgenic mice, there was a significant downregulation of mRNA levels of proinflammatory cytokines, namely TNF-α, IL-6 and IL-1β. Taken together, these data suggest that EchAMP has an antiinflammatory response and is effective against S. aureus and B. subtilis. We suggest that EchAMP may be a potential prophylactic protein against mastitis in dairy animals by expressing this gene in their mammary gland.
... Both families consist of small, positively charged peptides which directly target and kill a range of bacteria and fungi (Steinstraesser et al., 2011;Kosciuczuk et al., 2012). Cathelicidins are of particular interest, as the need protect immunologically naïve young within the pouch has encouraged the lineage specific expansion of the cathelicidin gene family in marsupials (Daly et al., 2008;Wang et al., 2011;Peel et al., 2016;Belov et al., 2007). This has resulted in a high number of cathelicidin genes that encode separate peptides which target and kill different pathogens (Peel et al., 2016(Peel et al., , 2017Wang et al., 2011). ...
... Cathelicidins are of particular interest, as the need protect immunologically naïve young within the pouch has encouraged the lineage specific expansion of the cathelicidin gene family in marsupials (Daly et al., 2008;Wang et al., 2011;Peel et al., 2016;Belov et al., 2007). This has resulted in a high number of cathelicidin genes that encode separate peptides which target and kill different pathogens (Peel et al., 2016(Peel et al., , 2017Wang et al., 2011). In comparison, the majority of eutherian mammals have a very small number of cathelicidins, with only a single gene in humans and mice (Agerberth et al., 1995;Gallo et al.,1997). ...
... Cathelicidins are of particular interest, as the need protect immunologically naïve young within the pouch has encouraged the lineage specific expansion of the cathelicidin gene family in marsupials (Daly et al., 2008;Wang et al., 2011;Peel et al., 2016;Belov et al., 2007). This has resulted in a high number of cathelicidin genes that encode separate peptides which target and kill different pathogens (Peel et al., 2016(Peel et al., , 2017Wang et al., 2011). In comparison, the majority of eutherian mammals have a very small number of cathelicidins, with only a single gene in humans and mice (Agerberth et al., 1995;Gallo et al.,1997). ...
... Pouch secretions include immunomodulators such as cathelicidin, dermcidin, eugenin and cystatin C (Wang et al. 2011;Edwards et al. 2012;Cheng and Belov 2017). Notably, Tasmanian devil pouch secretions include cathelicidins that kill human methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecalis (Peel et al. 2017), which may have future applications in human health. ...
... A cathelicidin expressed during early tammar lactation has been shown to kill multidrug-resistant isolates of P. aeruginosa, Klebsiella pneumoniae and Acinetobacter baumannii (Wang et al. 2011). The activity of marsupial cathelicidins against many multiple drug-resistant bacteria (Wang et al. 2011;Peel et al. 2017) may be exploited in future to treat humans with multidrug-resistant bacterial infections. ...
Marsupials have a very different reproductive strategy to eutherians. An Australian marsupial, the tammar wallaby (Macropus eugenii) has a very short pregnancy of about 26.5 days, with a comparatively long lactation of 300-350 days. The tammar mother gives birth to an altricial, approximately 400 mg young that spends the first 200 days postpartum (p.p.) in its mother's pouch, permanently (0-100 days p.p.; Phase 2A) and then intermittently (100-200 days p.p.; Phase 2B) attached to the teat. The beginning of Phase 3 marks the first exit from the pouch (akin to the birth of a precocious eutherian neonate) and the supplementation of milk with herbage. The marsupial mother progressively alters milk composition (proteins, fats and carbohydrates) and individual milk constituents throughout the lactation cycle to provide nutrients and immunological factors that are appropriate for the considerable physiological development and growth of her pouch young. This review explores the changes in tammar milk components that occur during the lactation cycle in conjunction with the development of the young.
... We quickly annotated the major histocompatibility complex (MHC) ) and other immune gene clusters within the genome (Wong et al. 2006), and discovered highly divergent immune genes, including cytokines . We also discovered novel antimicrobial peptides that had expanded within the genome via gene duplication (Wang et al. 2011;Peel et al. 2017). We have since shown that these independent gene expansions have occurred in different marsupial and monotreme lineages and that these peptides are expressed in the mammary gland, in the pouch (in marsupials that have a pouch) and skin of the young themselves (Peel et al. 2017). ...
... We also discovered novel antimicrobial peptides that had expanded within the genome via gene duplication (Wang et al. 2011;Peel et al. 2017). We have since shown that these independent gene expansions have occurred in different marsupial and monotreme lineages and that these peptides are expressed in the mammary gland, in the pouch (in marsupials that have a pouch) and skin of the young themselves (Peel et al. 2017). These peptides are powerful antimicrobials and hold great potential as novel antibiotics for humans and other animals. ...
The Tasmanian devil, an iconic carnivorous marsupial, is at risk of extinction due to a contagious cancer called devil facial tumour disease. Saving any species from extinction requires strong partnerships between government agencies, zoo bodies and academia. The Devil Tools & Tech project brought these groups together under a single banner to achieve a common goal. The project has strong leadership from women. Here we tell our personal stories as to how we came to be involved in saving the devil and emphasise the importance of strong networks for women to reach their full potential.
... Antimicrobial compounds expressed in the pouch skin likely contribute to changes in the pouch microbiome throughout lactation in marsupials [83,84], and may selectively eliminate pathogens via direct antibacterial activity [79,85,86]. genes, comparable to other marsupial genomes [58-60, 63, 87]. ...
Biodiversity is declining globally, and Australia has one of the worst extinction records for mammals. The development of sequencing technologies means that genomic approaches are now available as important tools for wildlife conservation and management. Despite this, genome sequences are available for only 5% of threatened Australian species. Here we report the first reference genome for the woylie (Bettongia penicillata ogilbyi), a critically endangered marsupial from Western Australia, and the first genome within the Potoroidae family. The woylie reference genome was generated using Pacific Biosciences HiFi long-reads, resulting in a 3.39 Gbp assembly with a scaffold N50 of 6.49 Mbp and 86.5% complete mammalian BUSCOs. Assembly of a global transcriptome from pouch skin, tongue, heart and blood RNA-seq reads was used to guide annotation with Fgenesh++, resulting in the annotation of 24,655 genes. The woylie reference genome is a valuable resource for conservation, management and investigations into disease-induced decline of this critically endangered marsupial.
... PhciCath2 and 5 cluster within a marsupial-specific clade, sister to that containing eutherian cathelicidins (Fig 2). Interestingly, PhciCath5 is located in the clade containing SahaCath3, 5 and 6, ModoCath4, and MaeuCath1 and 7 (Fig 2), all of which display antimicrobial activity [30,35,56]. Focusing on the conserved cathelin domain, the inclusion of partial koala sequences PhciCath7p to 10p does not influence the clustering of koala cathelicidins (S1 Fig). ...
Devastating fires in Australia over 2019-20 decimated native fauna and flora, including koalas. The resulting population bottleneck, combined with significant loss of habitat, increases the vulnerability of remaining koala populations to threats which include disease. Chlamydia is one disease which causes significant morbidity and mortality in koalas. The predominant pathogenic species, Chlamydia pecorum, causes severe ocular, urogenital and reproductive tract disease. In marsupials, including the koala, gene expansions of an antimicrobial peptide family known as cathelicidins have enabled protection of immunologically naïve pouch young during early development. We propose that koala cathelicidins are active against Chlamydia and other bacteria and fungi. Here we describe ten koala cathelicidins, five of which contained full length coding sequences that were widely expressed in tissues throughout the body. Focusing on these five, we investigate their antimicrobial activity against two koala C. pecorum isolates from distinct serovars; MarsBar and IPTaLE, as well as other bacteria and fungi. One cathelicidin, PhciCath5, inactivated C. pecorum IPTaLE and MarsBar elementary bodies and significantly reduced the number of inclusions compared to the control (p
... antimicrobial peptides Antimicrobial peptides (AMPs) are powerful host molecules that bore pores into and disintegrate bacterial membranes, disrupt microbial metabolism, and modulate the activities of immune cells ( Figure 1B; reviewed in Cole and Nizet 2016). AMPs emerged with Archaea approximately 4.3 billion years ago and the cells of vertebrate species have evolved to express different types of these peptides in a species-specific manner (Maxwell et al. 2003;Semple et al. 2003;Das et al. 2010;Parfrey et al. 2011;Notomista et al. 2015;Blodkamp et al. 2016;Peel et al. 2016Peel et al. , 2017. AMPs are small, 10-50 amino acids long, issued within and outside host cells and bind mainly to bacterial lipids via electrostatic charge (Cole and Nizet 2016). ...
Host immune tactics at the level of the single cell have a very large effect on disease progression and host survival. These cell-level defense mechanisms, known as cell-autonomous immunity, are among the most important determinants of human survival, yet are millions to billions of years old, inherited from our prokaryotic and single-celled ancestors. An understanding of how cell-autonomous immunity has evolved in primates is crucial to understanding the human evolutionary story, not only because multiple infectious agents thought to have strongly affected human genomic evolution are excellent manipulators of cell-autonomous immunity, but because these defenses are found in every cell in every physiological system. The ubiquity of cell-autonomous immunity highlights a biological reality not commonly addressed in human evolutionary studies—that pathogens can mediate the evolution of all body cells and, therefore, all body systems, affecting human evolution in a cell-type-specific fashion. Here we explore these very ancient tactics in light of evolutionarily important human pathogens and illustrate inter-primate differences in the potential of such defenses. Often considered an independent physiological system in human evolutionary biology, the immune system is ubiquitous, integrated into every other aspect of human physiology. It is, effectively, the entire organism. We argue, therefore, that immunity and pathogen-mediated natural selection are considerations in the examination of the evolution and function of any human physiological system or trait.
... It was demonstrated that ancient AMPs from Australian marsupial kill antibiotic-resistant pathogens efficiently compared to human cathelicidin LL-37 (Wang et al., 2011). Several studies demonstrated the antimicrobial effect of AMPs from marsupial mammals and how these peptides protect the marsupial pouch Peel et al., 2017). An effective AMP could also help the opossum to fight against T. cruzi when trapped in ETs as known for LL-37 in humans or PR-39 in pigs de Buhr et al., 2017). ...
Granulocytes mediate the first line of defense against infectious diseases in humans as well as animals and they are well known as multitasking cells. They can mediate antimicrobial activity by different strategies depending on the pathogen they encounter. Besides phagocytosis, a key strategy against extracellular pathogens is the formation of extracellular traps (ETs). Those ETs mainly consist of DNA decorated with antimicrobial components and mediate entrapment of various pathogens. In the last years, various studies described ET formation as response to bacteria, viruses and parasites e.g., Trypanosma (T.) cruzi. Nevertheless, it is not fully understood, if ET formation helps the immune system to eliminate intracellular parasites. The goal of this study was to analyze ET formation in response to the intracellular parasite Trypanosma (T.) cruzi by granulocytes derived from animals that serve as natural reservoir. Thus, we investigated the ET formation in two T. cruzi reservoirs, namely dogs as domestic animal and common opossums (Didelphis marsupialis) as wild animal. Granulocytes were harvested from fresh blood by density gradient centrifugation and afterwards incubated with T. cruzi. We conducted the analysis by determination of free DNA and immunofluorescence microscopy. Using both methods, we show that T. cruzi efficiently induces ET formation in granulocytes derived from common opossum as well as dog blood. Most ETs from both animal species as response to T. cruzi are decorated with the protease neutrophil elastase. Since T. cruzi is well known to circulate over years in both analyzed animals as reservoirs, it may be assumed that T. cruzi efficiently evades ET-mediated killing in those animals. Therefore, ETs may not play a major role in efficient elimination of the pathogen from the blood of dogs or common opossums as T. cruzi survives in niches of their body. The characterization of granulocytes in various animals and humans may be helpful to understand the anti-pathogenic capacity and overall role of ETs against zoonotic pathogens like T. cruzi.
Objective:
Infection is one of the main causes of death in clinical patients, and multi-drug resistance leads to ineffective treatment with conventional antibiotics. Therefore, it is imperative to develop new anti-infective drugs. Antimicrobial peptides cathelicidins are cationic host defense peptides found in many organisms. It has been demonstrated by in vivo and in vitro studies that antimicrobial peptides cathelicidins not only show broad-spectrum antibacterial activity and high sensitivity to drug-resistant bacteria, but also have a good guiding effect on the immune response. This paper summarizes the reports of antimicrobial peptides cathelicidins in recent years, highlighting their research achievements in antibiosis, anti-inflammatory, chemotaxis regulation and phagocytosis, providing new ideas for the treatment of infection-related diseases.
Tasmanian devil joeys, like other marsupials, are born at a very early stage of development, prior to the development of their adaptive immune system, yet survive in a pathogen-laden pouch and burrow. Antimicrobial peptides, called cathelicidins, which provide innate immune protection during early life, are expressed in the pouch lining, skin and milk of devil dams. These peptides are active against pathogens identified in the pouch microbiome. Of the six characterised cathelicidins, Saha-CATH5 and 6 have broad-spectrum antibacterial activity and are capable of killing problematic human pathogens including methicillin-resistant S. aureus and vancomycin-resistant E. faecalis, while Saha-CATH3 is active against fungi. Saha-CATH5 and 6 were toxic to human A549 cells at 500 μg/mL, which is over seven times the concentration required to kill pathogens. The remaining devil cathelicidins were not active against tested bacterial or fungal strains, but are widely expressed throughout the body, such as in immune tissues, in digestive, respiratory and reproductive tracts, and in the milk and pouch, which indicates that they are likely also important components of the devil immune system. Our results suggest cathelicidins play a role in protecting naive young during pouch life by passive immune transfer in the milk and may modulate pouch microbe populations to reduce potential pathogens.
Background
The Tasmanian devil, the world’s largest carnivorous marsupial, is at risk of extinction due to devil facial tumour disease (DFTD), a fatal contagious cancer. The Save the Tasmanian Devil Program has established an insurance population, which currently holds over 600 devils in captive facilities across Australia. Microbes are known to play a crucial role in the health and well-being of humans and other animals, and increasing evidence suggests that changes in the microbiota can influence various aspects of host physiology and development. To improve our understanding of devils and facilitate management and conservation of the species, we characterised the microbiome of wild devils and investigated differences in the composition of microbial community between captive and wild individuals.
Results
A total of 1,223,550 bacterial 16S ribosomal RNA (rRNA) sequences were generated via Roche 454 sequencing from 56 samples, including 17 gut, 15 skin, 18 pouch and 6 oral samples. The devil’s gut microbiome was dominated by Firmicutes and showed a high Firmicutes-to-Bacteroidetes ratio, which appears to be a common feature of many carnivorous mammals. Metabolisms of carbohydrates, amino acids, energy, cofactors and vitamins, nucleotides and lipids were predicted as the most prominent metabolic pathways that the devil's gut flora contributed to. The microbiota inside the female’s pouch outside lactation was highly similar to that of the skin, both co-dominated by Firmicutes and Proteobacteria. The oral microbiome had similar proportions of Proteobacteria, Bacteroidetes, Firmicutes and Fusobacteria.
Conclusions
Compositional differences were observed in all four types of microbiota between devils from captive and wild populations. Certain captive devils had significantly lower levels of gut bacterial diversity than wild individuals, and the two groups differed in the proportion of gut bacteria accounting for the metabolism of glycan, amino acids and cofactors and vitamins. Further studies are underway to investigate whether alterations in the microbiome of captive devils can have impacts on their ability to adapt and survive following re-introduction to the wild.
We report a high-quality draft of the genome sequence of the grey, short-tailed opossum (Monodelphis domestica). As the first metatherian ('marsupial') species to be sequenced, the opossum provides a unique perspective on the organization and evolution of mammalian genomes. Distinctive features of the opossum chromosomes provide support for recent theories about genome evolution and function, including a strong influence of biased gene conversion on nucleotide sequence composition, and a relationship between chromosomal characteristics and X chromosome inactivation. Comparison of opossum and eutherian genomes also reveals a sharp difference in evolutionary innovation between protein-coding and non-coding functional elements. True innovation in protein-coding genes seems to be relatively rare, with lineage-specific differences being largely due to diversification and rapid turnover in gene families involved in environmental interactions. In contrast, about 20% of eutherian conserved non-coding elements (CNEs) are recent inventions that postdate the divergence of Eutheria and Metatheria. A substantial proportion of these eutherian-specific CNEs arose from sequence inserted by transposable elements, pointing to transposons as a major creative force in the evolution of mammalian gene regulation.
Metagenomic analysis of 16S ribosomal RNA has been used to profile microbial communities at high resolution, and to examine their association with host diet or diseases. We examined the oral and gut microbiome composition of two captive koalas to determine whether bacterial communities are unusual in this species, given that their diet consists almost exclusively of Eucalyptus leaves. Despite a highly specialized diet, koala oral and gut microbiomes were similar in composition to the microbiomes from the same body regions of other mammals. Rectal swabs contained all of the diversity present in faecal samples, along with additional taxa, suggesting that faecal bacterial communities may merely subsample the gut bacterial diversity. Furthermore, the faecal microbiomes of the captive koalas were similar to those reported for wild koalas, suggesting that captivity may not compromise koala microbial health. Since koalas frequently suffer from ocular diseases caused by Chlamydia infection, we also examined the eye microbiome composition of two captive koalas, establishing the healthy baseline for this body part. The eye microbial community was very diverse, similar to other mammalian ocular microbiomes but with an unusually high representation of bacteria from the family Phyllobacteriaceae.
The influence of ion composition, pH, and peptide concentration on the conformation and activity of the 37-residue human antibacterial peptide LL-37 has been studied. At micromolar concentration in water, LL-37 exhibits a circular dichroism spectrum consistent with a disordered structure. The addition of 15 mm HCO3⁻, SO4²⁻, or CF3CO2⁻ causes the peptide to adopt a helical structure, with approximately equal efficiency, while 160 mm Cl⁻ is less efficient. A cooperative transition from disordered to helical structure is observed as the peptide concentration is increased, consistent with formation of an oligomer. The extent of α-helicity correlates with the antibacterial activity of LL-37 against both Gram-positive and Gram-negative bacteria. Two homologous peptides, FF-33 and SK-29, containing 4 and 8 residue deletions at the N terminus, respectively, require higher concentrations of anions for helix formation and are less active than LL-37 against Escherichia coli D21. Below pH 5, the helical content of LL-37 gradually decreases, and at pH 2 it is entirely disordered. In contrast, the helical structure is retained at pH over 13. The minimal inhibitory concentration of LL-37 againstE. coli is 5 μm, and at 13–25 μm the peptide is cytotoxic against several eukaryotic cells. In solutions containing the ion compositions of plasma, intracellular fluid, or interstitial fluid, LL-37 is helical, and hence it could pose a danger to human cells upon release. However, in the presence of human serum, the antibacterial and the cytotoxic activities of LL-37 are inhibited.
The increased prevalence of multidrug-resistant (MDR) bacteria in combination with the relatively limited development of new antibiotics presents a serious threat to public health. In chicken, especially Extended-Spectrum ß-Lactamase (ESBL) carrying Enterobacteriaceae are often asymptomatically present but can infect humans. Due to their broad range antimicrobial activity cathelicidins and other host defence peptides, are considered to be an attractive alternative to conventional antibiotics. In this study, the antimicrobial activity of three chicken cathelicidins against a broad array of multidrug resistant bacteria was determined. All three peptides showed high antibacterial activity independent of the presence of MDR characteristics. Induction experiments using S. aureus and K. pneumoniae showed that although an increase in resistance was initially observed, susceptibility towards chicken cathelicidins remained high and no major resistance was developed. The combined results underline the potential of chicken cathelicidins as a new alternative to antibiotics.
The antibacterial and cytotoxic activity of the human cathelicidin peptide LL-37 is inhibited by plasma. Because LL-37 does
not undergo rapid degradation in human plasma, we postulated that this inhibition results from binding of LL-37 to unidentified
proteins. An LL-37 binding plasma protein has now been isolated by affinity chromatography. SDS-polyacrylamide gel electrophoresis
of proteins that bound to an LL-37 column revealed one band with a molecular mass of about 26 kDa, and amino acid sequence
analysis identified the protein as apolipoprotein A-I (apoA-I). Biomolecular interaction analysis using surface plasmon resonance
showed that LL-37 and isolated apoA-I bind with an apparent K
d in the low micromolar range. 50 μm of apoA-I inhibits the antibacterial activity of 50 μm LL-37 by about 50% of the inhibition exhibited by plasma. In addition, anti-apoA-I IgG completely blocks the plasma inhibition
of LL-37 antibacterial activity up to a peptide concentration of 25 μm and blocks most of the plasma inhibition at higher LL-37 concentrations. These results indicate that apoA-I is the main LL-37
binding protein in human plasma and may work as a scavenger of LL-37, thus suggesting a novel mechanism involved in the regulation
of a cathelicidin peptide.
Cathelicidins are small, cationic, antimicrobial peptides found in humans and other species, including farm animals (cattle, horses, pigs, sheep, goats, chickens, rabbits and in some species of fish). These proteolytically activated peptides are part of the innate immune system of many vertebrates. These peptides show a broad spectrum of antimicrobial activity against bacteria, enveloped viruses and fungi. Apart from exerting direct antimicrobial effects, cathelicidins can also trigger specific defense responses in the host. Their roles in various pathophysiological conditions have been studied in mice and humans, but there are limited information about their expression sites and activities in livestock. The aim of the present review is to summarize current information about these antimicrobial peptides in farm animals, highlighting peptide expression sites, activities, and future applications for human and veterinary medicine.
A female platypus Ornithorhynchus
anatinus was intensively monitored with the use of video equipment in captivity at Healesville Sanctuary, Victoria, Australia to record feeding and behavioural changes that occurred as a result of only the second successful raising of young platypus (twins) in captivity. These results revealed the female to be inactive (not leaving the burrow) for periods up to 6 days, and mating occurred 15–21 days before eggs were laid. The nest was built over 5 nights between 5 and 9 days before the eggs were laid. The eggs were thought to have been laid during a 4-day period when the female was confined to her burrow. Once the eggs were laid there was a slow initial rate of feeding which sharply rose 20 days after the eggs were laid. The first time the female spent a day away from the young was 39 days after the eggs were laid, after which she decreasingly nested with them until she spent only 8% of the time with them when they emerged from the burrow, 131 and 136 days after the eggs are thought to have been laid. She rarely went in the nest after day 155, suggesting that the lactation period is c. 135–145 days, assuming 10 days for incubation. The time of emergence also corresponded with the peak in the female''s food consumption, which correlates well with the high demands of late lactation, with the daily food intake reaching c. 90–100% of body weight 140 days after the eggs are thought to have hatched. This captive breeding of the platypus has provided the first records of the gestation period, food consumption in relation to breeding stage, the time the female spends with her young during their development, and a partial growth curve of developing platypus.
Protein identification and analysis software performs a central role in the investigation of proteins from two-dimensional
(2-D) gels and mass spectrometry. For protein identification, the user matches certain empirically acquired information against
a protein database to define a protein as already known or as novel. For protein analysis, information in protein databases
can be used to predict certain properties about a protein, which can be useful for its empirical investigation. The two processes
are thus complementary. Although there are numerous programs available for those applications, we have developed a set of
original tools with a few main goals in mind. Specifically, these are:
1.
To utilize the extensive annotation available in the Swiss-Prot database (1) wherever possible, in particular the position-specific annotation in the Swiss-Prot feature tables to take into account
posttranslational modifications and protein processing.
2.
To develop tools specifically, but not exclusively, applicable to proteins prepared by twodimensional gel electrophoresis
and peptide mass fingerprinting experiments.
3.
To make all tools available on the World-Wide Web (WWW), and freely usable by the scientific community.
We present the genome sequence of the tammar wallaby, Macropus eugenii, which is a member of the kangaroo family and the first representative of the iconic hopping mammals that symbolize Australia to be sequenced. The tammar has many unusual biological characteristics, including the longest period of embryonic diapause of any mammal, extremely synchronized seasonal breeding and prolonged and sophisticated lactation within a well-defined pouch. Like other marsupials, it gives birth to highly altricial young, and has a small number of very large chromosomes, making it a valuable model for genomics, reproduction and development. RESULTS: The genome has been sequenced to 2 × coverage using Sanger sequencing, enhanced with additional next generation sequencing and the integration of extensive physical and linkage maps to build the genome assembly. We also sequenced the tammar transcriptome across many tissues and developmental time points. Our analyses of these data shed light on mammalian reproduction, development and genome evolution: there is innovation in reproductive and lactational genes, rapid evolution of germ cell genes, and incomplete, locus-specific X inactivation. We also observe novel retrotransposons and a highly rearranged major histocompatibility complex, with many class I genes located outside the complex. Novel microRNAs in the tammar HOX clusters uncover new potential mammalian HOX regulatory elements. CONCLUSIONS: Analyses of these resources enhance our understanding of marsupial gene evolution, identify marsupial-specific conserved non-coding elements and critical genes across a range of biological systems, including reproduction, development and immunity, and provide new insight into marsupial and mammalian biology and genome evolution.
The Tasmanian devil (Sarcophilus harrisii), the largest marsupial carnivore, is endangered due to a transmissible facial cancer spread by direct transfer of living cancer cells through biting. Here we describe the sequencing, assembly, and annotation of the Tasmanian devil genome and whole-genome sequences for two geographically distant subclones of the cancer. Genomic analysis suggests that the cancer first arose from a female Tasmanian devil and that the clone has subsequently genetically diverged during its spread across Tasmania. The devil cancer genome contains more than 17,000 somatic base substitution mutations and bears the imprint of a distinct mutational process. Genotyping of somatic mutations in 104 geographically and temporally distributed Tasmanian devil tumors reveals the pattern of evolution and spread of this parasitic clonal lineage, with evidence of a selective sweep in one geographical area and persistence of parallel lineages in other populations.
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Background
We present the genome sequence of the tammar wallaby, Macropus eugenii, which is a member of the kangaroo family and the first representative of the iconic hopping mammals that symbolize Australia to be sequenced. The tammar has many unusual biological characteristics, including the longest period of embryonic diapause of any mammal, extremely synchronized seasonal breeding and prolonged and sophisticated lactation within a well-defined pouch. Like other marsupials, it gives birth to highly altricial young, and has a small number of very large chromosomes, making it a valuable model for genomics, reproduction and development.
Results
The genome has been sequenced to 2 × coverage using Sanger sequencing, enhanced with additional next generation sequencing and the integration of extensive physical and linkage maps to build the genome assembly. We also sequenced the tammar transcriptome across many tissues and developmental time points. Our analyses of these data shed light on mammalian reproduction, development and genome evolution: there is innovation in reproductive and lactational genes, rapid evolution of germ cell genes, and incomplete, locus-specific X inactivation. We also observe novel retrotransposons and a highly rearranged major histocompatibility complex, with many class I genes located outside the complex. Novel microRNAs in the tammar HOX clusters uncover new potential mammalian HOX regulatory elements.
Conclusions
Analyses of these resources enhance our understanding of marsupial gene evolution, identify marsupial-specific conserved non-coding elements and critical genes across a range of biological systems, including reproduction, development and immunity, and provide new insight into marsupial and mammalian biology and genome evolution.
Erratum to: Genome sequence of an Australian kangaroo, Macropus eugenii, provides insight into the evolution of mammalian reproduction and development (Genome Biology 2011 12:414)
To overcome the increasing resistance of pathogens to existing antibiotics the 10×'20 Initiative declared the urgent need for a global commitment to develop 10 new antimicrobial drugs by the year 2020. Naturally occurring animal antibiotics are an obvious place to start. The recently sequenced genomes of mammals that are divergent from human and mouse, including the tammar wallaby and the platypus, provide an opportunity to discover novel antimicrobials. Marsupials and monotremes are ideal potential sources of new antimicrobials because they give birth to underdeveloped immunologically naïve young that develop outside the sterile confines of a uterus in harsh pathogen-laden environments. While their adaptive immune system develops innate immune factors produced either by the mother or by the young must play a key role in protecting the immune-compromised young. In this study we focus on the cathelicidins, a key family of antimicrobial peptide genes.
We identified 14 cathelicidin genes in the tammar wallaby genome and 8 in the platypus genome. The tammar genes were expressed in the mammary gland during early lactation before the adaptive immune system of the young develops, as well as in the skin of the pouch young. Both platypus and tammar peptides were effective in killing a broad range of bacterial pathogens. One potent peptide, expressed in the early stages of tammar lactation, effectively killed multidrug-resistant clinical isolates of Pseudomonas aeruginosa, Klebsiella pneumoniae and Acinetobacter baumannii.
Marsupial and monotreme young are protected by antimicrobial peptides that are potent, broad spectrum and salt resistant. The genomes of our distant relatives may hold the key for the development of novel drugs to combat multidrug-resistant pathogens.
To understand the evolutionary origins of our own immune system, we need to characterise the immune system of our distant relatives, the marsupials and monotremes. The recent sequencing of the genomes of two marsupials (opossum and tammar wallaby) and a monotreme (platypus) provides an opportunity to characterise the immune gene repertoires of these model organisms. This was required as many genes involved in immunity evolve rapidly and fail to be detected by automated gene annotation pipelines.
We have developed a database of immune genes from the tammar wallaby, red-necked wallaby, northern brown bandicoot, brush-tail possum, opossum, echidna and platypus. The resource contains 2,235 newly identified sequences and 3,197 sequences which had been described previously. This comprehensive dataset was built from a variety of sources, including EST projects and expert-curated gene predictions generated through a variety of methods including chained-BLAST and sensitive HMMER searches. To facilitate systems-based research we have grouped sequences based on broad Gene Ontology categories as well as by specific functional immune groups. Sequences can be extracted by keyword, gene name, protein domain and organism name. Users can also search the database using BLAST.
The Immunome Database for Marsupials and Monotremes (IDMM) is a comprehensive database of all known marsupial and monotreme immune genes. It provides a single point of reference for genomic and transcriptomic datasets. Data from other marsupial and monotreme species will be added to the database as it become available. This resource will be utilized by marsupial and monotreme immunologists as well as researchers interested in the evolution of mammalian immunity.
Production of antimicrobial peptides and proteins is an important means of host defense in eukaryotes. The larger antimicrobial
proteins, containing more than 100 amino acids, are often lytic enzymes, nutrient-binding proteins or contain sites that target
specific microbial macromolecules. The smaller antimicrobial peptides act largely by disrupting the structure or function
of microbial cell membranes. Hundreds of antimicrobial peptides have been found in the epithelial layers, phagocytic cells
and body fluids of multicellular animals, from mollusks to humans. Some antimicrobial peptides are produced constitutively,
others are induced in response to infection or inflammation. Studies of the regulation of antimicrobial peptide synthesis
in Drosophila have been particularly fruitful, and have provided a new paradigm for the analysis of mammalian host defense
responses. It now appears that the general patterns of antimicrobial responses of invertebrates have been preserved in vertebrates
(“innate immunity”) where they contribute to host defense both independently and in complex interplay with adaptive immunity.
Bactenecins are a class of arginine-rich antibacterial peptides of bovine neutrophil granules. Two bactenecins with approximate molecular weights of 5,000 and 7,000 designated Bac5 and Bac7, respectively, exert in vitro a potent bactericidal activity toward several gram-negative bacteria (R. Gennaro, B. Skerlavaj, and D. Romeo, Infect. Immun. 57:3142-3146, 1989). We have now found that this activity shows an inverse relationship to the ionic strength of the medium and is inhibited by divalent cations and greatly potentiated by lactoferrin. Under conditions supporting marked bactericidal activity, the two peptides cause a rapid increase in the permeability of both the outer and inner membranes of Escherichia coli, as shown by unmasking of periplasmic beta-lactamase and of cytoplasmic beta-galactosidase. In addition, the two bactenecins inhibit the respiration of E. coli and Klebsiella pneumoniae but not of Bac5- and Bac7-resistant Staphylococcus aureus. Furthermore, they induce a drop in ATP content in E. coli, K. pneumoniae, and Salmonella typhimurium and a marked decrease in the rates of transport and incorporation of [3H]leucine and [3H]uridine into E. coli protein and RNA, respectively. In general, all these effects become evident within 1 to 2 min and reach their maximal expression within about 5 min. Overall, these data strongly suggest that the decrease in bacterial viability is causally related to the increase in membrane permeability and the subsequent fall in respiration-linked proton motive force, with the attendant loss of cellular metabolites and macromolecular biosynthesis ability.
PR-39, a proline/arginine-rich peptide antibiotic, has been purified from pig intestine and later shown to originate in the bone marrow. Intending to isolate a clone for a human counterpart to PR-39, we synthesized a PCR probe derived from the PR-39 gene. However, when this probe was used to screen a human bone marrow cDNA library, eight clones were obtained with information for another putative human peptide antibiotic, designated FALL-39 after the first four residues. FALL-39 is a 39-residue peptide lacking cysteine and tryptophan. All human peptide antibiotics previously isolated (or predicted) belong to the defensin family and contain three disulfide bridges. The clone for prepro-FALL-39 encodes a cathelin-like precursor protein with 170 amino acid residues. We have postulated a dibasic processing site for the mature FALL-39 and chemically synthesized the putative peptide. In basal medium E, synthetic FALL-39 was highly active against Escherichia coli and Bacillus megaterium. Residues 13-34 in FALL-39 can be predicted to form a perfect amphiphatic helix, and CD spectra showed that medium E induced 30% helix formation in FALL-39. RNA blot analyses disclosed that the gene for FALL-39 is expressed mainly in human bone marrow and testis.
Cathelicidins are a family of myeloid antimicrobial peptide precursors that have been identified in several mammalian species
(Zanetti, M., Gennaro, R., and Romeo, D. (1995) FEBS Lett. 374, 1-5). Two novel bovine congeners have been deduced from cDNA. Their C-terminal sequences of 27 and 28 residues correspond
to putative antimicrobial peptides with a cationic N-terminal region predicted to assume an amphipathic α-helical conformation
followed by a hydrophobic C-terminal tail. Peptides corresponding to these sequences have been chemically synthesized and
shown to exert a potent antimicrobial activity against Gram-negative and Gram-positive bacteria, including methicillin-resistant
Staphylococcus aureus, and fungi. Both peptides are also cytotoxic to human erythrocytes and neutrophils, although at higher than microbicidal
concentrations. The target selectivity has been improved by synthesizing truncated analogues, comprising only the 18 N-terminal
residues, which show a great reduction in cytotoxic, but not in antimicrobial activity. The involvement of the C-terminal
hydrophobic tail in the cytotoxic activity has been further demonstrated by inducing a major loss of activity in an analogue
after replacing highly hydrophobic residues with more hydrophilic ones.
The influence of ion composition, pH, and peptide concentration on the conformation and activity of the 37-residue human antibacterial peptide LL-37 has been studied. At micromolar concentration in water, LL-37 exhibits a circular dichroism spectrum consistent with a disordered structure. The addition of 15 mM HCO3-, SO42-, or CF3CO2- causes the peptide to adopt a helical structure, with approximately equal efficiency, while 160 mM Cl- is less efficient. A cooperative transition from disordered to helical structure is observed as the peptide concentration is increased, consistent with formation of an oligomer. The extent of alpha-helicity correlates with the antibacterial activity of LL-37 against both Gram-positive and Gram-negative bacteria. Two homologous peptides, FF-33 and SK-29, containing 4 and 8 residue deletions at the N terminus, respectively, require higher concentrations of anions for helix formation and are less active than LL-37 against Escherichia coli D21. Below pH 5, the helical content of LL-37 gradually decreases, and at pH 2 it is entirely disordered. In contrast, the helical structure is retained at pH over 13. The minimal inhibitory concentration of LL-37 against E. coli is 5 microM, and at 13-25 microM the peptide is cytotoxic against several eukaryotic cells. In solutions containing the ion compositions of plasma, intracellular fluid, or interstitial fluid, LL-37 is helical, and hence it could pose a danger to human cells upon release. However, in the presence of human serum, the antibacterial and the cytotoxic activities of LL-37 are inhibited.
Endogenous antimicrobial peptides of the cathelicidin family contribute to innate immunity. The emergence of widespread antibiotic resistance in many commonly encountered bacteria requires the search for new bactericidal agents with therapeutic potential. Solid-phase synthesis was employed to prepare linear antimicrobial peptides found in cathelicidins of five mammals: human (FALL39/LL37), rabbit (CAP18), mouse (mCRAMP), rat (rCRAMP), and sheep (SMAP29 and SMAP34). These peptides were tested at ionic strengths of 25 and 175 mM against Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, and methicillin-resistant Staphylococcus aureus. Each peptide manifested activity against P. aeruginosa irrespective of the NaCl concentration. CAP18 and SMAP29 were the most effective peptides of the group against all test organisms under both low- and high-salt conditions. Select peptides of 15 to 21 residues, modeled on CAP18 (37 residues), retained activity against the gram-negative bacteria and methicillin-sensitive S. aureus, although the bactericidal activity was reduced compared to that of the parent peptide. In accordance with the behavior of the parent molecule, the truncated peptides adopted an alpha-helical structure in the presence of trifluoroethanol or lipopolysaccharide. The relationship between the bactericidal activity and several physiochemical properties of the cathelicidins was examined. The activities of the full-length peptides correlated positively with a predicted gradient of hydrophobicity along the peptide backbone and with net positive charge; they correlated inversely with relative abundance of anionic residues. The salt-resistant, antimicrobial properties of CAP18 and SMAP29 suggest that these peptides or congeneric structures have potential for the treatment of bacterial infections in normal and immunocompromised persons and individuals with cystic fibrosis.
Antimicrobial peptides have been evaluated in vitro and in vivo as alternatives to conventional antibiotics. Apart from being antimicrobial, the native human cathelicidin-derived peptide LL-37 (amino acids [aa] 104 to 140 of the human cathelicidin antimicrobial peptide) also binds and neutralizes bacterial lipopolysaccharide (LPS) and might therefore have beneficial effects in the treatment of septic shock. However, clinical trials have been hampered by indications of toxic effects of LL-37 on mammalian cells and evidence that its antimicrobial effects are inhibited by serum. For the present study, LL-37 was compared to two less hydrophobic fragments obtained by N-terminal truncation, named 106 (aa 106 to 140) and 110 (aa 110 to 140), and to a previously described more hydrophobic variant, the 18-mer LLKKK, concerning antimicrobial properties, lipopolysaccharide neutralization, toxicity against human erythrocytes and cultured vascular smooth muscle cells, chemotactic activity, and inhibition by serum. LL-37, fragments 106 and 110, and the 18-mer LLKKK inhibited the growth of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans in a radial diffusion assay, inhibited lipopolysaccharide-induced vascular nitric oxide production, and attracted neutrophil granulocytes similarly. While fragments 106 and 110 caused less hemolysis and DNA fragmentation in cultured cells than did LL-37, the 18-mer LLKKK induced severe hemolysis. The antibacterial effect of fragments 106 and 110 was not affected by serum, while the effect of LL-37 was reduced. We concluded that the removal of N-terminal hydrophobic amino acids from LL-37 decreases its cytotoxicity as well as its inhibition by serum without negatively affecting its antimicrobial or LPS-neutralizing action. Such LL-37-derived peptides may thus be beneficial for the treatment of patients with sepsis.
LL-37 is a human cationic host defense peptide that is present in the specific granules of neutrophils, produced by epithelial cells from a variety of tissues, and is upregulated during inflammation, infection, and injury. It has been proposed to have a variety of antimicrobial functions, including both direct antimicrobial activity and immunomodulatory functions. Using the TUNEL assay it was demonstrated that LL-37 induced apoptosis in vitro in the A549 human lung and 16 HBE4o- human airway epithelial cell lines, and in vivo in the murine airway. Peptide-induced apoptosis in vitro involved the activation of caspase pathways and was substantially inhibited by an inhibitor of caspase 3. Apoptosis was also inhibited by human serum, but not fetal bovine serum. Similarly, human but not fetal bovine serum inhibited the cellular internalization of LL-37 and the production of IL-8 in response to LL-37 treatment of epithelial cells. The protective effects of human serum were also observed with high-density lipoproteins but not by the core peptide apolipoprotein A1, providing one possible mechanism of human serum inhibition of apoptosis. We propose that LL-37-induced apoptosis of epithelial cells at low serum tissue sites may have a protective role against bacterial infection.
The availability of the first marsupial genome sequence has allowed us to characterize the immunome of the gray short-tailed opossum (Monodelphis domestica). Here we report the identification of key immune genes, including the highly divergent chemokines, defensins, cathelicidins, and Natural Killer cell receptors. It appears that the increase in complexity of the mammalian immune system occurred prior to the divergence of the marsupial and eutherian lineages approximately 180 million years ago. Genomes of ancestral mammals most likely contained all of the key mammalian immune gene families, with evolution on different continents, in the presence of different pathogens leading to lineage specific expansions and contractions, resulting in some minor differences in gene number and composition between different mammalian lineages. Gene expansion and extensive heterogeneity in opossum antimicrobial peptide genes may have evolved as a consequence of the newborn young needing to survive without an adaptive immune system in a pathogen laden environment. Given the similarities in the genomic architecture of the marsupial and eutherian immune systems, we propose that marsupials are ideal model organisms for the study of developmental immunology.
Marsupials differ from most other mammals in their method of reproduction, in that they have chosen, in an evolutionary sense, to develop lactation rather than placentation for the nurture of their young. The neonate is therefore born with a mixture of advanced and embryonic characters, and yet is readily accessible within the pouch, providing a unique system for the study of the ontogeny of various physiological and endocrinological parameters. Marsupials are therefore ideal animals for research into mammalian reproductive physiology. The results of this exciting new research are summarized in this book by two of the foremost workers in the field. Individual chapters analyse the genetic and hormonal control of sexual differentiation, male and female reproductive structures and their functions, the role of the corpus luteum in the oestrous cycle and pregnancy, the hormonal control of embryonic diapause and the role of the marsupial placenta in the development of the embryo. This book is more than just a straightforward review of marsupial reproduction for its detailed analyses and broad comparative coverage will attract mammalogists and reproductive physiologists with a wide range of research interests.
Over the past half a century research has revealed that marsupials – far from being ‘second class’ mammals – have adaptations for particular ways of life quite equal to their placental counterparts. Despite long separate evolution, there are extraordinary similarities in which marsupials have solved the challenges of living in such environments as deserts, alpine snowfields or tropical rainforests. Some can live on grass, some on pollen and others on leaves; some can glide, some can swim and others hop with extraordinary efficiency. In Life of Marsupials, one of the world’s leading experts explores the biology and evolution of this unusual group – with their extraordinary diversity of forms around the world – in Australia, New Guinea and South America. Joint winner of the 2005 Whitley Medal. Included in Choice Magazine's 2006 Outstanding Academic Titles list.
LL-37 is a human cationic host defense peptide that is present in the specific granules of neutrophils, produced by epithelial cells from a variety of tissues, and is upregulated during inflammation, infection, and injury. It has been proposed to have a variety of antimicrobial functions, including both direct antimicrobial activity and immunomodulatory functions. Using the TUNEL assay it was demonstrated that LL-37 induced apoptosis in vitro in the A549 human lung and 16HBE4o- human airway epithelial cell lines, and in vivo in the murine airway. Peptide-induced apoptosis in vitro involved the activation of caspase pathways and was substantially inhibited by an inhibitor of caspase 3. Apoptosis was also inhibited by human serum, but not fetal bovine serum. Similarly, human but not fetal bovine serum inhibited the cellular internalization of LL-37 and the production of IL-8 in response to LL-37 treatment of epithelial cells. The protective effects of human serum were also observed with high-density lipoproteins but not by the core peptide apolipoprotein All, providing one possible mechanism of human serum inhibition of apoptosis. We propose that LL-37-induced apoptosis of epithelial cells at low serum tissue sites may have a protective role against bacterial infection.
As a dietary source, the foliage of Eucalyptus spp. is low in available protein and carbohydrate while containing polyphenolic compounds that interfere with enzymatic digestion. To overcome this, the koala (Phascolarctos cinereus) has evolved a range of anatomical and physiological adaptations to assist with digestion and absorption of nutrients from this food source. Microbial fermentation of partially digested eucalyptus leaves is thought to be critical in this process, however, little is known about the composition and diversity of microorganisms that are associated with digestive health in this native species. In this study, we performed 16S rRNA gene pyrosequencing of caecum, colon and faecal pellet samples from two wild, free ranging, Queensland koalas. Our results reveal a highly complex and diverse ecosystem with considerable intra-individual variation. Although samples were dominated by sequences from the Bacteroidetes and Firmicutes phyla there was considerable variation at the genus level. This study is the first non-culture based microbiota analysis, using 454-amplicon pyrosequencing, and provides preliminary data to expand our understanding of the koala hindgut.
The sequencing of the platypus genome has spurred investigations into the characterisation of the monotreme immune response. As the most divergent of extant mammals, the characterisation of the monotreme immune repertoire allows us to trace the evolutionary history of immunity in mammals and provide insights into the immune gene complement of ancestral mammals. The immune system of monotremes has remained largely uncharacterised due to the lack of specific immunological reagents and limited access to animals for experimentation. Early immunological studies focussed on the anatomy and physiology of the lymphoid system in the platypus. More recent molecular studies have focussed on characterisation of individual immunoglobulin, T-cell receptor and MHC genes in both the platypus and short-beaked echidna. Here, we review the published literature on the monotreme immune gene repertoire and provide new data generated from genome analysis on cytokines, Fc receptors and immunoglobulins. We present an overview of key gene families responsible for innate and adaptive immunity including the cathelicidins, defensins, T-cell receptors and the major histocompatibility complex (MHC) Class I and Class II antigens. We comment on the usefulness of these sequences for future studies into immunity, health and disease in monotremes.
A computer program that progressively evaluates the hydrophilicity and hydrophobicity of a protein along its amino acid sequence has been devised. For this purpose, a hydropathy scale has been composed wherein the hydrophilic and hydrophobic properties of each of the 20 amino acid side-chains is taken into consideration. The scale is based on an amalgam of experimental observations derived from the literature. The program uses a moving-segment approach that continuously determines the average hydropathy within a segment of predetermined length as it advances through the sequence. The consecutive scores are plotted from the amino to the carboxy terminus. At the same time, a midpoint line is printed that corresponds to the grand average of the hydropathy of the amino acid compositions found in most of the sequenced proteins. In the case of soluble, globular proteins there is a remarkable correspondence between the interior portions of their sequence and the regions appearing on the hydrophobic side of the midpoint line, as well as the exterior portions and the regions on the hydrophilic side. The correlation was demonstrated by comparisons between the plotted values and known structures determined by crystallography. In the case of membrane-bound proteins, the portions of their sequences that are located within the lipid bilayer are also clearly delineated by large uninterrupted areas on the hydrophobic side of the midpoint line. As such, the membrane-spanning segments of these proteins can be identified by this procedure. Although the method is not unique and embodies principles that have long been appreciated, its simplicity and its graphic nature make it a very useful tool for the evaluation of protein structures.
Cathelicidins secreted in milk may be central to autocrine feedback in the mammary gland for optimal development in addition to conferring innate immunity to both the mammary gland and the neonate. This study exploits the unique reproductive strategy of the tammar wallaby (Macropus eugenii) model to analyse differential splicing of cathelicidin genes and to evaluate the bactericidal activity and effect of the protein on mammary epithelial cell proliferation. Two linear peptides, Con73 and Con218, derived from the heterogeneous carboxyl end of cathelicidin transcripts, MaeuCath1 and MaeuCath7 respectively, were evaluated for antimicrobial activity. Both Con73 and Con218 significantly inhibited the growth of Staphylococcus aureus, Pseudomonas aureginosa, Enterococcus faecalis and Salmonella enterica. In addition both MaeuCath1 and MaeuCath7 stimulated proliferation of primary tammar wallaby mammary epithelial cells (WallMEC). Lactation-phase specific alternate spliced transcripts were determined for MaeuCath1 showing utilisation of both antimicrobial and proliferative functions are required by the mammary gland and the suckled young. The study has shown for the first time that temporal regulation of milk cathelicidins may be crucial in antimicrobial protection of the mammary gland and suckled young and mammary cell proliferation.
Marsupial mammals, born in an extremely atricial state with no functional immune system, offer a unique opportunity to investigate both the developing microbiome and its relationship to that of the mother and the potential influence of this microbiome upon the development of the immune system. In this study we used a well-established marsupial model animal, Macropus eugenii, the tammar wallaby, to document the microbiome of three related sites: the maternal pouch and saliva, and the gastrointestinal tract (GIT) of the young animal. We used molecular-based methods, targeting the 16S rDNA gene to determine the bacterial diversity at these study sites. In the maternal pouch, 41 unique phylotypes, predominantly belonging to the phylum Actinobacteria, were detected, while in the saliva, 48 unique phylotypes were found that predominantly belonged to the phylum Proteobacteria. The GIT of the pouch young had a complex microbiome of 53 unique phylotypes, even though the pouch young were still permanently attached to the teat and had only been exposed to the external environment for a few minutes immediately after birth while making their way from the birth canal to the maternal pouch. Of these 53 phylotypes, only nine were detected at maternal sites. Overall, the majority of bacteria isolated were novel species (<97 % identity to known 16S rDNA sequences), and each study site (i.e. maternal pouch and saliva, and the GIT of the pouch young) possessed its own unique microbiome.
Antimicrobial peptides (AMPs) are widely expressed and rapidly induced at epithelial surfaces to repel assault from diverse infectious agents including bacteria, viruses, fungi and parasites. Much information suggests that AMPs act by mechanisms that extend beyond their capacity to serve as gene-encoded antibiotics. For example, some AMPs alter the properties of the mammalian membrane or interact with its receptors to influence diverse cellular processes including cytokine release, chemotaxis, antigen presentation, angiogenesis and wound healing. These functions complement their antimicrobial action and favor resolution of infection and repair of damaged epithelia. Opposing this, some microbes have evolved mechanisms to inactivate or avoid AMPs and subsequently become pathogens. Thus, AMPs are multifunctional molecules that have a central role in infection and inflammation.
An algorithm has been developed to improve the success rate in the prediction of the secondary structure of proteins by taking
into account the predicted class of the proteins. This method has been called the ‘double prediction method’ and consists
of a first prediction of the secondary structure from a new algorithm which uses parameters of the type described by Chou
and Fasman, and the prediction of the dass of the proteins from their amino acid composition. These two independent predictions
allow one to optimize the parameters calculated over the secondary structure database to provide the final prediction of secondary
structure. This method has been tested on 59 proteins in the database (i.e. 10 322 residues) and yields 72% success in class
prediction, 61.3% of residues correctly predicted for three states (helix, sheet and coil) and a good agreement between observed
and predicted contents in secondary structure.
A study has been made of the development of four lymphoid tissues from birth to maturity in the tammar wallaby Macropus eugenii--the cervical and thoracic thymus, lymph nodes and gut-associated lymphoid tissue (GALT). The development of these tissues in the tammar wallaby is similar to that in two other marsupials, the quokka Setonix brachyurus and the Virginian opossum Didelphis virginiana. Lymphocytes were first detected in the cervical thymus of the tammar at Day 2 post partum and in the thoracic thymus at Day 6. They were subsequently detected in lymph nodes at Day 4 and in the spleen by Day 12 but were not apparent in the GALT until around Day 90 post partum. By Day 21, the cervical thymus had developed distinct areas of cortex and medulla and Hassall's corpuscles were apparent. The maturation of other tissues followed with Hassall's corpuscles in the thoracic thymus by Day 30 and nodules and germinal centres in the lymph nodes by Day 90. Measurement of immunoglobulin G concentrations in the serum of young animals indicated a rise in titre around Day 90 post partum, correlating with the apparent maturation of the lymphoid tissues.
The airway surface is an important host defense against pulmonary infection. Secretion of proteins with antimicrobial activity from epithelial cells onto the airway surface represents an important component of this innate immune system. Defensins are the best characterized epithelial-derived peptide antibiotics. A member of another family of peptide antibiotics called cathelicidins recently was identified from human bone marrow. We show in this paper that this human peptide named LL-37/hCAP-18 also may play a role in innate immunity of the human lung. In situ hybridization localized high levels of LL-37/hCAP-18 RNA to surface epithelial cells of the conducting airway as well as serous and mucous cells of the submucosal glands. LL-37/hCAP-18 peptide with antimicrobial activity was partially purified from airway surface fluid from human lung and a human bronchial xenograft model. The synthetic peptide LL-37 demonstrated antibiotic activity against a number of Gram-negative and Gram-positive organisms including Pseudomonas aeruginosa; bacterial killing of LL-37 was sensitive to NaCl and was synergistic with lactoferrin and lysozyme. In summary, we show that LL-37/hCAP-18 is a peptide with broad antimicrobial activity that is secreted onto the airway surface from epithelial cells of the human lung.
Antimicrobial peptides are present in a wide range of species, from protozoa to man, as effector molecules of innate immunity. Several bovine precursors of antimicrobial peptides have recently been identified, as deduced from cDNA, and assigned to the cathelicidin family. Two of these are the proforms of the antimicrobial peptides BMAP-27 and BMAP-28, which share a similar amino acid sequence, structural conformation, and toxic activity toward several bacterial and fungal strains. Here we report that they are cytotoxic to human tumor cells and normal proliferating, but not resting, lymphocytes at concentrations comparable to those microbiocidal. This effect is primarily due to damage of plasma membrane integrity. A more detailed investigation of the U937 cell line revealed that a Ca2+ influx into the cytosol occurs in the early steps of permeabilization. The perturbation of the membrane structure and the Ca2+ influx are followed by programmed death. A similar apoptosis inducing effect is also observed on in vitro activated human lymphocytes.
Permeation of the cell membrane leading to cell death is a mechanism used by a large number of membrane-lytic peptides. Some are linear, mostly helical, and others contain one or more disulfide bonds forming beta-sheet or both beta-sheet and alpha-helix structures. They are all soluble in solution but when they reach the target membrane, conformational changes occur which let them associate with and lyse the membrane. Some lytic peptides are not cell-selective and lyse different microorganisms and normal mammalian cells, while others are specific to either type of cells. Despite extensive studies, the mode of action of membrane-lytic peptides is not fully understood and the basis for their selectivity towards specific target cells is not known. Many studies have shown that peptide-lipid interactions leading to membrane permeation play a major role in their activity. Membrane permeation by amphipathic alpha-helical peptides has been proposed to occur via one of two general mechanisms: (i) transmembrane pore formation via a 'barrel-stave' mechanism; and (ii) membrane destruction/solubilization via a 'carpet' mechanism. This review, which is focused on the different stages of membrane permeation induced by representatives of amphipathic alpha-helical antimicrobial and cell non-selective lytic peptides distinguishes between the 'carpet' mechanism, which holds for antimicrobial peptides versus the 'barrel-stave' mechanism, which holds for cell non-selective lytic peptides.
SMAP-29 is a cathelicidin-derived peptide deduced from sheep myeloid mRNA. The C-terminally amidated form of this peptide was chemically synthesized and shown to exert a potent antimicrobial activity. Antibiotic-resistant clinical isolates highly susceptible to this peptide include MRSA and VREF isolates, that are a major worldwide problem, and mucoid Pseudomonas aeruginosa associated with chronic respiratory inflammation in CF patients. In addition, SMAP-29 is also active against fungi, including Cryptococcus neoformans isolated from immunocompromised patients. SMAP-29 causes significant morphological alterations of the bacterial surfaces, as shown by scanning electron microscopy, and is also hemolytic against human, but not sheep erythrocytes. Its potent antimicrobial activity suggests that this peptide is an excellent candidate as a lead compound for the development of novel antiinfective agents.
Antimicrobial peptides are important host-defense molecules of innate immunity. Cathelicidins are a diverse family of potent, rapidly acting and broadly effective antimicrobial peptides, which are produced by a variety of cells. This review examines the classification, antimicrobial spectrum, mechanism of action, and regulation of cathelicidins.
Cationic antimicrobial peptides are observed throughout nature. In mammals they are observed both at epithelial surfaces and within the granules of phagocytic cells. They are an important component of innate defences, since in addition to their ability to kill microorganisms, they are able to modulate inflammatory responses. With respect to their ability to kill bacteria, it is very difficult to isolate resistant mutants. However there are a few known mechanisms of intrinsic resistance, including PhoPQ-dependent and other alterations in lipopolysaccharide structure that influence self promoted uptake, and protease-mediated resistance.
Antimicrobial peptides have been isolated and characterized from tissues and organisms representing virtually every kingdom and phylum, ranging from prokaryotes to humans. Yet, recurrent structural and functional themes in mechanisms of action and resistance are observed among peptides of widely diverse source and composition. Biochemical distinctions among the peptides themselves, target versus host cells, and the microenvironments in which these counterparts convene, likely provide for varying degrees of selective toxicity among diverse antimicrobial peptide types. Moreover, many antimicrobial peptides employ sophisticated and dynamic mechanisms of action to effect rapid and potent activities consistent with their likely roles in antimicrobial host defense. In balance, successful microbial pathogens have evolved multifaceted and effective countermeasures to avoid exposure to and subvert mechanisms of antimicrobial peptides. A clearer recognition of these opposing themes will significantly advance our understanding of how antimicrobial peptides function in defense against infection. Furthermore, this understanding may provide new models and strategies for developing novel antimicrobial agents, that may also augment immunity, restore potency or amplify the mechanisms of conventional antibiotics, and minimize antimicrobial resistance mechanisms among pathogens. From these perspectives, the intention of this review is to illustrate the contemporary structural and functional themes among mechanisms of antimicrobial peptide action and resistance.
The production of natural antibiotic peptides has emerged as an important mechanism of innate immunity in plants and animals. Defensins are diverse members of a large family of antimicrobial peptides, contributing to the antimicrobial action of granulocytes, mucosal host defence in the small intestine and epithelial host defence in the skin and elsewhere. This review, inspired by a spate of recent studies of defensins in human diseases and animal models, focuses on the biological function of defensins.
Antimicrobial peptides extracted from ovine neutrophils have potential to be high-value by-products of the lamb industry as, for example, a biopreservative for chilled lamb products. This work was carried out to determine the conditions in which ovine peptides are most effective and to assist in product development. The activities of three synthetic ovine-derived antimicrobial peptides tested were not significantly affected by pH or temperature. However, they exhibited decreased activity at high ionic strengths and in the presence of divalent cations. The three peptides worked better in combination than individually.
Antimicrobial peptides, such as cathelicidin, are an evolutionarily old defense system. However they have more complex actions than just simply their antimicrobial effects, including immunoregulation and interaction with the adaptive immune system. In this study we have characterized several novel cathelicidin-like peptides from the tammar wallaby (Macropus eugenii). The tammar cathelicidin-like (MaeuCath) mRNA were isolated based on the conservation of the cathelin-like amino terminus. Mature MaeuCath peptides were positively charged with hydrophobic carboxyl tails, features that are fundamental for antimicrobial function. MaeuCath1 was induced in tammar leukocytes in response to pathogen-associated molecular patterns from both gram positive and negative bacteria. In addition, we also examined the expression of MaeuCath1 in the primary and secondary lymphoid organs of the tammar neonate throughout early pouch life. The results from this study demonstrate the importance that MaeuCath1 may play in innate defense of the marsupial young, especially in the mucosal organs. Such expression of antimicrobial peptides may form part of the immune strategies of marsupials for neonatal survival during their post-partum development.
The bacterial diversity of the openings of the urogenital and anal tracts of the adult female tammar wallaby, Macropus eugenii, was determined in order to ascertain whether the physical proximity of the openings of these tracts within the cloaca affected the two populations of bacteria. Terminal restriction fragment length polymorphism (T-RFLP) analyses of 42 wallabies identified 81 different terminal fragments, indicative of diverse and complex microbiomes at these anatomical locations. Subsequent amplified rDNA restriction analysis (ARDRA) identified 72 phylotypes from the urogenital tract and 50 from the anal tract. Twenty-two of these phylotypes were common to both tracts. Phylogenetic analysis of sequenced 16S rDNA showed that 83 % of the phylotypes were unidentified species based on the premise that any sequence possessing <97 % homology to a known bacterial species or phylotype was novel. Thus, despite the close proximity of the openings of the urogenital and anal tracts within the cloaca, the two sites retained a diverse range of distinct bacteria, with only a small percentage of overlapping species.