Scedosporium Cell Wall: From Carbohydrate-Containing Structures to Host–Pathogen Interactions

Scedosporium species are filamentous fungi usually found in sewage and soil from human-impacted areas. They cause a wide range of diseases in humans, from superficial infections, such as mycetoma, to invasive and disseminated cases, especially associated in immunocompromised patients. Scedosporium species are also related to lung colonization in individuals presenting cystic fibrosis and are considered one of the most frequent fungal pathogens associated to this pathology. Scedosporium cell wall contains glycosylated molecules involved in important biological events related to virulence and pathogenicity and represents a significant source of antigens. Polysaccharides, peptidopolysaccharides, O-linked oligosaccharides and glycosphingolipids have been identified on the Scedosporium surface. Their primary structures were determined based on a combination of techniques including gas chromatography, ESI-MS, and ¹H and ¹³C nuclear magnetic resonance. Peptidorhamnnomannans are common cell wall components among Scedosporium species. Comparing different species, minor structural differences in the carbohydrate portions were detected which could be useful to understand variations in virulence observed among the species. N- and O-linked peptidorhamnomannans are major pathogen-associated molecular patterns and, along with α-glucans, play important roles in triggering host innate immunity. Glycosphingolipids, such as glucosylceramides, have highly conserved structures in Scedosporium species and are crucial for fungal growth and virulence. The present review presents current knowledge on structural and functional aspects of Scedosporium glycoconjugates that are relevant for understanding pathogenicity mechanisms and could contribute to the design of new agents capable of inhibiting growth and differentiation of Scedosporium species. Other cell components such as melanin and ectophosphatases will be also included.

     

Phenotypic Profiling of Scedosporium aurantiacum,an Opportunistic Pathogen Colonizing Human Lungs

Genotyping studies of Australian Scedosporium isolates have revealed the strong prevalence of a recently described species: Scedosporium aurantiacum. In addition to occurring in the environment, this fungus is also known to colonise the respiratory tracts of cystic fibrosis (CF) patients. A high throughput Phenotype Microarray (PM) analysis using 94 assorted substrates (sugars, amino acids, hexose-acids and carboxylic acids) was carried out for four isolates exhibiting different levels of virulence, determined using a Galleria mellonella infection model. A significant difference was observed in the substrate utilisation patterns of strains displaying differential virulence. For example, certain sugars such as sucrose (saccharose) were utilised only by low virulence strains whereas some sugar derivatives such as D-turanose promoted respiration only in the more virulent strains. Strains with a higher level of virulence also displayed flexibility and metabolic adaptability at two different temperature conditions tested (28 and 37°C). Phenotype microarray data were integrated with the whole-genome sequence data of S. aurantiacum to reconstruct a pathway map for the metabolism of selected substrates to further elucidate differences between the strains.     

Current Status in Diagnosis of Scedosporium Infections: What Is the Impact of New Molecular Methods?

Scedosporium species are increasingly encountered as fungal pathogens. Species identification is important due to species-specific differences in epidemiology, antifungal susceptibility and virulence. Histology and culture-based identification are hampered by their low sensitivity and specificity. The use of new selective media has improved the recovery rate from clinical samples. Molecular methods, including multiplex PCR, PCR-RFLP analysis, DNA sequencing, oligonucleotide arrays, real-time PCR, rolling circle amplification, are increasingly used for species identification. Most recently, Matrix-Assisted Laser Desorption-Time of Flight Mass Spectrometry has been successfully applied as a tool for rapid identification of clinically relevant Scedosporium species. This review aims to summarize the methods currently used to guide the clinical microbiology laboratory in the selection of the most appropriate identification techniques. This will aid the laboratory in making a fast and reliable diagnosis that enables the clinician to make correct treatment choices.     

Inmunopatología de las micosis invasivas por hongos filamentosos

Invasive fungal infections caused by filamentous fungi are devastating diseases that occur in patients with a variety of immunosuppressive conditions. This review focuses on the pathogenesis of the most important invasive mycosis in the human being caused by the filamentous fungi Aspergillus, Fusarium, Scedosporium and mucorales. The first contact between the mould and the patient, the host defense to different fungi, including the role of mucosa in the innate immune system, the whole innate immune recognition receptors, and the pathways connecting innate and adaptive immunity, as well as the virulence factors of fungi, are discussed in this paper.     

Clinical characteristics and epidemiology of pulmonary pseudallescheriasis

BackgroundSome members of the Pseudallescheria (anamorph Scedosporium) have emerged as an important cause of life-threatening infections in humans. These fungi may reach the lungs and bronchial tree causing a wide range of manifestations, from colonization of airways to deep pulmonary infections. Frequently, they may also disseminate to other organs, with a predilection for the brain. In otherwise healthy patients, the infection is characterized by non-invasive type involvement, while invasive and/or disseminated infections were mostly seen in immunocompromised patients.AimsWe reviewed all the available reports on Pseudallescheria/Scedosporium pulmonary infections, focusing on the geographical distribution, immune status of infected individuals, type of infections, clinical manifestations, treatment and outcome.Results and conclusionsThe main clinical manifestations of the 189 cases of pulmonary pseudallescheriasis reviewed were pneumonia (89), followed by fungus ball (26), and chest abscess (18). Some patients had more than one type of invasive pulmonary manifestations. Among patients with pneumonia, several cases of pneumonia associated with near-drowning (10/89, 11.2%) have also been reported in immunocompetent hosts. Major underlying conditions for non-invasive pulmonary infection were preexisting lung cavities and medical immunosuppression for invasive pulmonary infection. Saprobic airway colonization was mostly seen in patients with mucosal dysfunction, i.e. patients with cystic fibrosis. The mortality rate was closely related to the infection type, being 26.8% in non-invasive type (fungus balls) and 57.2% in invasive type.     

Gene Disruption in <Emphasis Type="Italic">Scedosporium aurantiacum</Emphasis>: Proof of Concept with the Disruption of <Emphasis Type="Italic">SODC</Emphasis> Gene Encoding a Cytosolic Cu,Zn-Superoxide Dismutase

Scedosporium species are opportunistic pathogens responsible for a large variety of infections in humans. An increasing occurrence was observed in patients with underlying conditions such as immunosuppression or cystic fibrosis. Indeed, the genus Scedosporium ranks the second among the filamentous fungi colonizing the respiratory tracts of the CF patients. To date, there is very scarce information on the pathogenic mechanisms, at least in part because of the limited genetic tools available. In the present study, we successfully developed an efficient transformation and targeted gene disruption approach on the species Scedosporium aurantiacum. The disruption cassette was constructed using double-joint PCR procedure, and resistance to hygromycin B as the selection marker. This proof of concept was performed on the functional gene SODC encoding the Cu,Zn-superoxide dismutase. Disruption of the SODC gene improved susceptibility of the fungus to oxidative stress. This technical advance should open new research areas and help to better understand the biology of Scedosporium species.     

Ecology of <Emphasis Type="Italic">Scedosporium</Emphasis> Species: Present Knowledge and Future Research

The genus Scedosporium, which comprises at least five clinically relevant species, i.e. Scedosporium apiospermum, Scedosporium boydii, Scedosporium aurantiacum, Scedosporium dehoogii and Scedosporium minutisporum, ranks the second among the filamentous fungi colonizing the airways of patients with cystic fibrosis (CF). This colonization of the airways is thought to contribute to the inflammatory reaction leading to a progressive deterioration of the lung function. Additionally, these colonizing fungi may lead to severe disseminated infections in case of lung transplantation. Therefore, considering the low susceptibility of Scedosporium species to all current antifungal drugs, preventive measures should be defined to reduce the risk of exposure to these fungi for non-colonized CF patients. With this in mind, several studies have been conducted to elucidate the ecology of these fungi and to define possible sources of patient contamination. This review will summarize the major outcomes of those studies, including: the clear demonstration that ecological niches of Scedosporium species are strongly impacted by human activities, and the ability of Scedosporium species to degrade aliphatic and aromatic pollutants which supports the high occurrence of these species in contaminated soils and polluted waters and makes them promising candidates for bioremediation purposes. Finally, prospects for future research in this field are proposed.     

Monitoring of Vibrio harveyi quorum sensing activity in real time during infection of brine shrimp larvae

Quorum sensing, bacterial cell-to-cell communication, has been linked to the virulence of pathogenic bacteria. Indeed, in vitro experiments have shown that many bacterial pathogens regulate the expression of virulence genes by this cell-to-cell communication process. Moreover, signal molecules have been detected in samples retrieved from infected hosts and quorum sensing disruption has been reported to result in reduced virulence in different host–pathogen systems. However, data on in vivo quorum sensing activity of pathogens during infection of a host are currently lacking. We previously reported that quorum sensing regulates the virulence of Vibrio harveyi in a standardised model system with gnotobiotic brine shrimp (Artemia franciscana) larvae. Here, we monitored quorum sensing activity in Vibrio harveyi during infection of the shrimp, using bioluminescence as a read-out. We found that wild-type Vibrio harveyi shows a strong increase in quorum sensing activity early during infection. In this respect, the bacteria behave remarkably similar in different larvae, despite the fact that only half of them survive the infection. Interestingly, when expressed per bacterial cell, Vibrio harveyi showed around 200-fold higher maximal quorum sensing-regulated bioluminescence when associated with larvae than in the culture water. Finally, the in vivo quorum sensing activity of mutants defective in the production of one of the three signal molecules is consistent with their virulence, with no detectable in vivo quorum sensing activity in AI-2- and CAI-1-deficient mutants. These results indicate that AI-2 and CAI-1 are the dominant signals during infection of brine shrimp.     

Insights into the social life and obscure side of Scedosporium/Lomentospora species: ubiquitous,emerging and multidrug-resistant opportunistic pathogens

The undeniable relevance of fungal infections caused by both yeasts and molds has considerably augmented over the last four decades, becoming a serious health public problem in hospitals worldwide. In this scenario, several fungal species have emerged as human pathogens able to cause diseases in both immucompromised and immunocompetent individuals. In this context, species belonging to the Scedosporium and Lomentospora genera are awakening global attention due to their ubiquitous distribution in human-impacted environments, multidrug–resistance profile and ability to cause severe and debilitating illnesses, which culminate in laborious and meticulous medical care, substantial treatment costs and high (and unacceptable!) morbidity and mortality rates. Together, these reasons have been stimulating the international scientific community to take a hard work in order to circumvent these undesirable situations. In this way, the researchers are trying to decipher the main physiopathological events behind the successful capacity of Scedosporium/Lomentospora species to escape from host immune attacks and to resist the available chemotherapeutic treatment options. Therefore, in an attempt to add some pieces to this puzzle, the present review compiles the main published data on taxonomy, genomic, ecology, diagnosis, epidemiology, clinical manifestations, relevant clinic-epidemiological statistics/metrics, current treatment options, promising alternative therapeutic approaches, expression of virulence attributes, in vitro interaction with phagocytic and non-phagocytic cells, in vivo animal infections and host immune defenses in the Scedosporium/Lomentospora field.     

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires'' disease, is an important human pathogen that infects and replicates within alveolar macrophages. Its virulence depends on the Dot/Icm type IV secretion system (T4SS), which is essential to establish a replication permissive vacuole known as the Legionella containing vacuole (LCV). L. pneumophila infection can be modeled in mice however most mouse strains are not permissive, leading to the search for novel infection models. We have recently shown that the larvae of the wax moth Galleria mellonella are suitable for investigation of L. pneumophila infection. G. mellonella is increasingly used as an infection model for human pathogens and a good correlation exists between virulence of several bacterial species in the insect and in mammalian models. A key component of the larvae''s immune defenses are hemocytes, professional phagocytes, which take up and destroy invaders. L. pneumophila is able to infect, form a LCV and replicate within these cells. Here we demonstrate protocols for analyzing L. pneumophila virulence in the G. mellonella model, including how to grow infectious L. pneumophila, pretreat the larvae with inhibitors, infect the larvae and how to extract infected cells for quantification and immunofluorescence microscopy. We also describe how to quantify bacterial replication and fitness in competition assays. These approaches allow for the rapid screening of mutants to determine factors important in L. pneumophila virulence, describing a new tool to aid our understanding of this complex pathogen.     

Pouring Salt on a Wound: Pseudomonas aeruginosa Virulence Factors Alter Na+ and Cl? Flux in the Lung

Pseudomonas aeruginosa is a ubiquitous opportunistic pathogen with multiple niches in the human body, including the lung. P. aeruginosa infections are particularly damaging or fatal for patients with ventilator-associated pneumonia, chronic obstructive pulmonary disease, and cystic fibrosis (CF). To establish an infection, P. aeruginosa relies on a suite of virulence factors, including lipopolysaccharide, phospholipases, exoproteases, phenazines, outer membrane vesicles, type III secreted effectors, flagella, and pili. These factors not only damage the epithelial cell lining but also induce changes in cell physiology and function such as cell shape, membrane permeability, and protein synthesis. While such virulence factors are important in initial infection, many become dysregulated or nonfunctional during the course of chronic infection. Recent work on the virulence factors alkaline protease (AprA) and CF transmembrane conductance regulator inhibitory factor (Cif) show that P. aeruginosa also perturbs epithelial ion transport and osmosis, which may be important for the long-term survival of this microbe in the lung. Here we discuss the literature regarding host physiology-altering virulence factors with a focus on Cif and AprA and their potential roles in chronic infection and immune evasion.     

Functional Dissection of Streptococcus pyogenes M5 Protein: the Hypervariable Region is Essential for Virulence

The surface-localized M protein of Streptococcus pyogenes is a major virulence factor that inhibits phagocytosis, as determined ex vivo. Because little is known about the role of M protein in vivo we analyzed the contribution of different M protein regions to virulence, using the fibrinogen (Fg)-binding M5 protein and a mouse model of acute invasive infection. This model was suitable, because M5 is required for mouse virulence and binds mouse and human Fg equally well, as shown here. Mixed infection experiments with wild type bacteria demonstrated that mutants lacking the N-terminal hypervariable region (HVR) or the Fg-binding B-repeat region were strongly attenuated, while a mutant lacking the conserved C-repeats was only slightly attenuated. Because the HVR of M5 is not required for phagocytosis resistance, our data imply that this HVR plays a major but unknown role during acute infection. The B-repeat region is required for phagocytosis resistance and specifically binds Fg, suggesting that it promotes virulence by binding Fg. However, B-repeat mutants were attenuated even in Fg-deficient mice, implying that the B-repeats may have a second function, in addition to Fg-binding. These data demonstrate that two distinct M5 regions, including the HVR, are essential to virulence during the early stages of an infection. In particular, our data provide the first in vivo evidence that the HVR of an M protein plays a major role in virulence, focusing interest on the molecular role of this region.     

Invasive scedosporiosis in lung transplant recipients: A nine-year retrospective study in a tertiary care hospital

BackgroundScedosporium species and Lomentospora prolificans (Sc/Lp) are emerging molds that cause invasive disease associated with a high mortality rate. After Aspergillus, these molds are the second filamentous fungi recovered in lung transplant (LT) recipients.AimsOur objective was to evaluate the incidence, risk factors and outcome of Sc/Lp infections in LT recipients at a tertiary care hospital with a national reference LT program.MethodsA nine-year retrospective study was conducted.ResultsDuring this period, 395 LT were performed. Positive cultures for Sc/Lp were obtained from twenty-one LT recipients. Twelve patients (incidence 3.04%) developed invasive scedosporiosis (IS). In 66.7% of the patients with IS the invasive infection was defined as a breakthrough one. The main sites of infection were lungs and paranasal sinuses. Most of the patients received combination antifungal therapy. The IS crude mortality rate after 30 days was 16.7%, and 33.3% after a year.ConclusionsOur study highlights improved survival rates associated with combination antifungal therapy in LT recipients and underlines the risk of breakthrough infections in patients with allograft dysfunction on nebulized lipidic amphotericin B prophylaxis. In addition to pretransplant colonization, acute or chronic organ dysfunctions seem to be the main risk factors for IS.     

The Influence of Infective Dose on the Virulence of a Generalist Pathogen in Rainbow Trout (Oncorhynchus mykiss) and Zebra Fish (Danio rerio)

Pathogen density and genetic diversity fluctuate in the outside-host environment during and between epidemics, affecting disease emergence and the severity and probability of infections. Although the importance of these factors for pathogen virulence and infection probability has been acknowledged, their interactive effects are not well understood. We studied how an infective dose in an environmentally transmitted opportunistic fish pathogen, Flavobacterium columnare, affects its virulence both in rainbow trout, which are frequently infected at fish farms, and in zebra fish, a host that is not naturally infected by F. columnare. We used previously isolated strains of confirmed high and low virulence in a single infection and in a co-infection. Infection success (measured as host morbidity) correlated positively with dose when the hosts were exposed to the high-virulence strain, but no response for the dose increase was found when the hosts were exposed to the low-virulence strain. Interestingly, the co-infection resulted in poorer infection success than the single infection with the high-virulence strain. The rainbow trout were more susceptible to the infection than the zebra fish but, in both species, the effects of the doses and the strains were qualitatively similar. We suggest that as an increase in dose can lead to increased host morbidity, both the interstrain interactions and differences in infectivity in different hosts may influence the severity and consequently the evolution of disease. Our results also confirm that the zebra fish is a good laboratory model to study F. columnare infection.     

Vibrio cholerae Hemolysin Is Required for Lethality,Developmental Delay,and Intestinal Vacuolation in Caenorhabditis elegans

Background

Cholera toxin (CT) and toxin-co-regulated pili (TCP) are the major virulence factors of Vibrio cholerae O1 and O139 strains that contribute to the pathogenesis of disease during devastating cholera pandemics. However, CT and TCP negative V. cholerae strains are still able to cause severe diarrheal disease in humans through mechanisms that are not well understood.

Methodology/Principal Findings

To determine the role of other virulence factors in V. cholerae pathogenesis, we used a CT and TCP independent infection model in the nematode Caenorhabditis elegans and identified the hemolysin A (hlyA) gene as a factor responsible for animal death and developmental delay. We demonstrated a correlation between the severity of infection in the nematode and the level of hemolytic activity in the V. cholerae biotypes. At the cellular level, V. cholerae infection induces formation of vacuoles in the intestinal cells in a hlyA dependent manner, consistent with the previous in vitro observations.

Conclusions/Significance

Our data strongly suggest that HlyA is a virulence factor in C. elegans infection leading to lethality and developmental delay presumably through intestinal cytopathic changes.     

Streptococcus pneumoniae Serine Protease HtrA,but Not SFP or PrtA,Is a Major Virulence Factor in Pneumonia

Streptococcus (S.) pneumoniae is a common causative pathogen in pneumonia. Serine protease orthologs expressed by a variety of bacteria have been found of importance for virulence. Previous studies have identified two serine proteases in S. pneumoniae, HtrA (high-temperature requirement A) and PrtA (cell wall-associated serine protease A), that contributed to virulence in models of pneumonia and intraperitoneal infection respectively. We here sought to identify additional S. pneumoniae serine proteases and determine their role in virulence. The S. pneumoniae D39 genome contains five putative serine proteases, of which HtrA, Subtilase Family Protein (SFP) and PrtA were selected for insertional mutagenesis because they are predicted to be secreted and surface exposed. Mutant D39 strains lacking serine proteases were constructed by in-frame insertion deletion mutagenesis. Pneumonia was induced by intranasal infection of mice with wild-type or mutant D39. After high dose infection, only D39ΔhtrA showed reduced virulence, as reflected by strongly reduced bacterial loads, diminished dissemination and decreased lung inflammation. D39ΔprtA induced significantly less lung inflammation together with smaller infiltrated lung surface, but without influencing bacterial loads. After low dose infection, D39ΔhtrA again showed strongly reduced bacterial loads; notably, pneumococcal burdens were also modestly lower in lungs after infection with D39Δsfp. These data confirm the important role for HtrA in S. pneumoniae virulence. PrtA contributes to lung damage in high dose pneumonia; it does not however contribute to bacterial outgrowth in pneumococcal pneumonia. SFP may facilitate S. pneumoniae growth after low dose infection.