Overview of microbial biofilms

As the success of this two-issue special section of the Journal of Industrial Microbiology attests, the study of microbial biofilms is truly burgeoning as the uniqueness and the importance of this mode of growth is increasingly recognized. Because of its universality the biofilm concept impacts virtually all of the subdivisions of Microbiology (including Medical, Dental, Agricultural, Industrial and Environmental) and these two issues incorporate contributions from authors in all of these disciplines. Some time ago we reasoned that bacteria cannot possibly be aware (sic) of their precise location, in terms of this spectrum of anthrocentric subspecialties, and that their behavior must be dictated by a standard set of phenotypic responses to environmental conditions in what must seem to them (sic) to be a continuum of very similar aquatic ecosystems. In this overview I will, therefore, stress the common features of microbial biofilms that we should bear in mind as we use this simple universal concept to seek to understand bacterial behavior in literally hundreds of aquatic ecosystems traditionally studied by dozens of subspecies of microbiologists reared in sharply different scientific and academic conventions.     

ras2 Controls morphogenesis,pheromone response,and pathogenicity in the fungal pathogen Ustilago maydis

Ustilago maydis, a pathogen of maize, is a useful model for the analysis of mating, pathogenicity, and the morphological transition between budding and filamentous growth in fungi. As in other fungi, these processes are regulated by conserved signaling mechanisms, including the cyclic AMP (cAMP)/protein kinase A (PKA) pathway and at least one mitogen-activated protein kinase (MAP kinase) pathway. A current challenge is to identify additional factors that lie downstream of the cAMP pathway and that influence morphogenesis in U. maydis. In this study, we identified suppressor mutations that restored budding growth to a constitutively filamentous mutant with a defect in the gene encoding a catalytic subunit of PKA. Complementation of one suppressor mutation unexpectedly identified the ras2 gene, which is predicted to encode a member of the well-conserved ras family of small GTP-binding proteins. Deletion of the ras2 gene in haploid cells altered cell morphology, eliminated pathogenicity on maize seedlings, and revealed a role in the production of aerial hyphae during mating. We also used an activated ras2 allele to demonstrate that Ras2 promotes pseudohyphal growth via a MAP kinase cascade involving the MAP kinase kinase Fuz7 and the MAP kinase Ubc3. Overall, our results reveal an additional level of crosstalk between the cAMP signaling pathway and a MAP kinase pathway influenced by Ras2.     

Characterisation of<Emphasis Type="Italic">fusarium graminearum</Emphasis> and<Emphasis Type="Italic">F. culmorum</Emphasis> isolates by mycotoxin production and aggressiveness to wheat

A total of 27Fusarium culmorum isolates from Germany and 41F. graminearum isolates from Kenya were investigated for aggressiveness and mycotoxin production on wheat ears. In addition, ergosterol content of the kernels from ears inoculated withF. graminearum was determined and theF. culmorum isolates were tested for mycotoxin productionin vitro. For both pathogens, isolates markedly differed in aggressiveness. 59% and 37% of theF. culmorum isolates produced NIV and DON, respectively,in vivo andin vitro. The DON-producing isolates also produced 3-acDONin vitro. The more aggressive isolates produced mainly DON while the less aggressive isolates produced mainly NIV. 12% and 85% of theF. graminearum isolates produced NIV and DON, respectively. The highly aggressive isolates produced higher amounts of DON, aggressiveness being highly correlated to DON content in the kernels. NIV-producing isolates were less aggressive. Ergosterol content of kernels was moderately correlated to aggressiveness but highly correlated to DON content. Disease severity was associated with kernel weight reduction.     

A Putative P-Type ATPase,Apt1, Is Involved in Stress Tolerance and Virulence in Cryptococcus neoformans

The export of virulence factors, such as the capsule polysaccharide, to the cell surface is a critical aspect of the pathogenicity of Cryptococcus neoformans. A view of capsule export via exocytosis and extracellular vesicles is emerging, but the molecular mechanisms underlying virulence factor transport pathways remain to be established. In this study, we characterized the APT1 gene, which encodes a predicted integral membrane P-type ATPase belonging to the type IV, Drs2 family of aminophospholipid translocases (flippases) (APTs). APTs maintain the phospholipid asymmetry that is critical in membrane fusion events for trafficking and in establishing cell polarity. Deletion of the APT1 gene resulted in phenotypes consistent with similar roles in C. neoformans. These included altered actin distribution, increased sensitivity to stress conditions (oxidative and nitrosative stress) and to trafficking inhibitors, such as brefeldin A and monensin, a reduction in exported acid phosphatase activity, and hypersensitivity to the antifungal drugs amphotericin B, fluconazole, and cinnamycin. However, there was no difference in growth, capsule size, or melanin production between the wild type and the apt1 mutant strains at either 30°C or 37°C. Despite the absence of an influence on these major virulence factors, Apt1 was required for survival during interactions with macrophages, and apt1 mutants exhibited attenuated virulence in a mouse inhalation model of cryptococcosis. Therefore, Apt1 contributes to virulence and the stress response in C. neoformans through apparent functions in membrane fusion and trafficking that do not influence the deposition of major virulence factors, such as capsule and melanin, outside the cell.The opportunistic fungal pathogen Cryptococcus neoformans causes life-threatening meningoencephalitis in immunocompromised individuals (44). One million cases of cryptococcosis are estimated to occur each year, and approximately two-thirds of these are fatal (43). Key virulence traits for the fungus include growth at the mammalian host temperature, production of a polysaccharide capsule, deposition of laccase-synthesized melanin in the cell wall, secretion of enzymes, and resistance to host defenses, such as oxidative and nitrosative killing (44).The polysaccharide capsule is a key virulence factor and is both cell associated and released during infection (4). The two species of polysaccharide in the capsule, an abundant glucuronoxylomannan (GXM) and a minor galactoxylomannan (GalXM), cause a number of deleterious effects in mammalian hosts (4, 44). Extracellular vesicles (exosomes) containing capsule polysaccharide are present in culture supernatants, in lysates of macrophages containing C. neoformans, and in association with fungal cells during murine infection (41, 49, 50, 54). These so-called “virulence factor delivery bags” are thought to pass through the cell wall to deliver material outside the cell (50). Proteomic analysis of the vesicles identified 76 proteins, and many of these are associated with virulence, including urease, laccase, heat shock proteins, superoxide dismutase, thiol-specific antioxidants, and catalases (49).The mechanisms of trafficking of capsule polysaccharide and laccase are being actively pursued. For example, analysis of a mutant with a defect in the exocyst GTPase Sec4/Rab8 (designated Sav1) revealed the accumulation of intracellular vesicles containing capsule polysaccharide, thus providing support for intracellular synthesis and secretion via exocytosis (60). In addition, reduced expression of the exocyst protein Sec6 due to RNA interference (RNAi) resulted in partial attenuation of virulence as well as defects in melanin production and the export of urease and soluble capsule polysaccharide (42). The RNAi strains were also completely defective in the production of extracellular exosomes but retained wild-type (WT) levels of cell-associated capsule. Trafficking of the laccase required for melanin production and virulence has also been examined. Hu et al. (25) showed that C. neoformans lacking Vps34 (vacuolar protein sorting 34) had a marked reduction in melanin formation, suggesting that laccase-containing vesicles are derived from the endocytic pathway. Overall, the current evidence suggests that exocytic, endocytic, and specialized extracellular vesicles mediate the export of capsule and other virulence factors in C. neoformans (42, 49, 60).We demonstrated previously that vesicle trafficking functions in C. neoformans are regulated by the cAMP signal transduction pathway, which also controls the elaboration of both the capsule and melanin (28). We found that treatment of C. neoformans with inhibitors of Golgi apparatus-mediated transport (e.g., brefeldin A or monensin) or with lithium chloride results in inhibition of capsule expression (28). In addition, we found that cAMP-dependent protein kinase regulated the expression of a predicted phospatidylethanolamine binding protein, Ova1, which negatively influences capsule and melanin formation. These findings focused our attention on the roles of intracellular trafficking functions and phospholipids in virulence factor expression.In the context of phospholipid trafficking, some aminophospholipid translocases within the P-type ATPases are known to play roles in fungal virulence. For example, the aminophospholipid translocase MgApt2 is required for exocytosis during plant infection by the rice blast pathogen Magnaporthe grisea (18). P-type ATPases are a large family of multitransmembrane domain, ATP-dependent transporters, and three subfamilies are found in eukaryotes (29): (i) heavy metal ion ATPases (e.g., copper transporters), (ii) non-heavy-metal ion ATPases (e.g., Ca²⁺, H⁺, Na⁺, and K⁺ ATPases), and (iii) aminophospholipid translocases (APTs/flippases of the type IV or Drs2 family). APTs maintain the asymmetrical distribution of aminophospholipids in membranes by translocating phosphatidylserine (PS) and/or phosphatidylethanolamine (PE) from one leaflet of the bilayer to the other. Phospholipid asymmetry is important in membrane fusion events (vesicle budding and docking) at the plasma membrane and in the trans-Golgi network (3). Thus, APTs are required for efficient Golgi function and play roles in both endocytosis and exocytosis. Some disorders in humans have been linked or attributed to genes from the APT subfamily, including familial intrahepatic cholestasis and Angelman syndrome (32, 55).Previously, we constructed a deletion of the APT1 gene, encoding a putative aminophospholipid translocase, as part of a study to examine disomy at chromosome 13 in C. neoformans (27). Our preliminary phenotypic analysis suggested a connection to nitrosative stress and prompted further investigation of virulence-related functions. In the present study, we show that Apt1 is functionally related to Drs2 in Saccharomyces cerevisiae and has roles in membrane trafficking and sensitivity to stress (oxidative and nitrosative) and drugs targeting ergosterol biosynthesis and secretion. Importantly, loss of Apt1 does not influence capsule and melanin formation, but the protein is required for intracellular growth in macrophages and for full virulence in mice.