Most studies on fungal biofilms have focused on Candida in yeasts and Aspergillus in mycelial fungi. To the authors’ knowledge, biofilm formation by zygomycetes has not been reported previously. In this study, the biofilm-forming capacity of Rhizopus oryzae, Lichtheimia corymbifera, Rhizomucor pusillus and Apophysomyces elegans was evaluated. At appropriate seeding spore densities, Rhp . oryzae (105 c.f.u. ml−1), L. corymbifera (104 c.f.u. ml−1) and Rhm. pusillus (104 c.f.u. ml−1) produced highly intertwined, adherent structures on flat-bottomed polystyrene microtitre plates after 24 h at 37 °C. The adhered fungal hyphae were encased in an extracellular matrix, as confirmed by phase-contrast and confocal microscopy. The thickness of Rhp. oryzae, L. corymbifera and Rhm. pusillus biofilms was 109.67±10.02, 242±23.07 and 197±9.0 µm (mean±sd), respectively. Biochemical characterization of the biofilm matrix indicated the presence of glucosamine, constituting 74.54–82.22 % of its dry weight, N-acetylglucosamine, glucose and proteins. Adherence and biofilm formation were not observed in A. elegans. Although A. elegans spores germinated at all three seeding densities tested (1×107, 1×106 and 1×105 c.f.u. ml−1), no significant difference was observed (P>0.05) between the A 490 of wells inoculated with A. elegans and the cut-off A 490 for biofilm detection. This study highlights the potential for biofilm formation by at least three medically important species of zygomycetes.
Regulators of membrane fusion play an important role in phagocytosis, as they regulate the focal delivery of endomembrane that is required for optimal internalization of large particles. During internalization of Leishmania promastigotes, the surface glycolipid lipophosphoglycan (LPG) is transferred to the macrophage membrane and modifies its fusogenic properties. In this study, we investigated the impact of LPG on the recruitment of the exocytosis regulator synaptotagmin V (Syt V) at the area of internalization and on the early steps of phagocytosis. Using Leishmania donovani LPG-defective mutants and LPG-coated particles, we established that LPG reduces the phagocytic capacity of macrophages and showed that it causes exclusion of Syt V from the nascent phagosome. Silencing of Syt V inhibited phagocytosis to the same extent as LPG, and these effects were not cumulative, consistent with a Syt V-dependent mechanism for the inhibition of phagocytosis by LPG. Previous work has revealed that LPG-mediated exclusion of Syt V from phagosomes prevents the recruitment of the vacuolar ATPase and acidification. Thus, whereas exclusion of Syt V from phagosomes in the process of formation may be beneficial for the creation of a hospitable intracellular niche, it reduces the phagocytic capacity of macrophages. We propose that the cost associated with a reduced internalization rate may be compensated by increased survival, and could lead to a greater overall parasite fitness.
Burkholderia pseudomallei is a facultative intracellular bacterial pathogen causing melioidosis, an often fatal infectious disease that is endemic in several tropical and subtropical areas around the world. We previously described a Ptk2 cell-based plaque assay screening system of B. pseudomallei transposon mutants that led to the identification of several novel virulence determinants. Using this approach we identified a mutant with reduced plaque formation in which the BPSL0918 gene was disrupted. BPSL0918 encodes a putative FK-506-binding protein (FKBP) representing a family of proteins that typically possess peptidyl-prolyl isomerase (PPIase) activity. A B. pseudomallei ΔBPSL0918 mutant showed a severely impaired ability to resist intracellular killing and to replicate within primary macrophages. Complementation of the mutant fully restored its ability to grow intracellularly. Moreover, B. pseudomallei ΔBPSL0918 was significantly attenuated in a murine model of infection. Structural modelling confirmed a modified FKBP fold of the BPSL0918-encoded protein but unlike virulence-associated FKBPs from other pathogenic bacteria, recombinant BPSL0918 protein did not possess PPIase activity in vitro. In accordance with this observation BPSL0918 exhibits several mutations in residues that have been proposed to mediate PPIase activity in other FKBPs. To our knowledge this B. pseudomallei FKBP represents the first example of this protein family which lacks PPIase activity but is important in intracellular infection of a bacterial pathogen.
Surface layer (S-layer) proteins are crystalline arrays of proteinaceous subunits that are present as the outermost component of the cell wall in several Lactobacillus species. The S-layer proteins have been shown to play a role in the antimicrobial activity of certain lactobacilli. However, it is not fully understood how the S-layer proteins exert this biological function. The aim of this study was to test the hypothesis that Lactobacillus acidophilus S-layer proteins antagonize Salmonella Typhimurium (S. Typhimurium) infection by protecting against F-actin cytoskeleton rearrangements and the activation of mitogen-activated protein kinase (MAPK) signalling pathways. Monolayer transepithelial electrical resistance (TER) was measured after S. Typhimurium infection in Caco-2 cultured human intestinal cells with L. acidophilus S-layer proteins. F-actin rearrangement and MAPK activation were also assessed by immunofluorescence staining or Western blotting. The results showed that when S. Typhimurium was co-incubated with S-layer proteins, the S. Typhimurium-induced Caco-2 cell F-actin rearrangement was reduced, and the S. Typhimurium-induced TER decrease and interleukin 8 (IL-8) secretion were attenuated. Additionally, L. acidophilus S-layer proteins could inhibit S. Typhimurium-induced phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun amino-terminal kinase (JNK) and p38. This study indicates that L. acidophilus S-layer proteins are able to inhibit S. Typhimurium infection through blocking S. Typhimurium-induced F-actin rearrangements and S. Typhimurium-induced ERK1/2, JNK and p38 activation in Caco-2 cells. These data provide a rationale for the use of lactobacillus S-layer proteins as therapeutic and preventative agents, at least in infectious diarrhoea.
The evolution of antibiotic resistance in pathogenic bacteria is a growing global health problem which is gradually making the treatment of infectious diseases less efficient. Antimicrobial peptides are small charged molecules found in organisms from the complete phylogenetic spectrum. The peptides are attractive candidates for novel drug development due to their activity against bacteria that are resistant to conventional antibiotics, and reports of peptide resistance are rare in the clinical setting. Paradoxically, many clinically relevant bacteria have mechanisms that can recognize and respond to the presence of cationic antimicrobial peptides (CAMPs) in the environment by changing the properties of the microbial surface thereby increasing the tolerance of the microbes towards the peptides. In Pseudomonas aeruginosa an essential component of this inducible tolerance mechanism is the lipopolysaccharide modification operon arnBCADTEF–PA3559 which encodes enzymes required for LPS alterations leading to increased antimicrobial peptide tolerance. The expression of the operon is induced by the presence of CAMPs in the environment but the molecular mechanisms underlying the cellular recognition of the peptides are poorly elucidated. In this work, we investigate the factors influencing arnB expression by transposon mutagenesis and arnB promoter green fluorescent protein reporters. We have identified a novel gene encoding a Mig-14-like protein that is required for recognition of the CAMPs colistin and Novispirin G10 by P. aeruginosa. Moreover, we show that this gene is also required for the formation of CAMP-tolerant subpopulations in P. aeruginosa hydrodynamic flow chamber biofilms.
In Fusarium verticillioides, a ubiquitous pathogen of maize, virulence and mycotoxigenesis are regulated in response to the types and amounts of carbohydrates present in maize kernels. In this study, we investigated the role of a putative hexokinase-encoding gene (HXK1) in growth, development and pathogenesis. A deletion mutant (Δhxk1) of HXK1 was not able to grow when supplied with fructose as the sole carbon source, and growth was impaired when glucose, sucrose or maltotriose was provided. Additionally, the Δhxk1 mutant produced unusual swollen hyphae when provided with fructose, but not glucose, as the sole carbon source. Moreover, the Δhxk1 mutant was impaired in fructose uptake, although glucose uptake was unaffected. On maize kernels, the Δhxk1 mutant was substantially less virulent than the wild-type, but virulence on maize stalks was not impaired, possibly indicating a metabolic response to tissue-specific differences in plant carbohydrate content. Finally, disruption of HXK1 had a pronounced effect on fungal metabolites produced during colonization of maize kernels; the Δhxk1 mutant produced approximately 50 % less trehalose and 80 % less fumonisin B1 (FB1) than the wild-type. The reduction in trehalose biosynthesis likely explains observations of increased sensitivity to osmotic stress in the Δhxk1 mutant. In summary, this study links early events in carbohydrate sensing and glycolysis to virulence and secondary metabolism in F. verticillioides, and thus provides a new foothold from which the genetic regulatory networks that underlie pathogenesis and mycotoxigenesis can be unravelled and defined.