Towards non‐invasive monitoring of pathogen–host interactions during Candida albicans biofilm formation using in vivo bioluminescence

Candida albicans is a major human fungal pathogen causing mucosal and deep tissue infections of which the majority is associated with biofilm formation on medical implants. Biofilms have a huge impact on public health, as fungal biofilms are highly resistant against most antimycotics. Animal models of biofilm formation are indispensable for improving our understanding of biofilm development inside the host, their antifungal resistance and their interaction with the host immune defence system. In currently used models, evaluation of biofilm development or the efficacy of antifungal treatment is limited to ex vivo analyses, requiring host sacrifice, which excludes longitudinal monitoring of dynamic processes during biofilm formation in the live host. In this study, we have demonstrated for the first time that non‐invasive, dynamic imaging and quantification of in vitro and in vivo C. albicans biofilm formation including morphogenesis from the yeast to hyphae state is feasible by using growth‐phase dependent bioluminescent C. albicans strains in a subcutaneous catheter model in rodents. We have shown the defect in biofilm formation of a bioluminescent bcr1 mutant strain. This approach has immediate applications for the screening and validation ofantimycotics under in vivo conditions, for studying host–biofilm interactions in different transgenic mouse models and for testing the virulence of luminescent C. albicans mutants, hereby contributing to a better understanding of the pathogenesis of biofilm‐associated yeast infections.     

Streptococcus pyogenes pili promote pharyngeal cell adhesion and biofilm formation

Group A Streptococcus (GAS, Streptococcus pyogenes) is a Gram-positive human pathogen responsible for several acute diseases and autoimmune sequelae that account for half a million deaths worldwide every year. GAS infections require the capacity of the pathogen to adhere to host tissues and assemble in cell aggregates. Furthermore, a role for biofilms in GAS pathogenesis has recently been proposed. Here we investigated the role of GAS pili in biofilm formation. We demonstrated that GAS pilus-negative mutants, in which the genes encoding either the pilus backbone structural protein or the sortase C1 have been deleted, showed an impaired capacity to attach to a pharyngeal cell line. The same mutants were much less efficient in forming cellular aggregates in liquid culture and microcolonies on human cells. Furthermore, mutant strains were incapable of producing the typical three-dimensional layer with bacterial microcolonies embedded in a carbohydrate polymeric matrix. Complemented mutants had an adhesion and aggregation phenotype similar to the wild-type strain. Finally, in vivo expression of pili was indirectly confirmed by demonstrating that most of the sera from human patients affected by GAS-mediated pharyngitis recognized recombinant pili proteins. These data support the role of pili in GAS adherence and colonization and suggest a general role of pili in all pathogenic streptococci.     

The group A streptococcal collagen‐like protein‐1, Scl1, mediates biofilm formation by targeting the extra domain A‐containing variant of cellular fibronectin expressed in wounded tissue

Wounds are known to serve as portals of entry for group A Streptococcus (GAS). Subsequent tissue colonization is mediated by interactions between GAS surface proteins and host extracellular matrix components. We recently reported that the streptococcal collagen‐like protein‐1, Scl1, selectively binds the cellular form of fibronectin (cFn) and also contributes to GAS biofilm formation on abiotic surfaces. One structural feature of cFn, which is predominantly expressed in response to tissue injury, is the presence of a spliced variant containing extra domain A (EDA/EIIIA). We now report that GAS biofilm formation is mediated by the Scl1 interaction with EDA‐containing cFn. Recombinant Scl1 proteins that bound cFn also bound recombinant EDA within the C‐C’ loop region recognized by the α₉β₁ integrin. The extracellular 2‐D matrix derived from human dermal fibroblasts supports GAS adherence and biofilm formation. Altogether, this work identifies and characterizes a novel molecular mechanism by which GAS utilizes Scl1 to specifically target an extracellular matrix component that is predominantly expressed at the site of injury in order to secure host tissue colonization.     

Dispersal of Group A streptococcal biofilms by the cysteine protease SpeB leads to increased disease severity in a murine model

Group A Streptococcus (GAS) is a Gram-positive human pathogen best known for causing pharyngeal and mild skin infections. However, in the 1980's there was an increase in severe GAS infections including cellulitis and deeper tissue infections like necrotizing fasciitis. Particularly striking about this elevation in the incidence of severe disease was that those most often affected were previously healthy individuals. Several groups have shown that changes in gene content or regulation, as with proteases, may contribute to severe disease; yet strains harboring these proteases continue to cause mild disease as well. We and others have shown that group A streptococci (MGAS5005) reside within biofilms both in vitro and in vivo. That is to say that the organism colonizes a host surface and forms a 3-dimensional community encased in a protective matrix of extracellular protein, DNA and polysaccharide(s). However, the mechanism of assembly or dispersal of these structures is unclear, as is the relationship of these structures to disease outcome. Recently we reported that allelic replacement of the streptococcal regulator srv resulted in constitutive production of the streptococcal cysteine protease SpeB. We further showed that the constitutive production of SpeB significantly decreased MGAS5005Δsrv biofilm formation in vitro. Here we show that mice infected with MGAS5005Δsrv had significantly larger lesion development than wild-type infected animals. Histopathology, Gram-staining and immunofluorescence link the increased lesion development with lack of disease containment, lack of biofilm formation, and readily detectable levels of SpeB in the tissue. Treatment of MGAS5005Δsrv infected lesions with a chemical inhibitor of SpeB significantly reduced lesion formation and disease spread to wild-type levels. Furthermore, inactivation of speB in the MGAS5005Δsrv background reduced lesion formation to wild-type levels. Taken together, these data suggest a mechanism by which GAS disease may transition from mild to severe through the Srv mediated dispersal of GAS biofilms.     

Recruited macrophages control dissemination of group A Streptococcus from infected soft tissues

Group A Streptococcus (GAS) causes diverse infections in humans, ranging from mild to life-threatening invasive diseases, such as necrotizing fasciitis (NF), a rapidly progressing deep tissue infection. Despite prompt treatments, NF remains a significant cause of morbidity and mortality, even in previously healthy individuals. The early recruitment of leukocytes is crucial to the outcome of NF; however, although the role of polymorphonuclear neutrophils (PMNs) in host defense against NF is well established, the role of recruited macrophages remains poorly defined. Using a cutaneous murine model mimicking human NF, we found that mice deficient in TNF-α were highly susceptible to s.c. infections with GAS, and a paucity of macrophages, but not PMNs, was demonstrated. To test whether the effects of TNF-α on the outcome of infection are mediated by macrophages/monocytes, we systemically depleted C57BL/6 mice of monocytes by pharmacological and genetic approaches. Systemic monocyte depletion substantially increased bacterial dissemination from soft tissues without affecting the number of recruited PMNs or altering the bacterial loads in soft tissues. Enhanced GAS dissemination could be reverted by either i.v. injection of monocytes or s.c. administration of peritoneal macrophages. These experiments demonstrated that recruited macrophages play a key role in defense against the extracellular pathogen GAS by limiting its spread from soft tissues.     

Adaptation of the group A Streptococcus adhesin Scl1 to bind fibronectin type III repeats within wound‐associated extracellular matrix: implications for cancer therapy

The human‐adapted pathogen group A Streptococcus (GAS) utilizes wounds as portals of entry into host tissue, wherein surface adhesins interact with the extracellular matrix, enabling bacterial colonization. The streptococcal collagen‐like protein 1 (Scl1) is a major adhesin of GAS that selectively binds to two fibronectin type III (FnIII) repeats within cellular fibronectin, specifically the alternatively spliced extra domains A and B, and the FnIII repeats within tenascin‐C. Binding to FnIII repeats was mediated through conserved structural determinants present within the Scl1 globular domain and facilitated GAS adherence and biofilm formation. Isoforms of cellular fibronectin that contain extra domains A and B, as well as tenascin‐C, are present for several days in the wound extracellular matrix. Scl1‐FnIII binding is therefore an example of GAS adaptation to the host's wound environment. Similarly, cellular fibronectin isoforms and tenascin‐C are present in the tumor microenvironment. Consistent with this, FnIII repeats mediate GAS attachment to and enhancement of biofilm formation on matrices deposited by cancer‐associated fibroblasts and osteosarcoma cells. These data collectively support the premise for utilization of the Scl1‐FnIII interaction as a novel method of anti‐neoplastic targeting in the tumor microenvironment.     

Nitric oxide‐mediated dispersal in single‐ and multi‐species biofilms of clinically and industrially relevant microorganisms

Strategies to induce biofilm dispersal are of interest due to their potential to prevent biofilm formation and biofilm‐related infections. Nitric oxide (NO), an important messenger molecule in biological systems, was previously identified as a signal for dispersal in biofilms of the model organism Pseudomonas aeruginosa. In the present study, the use of NO as an anti‐biofilm agent more broadly was assessed. Various NO donors, at concentrations estimated to generate NO levels in the picomolar and low nanomolar range, were tested on single‐species biofilms of relevant microorganisms and on multi‐species biofilms from water distribution and treatment systems. Nitric oxide‐induced dispersal was observed in all biofilms assessed, and the average reduction of total biofilm surface was 63%. Moreover, biofilms exposed to low doses of NO were more susceptible to antimicrobial treatments than untreated biofilms. For example, the efficacy of conventional chlorine treatments at removing multi‐species biofilms from water systems was increased by 20‐fold in biofilms treated with NO compared with untreated biofilms. These data suggest that combined treatments with NO may allow for novel and improved strategies to control biofilms and have widespread applications in many environmental, industrial and clinical settings.     

Biofilm formation by Pseudomonas aeruginosa in solid murine tumors - a novel model system

The ability of opportunistic bacterial pathogens to grow in biofilms is decisive in the pathogenesis of chronic infectious diseases. Growth within biofilms does not only protect the bacteria against the host immune system but also from the killing by antimicrobial agents. Here, we introduce a mouse model in which intravenously administered planktonic Pseudomonas aeruginosa bacteria are enriched in transplantable subcutaneous mouse tumors. Electron microscopy images provide evidence that such bacteria reside in the tumor tissue within biofilm structures. Immunohistology furthermore demonstrated that infection of the tumor tissue elicits a host response characterized by strong neutrophilic influx. Interestingly, the biofilm defective PA14 pqsA transposon mutant formed less biofilm in vivo and was more susceptible to clearance by intravenous ciprofloxacin treatment as compared to the wild-type control. In conclusion, we have established an experimentally tractable model that may serve to identify novel bacterial and host factors important for in vivo biofilm formation and to re-evaluate bactericidal and anti-biofilm effects of currently used and novel antibacterial compounds.     

Staphylococcus aureus biofilms prevent macrophage phagocytosis and attenuate inflammation in vivo

Biofilms are complex communities of bacteria encased in a matrix composed primarily of polysaccharides, extracellular DNA, and protein. Staphylococcus aureus can form biofilm infections, which are often debilitating due to their chronicity and recalcitrance to antibiotic therapy. Currently, the immune mechanisms elicited during biofilm growth and their impact on bacterial clearance remain to be defined. We used a mouse model of catheter-associated biofilm infection to assess the functional importance of TLR2 and TLR9 in the host immune response during biofilm formation, because ligands for both receptors are present within the biofilm. Interestingly, neither TLR2 nor TLR9 impacted bacterial density or inflammatory mediator secretion during biofilm growth in vivo, suggesting that S. aureus biofilms circumvent these traditional bacterial recognition pathways. Several potential mechanisms were identified to account for biofilm evasion of innate immunity, including significant reductions in IL-1β, TNF-α, CXCL2, and CCL2 expression during biofilm infection compared with the wound healing response elicited by sterile catheters, limited macrophage invasion into biofilms in vivo, and a skewing of the immune response away from a microbicidal phenotype as evidenced by decreases in inducible NO synthase expression concomitant with robust arginase-1 induction. Coculture studies of macrophages with S. aureus biofilms in vitro revealed that macrophages successful at biofilm invasion displayed limited phagocytosis and gene expression patterns reminiscent of alternatively activated M2 macrophages. Collectively, these findings demonstrate that S. aureus biofilms are capable of attenuating traditional host proinflammatory responses, which may explain why biofilm infections persist in an immunocompetent host.     

The vascular plant‐pathogenic bacterium Ralstonia solanacearum produces biofilms required for its virulence on the surfaces of tomato cells adjacent to intercellular spaces

The mechanism of colonization of intercellular spaces by the soil‐borne and vascular plant‐pathogenic bacterium Ralstonia solanacearum strain OE1‐1 after invasion into host plants remains unclear. To analyse the behaviour of OE1‐1 cells in intercellular spaces, tomato leaves with the lower epidermis layers excised after infiltration with OE1‐1 were observed under a scanning electron microscope. OE1‐1 cells formed microcolonies on the surfaces of tomato cells adjacent to intercellular spaces, and then aggregated surrounded by an extracellular matrix, forming mature biofilm structures. Furthermore, OE1‐1 cells produced mushroom‐type biofilms when incubated in fluids of apoplasts including intercellular spaces, but not xylem fluids from tomato plants. This is the first report of biofilm formation by R. solanacearum on host plant cells after invasion into intercellular spaces and mushroom‐type biofilms produced by R. solanacearum in vitro. Sugar application led to enhanced biofilm formation by OE1‐1. Mutation of lecM encoding a lectin, RS‐IIL, which reportedly exhibits affinity for these sugars, led to a significant decrease in biofilm formation. Colonization in intercellular spaces was significantly decreased in the lecM mutant, leading to a loss of virulence on tomato plants. Complementation of the lecM mutant with native lecM resulted in the recovery of mushroom‐type biofilms and virulence on tomato plants. Together, our findings indicate that OE1‐1 produces mature biofilms on the surfaces of tomato cells after invasion into intercellular spaces. RS‐IIL may contribute to biofilm formation by OE1‐1, which is required for OE1‐1 virulence.     

Microcolony and biofilm formation as a survival strategy for bacteria

Bacterial communities such as biofilms are widely recognized as being important for survival and persistence of bacteria in harsh environments. Mechanistic models of biofilm growth indicate that the way in which the surface is seeded can effect the morphology of simulated biofilms. Experimental studies indicate that genes which are important for chemotaxis also influence biofilm formation, perhaps by influencing aggregation on a surface. Understanding aggregation and microcolony formation could therefore help clarify factors influencing biofilm formation and illuminate how groups influence the fitness of bacteria. In this paper I develop an individual based model to examine how different behaviors involved in microcolony formation on a surface determine patterns of group sizes and link patterns to bacterial fitness.     

Cellulose production,activated by cyclic di‐GMP through BcsA and BcsZ,is a virulence factor and an essential determinant of the three‐dimensional architectures of biofilms formed by Erwinia amylovora Ea1189

Bacterial biofilms are multicellular aggregates encased in an extracellular matrix mainly composed of exopolysaccharides (EPSs), protein and nucleic acids, which determines the architecture of the biofilm. Erwinia amylovora Ea1189 forms a biofilm inside the xylem of its host, which results in vessel plugging and water transport impairment. The production of the EPSs amylovoran and levan is critical for the formation of a mature biofilm. In addition, cyclic dimeric GMP (c‐di‐GMP) has been reported to positively regulate amylovoran biosynthesis and biofilm formation in E. amylovora Ea1189. In this study, we demonstrate that cellulose is synthesized by E. amylovora Ea1189 and is a major modulator of the three‐dimensional characteristics of biofilms formed by this bacterium, and also contributes to virulence during systemic host invasion. In addition, we demonstrate that the activation of cellulose biosynthesis in E. amylovora is a c‐di‐GMP‐dependent process, through allosteric binding to the cellulose catalytic subunit BcsA. We also report that the endoglucanase BcsZ is a key player in c‐di‐GMP activation of cellulose biosynthesis. Our results provide evidence of the complex composition of the extracellular matrix produced by E. amylovora and the implications of cellulose biosynthesis in shaping the architecture of the biofilm and in the expression of one of the main virulence phenotypes of this pathogen.     

Drosophila melanogaster as an animal model for the study of Pseudomonas aeruginosa biofilm infections in vivo

Pseudomonas aeruginosa is an opportunistic pathogen capable of causing both acute and chronic infections in susceptible hosts. Chronic P. aeruginosa infections are thought to be caused by bacterial biofilms. Biofilms are highly structured, multicellular, microbial communities encased in an extracellular matrix that enable long-term survival in the host. The aim of this research was to develop an animal model that would allow an in vivo study of P. aeruginosa biofilm infections in a Drosophila melanogaster host. At 24 h post oral infection of Drosophila, P. aeruginosa biofilms localized to and were visualized in dissected Drosophila crops. These biofilms had a characteristic aggregate structure and an extracellular matrix composed of DNA and exopolysaccharide. P. aeruginosa cells recovered from in vivo grown biofilms had increased antibiotic resistance relative to planktonically grown cells. In vivo, biofilm formation was dependent on expression of the pel exopolysaccharide genes, as a pelB::lux mutant failed to form biofilms. The pelB::lux mutant was significantly more virulent than PAO1, while a hyperbiofilm strain (PAZHI3) demonstrated significantly less virulence than PAO1, as indicated by survival of infected flies at day 14 postinfection. Biofilm formation, by strains PAO1 and PAZHI3, in the crop was associated with induction of diptericin, cecropin A1 and drosomycin antimicrobial peptide gene expression 24 h postinfection. In contrast, infection with the non-biofilm forming strain pelB::lux resulted in decreased AMP gene expression in the fly. In summary, these results provide novel insights into host-pathogen interactions during P. aeruginosa oral infection of Drosophila and highlight the use of Drosophila as an infection model that permits the study of P. aeruginosa biofilms in vivo.