Cross-kingdom assemblies in human saliva show group-level surface mobility and emerging disease-promoting functions. These cross-kingdom multicellular assemblages were more resistant to antimicrobials and removal, and caused more extensive dental cavities than their single-species counterparts, according to research led by scientists at the University of Pennsylvania.
Meaning Fungi and bacteria form multicellular biofilms that cause many human infections. How these distinctive microbes act together spatiotemporally to coordinate disease-promoting functionality remains unstudied. Using real-time multiscale microscopy and computational analysis, we investigated the dynamics of fungal and bacterial interactions in human saliva and their biofilm development on tooth surfaces. We discover structured assemblages across realms that exhibit emerging functionalities to enhance colonization, survival, and collective surface growth. Further analysis revealed unexpected surface mobility at the group level with coordinated “jumping” and “walking” movements while growing continuously. These motile clusters of growing cells promote rapid spatial spread of both species across surfaces, leading to more extensive dental caries. Our findings show multicellular assemblies between kingdoms that act as supraorganisms with functionalities that cannot be achieved without co-assembly. |
Summary
Fungi and bacteria often participate in complex interactions, such as the formation of multicellular biofilms within the human body. Knowledge about how biofilms initiate and coalesce between kingdoms in higher-level communities and what functions different species carry out during biofilm formation remains limited. We found native state assemblies of Candida albicans (fungi) and Streptococcus mutans (bacteria) with highly structured arrangement in the saliva of patients with childhood caries.
Further analysis revealed that the bacterial groups are linked within a network of fungal yeasts, hyphae and exopolysaccharides, which bind to surfaces as a pre-assembled cell group. Cross-kingdom assemblages exhibit emergent functions, including increased growth rate and surface colonization, increased antimicrobial tolerance, and improved shear resistance, compared to either species alone. In particular, we found that interkingdom assemblages display a unique form of migratory spatial mobility that allows rapid spread of biofilms across surfaces and leads to enhanced and more extensive dental caries.
Using mutants, selective species inactivation, and selective matrix deletion, we demonstrate that enhanced stress resistance and surface mobility arise from the exopolymeric matrix and require the presence of both species in the assembly. Mobility is directed by fungal filamentation as the hyphae extend and contact the surface, lifting the assembly with a "forward hopping motion" .
Clusters of bacterial cells can "hitchhike" on this mobile unit while continuously growing, to spread three-dimensionally across the surface and fuse with other assemblies, promoting community expansion. Together, our results reveal a cross-kingdom assembly in human saliva that behaves like a supraorganism, with emerging disease-causing functionalities that cannot be achieved without co-assembly.
Comments
A cross-kingdom partnership between bacteria and fungi may result in the two coming together to form a "superorganism" with unusual strength and endurance. It may sound like the stuff of science fiction, but these microbial clusters are very much part of the here and now.
These assemblages, found in the saliva of young children with severe childhood caries, can effectively colonize the teeth. They were stickier, more resistant to antimicrobials, and harder to remove from teeth than bacteria or fungi alone, according to the research team, led by scientists at the University of Pennsylvania School of Dental Medicine.
What’s more, the assemblies unexpectedly sprout "limbs" that prompt them to "walk" and "jump" to spread rapidly on the tooth surface, even though each microbe alone is not mobile, the team reported in the journal Proceedings of the National Academy of Sciences .
“This started with a very simple, almost accidental discovery while looking at saliva samples from young children who developed aggressive tooth decay,” says Hyun (Michel) Koo, professor at Penn Dental Medicine and co-author of the paper. “Looking under the microscope, we noticed that bacteria and fungi formed these assemblies and developed movements that we never thought they would have: a mobility similar to walking and jumping. They have many of what we call "emerging features" that bring new benefits to this stack that they couldn’t achieve on their own. “It is almost like a new organism, a superorganism, with new functions.”
Cross-kingdom assemblies in human saliva behave as supraorganisms with new functionalities and disease-promoting activity. 1) C. albicans and S. mutans assemble into structured cell clusters in human saliva, which are strikingly similar to the native cross-kingdom aggregates found in the intact saliva of diseased patients. 2) Bacteria and fungi collectively colonize the surface as a structured cell group with higher binding affinity. 3) The assembly shows greater tolerance to shear stress and antimicrobials. 4) Assemblages behave as single units that grow faster than aggregates of a single species, spreading three-dimensionally and fusing with each other, resulting in high surface coverage. 5) Interkingdom assemblages display a novel mode of mobility at the migratory group level with forward movements and a hitchhiking growth mechanism during biofilm initiation that allows immobile bacteria to relocate after surface colonization, thereby promotes the spatial expansion of the biofilm on the surface. 6) Interkingdom assemblages cause extensive and severe damage to the tooth enamel surface.
Better (or worse) together
In the past, Koo’s lab focused on dental biofilm, or plaque, present in children with severe cavities, and found that both bacteria (Streptococcus mutans) and fungus (Candida albicans) contribute to the disease. Cavities, commonly known as cavities, arise when sugars in the diet are left to feed bacteria and fungi in the mouth, leading to acid-producing dental plaque that destroys enamel.
The new set of discoveries came when Zhi Ren, a postdoctoral fellow in Koo’s group, was using microscopy that allows scientists to visualize the behavior of living microbes in real time. The technique "opens up new possibilities for investigating the dynamics of complex biological processes," says Ren, first author of the paper and part of the first cohort of the NIDCR T90R90 postdoctoral training program within Penn’s Center for Innovation and Precision Dentistry.
After seeing the clumps of bacteria and fungi present in saliva samples, Ren, Koo and their colleagues were curious about how the clumps would behave once attached to the surface of a tooth. Thus began a series of experiments using live microscopy in real time to observe the process of attachment and eventual growth.
They created a laboratory system to recreate the formation of these assemblies, using bacteria, fungi and a tooth-like material, all incubated in human saliva. The platform allowed researchers to observe how groups came together and analyze the structure of the resulting assemblages. They found a highly organized structure with bacterial groups linked together in a complex network of fungal yeast and filament-like projections called hyphae, all entangled in an extracellular polymer, a glue-like material.
The team then tested the properties of these cross-kingdom assemblies once they colonized the tooth surface and found "surprising behaviors and emergent properties," Ren says, "including better adhesion to the surface, making them very sticky, and greater mechanical tolerance." and antimicrobial. making them difficult to eliminate or kill.”
Perhaps the most intriguing feature of the assemblages, the researchers say, was their mobility. "They showed ’jumping’ and ’walking’ movements while growing continuously," Ren says.
While some bacteria can propel themselves using appendages such as flagella, the microbial species in the current study are not motile. And unlike any known microbial motility, the assemblages used fungal hyphae to anchor themselves to the surface and then propel the entire superorganism forward, transporting the attached bacteria across the surface, Koo says, "like bacteria hitchhiking on fungi." ".
The microbial clusters moved fast and far, the researchers found. On the tooth-like surface, the team measured speeds of more than 40 microns per hour, similar to the speed of fibroblasts, a type of cell in the human body involved in wound healing. In the first hours of growth, the scientists observed the assemblies "jumping" more than 100 microns on the surface. "That’s more than 200 times their own body length," Ren says, "making them even better than most vertebrates, relative to body size. For example, tree frogs and grasshoppers can jump forward. about 50 and 20 times the length of their own body, respectively.”
Although the exact mechanisms are unknown, the ability of assemblages to "move as they grow," the researchers say, has one clear consequence: it allows them to rapidly colonize and spread to new surfaces. When the research team allowed the assemblies to adhere and grow on real human teeth in a laboratory model, they found more extensive tooth decay as a result of a rapidly spreading biofilm.
Treatment of diseases and biology in general.
Because these assemblies are found in saliva, targeting them early could be a therapeutic strategy to prevent childhood tooth decay, Koo says. “If you block this bond or disrupt the assembly before it reaches the tooth and causes damage, that could be a preventative strategy.”
And beyond applications for treating this specific disease, the researchers say, the new findings could be applicable to microbial biology in general. For example, aggregated organisms found in other biological fluids or aquatic ecosystems can similarly enhance surface colonization and growth to cause infectious diseases or environmental pollution.
"We saw these two distinct organisms come together as a new organic entity that gave each additional benefits and functions that the individual cells did not have on their own," Koo says. The findings could even shed light on the evolution of mutualism and multicellularity that enhances the survival and growth of individual organisms when they come together and work together as a unit in a given environment, the team notes.
"This discovery of a superorganism is really innovative and unexpected," says Knut Drescher of the University of Basel, co-author of the paper. “No one would have predicted this. Zhi accidentally stumbled upon this while keeping an open mind.”
