Experts comment on EMBO J | Fang Xianyang's group reveals the regulation mechanism that Staphylococcus Bap protein forms functional amyloid fibers through phase change and mediates the formation of biofilms

Comments | Liu Cong (Shanghai Intersection Center for Biochemistry, Chinese Academy of Sciences), Feng Yingang (Qingdao Institute of Bioenergy and Process, Chinese Academy of Sciences) The history of human civilization is a history of fighting against microbes.

Since 1928, the discovery of a series of antibiotics represented by penicillin (penicillin) has enabled many infections caused by pathogenic bacteria to be successfully treated.
People once thought that they saw the dawn of a complete victory over pathogenic bacteria [1].

However, for decades, due to the extensive use or abuse of broad-spectrum antibiotics and antibacterial drugs, a large number of resistant bacteria or fungi have emerged [2], posing a huge challenge to global public health security.

In the face of the threat of microorganisms, mankind has never stopped fighting.

Revealing new drug resistance mechanisms and finding new antibacterial targets has become an urgent need for the development of new anti-infective drugs.

Studies have shown that 80% of bacterial infections are related to the formation of biofilm [3].

Bacterial biofilm is a cell population with specific structure and function formed by the adhesion of extracellular matrix secreted by bacteria itself.

The main components of the extracellular matrix are extracellular polysaccharides, proteins, extracellular DNA (eDNA) and lipids [4].

The biofilm provides a mechanical barrier for bacteria, which can resist the host immune response and reduce the bacteria's sensitivity to antibiotics and disinfectants, thereby enhancing their ability to survive in the host and the environment; in addition, the inside of the biofilm is a kind of low oxygen, The dry environment with low pH, high carbon dioxide and lack of water, lack of nutrients, is not conducive to the growth and metabolism of bacteria.

Traditional antibiotics may have no effect on the bacteria in the mature biofilm due to the barrier function of the biofilm and the changes in the growth and metabolism of the bacteria in the biofilm.
The formation of the biofilm is closely related to the drug resistance of bacteria, and it also causes chronic infection and prolonged disease.
The main reason for non-healing.In recent years, nosocomial infections caused by multi-drug resistant Staphylococcus aureus have posed a great threat to human health.

Every year, nearly 1 million people worldwide die from bacterial infections that cannot be treated with traditional antibiotics.
Among them, the deaths caused by methicillin-resistant Staphylococcus aureus far exceed the total deaths caused by AIDS and tuberculosis [5].

The resistance of Staphylococcus aureus is often closely related to its formation of biofilm.

A cell surface protein called Biofilm associated proteins (Biofilm associated proteins), Bap, has been found in dairy cow-derived Staphylococcus aureus for a long time.
Bap plays an important role in mediating the formation of Staphylococcus biofilm [6] .

Bap is a multi-domain protein, the full-length protein contains 2276 amino acids [6].

Recent studies have shown that the N-terminus of Bap protein tends to aggregate to form functional amyloid fibers at low pH and low calcium, and promotes bacterial aggregation and biofilm formation [7].

However, the mechanism by which Bap protein regulates the formation of amyloid fibers and mediates the formation of biofilms in response to environmental changes is not clear.

On May 28, 2021, Fang Xianyang's group from School of Life Sciences, Tsinghua University published a research paper entitled Structural mechanism for modulation of functional amyloid and biofilm formation by Staphylococcal Bap protein switch in EMBO J.

This work resolved the 1.
9 Å high-resolution crystal structure of the N-terminal BSP domain of the Bap protein.
This structure presents a dumbbell-shaped fold.
The middle module (MM) connecting the N-terminal and C-terminal is composed of two new series connection The calcium ion binding motif composition. Although this motif was predicted to be EF-hand that can bind calcium ions, the crystal structure shows that, unlike the traditional EF-hand that forms the structure of Helix-Loop-Helix and only binds one calcium ion, this motif forms Loop- The structure of Helix-Loop also binds two calcium ions, so it is a new calcium ion binding motif.

Small-angle X-ray scattering (SAXS), nuclear magnetic resonance (NMR) and molecular dynamics simulation data show that the intermediate module undergoes a conformational transition from order to disorder in response to changes in calcium ion concentration.

Biochemical experiments in vitro and in vivo showed that the N-terminus of BSP undergoes liquid-liquid and liquid-solid phase transformations under acidic conditions (pH <= 4.
5) to form amyloid fibers and mediate the formation of biofilms.

This process is promoted by the intermediate module (MM) in a disordered conformation under the condition of low calcium ions, and the binding of calcium ions can promote the compact folding of the intermediate module and form an interaction network with the surrounding modules, thereby inhibiting the occurrence of phase separation and biofilm Formation.

Since many bacteria form biofilms through Bap, the discovery of the regulatory mechanism of Bap protein-mediated biofilm formation of Staphylococcus aureus will help to carry out related drug design.

Figure 1.
The regulatory mechanism of Bap to form amyloid fibers and mediate the formation of biofilms by Staphylococcus aureus.
The study has the following important findings: 1.
The N-terminus of BSP contains repeating tandem structural modules, which can be used as scaffold proteins to mediate the liquid phase.
Separated and aggregated into amyloid fibers through liquid-solid phase transformation.

This is the first bacterial cell surface protein discovered so far that can form functional amyloid fibers through phase separation, indicating that the phase separation process plays an important role in the formation of bacterial functional amyloid fibers.

2.
As a novel calcium-binding motif, the middle module of BSP has a completely different structure from a variety of calcium-binding motifs found in eukaryotes and prokaryotes in recent years.

The intermediate module undergoes a conformational transition between "disorder and order" by sensing changes in calcium ions and pH, giving bacteria the ability to respond to external environmental signals and survive under special conditions.

3.
Under the conditions of low pH and low calcium ion concentration, the various structural modules of BSP coordinate the formation of amyloid fibers and mediate the formation of Staphylococcus aureus biofilm.

Assistant Professor Fang Xianyang, School of Life Sciences, Tsinghua University, is the corresponding author of the article, 2016 doctoral student Ma Junfeng is the first author of the article, research assistant Cheng Xiang, 2017 doctoral student Xu Zhonghe, and graduated doctoral student Zhang Yikan made this topic Important contribution.

Original link: https:// Expert comment Liu Cong (Shanghai Intersection Center for Biology and Chemistry, Chinese Academy of Sciences) Protein amyloid aggregates were first discovered to be related to a variety of important human nerves The occurrence and development of degenerative diseases (such as Alzheimer's disease, Parkinson's disease, etc.
) are closely related.
Therefore, early studies believe that protein amyloid aggregation is generally pathologically toxic.

In recent years, more and more studies have shown that different proteins from humans, bacteria and other species can perform different normal physiological functions in the form of liquid-solid phase transformation to form amyloid fiber aggregates, such as: yeast cells Sup35 protein, RIPK3 in human cells, etc.

Among different types of bacteria, there is a class of biofilm-related proteins that can form functional amyloid fibers and further aggregate to form biofilms.

Biofilm plays a vital role in the surface adhesion of bacteria and resistance to environmental stress.

Therefore, studying the dynamic assembly and regulation mechanism of functional amyloid fibers has important theoretical and practical significance for understanding the formation of bacterial biofilms and the development of antibacterial drugs.

However, compared with pathological protein amyloid fibers, the research on functional protein amyloid is still relatively lagging.

Recently, Fang Xianyang’s research group from Tsinghua University reported on the molecular mechanism of the dynamic assembly of the biofilm-associated protein Bap derived from cow-derived Staphylococcus aureus to form functional amyloid aggregation.

This research work found that Bap spontaneously forms dynamic protein aggregates through liquid-liquid phase separation under acidic or low calcium ion conditions, and further assembles through liquid-solid phase conversion to form functional amyloid fiber aggregates.

It is suggested that protein liquid-liquid phase separation can be used as an important intermediate step for dynamic regulation of functional protein fibrosis.

The researchers further integrated a variety of different structural biology and computational biology methods to explain the structural basis of Bap's liquid-liquid separation and liquid-solid phase transformation caused by the conformational changes induced by pH and calcium ions.

This work is of great significance for understanding how external environmental factors induce protein phase separation and the formation of amyloid fibers.

In addition, this study provides a new idea for the design and development of small molecule inhibitors that regulate Bap phase separation and phase change.

It is worth pointing out that this study has made important findings in the study of Bap's natural conformation (non-aggregated state), but it also left many interesting questions.

For example: What is the conformation of Bap under liquid-liquid phase separation conditions and further assembly to form functional amyloid fibers? How does it transform from its natural conformation? How does the conformation of Bap in functional amyloid fibers determine its function? These key scientific questions are of great significance for understanding the structural basis of protein phase transition and phase separation and the difference between functional amyloid fibers and pathological amyloid fibers.
We look forward to further answers in the following research.

Expert comments Feng Yingang (Qingdao Institute of Bioenergy and Process, Chinese Academy of Sciences) Bacterial biofilm is a polysaccharide protein complex secreted by bacteria under special conditions, usually wrapped on the surface of bacteria and attached to a certain solid surface.

The bacteria that form the biofilm have a strong ability to resist harsh environments, and it is also one of the reasons why many pathogens are difficult to be eradicated in the body.
Therefore, the research of biofilm is not only important for understanding the interaction between microorganisms and the environment, but also in antibacterial There is also important value in the development of drugs. A research work recently published by Fang Xianyang’s research group at Tsinghua University in EMBO Journal revealed a new type of calcium binding module through the structure and biophysical study of the polymerizable region of the biofilm-associated protein (Bap) of Staphylococcus aureus It plays a key role in amyloid polymerization, liquid-liquid phase separation, and biofilm formation regulated by pH and calcium ions, thus proposing a new structural molecular mechanism for regulation of biofilm formation.

This work of Fang Xianyang's research group is not only of great significance in the study of biofilms, but also very distinctive in research methods and ideas.

This work started from the analysis of the crystal structure of the easily polymerizable region of Bap, and used a series of biophysical methods to gradually clarify the physiological function and molecular mechanism of this region.

In this series of experiments, the crystal structure of Bap easily polymerized region provided a high-resolution structural model, and a new type of double calcium ion binding module was discovered; through small-angle X-ray scattering (SAXS) and nuclear magnetic resonance (NMR) methods, it was revealed Calcium ion-dependent conformational changes in solution; measured by transmission electron microscopy and ThT fluorescence monitoring, measured pH and calcium ion-dependent amyloid fibril formation; fused GFP for fluorescence microscope observation, determined the liquid and liquid that determine pH and calcium ion regulation The key area of ​​the phase separation process; finally, the phenotype of the bacterial biofilm was measured by filling the mutants of the missing part of the Bap knockout bacteria to verify the key role of each area in the formation of the biofilm.

This series of experiments has gradually deepened from structure to function, fully demonstrating the advantages of various technologies, and reading it makes people feel very comfortable.

So far, X-ray crystallography is still the technology of choice for obtaining high-resolution protein structures; SAXS and NMR are the most ideal methods for studying the process of protein conformation changes in solution, and they are complementary: SAXS can better reflect global structural changes NMR can reflect structural changes at the atomic level; transmission electron microscopy is an ideal technology for observing supramolecular morphology such as amyloid fibers; fluorescence technology is very effective in studying protein phase transition processes and compartmentalization positioning.

All these biophysical technologies can reveal the functional mechanism of proteins from the deep to the atomic level, and the functional mechanisms revealed by these in vitro experiments need to be further verified by in vivo experiments, so that we can get an extremely deep understanding of the molecular mechanisms of important physiological processes.
.

The work published by Fang Xianyang's group in EMBO Journal provides an excellent example for the study of protein structure and function.

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