How Protein Moonlighting Impacts the Gut Microbiome

Oct. 2, 2023

Bifidobacterium longum under electron microscope.
Bifidobacterium longum under electron microscope.
Source: wikimedia Commons
In 1999, Constance J. Jeffery, Ph.D., now an associate professor at the University of Illinois, wrote that “the idea of 1 gene—1 protein—1 function has become too simple because increasing numbers of proteins are found to have 2 or more different functions.” Jeffery was referring to moonlighting proteins (MPs), a ubiquitous subset of proteins that made their debut well before the start of this century. As these biomolecules come into focus, one can understand Jeffery's excitement. These proteins are indeed special.

Evolving through gene sharing, MPs are made of single polypeptide chains that play multiple biological roles. Here we digest how such multifunctionality is impactful to both individual microbes, and their surrounding microbial consortia, especially in the highly dynamic environment of the gut.

What Are Moonlighting Proteins?

In the business world, moonlighting refers to someone who works more than 1 job, especially during late evening or at night. In the world of biochemistry, certain proteins are also known to “moonlight”—not necessarily based on circadian rhythms.

MPs are master multi-taskers that can coordinate multiple, independent cellular activities simultaneously, including primary functions (e.g., enzymatic catalysis) and secondary functions (e.g., signal transduction, transcription regulation and adhesion to target cells, amongst others). Many function as chaperones, the major class of MPs, and most are highly conserved and thought to have evolved from ancient enzymes.

Additionally, MPs are ubiquitous in many domains of life—from archaea, protists and plants to animals. This is especially significant for prokaryotes, like bacteria and archaea, as MP multifunctionality leads to less energy spent on metabolism. From DNA replication to various protein syntheses, energy that would have otherwise been required for producing 2 or more distinct proteins is conserved, providing the microbes with a better competitive edge for survival, growth and efficient resource utilization. Altogether, the roles of MPs are so essential that attempting whole gene inactivation will simply kill the organism.

Still, it is important to note that the cellular activities of a given MP are coordinated independently. If 1 function is inactivated, the gene can still enact its second function flawlessly. This puts the gene under 2 or more different selective constraints and means that MP functionality is influenced by protein localization as well as structure. Examples of how protein localization influences cell shape and the possibility to aggregate have been reported in Candida auris and Staphylococcus aureus.

How Are Moonlighting Proteins Acquired?

How do these genes acquire such multifunctionality in the first place? It is not from promiscuous enzymatic activity—where 1 site on the protein accommodates many molecules—gene splicing or gene fusions. Rather, MPs have sites on a single protein that have evolved through gene sharing to perform and maintain multiple roles without gene duplication or loss of primary function.

Understanding Moonlight Proteins Through Gut Microbes 

MPs impact cells and other organisms in their surrounding environments, making the gut microbiome, which holds within itself a world of microbe-microbe interactions, an ideal place to better dissect the role that MPs play in regulating interactions amongst microbial species. 

In the gut, the first prominent interaction between bacteria and host cell surfaces is adherence, as bacteria must establish their presence (colonize) in order to survive. Some bacteria, such as the anaerobic Bacteroidaceae and Bifidobacteriaceae, associate with the mucus layer of the gut (where it is the thickest: 30 to 300 µm) and use undigested complex carbohydrates from the small intestine for nutrition. As complex carbohydrates like mucin O-glycan break down, they become available for other microbes to eat. This cross-feeding encourages the intestinal microbial community to develop and prosper.
Moonlighting proteins, such as EF-Tu, can be found in the colon.
Moonlighting proteins, such as EF-Tu, can be found in the colon.
Source: Created by author using

The factors and mechanisms underlying the maintenance of microbial communities in the gut are still quite unexplored, yet studies indicate that MPs might play an important role. MPs are often secreted and will oscillate from catalysis to secondary functions, like signal transduction and transcriptional regulation, by localizing on target cell surfaces (for example, the gut lining). They adhere to epithelial cells, extracellular matrix proteins, glycocalyx and mucins not via receptors, but through ionic interactions in both commensal and pathogenic gut bacteria. 

Some MPs even act as probiotics. For example, MP GroEL (a molecular chaperone complex that facilitates correct folding of other proteins in the cell) intervenes with the aggregation of pathogen Helicobacter pylori by binding to mucin and epithelial cells in the stomach and duodenal areas. The enolase of Lactobacillus jensenii inhibits the adherence of Neisseria gonorrhoeae to epithelial cells, and the MP trigger factor protein (Tig) from Lactobacillus fermentum inhibits the adhesion of Enterococcus faecalisThese MP proteins enable the commensal bacteria to attach and interact with the host system, thus establishing gut homeostasis, while preventing the attachment of various pathogenic microorganisms.

Deeper analysis of 2 specific gut bacteria, has illuminated additional information about such moonlighting behavior.

Narrowing the Lens

Bifidobacterium longum and Bacteroides thetaiotaomicron are 2 common mucolytic gut commensals that are adept at overcoming the mucus barrier. B. longum usually tags along with its natural symbiont, B. thetaiotaomicron, and together, these microbes account for 20% of the human gut microbiome. 

Once B. longum and B. thetaiotaomicron have adhered to the mucin layer, they prompt the surrounding microflora to flourish using the cross-feeding principle described above, and they manage to maintain contact with the host with the help of MPs. These 2 anaerobic symbionts produce extracellular vesicles (EVs) containing cytoplasmic MPs that are constantly secreted in the intestine. In fact, the EVs of B. longum have at least 13 MPs, including ribosomal proteins, enolase, phosphoglycerate kinase (PGK), heat shock protein (Hsp) GroEL and elongation factor EF-Tu, to name a few.

GroEL and EF-Tu secreted from B. longum localize to the cell surface and use electrostatic interactions (characteristic of MPs) to enhance the adherence of B. thetaiotaomicron to host mucin. Notably, this interaction is different from anchoring adhesion that is seen in other single-function proteins, like fimbriae, which is irreversible. B. longum and B. thetaiotaomicron manage to adhere themselves together to the mucin, as well as aid in the colonization (adhesion) of other gut microbes. 

Thanks to the “benevolence” of these and other mucolytic microbes, the result is a readily available food source, with symbiosis favored over antagonism in the gut, and a constant interaction with host species.

Pinning the Location of MPs

The cellular origin of MPs is tough to pin down. Since they play non-specific and multiple roles within a cell of the host or the microbe, they challenge the notions of traditional protein function, and thus may be elusive to track or trace back to their original sources. Nevertheless, a few environmental triggers are known to cause the switch between MP functions, and they include interactions with DNA or RNA, a slight temperature change, change in oligomeric state of the protein, etc.

Despite these difficulties, a few methods are being pursued to identify MPs:
  • Studying the evolutionary history of proteins.
  • Examining the structural (especially protein surface) and sequence features.
  • Further comparing them with orthologous proteins with the same primary functions.
  • Developing and referring to databases.

What's Next for Moonlight Protein Research?

Scientists affirm that exploring this line of protein research has high prospects, especially when the MPs heavily influence the inner metabolism—not just in our guts, but beyond—via immunomodulation and biofilm formations in host and microbe alike.

So, what’s next? The origin of MPs has yet to be determined, and the universal roles of each MP in the gut microbiome yet to be clearly elucidated. Studying the evolutionary history and conservation of the MPs across various species through time could lead to fascinating insights. These are just a few questions, but many still remain unanswered, and as such this field shows a high potential for research and development.

Author: Tejaswini Petkar

Tejaswini Petkar
Tejaswini Petkar is a researcher at the Biomaterials, Drug Delivery & Nanotechnology Unit, Centre for Biomedical and Biomaterials Research (CBBR), at the University of Mauritius.