HMOs Increase the Ability of Bifidobacteria to Adhere to the Intestinal Epithelium

Adhering to the intestinal epithelial cells lining the gut is important for yielding the health benefits of  Bifidobacteria.  Here we explore the results of a recent study to find out if HMOs can increase the adherence of  Bifidobacterial  strains in the human gut.  

Content Outline

• Introduction
  • How do bacteria adhere to the intestinal wall?
• How was the study conducted?
• Do HMOs enhance Bifidobacteria adherence?
  • Single and multi-strain adherence to colonic cells
  • Identifying the colonization factors
  • Proteomic analysis
• Summary

Introduction

Bifidobacteria  is one of the most abundant gut commensals, accounting for up to 90% of the total population found in infant feces. They are also one of the early colonisers in the human colon and their huge array of health benefits have been associated with their ability to use human milk oligosaccharides (HMOs).

HMOs are crucial for health because they promote the growth of beneficial bacteria, including  Bifidobacteria,  and encourage the production of short-chain fatty acids (SCFAs) and other important metabolites. That makes them important prebiotics.

Because of its well-documented health benefits,  Bifidobacteria  is one of the most used and well-researched probiotics. When used in probiotic products,  Bifidobacteria  work in several ways to promote your health, including:

• modulating the composition of the gut microbiota
• stimulating the development of the immune system
• promoting and maintaining the integrity of the gut barrier
• fighting off potential pathogens

However, developing a successful  Bifidobacteria-containing probiotic doesn’t come without its challenges. For example, it must compete with other microbes for nourishment, survive the journey through the gastrointestinal tract, and effectively colonize the gut. One of the key differentiators for developing a probiotic-containing product is its ability to adhere to the intestinal wall.

Here we explore the results of a study by Walsh and colleagues (2023) which investigated the ability of HMOs to increase the adhesion of  Bifidobacteria  to mucus-secreting intestinal cells.

How do bacteria adhere to the intestinal wall?

Recent research has identified an array of bacterial features that are important in the interactions between microbes and their human hosts.

For probiotics like  Bifidobacteria  to colonise the gut they must resist the peristaltic motion in the gastrointestinal tract (GI), the involuntary relaxation and contraction of the digestive muscles that work to push food through the tract. To do this, bacteria must adhere or stick to the intestinal wall[i]. There are two types of bacterial adhesion:

1. Nonspecific:a reversible form of adhesion which relies on forces to mount onto the intestinal wall, but bacteria can easily be dislodged by peristalsis or environmental changes.
2. Specific:a process where bacteria attach to the intestinal mucosa via adhesins, specific recognition molecules that can be categorized into fimbrial or afimbrial adhesins[ii].

Evidence suggests that  Bifidobacteria  strains express hair-like structures called pili to help them adhere to intestinal mucus cells.

Figure 1.The image shows the process of afimbrial adhesions. B shows that Bifidobacterium longum binds to the intestinal epithelial cells via the afimbrial adhesin FimM which binds to mucin and fibronectin receptors on the cell's surface[iii].

How was the study conducted?

The study by Walsh et al (2023) was conducted to find out if human milk oligosaccharides (HMOs) could enhance the adhesion of  Bifidobacteria  to the intestinal epithelium. The study assessed the colonization-promoting benefits of HMOs on four commercial infant strains of  Bifidobacterium.  The strains were:

• B. bifidum  R0071
• B. infantis  R0033
• B. breve  M-16V
• B. infantis  M-63

All these strains have been shown to have strong immunomodulatory and protective benefits in vivo.An in vitro intestinal model for bacterial adhesion was set up using HT29-MTX cells, cells isolated from the human colon cancer cell line HT29[iv].

The bacteria strains were incubated with HMOs derived from human breast milk from donors. Proteomic analysis was carried out to determine the factors critical for enhancing adhesion to the colonic cell line.

Do HMOs enhance Bifidobacteriaadherence?

The simple answer is yes!

Some of the key findings from the study were:

• B. bifidum  R0071 had the highest adherence after S-HMO exposure
• B. infantis  R0033 had better adhesion than the M-63 strain after being exposed to the S-HMO
• A multi-strain Bifidobacteria mixture had increased adhesion compared to single strains following S-HMO treatment
• B. bifidumR0071 had more extensive and varied sortase-dependent adhesins and pili-related genes which may explain why it has superior adherence properties
• Proteome changes were evident, especially in B. infantiswhere HMO metabolism proteins increased
• HMO pre-treatment upregulated proteins involved in cell adhesion and surface appendages in B. bifidum
• Proteins important for the interaction with the mucus layer were upregulated following HMO exposure

Single and multi-strain adherence to colonic cells

The four Bifidobacteria strains were incubated with glucose, 2’-fucosyllactose (2’-FL), or secretor-HMO (S-HMO) and their ability to stick to the HT29-MTX cells was determined.

The adhesion of Bifidobacteria to the colon cells depends on the strain and the preferred source of nourishment. As shown in Figure 2. The lowest adherence was seen in B. breveM-16 V, particularly when glucose was the sole carbon source. The highest levels of adherence were visible in B.breveR0071 after being exposed to S-HMO.

Figure 2.The graph shows the adherence of four Bifidobacteria strains to a colonic cell line after being exposed to glucose control, breastmilk-derived HMOs (S-HMOs), and 2’-FL. B. bifidum R0071 and B. infantis R0033 were associated with significantly higher adherence than B. breve M-16 V. The four Bifidobacterium mix shows that S-HMO showed the greatest adherence by an average of 1.31-fold vs the control.

Overall, exposure to S-HMO (mix of various HMOs) significantly increased the adhesion of all Bifidobacteriastrains with B. bifidum R0071 showing the largest improvement. Between the two B. infantis strains, R0033 had the highest adhesion compared to B. infantis M-64 following exposure to S-HMO. Compared to the single strains, the multiple-strain mixture showed higher adhesion levels, particularly after S-HMO exposure.

Identifying the colonization factors

To identify the key factors responsible for the bacterial adherence differences, the genomes of the  Bifidobacterial  strains were surveyed for colonization-related factors. Using a literature search, the researchers identified 45 genes that could have potential roles in the survivability and persistence of Lactobacillus and Bifidobacteria in the gut.

Several components were identified that can interact with and adhere to mucus and surface glycans on the epithelial cell surface, such as:

• Outer membrane proteins
• Adhesins
• Capsules
• Flagella
• Fimbriae
• Pili

Research shows that pilus-like appendages on the bacterial surface are believed to be fundamental for  Bifidobacterial  colonization in the gut[v]. More recently, specific encoding-gene clusters have been identified in infant-derived  Bifidobacteria,  particularly:

1. a type IVb tight adherence (Tad) pilus encoding gene cluster in B. breve  UCC2003
2. sortase-dependent pilus gene clusters in B. breve  PRL2010

The study by Walsh and colleagues (2023) found genes with homology to the UCC2003 tadlocus in all four  Bifidobacteria  strains. A study by O’ Connell Motherway et al (2011) confirmed that the tad gene cluster is critical for the gut colonization of  Bifidobacteria.

Another functional component that is important in facilitating the crosstalk between bacteria and its host, particularly within the  Bifidobacteria  genus is specific enzymes called sortases. There are different types of sortases with Class C types being shown to be responsible for constructing pilus polymers. Although these are rare, the study found Class C fimbrial units in the R0033 which may explain why this strain of  B. infantis  showed greater adherence compared to its M-63 counterpart.

Proteomic analysis

The researchers also carried out a proteomic analysis on the four-strain Bifidobacterial strains after exposure to the colonic cell line. The results were compared to the control that had not been in contact with the colon cells. The effect of the S-HMO pre-treatment on colonization-associated pathways was also analysed by comparing the HMO-treated and untreated bacteria after being exposed to the HT29-MTX cells.

The results from the proteomic analysis are summarized below:

• 2191 bacterial proteins were identified
• When exposed to the colon cells, a significant change in the proteome was seen
• B. bifidum  was associated with increased abundance in 225 proteins post-cell exposure, including 27 related to colonization
• Pre-treatment with S-HMO shifted the proteome, mostly in B. infantis,where proteins linked to HMO metabolism increased
• Proteins in B. bifidumwith roles in cell adhesion and surface appendages were upregulated after being treated with HMOs

The specific proteins that were upregulated in  B. bifidum  included:

• Sortase type C-dependent pilus proteins (FimP, FimQ, Pil1)
• Moonlighting proteins, such as DnaK, GroES, and Lux, that are important for cell adhesion
• Sortase type E-dependent glycosyl hydrolases that are critical for interacting with the intestinal mucus layer

Overall, B. bifidum responded well to HMO pre-treatment with evidence that there was an increase in proteins linked to bacterial colonization and adhesion. Plus, the mucosal layer in the gut not only serves as a point of adhesion but is also an important nutrient source for many gut microbes including Akkermansia muciniphila and Eubacterium hallii, a vital producer of butyrate and propionate.

Studies have shown that B. bifidum is the only strain of Bifidobacteria that can break down mucin and release metabolites that can be used by other gut microbes[vi]. Therefore, because HMOs can enhance the colonization of B. bifidum this could also benefit other commensal gut bacteria, particularly via mucin cross-feeding activities

Summary

Overall, the study by Walsh and Co. (2023) shows that HMOs can increase the interaction between infant-derived Bifidobacteria strains and colonic epithelial cells, significantly increasing the adhesion of commercial Bifidobacteria strains.Therefore, shedding light on the importance of combining prebiotics, such as HMOs, with common probiotic bacteria strains, to enhance their effectiveness.

You can get ahead of the trend and bolster your colonic ecosystem with our PureHMO® range.

Written by: Leanne Edermaniger, M.Sc. Leanne is a professional science writer who specializes in human health and enjoys writing about all things related to the gut microbiome.  

Sources

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[ii] Walsh C, Owens RA, Bottacini F, Lane JA, van Sinderen D, Hickey RM. HMO-primed bifidobacteria exhibit enhanced ability to adhere to intestinal epithelial cells. Frontiers in Microbiology. 2023 Dec 15;14. doi:10.3389/fmicb.2023.1232173

[iii] Larissa Jank M. Exploring the impact of indole-3-lactate in the gut-brain axis and multiple sclerosis: Larissa Jank, MD [Internet]. Neurology live; 2023 [cited 2024 Jul 30]. Available from: https://www.neurologylive.com/view/exploring-impact-indole-3-lactate-gut-brain-axis-multiple-sclerosis-larissa-jank

[iv] Martínez-Maqueda D, Miralles B, Recio I. HT29 Cell Line. In: Verhoeckx K, Cotter P, López-Expósito I, et al., editors. The Impact of Food Bioactives on Health: in vitro and ex vivo models [Internet]. Cham (CH): Springer; 2015. Chapter 11. Available from: https://www.ncbi.nlm.nih.gov/books/NBK500137/ doi: 10.1007/978-3-319-16104-4_11

[v] Ligthart K, Belzer C, de Vos WM, Tytgat HLP. Bridging bacteria and the gut: Functional aspects of type IV pili. Trends in Microbiology. 2020 May;28(5):340–8. doi:10.1016/j.tim.2020.02.003

[vi] Bunesova V, Lacroix C, Schwab C. Mucin Cross-Feeding of Infant Bifidobacteria and Eubacterium hallii. Microb Ecol. 2018 Jan;75(1):228-238. doi: 10.1007/s00248-017-1037-4. Epub 2017 Jul 18. PMID: 28721502.