Rothia to the rescue

Gluten is problematic. Almost every paleo advocate agrees that wheat should be restricted in the diet because gliadin peptides generated by the uncomplete digestion of gluten produces highly reactive epitopes, which then can trigger a T-cell response in the gut. 

The main issue with the "gluten is bad for everyone" meme is that not all people develop gluten sensitivity or celiac disease. Many can tolerate great doses of wheat-based foods for years without serious health consequences. This is often attributed to lack of an environmental trigger which increases susceptibility to gliadin peptides (ie. inflammation). 

A recent paper (1) has found that two strains from the genus Rothia, namely R.mucilaginosa and R. aeria are capable of metabolizing gluten. These are oral microorganisms.

After isolation on a gluten agar media, these microbes were capable of hydrolizing YPQ tripeptides (which occur with high frequency in gluten sequences) and KPQ. Moreover, R.aeria degraded gliadin in vitro: 

SDS-PAGE of aliquots from the incubation mixture

The arrow shows the major protein constitutent in the gliadin mixture. As it can be seen, gliadin was progressively degrated (lanes 2-7, figure A). Shorter time intervals (lanes 2-7, figure B) show that almost 50% of gliadin was degraded in 30 minutes. Boiling bacterial suspensions abolished degradation (lanes 8 and 9, figure B). This suggests enzyme denaturation. Lanes 10-11 and 12-13 served as negative and positive controls, respectively. 

Proteolytic degradation of two problematic peptides (a-gliadin derived 33-mer and y-gliadin derived 26-mer) was compared between mammalian enzymes (pepsin, trypsin, chymotrypsin) and R.aeria:

RP-HPLC of sample aliquots

As it can be seen, chromatograms from all mammalian enzymes show the same pattern at 0 and 24h, showing no digestion of the peptides (A-C). In contrast, the sample containing R.aeria (WSA-8, figure D) showed the presence of different peaks earlier in the chromatogram, representing degradation fragments which elute earlier. At 2 hours, the peptide was completely degraded. 

These results show that enzymes present in R.aeria are capable of degrading gliadin and two peptides (33-mer and 26-mer) which are resistant to mammalian enzymes. Analysis by Mass Spectrometry determined that cleavage was made after QPQ and LPY for R. aeria and XPQ and LPY for R.mucilaginosa (X denotes any aminoacid). This is important because these tripeptides are part of the immunogenic epitopes contained within the 33-mer (glia-a9, glia-a2) and 26-mer peptides (glia-y2):

glia-a9: LQLQPFPQPQLPY

glia-a2: PQPQLPYPQPQLPY

glia-y2: PFPQQPQQP / PYPQQPQQP

It is worth noting that cleavage from both Rothia strains was observed also after Q residues along the 26-mer sequence. Repeated Q residues (along with P residues) are responsible for resistance to proteolysis by mammalian enzymes. 

Zymography at pH 7.0 showed that the putative gliadin-degrading enzymes had a molecular weight of approximately 75kDa and 70kDa for R.mucilaginosa and R.aeria, respectively. Further analysis on the activity of these enzymes at different pH revealed that enzymes from R.mucilaginosa were completely inactive at pH 3.0, while R.aeria maintained a weak enzymatic activity. The optimal pH determined was 7.0, and substrate hydrolysis rates declined in parallel with decreasing pH values from 7.0 to 4.0. At pH 3.0, R.aeria showed a much slower activity, while at pH 2.0, activity was completely abolished. 

Summary

- R.mucilaginosa and R.aeria were capable of degrading gliadin and immunogenic peptides in vitro.

- The enzymes present have an approximate molecular weight of 70-75kDa. 

- Degrading activity of R.aeria was maintained at pH 3.0. 

- Both species are normal colonizers of the oral cavity and other areas (R.mucilaginosa).

From the discussion (my bolds):

"R. aeria (...) is an oral colonizer [31]. R. mucilaginosa also primarily colonizes the oral cavity [33] but has furthermore been isolated from other body sites, including the upper respiratory tract and the duodenum [34,35,36]"

"Bacterial speciation of 2,247 clones recovered from 63 duodenal biopsies obtained from healthy and celiac patients showed that R. mucilaginosa comprised [aprox.] 6% of the clones and was present in [aprox.] 65% of the biopsies, identifying it as a true colonizer of the duodenum [36]."

The million dollar question:

"The discovery of salivary microorganisms degrading dietary proteins in vitro prompts the question to what extent such microorganisms play a role in food processing in vivo. During mastication (chewing) foods are mixed with whole saliva helping to accelerate the break-down by digestive enzymes during the residency time in the oral cavity. Oral microorganisms in the swallowed food bolus may or may not survive and/or continue to exert proteolytic activities during or after gastric passage. Our in vitro data with R. aeria show that its enzymes are not abolished at acidic pH values, and are optimally active under more basic pH conditions. In vivo, this could mean that during gastric passage the enzymes will neither be active nor destroyed, and that enzymatic reactivation would occur upon transfer to the duodenum.

With regard to duodenal Rothia enzyme activity, it is relevant that R. mucilaginosa gains a foothold in the duodenum [36]. This offers the intriguing possibility that Rothia may colonize the duodenum and perform proteolytic activities locally in conjunction with mammalian- derived enzymes to degrade gluten."

The study here presented is a follow-up of one published in 2010 by the same group (2) in which they found that oral bacteria were capable of degrading completely immunogenic gliadin peptides. 

Further studies should help elucidate detailed information about the enzymes responsible for gluten degradation by these bacteria. 

This relationship might offer a way in which we are evolving and adapting to foods introduced in the neolithic: not only by changes in genes and gene expression (ie. AMY1), but also by establishing new symbiotic relationships with microorganisms. 

Zamakhchari M, Wei G, Dewhirst F, Lee J, Schuppan D, Oppenheim FG, & Helmerhorst EJ (2011). Identification of rothia bacteria as gluten-degrading natural colonizers of the upper gastro-intestinal tract. PloS one, 6 (9) PMID: 21957450