Variable Proteins to Speed Evolution

 

In an earlier post from last month, we examined how phenotypic diversity of an organism, necessary for enhancing that organism’s adaptability to changes occurring in its prevailing environment could be better understood from the perspective of that organism representing a holobiont, a meta-organism of both the host together with the commensal microorganisms resident in the host. This expanded view allowed the incorporation of certain microbial genes along with the host genome in accounting for some phenotypic trait variation for which the host genome alone cannot account. An important example of these interactions comes from the study microorganism-derived immune modulation in cancer and the ability of those microorganisms to alter the cancer microenvironment.

Mutagenic Retrohoming Microorganisms of the human gastrointestinal (GI) tract are also able to quickly adapt  some of their own phenotypic characteristics through a process that has been termed mutagenic retrohoming. Diversity-generating retroelements (DGR) are unique segments of DNA consisting of a target gene that includes a set of variable codons termed a variable repeat (VR) which encode variations in protein, a template gene, termed a template repeat (TR) which is similar to the VR and a reverse transcriptase. During mutagenic retrohoming RNA transcripts of the TR become the substrate for that segment’s reverse transcriptase to produce a cDNA copy. The reverse transcriptase, however, is error-prone and selectively mismatches adenine nucleotides, replacing the adenine with any of the four nucleotides to alter the resulting codons, now hypermutated. The newly hypermutated DNA is then inserted into the VR swapping place with the parenteral sequences, but importantly leaving the reminder of the target gene unaffected. Below is a diagrammatic representation of mutagenic retohoming by DGRs.

In a report in Science 390.eadv2111 (2025), Macadangdang et. al identified DGRs occurring within the microbiota of the human GI tract, focusing on their frequent identification in Bacteroides species. Bacteroides frequently harbor DGRs in over two-thirds of isolates with the investigators identifying over 1100 distinct DGRs, an indication of their considerable potential in diversifying affected protein and a likely sign of the considerable selection these microorganisms find themselves under.  Their investigation of Bacteroides DGRs included a functional characterization, their mobility in spreading horizontally to other microorganisms, the mutational dynamics observed in response to competition and their vertical transmission in maternal-infant pairs.

Functional Characterization A principal major finding of the study was that the target for many of the identified DGRs were the variable proteins of the ligand binding region of adhesion proteins of bacterial pili. Pili are hair-like structures on the surface of bacteria which permit their adherence to structures of the GI tract such as mucus and thus promote that microorganism’s fixation and persistence. In particular, they noted the variable proteins targeted by the DGRs were responsible for a strategic region of the pilus, the proteins located at the tip of the pilus, an indication of their key role in affecting binding affinity of the pilus. The functional significance of this rapid protein diversifying capability in Bacteroides species thus is ability of Bacteroides to modify their pilus binding to one or another structures in the gut, allowing improved survival chances in the face of fluctuating gastrointestinal ecology.

Mobility through Horizontal Transmission DGRs also proved to be mobile, permitting the horizontal transmission of these sequences to recipient microorganisms. They demonstrated this by examining for the presence of a set of genes flanking the DGR, termed integrative and conjugation elements (ICE) surrounding DGRs of ten Bacteroides species. ICEs are known to promote improved survival by assisting in the transmission of factors such as those involved with colonization, metabolism, and antibiotic resistance. The investigators found sequence homology across almost one dozen DGR from these strains as an indication of likely horizontal transfer. Consistent with their observation of functional aspects of DGRs, pilin genes remained the principal target for these homologs.

Mutational Dynamics Evidence for DGR’s adaptive capability included an analysis of VR sequences which had undergone retrohoming in Bacteroides strains which had been exposed to differing competitive environments. To do this the investigators collected fecal samples from mice raised in either a germ-free environment or together with a collection of other non-Bacteroides strains to simulate the competition Bacteroides would ordinarily be under. DNA from these serial collections over a two-week period was isolated, the VR sequences from each sample were then barcoded, amplified by PCR and then deeply sequenced.  The amplified VR sequences could be compared across the sample allowing calculation of  Shannon entropy. This is a statistical measure of each VR collection as an estimate of the degree in which sequences were divergent from one another over the time of the collections. A high degree of entropy would be consistent with the active presence of mutagenic retrohoming producing a high degree of variation. Lower Shannon entropy measures would be consistent with the presence of purifying selection, that is positive selection in response to an environmental cue.

In mice raised in either a germ-free or monocolonized environment, VRs demonstrated considerable divergence with high VR entropy but in a pattern which seemed random. Bacteroides strains however raised under co-colonization conditions were found to exhibit a marked drop in the entropy in which almost one-half of VR sequences being derived from two or three VRs. This convergence is an indication of positive selection as protein vaiants of lower biooding affinity were gradually replaced by more adaptive lilus binding. DGR activity then was being focused on generating adaptive proteins to address the competition for ligand binding among the microbiota. Interestingly, they point out that the convergence of diversified proteins in response to selection was being achieved though the DNA sequences generating these proteins were different. DGR hypermutation was thus sensitive to those prevailing environmental conditions of the mouse gut.

Maternal-Infant Transmission The investigation also examined the possibility of vertical transmission of DGRs in an accompanying study of 144 maternal-infant pairs where metagenomic sequencing of DNA retrieved data from fecal samples obtained at birth and then up to one year of age from the pairs. They identified over 2700 unique DGR from that data set including 388 instances in which they found sequence homology between maternal and infant identified DGRs. For infants born following vaginal birth they found an overall higher number of DGRs which were detected on average at an earlier time than from the DGRs from infants born through caesarian section. This is as would be expected if conveyance of the microbial-derived DGRs was enhanced though transvaginal passage. If DGRs function to speed the adaptive response of Bacteroides adhesion molecules for instance, it would might be an advantage in establishing a stable GI microbial environment during early infancy in the maturing gastrointestinal tract. Below is a graphic illustration summarizing the principal findings of Macadangdang et. al.

Given our emerging understanding of microbe-induced enhancement of anticancer immunotherapy and knowing earlier data identifying Bacteroides Fragilis as providing such enhancement it is tempting to speculate on this microbe’s special rapid adaptability of surface adhesion capabilities that might contribute to that increase. Recall how in China Bacteroides Fragilis is used as a probiotic given for instances of GI dysbiosis. Dysbiosis occurs when there is a loss of beneficial bacteria with an accompanying increase in harmful bacteria leading to chronic inflammation. An organism such as Bacteroides, a common GI commensal known to be present in over 60% of studied individuals, might be acting to stabilize the gut mucosa through mucus or biofilm binding favoring a protective environment to predominate.

Rapid Protein Variation Across Kingdoms DGRs acting to induce highly targeted mutagenesis in the coding regions of variable microbial proteins are reminiscent of other molecular mechanisms to augment phenotypic variation, notably B-cell immunoglobulin synthesis, the central means of diversifying an adaptive humoral immune response. Here in the germinal centers of lymph nodes, hypermutation of the variable regions of light chain and heavy chains create a set of immunoglobulin molecules in which those B-cell clones producing the highest affinity for an antigen will be selected, what is termed affinity maturation. In both instances, microbial cells and B-cells have developed a acute means of generating protein diversity to actively respond to the always fluctuating environment where they live, an example of nature using analogous mechanisms across kingdoms to meet this challenge.