Host defense functions of the epididymal amyloid matrix.

The epididymal lumen is an immunologically distinct environment. It maintains tolerance for the naturally antigenic spermatozoa to allow their maturation into functional cells while simultaneously defending against pathogens that can ascend the male tract and cause infertility. We previously demonstrated that a nonpathological amyloid matrix that includes several cystatin-related epididymal spermatogenic (CRES) subgroup family members is distributed throughout the mouse epididymal lumen but its function was unknown. Here, we reveal a role for the epididymal amyloid matrix in host defense and demonstrate that the CRES amyloids and CD-1 mouse epididymal amyloid matrix exhibit potent antimicrobial activity against bacterial strains that commonly cause epididymal infections in men. We show the CRES and epididymal amyloids use several defense mechanisms including bacterial trapping, disruption of bacterial membranes and promotion of unique bacterial ghost-like structures. Remarkably, these antimicrobial actions varied depending on the bacterial strain indicating CRES amyloids and the epididymal amyloids elicit strain-specific host defense responses. We also demonstrate that the CRES monomer and immature assemblies of the epididymal amyloid transitioned into advanced structures in the presence of bacteria, suggesting their amyloid-forming/shape-shifting properties allows for a rapid reaction to a pathogen and provides an inherent plasticity in their host defense response. Together, our studies reveal new mechanistic insight into how the male reproductive tract defends against pathogens. Future studies using a mouse model for human epididymitis are needed to establish the epididymal amyloid responses to pathogens in vivo. Broadly, our studies provide an example of why nature has maintained the amyloid fold throughout evolution.

The mouse epididymal amyloid matrix is a mammalian counterpart of a bacterial biofilm.

The mouse epididymis is a long tubule connecting the testis to the vas deferens. Its primary functions are to mature spermatozoa into motile and fertile cells and to protect them from pathogens that ascend the male tract. We previously demonstrated that a functional extracellular amyloid matrix surrounds spermatozoa in the epididymal lumen and has host defense functions, properties not unlike that of an extracellular biofilm that encloses and protects a bacterial community. Here we show the epididymal amyloid matrix also structurally resembles a biofilm by containing eDNA, eRNA, and mucin-like polysaccharides. Further these structural components exhibit comparable behaviors and perform functions such as their counterparts in bacterial biofilms. Our studies suggest that nature has used the ancient building blocks of bacterial biofilms to form an analogous structure that nurtures and protects the mammalian male germline.

Host defense amyloids: Biosensors of the immune system?

There is considerable evidence showing that highly ordered aggregate structures known as amyloids carry out essential biological roles in species ranging from bacteria to humans. Indeed, many antimicrobial peptides/proteins form amyloids to carry out their host defense functions and many amyloids are antimicrobial. The similarity of host defense amyloids from bacterial biofilms to the mammalian epididymal amyloid matrix implies highly conserved host defense structures/functions. With an emphasis on the epididymal amyloid matrix, here we review the common properties of host defense amyloids including unique traits that would allow them to function as powerful biosensors of the immune system.

The Functional Mammalian CRES (Cystatin-Related Epididymal Spermatogenic) Amyloid is Antiparallel ß-Sheet Rich and Forms a Metastable Oligomer During Assembly.

An amyloid matrix composed of several family 2 cystatins, including the reproductive cystatin CRES, is an integral structure in the mouse epididymal lumen and has proposed functions in sperm maturation and protection. Understanding how CRES amyloid assembles in vitro may provide clues on how the epididymal amyloid matrix forms in vivo. We therefore purified full-length CRES under nondenaturing conditions and followed its aggregation from monomer to amyloid under conditions that may approximate those in the epididymal lumen. CRES transitioned into a metastable oligomer that was resistant to aggregation and only over extended time formed higher-ordered amyloids. High protein concentrations facilitated oligomer assembly and also were required to maintain the metastable state since following dilution the oligomer was no longer detected. Similar to other amyloid precursors, the formation of CRES amyloids correlated with a loss of α-helix and a gain of ß-sheet content. However, CRES is unique in that its amyloids are rich in antiparallel ß-sheets instead of the more common parallel ß-sheets. Taken together, our studies suggest that early metastable oligomers may serve as building blocks for functional amyloid assembly and further reveal that antiparallel ß-sheet-rich amyloids can be functional forms.

Functional Amyloids in Reproduction.

Amyloids are traditionally considered pathological protein aggregates that play causative roles in neurodegenerative disease, diabetes and prionopathies. However, increasing evidence indicates that in many biological systems nonpathological amyloids are formed for functional purposes. In this review, we will specifically describe amyloids that carry out biological roles in sexual reproduction including the processes of gametogenesis, germline specification, sperm maturation and fertilization. Several of these functional amyloids are evolutionarily conserved across several taxa, including human, emphasizing the critical role amyloids perform in reproduction. Evidence will also be presented suggesting that, if altered, some functional amyloids may become pathological.