Candida albicans mannans mediate Streptococcus mutans exoenzyme GtfB binding to modulate cross-kingdom biofilm development in vivo

Candida albicans is frequently detected with heavy infection by Streptococcus mutans in plaque-biofilms from children with early-childhood caries (ECC). This cross-kingdom biofilm contains an extensive matrix of extracellular α-glucans that is produced by an exoenzyme (GtfB) secreted by S. mutans. Here, we report that mannans located on the outer surface of C. albicans cell-wall mediates GtfB binding, enhancing glucan-matrix production and modulating bacterial-fungal association within biofilms formed in vivo. Using single-molecule atomic force microscopy, we determined that GtfB binds with remarkable affinity to mannans and to the C. albicans surface, forming a highly stable and strong bond (1–2 nN). However, GtfB binding properties to C. albicans was compromised in strains defective in O-mannan (pmt4ΔΔ) or N-mannan outer chain (och1ΔΔ). In particular, the binding strength of GtfB on och1ΔΔ strain was severely disrupted (>3-fold reduction vs. parental strain). In turn, the GtfB amount on the fungal surface was significantly reduced, and the ability of C. albicans mutant strains to develop mixed-species biofilms with S. mutans was impaired. This phenotype was independent of hyphae or established fungal-biofilm regulators (EFG1, BCR1). Notably, the mechanical stability of the defective biofilms was weakened, resulting in near complete biomass removal by shear forces. In addition, these in vitro findings were confirmed in vivo using a rodent biofilm model. Specifically, we observed that C. albicans och1ΔΔ was unable to form cross-kingdom biofilms on the tooth surface of rats co-infected with S. mutans. Likewise, co-infection with S. mutans defective in GtfB was also incapable of forming mixed-species biofilms. Taken together, the data support a mechanism whereby S. mutans-secreted GtfB binds to the mannan layer of C. albicans to promote extracellular matrix formation and their co-existence within biofilms. Enhanced understanding of GtfB-Candida interactions may provide new perspectives for devising effective therapies to disrupt this cross-kingdom relationship associated with an important childhood oral disease.白色念珠菌（Candida albicans）常与变形链球菌（Streptococcus mutans）的重度感染一同存在于儿童早期龋病（early-childhood caries, ECC）患儿的菌斑生物膜内。该跨王国生物膜含有丰富的细胞外α-葡聚糖基质，此类基质由变形链球菌分泌的胞外酶（exoenzyme）GtfB催化合成。本研究发现，位于白色念珠菌细胞壁外表面的甘露聚糖（mannans）可介导GtfB结合，进而增强葡聚糖基质生成，并调控体内（in vivo）形成的生物膜内的细菌-真菌共生关系。借助单分子原子力显微镜，我们测定发现GtfB与甘露聚糖及白色念珠菌表面具有极高的结合亲和力，可形成高度稳定且牢固的键合（结合力达1–2 nN）。然而，在O-甘露聚糖（O-mannan）缺陷菌株（pmt4ΔΔ）或N-甘露聚糖外侧链（N-mannan outer chain）缺陷菌株（och1ΔΔ）中，GtfB与白色念珠菌的结合特性会受到削弱。尤为关键的是，och1ΔΔ菌株上GtfB的结合强度受到严重破坏（相较于亲本菌株（parental strain），结合强度降低超过3倍）。随之而来的是，真菌表面的GtfB含量显著降低，且白色念珠菌突变株与变形链球菌形成混合物种生物膜的能力受损。该表型与菌丝形成或已明确的真菌生物膜调控因子（EFG1、BCR1）无关。值得注意的是，缺陷型生物膜的机械稳定性有所减弱，最终导致其生物量在剪切力（shear forces）作用下几乎完全被移除。此外，本研究借助啮齿类动物生物膜模型，在体内（in vivo）验证了上述体外（in vitro）实验结果。具体而言，本研究观察到，在与变形链球菌共感染的大鼠牙齿表面，白色念珠菌och1ΔΔ突变株无法形成跨王国生物膜。同理，若采用GtfB缺陷型变形链球菌进行共感染，同样无法形成混合物种生物膜。综上，本研究数据支持如下作用机制：变形链球菌分泌的GtfB可结合至白色念珠菌的甘露聚糖层，从而促进细胞外基质形成并维持二者在生物膜内的共生关系。深入理解GtfB与念珠菌的相互作用，可为靶向干预这一与重要儿童口腔疾病相关的跨王国共生关系提供新的治疗思路。