The Role of the Extracellular Matrix components of Salmonella enterica serovar Typhimurium Biofilms in Biofilm Formation and Architecture via in vitro analysis

Abstract

Biofilms are aggregates of bacterial cells enclosed within a self-generated extracellular matrix, forming a three-dimensional structure that allows the bacteria cells to firmly attach to both living and non-living surfaces. The extracellular matrix of Salmonella enterica serovar Typhimurium (STm) biofilms is composed of curli, cellulose, BapA, O-antigen capsule, colanic acid and extracellular DNA (eDNA) and protects the resident bacteria from diverse environmental stresses such as antibiotics and the host immune response. Studies have shown that curli, which accounts for 85% of the extracellular matrix, plays a significant role in the establishment of the matrix. Curli interacts with eDNA and cellulose to create a mesh-like network that stabilizes the biofilm, aids in surface adherence, and confers elasticity to the biofilm. Although curli, cellulose, eDNA, and their interactions have been studied, it is not clear how BapA, O-antigen capsule, and colanic acid contribute to Salmonella biofilm structure. Here, we examined the individual role of the matrix components utilizing various isogenic mutants for curli (csgBA), cellulose (bcsE, bcsA), enterobacterial common antigen (wecA), BapA protein (bapA), colanic acid (wza-wcaM), O-antigen capsule (yihQ) and csgD, a master regulator for biofilm formation. We characterized the colony biofilm morphology by Congo red-Coomassie blue staining and the biomass in pellicle biofilms by crystal violet assay. Next, we utilized a computational 4D approach that was developed in collaboration with the Quiesser and Buttaro Labs to identify the contribution of each matrix component into the material properties of the biofilm. Finally, by utilizing the above-mentioned assays, we explored the influence of different culture media on biofilm formation and architecture comparing Tryptic Soy broth (TSB) and No salt Luria Broth (LB No salt) media for biofilm growth. In our studies, we found that only curli-containing strains maintained the ability to form biofilms comparable to wildtype STm and irrespective of the media condition, indicating a key role for curli in the establishment of biofilms. However, when characterizing the physical properties of the biofilms utilizing the 4D assay, we saw a different phenotype between the two culture media conditions. We observed that when biofilms were formed in LB No salt culture condition, curli was the most significant contributor to the rigidity of biofilms such that, in the absence of curli, biofilms were more fluid-like and less rigid when compared to curli-containing strains. In TSB media, curli production was decreased although there was no loss of biofilm rigidity. Overall, our data suggests that different culture conditions could lead to different material properties of STm biofilm matrix, which in turn affects the physical properties such as the rigidity of the biofilms. Additionally, the biofilm matrix components may play redundant roles and another component could compensate for the loss of curli.