The following article has been adapted from a poster by Cayman Chemical presented at ASM Microbe 2025. Download the original poster.

Introduction

Urinary tract infections (UTIs) represent a significant healthcare burden, affecting approximately 150 million people annually. Proteus mirabilis is the causative agent of up to 44% of catheter-associated UTIs, with an estimated 48% of strains exhibiting antibiotic resistance. P. mirabilis utilizes an arsenal of pathogenic strategies including swarming motility, urease production, and fimbriae and biofilm formation.

Biofilms are particularly relevant in clinical applications as they enhance antibiotic resistance and diminish host defense mechanisms, leading to recurrent infections. Cyclic dinucleotides (CDNs) play a crucial role in biofilm formation by regulating the transition between motile and sessile lifestyles. They modulate the synthesis and secretion of adhesins, polysaccharides, and DNA, all of which contribute to the structural integrity and mechanical resilience of the biofilm's extracellular matrix.

Understanding how CDNs are regulated could reveal novel strategies for disrupting biofilm formation and mitigating persistent infections caused by P. mirabilis and other bacteria.

Biofilm Formation

In this study, we explored the regulatory network underlying biofilm formation in P. mirabilis HI4320. Utilizing BV-BRC and BioCyc databases, we identified 14 genes with sequence similarity to enzymes implicated in dinucleotide synthesis or its regulation. P. mirabilis strains with transposon mutations corresponding to these genes were assayed for their ability to form biofilms under various conditions using a peg lid-based biofilm formation and staining assay (Figures 1 & 2). Briefly, cultures were incubated statically in a 96-well plate at 37°C for 24 hours, allowing biofilms to form on pegs attached to the lid and immersed in the medium. Following incubation, the relative levels of biofilm biomass on the pegs were determined using crystal violet staining (Figure 1).

Figure 1. Peg lid-based biofilm staining workflow. This workflow has been adapted to create Cayman's new Biofilm Formation and Staining Kit (Item No. 502869). Learn more about the kit in our New Product Spotlight.

Several mutants exhibited notable deficiencies in biofilm formation across the tested conditions. Specifically, transposon mutations in bcsA, crp, bcsA2, bcsB2, PMI3101, PMI3179, and PMI3508 exhibited reduced biofilm formation in one or more media types relative to the wild-type (WT) strain. In contrast, mutations in moaC, PMI1203, and spoT led to an increase in biofilm formation in LB medium compared to WT (Figure 2).

Figure 2. A peg lid-based assay identified biofilm abnormalities in P. mirabilis transposon mutants. Overnight cultures of transposon mutants were normalized and inoculated into a 96-well plate at a final OD₆₀₀ of 0.05 in (A) Tryptic Soy Broth (TSB), (B) LB, and (C) LB without salt. Plates were covered with a 96-peg lid and incubated statically at 37°C for 24 hours. Biofilm formation was quantified using the staining protocol described previously and normalized to WT. (D) A heat map showing the summary of results. *= p <0.05; N=3

Quantification of Cyclic Dinucleotides via Competitive ELISA

We next sought to examine the interconnections between biofilm phenotypes and their regulation by CDNs. Using Cayman's competitive ELISA kits, production of the CDNs cyclic di-GMP, cyclic di-AMP, 3′2′-cGAMP, and 3′3′-cGAMP by P. mirabilis was quantified over a period of 72 hours under biofilm-forming conditions in either TSB or LB medium (Figure 3). Cyclic di-GMP production peaked in TSB at 72 hours whereas other CDNs peaked significantly earlier.

Figure 3. Kinetics of CDN production in P. mirabilis. (A) Competitive ELISA schematic. (B-E) Overnight P. mirabilis cultures were normalized to an OD of 1.0, inoculated into the assay plate at a final OD of 0.05, and incubated statically at 37°C for 72 hours. At the indicated timepoints, culture media were collected and CDN levels were quantified by competitive ELISA: (B) Cyclic di-AMP ELISA Kit (Item No. 501960); (C) Cyclic di-GMP ELISA Kit (Item No. 501780); (D) 3'2'-cGAMP ELISA Kit (Item No. 502340); and (E) 3'3'-cGAMP ELISA Kit (Item No. 502130). N=2

Transposon mutants PMI3508, bcsA2, PMI3101, cpdB, and moaC exhibited an increase in CDN production, while mutations in PMI2841 and bcsA showed reduced CDN production compared to WT (Figure 4). Interestingly, the PMI3101 mutant displayed reduced biofilm biomass despite elevated cyclic di-GMP levels in LB medium, suggesting that additional factors beyond CDN concentration influence biofilm formation in this mutant. Taken together with the biofilm formation results, these findings indicate insertion mutants in moaC and bcsA generally conform to the paradigm in which biofilm biomass is positively correlated with CDN levels. Exceptions such as PMI3101 highlight the complexity of biofilm regulation in P. mirabilis.

Figure 4. CDN production significantly differed from WT in seven transposon mutant strains. (A-D) Overnight cultures were normalized to OD 1.0, inoculated into the assay plate at final OD 0.05, and incubated statically at 37°C for 72 hours. *= p <0.05; N=2

Swarming Motility

The transition between motile and sessile phenotypes is governed by interconnected signaling pathways and the control of these pathways is a critical aspect of pathogenesis, particularly in the urinary tract. To assess swarming motility, overnight P. mirabilis cultures were normalized to an OD₆₀₀ of 1.0 in PBS, and 5 µl was spotted onto a swarm agar plate (per liter: 10 g tryptone, 10 g NaCl, 5 g yeast extract, 15 g agar). Once dry, the plates were incubated for 18 hours at 30°C, after which swarm ring diameters were measured (Figure 5). Mutations in crp and cyaA resulted in decreased swarming motility relative to WT, whereas mutations in cpdB, moaC, and spoT led to increased motility. Taken together with the effects on biofilm formation presented in Figure 2, the phenotypes observed for crp, moaC, and spoT mutants suggest a regulatory network in which multiple pathways modulate motility and biofilm formation.

Figure 5. Mutation of crp and cyaA inhibited swarming motility. (A) Swarm diameters of P. mirabilis transposon mutants. (B) Representative images. *= p <0.05; N=3

HEK293T/17 Cell Adherence

As bacteria ascend the urinary tract from the urethra to the bladder, kidneys, and ultimately the bloodstream, they encounter niches where adherence is more advantageous than motility. To determine if mutations affect the ability of P. mirabilis to adhere to human kidney cells, overnight cultures were first normalized to an OD₆₀₀ of 1.0 in PBS and seeded at a multiplicity of infection (MOI) of 200 onto confluent HEK293T/17 cells. Mammalian and bacterial cells were incubated together in DMEM for 2 hours at 37°C with 5% CO₂. Following incubation, mammalian cells were gently washed with PBS, lysed with 0.5% Triton X-100, and bacterial colony forming units (CFU) were determined (Figure 6). CFU values were normalized to total protein levels and displayed relative to WT. Mutations in crp, bcsA2, and cyaA led to an increase in adherence of P. mirabilis to human kidney cells. The swarming motility defects in crp and cyaA mutants are consistent with these adherence phenotypes.

Figure 6. Adherence of P. mirabilis to human kidney cells. Confluent HEK293T/17 cells were infected with P. mirabilis cultures normalized in PBS to an MOI of 200. Following 2 hours of incubation at 37°C with 5% CO₂, cells were lysed with Triton X-100 and plated to determine CFU. Total protein in each lysate was determined by using a BCA assay. CFU counts were normalized to total protein and shown relative to WT. *= p <0.05; N=3

Conclusions

Biofilm formation is a key virulence factor of P. mirabilis during urinary tract infection, particularly in catheterized patients. This study provides insights into the regulation of biofilm production and highlights the complex interplay between sessile lifestyles and motile states, which share overlapping signaling pathways.

• Mutation of moaC, which encodes a putative phosphodiesterase, followed the canonical relationship where increased biofilm production was associated with elevated CDN concentration, however the observed increase in swarming motility was unexpected.
• Other genes reveal more intricate uncharacterized regulatory networks; for example, transposon mutations in the highly conserved transcriptional regulator crp and adenylate cyclase cyaA both exhibited defects in swarming motility and enhanced cellular attachment, however, crp was biofilm deficient and cyaA was not.
• Notably, insertions in three members of the bcs operon (bcsA2, bcsB2, and PMI3101), annotated as important for cellulose biosynthesis, displayed defects in biofilm formation in multiple culture media types despite increased cyclic di-GMP levels in bcsA2 and PMI3101.

Collectively, these genes represent promising targets for further mechanistic studies towards the development of anti-biofilm therapeutics.

Acknowledgements

We thank the Anderson and Pearson labs of the University of Michigan for the gift of P. mirabilis transposon mutant strains.

Cayman Products Used in this Work

• Cyclic di-AMP ELISA Kit
  

• Cyclic di-GMP ELISA Kit

• 3'2'-cGAMP ELISA Kit

• 3'3'-cGAMP ELISA Kit
  

New Product

Biofilm Formation and Staining Kit

Based on the biofilm quantification assay presented in this work, Cayman has developed the new Biofilm Formation and Staining Kit. This kit provides a straightforward, semi-quantitative method for evaluating bacterial biofilm formation. It allows for rapid screening of mutations and/or compounds that modulate biofilm formation with less time and variability than traditional staining methods.

Read our New Product Spotlight to learn more about this kit.

You May Also Be Interested In

References

1. Pearson, M.M., Sebaihia, M., Churcher, C., et al. Complete genome sequence of uropathogenic Proteus mirabilis, a master of both adherence and motility. J. Bacteriol. 190(11), 4027-4037 (2008).

2. Pearson, M.M., Pahil, S., Forsyth, V.S., et al. Construction of an ordered transposon library for uropathogenic Proteus mirabilis HI4320. Microbiol. Spectr. 10(6), e0314222 (2022).    

3. Olson, R.D., Assaf, R., Brettin, T., et al. Introducing the bacterial and viral bioinformatics resource center (BV-BCR): A resource combining PATRIC, IRD and ViPR. Nucleic Acids Res. 51(D1), D678-D689 (2022).

4. Karp, P.D., Billington, R., Caspi, R., et al. The BioCyc collection of microbial genomes and metabolic pathways. Brief. Bioinform. 20(4),1085-1093 (2019).

5. Pearson, M.M. Culture methods for Proteus mirabilis. 2021, 5-13 (2019).