ABS 051: Glycerophosphodiesterase F's Association with Virulence in Pathogenic Mycoplasma pneumoniae

Patrick L. Pagano ¹ , Jeremy M. Miller ¹ , Arlind B. Mara ¹ , Edan R. Tulman ¹ , Steven M. Szczepanek ¹ , Steven J. Geary ¹

¹ University of Connecticut, Storrs, Connecticut

Van Wickle (2025) Volume 1, ABS051

Introduction: Mycoplasma pneumoniae (Mp) is a leading cause of atypical pneumonia, responsible for around 2 million infections and 100,000 hospitalizations in the U.S. annually. While its clinical impact is well recognized, the molecular mechanisms underlying Mp virulence remain poorly understood. Glycerol metabolism has been linked to pathogenicity in related Mycoplasma species, prompting investigation of GlpF—a gene encoding a glycerol transporter—in Mp pathogenesis.

Using transposon mutagenesis, we disrupted GlpF in Mp strain PI-1428, producing mutant PM276. Mice were infected intranasally with either PM276 or wild-type (WT) Mp, and bacterial burden and immune responses were evaluated. Despite similar infection titers, significantly fewer bacteria were recovered from the lungs and bronchoalveolar lavage fluid (BALF) of PM276-infected mice. Flow cytometry revealed notable reductions in both the absolute and relative numbers of neutrophils, alongside changes in the broader inflammatory cell landscape.

Dimensionality reduction via t-SNE further highlighted altered immune profiles in mutant-infected mice. Neutrophil frequencies were used to project lung histopathology, indicating substantially less tissue damage with PM276 infection. These findings suggest that GlpF is important for full Mp virulence, likely by facilitating glycerol uptake required for downstream hydrogen peroxide production via GlpO.
In conclusion, GlpF plays a central role in Mp pathogenicity and shapes the host inflammatory response. Future work targeting GlpO may clarify the metabolic pathway’s contribution to virulence.

Methods: We screened a transposon mutant library of Mycoplasma pneumoniae strain PI1428 using PCR to isolate mutants with insertions in the glpF gene. Whole genome sequencing confirmed transposon insertion sites and mutant isogeneity. To evaluate the role of glpF in virulence, we used an established murine infection model. Eight-week-old female BALB/c mice were infected intranasally, and lungs and bronchoalveolar lavage fluid (BALF) were collected four days post-infection. Airway inflammation was assessed through differential leukocyte counts, histopathological scoring, and bacterial burden analysis, allowing us to compare immune responses and pathogenicity between the glpF mutant and wild-type strains.

Results: Following extensive screening and whole genome sequencing, a single mutant—designated mutant 276—was identified with a transposon insertion near the 3′ end of glpF, likely disrupting the final 24 of 264 amino acids. We evaluated its virulence using a mouse infection model. Bronchoalveolar lavage fluid (BALF) analysis showed significantly higher leukocyte counts in wild-type (WT)–infected mice compared to those infected with the glpF mutant. This difference was primarily due to elevated neutrophil levels in WT infections, which are associated with more severe lung pathology and disease in Mycoplasma pneumoniae infection.

Discussion: Collectively, these data indicate that the glpF mutant strain of Mp is attenuated in its in vivo virulence in the mouse model of disease, implicating glpF in Mp pathogenesis. Further studies are required to assess the mechanisms by which glpF serves as a virulence determinant.