Streptococcus pneumoniae (Spn) is a common bacterial co-infection in patients with respiratory viral infections, including influenza and SARS-CoV-2, and is associated with increased morbidity and mortality. However, the mechanisms underlying the severe disease caused by SARS-CoV-2 and Spn co-infection remain poorly understood. Here, we show that Spn infection 3 days after infection with mouse-adapted SARS-CoV-2 MA10 markedly increased morbidity and mortality in wild-type mice. In contrast, SARS-CoV-2 infection 3 days after Spn infection did not increase mortality, demonstrating that disease severity depends on the sequence of infection. Increased susceptibility to Spn following SARS-CoV-2 infection was associated with enhanced viral burden, exacerbated lung pathology, pronounced neutrophil accumulation, and increased expression of IFN-β and the neutrophil-recruiting chemokine CXCL2. To determine whether pneumolysin (PLY), a major Spn virulence factor, contributes to superinfection-induced pathology, we infected mice with a PLY-deficient Spn strain. Unexpectedly, PLY deficiency further increased mortality and lung pathology without altering bacterial or viral titers. Immunofluorescence analysis revealed increased accumulation of neutrophils and macrophages in the lungs of mice co-infected with PLY-deficient Spn. These results indicate that Spn superinfection exacerbates SARS-CoV-2-induced lung disease, whereas PLY unexpectedly attenuates disease severity. We propose that PLY limits immunopathology during SARS-CoV-2–Spn superinfection by restraining macrophage- and neutrophil-mediated inflammation.

Mycoplasma pneumoniae is a primary cause of bacterial community-acquired pneumonia. One of its key virulence factors, the community-acquired respiratory distress syndrome (CARDS) toxin, replicates most clinical symptoms of the infection. Although CARDS toxin exhibits multi-receptor binding affinity, the mechanisms governing its cellular entry remain poorly understood. In this study, we characterized the toxin entry pathway mediated by the host protein annexin A2 (AnxA2). Using flow cytometry and biotinylated membrane protein analysis, we demonstrate that CARDS toxin induces a rapid, transient translocation of AnxA2 to the plasma membrane, peaking at 15 min post-exposure. This recruitment occurs via a phosphorylation-independent mechanism, distinguishing it from the Src-dependent recruitment typically observed in other physiological contexts. Co-immunoprecipitation (co-IP) of the clathrin heavy chain revealed an enhanced AnxA2-clathrin association that accelerates clathrin-mediated endocytosis of toxin. Endosomal fractionation, immunofluorescence, and co-IP studies confirmed the time-dependent co-localization and physical interaction of CARDS toxin with AnxA2 within early endosomes. Furthermore, CRISPR/Cas9-mediated knockout of AnxA2 in A549 cells demonstrated that this protein is essential for the internalization of CARDS toxin; its absence not only reduces the toxin binding but also effectively impairs the rapid cellular uptake required to trigger the downstream cytopathic effects and vacuolation. These results establish that CARDS toxin modulates host surface remodeling through AnxA2 cell surface translocation to facilitate efficient cellular entry, identifying a rate-limiting step in M. pneumoniae pathogenesis.