S. aureus enhances replication of SARS-CoV-2 in vitro through the bacterial iron-regulated surface determinant protein A

By Bhavana KunkalikarJan 17 2023Reviewed by Danielle Ellis, B.Sc.

In a recent study published in iScience, researchers assessed the impact of Staphylococcus aureus (S. aureus) on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication.

Background

S. aureus has been identified as a common secondary bacterial coinfection agent for different respiratory viruses. Several investigations have revealed that coinfection with the influenza A virus (IAV) led to more severe dysregulation of the immune system, such as the destruction of alveolar macrophages.

Studies have also described the on-immune mechanisms at the molecular level as IAV infection increases the adherence of S. aureus to epithelial cells and elevates intracellular bacterial reproduction.

In contrast, the S. aureus protein lipase 1 promotes IAV replication within primary cells by positively controlling infectious particle release.

According to the closeness of clinical presentation and frequency of coinfections, the present study hypothesized that events during coinfection between IAV and S. aureus and CoV-2 and S. aureus would be comparable.

About the study

In the present study, researchers analyzed the relationship between S. aureus and CoV-2 and found that S. aureus increases the in vitro CoV-2 replication via the iron-regulated surface determinant protein A (IsdA).

IAV infection is evidenced to increase the adherence and multiplication of S. aureus within epithelial cells. Therefore, the team aimed to examine whether CoV-2-infected cells impact S. aureus adhesion. This was achieved by constructing an in vitro coinfection model that evaluated the replication kinetics of the two viruses.

The team utilized the CoV-2 Wuhan isolate, the methicillin-resistant Staphylococcus aureus (MRSA) strain USA300 LAC, along with a bacterial mutant devoid of fibronectin-binding proteins (FnbAB), which are necessary for S. aureus invasion into epithelial cells. Bacteria numbers were estimated six, eight, and 20 hours after an invasion.

The team investigated whether S. aureus cells alone were sufficient to promote CoV-2 proliferation or if the bacteria must be alive. This was determined by assessing viral replication in the presence of wild-type (WT) S. aureus or an equivalent amount of heat-killed bacteria.

Furthermore, virus-infected cells were co-cultured with bacterial mutants that did not have one or more of the main S. aureus proteins typically observed in the bacterial supernatant. Using reverse transcription–polymerase chain reaction (RT-PCR), the team analyzed transcription 12 hours after viral infection in the absence and presence of bacteria.

Results

The study results showed that S. aureus attached effectively to Vero E6 cells, although a previous CoV-2 infection had an insignificant effect on bacterial adherence. The invasion of Vero E6 cells by S. aureus relied on FnbAB presence. However, there were no changes in invasion rates between uninfected cells and those infected with CoV-2.

The rate of bacterial replication and the amount of bacterial growth did not change between uninfected and CoV-2-infected cells. At both 12 hours and 24 hours post-infection, when the CoV-2 titer was assessed, S. aureus enhanced the number of infectious virus particles. The data showed that infection with CoV-2 had no discernible effect on the interaction between S. aureus and epithelial cells, yet, S. aureus promoted CoV-2 replication within Vero E6 epithelial cells.

In contrast to the presence of wild-type bacteria, which increased viral proliferation by a factor of 10, exposure to heat-killed bacteria had no effect. Simultaneously, the team noted that the WT bacteria numbers grew by more than 2-log in 24 hours.

Moreover, when various S. aureus strains, such as the avirulent laboratory strain RN4220, were evaluated, the team observed comparable pro-viral activity levels and bacterial multiplication, indicating that this impact was not exclusive to USA300. Based on these results, the researchers hypothesized that the pro-viral activity was caused by either an elevation in the bacteria number over 24 hours or a component created during bacterial development.

The team noted that except for the sortase A mutant (srtA), all bacterial mutants maintained pro-viral activity. Sortase A, an endopeptidase, covalently binds "cell-wall anchored" (CWA) proteins and bacterial cell peptidoglycans together, resulting in their presentation on the cell surface. The proviral activity of specific mutants of the 18 proteins expressed by the strain USA300 showed that only three of these mutants, namely isdA, isdB, and sdrC, preserved pro-viral activity.

To investigate the function of these three genes, each mutant was supplemented with full-length genes on a plasmid and retested for viral activity. This revealed that only the addition of isdA revived the phenotype, despite all the strains growing at the same rate. Additionally, the team discovered that coinfection led to a slight elevation in Janus kinase 1 (JAK1) transcripts when exposed to the WT S. aureus but that JAK2, JAK3, and tyrosine kinase 2 (TYK2) transcripts were unaffected.

Conclusion

Overall, the study findings provided evidence of a novel connection of CoV-2 with co-infecting bacteria, as well as direct molecular interactions between a CoV and bacteria. The discovery of IsdA as a pro-viral factor demonstrated the need for extensive research to understand the interactions occurring between particular bacterial proteins and respiratory viruses.