A Study Of SUMOylation During Mycoplasma Pneumoniae Infection

Objective:Mycoplasmas are the smallest microorganism which does not have a rigid cell wall, but it’s very resistant to a great number of antibiotics, causing kinds of diseases. One of the asthma-associated mycoplasma is Mycoplasma pneumoniae. Cytadherence of M. pneumoniae on the respiratory tract mucosa lead to a variety of effects, inducing the local inflammation and stimulate the host immune system to clearance the pathogen by activation MyD88-NF-κB pathway in macrophages.SUMOylation is one of the most important protein postranslational modificaion, which is an reverse process, including activation, conjugation, ligation and desumoylation. The E1-activating enzyme (Aos1/Uba2, or SAE1/SAE2) form a thioester bond between C173and C terminal of mature SUMO, the E2-conjugating enzyme (UBC9), form E2-SUMO mediated body, and enzyme-E3ligase is often involved in this process. The member of SENP(SUMO/sentrin-specific protease) ensure reversibility of the SUMOyaliton. SUMOylation can be regulated by many factors, including hydrogen peroxide, nitric oxide, and play an important role during pathogen infections.NF-κB is widely used by eukaryotic cells as a regulator of genes that control cell proliferation and cell survival. The transcription factor NF-κB signaling pathway plays an important role in immune and inflammatory responses, cellular growth, and apoptosis. Activation of NF-κB is achieved by ubiquitination and proteasome-mediated degradation of κB, which allows the NF-κB to tanslocate into nucleus and promote transcription of many inflammatory cytokines for clearance the pathogens. But the SUMOylation of iκB acts antagonistically to generate proteins to resistant to degradation, and inhibition NF-κB pathway.Our objective of this study is to reveal the function of SUMOylation during the M. pneumoniae infection and provide new direction and new ideas for the study of pathogenesis.MethodsA. Real time PCR was used for detection of the human monocytic THP-1cells clear M. pneumoniae. B. Cell counts and LDH quantification were to observe the effect of Mp on the growth of THP-1in vitro.C. Flow cytometric analysis were used for the detection of THP-1cell apoptosis.D. Western blot techniques was to investigate SUMO expression.E. Using ELIS A to detective the expression of cytokine TNF-α.ResultsA. The concentration of M. pneumoniae DNA decreased until24hr but appeared to increase at subsequent time points. This phenomenon indicated that THP-1cells are able to clear M. pneumoniae early in infection.B. Cell counting results showed that, with the increase of Mp concentration, the more cells decreased, and the difference was statistically significant.C. The LDH (lactate dehydrogenase) results also show that THP-1cell membranes are damaged after M. pneumoniae infection (p＜0.05).D. Flow cytometric analysis showed that apoptosis of THP-1cells was significantly activated after M. pneumoniae infection. The ratio of apoptotic to non-apoptotic cells increased between24hr and48hr infection time points.E. The states of conjugative SUMO1dramatically change in THP-1cells after0hr,8hr,24hr and48hr M. pneumoniae infections. As the infection time increased, the quantity of conjugative SUMO1increased as well, suggesting that SUMOylation plays an important role during M. pneumoniae infection.ConclusionsIn this study, we proved that M. pneumoniae can both promote apoptosis and decrease activation by increasing iκB SUMOylation to evade clearance by THP-1cells. In this way, M. pneumoniae can successfully evade the body’s immune system.