THE ROLE OF THE HTRA PROTEASE IN THE PNEUMOCOCCAL SURVIVAL MECHANISM IN HOST CELLS

ZAPPIA, VICTORIA E.; CORTES, PAULO; HERNANDEZ MORFA, MIRELYS; NADIA B. OLIVERO; JAIME, ANDREA; ECHENIQUE J

Mendoza

Congreso; Reuniòn Anual de la Sociedad Argentina de nvestigacion en Bioquimica y Biol Molecular; 2022

SAIB

Streptococcus pneumoniae is an important human pathogen and is the causal agent of a variety of diseases, such as otitis media, sinusitis, pneumonia, and meningitis, especially among children. While causing infection, the pneumococcus encounters oxygen and its reactive derivatives at different concentrations depending on the stage of the infection process. Understanding how the bacterium copes with reactive oxygen species in the host could facilitate the development of anti-infective agents against S. pneumoniae.HtrA (high-temperature requirement A) is a heat shock-induced serine protease that has both chaperone and proteolytic activities. It has been reported that HtrA could also have a role in the oxidative stress response of S. pneumoniae. Strains lacking the gene that encodes for HtrA showed decreased sensibility to exogenous hydrogen peroxide exposure, and that this phenotype could be restored with antioxidants. To further investigate the role of HtrA in the oxidative stress response, we tested its capacity to survive the oxidative stress found during intracellular survival in host cells. We created the ∆htrA mutant using deletion mutagenesis, and we confirmed that the mutant ∆htrA was more sensitive to exogenous H2O2 than the wt strain, as reported, being HtrA involved in the oxidative stress response of S. pneumoniae.Moreover, it has been described that CiaRH, a pneumococcal two-component system, is the main regulator of HtrA. To determine the effect of oxidative stress in the induction of htrA, qPCR was used to quantify the relative expression level of this gene in both the wt and ∆ciaR strains. When exposed to 20mM H2O2, htrA transcripts showed a 3-fold increment in the wt strain. Although the ciaR mutation negatively impacted the expression of htrA compared to the wt strain, it still retained the capacity to increase its expression in response to H2O2, suggesting additional mechanisms involved. On another hand, intracellular survival of pneumococcal mutants was measured in A549 pneumocytes and RAW 264.7 macrophages. We found significantly decreased survival of the ∆htrA and ΔciaR mutants in both cell types, being even more predominant in RAW 264.7 cells, probably because ROS levels are much higher in macrophages than in pneumocytes. When A549 and RAW 264.7 cells were previously treated with NAC (a known ROS inhibitor), the survival of ∆htrA was similar to the wt strain, suggesting that this protein plays a key role in the defense of the oxidative stress response in S. pneumoniae.We propose that HtrA, which is part of the oxidative stress response in S. pneumoniae, has a relevant role in the pneumococcal survival mechanism in host cells. Thus, we propose that it could be a potentially good candidate for the development of novel therapeutics in the fight against pneumococcal disease.