Streptococcus pneumoniae, or the pneumococcus, is a normal inhabitant of nasopharynx, but under certain conditions, it becomes in one of the main bacterial pathogens involved in human health. The pneumococcus is the causal agent of human infections such as sinusitis, otitis, bacteremia, pneumonia and meningitis, causing more than 1.5 million of deaths annually, mainly in children under 5-y-o. The pneumococcus expresses diverse virulence factors that allows achieving different steps during the bacterial infection, such as adhesion, colonization, invasion and tissue damage. Gene expression is regulated by environmental conditions and by the specific pathogenicity of each strain. These environmental changes are sensed by signal transduction systems that triggers an adaptive response mediating gene regulation.
At present, we are studying different signal transduction systems, particularly those named two-component systems (TCS), that the pneumococcus use to adapt its metabolism and virulence factors to environmental stress. Bacteria must to overcome these stress conditions in culture media or inside eukaryotic cells. In our lab, we use molecular biology methods to construct deletion, insertion or point mutants of genes that encode these TCS and allow analyzing the impact of these mutations on determined phenotypes, as well as identifying genes that are regulated by these TCS. In addition, we use methodology related to cellular biology to localize fluorescent proteins fused to certain proteins of our interest by fluorescent or confocal microscopy. We have also established an S. pneumoniae infection model in pneumocytes to elucidate the survival mechanism of this pathogen at intracellular level.
1) Gene regulation involved in intracellular life of S. pneumoniae in pneumocytes
Previously, we described that certain processes, known as acid tolerance response (survival at acidic pH) and autolysis (programmed cell death), are induced in response to acidic stress. The activation of these mechanisms is regulated by two TCS that present antagonistic roles. We also found that phenotypes of each TCS mutant showed similar phenotypes in both chemically defined media and intracellular survival in pneumocytes. We are working on the biochemical and molecular characterization of two TCS to identify genes involved in these adaptive mechanisms that allows S. pneumoniae to survive inside pneumocytes. (Fig. 1)
2) Characterization of a synergistic mechanism between S. pneumoniae and influenza A in a coinfection model in pneumocytes.
The human flu is an infectious disease caused by influenza A virus (IAV), one of the main viral pathogens in human health due to its pandemic potential, During the XX century, IAV caused more the 50 millions of deaths, and last pandemic outbreak caused more than 750000 deaths in 2009. IAV normally produces an infection in respiratory tract; however, approximately a 20% of the flu-infected patients develops secondary bacterial infections that complicates the flu symptoms and antimicrobial treatment. S. pneumoniae is the main bacterial pathogen that causes these type of secondary infections. Other research groups have studied particular aspects of this cooperative synergism. In our lab, we have established a co-infection model in pneumocytes, ad we demonstrated that, in flu-infected pneumocytes, S. pneumoniae was able to replicate intracellularly two-fold higher that in non-infected cells. At present, we are trying to identify which viral, bacterial or eukaryotic factors contributes to this synergistic mechanism that appear to be relevant to understand the IAV and S. pneumoniae pathogenesis (Fig. 2).
3) Identification of proteins involved in the cell division mechanism of S. pneumoniae
Previously, we described a compensatory evolution mechanism between pbp mutations. The pbp genes encodes penicillin-binding proteins that synthetize peptidoglycan, but point mutations in the catalytic domain confers penicillin resistance among clinical strains. We observed that pbp2b mutations produce alterations in cell shape and cell division in S. pneumoniae. To understand the PBP2b contribution to the cell division mechanism, we are analyzing the protein-protein interaction between PBP2b with other proteins that belong to the pneumococcal divisome, as well as the phenotype of specific mutants of divisome genes. To localize these proteins, we develop molecular tools to fuse these proteins to fluorescent proteins (GFP, CFP, mCherry, CitrineFP) to express them in S. pneumoniae under the control of inducible promoters. To understand the interaction between divisome proteins is important for the development of new antimicrobials that could block the cell division mechanism of the pneumococcus. (Fig. 3)
- Cortes PR, Chiapello LS, Dib D, Herrero MV, Nuncira CT, De Petris C and J Echenique (2016). Coinfection of Leishmania (Viannia) braziliensis and S. pneumoniae in multiple cutaneous lesions. PLoS Neglected Trop Dis. 10;10(3):e0004388.
- Paulo R. Cortes, Germán E. Piñas, Melina Cian, Nubia Yandar and José Echenique. Stress-Triggered Signaling Affecting Survival or Suicide of Streptococcus pneumoniae. International Journal of Medical Microbiology (2014).
- Chockalingam AK, Hickman D, Pena L, Ye J, Ferrero A, Echenique JR, Chen H, Sutton T, Perez DR (2012). Deletions in the neuraminidase stalk region of H2N2 and H9N2 avian influenza subtypes do not affect post-influenza secondary bacterial pneumonia. Journal of Virology. 86(7):3564-75.
- Andrea Albarracín Orio, Germán E. Piñas; Paulo R. Cortes and José Echenique (2011). Compensatory evolution of pbp mutations ameliorates fitness costs of β-lactam resistance in Streptococcus pneumoniae. PLoS Pathogens, 7(2): e1002000.
- Cortes Paulo R., Germán E. Piñas, Andrea Albarracín Orio, José Echenique. (2008). Subinhibitory concentrations of penicillin increase the mutation rate to optochin resistance in Streptococcus pneumoniae. Journal of Antimicrobial Chemotherapy, 62(5):973-7.
- Albarracín Orio Andrea, Paulo R. Cortes, Miguel Tregnaghi, Germán E. Piñas, José Echenique. (2008). Characterization of 14 serotype variants of the pneumococcal Spain9V-3 clone spread in Cordoba, Argentina. Journal of Medical Microbiology, 57 (8): 992-999.
- Cortes Paulo R., Andrea G. Albarracín Orio, Mabel Regueira, Germán E. Piñas and José Echenique. (2008). Characterization of in vitro-generated and clinical optochin-resistant strains of Streptococcus pneumoniae isolated from Argentina. Journal of Clinical Microbiology, 46 (6):1930-34.
- Piñas Germán E., Paulo R. Cortes, Andrea G. Albarracín Orio and José Echenique (2008). Acidic stress induces autolysis by a CSP-independent ComE pathway in Streptococcus pneumoniae. Microbiology (SGM), 154(5):1300-1308..
- Saka, HA, C Bidinost, C Sola, P Carranza, S Ortiz, JR Echenique and JL Bocco. (2008). Vibrio cholerae is essential for high enterotoxicity and apoptosis induction produced by a cholera toxin gene-negative V. cholerae non-O1, non-0139 strain. Microbial Pathogenesis, 44(2):118-28.
- Reviglio VE, Sambuelli RH, Olmedo A, Falco M, Echenique J, O'Brien TP, Kuo IC. (2007). Secretory leukocyte protease inhibitor is an inducible antimicrobial peptide expressed in Staphylococcus aureus endophthalmitis. Mediators Inflamm. 2007:93857.
- Ame, MV; Echenique, JR; Pflugmacher, S and Wunderlin, DA. (2006). Degradation Of Microcystin-RR by Sphingomonas sp. Isolated From San Roque Reservoir (Córdoba – Argentina). Biodegradation, 17: 447-455.
- Monferrán MV, Echenique JR and DA Wunderlin (2005). Degradation of Chlorobenzenes by a strain of Acidovorax avenae Isolated from a Polluted Aquifer. Chemosphere, 61(1):98-106.
- Novakova L, Saskova L, Pallova P, Janecek J, Novotna J, Ulrych A, Echenique J, Trombe MC, Branny P. (2005) Characterization of a eukaryotic type serine/threonine protein kinase and protein phosphatase of Streptococcus pneumoniae and identification of kinase substrates. FEBS J. (ex Eur J Biochem) 272(5):1243-1254.
- Echenique JR, Kadioglu A, Romao S, Andrew P and MC Trombe. (2004) The serine/threonine kinase, StkP, positively controls virulence and competence in Streptococcus pneumoniae. Infection and Immunity, 72: 2434-2437.
- Kadioglu A, Echenique JR, Manco S, Trombe MC and Andrew P (2003) The MicAB two-component system is involved in virulence of Streptococcus pneumoniae. Infection and Immunity, 71: 6676–6679.
- Echenique, JR., Caleb Dorsey, Luis C. Patrito, Alejandro Petroni, Marcelo E. Tolmasky, and Luis A. Actis (2001) Acinetobacter baumannii has two genes encoding glutathione-dependent formaldehyde dehydrogenase: evidence for differential regulation in response to iron. Microbiology (SGM). 147:2805-2815.
- Echenique JR and Marie-Claude Trombe (2001). Competence repression under oxygen limitation through the two-component MicAB signal-transducing System in Streptococcus pneumoniae and Involvement of the PAS Domain of MicB. Journal of Bacteriology;183(15) 4599-4608.
- Echenique JR. and Marie-C. Trombe (2001). Competence modulation by the NADH oxidase of S. pneumoniae involves signal transduction. Journal of Bacteriology 183(2):768-72.
- Echenique, JR.; S. Chapuy-Regaud and Trombe, MC (2000). Competence regulation by oxygen in Streptococcus pneumoniae: Involvement of ciaRH and comCDE. Molecular Microbiology 36(3):688-96.
- Juarez CP, Muino JC, Guglielmone H, Sambuelli R, Echenique JR, Hernandez M, Luna JD (2000). Experimental retinopathy of prematurity: angiostatic inhibition by nimodipine, ginkgo-biloba, and dipyridamole,and response to different growth factors. Eur J Ophthalmol 10:51-59..
- Echenique, JR., Arienti, H., Tolmasky, M., Read, R., Staneloni,R., Crosa, J. and Actis, L. (199) Characterization of a High Affinity Iron Transport System in Acinetobacter baumannii. Journal of Bacteriology, 170:7670-7679.