Characterization of the PhoP/PhoQ system in S. marcescens and its role in pathogenesis.

JULIETA BARCHIESI; MARÍA E. CASTELLI; ELEONORA GARCÍA VÉSCOVI

Villa Carlos Paz

Congreso; VI Congreso Argentino de Microbiología General (SAMIGE); 2009

The opportunistic human pathogen Serratia marcescens is a Gram-negative bacterium that causes disease in a wide range of both invertebrate and vertebrate hosts and in plants. S. marcescens is a significant cause of hospital-acquired infection, especially in patients with impaired immunity. As many S. marcescens strains are also resistant to multiple antibiotics, it represents a growing problem for public health. However, relatively little is known about the factors that contribute to S. marcescens pathogenesis within its host. In our laboratory, with the aim of finding regulatory mechanisms that would participate in S. marcescens pathogenesis, we undertook the characterization of PhoP/PhoQ two component system. This system controls transcription of key virulence genes in diverse bacterial pathogens. In order to study the physiological role of phoP in S. marcescens, we have constructed a phoP mutant strain. This mutant exhibited impaired growth in minimal broth limited in Mg2+, in acid pH, and showed increased sensitivity to antimicrobial peptides than the wild type strain. Furthermore, the phoP strain was attenuated in the survival inside epithelial cells. These results suggest that the phoP gene is required for S. marcescens invasion of epithelial cells. In addition, beta-galactosidase assays revealed that phoP transcription was modulated by the Mg2+ and Polimixin B levels, predicted to be environmental signals detected by the system. To further explore the PhoP regulon, we perform a random mutagenesis strategy selecting for Mg2+ transcriptional regulated clones. One of the novel PhoPactivated genes identified was mgtE. MgtE is a Mg2+ transport protein and it has never been reported before as a PhoP regulated gene. Conversely, MgtA and MgtB, members of another Mg2+ transport family, have been extensively characterized as PhoP regulon member in Salmonella. This result adds a new Mg2+ transporter as a PhoP target, reinforcing the importance of PhoP/PhoQ system in Mg2+ homeostasis control. On the other hand, we carried out an in silico search of PhoP binding sites in the S. marcescens genome by MEME/MAST programs. We identified several predicted gene members of the PhoP regulon, such as phoP, mgtA, mgtCB and pmrG. The other PhoP binding sites identified corresponded to genes previously uncharacterized as PhoP regulon members in others bacteria. Among them, genes implied in copper resistance (cueR), citrate metabolism (citB and citC), oxidative stress resistance and antibiotics resistance (ramA). To our knowledge, this represents the first study of the virulence PhoP/PhoQ system in S. marcescens.