EepR Mediates Secreted-Protein Production, Desiccation Survival, and Proliferation in a Corneal Infection Model

Abstract

Serratia marcescens is a soil-and water-derived bacterium that secretes several host-directed factors and causes hospital infections and community-acquired ocular infections. The putative two-component regulatory system composed of EepR and EepS regulates hemolysis and swarming motility through transcriptional control of the swrW gene and pigment production through control of the pigA-pigN operon. Here, we identify and characterize a role for EepR in regulation of exoenzyme production, stress survival, cytotoxicity to human epithelial cells, and virulence. Genetic analysis supports the model that EepR is in a common pathway with the widely conserved cyclic-AMP receptor protein that regulates protease production. Together, these data introduce a novel regulator of host-pathogen interactions and secreted-protein production. Secreted enzymes such as proteases can act as immune system and physiology modulators and virulence factors during infection by a wide variety of pathogens (1-5). Serratia marcescens is a Gram-negative coccobacillus bacterium isolated from a variety of environmental niches and the human gut. It is an important opportunistic pathogen in hospitals (6-9), and isolates collected in intensive care units became more resistant to antibiotics between 2004 and 2009 (10). S. marcescens is also a leading contaminant of contact lens cases and contact lenses and can cause vision-threatening microbial keratitis and other ocular infections (11-13). S. marcescens synthesizes several secreted enzymes, including proteases, a potent nuclease, chitinases, and other enzymes that have been used for biotechnological applications or have been implicated in cytotoxicity to mammalian cells and pathogenesis (14-16). However, the regulation of these enzymes is poorly understood.Known regulators of S. marcescens proteolysis include the crp gene that encodes the cyclic-AMP (cAMP) receptor protein CRP. Mutation of crp yields bacteria with elevated production of the protease serralysin (PrtS; also known as PrtA) and a loss of secreted-chitinase and -lipase activities (17). Matsuyama and colleagues observed reduced expression of extracellular protease activity when the hexS gene, an lhrA homolog, was expressed from a multicopy plasmid, suggesting a negative regulatory role for this protein (63).Extracellular proteases, such as the S. marcescens PrtS metalloprotease, are considered important virulence factors. In vitro studies indicate that serralysin is cytotoxic to a variety of cell types (16,21,22). PrtS contributed to tissue damage in corneal infections using a rabbit model (23), altered immune cell interactions using a silkworm model (20,24), and enhanced secondary infections by a respiratory virus in mice (25). Those studies provided the impetus to determine regulators of S. marcescens protease production.A putative regulatory system that includes the response regulator-like protein EepR and a putative hybrid-histidine kinase, EepS, was recently identified for its role in controlling expression of swarming motility, pigme...

SlpE is a calcium-dependent cytotoxic metalloprotease associated with clinical isolates of Serratia marcescens

“…The transcription factor EepR was recently shown to regulate the prtS and slpB proteases in a positive manner [7]. We tested whether slpE expression was similarly diminished in a Δ eepR strain isogenic to K904 using quantitative RT-PCR.…”

“…This suggests that SM6 has one cytotoxic protease compared to K904, which has PrtS, SlpB and SlpE, or that the mutation was in a positive regulator of multiple proteases, as the SM6 protease mutation was never mapped and could have been in a transcriptional regulator. Such regulators may exist, as the EepR gene that positively regulates prtS and slpB expression [7] was postulated to regulate slpE expression. Consistent with this prediction, we observed that slpE expression was highly reduced in an eepR mutant strain.…”

Repurposing anti-diabetic drug “Sitagliptin” as a novel virulence attenuating agent in Serratia marcescens

“…The tripyrrole red QS controlled pigment, prodigiosin, is synthesized by Serratia through expression of the prodigiosin biosynthetic operon, pigA-N [46,47]. It was found that sitagliptin significantly reduced the expression of pigB gene.…”

Biologically active pigment and ShlA cytolysin of Serratia marcescens induce autophagy in a human ocular surface cell line

“…Mutations in two different genes that confer major pleiotropic effects on S. marcescens behavior, eepR and gumB, prevented bacterial activation of autophagy. The eepR gene is a transcription factor that is required for wild-type levels bacterial proliferation in a rabbit keratitis model as well as positive regulation of secondary metabolites such as prodigiosin and serratamolide [24,25]. The gumB gene codes for a stress response signal transmitting protein that positively regulates prodigiosin and serratamolide, and is necessary for production of the ShlA, ShlB, and flagellin [20,28].…”

“…Our previous study demonstrated that secretomes could be heated at 95°C and still induce autophagy [18]. Well-defined S. marcescens strains with deletion mutations in the eepR and gumB genes were tested for loss of autophagy induction because these genes have a conserved and broad impact on S. marcescens biology [20,24,25,28,36]. The EepR and GumB genes regulate expression of multiple secreted factors including heat-stable secondary metabolites such as the biologically active pigment prodigiosin and the hemolytic and antimicrobial biosurfactant serratamolide.…”