Serratia marcescens has long been recognized as an important opportunistic pathogen, but the underlying pathogenesis mechanism is not completely clear. Here, we report a key pathogenesis pathway in S. marcescens comprising the RssAB two-component system and its downstream elements, FlhDC and the dominant virulence factor hemolysin ShlBA. Expression of shlBA is under the positive control of FlhDC, which is repressed by RssAB signaling. At 37°C, functional RssAB inhibits swarming, represses hemolysin production, and promotes S. marcescens biofilm formation. In comparison, when rssBA is deleted, S. marcescens displays aberrant multicellularity favoring motile swarming with unbridled hemolysin production. Cellular and animal infection models further demonstrate that loss of rssBA transforms this opportunistic pathogen into hypervirulent phenotypes, leading to extensive inflammatory responses coupled with destructive and systemic infection. Hemolysin production is essential in this context. Collectively, a major virulence regulatory pathway is identified in S. marcescens.The Gram-negative bacterium Serratia marcescens is an important opportunistic pathogen that causes a wide range of diseases and clinical presentations with high morbidity (25). S. marcescens frequently causes outbreaks in intensive and neonatal care units, and the occurrence of multiple-antibioticresistant strains has further exacerbated clinical treatment difficulties (3, 39). Despite years of study, the mechanism of pathogenesis of S. marcescens and why it behaves as an opportunistic pathogen remain poorly understood. Unraveling the underlying mechanism of pathogenesis is thus very important for developing strategies to prevent and treat S. marcescens infection.The hemolysin ShlA was shown to be a dominant virulence factor in S. marcescens pathogenesis using a murine lung infection model (35). ShlA is responsible for the hemolytic and cytotoxic effects on erythrocytes and cultured cells, with the aid of an outer membrane protein, ShlB (28,29,47,53,54). ShlA also contributes to the release of inflammatory mediators, increases uropathogenicity, and triggers microtubule-dependent invasion of S. marcescens into epithelial cells (27,30,34,40). However, the mechanism by which the expression of shlA is regulated, especially in response to any bacterial signaling system control, remains uncharacterized. Only one reported study has indicated that iron is involved in the regulation of shlBA expression in S. marcescens (46). S. marcescens exhibits swarming, which is recognized as a highly coordinated multicellular surface migration behavior (24,51,62) that is correlated with virulence capability, antibiotic resistance, and hemolysin production in other bacteria (1,17,44). S. marcescens swarms on Luria-Bertani (LB) agar surfaces at 30°C, but not at 37°C (36). Our previous studies showed that activation of a bacterial two-component system, RssAB, comprising a sensor kinase, RssA, and a response regulator, RssB, inhibited swarming and reduced hemolysin productio...

The protein pirin, which is involved in a variety of biological processes, is conserved from prokaryotic microorganisms, fungi, and plants to mammals. It acts as a transcriptional cofactor or an apoptosis-related protein in mammals and is involved in seed germination and seedling development in plants. In prokaryotes, while pirin is stress induced in cyanobacteria and may act as a quercetinase in Escherichia coli, the functions of pirin orthologs remain mostly uncharacterized. We show that the Serratia marcescens pirin (pirin Sm ) gene encodes an ortholog of pirin protein. Protein pull-down and bacterial two-hybrid assays followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrospray ionization-tandem mass spectrometry analyses showed the pyruvate dehydrogenase (PDH) E1 subunit as a component interacting with the pirin Sm gene. Functional analyses showed that both PDH E1 subunit activity and PDH enzyme complex activity are inhibited by the pirin Sm gene in S. marcescens CH-1. The S. marcescens CH-1 pirin Sm gene was subsequently mutated by insertion-deletion homologous recombination. Accordingly, the PDH E1 and PDH enzyme complex activities and cellular ATP concentration increased up to 250%, 140%, and 220%, respectively, in the S. marcescens CH-1 pirin Sm mutant. Concomitantly, the cellular NADH/NAD ؉ ratio increased in the pirin Sm mutant, indicating increased tricarboxylic acid (TCA) cycle activity. Our results show that the pirin Sm gene plays a regulatory role in the process of pyruvate catabolism to acetyl coenzyme A through interaction with the PDH E1 subunit and inhibiting PDH enzyme complex activity in S. marcescens CH-1, and they suggest that pirin Sm is an important protein involved in determining the direction of pyruvate metabolism towards either the TCA cycle or the fermentation pathways.The protein pirin is widely found in mammals, plants, fungi, and also prokaryotic organisms (32). While the cellular functions of pirin show diversity and pirin homologs play important roles in a number of different biological processes, cellular localization of pirin is not restricted to specific compartments. In eukaryotes, pirin was initially isolated through a yeast twohybrid screen from the HeLa cell cDNA library and is localized within cell nuclei; it acts as an interactor with nuclear factor

Quorum-sensing systems that have been widely identified in bacteria play important roles in the regulation of bacterial multicellular behavior by which bacteria sense population density to control various biological functions, including virulence. One characteristic of the luxIR quorum-sensing genes is their diverse and discontinuous distribution among proteobacteria. Here we report that the spnIR quorum-sensing system identified in the enterobacterium Serratia marcescens strain SS-1 is carried in a transposon, TnTIR, which has common characteristics of Tn3 family transposons and is mobile between chromosomes and plasmids of different enterobacterial hosts. SpnIR functions in the new host and was shown to negatively regulate the TnTIR transposition frequency. This finding may help reveal the horizontal transfer and evolutionary mechanism of quorum-sensing genes and alter the way that we perceive regulation of bacterial multicellular behavior.

Our previous study had identified a pair of potential two-component signal transduction proteins, RssARssB, involved in the regulation of Serratia marcescens swarming. When mutated, both rssA and rssB mutants showed precocious swarming phenotypes on LB swarming agar, whereby swarming not only occurred at 37°C but also initiated on a surface of higher agar concentration and more rapidly than did the parent strain at 30°C. In this study, we further show that the predicted sensor kinase RssA and the response regulator RssB bear characteristics of components of the phosphorelay signaling system. In vitro phosphorylation and site-directed mutagenesis assays showed that phosphorylated RssA transfers the phosphate group to RssB and that histidine 248 and aspartate 51 are essential amino acid residues involved in the phosphotransfer reactions in RssA and RssB, respectively. Accordingly, while wild-type rssA could, the mutated rssA(H248A) in trans could not complement the precocious swarming phenotype of the rssA mutant. Although RssA-RssB regulates expressions of shlA and ygfF of S. marcescens (ygfF Sm ), in vitro DNA-binding assays showed that the phosphorylated RssB did not bind directly to the promoter regions of these two genes but bound to its own rssB promoter. Subsequent assays located the RssB binding site within a 63-bp rssB promoter DNA region and confirmed a direct negative autoregulation of the RssA-RssB signaling pathway. These results suggest that when activated, RssA-RssB acts as a negative regulator for controlling the initiation of S. marcescens swarming.To unravel the underlying regulatory mechanism of Serratia marcescens swarming, we had utilized a mini-Tn5 mutagenesis approach to discover a group of S. marcescens mutant strains that demonstrated precocious swarming at both 30 and 37°C (16). A pair of potential bacterial two-component signal transduction proteins (11, 27), RssA-RssB, had been identified as involved in the regulation of Serratia swarming. Further studies suggested that either saturated fatty acids or temperature shift sensed by RssA and RssB influence swarming behavior through changing the cellular fatty acid profile and altering the ratio of saturated fatty acids to unsaturated fatty acids (16). The negative regulatory effect of certain fatty acids on bacterial swarming was also observed in Proteus mirabilis (17), suggesting the existence of a common regulatory pathway in bacterial swarming.Both rssA and rssB knockout mutants showed similar precocious swarming behaviors (16). To further analyze the biochemical property of this two-component system and understand the underlying mechanism by which this two-component system regulates swarming mobility, the phosphorelay between RssA and RssB during signal transduction was studied, and the interaction between RssB and the regulated target DNA fragments are characterized in this report. Our results (i) show that RssA and RssB are two-component signal transduction proteins involved in phosphorelay reactions and (ii) provide evidence of negat...

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