Enterobacterial Common Antigen Integrity Is a Checkpoint for Flagellar Biogenesis inSerratia marcescens

Abstract

Serratia marcescens strains are ubiquitous bacteria isolated from environmental niches, such as soil, water, and air, and also constitute emergent nosocomial opportunistic pathogens. Among the numerous extracellular factors that S. marcescens is able to produce, the PhlA phospholipase is the only described exoprotein secreted by the flagellar apparatus while simultaneously being a member of the flagellar regulon. To gain insight into the regulatory mechanism that couples PhlA and flagellar expression, we conducted a generalized insertional mutagenesis and screened for PhlA-deficient strains. We found that three independent mutations in the wec cluster, which impaired the assembly of enterobacterial common antigen (ECA), provoked the inhibition of PhlA expression. Swimming and swarming assays showed that in these strains, motility was severely affected. Microscopic examination and flagellin immunodetection demonstrated that a strong defect in flagellum expression was responsible for the reduced motility in the wec mutant strains. Furthermore, we determined that in the ECA-defective strains, the transcriptional cascade that controls flagellar assembly was turned off due to the down-regulation of flhDC expression. These findings provide a new perspective on the physiological role of the ECA, providing evidence that in S. marcescens, its biosynthesis conditions the expression of the flagellar regulon.Serratia marcescens is an opportunistic human pathogen associated with urinary and respiratory tract as well as wound and eye infections, endocarditis, osteomyelitis, meningitis, and septicemia. Immunocompromised people and newborns are the most affected hosts. The incidence of S. marcescens infection has increased over the last years, mainly due to the acquisition of multiple antibiotic resistance (19,20). S. marcescens produces numerous extracellular factors, including hemolysin, esterase, DNase, chloroperoxidase, S-layer, lipases, proteases, chitinases, and siderophohore (21,24,26,27,31,38,44,45). These factors are predicted to play a role in bacterial environmental adaptive capacity, in either nonhost or host environments, contributing to its ambient persistence and to its pathogenic potential. In addition, an extracellular phospholipase named PhlA in Serratia liquefaciens MG1 (recently reclassified as S. marcescens MG1) and PlaA in Serratia sp. strain MK1 has been described previously (16, 48). The phlAB (or plaAS) locus codes for the PhlA (PlaA) phospholipase and for an accessory protein (PhlB/PlaS) that prevents the intracellular phospholipase enzymatic action. Interestingly, it has been shown not only that the phlAB promoter region displays homology to class III promoters of the flagellar transcriptional cascade controlled by the FliA sigma factor (15) but also that PhlA secretion depends on the integrity of the type III flagellar system export apparatus (15-17).The flagellar structure has its dedicated type III protein export embedded apparatus that translocates proteins involved in the self-assembly process an...

Loss of the WaaL O-Antigen Ligase Prevents Surface Activation of the Flagellar Gene Cascade in Proteus mirabilis

“…Therefore, O-antigen, but not ECA, is required for swarming. This contrasts to the proposed role of ECA in Serratia marcescens, where it was necessary for swarming and flhDC activation and was suggested to act as a checkpoint for flhDC activation (9). An analogous role for O antigen in P. mirabilis seems unlikely, since liquid-grown cultures at early stationary phase do not upregulate flhDC but have complete O antigen (data not shown).…”

“…In E. coli, WaaL can link either O-antigen or ECA subunits to the lipid A::core (27,45). In S. marcescens ECA, completion acts as a checkpoint for flhDC activation (9). To test whether the lack of ECA or O antigen due to the waaL mutation was causing the swarming defect, a mutation in wzyE (PMI3326) encoding the ECA polymerase was made in PM7002.…”

“…In Serratia marcescens, completed ECA was needed for the upregulation of the master regulator flhDC. Without functional ECA, swarming motility was abolished, while swimming motility was only decreased (9). Finally, recent studies in E. coli indicate that functional LPS is required for both swimming and swarming motility (18).…”

Enterobacterial common antigen and O-specific polysaccharide coexist in the lipopolysaccharide of Yersinia enterocolitica serotype O : 3

“…low pH, bile salts, and defense mechanisms from the eukaryotic host (Barua et al, 2002;Danese et al, 1998;Ramos-Morales et al, 2003). It was also proposed that ECA may be required for proper formation of flagella and motility of Serratia marcescens (Castelli et al, 2008).…”

mgtAExpression Is Induced by Rob Overexpression and Mediates aSalmonella entericaResistance Phenotype

“…Total RNA was extracted from lateexponential-phase cultures (optical density at 630 nm ϭ 0.9) grown in LB at 37°C with 100 g ml Ϫ1 ampicillin and 0.1 mM IPTG as previously described (5). A final RNA cleanup step with an RNeasy kit (Qiagen), used according to the manufacturer's protocol, was included.…”