Effect of iacP Mutation on Flagellar Phase Variation in Salmonella enterica Serovar Typhimurium Strain UK-1

Abstract

c Flagella are surface appendages that are important for bacterial motility and invasion of host cells. Two flagellin subunits in Salmonella enterica serovar Typhimurium, FliC and FljB, are alternatively expressed by a site-specific DNA inversion mechanism called flagellar phase variation. Although this inversion mechanism is understood at the molecular level, the key factor controlling the expression of the two flagellin subunits has not been determined. In this study, we found that a putative acyl carrier protein, IacP, affects flagellar phase variation in S. Typhimurium strain UK-1 under Salmonella pathogenicity island 1 (SPI1)-inducing conditions. Liquid chromatography-mass spectrometry analysis of the secreted proteins from S. Typhimurium determined that the amount of FljB secreted was significantly higher in the iacP mutant strain, a finding confirmed by Western blot analysis. Northern blotting, quantitative PCR, and microarray data showed that the level of FljB in the iacP mutant strain was regulated at the transcriptional level, although the transcription and expression of the fliC gene were independent of IacP. FljB production was abolished by the deletion of the Hin DNA invertase but could be restored by the introduction of a plasmid carrying the hin gene. We also found that in the iacP mutant strain, the orientation of the invertible H segment is in the FljB-expressing phase. Furthermore, electron microscopy observations indicated that the iacP mutant strain had more flagella per cell than the wildtype strain. These results suggest that IacP is associated with flagellar phase switching under SPI1-inducing conditions. F lagella play crucial roles in bacterial motility, chemotactic behavior, and host cell invasion as a virulence determinant (1). Salmonella enterica serovar Typhimurium has 5 to 10 flagella emanating from the bacterial surface. The individual flagellum is a complex structure composed of three basic parts: a basal body, a hook, and a filament (22). The flagellar filament consists of thousands of flagellin protein monomers, which are polymerized into a helical structure that can be 10 m long. S. Typhimurium possesses two genetically distinct flagellin subunits, FliC (phase 1 flagellin) and FljB (phase 2 flagellin), which are alternatively expressed via a site-specific DNA inversion process (19,30). When the 1-kb upstream region of the fljB gene (H segment) is inverted by the DNA invertase Hin, the promoter for the fljB gene is properly oriented for the transcription of the fljB-fljA operon. Because FljA is responsible for the posttranscriptional and posttranslational inhibition of FliC expression (2, 38), only FljB flagellin is produced. Although the molecular mechanism of the Hin-mediated DNA inversion mechanism has been studied intensively (6, 24, 28), the question of which cellular signals control the flagellinswitching mechanism and the frequency of DNA inversion remains unclear.Salmonella pathogenicity island 1 (SPI1) encodes a type III secretion system (T3SS) that is required for the ...

Enhancement of Host Immune Responses by Oral Vaccination to Salmonella enterica serovar Typhimurium Harboring Both FliC and FljB Flagella

“…Yang et al observed that the flagellar overexpression attenuates Salmonella virulence and elicits an elevated immune response via flagellin adjuvant, thereby conferring robust protection against Salmonellosis [8]. We previously found that the expression level of the fljB gene was higher in the iacP mutant strain grown under SPI1-inducing conditions, although the expression of the fliC gene was independent of IacP [7]. As expected, the iacP mutant strain produced more flagella than the wild-type strain.…”

“…Bacterial flagella are appendages on the cell surface that are required for the motility and chemotaxis of bacterial pathogens and for epithelial cell invasion as a virulence factor [6,7]. Flagellins, a key component of the bacterial flagella, are recognized as PAMPs in host cells, through binding with TLR5 and NLRC4.…”

Posttranslational Maturation of the Invasion Acyl Carrier Protein of Salmonella enterica Serovar Typhimurium Requires an Essential Phosphopantetheinyl Transferase of the Fatty Acid Biosynthesis Pathway

“…The use of an essential function as the one performed by AcpS may ensure that the bacterial pathogen maintains the capacity to mature the virulence-associated protein IacP. As mentioned earlier, IacP has been proposed to promote the secretion of the protein effectors SopA, SopB, and SopD (16) and to influence the Hin DNA invertase-mediated flagellar phase variation (17). However, in light of what is known about the mode of action of ACPs, it is unclear at this stage how IacP would regulate these events.…”

“…The proposed explanation for this phenotype was that IacP might promote secretion and translocation of specific protein effectors: SopA, SopB, and SopD (16). It has also been reported that IacP might be involved in the regulation of flagellin expression (17). In the two studies, the conserved serine 38 residue was required for the observed roles of IacP, suggesting that maturation was occurring on IacP and was necessary for its activity.…”

The Bacterial iprA Gene Is Conserved across Enterobacteriaceae, Is Involved in Oxidative Stress Resistance, and Influences Gene Expression in Salmonella enterica Serovar Typhimurium

“…Confirmation of flagellin phase switching was performed at both the protein and DNA level, as described previously (34)(35)(36). The primers used to assay genomic DNA for flagellin phase are indicated in Table S3 in the supplemental material.…”