Single Cell Analysis of Bistable Expression of Pathogenicity Island 1 and the Flagellar Regulon in Salmonella enterica

Bistable expression of the Salmonella enterica pathogenicity island 1 (SPI-1) and the flagellar network (Flag) has been described previously. In this study, simultaneous monitoring of OFF and ON states in SPI-1 and in the flagellar regulon reveals independent switching, with concomitant formation of four subpopulations: SPI-1OFF FlagOFF, SPI-1OFF FlagON, SPI-1ON FlagOFF, and SPI-1ON FlagON. Invasion assays upon cell sorting show that none of the four subpopulations is highly invasive, thus raising the possibility that FlagOFF cells might contribute to optimal invasion as previously proposed for SPI-1OFF cells. Time lapse microscopy observation indicates that expression of the flagellar regulon contributes to the growth impairment previously described in SPI-1ON cells. As a consequence, growth resumption in SPI-1ON FlagON cells requires switching to both SPI-1OFF and FlagOFF states.

Clarithromycin Exerts an Antibiofilm Effect against Salmonella typhimurium rdar Biofilm Formation, and Transforms the Physiology towards an Apparent Oxygen-depleted Energy and Carbon Metabolism

ABSTRACTUpon biofilm formation, production of extracellular matrix components and alteration in physiology and metabolism allows bacteria to build up multicellular communities which can facilitate nutrient acquisition during unfavorable conditions and provide protection towards various forms of environmental stresses to individual cells. Thus, bacterial cells become tolerant against antimicrobials and the immune system within biofilms. In the current study, we evaluated the antibiofilm activity of the macrolides clarithromycin and azithromycin. Clarithromycin showed antibiofilm activity against rdar (red, dry and rough) biofilm formation of the gastrointestinal pathogen Salmonella typhimurium ATCC14028 Nalr at 1.56 μM subinhibitory concentration in standing culture and dissolved cell aggregates at 15 μM in a microaerophilic environment suggesting that the oxygen level affects the activity of the drug. Treatment with clarithromycin significantly decreased transcription and production of the rdar biofilm activator CsgD, with biofilm genes such as csgB and adrA to be consistently downregulated. While fliA and other flagellar regulon genes were upregulated, apparent motility was downregulated. RNA sequencing showed a holistic cell response upon clarithromycin exposure, whereby not only genes involved in the biofilm-related regulatory pathways, but also genes that likely contribute to intrinsic antimicrobial resistance, and the heat shock stress response were differentially regulated. Most significantly, clarithromycin exposure shifts the cells towards an apparent oxygen- and energy-depleted status, whereby the metabolism that channels into oxidative phosphorylation is downregulated, and energy gain by degradation of propane 1,2-diol, ethanolamine and L-arginine catabolism is upregulated. This initial analysis will allow the subsequent identification of novel intrinsic antimicrobial resistance determinants.

Under Elevated c-di-GMP in Escherichia coli, YcgR Alters Flagellar Motor Bias and Speed Sequentially, with Additional Negative Control of the Flagellar Regulon via the Adaptor Protein RssB

Flagellum-driven motility has been studied in E. coli and Salmonella for nearly half a century. Over 60 genes control flagellar assembly and function. The expression of these genes is regulated at multiple levels in response to a variety of environmental signals. Cues that elevate c-di-GMP levels, however, inhibit motility by direct binding of the effector YcgR to the flagellar motor. In this study conducted mainly in E. coli, we show that YcgR is the only effector of motor control and tease out the order of YcgR-mediated events. In addition, we find that the σS regulator protein RssB contributes to negative regulation of flagellar gene expression when c-di-GMP levels are elevated.

Functional Analysis of the Alternative Sigma-28 Factor FliA and Its Anti-Sigma Factor FlgM of the Nonflagellated Legionella Species L. oakridgensis

ABSTRACT Legionella oakridgensis causes Legionnaires' disease but is known to be less virulent than Legionella pneumophila. L. oakridgensis is one of the Legionella species that is nonflagellated. The genes of the flagellar regulon are absent, except those encoding the alternative sigma-28 factor (FliA) and its anti-sigma-28 factor (FlgM). Similar to L. oakridgensis, Legionella adelaidensis and Legionella londiniensis, located in the same phylogenetic clade, have no flagellar regulon, although both are positive for fliA and flgM. Here, we investigated the role and function of both genes to better understand the role of FliA, the positive regulator of flagellin expression, in nonflagellated strains. We demonstrated that the FliA gene of L. oakridgensis encodes a functional sigma-28 factor that enables the transcription start from the sigma-28-dependent promoter site. The investigations have shown that FliA is necessary for full fitness of L. oakridgensis. Interestingly, expression of FliA-dependent genes depends on the growth phase and temperature, as already shown for L. pneumophila strains that are flagellated. In addition, we demonstrated that FlgM is a negative regulator of FliA-dependent gene expression. FlgM seems to be degraded in a growth-phase- and temperature-dependent manner, instead of being exported into the medium as reported for most bacteria. The degradation of FlgM leads to an increase of FliA activity. IMPORTANCE A less virulent Legionella species, L. oakridgensis, causes Legionnaires' disease and is known to not have flagella, even though L. oakridgensis has the regulator of flagellin expression (FliA). This protein has been shown to be involved in the expression of virulence factors. Thus, the strain was chosen for use in this investigation to search for FliA target genes and to identify putative virulence factors of L. oakridgensis. One of the five major target genes of FliA identified here encodes the anti-FliA sigma factor FlgM. Interestingly, in contrast to most homologs in other bacteria, FlgM in L. oakridgensis seems not to be transported from the cell so that FliA gets activated. In L. oakridgensis, FlgM seems to be degraded by protease activities.

Modulation of Toxin Production by the Flagellar Regulon in Clostridium difficile

ABSTRACTWe show in this study that toxin production inClostridium difficileis altered in cells which can no longer form flagellar filaments. The impact of inactivation offliC,CD0240,fliF,fliG,fliM, andflhB-fliRflagellar genes upon toxin levels in culture supernatants was assessed using cell-based cytotoxicity assay, proteomics, immunoassay, and immunoblotting approaches. Each of these showed that toxin levels in supernatants were significantly increased in afliCmutant compared to that in theC. difficile630 parent strain. In contrast, the toxin levels in supernatants secreted from other flagellar mutants were significantly reduced compared with that in the parentalC. difficile630 strain. Transcriptional analysis of the pathogenicity locus genes (tcdR,tcdB,tcdE, andtcdA) revealed a significant increase of all four genes in thefliCmutant strain, while transcription of all four genes was significantly reduced infliM,fliF,fliG, andflhB-fliRmutants. These results demonstrate that toxin transcription inC. difficileis modulated by the flagellar regulon. More significantly, mutant strains showed a corresponding change in virulence compared to the 630 parent strain when tested in a hamster model ofC. difficileinfection. This is the first demonstration of differential flagellum-related transcriptional regulation of toxin production inC. difficileand provides evidence for elaborate regulatory networks for virulence genes inC. difficile.