Gre factors-mediated control of hilD transcription is essential for the invasion of epithelial cells by Salmonella enterica serovar Typhimurium.

The invasion of epithelial cells by Salmonella enterica serovar Typhimurium is a very tightly regulated process. Signaling cascades triggered by different environmental and physiological signals converge to control HilD, an AraC regulator that coordinates the expression of several virulence factors. The expression of hilD is modulated at several steps of the expression process. Here, we report that the invasion of epithelial cells by S. Typhimurium strains lacking the Gre factors, GreA and GreB, is impaired. By interacting with the RNA polymerase secondary channel, the Gre factors prevent backtracking of paused complexes to avoid arrest during transcriptional elongation. Our results indicate that the Gre factors are required for the expression of the bacterial factors needed for epithelial cell invasion by modulating expression of HilD. This regulation does not occur at transcription initiation and depends on the capacity of the Gre factors to prevent backtracking of the RNA polymerase. Remarkably, genetic analyses indicate that the 3'-untranslated region (UTR) of hilD is required for Gre-mediated regulation of hilD expression. Our data provide new insight into the complex regulation of S. Typhimurium virulence and highlight the role of the hilD 3'-UTR as a regulatory motif.

Identification and taxonomic characterization of Bordetella pseudohinzii sp. nov. isolated from laboratory-raised mice.

Bordetella hinzii is known to cause respiratory disease in poultry and has been associated with a variety of infections in immunocompromised humans. In addition, there are several reports of B. hinzii infections in laboratory-raised mice. Here we sequenced and analysed the complete genome sequences of multiple B. hinzii-like isolates, obtained from vendor-supplied C57BL/6 mice in animal research facilities on different continents, and we determined their taxonomic relationship to other Bordetella species. The whole-genome based and 16S rRNA gene based phylogenies each identified two separate clades in B. hinzii, one was composed of strains isolated from poultry, humans and a rabbit whereas the other clade was restricted to isolates from mice. Distinctly different estimated DNA-DNA hybridization values, average nucleotide identity scores, gene content, metabolic profiles and host specificity all provide compelling evidence for delineation of the two species, B. hinzii - from poultry, humans and rabbit - and Bordetella pseudohinzii sp. nov. type strain 8-296-03T (=NRRL B-59942T=NCTC 13808T) that infect mice.

BcsZ inhibits biofilm phenotypes and promotes virulence by blocking cellulose production in Salmonella enterica serovar Typhimurium.

BACKGROUND: Cellulose, a 1,4 beta-glucan polysaccharide, is produced by a variety of organisms including bacteria. Although the production of cellulose has a high biological, ecological and economical impact, regulatory mechanisms of cellulose biosynthesis are mostly unknown. Family eight cellulases are regularly associated with cellulose biosynthesis operons in bacteria; however, their function is poorly characterized. In this study, we analysed the role of the cellulase BcsZ encoded by the bcsABZC cellulose biosynthesis operon of Salmonella enterica serovar Typhimurium (S. Typhimurium) in biofilm related behavior. We also investigated the involvement of BcsZ in pathogenesis of S. Typhimurium including a murine typhoid fever infection model. RESULT: In S. Typhimurium, cellulase BcsZ with a putative periplasmic location negatively regulates cellulose biosynthesis. Moreover, as assessed with a non-polar mutant, BcsZ affects cellulose-associated phenotypes such as the rdar biofilm morphotype, cell clumping, biofilm formation, pellicle formation and flagella-dependent motility. Strikingly, although upregulation of cellulose biosynthesis was not observed on agar plate medium at 37 °C, BcsZ is required for efficient pathogen-host interaction. Key virulence phenotypes of S. Typhimurium such as invasion of epithelial cells and proliferation in macrophages were positively regulated by BcsZ. Further on, a bcsZ mutant was outcompeted by the wild type in organ colonization in the murine typhoid fever infection model. Selected phenotypes were relieved upon deletion of the cellulose synthase BcsA and/or the central biofilm activator CsgD. CONCLUSION: Although the protein scaffold has an additional physiological role, our findings indicate that the catalytic activity of BcsZ effectively downregulates CsgD activated cellulose biosynthesis. Repression of cellulose production by BcsZ subsequently enables Salmonella to efficiently colonize the host.

Dissecting the cyclic di-guanylate monophosphate signalling network regulating motility in Salmonella enterica serovar Typhimurium.

Flagella-mediated swimming and swarming motility in Salmonella enterica serovar Typhimurium is intercalated with the cyclic di-guanylate monophosphate (c-di-GMP) signalling network. In this study, we identified the GGDEF domain proteins STM2672, STM4551 and STM1987 as key di-guanylate cyclases involved in regulation of motility in a ΔyhjH phosphodiesterase gene deletion mutant with elevated c-di-GMP levels inhibiting motility. Surprisingly, these di-guanylate cyclases distinctively inhibited motility through the c-di-GMP receptors YcgR and the cellulose synthase BcsA, whereby STM2672 corresponded to YcgR, STM1987 to BcsA and STM4551 to both receptors. Although downregulation of motility is believed to prepare the bacterial cells for surface adhesion and biofilm formation, the major biofilm regulator CsgD of S. sv. Typhimurium was not involved in the regulation of swimming or swarming motility. Together with previously identified c-di-GMP networks regulating flagella-related phenotypes, flagella biosynthesis is a major target of c-di-GMP signalling in S. sv. Typhimurium.

Regulation of biofilm formation in Salmonella enterica serovar Typhimurium.

In animals, plants and the environment, Salmonella enterica serovar Typhimurium forms the red dry and rough (rdar) biofilm characterized by extracellular matrix components curli and cellulose. With complex expression control by at least ten transcription factors, the bistably expressed orphan response regulator CsgD directs rdar morphotype development. CsgD expression is an integral part of the Hfq regulon and the complex cyclic diguanosine monophosphate signaling network partially controlled by the global RNA-binding protein CsrA. Cell wall turnover and the periplasmic redox status regulate csgD expression on a post-transcriptional level by unknown mechanisms. Furthermore, phosphorylation of CsgD is a potential inactivation and degradation signal in biofilm dissolution. Including complex incoherent feed-forward loops, regulation of biofilm formation versus motility and virulence is of recognized complexity.

The effects of codon context on in vivo translation speed.

We developed a bacterial genetic system based on translation of the his operon leader peptide gene to determine the relative speed at which the ribosome reads single or multiple codons in vivo. Low frequency effects of so-called "silent" codon changes and codon neighbor (context) effects could be measured using this assay. An advantage of this system is that translation speed is unaffected by the primary sequence of the His leader peptide. We show that the apparent speed at which ribosomes translate synonymous codons can vary substantially even for synonymous codons read by the same tRNA species. Assaying translation through codon pairs for the 5'- and 3'- side positioning of the 64 codons relative to a specific codon revealed that the codon-pair orientation significantly affected in vivo translation speed. Codon pairs with rare arginine codons and successive proline codons were among the slowest codon pairs translated in vivo. This system allowed us to determine the effects of different factors on in vivo translation speed including Shine-Dalgarno sequence, rate of dipeptide bond formation, codon context, and charged tRNA levels.

The EAL-like protein STM1697 regulates virulence phenotypes, motility and biofilm formation in Salmonella typhimurium.

The ubiquitous second messenger c-di-GMP regulates the switching of bacterial lifestyles from motility to sessility and acute to chronic virulence to adjust bacterial fitness to altered environmental conditions. Conventionally, EAL proteins being c-di-GMP phosphodiesterases promote motility and acute virulence phenotypes such as invasion into epithelial cells and inhibit biofilm formation. We report here that in contradiction, the EAL-like protein STM1697 of Salmonella typhimurium suppresses motility, invasion into HT-29 epithelial cell line and secretion of the type three secretion system 1 effector protein SipA, whereas it promotes rdar biofilm formation and CsgD expression. STM1697 can, however, functionally replace the EAL-like protein STM1344 and vice versa, whereby both proteins neither degrade nor bind c-di-GMP. Like STM1344, STM1697 suppresses the transcription of class 2 and class 3 flagella regulon genes by binding to FlhD, a component of the master regulator of the flagella regulon FlhD4 C2 and act additively under numerous conditions. Interestingly, the interaction interface of STM1697 with FlhD2 is distinct from its paralogue STM1344. We predict that the stand alone EAL domain proteins STM1697 and STM1344 belong to a subclass of EAL domain proteins in S. typhimurium, which are all involved in motility, biofilm and virulence regulation through interaction with proteins that regulate flagella function.

Hfq and Hfq-dependent small RNAs are major contributors to multicellular development in Salmonella enterica serovar Typhimurium.

The RNA chaperone Hfq and its associated small RNAs (sRNAs) regulate a variety of phenotypes in bacteria. In this work, we show that Hfq is a master regulator of biofilm formation in Salmonella enterica serovar Typhimurium. Hfq and two Hfq-dependent sRNAs (ArcZ and SdsR) are required for rdar morphotype expression in S. typhimurium. Hfq controls rdar biofilm formation through the major biofilm regulator CsgD. While csgD mRNA steady-state levels are altered in a sdsR mutant, ArcZ seems to work mainly at the post-transcriptional level. Overexpression of ArcZ complemented rdar morphotype formation of an hfq mutant under plate-grown conditions. Although ArcZ activates rpoS expression, its effect on csgD expression is mainly independent of RpoS. ArcZ does not only regulate rdar morphotype expression, but also the transition between sessility and motility and the timing of type 1 fimbriae vs. curli fimbriae surface-attachment at ambient temperature. Consequently, ArcZ is a major regulator of rdar biofilm development.

Complex c-di-GMP signaling networks mediate transition between virulence properties and biofilm formation in Salmonella enterica serovar Typhimurium.

Upon Salmonella enterica serovar Typhimurium infection of the gut, an early line of defense is the gastrointestinal epithelium which senses the pathogen and intrusion along the epithelial barrier is one of the first events towards disease. Recently, we showed that high intracellular amounts of the secondary messenger c-di-GMP in S. typhimurium inhibited invasion and abolished induction of a pro-inflammatory immune response in the colonic epithelial cell line HT-29 suggesting regulation of transition between biofilm formation and virulence by c-di-GMP in the intestine. Here we show that highly complex c-di-GMP signaling networks consisting of distinct groups of c-di-GMP synthesizing and degrading proteins modulate the virulence phenotypes invasion, IL-8 production and in vivo colonization in the streptomycin-treated mouse model implying a spatial and timely modulation of virulence properties in S. typhimurium by c-di-GMP signaling. Inhibition of the invasion and IL-8 induction phenotype by c-di-GMP (partially) requires the major biofilm activator CsgD and/or BcsA, the synthase for the extracellular matrix component cellulose. Inhibition of the invasion phenotype is associated with inhibition of secretion of the type three secretion system effector protein SipA, which requires c-di-GMP metabolizing proteins, but not their catalytic activity. Our findings show that c-di-GMP signaling is at least equally important in the regulation of Salmonella-host interaction as in the regulation of biofilm formation at ambient temperature.