Selective Purification of Recombinant Neuroactive Peptides Using the Flagellar Type III Secretion System

The structure, assembly, and function of the bacterial flagellum involves about 60 different proteins, many of which are selectively secreted via a specific type III secretion system (T3SS) (J. Frye et al., J. Bacteriol. 188:2233–2243, 2006). The T3SS is reported to secrete proteins at rates of up to 10,000 amino acid residues per second. In this work, we showed that the flagellar T3SS of Salmonella enterica serovar Typhimurium could be manipulated to export recombinant nonflagellar proteins through the flagellum and into the surrounding medium. We translationally fused various neuroactive peptides and proteins from snails, spiders, snakes, sea anemone, and bacteria to the flagellar secretion substrate FlgM. We found that all tested peptides of various sizes were secreted via the bacterial flagellar T3SS. We subsequently purified the recombinant μ-conotoxin SIIIA (rSIIIA) from Conus striatus by affinity chromatography and confirmed that T3SS-derived rSIIIA inhibited mammalian voltage-gated sodium channel NaV1.2 comparably to chemically synthesized SIIIA

Commensal-Induced Regulatory T Cells Mediate Protection against Pathogen-Stimulated NF-κB Activation

Host defence against infection requires a range of innate and adaptive immune responses that may lead to tissue damage. Such immune-mediated pathologies can be controlled with appropriate T regulatory (Treg) activity. The aim of the present study was to determine the influence of gut microbiota composition on Treg cellular activity and NF-κB activation associated with infection. Mice consumed the commensal microbe Bifidobacterium infantis 35624 followed by infection with Salmonella typhimurium or injection with LPS. In vivo NF-κB activation was quantified using biophotonic imaging. CD4+CD25+Foxp3+ T cell phenotypes and cytokine levels were assessed using flow cytometry while CD4+ T cells were isolated using magnetic beads for adoptive transfer to naïve animals. In vivo imaging revealed profound inhibition of infection and LPS induced NF-κB activity that preceded a reduction in S. typhimurium numbers and murine sickness behaviour scores in B. infantis–fed mice. In addition, pro-inflammatory cytokine secretion, T cell proliferation, and dendritic cell co-stimulatory molecule expression were significantly reduced. In contrast, CD4+CD25+Foxp3+ T cell numbers were significantly increased in the mucosa and spleen of mice fed B. infantis. Adoptive transfer of CD4+CD25+ T cells transferred the NF-κB inhibitory activity. Consumption of a single commensal micro-organism drives the generation and function of Treg cells which control excessive NF-κB activation in vivo. These cellular interactions provide the basis for a more complete understanding of the commensal-host-pathogen trilogue that contribute to host homeostatic mechanisms underpinning protection against aberrant activation of the innate immune system in response to a translocating pathogen or systemic LPS

The RNA chaperone Hfq is essential for the virulence of Salmonella typhimurium

The RNA chaperone, Hfq, plays a diverse role in bacterial physiology beyond its original role as a host factor required for replication of Qβ RNA bacteriophage. In this study, we show that Hfq is involved in the expression and secretion of virulence factors in the facultative intracellular pathogen, Salmonella typhimurium. A Salmonella hfq deletion strain is highly attenuated in mice after both oral and intraperitoneal infection, and shows a severe defect in invasion of epithelial cells and a growth defect in both epithelial cells and macrophages in vitro. Surprisingly, we find that these phenotypes are largely independent of the previously reported requirement of Hfq for expression of the stationary phase sigma factor, RpoS. Our results implicate Hfq as a key regulator of multiple aspects of virulence including regulation of motility and outer membrane protein (OmpD) expression in addition to invasion and intracellular growth. These pleiotropic effects are suggested to involve a network of regulatory small non-coding RNAs, placing Hfq at the centre of post-transcriptional regulation of virulence gene expression in Salmonella. In addition, the hfq mutation appears to cause a chronic activation of the RpoE-mediated envelope stress response which is likely due to a misregulation of membrane protein expression

The Fungal Pathogen Candida albicans Autoinduces Hyphal Morphogenesis by Raising Extracellular pH

pH homeostasis is critical for all organisms; in the fungal pathogen Candida albicans, pH adaptation is critical for virulence in distinct host niches. We demonstrate that beyond adaptation, C. albicans actively neutralizes the environment from either acidic or alkaline pHs. Under acidic conditions, this species can raise the pH from 4 to >7 in less than 12 h, resulting in autoinduction of the yeast-hyphal transition, a critical virulence trait. Extracellular alkalinization has been reported to occur in several fungal species, but under the specific conditions that we describe, the phenomenon is more rapid than previously observed. Alkalinization is linked to carbon deprivation, as it occurs in glucose-poor media and requires exogenous amino acids. These conditions are similar to those predicted to exist inside phagocytic cells, and we find a strong correlation between the use of amino acids as a cellular carbon source and the degree of alkalinization. Genetic and genomic approaches indicate an emphasis on amino acid uptake and catabolism in alkalinizing cells. Mutations in four genes, STP2, a transcription factor regulating amino acid permeases, ACH1 (acetyl-coenzyme A [acetyl-CoA] hydrolase), DUR1,2 (urea amidolyase), and ATO5, a putative ammonia transporter, abolish or delay neutralization. The pH changes are the result of the extrusion of ammonia, as observed in other fungi. We propose that nutrient-deprived C. albicans cells catabolize amino acids as a carbon source, excreting the amino nitrogen as ammonia to raise environmental pH and stimulate morphogenesis, thus directly contributing to pathogenesis

Analysis of Pools of Targeted Salmonella Deletion Mutants Identifies Novel Genes Affecting Fitness during Competitive Infection in Mice

Pools of mutants of minimal complexity but maximal coverage of genes of interest facilitate screening for genes under selection in a particular environment. We constructed individual deletion mutants in 1,023 Salmonella enterica serovar Typhimurium genes, including almost all genes found in Salmonella but not in related genera. All mutations were confirmed simultaneously using a novel amplification strategy to produce labeled RNA from a T7 RNA polymerase promoter, introduced during the construction of each mutant, followed by hybridization of this labeled RNA to a Typhimurium genome tiling array. To demonstrate the ability to identify fitness phenotypes using our pool of mutants, the pool was subjected to selection by intraperitoneal injection into BALB/c mice and subsequent recovery from spleens. Changes in the representation of each mutant were monitored using T7 transcripts hybridized to a novel inexpensive minimal microarray. Among the top 120 statistically significant spleen colonization phenotypes, more than 40 were mutations in genes with no previously known role in this model. Fifteen phenotypes were tested using individual mutants in competitive assays of intraperitoneal infection in mice and eleven were confirmed, including the first two examples of attenuation for sRNA mutants in Salmonella. We refer to the method as Array-based analysis of cistrons under selection (ABACUS)

Unprecedented high-resolution view of bacterial operon architecture revealed by RNA sequencing

We analyzed the transcriptome of Escherichia coli K-12 by strand-specific RNA sequencing at single-nucleotide resolution during steady-state (logarithmic-phase) growth and upon entry into stationary phase in glucose minimal medium. To generate high-resolution transcriptome maps, we developed an organizational schema which showed that in practice only three features are required to define operon architecture: the promoter, terminator, and deep RNA sequence read coverage. We precisely annotated 2,122 promoters and 1,774 terminators, defining 1,510 operons with an average of 1.98 genes per operon. Our analyses revealed an unprecedented view of E. coli operon architecture. A large proportion (36%) of operons are complex with internal promoters or terminators that generate multiple transcription units. For 43% of operons, we observed differential expression of polycistronic genes, despite being in the same operons, indicating that E. coli operon architecture allows fine-tuning of gene expression. We found that 276 of 370 convergent operons terminate inefficiently, generating complementary 3′ transcript ends which overlap on average by 286 nucleotides, and 136 of 388 divergent operons have promoters arranged such that their 5′ ends overlap on average by 168 nucleotides. We found 89 antisense transcripts of 397-nucleotide average length, 7 unannotated transcripts within intergenic regions, and 18 sense transcripts that completely overlap operons on the opposite strand. Of 519 overlapping transcripts, 75% correspond to sequences that are highly conserved in E. coli (>50 genomes). Our data extend recent studies showing unexpected transcriptome complexity in several bacteria and suggest that antisense RNA regulation is widespread.Ye

Phosphate-independent controls of bacterial alkaline phosphatase synthesis in Escherichia coli K-12

The synthesis of bacterial alkaline phosphatase (Bap) is normally induced several hundredfold when phosphate is growth limiting. This control requires both products of the phoBR operon. PhoB is a transcriptional activator, and PhoR is a sensory protein that detects environmental phosphate levels. Earlier studies showed that in phoR mutants phoA transcription is no longer controlled by phosphate. Instead, its expression is regulated by the creABCD (formerly called phoM) operon. phoR mutants with a wild-type creABCD operon show induced synthesis of Bap on glucose medium, and an alternating pattern of Bap synthesis, termed Bap clonal variation, on TYE agar. Point mutations in the creC gene confer a Bap\sp- phenotype. However, deletion mutations can confer either a Bap\sp- or clonal variation phenotype, depending on whether neighboring genes are deleted and the strain background. Because strains with certain creABCD deletions are Bap variable, this suggested that gene products other than CreC may be involved in regulation of Bap synthesis. To determine which additional genes in the creABCD region are involved, this region was mutagenized with the transposon Tn5, and these insertions were used to construct new deletions. The results showed that (i) strains with non-polar insertions in the creA, creB, and creD genes show normal regulation of Bap synthesis; (ii) a strain with a deletion removing the creA and creB genes shows a Bap induced phenotype; (iii) strains with simple mutations in the creC gene are Bap\sp-; (iv) strains with insertions in the neighboring arcA gene are Bap induced; and (v) strains with deletions that remove both the creC and arcA genes are Bap\sp- on glucose and show the Bap clonal variation phenotype on TYE agar. Models are presented to explain how the products of the creABCD operon and the arcA gene regulate Bap synthesis. Several lines of evidence indicate that one or more additional controls regulate bap synthesis in a PhoR-and CreC-independent manner. As described above, arcA mutations restore Bap synthesis in phoR $\Delta$ (creABCD) mutants. We found that an ompR mutation also restored Bap synthesis in phoR $\Delta$ (creABCD) mutants. Furthermore, phoR $\Delta$ (creABCD) mutants synthesize Bap when plated on TYEG agar. In order to study Pi-independent control of Bap synthesis, 74 transposon-induced mutants were isolated with altered Bap phenotypes. Thirty-two Bap\sp- mutants were shown to have insertions in the phoA or phoB genes. The remaining 42 mutants were placed into one of 19 mutant classes based on their Bap phenotypes or linkage data. Many insertions resulted in the induction of a phoR- and CreC-independent control of Bap synthesis, whereas some mutants eliminated the phoR- and CreC-independent Bap synthesis on TYEG agar. Representative insertions from several mutant classes were cloned and the DNA sequence of their fusion junctions were determined. Based on sequence and/or linkage data, the sites of the insertions for 11 mutants classes were identified. Interestingly, 4 mutants that conferred a Bap induced phenotype had insertions in the ackA gene, for acetate kinase. Based on subsequent experiments with these mutants, we conclude that one phoR- and CreC-independent control of Bap synthesis responds to acetyl phosphate or a closely related metabolite. Several other mutations that alter Bap synthesis in a phoR $\Delta$(creABCD) mutant may affect Bap synthesis indirectly by altering the level of acetyl phosphate

TnphoA and TnphoA' elements for making and switching fusions for study of transcription, translation, and cell surface localization.

We describe a set of elements based on the transposon TnphoA for making transcriptional fusions to the lacZ gene and for making translational fusions to the phoA or lacZ structural gene. Each element can be switched, one for another, by homologous recombination, thereby allowing testing for transcription, translation, or cell surface localization determinants at the same site within a gene. We describe three kinds of elements for making each fusion type. Two kinds are transposition proficient (Tnp+): one encodes kanamycin resistance, and the other encodes tetracycline resistance. The third kind is transposition defective (Tnp-) and encodes kanamycin resistance. In addition, we describe one Tnp- element that has no reporter gene and encodes chloramphenicol resistance; this element is used primarily as a tool to aid in switching fusions. Switching is efficient because each element has in common 254 bp of DNA at the phoA end and 187 bp (or more) of DNA at the IS50R end of TnphoA, and switching is straightforward because individual elements encode different drug resistances. Thus, switched recombinants can be selected as drug-resistant transductants, and they can be recognized as ones that have lost the parental drug resistance and fusion phenotype. Further, switching Tnp+ elements to Tnp- elements reduces problems due to transposition that can arise in P1 crosses or cloning experiments. Some TnphoA and TnphoA' elements cause polar mutations, while others provide an outward promoter for downstream transcription. This feature is especially useful in the determination of operon structures. Strategies for the use of TnphoA and TnphoA' elements in gene analysis are also described

Involvement of phosphotransacetylase, acetate kinase, and acetyl phosphate synthesis in control of the phosphate regulon in Escherichia coli.

Two controls of the phosphate (PHO) regulon require sensor proteins that are protein kinases that phosphorylate the regulator, PhoB, which in turn activates transcription only when phosphorylated. Pi control requires the Pi sensor PhoR; the other control is Pi independent and requires the sensor CreC (formerly called PhoM). Here we describe an additional control of the PHO regulon which is Pi independent and requires neither PhoR nor CreC. This control is regulated by a two-step pathway in carbon metabolism in which acetyl coenzyme A, Pi, and ADP are converted into acetate, coenzyme A, and ATP via the enzymes phosphotransacetylase (Pta) and acetate kinase (AckA). It responds to the synthesis of acetyl phosphate, an intermediate in the Pta-AckA pathway. Since the synthesis of acetyl phosphate via this pathway leads to the incorporation of Pi into ATP, the primary phosphoryl donor in metabolism, we propose that a regulatory coupling(s) may exist between the PHO regulon, which encodes genes for Pi uptake, and genes for enzymes in central metabolism for incorporation of Pi into ATP. Regulatory interactions of this sort may be important in global control. Further, it provides a functional basis for the concept of cross-regulation in the PHO regulon. This is also the first evidence that acetyl phosphate may have a role as an effector of gene regulation