Matrix stiffness regulates endosomal escape of uropathogenic E. coli.

Uropathogenic E. coli (UPEC) infection in vivo is characterized by invasion of bladder umbrella epithelial cells followed by endosomal escape and proliferation in the cytoplasm to form intracellular bacterial communities. By contrast, UPEC infection in tissue culture models results in bacteria being trapped within Lamp1-positive endosomes where proliferation is limited. Pharmacological disruption of the actin cytoskeleton has been shown to facilitate UPEC endosomal escape in vitro and extracellular matrix stiffness is a well-characterized physiological regulator of actin dynamics; therefore, we hypothesized that substrate stiffness may play a role in UPEC endosomal escape. Using functionalized polyacrylamide substrates, we found that at physiological stiffness, UPEC escaped the endosome and proliferated rapidly in the cytoplasm of bladder epithelial cells. Dissection of the cytoskeletal signaling pathway demonstrated that inhibition of the Rho GTPase RhoB or its effector PRK1 was sufficient to increase cytoplasmic bacterial growth and that RhoB protein level was significantly reduced at physiological stiffness. Our data suggest that tissue stiffness is a critical regulator of intracellular bacterial growth. Due to the ease of doing genetic and pharmacological manipulations in cell culture, this model system may provide a useful tool for performing mechanistic studies on the intracellular life cycle of uropathogens.

Enhanced expression of the bgl operon of Escherichia coli in the stationary phase.

The bgl operon is silent and uninducible in wild-type strains of Escherichia coli and requires mutational activation for optimal expression. We show that transcription from the wild-type and the activated bgl promoter exhibits a growth phase-dependent enhancement that is highest in the stationary phase. We have assessed the effect of mutations in rpoS, crl, hns, leuO and bglJ, known to regulate bgl expression, on the growth phase-dependent increase in bgl activity. These studies show that this increase is greater in the absence of wild-type rpoS and/or crl. Our studies also indicate that while BglJ has a moderate effect on the expression of the bgl operon in the stationary phase in the absence of rpoS/crl, the modest increase in LeuO concentration in the stationary phase is insufficient to affect transcription from the bgl promoter. Measurements of the fitness of strains carrying the wild type or a null allele of crl showed that, while the strain deleted for crl exhibited a growth advantage over the crl+ strain in an rpoS+ background, it showed a low-level disadvantage in the presence of an rpoS allele that results in attenuated RpoS expression. Possible physiological implications of these results are discussed.

Perspective: Adhesion Mediated Signal Transduction in Bacterial Pathogens.

During the infection process, pathogenic bacteria undergo large-scale transcriptional changes to promote virulence and increase intrahost survival. While much of this reprogramming occurs in response to changes in chemical environment, such as nutrient availability and pH, there is increasing evidence that adhesion to host-tissue can also trigger signal transduction pathways resulting in differential gene expression. Determining the molecular mechanisms of adhesion-mediated signaling requires disentangling the contributions of chemical and mechanical stimuli. Here we highlight recent work demonstrating that surface attachment drives a transcriptional response in bacterial pathogens, including uropathogenic Escherichia coli (E. coli), and discuss the complexity of experimental design when dissecting the specific role of adhesion-mediated signaling during infection.

Identification of novel stage-specific genetic requirements through whole genome transcription profiling of Vibrio cholerae biofilm development.

Bacterial biofilm formation has been described as a developmental process. This process may be divided into three stages: the planktonic stage, the monolayer stage and the biofilm stage. Bacteria in the planktonic stage are not attached to each other or to a surface; bacteria in the monolayer stage are attached to surfaces as single cells; and bacteria in the biofilm stage are attached to surfaces as cellular aggregates. In a study limited to the Vibrio cholerae flaA, mshA and vps genes, we previously demonstrated that transcription in monolayer cells is distinct from that in biofilm cells and that the genetic requirements of monolayer formation are distinct from those of biofilm formation. In this work, we sought to identify additional stage-specific genetic requirements through microarray analysis of the V. cholerae transcriptome during biofilm development. These studies demonstrated unique patterns of transcription in the planktonic, monolayer and biofilm stages of biofilm development. Based on our microarray results, we selected cheY-3 as well as two previously uncharacterized genes, bap1 and leuO, for targeted mutation. The DeltacheY-3 mutant displayed a defect in monolayer but not biofilm formation, suggesting that chemotaxis plays a stage-specific role in formation of the V. cholerae monolayer. Mutants carrying deletions in bap1 and leuO formed monolayers that were indistinguishable from those formed by wild-type V. cholerae. In contrast, these mutants displayed greatly decreased biofilm accumulation. Our microarray analyses document modulation of the transcriptome of V. cholerae as it progresses through the stages in biofilm development. These studies demonstrate that microarray analysis of the transcriptome of biofilm development may greatly accelerate the discovery of novel targets for stage-specific inhibition of biofilm development.

Differential spectrum of mutations that activate the Escherichia coli bgl operon in an rpoS genetic background.

The bgl promoter is silent in wild-type Escherichia coli under standard laboratory conditions, and as a result, cells exhibit a beta-glucoside-negative (Bgl-) phenotype. Silencing is brought about by negative elements that flank the promoter and include DNA structural elements and sequences that interact with the nucleoid-associated protein H-NS. Mutations that confer a Bgl+ phenotype arise spontaneously at a detectable frequency. Transposition of DNA insertion elements within the regulatory locus, bglR, constitutes the major class of activating mutations identified in laboratory cultures. The rpoS-encoded sigmaS, the stationary-phase sigma factor, is involved in both physiological as well as genetic changes that occur in the cell under stationary-state conditions. In an attempt to see if the rpoS status of the cell influences the nature of the mutations that activate the bgl promoter, we analyzed spontaneously arising Bgl+ mutants in rpoS+ and rpoS genetic backgrounds. We show that the spectrum of activating mutations in rpoS cells is different from that in rpoS+ cells. Unlike rpoS+ cells, where insertions in bglR are the predominant activating mutations, mutations in hns make up the majority in rpoS cells. The physiological significance of these differences is discussed in the context of survival of natural populations of E. coli.

Genetic evidence that the Vibrio cholerae monolayer is a distinct stage in biofilm development.

Biofilm development is conceived as a developmental process in which free swimming cells attach to a surface, first transiently and then permanently, as a single layer. This monolayer of immobilized cells gives rise to larger cell clusters that eventually develop into the biofilm, a three-dimensional structure consisting of large pillars of bacteria interspersed with water channels. Previous studies have shown that efficient development of the Vibrio cholerae biofilm requires a combination of pili, flagella and exopolysaccharide. Little is known, however, regarding the requirements for monolayer formation by wild-type V. cholerae. In this work, we have isolated the wild-type V. cholerae monolayer and demonstrated that the environmental signals, bacterial structures, and transcription profiles that induce and stabilize the monolayer state are unique. Cells in a monolayer are specialized to maintain their attachment to a surface. The surface itself activates mannose-sensitive haemagglutinin type IV pilus (MSHA)-mediated attachment, which is accompanied by repression of flagellar gene transcription. In contrast, cells in a biofilm are specialized to maintain intercellular contacts. Progression to this stage occurs when exopolysaccharide synthesis is induced by environmental monosaccharides. We propose a model for biofilm development in natural environments in which cells form a stable monolayer on a surface. As biotic surfaces are degraded with subsequent release of carbohydrates, the monolayer develops into a biofilm.