Salmonella biofilms program innate immunity for persistence in Caenorhabditis elegans.

The adaptive in vivo mechanisms underlying the switch in Salmonella enterica lifestyles from the infectious form to a dormant form remain unknown. We employed Caenorhabditis elegans as a heterologous host to understand the temporal dynamics of Salmonella pathogenesis and to identify its lifestyle form in vivo. We discovered that Salmonella exists as sessile aggregates, or in vivo biofilms, in the persistently infected C. elegans gut. In the absence of in vivo biofilms, Salmonella killed the host more rapidly by actively inhibiting innate immune pathways. Regulatory cross-talk between two major Salmonella pathogenicity islands, SPI-1 and SPI-2, was responsible for biofilm-induced changes in host physiology during persistent infection. Thus, biofilm formation is a survival strategy in long-term infections, as prolonging host survival is beneficial for the parasitic lifestyle.

To ∼P or Not to ∼P? Non-canonical activation by two-component response regulators.

Bacteria sense and respond to their environment through the use of two-component regulatory systems. The ability to adapt to a wide range of environmental stresses is directly related to the number of two-component systems an organism possesses. Recent advances in this area have identified numerous variations on the archetype systems that employ a sensor kinase and a response regulator. It is now evident that many orphan regulators that lack cognate kinases do not rely on phosphorylation for activation and new roles for unphosphorylated response regulators have been identified. The significance of recent findings and suggestions for further research are discussed.

RpoS activates Salmonella Typhi biofilms and drives persistence in a Zebrafish model.

The development of strategies for targeting the asymptomatic carriage of Salmonella Typhi in chronic typhoid patients has suffered owing to our basic lack of understanding of the molecular mechanisms that enable the formation of S. Typhi biofilms. Traditionally, studies have relied on cholesterol-attached biofilms formed by a closely related serovar, Typhimurium, to mimic multicellular Typhi communities formed on human gallstones. In long-term infections, S. Typhi adopts the biofilm lifestyle to persist in vivo and survive in the carrier state, ultimately leading to the spread of infections via the fecal-oral route of transmission. In the present work, we studied S. Typhi biofilms directly, applied targeted as well as genome-wide genetic approaches to uncover unique biofilm components that do not conform to the CsgD-dependent pathway as established in S. Typhimurium. We adopted a genome-wide Tn5 mutation screen in S. Typhi in gallstone-mimicking conditions and generated New Generation Sequencing libraries based on the ClickSeq technology to identify the key regulators, IraP and RpoS, and the matrix components as Sth fimbriae, Vi capsule and lipopolysaccharide. We discovered that the starvation sigma factor, RpoS, was required for the transcriptional activation of matrix-encoding genes in vitro, and for S. Typhi colonization in persistent infections in vivo, using a heterologous fish larval model. Overall, our work established a novel RpoS-driven paradigm for the formation of cholesterol-attached Typhi biofilms and emphasized the role(s) of stress signaling pathways for adaptation in chronic infections.

Diverse pathways for salicin utilization in Shigella sonnei and Escherichia coli carrying an impaired bgl operon.

Utilization of the aryl-ß-glucosides salicin or arbutin in most wild-type strains of E. coli is achieved by a single-step mutational activation of the bgl operon. Shigella sonnei, a branch of the diverse E. coli strain tree, requires two sequential mutational steps for achieving salicin utilization as the bglB gene, encoding the phospho-ß-glucosidase B, harbors an inactivating insertion. We show that in a natural isolate of S. sonnei, transcriptional activation of the gene SSO1595, encoding a phospho-ß-glucosidase, enables salicin utilization with the permease function being provided by the activated bgl operon. SSO1595 is absent in most commensal strains of E. coli, but is present in extra-intestinal pathogens as bgcA, a component of the bgc operon that enables ß-glucoside utilization at low temperature. Salicin utilization in an E. coli bglB laboratory strain also requires a two-step activation process leading to expression of BglF, the PTS-associated permease encoded by the bgl operon and AscB, the phospho-ß-glucosidase B encoded by the silent asc operon. BglF function is needed since AscF is unable to transport ß-glucosides as it lacks the IIA domain involved in phopho-relay. Activation of the asc operon in the Sal(+) mutant is by a promoter-up mutation and the activated operon is subject to induction. The pathway to achieve salicin utilization is therefore diverse in these two evolutionarily related organisms; however, both show cooperation between two silent genetic systems to achieve a new metabolic capability under selection.

Switching Lifestyles Is an in vivo Adaptive Strategy of Bacterial Pathogens.

Gram-positive and Gram-negative pathogens exist as planktonic cells only at limited times during their life cycle. In response to environmental signals such as temperature, pH, osmolality, and nutrient availability, pathogenic bacteria can adopt varied cellular fates, which involves the activation of virulence gene programs and/or the induction of a sessile lifestyle to form multicellular surface-attached communities. In Salmonella, SsrB is the response regulator which governs the lifestyle switch from an intracellular virulent state to form dormant biofilms in chronically infected hosts. Using the Salmonella lifestyle switch as a paradigm, we herein compare how other pathogens alter their lifestyles to enable survival, colonization and persistence in response to different environmental cues. It is evident that lifestyle switching often involves transcriptional regulators and their modification as highlighted here. Phenotypic heterogeneity resulting from stochastic cellular processes can also drive lifestyle variation among members of a population, although this subject is not considered in the present review.

Identification of Biofilm Inhibitors by Screening Combinatorial Libraries of Metal Oxide Thin Films.

With the rise in nosocomial infections worldwide, research on materials with an intrinsic ability to inhibit biofilm formation has been generating a great deal of interest. In the present work, we describe how thin film material libraries generated by pulsed laser deposition can be used for simultaneously screening several novel metal oxide mixtures that inhibit biofilm formation in a common human pathogen, Salmonella enterica serovar Typhimurium. We discovered that in a material library constructed using two metal oxides, the net effect on biofilm formation can be modeled as an addition of the activities of the individual oxides weighted to their relative composition at that particular point on the library. In contrast, for similar material libraries constructed using three metal oxides, there was a nonlinear relation between the amount of dominant metal oxide and the formation of Salmonella biofilms. This nonlinearity resulted in several useful metal oxide combinations that were not expected from the weighted average predictions. Our novel application will lead to the discovery of additional alternatives for creating antimicrobial surfaces.

The horizontally-acquired response regulator SsrB drives a Salmonella lifestyle switch by relieving biofilm silencing.

A common strategy by which bacterial pathogens reside in humans is by shifting from a virulent lifestyle, (systemic infection), to a dormant carrier state. Two major serovars of Salmonella enterica, Typhi and Typhimurium, have evolved a two-component regulatory system to exist inside Salmonella-containing vacuoles in the macrophage, as well as to persist as asymptomatic biofilms in the gallbladder. Here we present evidence that SsrB, a transcriptional regulator encoded on the SPI-2 pathogenicity-island, determines the switch between these two lifestyles by controlling ancestral and horizontally-acquired genes. In the acidic macrophage vacuole, the kinase SsrA phosphorylates SsrB, and SsrB~P relieves silencing of virulence genes and activates their transcription. In the absence of SsrA, unphosphorylated SsrB directs transcription of factors required for biofilm formation specifically by activating csgD (agfD), the master biofilm regulator by disrupting the silenced, H-NS-bound promoter. Anti-silencing mechanisms thus control the switch between opposing lifestyles.