Intrinsic and Extrinsic Regulation of Type III Secretion Gene Expression in Pseudomonas Aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen that is particularly problematic in the healthcare setting where it is a frequent cause of pneumonia, bloodstream, and urinary tract infections. An important determinant of P. aeruginosa virulence is a type III secretion system (T3SS). T3SS-dependent intoxication is a complex process that minimally requires binding of P. aeruginosa to host cells, injection of the cytotoxic effector proteins through the host cell plasma membrane, and induction of T3SS gene expression. The latter process, referred to as contact-dependent expression, involves a well-characterized regulatory cascade that activates T3SS gene expression in response to host cell contact. Although host cell contact is a primary activating signal for T3SS gene expression, the involvement of multiple membrane-bound regulatory systems indicates that additional environmental signals also play a role in controlling expression of the T3SS. These regulatory systems coordinate T3SS gene expression with many other cellular activities including motility, mucoidy, polysaccharide production, and biofilm formation. The signals to which the organism responds are poorly understood but many seem to be coupled to the metabolic state of the cell and integrated within a master circuit that assimilates informational signals from endogenous and exogenous sources. Herein we review progress toward unraveling this complex circuitry, provide analysis of the current knowledge gaps, and highlight potential areas for future studies. Complete understanding of the regulatory networks that control T3SS gene expression will maximize opportunities for the development of strategies to treat P. aeruginosa infections

Comparative genetics of seven plants endemic to Florida’s Lake Wales Ridge

Here we submit that mathematical tools used in population viability analysis can be used in conjunction with floristic and faunistic surveys to predict changes in biogeographic range. We illustrate our point by summarizing the results of a demographic study of Lobelia boykinii. In this study we used deterministic and stochastic matrix models to estimate the growth rate and to predict the time to extinction for three populations growing in the Carolina bays. The stochastic model better discriminated among the fates of the three populations. It predicted extinction for two populations in the next 25 years but no extinction of the third population for at least 50 years. Probability of extinction is likely correlated with hydrologic regime and fire frequency of the bay in which a population is found. The stochastic model could be combined with information about the geographic distribution of L. boykinii habitats to predict short-term biogeographic change

Genetic Variation in Past and Current Landscapes: Conservation Implications Based on Six Endemic Florida Scrub Plants

If genetic variation is often positively correlated with population sizes and the presence of nearby populations and suitable habitats, landscape proxies could inform conservation decisions without genetic analyses. For six Florida scrub endemic plants (Dicerandra frutescens, Eryngium cuneifolium, Hypericum cumulicola, Liatris ohlingerae, Nolina brittoniana, and Warea carteri), we relate two measures of genetic variation, expected heterozygosity and alleles per polymorphic locus (APL), to population size and landscape variables. Presettlement areas were estimated based on soil preferences and GIS soils maps. Four species showed no genetic patterns related to population or landscape factors. The other two species showed significant but inconsistent patterns. For Liatris ohlingerae, APL was negatively related to population density and weakly, positively related to remaining presettlement habitat within 32 km. For Nolina brittoniana, APL increased with population size. The rather weak effects of population area/size and both past and current landscape structures suggest that genetic variation needs to be directly measured and not inferred for conservation planning

The Pseudomonas aeruginosa Magnesium Transporter MgtE Inhibits Transcription of the Type III Secretion System

Pseudomonas aeruginosa is an opportunistic pathogen that causes life-long pneumonia in individuals with cystic fibrosis (CF). These long-term infections are maintained by bacterial biofilm formation in the CF lung. We have recently developed a model of P. aeruginosa biofilm formation on cultured CF airway epithelial cells. Using this model, we discovered that mutation of a putative magnesium transporter gene, called mgtE, led to increased cytotoxicity of P. aeruginosa toward epithelial cells. This altered toxicity appeared to be dependent upon expression of the type III secretion system (T3SS). In this study, we found that mutation of mgtE results in increased T3SS gene transcription. Through epistasis analyses, we discovered that MgtE influences the ExsE-ExsC-ExsD-ExsA gene regulatory system of T3SS by either directly or indirectly inhibiting ExsA activity. While variations in calcium levels modulate T3SS gene expression in P. aeruginosa, we found that addition of exogenous magnesium did not inhibit T3SS activity. Furthermore, mgtE variants that were defective for magnesium transport could still complement the cytotoxicity effect. Thus, the magnesium transport function of MgtE does not fully explain the regulatory effects of MgtE on cytotoxicity. Overall, our results indicate that MgtE modulates expression of T3SS genes

The Impact of ExoS on Pseudomonas Aeruginosa Internalization by Epithelial Cells Is Independent of fleQ and Correlates with Bistability of Type Three Secretion System Gene Expression

Pseudomonas aeruginosa is internalized into multiple types of epithelial cell in vitro and in vivo and yet is often regarded as an exclusively extracellular pathogen. Paradoxically, ExoS, a type three secretion system (T3SS) effector, has antiphagocytic activities but is required for intracellular survival of P. aeruginosaand its occupation of bleb niches in epithelial cells. Here, we addressed mechanisms for this dichotomy using invasive (ExoS-expressing) P. aeruginosaand corresponding effector-null isogenic T3SS mutants, effector-null mutants of cytotoxic P. aeruginosa with and without ExoS transformation, antibiotic exclusion assays, and imaging using a T3SS-GFP reporter. Except for effector-null PA103, all strains were internalized while encoding ExoS. Intracellular bacteria showed T3SS activation that continued in replicating daughter cells. Correcting the fleQmutation in effector-null PA103 promoted internalization by \u3e10-fold with or without ExoS. Conversely, mutating fleQ in PAO1 reduced internalization by \u3e10-fold, also with or without ExoS. Effector-null PA103 remained less well internalized than PAO1 matched for fleQ status, but only with ExoS expression, suggesting additional differences between these strains. Quantifying T3SS activation using GFP fluorescence and quantitative reverse transcription-PCR (qRT-PCR) showed that T3SS expression was hyperinducible for strain PA103ΔexoUT versus other isolates and was unrelated to fleQ status. These findings support the principle that P. aeruginosa is not exclusively an extracellular pathogen, with internalization influenced by the relative proportions of T3SS-positive and T3SS-negative bacteria in the population during host cell interaction. These data also challenge current thinking about T3SS effector delivery into host cells and suggest that T3SS bistability is an important consideration in studying P. aeruginosa pathogenesis. IMPORTANCE P. aeruginosa is often referred to as an extracellular pathogen, despite its demonstrated capacity to invade and survive within host cells. Fueling the confusion, P. aeruginosa encodes T3SS effectors with anti-internalization activity that, paradoxically, play critical roles in intracellular survival. Here, we sought to address why ExoS does not prevent internalization of the P. aeruginosastrains that natively encode it. Results showed that ExoS exerted unusually strong anti-internalization activity under conditions of expression in the effector-null background of strain PA103, often used to study T3SS effector activity. Inhibition of internalization was associated with T3SS hyperinducibility and ExoS delivery. PA103 fleQ mutation, preventing flagellar assembly, further reduced internalization but did so independently of ExoS. The results revealed intracellular T3SS expression by all strains and suggested that T3SS bistability influences P. aeruginosa internalization. These findings reconcile controversies in the literature surrounding P. aeruginosa internalization and support the principle that P. aeruginosa is not exclusively an extracellular pathogen

Vfr Directly Activates exsA Transcription To Regulate Expression of the Pseudomonas aeruginosa Type III Secretion System

ABSTRACT The Pseudomonas aeruginosa cyclic AMP (cAMP)-Vfr system (CVS) is a global regulator of virulence gene expression. Regulatory targets include type IV pili, secreted proteases, and the type III secretion system (T3SS). The mechanism by which CVS regulates T3SS gene expression remains undefined. Single-cell expression studies previously found that only a portion of the cells within a population express the T3SS under inducing conditions, a property known as bistability. We now report that bistability is altered in a vfr mutant, wherein a substantially smaller fraction of the cells express the T3SS relative to the parental strain. Since bistability usually involves positive-feedback loops, we tested the hypothesis that virulence factor regulator (Vfr) regulates the expression of exsA . ExsA is the central regulator of T3SS gene expression and autoregulates its own expression. Although exsA is the last gene of the exsCEBA polycistronic mRNA, we demonstrate that Vfr directly activates exsA transcription from a second promoter (P exsA ) located immediately upstream of exsA . P exsA promoter activity is entirely Vfr dependent. Direct binding of Vfr to a P exsA promoter probe was demonstrated by electrophoretic mobility shift assays, and DNase I footprinting revealed an area of protection that coincides with a putative Vfr consensus-binding site. Mutagenesis of that site disrupted Vfr binding and P exsA promoter activity. We conclude that Vfr contributes to T3SS gene expression through activation of the P exsA promoter, which is internal to the previously characterized exsCEBA operon. IMPORTANCE Vfr is a cAMP-dependent DNA-binding protein that functions as a global regulator of virulence gene expression in Pseudomonas aeruginosa . Regulation by Vfr allows for the coordinate production of related virulence functions, such as type IV pili and type III secretion, required for adherence to and intoxication of host cells, respectively. Although the molecular mechanism of Vfr regulation has been defined for many target genes, a direct link between Vfr and T3SS gene expression had not been established. In the present study, we report that Vfr directly controls exsA transcription, the master regulator of T3SS gene expression, from a newly identified promoter located immediately upstream of exsA

Primary and Secondary Sequence Structure Requirements for Recognition and Discrimination of Target RNAs by Pseudomonas aeruginosa RsmA and RsmF

ABSTRACT CsrA family RNA-binding proteins are widely distributed in bacteria and regulate gene expression at the posttranscriptional level. Pseudomonas aeruginosa has a canonical member of the CsrA family (RsmA) and a novel, structurally distinct variant (RsmF). To better understand RsmF binding properties, we performed parallel systematic evolution of ligands by exponential enrichment (SELEX) experiments for RsmA and RsmF. The initial target library consisted of 62-nucleotide (nt) RNA transcripts with central cores randomized at 15 sequential positions. Most targets selected by RsmA and RsmF were the expected size and shared a common consensus sequence (CANGGAYG) that was positioned in a hexaloop region of the stem-loop structure. RsmA and RsmF also selected for longer targets (≥96 nt) that were likely generated by rare PCR errors. Most of the long targets contained two consensus-binding sites. Representative short (single consensus site) and long (two consensus sites) targets were tested for RsmA and RsmF binding. Whereas RsmA bound the short targets with high affinity, RsmF was unable to bind the same targets. RsmA and RsmF both bound the long targets. Mutation of either consensus GGA site in the long targets reduced or eliminated RsmF binding, suggesting a requirement for two tandem binding sites. Conversely, RsmA bound long targets containing only a single GGA site with unaltered affinity. The RsmF requirement for two binding sites was confirmed with tssA1 , an in vivo regulatory target of RsmA and RsmF. Our findings suggest that RsmF binding requires two GGA-containing sites, while RsmA binding requirements are less stringent. IMPORTANCE The CsrA family of RNA-binding proteins is widely conserved in bacteria and plays important roles in the posttranscriptional regulation of protein synthesis. P. aeruginosa has two CsrA proteins, RsmA and RsmF. Although RsmA and RsmF share a few RNA targets, RsmF is unable to bind to other targets recognized by RsmA. The goal of the present study was to better understand the basis for differential binding by RsmF. Our data indicate that RsmF binding requires target RNAs with two consensus-binding sites, while RsmA recognizes targets with just a single binding site. This information should prove useful to future efforts to define the RsmF regulon and its contribution to P. aeruginosa physiology and virulence

The AlgZR Two-Component System Recalibrates the RsmAYZ Posttranscriptional Regulatory System To Inhibit Expression of the Pseudomonas aeruginosa Type III Secretion System

Pseudomonas aeruginosa causes chronic airway infections in cystic fibrosis (CF) patients. A classic feature of CF airway isolates is the mucoid phenotype. Mucoidy arises through mutation of the mucA anti-sigma factor and subsequent activation of the AlgU regulon. Inactivation of mucA also results in reduced expression of the Vfr transcription factor. Vfr regulates several important virulence factors, including a type III secretion system (T3SS). In the present study, we report that ExsA expression, the master regulator of T3SS gene expression, is further reduced in mucA mutants through a Vfr-independent mechanism involving the RsmAYZ regulatory system. RsmA is an RNA binding protein required for T3SS gene expression. Genetic experiments suggest that the AlgZR two-component system, part of the AlgU regulon, inhibits ExsA expression by increasing the expression of RsmY and RsmZ, two small noncoding RNAs that sequester RsmA from target mRNAs. Epistasis analyses revealed that increasing the concentration of free RsmA, through either rsmYZ deletion or increased RsmA expression, partially restored T3SS gene expression in the mucA mutant. Furthermore, increasing RsmA availability in combination with Vfr complementation fully restored T3SS expression. Recalibration of the RsmAYZ system by AlgZR, however, did not alter the expression of other selected RsmA-dependent targets. We account for this observation by showing that ExsA expression is more sensitive to changes in free RsmA than other members of the RsmA regulon. Together, these data indicate that recalibration of the RsmAYZ system partially accounts for reduced T3SS gene expression in mucA mutants

Pressure reversal study through tensile tests

This paper is a summary of the results from a study of the variables related to pressure reversal and was sponsored by the US Department of Transportation, Office of Pipeline Safety. The circumferential pipe stress, which is the most significant variable in pressure reversal, was examined by using tensile specimens and then relating the results to pressurized pipe. A model is proposed that gives some insight into how pressure reversal can be minimized when a section of pipe is being hydrotested. Twenty tensile specimens from X-42 electric resistance welded (ERW) pipe and twenty specimens from X-52 ERW pipe were tested. Each specimen had a machined flaw. The flaw regions were monitored using strain gages and photoelasticity. These tensile tests represent the first phase of a research effort to examine and understand the variables related to pressure reversal. The second phase of this effort will be with pipe specimens and presently is in progress