Pseudomonas aeruginosa, an important nosocomial pathogen of humans, expresses a type III secretion system that is required for virulence. Previous studies demonstrated that the lung-virulent strain PA103 has the capacity to be either cytotoxic or invasive. Analyses of mutants suggest that PA103 delivers a negative regulator of invasion, or anti-internalization factor, to host cells via a type III secretion system. In this work we show that the type III secreted protein ExoT inhibits the internalization of PA103 by polarized epithelial cells (Madin-Darby canine kidney cells) and J774.1 macrophage-like cells. ExoS, which is closely related to ExoT but has additional ADP-ribosylating activity, can substitute for ExoT as an anti-internalization factor. ExoT contains a signature arginine finger domain found in GTPase-activating proteins. Mutation of the conserved arginine in ExoT diminished its anti-internalization activity and altered its ability to disrupt the actin cytoskeleton. Cell fractionation experiments showed that ExoT is translocated into host cells and that mutation of the arginine finger did not disrupt translocation. In a mouse model of acute pneumonia, PA103DeltaUDeltaT reached the lungs as efficiently as PA103DeltaU but showed reduced colonization of the liver. This finding suggests that the ability to resist internalization may be important for virulence in vivo.

SummaryBacterial cytochrome cbb 3 oxidases are members of the haeme-copper oxidase superfamily that are important for energy conservation by a variety of proteobacteria under oxygen-limiting conditions. The opportunistic pathogen Pseudomonas aeruginosa is unusual in possessing two operons that each potentially encode a cbb 3 oxidase ( cbb 3 -1 or cbb 3 -2). Our results demonstrate that, unlike typical enzymes of this class, the cbb 3 -1 oxidase has an important metabolic function at high oxygen tensions. In highly aerated cultures, cbb 3 -1 abundance and expression were greater than that of cbb 3 -2, and only loss of cbb 3 -1 influenced growth. Also, the activity of cbb 3 -1, not cbb 3 -2, inhibited expression of the alternative oxidase CioAB and thus influenced a signal transduction pathway much like that found in the a a a a -proteobacterium Rhodobacter sphaeroides . Cbb 3 -2 appeared to play a more significant role under oxygen limitation by nature of its increased abundance and expression compared to highly aerated cultures, and the regulation of the cbb 3 -2 operon by the putative iron-sulphur protein Anr. These results indicate that each of the two P. aeruginosa cbb 3 isoforms have assumed specialized energetic and regulatory roles.

In patients with suspected fungal pneumonia, an Aspergillus secondary metabolite signature in breath can identify individuals with IA. These results provide proof-of-concept that direct detection of exogenous fungal metabolites in breath can be used as a novel, noninvasive, pathogen-specific approach to identifying the precise microbial cause of pneumonia.

SummaryVirulence of Pseudomonas aeruginosa involves the co-ordinate expression of a range of factors including type IV pili (tfp), the type III secretion system (TTSS) and quorum sensing. Tfp are required for twitching motility, efficient biofilm formation, and for adhesion and type III secretion (TTS)-mediated damage to mammalian cells. We describe a novel gene ( fimL ) that is required for tfp biogenesis and function, for TTS and for normal biofilm development in P. aeruginosa . The predicted product of fimL is homologous to the Nterminal domain of ChpA, except that its putative histidine and threonine phosphotransfer sites have been replaced with glutamine. fimL mutants resemble vfr mutants in many aspects including increased autolysis, reduced levels of surface-assembled tfp and diminished production of type III secreted effectors. Expression of vfr in trans can complement fimL mutants. vfr transcription and production is reduced in fimL mutants whereas cAMP levels are unaffected. Deletion and insertion mutants of fimL frequently revert to wild-type phenotypes suggesting that an extragenic suppressor mutation is able to overcome the loss of fimL . vfr transcription and production, as well as cAMP levels, are elevated in these revertants, while Pseudomonas quinolone signal (PQS) production is reduced. These results suggest that the site(s) of spontaneous mutation is in a gene(s) which lies upstream of vfr transcription, cAMP, production, and PQS synthesis. Our studies indicate that Vfr and FimL are components of intersecting pathways that control twitching motility, TTSS and autolysis in P. aeruginosa .

Anoxygenic photosynthetic growth of Rhodobacter sphaeroides, a member of the ␣ subclass of the class Proteobacteria, requires the response regulator PrrA. PrrA and the sensor kinase PrrB are part of a twocomponent signaling pathway that influences a wide range of processes under oxygen-limited conditions. In this work we characterized the pathway of transcription activation by PrrB and PrrA by purifying these proteins, analyzing them in vitro, and characterizing a mutant PrrA protein in vivo and in vitro. When purified, a soluble transmitter domain of PrrB (cPrrB) could autophosphorylate, rapidly transfer phosphate to PrrA, and stimulate dephosphorylation of phospho-PrrA. Unphosphorylated PrrA activated transcription from a target cytochrome c 2 gene (cycA) promoter, P2, which contained sequences from ؊73 to ؉22 relative to the transcription initiation site. However, phosphorylation of PrrA increased its activity since activation of cycA P2 was enhanced up to 15-fold by treatment with the low-molecular-weight phosphodonor acetyl phosphate. A mutant PrrA protein containing a single amino acid substitution in the presumed phosphoacceptor site (PrrA-D63A) was not phosphorylated in vitro but also was not able to stimulate cycA P2 transcription. PrrA-D63A also had no apparent in vivo activity, demonstrating that aspartate 63 is necessary both for the function of PrrA and for its phosphorylation-dependent activation. The cellular level of wild-type PrrA was negatively autoregulated so that less PrrA was present in the absence of oxygen, conditions in which the activities of many PrrA target genes increase. PrrA-D63A failed to repress expression of the prrA gene under anaerobic conditions, suggesting that this single amino acid change also eliminated PrrA function in vivo.

Summary The facultative anaerobe Pseudomonas aeruginosa has multiple aerobic electron transport pathways, one of which is terminated by a cyanide‐insensitive oxidase (CIO). This study characterizes a P. aeruginosa two‐component system that regulates CIO production. The response regulator of this system (RoxR) has significant amino acid sequence similarity to PrrA of Rhodobacter sphaeroides and related proteins in other α‐proteobacteria. In heterologous complementation analysis, R. sphaeroides PrrA rescued the growth defect of a P. aeruginosa mutant lacking RoxR, and RoxR enabled photosynthetic growth of an R. sphaeroides PrrA mutant. Also, RoxR could substitute for PrrA in activating transcription in vitro, demonstrating that these proteins are functional homologues. P. aeruginosa strains lacking RoxR or the sensor kinase (RoxS) were more sensitive than wild type to the respiratory inhibitors cyanide and azide. The phenotypes of these mutant strains correlated with reduced cyanide‐insensitive O2 utilization and less cyanide‐dependent expression of the locus encoding the CIO (cioAB). The ability of purified RoxR to bind to the cioAB promoter region also suggests that this protein acts directly to regulate cioAB transcription. Therefore, RoxR appears to play a role in regulating the transcription of loci for P. aeruginosa energy‐generating enzymes similar to that of its homologues in α‐proteobacteria.

Type IV pili of the opportunistic pathogen Pseudomonas aeruginosa mediate twitching motility and act as receptors for bacteriophage infection. They are also important bacterial adhesins, and nonpiliated mutants of P. aeruginosa have been shown to cause less epithelial cell damage in vitro and have decreased virulence in animal models. This finding raises the question as to whether the reduction in cytotoxicity and virulence of nonpiliated P. aeruginosa mutants are primarily due to defects in cell adhesion or loss of twitching motility, or both. This work describes the role of PilT and PilU, putative nucleotide-binding proteins involved in pili function, in mediating epithelial cell injury in vitro and virulence in vivo. Mutants of pilT and pilU retain surface pili but have lost twitching motility. In three different epithelial cell lines, pilT or pilU mutants of the strain PAK caused less cytotoxicity than the wild-type strain but more than isogenic, nonpiliated pilA or rpoN mutants. ThepilT and pilU mutants also showed reduced association with these same epithelial cell lines compared both to the wild type, and surprisingly, to a pilA mutant. In a mouse model of acute pneumonia, the pilT and pilUmutants showed decreased colonization of the liver but not of the lung relative to the parental strain, though they exhibited no change in the ability to cause mortality. These results demonstrate that pilus function mediated by PilT and PilU is required for in vitro adherence and cytotoxicity toward epithelial cells and is important in virulence in vivo.

The drug development pipeline is severely limited by a lack of reliable tools for prediction of human clinical safety and efficacy profiles for compounds at the pre-clinical stage. Here we present the design and implementation of a platform technology comprising multiple human cell-based tissue models in a portable and reconfigurable format that supports individual organ function and crosstalk for periods of up to several weeks. Organ perfusion and crosstalk are enabled by a precision flow control technology based on electromagnetic actuators embedded in an arrayed format on a microfluidic platform. We demonstrate two parallel circuits of connected airway and liver modules on a platform containing 62 electromagnetic microactuators, with precise and controlled flow rates as well as functional biological metrics over a two week time course. Technical advancements enabled by this platform include the use of non-sorptive construction materials, enhanced scalability, portability, flow control, and usability relative to conventional flow control modes (such as capillary action, pressure heads, or pneumatic air lines), and a reconfigurable and modular organ model format with common fluidic port architecture. We demonstrate stable biological function for multiple pairs of airway-liver models for periods of 2 weeks in the platform, with precise control over fluid levels, temperature, flow rate and oxygenation in order to support relevant use cases involving drug toxicity, efficacy testing, and organ-organ interaction.