Candida albicans Isolates from the Gut of Critically Ill Patients Respond to Phosphate Limitation by Expressing Filaments and a Lethal Phenotype

Candida albicans is an opportunistic pathogen that proliferates in the intestinal tract of critically ill patients where it continues to be a major cause of infectious-related mortality. The precise cues that shift intestinal C. albicans from its ubiquitous indolent colonizing yeast form to an invasive and lethal filamentous form remain unknown. We have previously shown that severe phosphate depletion develops in the intestinal tract during extreme physiologic stress and plays a major role in shifting intestinal Pseudomonas aeruginosa to express a lethal phenotype via conserved phosphosensory-phosphoregulatory systems. Here we studied whether phosphate dependent virulence expression could be similarly demonstrated for C. albicans. C. albicans isolates from the stool of critically ill patients and laboratory prototype strains (SC5314, BWP17, SN152) were evaluated for morphotype transformation and lethality against C. elegans and mice during exposure to phosphate limitation. Isolates ICU1 and ICU12 were able to filament and kill C. elegans in a phosphate dependent manner. In a mouse model of intestinal phosphate depletion (30% hepatectomy), direct intestinal inoculation of C. albicans caused mortality that was prevented by oral phosphate supplementation. Prototype strains displayed limited responses to phosphate limitation; however, the pho4Δ mutant displayed extensive filamentation during low phosphate conditions compared to its isogenic parent strain SN152, suggesting that mutation in the transcriptional factor Pho4p may sensitize C. albicans to phosphate limitation. Extensive filamentation was also observed in strain ICU12 suggesting that this strain is also sensitized to phosphate limitation. Analysis of the sequence of PHO4 in strain ICU12, its transcriptional response to phosphate limitation, and phosphatase assays confirmed that ICU12 demonstrates a profound response to phosphate limitation. The emergence of strains of C. albicans with marked responsiveness to phosphate limitation may represent a fitness adaptation to the complex and nutrient scarce environment typical of the gut of a critically ill patient

Pseudomonas aeruginosa Overrides the Virulence Inducing Effect of Opioids When It Senses an Abundance of Phosphate

The gut during critical illness represents a complex ecology dominated by the presence of healthcare associated pathogens, nutrient scarce conditions, and compensatory host stress signals. We have previously identified key environmental cues, opioids and phosphate depletion that independently activate the virulence of Pseudomonas aeruginosa. Opioids induce quinolone signal production (PQS), whereas phosphate depletion leads to a triangulated response between MvfR-PQS, pyoverdin, and phosphosensory/phosphoregulatory systems (PstS-PhoB). Yet how P. aeruginosa manages its response to opioids during nutrient scarce conditions when growth is limited and a quorum is unlikely to be achieved is important in the context of pathogenesis in gut during stress. To mimic this environment, we created nutrient poor conditions and exposed P. aeruginosa PAO1 to the specific k-opioid receptor agonist U-50,488. Bacterial cells exposed to the k-opioid expressed a striking increase in virulence- and multi-drug resistance-related genes that correlated to a lethal phenotype in C. elegans killing assays. Under these conditions, HHQ, a precursor of PQS, rather than PQS itself, became the main inducer for pqsABCDE operon expression. P. aeruginosa virulence expression in response to k-opioids required PqsE since ΔPqsE was attenuated in its ability to activate virulence- and efflux pumps-related genes. Extracellular inorganic phosphate completely changed the transcriptional response of PAO1 to the k- opioid preventing pqsABCDE expression, the activation of multiple virulence- and efflux pumps-related genes, and the ability of P. aeruginosa to kill C. elegans. These results indicate that when P. aeruginosa senses resource abundance in the form of phosphate, it overrides its response to compensatory host signals such as opioids to express a virulent and lethal phenotype. These studies confirm a central role for phosphate in P. aeruginosa virulence that might be exploited to design novel anti- virulence strategies

Induction of aromatic ring: cleavage dioxygenases in Stenotrophomonas maltophilia strain KB2 in cometabolic systems

Stenotrophomonas maltophilia KB2 is known to produce different enzymes of dioxygenase family. The aim of our studies was to determine activity of these enzymes after induction by benzoic acids in cometabolic systems with nitrophenols. We have shown that under cometabolic conditions KB2 strain degraded 0.25–0.4 mM of nitrophenols after 14 days of incubation. Simultaneously degradation of 3 mM of growth substrate during 1–3 days was observed depending on substrate as well as cometabolite used. From cometabolic systems with nitrophenols as cometabolites and 3,4-dihydroxybenzoate as a growth substrate, dioxygenases with the highest activity of protocatechuate 3,4-dioxygenase were isolated. Activity of catechol 1,2- dioxygenase and protocatechuate 4,5-dioxygenase was not observed. Catechol 2,3-dioxygenase was active only in cultures with 4-nitrophenol. Ability of KB2 strain to induce and synthesize various dioxygenases depending on substrate present in medium makes this strain useful in bioremediation of sites contaminated with different aromatic compounds

Structural basis for native agonist and synthetic inhibitor recognition by the Pseudomonas aeruginosa quorum sensing regulator PqsR (MvfR)

Bacterial populations co-ordinate gene expression collectively through quorum sensing (QS), a cell-to-cell communication mechanism employing diffusible signal molecules. The LysR-type transcriptional regulator (LTTR) protein PqsR (MvfR) is a key component of alkyl-quinolone (AQ)-dependent QS in Pseudomonas aeruginosa. PqsR is activated by 2-alkyl-4-quinolones including the Pseudomonas quinolone signal (PQS; 2-heptyl-3-hydroxy-4(1H)-quinolone), its precursor 2-heptyl-4- hydroxyquinoline (HHQ) and their C9 congeners, 2-nonyl-3-hydroxy-4(1H)-quinolone (C9-PQS) and 2-nonyl-4-hydroxyquinoline (NHQ). These drive the autoinduction of AQ biosynthesis and the up-regulation of key virulence determinants as a function of bacterial population density. Consequently, PqsR constitutes a potential target for novel antibacterial agents which attenuate infection through the blockade of virulence. Here we present the crystal structures of the PqsR co-inducer binding domain (CBD) and a complex with the native agonist NHQ. We show that the structure of the PqsR CBD has an unusually large ligand-binding pocket in which a native AQ agonist is stabilized entirely by hydrophobic interactions. Through a ligand-based design strategy we synthesized and evaluated a series of 50 AQ and novel quinazolinone (QZN) analogues and measured the impact on AQ biosynthesis, virulence gene expression and biofilm development. The simple exchange of two isosteres (OH for NH2) switches a QZN agonist to an antagonist with a concomitant impact on the induction of bacterial virulence factor production. We also determined the complex crystal structure of a QZN antagonist bound to PqsR revealing a similar orientation in the ligand binding pocket to the native agonist NHQ. This structure represents the first description of an LTTR-antagonist complex. Overall these studies present novel insights into LTTR ligand binding and ligand-based drug design and provide a chemical scaffold for further anti-P. aeruginosa virulence drug development by targeting the AQ receptor PqsR

Local Inflammation Induces Complement Crosstalk Which Amplifies the Antimicrobial Response

By eliciting inflammatory responses, the human immunosurveillance system notably combats invading pathogens, during which acute phase proteins (CRP and cytokines) are elevated markedly. However, the Pseudomonas aeruginosa is a persistent opportunistic pathogen prevalent at the site of local inflammation, and its acquisition of multiple antibiotic-resistance factors poses grave challenges to patient healthcare management. Using blood samples from infected patients, we demonstrate that P. aeruginosa is effectively killed in the plasma under defined local infection-inflammation condition, where slight acidosis and reduced calcium levels (pH 6.5, 2 mM calcium) typically prevail. We showed that this powerful antimicrobial activity is provoked by crosstalk between two plasma proteins; CRP∶L-ficolin interaction led to communication between the complement classical and lectin pathways from which two amplification events emerged. Assays for C4 deposition, phagocytosis, and protein competition consistently proved the functional significance of the amplification pathways in boosting complement-mediated antimicrobial activity. The infection-inflammation condition induced a 100-fold increase in CRP∶L-ficolin interaction in a pH- and calcium-sensitive manner. We conclude that the infection-induced local inflammatory conditions trigger a strong interaction between CRP∶L-ficolin, eliciting complement-amplification pathways which are autonomous and which co-exist with and reinforce the classical and lectin pathways. Our findings provide new insights into the host immune response to P. aeruginosa infection under pathological conditions and the potential development of new therapeutic strategies against bacterial infection

A eukaryotic-type signalling system of Pseudomonas aeruginosa contributes to oxidative stress resistance, intracellular survival and virulence

<p>Abstract</p> <p>Background</p> <p>The genome of <it>Pseudomonas aeruginosa </it>contains at least three genes encoding eukaryotic-type Ser/Thr protein kinases, one of which, <it>ppkA</it>, has been implicated in <it>P. aeruginosa </it>virulence. Together with the adjacent <it>pppA </it>phosphatase gene, they belong to the type VI secretion system (H1-T6SS) locus, which is important for bacterial pathogenesis. To determine the biological function of this protein pair, we prepared a <it>pppA-ppkA </it>double mutant and characterised its phenotype and transcriptomic profiles.</p> <p>Results</p> <p>Phenotypic studies revealed that the mutant grew slower than the wild-type strain in minimal media and exhibited reduced secretion of pyoverdine. In addition, the mutant had altered sensitivity to oxidative and hyperosmotic stress conditions. Consequently, mutant cells had an impaired ability to survive in murine macrophages and an attenuated virulence in the plant model of infection. Whole-genome transcriptome analysis revealed that <it>pppA-ppkA </it>deletion affects the expression of oxidative stress-responsive genes, stationary phase σ-factor RpoS-regulated genes, and quorum-sensing regulons. The transcriptome of the <it>pppA-ppkA </it>mutant was also analysed under conditions of oxidative stress and showed an impaired response to the stress, manifested by a weaker induction of stress adaptation genes as well as the genes of the SOS regulon. In addition, expression of either RpoS-regulated genes or quorum-sensing-dependent genes was also affected. Complementation analysis confirmed that the transcription levels of the differentially expressed genes were specifically restored when the <it>pppA </it>and <it>ppkA </it>genes were expressed ectopically.</p> <p>Conclusions</p> <p>Our results suggest that in addition to its crucial role in controlling the activity of <it>P. aeruginosa </it>H1-T6SS at the post-translational level, the PppA-PpkA pair also affects the transcription of stress-responsive genes. Based on these data, it is likely that the reduced virulence of the mutant strain results from an impaired ability to survive in the host due to the limited response to stress conditions.</p

The role of P2 receptors in controlling infections by intracellular pathogens

A growing number of studies have demonstrated the importance of ATPe-signalling via P2 receptors as an important component of the inflammatory response to infection. More recent studies have shown that ATPe can also have a direct effect on infection by intracellular pathogens, by modulating membrane trafficking in cells that contain vacuoles that harbour intracellular pathogens, such as mycobacteria and chlamydiae. A conserved mechanism appears to be involved in controlling infection by both of these pathogens, as a role for phospholipase D in inducing fusion between lysosomes and the vacuoles has been demonstrated. Other P2-dependent mechanisms are most likely operative in the cases of pathogens, such as Leishmania, which survive in an acidic phagolysosomal-like compartment. ATPe may function as a ‘danger signal–that alerts the immune system to the presence of intracellular pathogens that damage the host cell, while different intracellular pathogens have evolved enzymes or other mechanisms to inhibit ATPe-mediated signalling, which should, thus, be viewed as virulence factors for these pathogens

Spatioregional assessment of the gut microbiota in experimental necrotizing pancreatitis

BACKGROUND: Infectious complications following experimental pancreatitis involve major disruptions in the gut microbiota. The aim of this study was to characterize this disruption by examining the spatioregional distribution in microbial community structure and function following experimental pancreatitis associated with pancreatic infection. METHODS: Mice were subjected to infusion of the pancreatic duct with either taurocholate to induce necrotizing pancreatitis or normal saline (control group). The spatial (lumen versus mucosa) and regional composition and function of the microbiota from the duodenum, ileum, caecum, colon, pancreas and blood were evaluated using 16S rRNA gene amplicon sequencing. RESULTS: Mice that developed necrotizing pancreatitis demonstrated a decrease in microbial richness and significantly altered microbiota in distal parts of the gastrointestinal tract, compared with controls. Among the most differentially increased taxa were the mucus-degrading Akkermansia muciniphila, and there was a decrease of butyrate-producing bacteria following pancreatitis. Application of the SourceTracker tool to the generated metadata indicated that the duodenum was the most probable source of bacteria that subsequently infected pancreatic tissue in this model. The functional prediction annotation using pathway analyses indicated a diminished capacity of the caecal microbiota to metabolize carbohydrate, and fatty and amino acids. DISCUSSION: The distal gut microbiota was significantly impacted in this model of experimental necrotizing pancreatitis. Data suggest that the duodenal microbiota might also play a role in bacterial translation and secondary infections