Characterization of the Entner-Doudoroff pathway in Pseudomonas aeruginosa catheter-associated urinary tract infections.

Pseudomonas aeruginosa is an opportunistic nosocomial pathogen responsible for a subset of catheter-associated urinary tract infections (CAUTI). In a murine model of P. aeruginosa CAUTI, we previously demonstrated that urea within urine suppresses quorum sensing and induces the Entner-Doudoroff (E-D) pathway. The E-D pathway consists of the genes zwf, pgl, edd, and eda. Zwf and Pgl convert glucose-6-phosphate into 6-phosphogluconate. Edd hydrolyzes 6-phosphogluconate to 2-keto-3-deoxy-6-phosphogluconate (KDPG). Finally, Eda cleaves KDPG to glyceraldehyde-3-phosphate and pyruvate, which enters the citric acid cycle. Here, we generated in-frame E-D mutants in the strain PA14 and assessed their growth phenotypes on chemically defined and complex media. These E-D mutants have a growth defect when grown on glucose or gluconate as the sole carbon source, which is similar to results previously reported for PAO1 mutants lacking E-D genes. RNA-sequencing following short exposure to urine revealed minimal gene regulation differences compared to the wild type. In a murine CAUTI model, virulence testing of E-D mutants revealed that two mutants lacking zwf and pgl showed minor fitness defects. Infection with the ∆pgl strain exhibited a 20% increase in host survival, and the ∆zwf strain displayed decreased colonization of the catheter and kidneys. Consequently, our findings suggest that the E-D pathway in P. aeruginosa is dispensable in this model of CAUTI. IMPORTANCE Prior studies have shown that the Entner-Doudoroff pathway is up-regulated when Pseudomonas aeruginosa is grown in urine. Pseudomonads use the Entner-Doudoroff (E-D) pathway to metabolize glucose instead of glycolysis, which led us to ask whether this pathway is required for urinary tract infection. Here, single-deletion mutants of each gene in the pathway were tested for growth on chemically defined media with single-carbon sources as well as complex media. The effect of each mutant on global gene expression in laboratory media and urine was characterized. The virulence of these mutants in a murine model of catheter-associated urinary tract infection revealed that these mutants had similar levels of colonization indicating that glucose is not the primary carbon source utilized in the urinary tract.

Characterization of the Entner-Douderoff Pathway in Pseudomonas aeruginosa Catheter-associated Urinary Tract Infections.

Pseudomonas aeruginosa is an opportunistic nosocomial pathogen responsible for catheter-associated urinary tract infections (CAUTI). In a murine model of P. aeruginosa CAUTI, we previously demonstrated that urea within urine suppresses quorum sensing and induces the Entner-Douderoff (E-D) pathway. The E-D pathway consists of the genes zwf, pgl, edd, and eda. Zwf and Pgl convert glucose-6-phosphate into 6-phosphogluconate. Edd hydrolyzes 6-phosphogluconate to 2-keto-3-deoxy-6-phosphogluconate (KDPG). Finally, Eda cleaves KDPG to glyceraldehyde-3-phosphate and pyruvate, which enters the citric acid cycle. Here, we generated in-frame E-D mutants in strain PA14 and assessed their growth phenotypes on chemically defined media. These E-D mutants have a growth defect when grown on glucose or gluconate as sole carbon source which are similar to results previously reported for PAO1 mutants lacking E-D genes. RNA-sequencing following short exposure to urine revealed minimal gene regulation differences compared to the wild type. In a murine CAUTI model, virulence testing of E-D mutants revealed that two mutants lacking zwf and pgl showed minor fitness defects. Infection with the ∆pgl strain exhibited a 20% increase in host survival, and the ∆zwf strain displayed decreased colonization of the catheter and kidneys. Consequently, our findings suggest that the E-D pathway in P. aeruginosa is dispensable in this model of CAUTI.

Catheter-associated urinary tract infection by Pseudomonas aeruginosa progresses through acute and chronic phases of infection.

Healthcare-associated infections are major causes of complications that lead to extended hospital stays and significant medical costs. The use of medical devices, including catheters, increases the risk of bacterial colonization and infection through the presence of a foreign surface. Two outcomes are observed for catheterized patients: catheter-associated asymptomatic bacteriuria and catheter-associated urinary tract infection (CAUTI). However, the relationship between these two events remains unclear. To understand this relationship, we studied a murine model of Pseudomonas aeruginosa CAUTI. In this model, we also observe two outcomes in infected animals: acute symptoms that is associated with CAUTI and chronic colonization that is associated with asymptomatic bacteriuria. The timing of the acute outcome takes place in the first week of infection, whereas chronic colonization occurs in the second week of infection. We further showed that mutants lacking genes encoding type III secretion system (T3SS), T3SS effector proteins, T3SS injection pore, or T3SS transcriptional activation all fail to cause acute symptoms of CAUTI. Nonetheless, all mutants defective for T3SS colonized the catheter and bladders at levels similar to the parental strain. In contrast, through induction of the T3SS master regulator ExsA, all infected animals showed acute phenotypes with bacteremia. Our results demonstrated that the acute symptoms, which are analogous to CAUTI, and chronic colonization, which is analogous to asymptomatic bacteriuria, are independent events that require distinct bacterial virulence factors. Experimental delineation of asymptomatic bacteriuria and CAUTI informs different strategies for the treatment and intervention of device-associated infections.

Patatin-like phospholipase CapV in Escherichia coli - morphological and physiological effects of one amino acid substitution.

In rod-shaped bacteria, morphological plasticity occurs in response to stress, which blocks cell division to promote filamentation. We demonstrate here that overexpression of the patatin-like phospholipase variant CapVQ329R, but not CapV, causes pronounced sulA-independent pyridoxine-inhibited cell filamentation in the Escherichia coli K-12-derivative MG1655 associated with restriction of flagella production and swimming motility. Conserved amino acids in canonical patatin-like phospholipase A motifs, but not the nucleophilic serine, are required to mediate CapVQ329R phenotypes. Furthermore, CapVQ329R production substantially alters the lipidome and colony morphotype including rdar biofilm formation with modulation of the production of the biofilm activator CsgD, and affects additional bacterial traits such as the efficiency of phage infection and antimicrobial susceptibility. Moreover, genetically diverse commensal and pathogenic E. coli strains and Salmonella typhimurium responded with cell filamentation and modulation in colony morphotype formation to CapVQ329R expression. In conclusion, this work identifies the CapV variant CapVQ329R as a pleiotropic regulator, emphasizes a scaffold function for patatin-like phospholipases, and highlights the impact of the substitution of a single conserved amino acid for protein functionality and alteration of host physiology.

Interaction of Staphylococcus aureus and Host Cells upon Infection of Bronchial Epithelium during Different Stages of Regeneration.

The primary barrier that protects our lungs against infection by pathogens is a tightly sealed layer of epithelial cells. When the integrity of this barrier is disrupted as a consequence of chronic pulmonary diseases or viral insults, bacterial pathogens will gain access to underlying tissues. A major pathogen that can take advantage of such conditions is Staphylococcus aureus, thereby causing severe pneumonia. In this study, we investigated how S. aureus responds to different conditions of the human epithelium, especially nonpolarization and fibrogenesis during regeneration using an in vitro infection model. The infective process was monitored by quantification of the epithelial cell and bacterial populations, fluorescence microscopy, and mass spectrometry. The results uncover differences in bacterial internalization and population dynamics that correlate with the outcome of infection. Protein profiling reveals that, irrespective of the polarization state of the epithelial cells, the invading bacteria mount similar responses to adapt to the intracellular milieu. Remarkably, a bacterial adaptation that was associated with the regeneration state of the epithelial cells concerned the early upregulation of proteins controlled by the redox-responsive regulator Rex when bacteria were confronted with a polarized cell layer. This is indicative of the modulation of the bacterial cytoplasmic redox state to maintain homeostasis early during infection even before internalization. Our present observations provide a deeper insight into how S. aureus can take advantage of a breached epithelial barrier and show that infected epithelial cells have limited ability to respond adequately to staphylococcal insults.

Molecular mechanisms of probiotic prevention of antibiotic-associated diarrhea.

Antibiotic-associated diarrhea (AAD) is a common and unintended adverse effect of antibiotic treatment. It is characterized by the disruption of the gut microbiota, decreased intestinal short chain fatty acid (SCFA) concentrations, accumulation of luminal carbohydrates and colonic bile acids, altered water absorption, and ultimately diarrhea. Probiotics were shown to prevent AAD in numerous clinical trials. This review examines what is currently known about how probiotics reduce the risk for AAD via modulating the gut microbiota, altering nutrient and bile acid metabolism, inducing epithelial solute transporter activity, supporting intestinal barrier function, and influencing the immune system. Although probiotics are frequently prescribed with antibiotic use, mechanistic evidence verifying how they confer protection against AAD is extremely limited. This information is urgently needed for improving recommendations for sustaining probiotic development and for implementing probiotics in clinical settings.

Heterogeneous antimicrobial activity in broncho-alveolar aspirates from mechanically ventilated intensive care unit patients.

Pneumonia is an infection of the lungs, where the alveoli in the affected area are filled with pus and fluid. Although ventilated patients are at risk, not all ventilated patients develop pneumonia. This suggests that the sputum environment may possess antimicrobial activities. Despite the generally acknowledged importance of antimicrobial activity in protecting the human lung against infections, this has not been systematically assessed to date. Therefore, the objective of the present study was to measure antimicrobial activity in broncho-alveolar aspirate ('sputum") samples from patients in an intensive care unit (ICU) and to correlate the detected antimicrobial activity with antibiotic levels, the sputum microbiome, and the respective patients' characteristics. To this end, clinical metadata and sputum were collected from 53 mechanically ventilated ICU patients. The antimicrobial activity of sputum samples was tested against Streptococcus pneumoniae, Staphylococcus aureus and Streptococcus anginosus. Here we show that sputa collected from different patients presented a high degree of variation in antimicrobial activity, which can be partially attributed to antibiotic therapy. The sputum microbiome, although potentially capable of producing antimicrobial agents, seemed to contribute in a minor way, if any, to the antimicrobial activity of sputum. Remarkably, despite its potentially protective effect, the level of antimicrobial activity in the investigated sputa correlated inversely with patient outcome, most likely because disease severity outweighed the beneficial antimicrobial activities.

Metabolic Cross-talk Between Human Bronchial Epithelial Cells and Internalized Staphylococcus aureus as a Driver for Infection.

Staphylococcus aureus is infamous for causing recurrent infections of the human respiratory tract. This is a consequence of its ability to adapt to different niches, including the intracellular milieu of lung epithelial cells. To understand the dynamic interplay between epithelial cells and the intracellular pathogen, we dissected their interactions over 4 days by mass spectrometry. Additionally, we investigated the dynamics of infection through live cell imaging, immunofluorescence and electron microscopy. The results highlight a major role of often overlooked temporal changes in the bacterial and host metabolism, triggered by fierce competition over limited resources. Remarkably, replicating bacteria reside predominantly within membrane-enclosed compartments and induce apoptosis of the host within ∼24 h post infection. Surviving infected host cells carry a subpopulation of non-replicating bacteria in the cytoplasm that persists. Altogether, we conclude that, besides the production of virulence factors by bacteria, it is the way in which intracellular resources are used, and how host and intracellular bacteria subsequently adapt to each other that determines the ultimate outcome of the infectious process.

Metabolic niche adaptation of community- and hospital-associated methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) originally emerged in nosocomial settings and has subsequently spread into the community. In turn, community-associated (CA) MRSA lineages are nowadays introduced from the community into hospitals where they can cause hospital-associated (HA) infections. This raises the question of how the CA-MRSA lineages adapt to the hospital environment. Previous studies implicated particular virulence factors in the CA-behaviour of MRSA. However, we hypothesized that physiological changes may also impact staphylococcal epidemiology. With the aim to identify potential metabolic adaptations, we comparatively profiled the cytosolic proteomes of CA- and HA-isolates from the USA300 lineage that was originally identified as CA-MRSA. Interestingly, enzymes for gluconeogenesis, the tricarboxylic acid cycle and biosynthesis of amino acids are up-regulated in the investigated CA-MRSA isolates, while enzymes for glycolysis and the pentose phosphate pathway are up-regulated in the HA-MRSA isolates. Of note, these data apparently match with the clinical presentation of each group. These observations spark interest in central carbon metabolism as a key driver for adaptations that streamline MRSA for propagation in the community or the hospital.

O-5S quantitative real-time PCR: a new diagnostic tool for laboratory confirmation of human onchocerciasis.

BACKGROUND: Onchocerciasis is a parasitic disease caused by the filarial nematode Onchocerca volvulus. In endemic areas, the diagnosis is commonly confirmed by microscopic examination of skin snip samples, though this technique is considered to have low sensitivity. The available melting-curve based quantitative real-time PCR (qPCR) using degenerated primers targeting the O-150 repeat of O. volvulus was considered insufficient for confirming the individual diagnosis, especially in elimination studies. This study aimed to improve detection of O. volvulus DNA in clinical samples through the development of a highly sensitive qPCR assay. METHODS: A novel hydrolysis probe based qPCR assay was designed targeting the specific sequence of the O. volvulus O-5S rRNA gene. A total of 200 clinically suspected onchocerciasis cases were included from Goma district in South-west Ethiopia, from October 2012 through May 2013. Skin snip samples were collected and subjected to microscopy, O-150 qPCR, and the novel O-5S qPCR. RESULTS: Among the 200 individuals, 133 patients tested positive (positivity rate of 66.5%) and 67 negative by O-5S qPCR, 74 tested positive by microscopy (37.0%) and 78 tested positive by O-150 qPCR (39.0%). Among the 133 O-5S qPCR positive individuals, microscopy and O-150 qPCR detected 55.6 and 59.4% patients, respectively, implying a higher sensitivity of O-5S qPCR than microscopy and O-150 qPCR. None of the 67 individuals who tested negative by O-5S qPCR tested positive by microscopy or O-150 qPCR, implying 100% specificity of the newly designed O-5S qPCR assay. CONCLUSIONS: The novel O-5S qPCR assay is more sensitive than both microscopic examination and the existing O-150 qPCR for the detection of O. volvulus from skin snip samples. The newly designed assay is an important step towards appropriate individual diagnosis and control of onchocerciasis.

Signatures of cytoplasmic proteins in the exoproteome distinguish community- and hospital-associated methicillin-resistant Staphylococcus aureus USA300 lineages.

Methicillin-resistant Staphylococcus aureus (MRSA) is the common name for a heterogeneous group of highly drug-resistant staphylococci. Two major MRSA classes are distinguished based on epidemiology, namely community-associated (CA) and hospital-associated (HA) MRSA. Notably, the distinction of CA- and HA-MRSA based on molecular traits remains difficult due to the high genomic plasticity of S. aureus. Here we sought to pinpoint global distinguishing features of CA- and HA-MRSA through a comparative genome and proteome analysis of the notorious MRSA lineage USA300. We show for the first time that CA- and HA-MRSA isolates can be distinguished by 2 distinct extracellular protein abundance clusters that are predictive not only for epidemiologic behavior, but also for their growth and survival within epithelial cells. This 'exoproteome profiling' also groups more distantly related HA-MRSA isolates into the HA exoproteome cluster. Comparative genome analysis suggests that these distinctive features of CA- and HA-MRSA isolates relate predominantly to the accessory genome. Intriguingly, the identified exoproteome clusters differ in the relative abundance of typical cytoplasmic proteins, suggesting that signatures of cytoplasmic proteins in the exoproteome represent a new distinguishing feature of CA- and HA-MRSA. Our comparative genome and proteome analysis focuses attention on potentially distinctive roles of 'liberated' cytoplasmic proteins in the epidemiology and intracellular survival of CA- and HA-MRSA isolates. Such extracellular cytoplasmic proteins were recently invoked in staphylococcal virulence, but their implication in the epidemiology of MRSA is unprecedented.

HDAC1-3 inhibitor MS-275 enhances IL10 expression in RAW264.7 macrophages and reduces cigarette smoke-induced airway inflammation in mice.

Chronic obstructive pulmonary disease (COPD) constitutes a major health burden. Studying underlying molecular mechanisms could lead to new therapeutic targets. Macrophages are orchestrators of COPD, by releasing pro-inflammatory cytokines. This process relies on transcription factors such as NF-κB, among others. NF-κB is regulated by lysine acetylation; a post-translational modification installed by histone acetyltransferases and removed by histone deacetylases (HDACs). We hypothesized that small molecule HDAC inhibitors (HDACi) targeting class I HDACs members that can regulate NF-κB could attenuate inflammatory responses in COPD via modulation of the NF-κB signaling output. MS-275 is an isoform-selective inhibitor of HDAC1-3. In precision-cut lung slices and RAW264.7 macrophages, MS-275 upregulated the expression of both pro- and anti-inflammatory genes, implying mixed effects. Interestingly, anti-inflammatory IL10 expression was upregulated in these model systems. In the macrophages, this was associated with increased NF-κB activity, acetylation, nuclear translocation, and binding to the IL10 promoter. Importantly, in an in vivo model of cigarette smoke-exposed C57Bl/6 mice, MS-275 robustly attenuated inflammatory expression of KC and neutrophil influx in the lungs. This study highlights for the first time the potential of isoform-selective HDACi for the treatment of inflammatory lung diseases like COPD.