Characterization of Early-Phase Neutrophil Extracellular Traps in Urinary Tract Infections.

Neutrophils have an important role in the antimicrobial defense and resolution of urinary tract infections (UTIs). Our research suggests that a mechanism known as neutrophil extracellular trap (NET) formation is a defense strategy to combat pathogens that have invaded the urinary tract. A set of human urine specimens with very high neutrophil counts had microscopic evidence of cellular aggregation and lysis. Deoxyribonuclease I (DNase) treatment resulted in disaggregation of such structures, release of DNA fragments and a proteome enriched in histones and azurophilic granule effectors whose quantitative composition was similar to that of previously described in vitro-formed NETs. The effector proteins were further enriched in DNA-protein complexes isolated in native PAGE gels. Immunofluorescence microscopy revealed a flattened morphology of neutrophils associated with decondensed chromatin, remnants of granules in the cell periphery, and myeloperoxidase co-localized with extracellular DNA, features consistent with early-phase NETs. Nuclear staining revealed that a considerable fraction of bacterial cells in these structures were dead. The proteomes of two pathogens, Staphylococcus aureus and Escherichia coli, were indicative of adaptive responses to early-phase NETs, specifically the release of virulence factors and arrest of ribosomal protein synthesis. Finally, we discovered patterns of proteolysis consistent with widespread cleavage of proteins by neutrophil elastase, proteinase 3 and cathepsin G and evidence of citrullination in many nuclear proteins.

Protein signatures from blood plasma and urine suggest changes in vascular function and IL-12 signaling in elderly with a history of chronic diseases compared with an age-matched healthy cohort.

Key processes characterizing human aging are immunosenescence and inflammaging. The capacity of the immune system to adequately respond to external perturbations (e.g., pathogens, injuries, and biochemical irritants) and to repair somatic mutations that may cause cancers or cellular senescence declines. An important goal remains to identify genetic or biochemical, predictive biomarkers for healthy aging. We recruited two cohorts in the age range 70 to 82, one afflicted by chronic illnesses (non-healthy aging, NHA) and the other in good health (healthy aging, HA). NHA criteria included major cardiovascular, neurodegenerative, and chronic pulmonary diseases, diabetes, and cancers. Quantitative analysis of forty proinflammatory cytokines in blood plasma and more than 500 proteins in urine was performed to identify candidate biomarkers for and biological pathway implications of healthy aging. Nine cytokines revealed lower quantities in blood plasma for the NHA compared with the HA groups (fold change > 1.5; p value < 0.025) including IL-12p40 and IL-12p70. We note that, sampling at two timepoints, intra-individual cytokine abundance patterns clustered in 86% of all 60 cases, indicative of person-specific, highly controlled multi-cytokine signatures in blood plasma. Twenty-three urinary proteins were differentially abundant (HA versus NHA; fold change > 1.5; p value < 0.01). Among the proteins increased in abundance in the HA cohort were glycoprotein MUC18, ephrin type-B receptor 4, matrix remodeling-associated protein 8, angiopoietin-related protein 2, K-cadherin, and plasma protease C1 inhibitor. These proteins have been linked to the extracellular matrix, cell adhesion, and vascular remodeling and repair processes. In silico network analysis identified the regulation of coagulation, antimicrobial humoral immune responses, and the IL-12 signaling pathway as enriched GO terms. To validate links of these preliminary biomarkers and IL-12 signaling with healthy aging, clinical studies using larger cohorts and functional characterization of the genes/proteins in cellular models of aging need to be conducted.

Urethral Catheter Biofilms Reveal Plasticity in Bacterial Composition and Metabolism and Withstand Host Immune Defenses in Hypoxic Environment.

Biofilms composed of multiple microorganisms colonize the surfaces of indwelling urethral catheters that are used serially by neurogenic bladder patients and cause chronic infections. Well-adapted pathogens in this niche are Escherichia coli, Proteus, and Enterococcus spp., species that cycle through adhesion and multilayered cell growth, trigger host immune responses, are starved off nutrients, and then disperse. Viable microbial foci retained in the urinary tract recolonize catheter surfaces. The molecular adaptations of bacteria in catheter biofilms (CBs) are not well-understood, promising new insights into this pathology based on host and microbial meta-omics analyses from clinical specimens. We examined catheters from nine neurogenic bladder patients longitudinally over up to 6 months. Taxonomic analyses from 16S rRNA gene sequencing and liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomics revealed that 95% of all catheter and corresponding urinary pellet (UP) samples contained bacteria. CB biomasses were dominated by Enterobacteriaceae spp. and often accompanied by lactic acid and anaerobic bacteria. Systemic antibiotic drug treatments of patients resulted in either transient or lasting microbial community perturbations. Neutrophil effector proteins were abundant not only in UP but also CB samples, indicating their penetration of biofilm surfaces. In the context of one patient who advanced to a kidney infection, Proteus mirabilis proteomic data suggested a combination of factors associated with this disease complication: CB biomasses were high; the bacteria produced urease alkalinizing the pH and triggering urinary salt deposition on luminal catheter surfaces; P. mirabilis utilized energy-producing respiratory systems more than in CBs from other patients. The NADH:quinone oxidoreductase II (Nqr), a Na+ translocating enzyme not operating as a proton pump, and the nitrate reductase A (Nar) equipped the pathogen with electron transport chains promoting growth under hypoxic conditions. Both P. mirabilis and E. coli featured repertoires of transition metal ion acquisition systems in response to human host-mediated iron and zinc sequestration. We discovered a new drug target, the Nqr respiratory system, whose deactivation may compromise P. mirabilis growth in a basic pH milieu. Animal models would not allow such molecular-level insights into polymicrobial biofilm metabolism and interactions because the complexity cannot be replicated.

Protein and Microbial Biomarkers in Sputum Discern Acute and Latent Tuberculosis in Investigation of Pastoral Ethiopian Cohort.

Differential diagnosis of tuberculosis (TB) and latent TB infection (LTBI) remains a public health priority in high TB burden countries. Pulmonary TB is diagnosed by sputum smear microscopy, chest X-rays, and PCR tests for distinct Mycobacterium tuberculosis (Mtb) genes. Clinical tests to diagnose LTBI rely on immune cell stimulation in blood plasma with TB-specific antigens followed by measurements of interferon-γ concentrations. The latter is an important cytokine for cellular immune responses against Mtb in infected lung tissues. Sputum smear microscopy and chest X-rays are not sufficiently sensitive while both PCR and interferon-γ release assays are expensive. Alternative biomarkers for the development of diagnostic tests to discern TB disease states are desirable. This study's objective was to discover sputum diagnostic biomarker candidates from the analysis of samples from 161 human subjects including TB patients, individuals with LTBI, negative community controls (NCC) from the province South Omo, a pastoral region in Ethiopia. We analyzed 16S rRNA gene-based bacterial taxonomies and proteomic profiles. The sputum microbiota did not reveal statistically significant differences in α-diversity comparing the cohorts. The genus Mycobacterium, representing Mtb, was only identified for the TB group which also featured reduced abundance of the genus Rothia in comparison with the LTBI and NCC groups. Rothia is a respiratory tract commensal and may be sensitive to the inflammatory milieu generated by infection with Mtb. Proteomic data supported innate immune responses against the pathogen in subjects with pulmonary TB. Ferritin, an iron storage protein released by damaged host cells, was markedly increased in abundance in TB sputum compared to the LTBI and NCC groups, along with the α-1-acid glycoproteins ORM1 and ORM2. These proteins are acute phase reactants and inhibit excessive neutrophil activation. Proteomic data highlight the effector roles of neutrophils in the anti-Mtb response which was not observed for LTBI cases. Less abundant in the sputum of the LTBI group, compared to the NCC group, were two immunomodulatory proteins, mitochondrial TSPO and the extracellular ribonuclease T2. If validated, these proteins are of interest as new biomarkers for diagnosis of LTBI.

Aerococcus urinae and Globicatella sanguinis Persist in Polymicrobial Urethral Catheter Biofilms Examined in Longitudinal Profiles at the Proteomic Level.

Aerococcus urinae (Au) and Globicatella sanguinis (Gs) are gram-positive bacteria belonging to the family Aerococcaceae and colonize the human immunocompromised and catheterized urinary tract. We identified both pathogens in polymicrobial urethral catheter biofilms (CBs) with a combination of 16S rDNA sequencing, proteomic analyses, and microbial cultures. Longitudinal sampling of biofilms from serially replaced catheters revealed that each species persisted in the urinary tract of a patient in cohabitation with 1 or more gram-negative uropathogens. The Gs and Au proteomes revealed active glycolytic, heterolactic fermentation, and peptide catabolic energy metabolism pathways in an anaerobic milieu. A few phosphotransferase system (PTS)-based sugar uptake and oligopeptide ABC transport systems were highly expressed, indicating adaptations to the supply of nutrients in urine and from exfoliating squamous epithelial and urothelial cells. Differences in the Au vs Gs metabolisms pertained to citrate lyase and utilization and storage of glycogen (evident only in Gs proteomes) and to the enzyme Xfp that degrades d-xylulose-5'-phosphate and the biosynthetic pathways for 2 protein cofactors, pyridoxal 6'-phosphate and 4'-phosphopantothenate (expressed only in Au proteomes). A predicted ZnuA-like transition metal ion uptake system was identified for Gs while Au expressed 2 LPXTG-anchored surface proteins, one of which had a predicted pilin D adhesion motif. While these proteins may contribute to fitness and virulence in the human host, it cannot be ruled out that Au and Gs fill a niche in polymicrobial biofilms without being the direct cause of injury in urothelial tissues.

Actinobaculum massiliense Proteome Profiled in Polymicrobial Urethral Catheter Biofilms.

Actinobaculum massiliense, a Gram-positive anaerobic coccoid rod colonizing the human urinary tract, belongs to the taxonomic class of Actinobacteria. We identified A. massiliense as a cohabitant of urethral catheter biofilms (CB). The CBs also harbored more common uropathogens, such as Proteus mirabilis and Aerococcus urinae, supporting the notion that A. massiliense is adapted to a life style in polymicrobial biofilms. We isolated a clinical strain from a blood agar colony and used 16S rRNA gene sequencing and shotgun proteomics to confirm its identity as A. massiliense. We characterized this species by quantitatively comparing the bacterial proteome derived from in vitro growth with that of four clinical samples. The functional relevance of proteins with emphasis on nutrient import and the response to hostile host conditions, showing evidence of neutrophil infiltration, was analyzed. Two putative subtilisin-like proteases and a heme/oligopeptide transporter were abundant in vivo and are likely important for survival and fitness in the biofilm. Proteins facilitating uptake of xylose/glucuronate and oligopeptides, also highly expressed in vivo, may feed metabolites into mixed acid fermentation and peptidolysis pathways, respectively, to generate energy. A polyketide synthase predicted to generate a secondary metabolite that interacts with either the human host or co-colonizing microbes was also identified. The product of the PKS enzyme may contribute to A. massiliense fitness and persistence in the CBs.

Sequence, annotation, and analysis of synteny between rice chromosome 3 and diverged grass species.

Rice (Oryza sativa L.) chromosome 3 is evolutionarily conserved across the cultivated cereals and shares large blocks of synteny with maize and sorghum, which diverged from rice more than 50 million years ago. To begin to completely understand this chromosome, we sequenced, finished, and annotated 36.1 Mb ( approximately 97%) from O. sativa subsp. japonica cv Nipponbare. Annotation features of the chromosome include 5915 genes, of which 913 are related to transposable elements. A putative function could be assigned to 3064 genes, with another 757 genes annotated as expressed, leaving 2094 that encode hypothetical proteins. Similarity searches against the proteome of Arabidopsis thaliana revealed putative homologs for 67% of the chromosome 3 proteins. Further searches of a nonredundant amino acid database, the Pfam domain database, plant Expressed Sequence Tags, and genomic assemblies from sorghum and maize revealed only 853 nontransposable element related proteins from chromosome 3 that lacked similarity to other known sequences. Interestingly, 426 of these have a paralog within the rice genome. A comparative physical map of the wild progenitor species, Oryza nivara, with japonica chromosome 3 revealed a high degree of sequence identity and synteny between these two species, which diverged approximately 10,000 years ago. Although no major rearrangements were detected, the deduced size of the O. nivara chromosome 3 was 21% smaller than that of japonica. Synteny between rice and other cereals using an integrated maize physical map and wheat genetic map was strikingly high, further supporting the use of rice and, in particular, chromosome 3, as a model for comparative studies among the cereals.