Comprehensive Metaproteomic Analyses of Urine in the Presence and Absence of Neutrophil-Associated Inflammation in the Urinary Tract

Yoshida, Yu; Sikorski, Patricia; Smith, Madeline; Bowman-Gholston, Cynthia; Cacciabeve, Nicolas; Nelson, Karen E.; Pieper, Rembert

doi:10.7150/thno.16086

Cited by 32 publications

(38 citation statements)

References 63 publications

(75 reference statements)

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“…Urine sample was processed following protocols published previously [32]. In brief, around 20 ml urine were first concentrated using Amicon Ultra-15 centrifugal filter device (10 kDa MWCO, Fisher Scientific).…”

Section: Methodsmentioning

confidence: 99%

“…The following processing steps were similar to the procedure described above. As a note, the urine specimens were deidentified and considered as medical waste from an on-going project [32]. Therefore, the study was exempted from human subject protocols and bioethical review [32].…”

Section: Methodsmentioning

confidence: 99%

“…For protein quantification, the MaxQuant and Andromeda software suite (version 1.4.2.0) was used. We accepted most of the default settings provided in this software [32]. Both the label-free quantitation (LFQ) and the intensity-based absolute quantitation (iBAQ) tools were enabled.…”

Section: Methodsmentioning

confidence: 99%

“…In this study, we use different type of sample sources, including Klebsiella pneumoniae whole cell lysate and human urine samples, to systemically investigate the possible application. Our rationale to choose these samples is to gain insights into several ongoing projects that are related to urine proteomics and urinary pathogens [31, 32]. We compare their performance with single FASP, and attempt to demonstrate their broad application for quantitative proteome analyses.…”

Section: Introductionmentioning

confidence: 99%

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Quick 96FASP for high throughput quantitative proteome analysis

Bekele

Pieper

2017

Journal of Proteomics

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Filter aided sample preparation (FASP) is becoming a central method for proteomic sample cleanup and peptide generation prior to LC-MS analysis. We previously adapted this method to a 96-well filter plate, and applied to prepare protein digests from cell lysate and body fluid samples in a high throughput quantitative manner. While the 96FASP approach is scalable and can handle multiple samples simultaneously, two key advantages compared to single FASP, it is also time-consuming. The centrifugation-based liquid transfer on the filter plate takes 3~5 times longer than single filter. To address this limitation, we now present a quick 96FASP (named q96FASP) approach that, relying on the use of filter membranes with a large MWCO size (~30 kDa), significantly reduces centrifugal times. We show that q96FASP allows the generation of protein digests derived from whole cell lysates and body fluids in a quality similar to that of the single FASP method. Processing a sample in multiple wells in parallel, we observed excellent experimental repeatability by label-free quantitation approach. We conclude that the q96FASP approach promises to be a promising cost- and time-effective method for shotgun proteomics and will be particularly useful in large scale biomarker discovery studies.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Quick 96FASP for high throughput quantitative proteome analysis

Bekele

Pieper

2017

Journal of Proteomics

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“…Neutrophils are the main type of immune cells attacking and phagocytosing microbial intruders (13). Pathogen clearance results from the activities of neutrophil granular effector proteins and reactive oxygen species (ROS) as well as extracellular trapping (14, 15). Investigations of E. coli have implicated the neutrophil cyclooxygenase-2 in the pathogenesis of recurrent UTI (16).…”

Section: Introductionmentioning

confidence: 99%

Nutritional Immunity and Antibiotic Drug Treatments Influence Microbial Composition but Fail to Eliminate Urethral Catheter Biofilms in Recurrently Catheterized Patients

Singh

Tsitrin

et al. 2019

Preprint

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21Polymicrobial biofilms that form on indwelling urethral catheters used by neurogenic 22 bladder patients are known to recur following catheter replacements. Uropathogens dominate in 23 catheter biofilms (CBs), grow and disperse as multi-cellular aggregates. Their microbial 24 complexity, the characteristics of host immune responses and the molecular crosstalk in this 25 ecosystem are incompletely understood. By surveying eight patients over up to six months with 26 meta-omics analysis methods, we shed new light on the longitudinal microbial dynamics in CBs 27 and the microbial-host crosstalk. There was evidence of chronic innate immune responses in all 28 patients. Pathogens dominated the microbial contents. Proteus mirabilis often out-competed other 29 species in cases of salt encrustation of catheters. The examination of proteomes in CBs and 30 associated urinary pellets revealed many abundant bacterial systems for transition metal ion (TMI) 31 acquisition. TMIs are sequestered by effector proteins released by activated neutrophils and 32 urothelial cells, such as lactotransferrin and calgranulins, which were abundant in the host 33 proteomes. We identified positive quantitative correlations among systems responsible for 34 siderophore biosynthesis, TMI/siderophore uptake and TMI cellular import in bacterial species, 35 suggesting competition for TMIs to support their metabolism and growth in CBs. Enterococcus 36 faecalis was prevalent as a cohabitant of CBs and expressed three lipoproteins with apparent TMI 37 acquisition functions. Fastidious anaerobic bacteria such as Veillonella, Actinobaculum, and 38 Bifidobacterium grew in CB communities that appeared to be oxygen starved. Finally, antibiotic 39 drug treatments were shown to influence microbial composition of CBs but failed to prevent re-40 colonization of urethral catheters with persisting and/or drug-resistant newly emerging pathogens. 41 42 43 44 45 46 Urethral catheter-associated urinary tract infection (CAUTI) is the most common type of 47 complicated UTI. CAUTIs have a higher risk of recurrence, pyelonephritis and bacteremia than 48 uncomplicated UTIs in nosocomial environments (1-3). Asymptomatic cases are usually 49 diagnosed as catheter-associated asymptomatic bacteriuria (CAASB). The use of nearly 100 50 million urethral catheters per year worldwide, the 3% to 10% incidence of bacteriuria over 24 51 hours following patient catheterization and an average bladder catheter insertion time of 72 h (2) 52 suggest an estimated 9 to 27 million CAUTI cases per year globally. Among the most common 53 causes are Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus mirabilis, 54 Enterococcus and Candida spp. (1, 3, 4). Indwelling Foley catheters are often used by patients 55 with anatomical urinary tract abnormalities and neurogenic bladder syndrome and retained in the 56 urinary tract for one week or longer. Microbial colonization is difficult to avoid even when catheters 57 are regularly replaced, and antibi...

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Using Proteomics to Identify Inflammation During Urinary Tract Infection

Yoshida

Pieper

2019

Methods in Molecular Biology

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Comprehensive Metaproteomic Analyses of Urine in the Presence and Absence of Neutrophil-Associated Inflammation in the Urinary Tract

Cited by 32 publications

References 63 publications

Quick 96FASP for high throughput quantitative proteome analysis

Quick 96FASP for high throughput quantitative proteome analysis

Nutritional Immunity and Antibiotic Drug Treatments Influence Microbial Composition but Fail to Eliminate Urethral Catheter Biofilms in Recurrently Catheterized Patients

Using Proteomics to Identify Inflammation During Urinary Tract Infection

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