Shih-Ting Huang

Integrated next-generation sequencing of 16S rDNA and metaproteomics differentiate the healthy urine microbiome from asymptomatic bacteriuria in neuropathic bladder associated with spinal cord injury

BackgroundClinical dogma is that healthy urine is sterile and the presence of bacteria with an inflammatory response is indicative of urinary tract infection (UTI). Asymptomatic bacteriuria (ABU) represents the state in which bacteria are present but the inflammatory response is negligible. Differentiating ABU from UTI is diagnostically challenging, but critical because overtreatment of ABU can perpetuate antimicrobial resistance while undertreatment of UTI can result in increased morbidity and mortality. In this study, we describe key characteristics of the healthy and ABU urine microbiomes utilizing 16S rRNA gene (16S rDNA) sequencing and metaproteomics, with the future goal of utilizing this information to personalize the treatment of UTI based on key individual characteristics.MethodsA cross-sectional study of 26 healthy controls and 27 healthy subjects at risk for ABU due to spinal cord injury-related neuropathic bladder (NB) was conducted. Of the 27 subjects with NB, 8 voided normally, 8 utilized intermittent catheterization, and 11 utilized indwelling Foley urethral catheterization for bladder drainage. Urine was obtained by clean catch in voiders, or directly from the catheter in subjects utilizing catheters. Urinalysis, urine culture and 16S rDNA sequencing were performed on all samples, with metaproteomic analysis performed on a subsample.ResultsA total of 589454 quality-filtered 16S rDNA sequence reads were processed through a NextGen 16S rDNA analysis pipeline. Urine microbiomes differ by normal bladder function vs. NB, gender, type of bladder catheter utilized, and duration of NB. The top ten bacterial taxa showing the most relative abundance and change among samples were Lactobacillales, Enterobacteriales, Actinomycetales, Bacillales, Clostridiales, Bacteroidales, Burkholderiales, Pseudomonadales, Bifidobacteriales and Coriobacteriales. Metaproteomics confirmed the 16S rDNA results, and functional human protein-pathogen interactions were noted in subjects where host defenses were initiated.ConclusionsCounter to clinical belief, healthy urine is not sterile. The healthy urine microbiome is characterized by a preponderance of Lactobacillales in women and Corynebacterium in men. The presence and duration of NB and method of urinary catheterization alter the healthy urine microbiome. An integrated approach of 16S rDNA sequencing with metaproteomics improves our understanding of healthy urine and facilitates a more personalized approach to prevention and treatment of infection.

Temperature and growth phase influence the outer-membrane proteome and the expression of a type VI secretion system in Yersinia pestis.

Yersinia pestis cells were grown in vitro at 26 and 37 degrees C, the ambient temperatures of its flea vector and its mammalian hosts, respectively, and subjected to subcellular fractionation. Abundance changes at 26 vs 37 degrees C were observed for many outer-membrane (OM) proteins. The cell adhesion protein Ail (y1324) and three putative small beta-barrel OM proteins (y1795, y2167 and y4083) were strongly increased at 37 degrees C. The Ail/Lom family protein y1682 (OmpX) was strongly increased at 26 degrees C. Several porins and TonB-dependent receptors, which control small molecule transport through the OM, were also altered in abundance in a temperature-dependent manner. These marked differences in the composition of the OM proteome are probably important for the adaptation of Y. pestis to its in vivo life stages. Thirteen proteins that appear to be part of an intact type VI secretion system (T6SS) were identified in membrane fractions of stationary-phase cells grown at 26 degrees C, but not at 37 degrees C. The corresponding genes are clustered in the Y. pestis KIM gene locus y3658-y3677. The proteins y3674 and y3675 were particularly abundant and co-fractionated in a Mr range indicative of participation in a multi-subunit complex. The soluble haemolysin-coregulated protein y3673 was even more abundant. Its release into the extracellular medium was triggered by treatment of Y. pestis cells with trypsin. Proteases and other stress-response-inducing factors may constitute environmental cues resulting in the activation of the T6SS in Y. pestis.

A proteogenomic update to Yersinia: enhancing genome annotation.

BackgroundModern biomedical research depends on a complete and accurate proteome. With the widespread adoption of new sequencing technologies, genome sequences are generated at a near exponential rate, diminishing the time and effort that can be invested in genome annotation. The resulting gene set contains numerous errors in even the most basic form of annotation: the primary structure of the proteins.ResultsThe application of experimental proteomics data to genome annotation, called proteogenomics, can quickly and efficiently discover misannotations, yielding a more accurate and complete genome annotation. We present a comprehensive proteogenomic analysis of the plague bacterium, Yersinia pestis KIM. We discover non-annotated genes, correct protein boundaries, remove spuriously annotated ORFs, and make major advances towards accurate identification of signal peptides. Finally, we apply our data to 21 other Yersinia genomes, correcting and enhancing their annotations.ConclusionsIn total, 141 gene models were altered and have been updated in RefSeq and Genbank, which can be accessed seamlessly through any NCBI tool (e.g. blast) or downloaded directly. Along with the improved gene models we discover new, more accurate means of identifying signal peptides in proteomics data.

The Shigella dysenteriae serotype 1 proteome, profiled in the host intestinal environment, reveals major metabolic modifications and increased expression of invasive proteins

Shigella dysenteriae serotype 1 (SD1) causes the most severe form of epidemic bacillary dysentery. We present the first comprehensive proteome analysis of this pathogen, profiling proteins from bacteria cultured in vitro and bacterial isolates from the large bowel of infected gnotobiotic piglets (in vivo). Overall, 1061 distinct gene products were identified. Differential display analysis revealed that SD1 cells switched to an anaerobic energy metabolism in vivo. High in vivo abundances of amino acid decarboxylases (GadB and AdiA) which enhance pH homeostasis in the cytoplasm and protein disaggregation chaperones (HdeA, HdeB and ClpB) were indicative of a coordinated bacterial survival response to acid stress. Several type III secretion system effectors were increased in abundance in vivo, including OspF, IpaC and IpaD. These proteins are implicated in invasion of colonocytes and subversion of the host immune response in S. flexneri. These observations likely reflect an adaptive response of SD1 to the hostile host environment. Seven proteins, among them the type III secretion system effectors OspC2 and IpaB, were detected as antigens in Western blots using piglet antisera. The outer membrane protein OmpA, the heat shock protein HtpG and OspC2 represent novel SD1 subunit vaccine candidates and drug targets.

Comparison of two label-free global quantitation methods, APEX and 2D gel electrophoresis, applied to the Shigella dysenteriae proteome

The in vitro stationary phase proteome of the human pathogen Shigella dysenteriae serotype 1 (SD1) was quantitatively analyzed in Coomassie Blue G250 (CBB)-stained 2D gels. More than four hundred and fifty proteins, of which 271 were associated with distinct gel spots, were identified. In parallel, we employed 2D-LC-MS/MS followed by the label-free computationally modified spectral counting method APEX for absolute protein expression measurements. Of the 4502 genome-predicted SD1 proteins, 1148 proteins were identified with a false positive discovery rate of 5% and quantitated using 2D-LC-MS/MS and APEX. The dynamic range of the APEX method was approximately one order of magnitude higher than that of CBB-stained spot intensity quantitation. A squared Pearson correlation analysis revealed a reasonably good correlation (R2= 0.67) for protein quantities surveyed by both methods. The correlation was decreased for protein subsets with specific physicochemical properties, such as low Mr values and high hydropathy scores. Stoichiometric ratios of subunits of protein complexes characterized in E. coli were compared with APEX quantitative ratios of orthologous SD1 protein complexes. A high correlation was observed for subunits of soluble cellular protein complexes in several cases, demonstrating versatile applications of the APEX method in quantitative proteomics.

Proteomic analysis of iron acquisition, metabolic and regulatory responses of Yersinia pestis to iron starvation

BackgroundThe Gram-negative bacterium Yersinia pestis is the causative agent of the bubonic plague. Efficient iron acquisition systems are critical to the ability of Y. pestis to infect, spread and grow in mammalian hosts, because iron is sequestered and is considered part of the innate host immune defence against invading pathogens. We used a proteomic approach to determine expression changes of iron uptake systems and intracellular consequences of iron deficiency in the Y. pestis strain KIM6+ at two physiologically relevant temperatures (26°C and 37°C).ResultsDifferential protein display was performed for three Y. pestis subcellular fractions. Five characterized Y. pestis iron/siderophore acquisition systems (Ybt, Yfe, Yfu, Yiu and Hmu) and a putative iron/chelate outer membrane receptor (Y0850) were increased in abundance in iron-starved cells. The iron-sulfur (Fe-S) cluster assembly system Suf, adapted to oxidative stress and iron starvation in E. coli, was also more abundant, suggesting functional activity of Suf in Y. pestis under iron-limiting conditions. Metabolic and reactive oxygen-deactivating enzymes dependent on Fe-S clusters or other iron cofactors were decreased in abundance in iron-depleted cells. This data was consistent with lower activities of aconitase and catalase in iron-starved vs. iron-rich cells. In contrast, pyruvate oxidase B which metabolizes pyruvate via electron transfer to ubiquinone-8 for direct utilization in the respiratory chain was strongly increased in abundance and activity in iron-depleted cells.ConclusionsMany protein abundance differences were indicative of the important regulatory role of the ferric uptake regulator Fur. Iron deficiency seems to result in a coordinated shift from iron-utilizing to iron-independent biochemical pathways in the cytoplasm of Y. pestis. With growth temperature as an additional variable in proteomic comparisons of the Y. pestis fractions (26°C and 37°C), there was little evidence for temperature-specific adaptation processes to iron starvation.

Characterizing the dynamic nature of the Yersinia pestis periplasmic proteome in response to nutrient exhaustion and temperature change

The periplasmic proteome of Yersinia pestis strain KIM6+ was characterized using differential 2‐DE display of proteins isolated from several subcellular fractions. Circa 160 proteins were designated as periplasmic, including 62 (putative) solute‐binding proteins of ATP‐binding cassette (ABC) transporters (SBPs) and 46 (putative) metabolic enzymes. More than 30 SBPs were significantly increased in abundance during stationary phase cell growth, compared to the exponential phase. The data suggest that nutrient exhaustion in the stationary phase triggers cellular responses resulting in the induced expression of numerous ABC transporters, which are responsible for the import of solutes/nutrients. Limited availability of inorganic phosphate (Pi) also caused dramatic proteomic changes. Nine proteins were functionally linked to the mobilization and import of three small molecules (Pi, phosphonate and glycerol‐3‐phosphate) and accounted for nearly half of the total protein mass in the periplasm of Pi‐starved cells. When cells were grown at 26°C versus 37°C, corresponding to ambient temperatures in the flea vector and mammalian hosts, respectively, several periplasmic proteins with no known roles in the Y. pestis life cycle were strongly altered in abundance. This included a putative nitrate/sulfonate/bicarbonate‐specific SBP (Y1004), encoded by the virulence‐associated plasmid pMT1 and increased in abundance at 37°C.

Analysis of the Proteome of Intracellular Shigella flexneri Reveals Pathways Important for Intracellular Growth

ABSTRACT Global proteomic analysis was performed with Shigella flexneri strain 2457T in association with three distinct growth environments: S. flexneri growing in broth (in vitro), S. flexneri growing within epithelial cell cytoplasm (intracellular), and S. flexneri that were cultured with, but did not invade, Henle cells (extracellular). Compared to in vitro and extracellular bacteria, intracellular bacteria had increased levels of proteins required for invasion and cell-to-cell spread, including Ipa, Mxi, and Ics proteins. Changes in metabolic pathways in response to the intracellular environment also were evident. There was an increase in glycogen biosynthesis enzymes, altered expression of sugar transporters, and a reduced amount of the carbon storage regulator CsrA. Mixed acid fermentation enzymes were highly expressed intracellularly, while tricarboxylic acid (TCA) cycle oxidoreductive enzymes and most electron transport chain proteins, except CydAB, were markedly decreased. This suggested that fermentation and the CydAB system primarily sustain energy generation intracellularly. Elevated levels of PntAB, which is responsible for NADPH regeneration, suggested a shortage of reducing factors for ATP synthesis. These metabolic changes likely reflect changes in available carbon sources, oxygen levels, and iron availability. Intracellular bacteria showed strong evidence of iron starvation. Iron acquisition systems (Iut, Sit, FhuA, and Feo) and the iron starvation, stress-associated Fe-S cluster assembly (Suf) protein were markedly increased in abundance. Mutational analysis confirmed that the mixed-acid fermentation pathway was required for wild-type intracellular growth and spread of S. flexneri. Thus, iron stress and changes in carbon metabolism may be key factors in the S. flexneri transition from the extra- to the intracellular milieu.

Integral and peripheral association of proteins and protein complexes with Yersinia pestis inner and outer membranes

Yersinia pestis proteins were sequentially extracted from crude membranes with a high salt buffer (2.5 M NaBr), an alkaline solution (180 mM Na2CO3, pH 11.3) and membrane denaturants (8 M urea, 2 M thiourea and 1% amidosulfobetaine-14). Separation of proteins by 2D gel electrophoresis was followed by identification of more than 600 gene products by MS. Data from differential 2D gel display experiments, comparing protein abundances in cytoplasmic, periplasmic and all three membrane fractions, were used to assign proteins found in the membrane fractions to three protein categories: (i) integral membrane proteins and peripheral membrane proteins with low solubility in aqueous solutions (220 entries); (ii) peripheral membrane proteins with moderate to high solubility in aqueous solutions (127 entries); (iii) cytoplasmic or ribosomal membrane-contaminating proteins (80 entries). Thirty-one proteins were experimentally associated with the outer membrane (OM). Circa 50 proteins thought to be part of membrane-localized, multi-subunit complexes were identified in high Mr fractions of membrane extracts via size exclusion chromatography. This data supported biologically meaningful assignments of many proteins to the membrane periphery. Since only 32 inner membrane (IM) proteins with two or more predicted transmembrane domains (TMDs) were profiled in 2D gels, we resorted to a proteomic analysis by 2D-LC-MS/MS. Ninety-four additional IM proteins with two or more TMDs were identified. The total number of proteins associated with Y. pestis membranes increased to 456 and included representatives of all six β-barrel OM protein families and 25 distinct IM transporter families.

Characterizing the Escherichia coli O157:H7 proteome including protein associations with higher order assemblies.

Background The recent outbreak of severe infections with Shiga toxin (Stx) producing Escherichia coli (STEC) serotype O104:H4 highlights the need to understand horizontal gene transfer among E. coli strains, identify novel virulence factors and elucidate their pathogenesis. Quantitative shotgun proteomics can contribute to such objectives, allowing insights into the part of the genome translated into proteins and the connectivity of biochemical pathways and higher order assemblies of proteins at the subcellular level. Methodology/Principal Findings We examined protein profiles in cell lysate fractions of STEC strain 86-24 (serotype O157:H7), following growth in cell culture or bacterial isolation from intestines of infected piglets, in the context of functionally and structurally characterized biochemical pathways of E. coli. Protein solubilization in the presence of Triton X-100, EDTA and high salt was followed by size exclusion chromatography into the approximate Mr ranges greater than 280 kDa, 280-80 kDa and 80-10 kDa. Peptide mixtures resulting from these and the insoluble fraction were analyzed by quantitative 2D-LC-nESI-MS/MS. Of the 2521 proteins identified at a 1% false discovery rate, representing 47% of all predicted E. coli O157:H7 gene products, the majority of integral membrane proteins were enriched in the high Mr fraction. Hundreds of proteins were enriched in a Mr range higher than that predicted for a monomer supporting their participation in protein complexes. The insoluble STEC fraction revealed enrichment of aggregation-prone proteins, including many that are part of large structure/function entities such as the ribosome, cytoskeleton and O-antigen biosynthesis cluster. Significance Nearly all E. coli O157:H7 proteins encoded by prophage regions were expressed at low abundance levels or not detected. Comparative quantitative analyses of proteins from distinct cell lysate fractions allowed us to associate uncharacterized proteins with membrane attachment, potential participation in stable protein complexes, and susceptibility to aggregation as part of larger structural assemblies.