Laser Capture Microdissection-Assisted Protein Biomarker Discovery from Coccidioides-Infected Lung Tissue.

Laser capture microdissection (LCM) coupled to label-free quantitative mass spectrometry is a viable strategy to identify biomarkers from infected tissues. In this study, LCM was employed to take a "snapshot" of proteins produced in vivo during Coccidiodies spp. infection in human lungs. Proteomic analysis of LCM lung sections revealed hundreds of hosts and Coccidioidal proteins. Twenty-seven highly abundant Coccidioides spp. proteins were identified which do not share significant sequence orthology with human proteins. Three of the 27 Coccidioidal proteins are also potential Coccidoides-specific biomarkers, as they also do not share sequence homology to any other pathogenic fungus or microbe. Gene ontology analysis of the 27 biomarker candidate proteins revealed enriched hydrolase activity and increased purine and carbohydrate metabolism functions. Finally, we provide proteomic evidence that all 27 biomarker candidates are produced by the fungus when grown in vitro in a media- and growth-phase dependent manner.

Carbo-loading in Coccidioides spp.: A quantitative analysis of CAZyme abundance and resulting glycan populations.

Coccidioides spp. are important pneumonia-causing pathogens of the American southwest, but little is known about their glycobiology and how their glycosylations differ from other pneumonia-causing fungi. There is mounting preliminary evidence to suggest genus or even species-specific glycosylations in the fungal kingdom due to the presence of unique carbohydrate-active enzymes (CAZymes) in fungal genomes (Deshpande et al. 2008, Glycobiology, 18(8), 626-637; Karkowska-Kuleta and Kozik 2015, Acta Biochim Pol., 62(3), 339-351). If Coccidioides spp.-specific glycans can be identified, it may be possible to exploit these differences to develop more specific diagnostic approaches and more effective therapeutics. Herein, we i) mined Coccidioides spp. and other pathogenic fungal genomes to identify CAZymes specific for Coccidioides spp., ii) proteomically determined the Coccidioides spp. "CAZome" produced in vivo and in vitro, and iii) utilized glycomics to differentiate Coccidioides genus-specific N-glycans from other pathogenic fungi. As far as we are aware, this is the first proteomic and glycomic comparison of the N-glycomes and CAZomes of different fungal genera during infection in human hosts.

Coccidioidomycosis Detection Using Targeted Plasma and Urine Metabolic Profiling.

Coccidioidomycosis, also known as Valley fever (VF), is a potentially lethal fungal infection that results in more than 200 deaths per year in the United States. Despite the important role of metabolic processes in the molecular pathogenesis of VF, robust metabolic markers to enable effective screening, rapid diagnosis, accurate surveillance, and therapeutic monitoring of VF are still lacking. We present a targeted liquid chromatography-tandem mass spectrometry-based metabolic profiling approach for identifying metabolic marker candidates that could enable rapid, highly sensitive, and specific VF detection. Using this targeted approach, 207 plasma metabolites and 231 urinary metabolites from many metabolic pathways of potential biological significance were reliably detected and monitored in 147 samples taken from two groups of subjects (48 VF patients and 99 non-VF controls). The results of our univariate significance testing and multivariate model development informed the construction of a three-metabolite panel of potential plasma biomarkers and a nine-metabolite panel of potential urinary biomarkers. Receiver operating characteristic curves generated based on orthogonal partial least-squares-discriminant analysis models showed excellent classification performance, with 94.4% sensitivity and 97.6% specificity for plasma metabolites. Urine metabolites were less accurate, demonstrating 89.7% sensitivity and 88.1% specificity. Enrichment, pathway, and network analyses revealed significant disturbances in glycine and serine metabolism, in both plasma and urine samples. To the best of our knowledge, this is the first study aiming to discover novel metabolite markers of VF, which could achieve accurate diagnosis within 24 h. The results expand the basic knowledge of the metabolome related to VF and potentially reveal pathways or markers that could be therapeutically targeted. This study also provides a promising basis for the development of larger multisite projects to validate our findings across population groups and further advance the development of better clinical care for VF patients.

Evaluation of Virex® II 256 and Virex® Tb as Disinfectants of the Dimorphic Fungi Coccidioides immitis and Coccidioides posadasii.

To date, limited published data exists regarding the efficacy of commonly used disinfectants in inactivating the Risk Group 3 dimorphic fungal pathogens, Coccidioides immitis and Coccidioides posadasii. Newer generation quaternary ammonium compounds, like Virex® II 256 and Virex® Tb, have not been previously evaluated. Herein, these disinfectants are evaluated against 10% bleach and 70% ethanol, for their ability to inactivate 5×107 arthroconidial spores of C. immitis RS or C. posadasii strain Silveira within 2, 5, 10 or 20 minutes contact time in aqueous solution. Evidence is provided that both Virex® II 256 and Virex® Tb are highly effective alternatives to 10% bleach or 70% ethanol for the disinfection of 5×107 arthroconidia of Coccidioides spp. within 2 minutes of contact time. 70% ethanol was seen as less effective in killing C.immitis RS arthroconidia and both 70% ethanol and 10% bleach were seen as less effective than the other disinfectants in killing C. posadasii strain Silveira, as longer contact times were required to completely inactivate the same number of arthroconidia.

Proteogenomic Re-Annotation of Coccidioides posadasii Strain Silveira.

The aims of this study are to provide protein-based evidence upon which to reannotate the genome of Coccidiodes posadasii, one of two closely related species of Coccidioides, a dimorphic fungal pathogen that causes coccidioidomycosis, also called Valley fever. Proteins present in lysates and filtrates of in vitro grown mycelia and parasitic phase spherules from C. posadasii strain Silveira are analyzed using a GeLC-MS/MS method. Acquired spectra are processed with a proteogenomics workflow comprising a Silveira proteome database, a six-frame translation of the Silveira genome and an ab initio gene prediction tool prior to validation against published ESTs. This study provides evidence for 837 genes expressed at the protein level, of which 169 proteins (20.2%) are putative proteins and 103 (12.3%) are not annotated in the Silveira genome. Additionally, 275 novel peptides are derived from intragenic regions of the genome and 13 from intergenic regions, resulting in 172 gene refinements. Additionally, we are the first group to report translationally active retrotransposon elements in a Coccidioides spp. Our study reveals that the currently annotated genome of C. posadasii str. Silveira needs refinement, which is likely to be the case for many nonmodel organisms.

Novel vaccine antigen combinations elicit protective immune responses against Escherichia coli sepsis.

Systemic infections caused by extraintestinal pathogenic Escherichia coli (ExPEC) have emerged as the most common community-onset bacterial infections and are major causes of nosocomial infections worldwide. The management of ExPEC infections has been complicated by the heterogeneity of ExPEC strains and the emergence of antibiotic resistance, thus their prevention through vaccination would be beneficial. The protective efficacy of four common ExPEC antigen candidates composed of common pilus antigens EcpA and EcpD and iron uptake proteins IutA and IroN, were tested by both active and passive immunization in lethal and non-lethal murine models of sepsis. Additionally, antibody raised to a synthetic form of a conserved surface polysaccharide, ß-(1-6)-linked poly-N-acetylglucosamine (dPNAG) containing 9 monomers of (non-acetylated) glucosamine (9GlcNH2) conjugated to tetanus toxoid TT (9GlcNH2-TT) was tested in passive immunization protocols. Active immunization of mice with recombinant antigens EcpA, EcpD, IutA, or IroN elicited high levels of total IgG antibody of IgG1/IgG2a isotypes, and were determined to be highly protective against E. coli infection in lethal and non-lethal sepsis challenges. Moreover, passive immunization against these four antigens resulted in significant reductions of bacteria in internal organs and blood of the mice, especially when the challenge strain was grown in iron-restricted media. Inclusion of antibodies to PNAG increased the efficacy of the passive immunization under conditions where the challenge bacteria were grown in LB medium but not in iron-restricted media. The information and data presented are the first step toward the development of a broadly protective vaccine against sepsis-causing E. coli strains.

Zoonotic potential of Escherichia coli isolates from retail chicken meat products and eggs.

Chicken products are suspected as a source of extraintestinal pathogenic Escherichia coli (ExPEC), which causes diseases in humans. The zoonotic risk to humans from chicken-source E. coli is not fully elucidated. To clarify the zoonotic risk posed by ExPEC in chicken products and to fill existing knowledge gaps regarding ExPEC zoonosis, we evaluated the prevalence of ExPEC on shell eggs and compared virulence-associated phenotypes between ExPEC and non-ExPEC isolates from both chicken meat and eggs. The prevalence of ExPEC among egg-source isolates was low, i.e., 5/108 (4.7%). Based on combined genotypic and phenotypic screening results, multiple human and avian pathotypes were represented among the chicken-source ExPEC isolates, including avian-pathogenic E. coli (APEC), uropathogenic E. coli (UPEC), neonatal meningitis E. coli (NMEC), and sepsis-associated E. coli (SEPEC), as well as an undefined ExPEC group, which included isolates with fewer virulence factors than the APEC, UPEC, and NMEC isolates. These findings document a substantial prevalence of human-pathogenic ExPEC-associated genes and phenotypes among E. coli isolates from retail chicken products and identify key virulence traits that could be used for screening.

Evaluation of the prevalence and production of Escherichia coli common pilus among avian pathogenic E. coli and its role in virulence.

Avian pathogenic Escherichia coli (APEC) strains cause systemic and localized infections in poultry, jointly termed colibacillosis. Avian colibacillosis is responsible for significant economic losses to the poultry industry due to disease treatment, decrease in growth rate and egg production, and mortality. APEC are also considered a potential zoonotic risk for humans. Fully elucidating the virulence and zoonotic potential of APEC is key for designing successful strategies against their infections and their transmission. Herein, we investigated the prevalence of a newly discovered E. coli common pilus (ECP) for the subunit protein of the ECP pilus (ecpA) and ECP expression amongst APEC strains as well as the role of ECP in virulence. A PCR-based ecpA survey of a collection of 167 APEC strains has shown that 76% (127/167) were ecpA+. An immunofluorescence assay using anti-EcpA antibodies, revealed that among the ecpA+ strains, 37.8% (48/127) expressed ECP when grown in DMEM +0.5% Mannose in contact with HeLa cells at 37°C and/or in biofilm at 28°C; 35.4% (17/48) expressed ECP in both conditions and 64.6% (31/48) expressed ECP in biofilm only. We determined that the ecp operon in the APEC strain χ7122 (ecpA+, ECP-) was not truncated; the failure to detect ECP in some strains possessing non-truncated ecp genes might be attributed to differential regulatory mechanisms between strains that respond to specific environmental signals. To evaluate the role of ECP in the virulence of APEC, we generated ecpA and/or ecpD-deficient mutants from the strain χ7503 (ecpA+, ECP+). Deletion of ecpA and/or ecpD abolished ECP synthesis and expression, and reduced biofilm formation and motility in vitro and virulence in vivo. All together our data show that ecpA is highly prevalent among APEC isolates and its expression could be differentially regulated in these strains, and that ECP plays a role in the virulence of APEC.