Proteomic Analysis of Antibiotic Susceptibility in Enterobacteriaceae.

Mass spectrometry is a sensitive and specific analytical technique that is capable of providing qualitative and quantitative data to resolve the protein elements of biochemical pathways that are altered by antibiotics. Here we present methods to study antibiotic susceptibility by changes in protein abundance, as exemplified by Klebsiella pneumoniae, a Gram-negative pathogen that colonizes mucosal surfaces of the human gastrointestinal and respiratory tracts. Cultured bacteria are exposed to antibiotics, the total proteomes of collected cell pellets are converted to complex peptide mixtures by filter-aided sample preparation (FASP), and the peptides are further processed by an optimized desalting procedure. A mixture of peptides from Klebsiella pneumoniae proteomes are analyzed by high-resolution mass spectrometry (MS) that is coupled to sensitive and comprehensive nano-liquid chromatography (nano-LC). The generic method described here for the identification and quantification of the proteome will provide a snapshot of differential protein abundances resulting from antimicrobial sensitivities, which can be used to model directed perturbations of the global system and to select targets of specific interest for further study.

Binding Sites of Anti-Lcr V Monoclonal Antibodies Are More Critical than the Avidities and Affinities for Passive Protection against Yersinia pestis Infection in a Bubonic Plague Model.

Plague is a zoonotic disease that is caused by Yersinia pestis. Monoclonal antibodies (mAbs) that bind to the V-antigen, a virulence factor that is produced by Y. pestis, can passively protect mice from plague. An analysis of protective mAbs that bind to V-antigen was made to assess binding sites, avidities, and affinities. Anti-V mAbs were screened for their efficacy in a murine model of plague. Antigen-binding sites of protective V mAbs were determined with a linear peptide library, V-antigen fragment, competitive binding, and surface plasmon resonance. The avidities to the V-antigen was determined by ELISA, and affinities of the mAbs to the V-antigen were determined by surface plasmon resonance. The most protective mAb 7.3 bound to a unique conformational site on the V-antigen, while a less protective mAb bound to a different conformational site located on the same V-antigen fragment as mAb 7.3. The avidity of mAb 7.3 for the V-antigen was neither the strongest overall nor did it have the highest affinity for the V-antigen. The binding site of the most protective mAb was critical in its ability to protect against a lethal plague challenge.

An increase in p62/NBR1 levels in melioidosis patients of Sri Lanka exhibit a characteristic of potential host biomarker.

Introduction. Melioidosis, caused by Burkholderia pseudomallei, in endemic areas, poses a challenge for treating the diseased populations without accurate diagnosis, and the disease-specific biomarkers linked with the infection have yet to be reported. Due to the invasive nature of the causative agent, Burkholderia pseudomallei, host innate effector mechanisms, including autophagy are known to be activated, resulting in differential expression of cellular proteins and immune markers. Identification of a disease-specific biomarker associated with B. pseudomallei infection will be helpful to facilitate rapid confirmation of melioidosis, which would enable early treatment and therapeutic success.Aim. We aimed to assess the levels of a host autophagy component, p62/NBR1, which function as a cargo-receptor in the process of autophagy activation leading to the degradation of ubiquitin-coated intracellular bacteria in which p62/NBR1 itself is degraded in the clearance of the pathogen. We further probed the extent of intracellular p62/NBR1 degradation and assessed its potential as a melioidosis biomarker.Methodology. We analysed peripheral blood mononuclear cell (PBMC) lysates using an ELISA-based assay for detecting cytosolic autophagy-related proteins p62/NBR1. We measured p62/NBR1 levels in diseased (confirmed B. pseudomallei infection) and non -diseased populations and utilized receiver operating characteristic (ROC) curve and max Youden index analysis for evaluating potential disease biomarker characteristics.Results. Our results revealed a three to fivefold increase in p62/NBR1 levels confirmed melioidosis cases compared to uninfected healthy donors. Comparable to p62/NBR1, levels of cytosolic LC3-I levels also increased, whereas the levels of degraded membrane bound form LC3-II was low, suggesting autophagy deficiency. Proinflammatory serum cytokine response, particularly IL-6, was consistently higher alongside B. pseudomallei infection in comparison to healthy controls.Conclusions. ROC curve and max Youden index analysis suggest that increased p62/NBR1 levels in diseased populations display characteristics of a potential disease biomarker in melioidosis and illustrates that an elevated p62/NBR1 level, in conjunction with B. pseudomallei infection associated with autophagy deficiency.

Decreased Antibiotic Susceptibility Driven by Global Remodeling of the Klebsiella pneumoniae Proteome.

Bacteria can circumvent the effect of antibiotics by transitioning to a poorly understood physiological state that does not involve conventional genetic elements of resistance. Here we examine antibiotic susceptibility with a Class A ß-lactamase+ invasive strain of Klebsiella pneumoniae that was isolated from a lethal outbreak within laboratory colonies of Chlorocebus aethiops sabaeus monkeys. Bacterial responses to the ribosomal synthesis inhibitors streptomycin and doxycycline resulted in distinct proteomic adjustments that facilitated decreased susceptibility to each antibiotic. Drug-specific changes to proteomes included proteins for receptor-mediated membrane transport and sugar utilization, central metabolism, and capsule production, whereas mechanisms common to both antibiotics included elevated scavenging of reactive oxygen species and turnover of misfolded proteins. Resistance to combined antibiotics presented integrated adjustments to protein levels as well as unique drug-specific proteomic features. Our results demonstrate that dampening of Klebsiella pneumoniae susceptibility involves global remodeling of the bacterial proteome to counter the effects of antibiotics and stabilize growth.

Targeting Protein-Protein Interactions of Tyrosine Phosphatases with Microarrayed Fragment Libraries Displayed on Phosphopeptide Substrate Scaffolds.

Chemical library screening approaches that focus exclusively on catalytic events may overlook unique effects of protein-protein interactions that can be exploited for development of specific inhibitors. Phosphotyrosyl (pTyr) residues embedded in peptide motifs comprise minimal recognition elements that determine the substrate specificity of protein tyrosine phosphatases (PTPases). We incorporated aminooxy-containing amino acid residues into a 7-residue epidermal growth factor receptor (EGFR) derived phosphotyrosine-containing peptide and subjected the peptides to solution-phase oxime diversification by reacting with aldehyde-bearing druglike functionalities. The pTyr residue remained unmodified. The resulting derivatized peptide library was printed in microarrays on nitrocellulose-coated glass surfaces for assessment of PTPase catalytic activity or on gold monolayers for analysis of kinetic interactions by surface plasmon resonance (SPR). Focusing on amino acid positions and chemical features, we first analyzed dephosphorylation of the peptide pTyr residues within the microarrayed library by the human dual-specificity phosphatases (DUSP) DUSP14 and DUSP22, as well as by PTPases from poxviruses (VH1) and Yersinia pestis (YopH). In order to identify the highest affinity oxime motifs, the binding interactions of the most active derivatized phosphopeptides were examined by SPR using noncatalytic PTPase mutants. On the basis of high-affinity oxime fragments identified by the two-step catalytic and SPR-based microarray screens, low-molecular-weight nonphosphate-containing peptides were designed to inhibit PTP catalysis at low micromolar concentrations.

A Smartphone-Based Rapid Telemonitoring System for Ebola and Marburg Disease Surveillance.

We have developed a digital and multiplexed platform for the rapid detection and telemonitoring of infections caused by Ebola and Marburg filoviruses. The system includes a flow cell assay cartridge that captures specific antibodies with microarrayed recombinant antigens from all six species of filovirus, and a smartphone fluorescent reader for high-performance interpretation of test results. Multiplexed viral proteins, which are expandable to include greater numbers of probes, were incorporated to obtain highest confidence results by cross-correlation, and a custom smartphone application was developed for data analysis, interpretation, and communication. The smartphone reader utilizes an opto-electro-mechanical hardware attachment that snaps at the back of a Motorola smartphone and provides a user interface to manage the operation, acquire test results, and communicate with cloud service. The application controls the hardware attachment to turn on LEDs and digitally record the optically enhanced images. Assay processing time is approximately 20 min for microliter amounts of blood, and test results are digitally processed and displayed within 15 s. Furthermore, a secure cloud service was developed for the telemonitoring of test results generated by the smartphone readers in the field. Assay system results were tested with sera from nonhuman primates that received a live attenuated EBOV vaccine. This integrated system will provide a rapid, reliable, and digital solution to prevent the rapid overwhelming of medical systems and resources during EVD or MVD outbreaks. Further, this disease-monitoring system will be useful in resource-limited countries where there is a need for dispersed laboratory analysis of recent or active infections.

An increase in intracellular p62/NBR1 and persistence of Burkholderia mallei and B. pseudomallei in infected mice linked to autophagy deficiency.

INTRODUCTION: Burkholderia mallei (B. mallei) and Burkholderia pseudomallei (B. pseudomallei), causative agents of glanders and melioidosis, respectively, are invasive intracellular pathogens that actively multiply in phagocytic and non-phagocytic cells. Activation of cell-autonomous autophagy mechanism eliminate intracellular pathogens in which p62 a cytosolic cargo protein is selectively degraded, and an accumulation of this marker occurs if autophagy is deficient. Recurrent, relapsed and reinfection of B. pseudomallei in melioidosis patients in endemic area indicative of lack of complete of clearance and persistence of the pathogen. Reasoning that abundance in the levels of p62 may provide an indication of the intracellular infection, we sought to examine whether increase in intracellular p62 and bacterial burden with Burkholderia infection are linked to autophagy deficiency. METHODS: In this study, we investigated cell culture and mouse models of disease to identify an association between autophagy biomarkers (p62/NBR1) accumulation and intracellular persistence of B. mallei and B. pseudomallei. RESULTS: We demonstrate, that elevated levels of intracellular p62/NBR1 correlated with bacterial persistence, while pre-treatment with a pharmacological inducer of autophagy, rapamycin, reduced both intracellular p62, and bacterial survival. Our results showed an elevated p62 levels (2-5 fold) in spleen and liver cells of Burkholderia-infected BALB/c mice, as well as in spleen cells of Burkholderia-infected C57BL/6 mice, suggesting that an increase in p62/NBR1 was due to an autophagy deficiency. Similar to p62, cytosolic LC3-I levels were also elevated, while the characteristic conversion to the autophagosome-associated membrane bound form LC3-II was low in spleens of the infected mice further supporting the conclusion that autophagy was deficient. CONCLUSION: Taken together, our results suggest that an increase in intracellular p62/NBR1 may be a potential host cell biomarker of B. mallei or B. pseudomallei infections, and identifying autophagy manipulation may potentially aid to therapeutic approach for complete clearance of the pathogen.

High-resolution crystal structures of the D1 and D2 domains of protein tyrosine phosphatase epsilon for structure-based drug design.

Here, new crystal structures are presented of the isolated membrane-proximal D1 and distal D2 domains of protein tyrosine phosphatase epsilon (PTPℇ), a protein tyrosine phosphatase that has been shown to play a positive role in the survival of human breast cancer cells. A triple mutant of the PTPℇ D2 domain (A455N/V457Y/E597D) was also constructed to reconstitute the residues of the PTPℇ D1 catalytic domain that are important for phosphatase activity, resulting in only a slight increase in the phosphatase activity compared with the native D2 protein. The structures reported here are of sufficient resolution for structure-based drug design, and a microarray-based assay for high-throughput screening to identify small-molecule inhibitors of the PTPℇ D1 domain is also described.

Impact of Dengue Virus Serotype 2 Strain Diversity on Serological Immune Responses to Dengue.

Dengue is a mosquito-borne disease caused by four dengue virus serotypes (DENV1-4) that are loosely categorized by sequence commonalities and antibody recognition profiles. The highly variable envelope protein (E) that is prominently displayed on the surface of DENV is an essential component of vaccines currently under development, yet the impact of using single strains to represent each serotype in tetravalent vaccines has not been adequately studied. We synthesized chimeric E by replacing highly variable residues from a dengue virus serotype 2 vaccine strain (PUO-218) with those from 16 DENV2 lineages spanning 60 years of antigen evolution. Examining sera from human and rhesus macaques challenged with single strains of DENV2, antibody-E interactions were markedly inhibited or enhanced by residues mainly focused within a 480 Å2 footprint displayed on the E backbone. The striking impact of E diversity on polyclonal immune responses suggests that frequent antigen updates may be necessary for vaccines to counter shifts in circulating strains.

Leaf-Encapsulated Vaccines: Agroinfiltration and Transient Expression of the Antigen Staphylococcal Endotoxin B in Radish Leaves.

Transgene introgression is a major concern associated with transgenic plant-based vaccines. Agroinfiltration can be used to selectively transform nonreproductive organs and avoid introgression. Here, we introduce a new vaccine modality in which Staphylococcal enterotoxin B (SEB) genes are agroinfiltrated into radishes (Raphanw sativus L.), resulting in transient expression and accumulation of SEB in planta. This approach can simultaneously express multiple antigens in a single leaf. Furthermore, the potential of high-throughput vaccine production was demonstrated by simultaneously agroinfiltrating multiple radish leaves using a multichannel pipette. The expression of SEB was detectable in two leaf cell types (epidermal and guard cells) in agroinfiltrated leaves. ICR mice intranasally immunized with homogenized leaves agroinfiltrated with SEB elicited detectable antibody to SEB and displayed protection against SEB-induced interferon-gamma (IFN-γ) production. The concept of encapsulating antigens in leaves rather than purifying them for immunization may facilitate rapid vaccine production during an epidemic disease.

Human Antibody Responses to Emerging Mayaro Virus and Cocirculating Alphavirus Infections Examined by Using Structural Proteins from Nine New and Old World Lineages.

Mayaro virus (MAYV), Venezuelan equine encephalitis virus (VEEV), and chikungunya virus (CHIKV) are vector-borne alphaviruses that cocirculate in South America. Human infections by these viruses are frequently underdiagnosed or misdiagnosed, especially in areas with high dengue virus endemicity. Disease may progress to debilitating arthralgia (MAYV, CHIKV), encephalitis (VEEV), and death. Few standardized serological assays exist for specific human alphavirus infection detection, and antigen cross-reactivity can be problematic. Therefore, serological platforms that aid in the specific detection of multiple alphavirus infections will greatly expand disease surveillance for these emerging infections. In this study, serum samples from South American patients with PCR- and/or isolation-confirmed infections caused by MAYV, VEEV, and CHIKV were examined by using a protein microarray assembled with recombinant capsid, envelope protein 1 (E1), and E2 from nine New and Old World alphaviruses. Notably, specific antibody recognition of E1 was observed only with MAYV infections, whereas E2 was specifically targeted by antibodies from all of the alphavirus infections investigated, with evidence of cross-reactivity to E2 of o'nyong-nyong virus only in CHIKV-infected patient serum samples. Our findings suggest that alphavirus structural protein microarrays can distinguish infections caused by MAYV, VEEV, and CHIKV and that this multiplexed serological platform could be useful for high-throughput disease surveillance. IMPORTANCE Mayaro, chikungunya, and Venezuelan equine encephalitis viruses are closely related alphaviruses that are spread by mosquitos, causing diseases that produce similar influenza-like symptoms or more severe illnesses. Moreover, alphavirus infection symptoms can be similar to those of dengue or Zika disease, leading to underreporting of cases and potential misdiagnoses. New methods that can be used to detect antibody responses to multiple alphaviruses within the same assay would greatly aid disease surveillance efforts. However, possible antibody cross-reactivity between viruses can reduce the quality of laboratory results. Our results demonstrate that antibody responses to multiple alphaviruses can be specifically quantified within the same assay by using selected recombinant protein antigens and further show that Mayaro virus infections result in unique responses to viral envelope proteins.

Antibiotic-dependent perturbations of extended spectrum beta-lactamase producing Klebsiella pneumoniae proteome.

Extended spectrum beta-lactamase producing Klebsiella pneumoniae (ESBL-KP) causes life-threatening infections in susceptible and immuno-compromised individuals. Because of the emergence of multidrug resistance and tolerance, it is crucial to better understand the mechanisms by which ESBL-KP can adapt to antibiotic stress. The aim of this study was to provide an overview of the global proteome changes occurring in ESBL-KP in response to sub-lethal concentrations of the antibiotics doxycycline (DC, bacteriostatic) and streptomycin (SM, bactericidal), which both impair ribosomal synthesis of bacterial proteins. These results represent the greatest experimental coverage of the ESBL-KP proteome yet described. The 1538 proteins, representing 30% of the 5126 predicted KP gene products were identified from the combined experimental groups. Antibiotic stress resulted in significantly elevated levels of 42 proteins for DC and 55 for SM treatments, whereas 53 proteins were reduced for DC- and six for SM-treated bacteria. Specifically, the ESBL-KP response to DC was accompanied by the reduced levels of the porins LamB, CirA, FepA, and OmpC. In contrast to DC, the stress response to SM demonstrated a dramatic increase in the peroxidase detoxification pathway proteins PutA, KatG, KatE, and Dps, which prevent harmful hydroxyl radical formation. The results from this proteomic study are important for understanding adaptive responses to antibiotics, and may provide novel targets for the development of new therapeutic strategies.

Antibody Responses to Zika Virus Infections in Environments of Flavivirus Endemicity.

Zika virus (ZIKV) infections occur in areas where dengue virus (DENV), West Nile virus (WNV), yellow fever virus (YFV), and other viruses of the genus Flavivirus cocirculate. The envelope (E) proteins of these closely related flaviviruses induce specific long-term immunity, yet subsequent infections are associated with cross-reactive antibody responses that may enhance disease susceptibility and severity. To gain a better understanding of ZIKV infections against a background of similar viral diseases, we examined serological immune responses to ZIKV, WNV, DENV, and YFV infections of humans and nonhuman primates (NHPs). Using printed microarrays, we detected very specific antibody responses to primary infections with probes of recombinant E proteins from 15 species and lineages of flaviviruses pathogenic to humans, while high cross-reactivity between ZIKV and DENV was observed with 11 printed native viruses. Notably, antibodies from human primary ZIKV or secondary DENV infections that occurred in areas where flavivirus is endemic broadly recognized E proteins from many flaviviruses, especially DENV, indicating a strong influence of infection history on immune responses. A predictive algorithm was used to tentatively identify previous encounters with specific flaviviruses based on serum antibody interactions with the multispecies panel of E proteins. These results illustrate the potential impact of exposure to related viruses on the outcome of ZIKV infection and offer considerations for development of vaccines and diagnostics.

Calprotectin as a Biomarker for Melioidosis Disease Progression and Management.

Melioidosis is a neglected tropical disease that is caused by the bacterium Burkholderia pseudomallei and is underreported in many countries where the disease is endemic. A long and costly administration of antibiotics is needed to clear infections, and there is an unmet need for biomarkers to guide antibiotic treatment and increase the number of patients that complete therapy. We identified calprotectin as a lead biomarker of B. pseudomallei infections and examined correlations between this serum protein and the antibiotic treatment outcomes of patients with melioidosis. Serum levels of calprotectin and C-reactive protein were significantly higher in patients with melioidosis and nonmelioidosis sepsis than in healthy controls. Median calprotectin levels were higher in patients with melioidosis than in those with nonmelioidosis sepsis, whereas C-reactive protein levels were similar in both groups. Notably, intensive intravenous antibiotic treatment of patients with melioidosis resulted in lower levels of calprotectin and C-reactive protein (P < 0.0001), coinciding with recovery. The median percent reduction of calprotectin and C-reactive protein was 71% for both biomarkers after antibacterial therapy. In contrast, we found no significant differences in calreticulin levels between the two melioidosis treatment phases. Thus, reductions in serum calprotectin levels were linked to therapeutic responses to antibiotics. Our results suggest that calprotectin may be a sensitive indicator of melioidosis disease activity and illustrate the potential utility of this biomarker in guiding the duration of antibiotic therapy.

Cell-free Determination of Binary Complexes That Comprise Extended Protein-Protein Interaction Networks of Yersinia pestis.

Binary protein interactions form the basic building blocks of molecular networks and dynamic assemblies that control all cellular functions of bacteria. Although these protein interactions are a potential source of targets for the development of new antibiotics, few high-confidence data sets are available for the large proteomes of most pathogenic bacteria. We used a library of recombinant proteins from the plague bacterium Yersinia pestis to probe planar microarrays of immobilized proteins that represented ∼85% (3552 proteins) of the bacterial proteome, resulting in >77,000 experimentally determined binary interactions. Moderate (KD ∼µm) to high-affinity (KD ∼nm) interactions were characterized for >1600 binary complexes by surface plasmon resonance imaging of microarrayed proteins. Core binary interactions that were in common with other gram-negative bacteria were identified from the results of both microarray methods. Clustering of proteins within the interaction network by function revealed statistically enriched complexes and pathways involved in replication, biosynthesis, virulence, metabolism, and other diverse biological processes. The interaction pathways included many proteins with no previously known function. Further, a large assembly of proteins linked to transcription and translation were contained within highly interconnected subregions of the network. The two-tiered microarray approach used here is an innovative method for detecting binary interactions, and the resulting data will serve as a critical resource for the analysis of protein interaction networks that function within an important human pathogen.

Human Survivors of Disease Outbreaks Caused by Ebola or Marburg Virus Exhibit Cross-Reactive and Long-Lived Antibody Responses.

A detailed understanding of serological immune responses to Ebola and Marburg virus infections will facilitate the development of effective diagnostic methods, therapeutics, and vaccines. We examined antibodies from Ebola or Marburg survivors 1 to 14 years after recovery from disease, by using a microarray that displayed recombinant nucleoprotein (NP), viral protein 40 (VP40), envelope glycoprotein (GP), and inactivated whole virions from six species of filoviruses. All three outbreak cohorts exhibited significant antibody responses to antigens from the original infecting species and a pattern of additional filoviruses that varied by outbreak. NP was the most cross-reactive antigen, while GP was the most specific. Antibodies from survivors of infections by Marburg marburgvirus (MARV) species were least cross-reactive, while those from survivors of infections by Sudan virus (SUDV) species exhibited the highest cross-reactivity. Based on results revealed by the protein microarray, persistent levels of antibodies to GP, NP, and VP40 were maintained for up to 14 years after infection, and survival of infection caused by one species imparted cross-reactive antibody responses to other filoviruses.

Povidone Iodine Ointment Application to the Vaccination Site Does Not Alter Immunoglobulin G Antibody Response to Smallpox Vaccine.

U.S. military personnel deployed to high-risk areas receive the live vaccinia virus (VACV) smallpox vaccine ACAM2000. VACV shedding from the vaccination site can result in autoinoculation and contact transmission. We previously found that the application of povidone iodine ointment (PIO) to the scarification site reduced viral shedding without altering the antibody response, as measured by plaque reduction neutralization or enzyme-linked immunosorbent assays. In this study, we used protein microarray assays to measure the amount of immunoglobulin G antibody bound to (1) ACAM2000 itself and (2) individual VACV antigens that are present within ACAM2000. We assessed antibody binding in sera from primary smallpox vaccinees who applied PIO to the scarification site beginning on day 7 (PIO group) and from those who did not apply PIO (control group). In both cohorts, the postvaccination antibody response-in terms of antibody binding, both to ACAM2000 and to 11 individual VACV antigens-was significantly greater than the prevaccination response (all p < 0.0001). The postvaccination antibody binding levels of vaccinees in the PIO group did not differ from those of control vaccinees. These findings further support the topical application of PIO, starting on day 7, to reduce the viral shedding associated with smallpox vaccination.

Phosphotyrosine Substrate Sequence Motifs for Dual Specificity Phosphatases.

Protein tyrosine phosphatases dephosphorylate tyrosine residues of proteins, whereas, dual specificity phosphatases (DUSPs) are a subgroup of protein tyrosine phosphatases that dephosphorylate not only Tyr(P) residue, but also the Ser(P) and Thr(P) residues of proteins. The DUSPs are linked to the regulation of many cellular functions and signaling pathways. Though many cellular targets of DUSPs are known, the relationship between catalytic activity and substrate specificity is poorly defined. We investigated the interactions of peptide substrates with select DUSPs of four types: MAP kinases (DUSP1 and DUSP7), atypical (DUSP3, DUSP14, DUSP22 and DUSP27), viral (variola VH1), and Cdc25 (A-C). Phosphatase recognition sites were experimentally determined by measuring dephosphorylation of 6,218 microarrayed Tyr(P) peptides representing confirmed and theoretical phosphorylation motifs from the cellular proteome. A broad continuum of dephosphorylation was observed across the microarrayed peptide substrates for all phosphatases, suggesting a complex relationship between substrate sequence recognition and optimal activity. Further analysis of peptide dephosphorylation by hierarchical clustering indicated that DUSPs could be organized by substrate sequence motifs, and peptide-specificities by phylogenetic relationships among the catalytic domains. The most highly dephosphorylated peptides represented proteins from 29 cell-signaling pathways, greatly expanding the list of potential targets of DUSPs. These newly identified DUSP substrates will be important for examining structure-activity relationships with physiologically relevant targets.

Serum biomarkers of Burkholderia mallei infection elucidated by proteomic imaging of skin and lung abscesses.

BACKGROUND: The bacterium Burkholderia mallei is the etiological agent of glanders, a highly contagious, often fatal zoonotic infectious disease that is also a biodefense concern. Clinical laboratory assays that analyze blood or other biological fluids are the highest priority because these specimens can be collected with minimal risk to the patient. However, progress in developing sensitive assays for monitoring B. mallei infection is hampered by a shortage of useful biomarkers. RESULTS: Reasoning that there should be a strong correlation between the proteomes of infected tissues and circulating serum, we employed imaging mass spectrometry (IMS) of thin-sectioned tissues from Chlorocebus aethiops (African green) monkeys infected with B. mallei to localize host and pathogen proteins that were associated with abscesses. Using laser-capture microdissection of specific regions identified by IMS and histology within the tissue sections, a more extensive proteomic analysis was performed by a technique that combined the physical separation capabilities of liquid chromatography (LC) with the sensitive mass analysis capabilities of mass spectrometry (LC-MS/MS). By examining standard formalin-fixed, paraffin-embedded tissue sections, this strategy resulted in the identification of several proteins that were associated with lung and skin abscesses, including the host protein calprotectin and the pathogen protein GroEL. Elevated levels of calprotectin detected by ELISA and antibody responses to GroEL, measured by a microarray of the bacterial proteome, were subsequently detected in the sera of C. aethiops, Macaca mulatta, and Macaca fascicularis primates infected with B. mallei. CONCLUSIONS: Our results demonstrate that a combination of multidimensional MS analysis of traditional histology specimens with high-content protein microarrays can be used to discover lead pairs of host-pathogen biomarkers of infection that are identifiable in biological fluids.

Characterization of cellular immune response and innate immune signaling in human and nonhuman primate primary mononuclear cells exposed to Burkholderia mallei.

Burkholderia pseudomallei infection causes melioidosis and is often characterized by severe sepsis. Although rare in humans, Burkholderia mallei has caused infections in laboratory workers, and the early innate cellular response to B. mallei in human and nonhuman primates has not been characterized. In this study, we examined the primary cellular immune response to B. mallei in PBMC cultures of non-human primates (NHPs), Chlorocebus aethiops (African Green Monkeys), Macaca fascicularis (Cynomolgus macaque), and Macaca mulatta (Rhesus macaque) and humans. Our results demonstrated that B. mallei elicited strong primary pro-inflammatory cytokines (IFN-γ, TNF-α, IL-1ß, and IL-6) equivalent to the levels of B. pseudomallei in primary PBMC cultures of NHPs and humans. When we examined IL-1ß and other cytokine responses by comparison to Escherichia coli LPS, African Green Monkeys appears to be most responsive to B. mallei than Cynomolgus or Rhesus. Characterization of the immune signaling mechanism for cellular response was conducted by using a ligand induced cell-based reporter assay, and our results demonstrated that MyD88 mediated signaling contributed to the B. mallei and B. pseudomallei induced pro-inflammatory responses. Notably, the induced reporter activity with B. mallei, B. pseudomallei, or purified LPS from these pathogens was inhibited and cytokine production was attenuated by a MyD88 inhibitor. Together, these results show that in the scenario of severe hyper-inflammatory responses to B. mallei infection, MyD88 targeted therapeutic intervention may be a successful strategy for therapy.