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.

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

Plague is a zoonotic disease that is caused by . Monoclonal antibodies (mAbs) that bind to the V-antigen, a virulence factor that is produced by , 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.

Decreased Antibiotic Susceptibility Driven by Global Remodeling of the 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 β-lactamase+ invasive strain of that was isolated from a lethal outbreak within laboratory colonies of monkeys. Bacterial responses to the ribosomal synthesis inhibitors streptomycin and doxycycline resulted in distinct proteomic adjustments that facilitated decreased susceptibility to each antibiotic.

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.

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 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).

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.

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.

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.

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.

Human antibody responses to the polyclonal Dryvax vaccine for smallpox prevention can be distinguished from responses to the monoclonal replacement vaccine ACAM2000.

Dryvax (Wyeth Laboratories, Inc., Marietta, PA) is representative of the vaccinia virus preparations that were previously used for preventing smallpox. While Dryvax was highly effective, the national supply stocks were depleted, and there were manufacturing concerns regarding sterility and the clonal heterogeneity of the vaccine.

Proteomic basis of the antibody response to monkeypox virus infection examined in cynomolgus macaques and a comparison to human smallpox vaccination.

Monkeypox is a zoonotic viral disease that occurs primarily in Central and West Africa. A recent outbreak in the United States heightened public health concerns for susceptible human populations. Vaccinating with vaccinia virus to prevent smallpox is also effective for monkeypox due to a high degree of sequence conservation.

Extensive antibody cross-reactivity among infectious gram-negative bacteria revealed by proteome microarray analysis.

Antibodies provide a sensitive indicator of proteins displayed by bacteria during sepsis. Because signals produced by infection are naturally amplified during the antibody response, host immunity can be used to identify biomarkers for proteins that are present at levels currently below detectable limits. We developed a microarray comprising approximately 70% of the 4066 proteins contained within the Yersinia pestis proteome to identify antibody biomarkers distinguishing plague from infections caused by other bacterial pathogens that may initially present similar clinical symptoms.

Analysis of the human immune response to vaccinia by use of a novel protein microarray suggests that antibodies recognize less than 10% of the total viral proteome.

Control of smallpox by mass vaccination was one of the most effective public health measures ever employed for eradicating a devastating infectious disease. However, new methods are needed for monitoring smallpox immunity within current vulnerable populations, and for the development of replacement vaccines for use by immunocompromized or low-responding individuals. As a measure for achieving this goal, we developed a protein microarray of the vaccinia virus proteome by using high-throughput baculovirus expression and purification of individual elements.