Antibodies in Action: Role of Human Opsonins in Killing Salmonella enterica Serovar Typhi

ABSTRACTAlthough vaccines have been available for over a century, a correlate of protection for typhoid fever has yet to be identified. Antibodies are produced in response to typhoid infection and vaccination and are generally used as the gold standard for determining vaccine immunogenicity, even though their role in clearance ofSalmonella entericaserovar Typhi infections is poorly defined. Here, we describe the first functional characterization ofS.Typhi-specific antibodies following vaccination with a new vaccine, M01ZH09 (Ty2 ΔaroCΔssaV). We determined that postvaccination sera increased the uptake of wild-typeS.Typhi by human macrophages up to 2.3-fold relative to prevaccination (day 0) or placebo samples. These results were recapitulated using immunoglobulins purified from postvaccination serum, demonstrating that antibodies were largely responsible for increases in uptake. Imaging verified that macrophages internalized 2- to 9.5-fold moreS.Typhi when the bacteria were opsonized with postvaccination sera than when the bacteria were opsonized with day 0 or placebo sera. Once inside macrophages, the survival ofS.Typhi was reduced as much as 50% when opsonized with postvaccination sera relative to day 0 or placebo serum samples. Lastly, bactericidal assays indicated that antibodies generated postvaccination were recognized by complement factors and assisted in killingS.Typhi: mean postvaccination bactericidal antibody titers were higher at all time points than placebo and day 0 titers. These data clearly demonstrate that there are at least two mechanisms by which antibodies facilitate killing ofS.Typhi. Future work could lead to improved immunogenicity tests associated with vaccine efficacy and the identification of correlates of protection against typhoid fever.

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Characterization of Epidemic IncI1-Iγ Plasmids Harboring Ambler Class A and C Genes in Escherichia coli and Salmonella enterica from Animals and Humans

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.05006-14 ◽

2015 ◽

Vol 59 (9) ◽

pp. 5357-5365 ◽

Cited By ~ 36

Author(s):

Hilde Smith ◽

Alex Bossers ◽

Frank Harders ◽

Guanghui Wu ◽

Neil Woodford ◽

...

Keyword(s):

Escherichia Coli ◽

Salmonella Enterica ◽

Phylogenetic Trees ◽

Functional Study ◽

Content Type ◽

Gene Associations ◽

Genes Encoding ◽

Marked Resistance

ABSTRACTThe aim of the study was to identify the plasmid-encoded factors contributing to the emergence and spread of epidemic IncI1-Iγ plasmids obtained fromEscherichia coliandSalmonella entericaisolates from animal and human reservoirs. For this, 251 IncI1-Iγ plasmids carrying various extended-spectrum β-lactamase (ESBL) or AmpC β-lactamase genes were compared using plasmid multilocus sequence typing (pMLST). Thirty-two of these plasmids belonging to different pMLST types were sequenced using Roche 454 and Illumina platforms. Epidemic IncI1-Iγ plasmids could be assigned to various dominant clades, whereas rarely detected plasmids clustered together as a distinct clade. Similar phylogenetic trees were obtained using only the plasmid backbone sequences, showing that the differences observed between the plasmids belonging to distinct clades resulted mainly from differences between their backbone sequences. Plasmids belonging to the various clades differed particularly in the presence/absence of genes encoding partitioning and addiction systems, which contribute to stable inheritance during cell division and plasmid maintenance. Despite this, plasmids belonging to the various phylogenetic clades also showed marked resistance gene associations, indicating the circulation of successful plasmid-gene combinations. The variation intraYandexcAgenes found in IncI1-Iγ plasmids is conserved within pMLST sequence types and plays a role in incompatibility, although functional study is needed to elucidate the role of these genes in plasmid epidemiology.

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Role of Antilipopolysaccharide Antibodies in Serum Bactericidal Activity against Salmonella enterica Serovar Typhimurium in Healthy Adults and Children in the United States

Clinical and Vaccine Immunology ◽

10.1128/cvi.00289-13 ◽

2013 ◽

Vol 20 (10) ◽

pp. 1491-1498 ◽

Cited By ~ 36

Author(s):

Estela Trebicka ◽

Susan Jacob ◽

Waheed Pirzai ◽

Bryan P. Hurley ◽

Bobby J. Cherayil

Keyword(s):

United States ◽

Bactericidal Activity ◽

Salmonella Enterica ◽

The United States ◽

Healthy Children ◽

Healthy Individuals ◽

Serum Samples ◽

Content Type ◽

Serovar Typhimurium

ABSTRACTRecent observations from Africa have rekindled interest in the role of serum bactericidal antibodies in protecting against systemic infection withSalmonella entericaserovar Typhimurium. To determine whether the findings are applicable to other populations, we analyzed serum samples collected from healthy individuals in the United States. We found that all but 1 of the 49 adult samples tested had robust bactericidal activity againstS. Typhimurium in a standardin vitroassay. The activity was dependent on complement and could be reproduced by immunoglobulin G (IgG) purified from the sera. The bactericidal activity was inhibited by competition with soluble lipopolysaccharide (LPS) fromS. Typhimurium but not fromEscherichia coli, consistent with recognition of a determinant in the O-antigen polysaccharide. Sera from healthy children aged 10 to 48 months also had bactericidal activity, although it was significantly less than in the adults, correlating with lower levels of LPS-specific IgM and IgG. The lone sample in our collection that lacked bactericidal activity was able to inhibit killing ofS. Typhimurium by the other sera. The inhibition correlated with the presence of an LPS-specific IgM and was associated with decreased complement deposition on the bacterial surface. Our results indicate that healthy individuals can have circulating antibodies to LPS that either mediate or inhibit killing ofS. Typhimurium. The findings contrast with the observations from Africa, which linked bactericidal activity to antibodies against anS. Typhimurium outer membrane protein and correlated the presence of inhibitory anti-LPS antibodies with human immunodeficiency virus infection.

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Functional Characterization of Burkholderia pseudomallei Trimeric Autotransporters

Infection and Immunity ◽

10.1128/iai.00526-13 ◽

2013 ◽

Vol 81 (8) ◽

pp. 2788-2799 ◽

Cited By ~ 15

Author(s):

Cristine G. Campos ◽

Matthew S. Byrd ◽

Peggy A. Cotter

Keyword(s):

Burkholderia Pseudomallei ◽

Functional Characterization ◽

A549 Cells ◽

Content Type ◽

Tier 1 ◽

Type V ◽

Mouse Model Of Infection ◽

Encoding Genes

ABSTRACTBurkholderia pseudomalleiis a tier 1 select agent and the causative agent of melioidosis, a severe and often fatal disease with symptoms ranging from acute pneumonia and septic shock to a chronic infection characterized by abscess formation in the lungs, liver, and spleen. Autotransporters (ATs) are exoproteins belonging to the type V secretion system family, with many playing roles in pathogenesis. The genome ofB. pseudomalleistrain 1026b encodes nine putative trimeric AT proteins, of which only four have been described. Using a bioinformatic approach, we annotated putative domains within each trimeric AT protein, excluding the well-studied BimA protein, and found short repeated sequences unique toBurkholderiaspecies, as well as an unexpectedly large proportion of ATs with extended signal peptide regions (ESPRs). To characterize the role of trimeric ATs in pathogenesis, we constructed disruption or deletion mutations in each of eight AT-encoding genes and evaluated the resulting strains for adherence to, invasion of, and plaque formation in A549 cells. The majority of the ATs (and/or the proteins encoded downstream) contributed to adherence to and efficient invasion of A549 cells. Using a BALB/c mouse model of infection, we determined the contributions of each AT to bacterial burdens in the lungs, liver, and spleen. At 48 h postinoculation, only one strain, Bp340::pDbpaC, demonstrated a defect in dissemination and/or survival in the liver, indicating that BpaC is required for wild-type virulence in this model.

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Seroprevalence of Antibody-Mediated, Complement-Dependent Opsonophagocytic Activity against Neisseria meningitidis Serogroup B in England

Clinical and Vaccine Immunology ◽

10.1128/cvi.00100-15 ◽

2015 ◽

Vol 22 (5) ◽

pp. 503-509 ◽

Cited By ~ 5

Author(s):

Holly E. Humphries ◽

Charlotte Brookes ◽

Lauren Allen ◽

Eeva Kuisma ◽

Andrew Gorringe ◽

...

Keyword(s):

Neisseria Meningitidis ◽

Bactericidal Activity ◽

Disease Incidence ◽

Age Group ◽

Serum Samples ◽

Complex Situation ◽

Content Type ◽

Serum Bactericidal Activity ◽

Bactericidal Antibody ◽

Antibody Titers

ABSTRACTThe correlate of protection for the licensure of meningococcal vaccines is serum bactericidal activity. However, evidence indicates that a complex situation and other mechanisms, such as antibody-mediated, complement-dependent opsonophagocytosis (OP), may play a role in protection and should be investigated in order to understand immunity to this disease. In this study, a high-throughput flow cytometric opsonophagocytic assay (OPA) was optimized. The assay measures the presence of killed fluorescently labeledNeisseria meningitidiswithin human granulocytes (differentiated HL60 cells) by flow cytometry, using IgG-depleted pooled human plasma as an exogenous source of complement. This method was found to be reliable and correlated with the results of an opsonophagocytic killing assay. The OPA was used to measure OP activity in 1,878 serum samples from individuals ranging from 0 to 99 years of age againstN. meningitidisstrain NZ98/254 (B:4:P1.7-2,4). The levels of OP activity in individual serum samples varied greatly. OP activity showed an initial peak in the 6- to 12-month age group corresponding to a peak in disease incidence. The OP activity dropped in childhood until the late teenage years, although there was still a higher percentage of individuals with OP activity than with protective bactericidal antibody titers. OP activity reached a peak in the 30- to 39-year age group and then declined. This later peak in OP activity did not coincide with the young adults in whom peak serum bactericidal activity and disease incidence occurred. The demonstration of OP activity when disease incidence is low and when protective bactericidal antibody titers are not detected may indicate a role for OP in protection from meningococcal disease in these age groups.

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An Inducible and Secreted Eukaryote-Like Serine/Threonine Kinase of Salmonella enterica Serovar Typhi Promotes Intracellular Survival and Pathogenesis

Infection and Immunity ◽

10.1128/iai.02521-14 ◽

2014 ◽

Vol 83 (2) ◽

pp. 522-533 ◽

Cited By ~ 4

Author(s):

Nagaraja Theeya ◽

Atri Ta ◽

Sayan Das ◽

Rahul S. Mandal ◽

Oishee Chakrabarti ◽

...

Keyword(s):

Salmonella Enterica ◽

Functional Characterization ◽

Systemic Infection ◽

Serine Threonine Kinase ◽

Content Type ◽

Threonine Kinase ◽

Universal Substrate

Eukaryote-like serine/threonine kinases (eSTKs) constitute an important family of bacterial virulence factors. Genome analysis had predicted putative eSTKs inSalmonella entericaserovar Typhi, although their functional characterization and the elucidation of their role in pathogenesis are still awaited. We show here that the primary sequence and secondary structure of thet4519locus ofSalmonellaTyphi Ty2 have all the signatures of eukaryotic superfamily kinases.t4519encodes a ∼39-kDa protein (T4519), which shows serine/threonine kinase activitiesin vitro. Recombinant T4519 (rT4519) is autophosphorylated and phosphorylates the universal substrate myelin basic protein. Infection of macrophages results in decreased viability of the mutant (Ty2Δt4519) strain, which is reversed by gene complementation. Moreover, reactive oxygen species produced by the macrophages signal to the bacteria to induce T4519, which is translocated to the host cell cytoplasm. That T4519 may target a host substrate(s) is further supported by the activation of host cellular signaling pathways and the induction of cytokines/chemokines. Finally, the role of T4519 in the pathogenesis ofSalmonellaTyphi is underscored by the significantly decreased mortality of mice infected with the Ty2Δt4519strain and the fact that the competitive index of this strain for causing systemic infection is 0.25% that of the wild-type strain. This study characterizes the first eSTK ofSalmonellaTyphi and demonstrates its role in promoting phagosomal survival of the bacteria within macrophages, which is a key determinant of pathogenesis. This, to the best of our knowledge, is the first study to describe the essential role of eSTKs in thein vivopathogenesis ofSalmonellaspp.

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Cloning and functional characterization of the smooth muscle ether-a-go-go-related gene K+ channel. Potential role of a conserved amino acid substitution in the S4 region. Vol. 278 (2003) 2503-2514

Journal of Biological Chemistry ◽

10.1016/s0021-9258(20)67993-5 ◽

2004 ◽

Vol 279 (46) ◽

pp. 48486

Author(s):

Fouzia Shoeb ◽

Anna P. Malykhina ◽

Hamid I. Akbarali

Keyword(s):

Smooth Muscle ◽

Amino Acid ◽

Amino Acid Substitution ◽

Potential Role ◽

Functional Characterization ◽

K Channel ◽

Related Gene ◽

Channel Potential

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Functional characterization of soybean (Glycine max) DIRIGENT genes reveals an important role of GmDIR27 in the regulation of pod dehiscence

Genomics ◽

10.1016/j.ygeno.2020.10.033 ◽

2020 ◽

Author(s):

Xiaofei Ma ◽

Wenying Xu ◽

Tong Liu ◽

Ruying Chen ◽

Hong Zhu ◽

...

Keyword(s):

Glycine Max ◽

Functional Characterization ◽

Pod Dehiscence

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Molecular and Functional Characterization of a ToxR-Regulated Lipoprotein from a Clinical Isolate of Aeromonas hydrophila

Infection and Immunity ◽

10.1128/iai.00402-06 ◽

2006 ◽

Vol 74 (7) ◽

pp. 3742-3755 ◽

Cited By ~ 22

Author(s):

Lakshmi Pillai ◽

Jian Sha ◽

Tatiana E. Erova ◽

Amin A. Fadl ◽

Bijay K. Khajanchi ◽

...

Keyword(s):

Virulence Factor ◽

Aeromonas Hydrophila ◽

Functional Characterization ◽

Affinity Column ◽

Serum Resistance ◽

Classical Pathway ◽

Dependent Manner ◽

T7 Promoter

ABSTRACT Human diseases caused by species of Aeromonas have been classified into two major groups: septicemia and gastroenteritis. In this study, we reported the molecular and functional characterization of a new virulence factor, ToxR-regulated lipoprotein, or TagA, from a diarrheal isolate, SSU, of Aeromonas hydrophila. The tagA gene of A. hydrophila exhibited 60% identity with that of a recently identified stcE gene from Escherichia coli O157:H7, which encoded a protein (StcE) that provided serum resistance to the bacterium and prevented erythrocyte lysis by controlling classical pathway of complement activation by cleaving the complement C1-esterase inhibitor (C1-INH). We purified A. hydrophila TagA as a histidine-tagged fusion protein (rTagA) from E. coli DE3 strain using a T7 promoter-based pET30 expression vector and nickel affinity column chromatography. rTagA cleaved C1-INH in a time-dependent manner. The tagA isogenic mutant of A. hydrophila, unlike its corresponding wild-type (WT) or the complemented strain, was unable to cleave C1-INH, which is required to potentiate the C1-INH-mediated lysis of host and bacterial cells. We indeed demonstrated colocalization of C1-INH and TagA on the bacterial surface by confocal fluorescence microscopy, which ultimately resulted in increased serum resistance of the WT bacterium. Likewise, we delineated the role of TagA in contributing to the enhanced ability of C1-INH to inhibit the classical complement-mediated lysis of erythrocytes. Importantly, we provided evidence that the tagA mutant was significantly less virulent in a mouse model of infection (60%) than the WT bacterium at two 50% lethal doses, which resulted in 100% mortality within 48 h. Taken together, our data provided new information on the role of TagA as a virulence factor in bacterial pathogenesis. This is the first report of TagA characterization from any species of Aeromonas.

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Functional Characterization of Spliceosomal Introns and Identification of U2, U4, and U5 snRNAs in the Deep-Branching Eukaryote Entamoeba histolytica

Eukaryotic Cell ◽

10.1128/ec.00059-07 ◽

2007 ◽

Vol 6 (6) ◽

pp. 940-948 ◽

Cited By ~ 16

Author(s):

Carrie A. Davis ◽

Michael P. S. Brown ◽

Upinder Singh

Keyword(s):

Entamoeba Histolytica ◽

Splice Site ◽

Expressed Sequence Tag ◽

Functional Characterization ◽

Molecular Evidence ◽

Spliceosomal Introns ◽

Accurate Expression ◽

Eukaryotic Organisms

ABSTRACT Pre-mRNA splicing is essential to ensure accurate expression of many genes in eukaryotic organisms. In Entamoeba histolytica, a deep-branching eukaryote, approximately 30% of the annotated genes are predicted to contain introns; however, the accuracy of these predictions has not been tested. In this study, we mined an expressed sequence tag (EST) library representing 7% of amoebic genes and found evidence supporting splicing of 60% of the testable intron predictions, the majority of which contain a GUUUGU 5′ splice site and a UAG 3′ splice site. Additionally, we identified several splice site misannotations, evidence for the existence of 30 novel introns in previously annotated genes, and identified novel genes through uncovering their spliced ESTs. Finally, we provided molecular evidence for the E. histolytica U2, U4, and U5 snRNAs. These data lay the foundation for further dissection of the role of RNA processing in E. histolytica gene expression.

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