scholarly journals Multiple Ehrlichia chaffeensis Genes Critical for Its Persistent Infection in a Vertebrate Host Are Identified by Random Mutagenesis Coupled with In Vivo Infection Assessment

2020 ◽  
Vol 88 (10) ◽  
Author(s):  
Ying Wang ◽  
Arathy D. S. Nair ◽  
Andy Alhassan ◽  
Deborah C. Jaworski ◽  
Huitao Liu ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Fatty Acid Biosynthesis ◽  
Vaccine Development ◽  
Efflux Pump ◽  
Outer Membrane Proteins ◽  
Random Mutagenesis ◽  
Ehrlichia Chaffeensis ◽  
Content Type ◽  
Obligate Intracellular

ABSTRACT Ehrlichia chaffeensis, a tick-transmitted obligate intracellular rickettsial agent, causes human monocytic ehrlichiosis. In recent reports, we described substantial advances in developing random and targeted gene disruption methods to investigate the functions of E. chaffeensis genes. We reported earlier that the Himar1 transposon-based random mutagenesis is a valuable tool in defining E. chaffeensis genes critical for its persistent growth in vivo in reservoir and incidental hosts. The method also aided in extending studies focused on vaccine development and immunity. Here, we describe the generation and mapping of 55 new mutations. To define the critical nature of the bacterial genes, infection experiments were carried out in the canine host with pools of mutant organisms. Infection evaluation in the physiologically relevant host by molecular assays and by xenodiagnoses allowed the identification of many proteins critical for the pathogen’s persistent in vivo growth. Genes encoding proteins involved in biotin biosynthesis, protein synthesis and fatty acid biosynthesis, DNA repair, electron transfer, and a component of a multidrug resistance (MDR) efflux pump were concluded to be essential for the pathogen’s in vivo growth. Three known immunodominant membrane proteins, i.e., two 28-kDa outer membrane proteins (P28/OMP) and a 120-kDa surface protein, were also recognized as necessary for the pathogen’s obligate intracellular life cycle. The discovery of many E. chaffeensis proteins crucial for its continuous in vivo growth will serve as a major resource for investigations aimed at defining pathogenesis and developing novel therapeutics for this and related pathogens of the rickettsial family Anaplasmataceae.

scholarly journals Genetic Similarity of Gonococcal Homologs to Meningococcal Outer Membrane Proteins of Serogroup B Vaccine

mBio ◽  
2019 ◽  
Vol 10 (5) ◽  
Author(s):  
Henju Marjuki ◽  
Nadav Topaz ◽  
Sandeep J. Joseph ◽  
Kim M. Gernert ◽  
Ellen N. Kersh ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Vaccine Development ◽  
Membrane Vesicle ◽  
Outer Membrane Proteins ◽  
The United States ◽  
Cross Protection ◽  
Outer Membrane Vesicle ◽  
Content Type ◽  
Sequence Identity

ABSTRACT The human pathogens Neisseria gonorrhoeae and Neisseria meningitidis share high genome identity. Retrospective analysis of surveillance data from New Zealand indicates the potential cross-protective effect of outer membrane vesicle (OMV) meningococcal serogroup B vaccine (MeNZB) against N. gonorrhoeae. A licensed OMV-based MenB vaccine, MenB-4C, consists of a recombinant FHbp, NhbA, NadA, and the MeNZB OMV. Previous work has identified several abundantly expressed outer membrane proteins (OMPs) as major components of the MenB-4C OMV with high sequence similarity between N. gonorrhoeae and N. meningitidis, suggesting a mechanism for cross-protection. To build off these findings, we performed comparative genomic analysis on 970 recent N. gonorrhoeae isolates collected through a U.S surveillance system against N. meningitidis serogroup B (NmB) reference sequences. We identified 1,525 proteins that were common to both Neisseria species, of which 57 proteins were predicted to be OMPs using in silico methods. Among the MenB-4C antigens, NhbA showed moderate sequence identity (73%) to the respective gonococcal homolog, was highly conserved within N. gonorrhoeae, and was predicted to be surface expressed. In contrast, the gonococcal FHbp was predicted not to be surface expressed, while NadA was absent in all N. gonorrhoeae isolates. Our work confirmed recent observations (E. A. Semchenko, A. Tan, R. Borrow, and K. L. Seib, Clin Infect Dis, 2018, https://doi.org/10.1093/cid/ciy1061) and describes homologous OMPs from a large panel of epidemiologically relevant N. gonorrhoeae strains in the United States against NmB reference strains. Based on our results, we report a set of OMPs that may contribute to the previously observed cross-protection and provide potential antigen targets to guide the next steps in gonorrhea vaccine development. IMPORTANCE Gonorrhea, a sexually transmitted disease, causes substantial global morbidity and economic burden. New prevention and control measures for this disease are urgently needed, as strains resistant to almost all classes of antibiotics available for treatment have emerged. Previous reports demonstrate that cross-protection from gonococcal infections may be conferred by meningococcal serogroup B (MenB) outer membrane vesicle (OMV)-based vaccines. Among 1,525 common proteins shared across the genomes of both N. gonorrhoeae and N. meningitidis, 57 proteins were predicted to be surface expressed (outer membrane proteins [OMPs]) and thus preferred targets for vaccine development. The majority of these OMPs showed high sequence identity between the 2 bacterial species. Our results provide valuable insight into the meningococcal antigens present in the current OMV-containing MenB-4C vaccine that may contribute to cross-protection against gonorrhea and may inform next steps in gonorrhea vaccine development.

scholarly journals Linkage between Anaplasma marginale Outer Membrane Proteins Enhances Immunogenicity but Is Not Required for Protection from Challenge

2013 ◽  
Vol 20 (5) ◽  
pp. 651-656 ◽  
Author(s):  
Susan M. Noh ◽  
Joshua E. Turse ◽  
Wendy C. Brown ◽  
Junzo Norimine ◽  
Guy H. Palmer
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Protective Immunity ◽  
Bacterial Infections ◽  
Vaccine Development ◽  
Outer Membrane Proteins ◽  
Anaplasma Marginale ◽  
Anamnestic Response ◽  
Covalent Bonds ◽  
Content Type

ABSTRACTThe prevention of bacterial infections via immunization presents particular challenges. While outer membrane extracts are often protective, they are difficult and expensive to isolate and standardize and thus are often impractical for development and implementation in vaccination programs. In contrast, individual proteins, which are easily adapted for use in subunit vaccines, tend to be poorly protective. Consequently, identification of the specific characteristics of outer membrane-based immunogens, in terms of the antigen contents and contexts that are required for protective immunity, represents a major gap in the knowledge needed for bacterial vaccine development. Using as a modelAnaplasma marginale, a persistent tick-borne bacterial pathogen of cattle, we tested two sets of immunogens to determine whether membrane context affected immunogenicity and the capacity to induce protection. The first immunogen was composed of a complex of outer membrane proteins linked by covalent bonds and known to be protective. The second immunogen was derived directly from the first one, but the proteins were individualized rather than linked. The antibody response induced by the linked immunogen was much greater than that induced by the unlinked immunogen. However, both immunogens induced protective immunity and an anamnestic response. These findings suggest that individual proteins or combinations of proteins can be successfully tested for the ability to induce protective immunity with less regard for overall membrane context. Once protective antigens are identified, immunogenicity could be enhanced by cross-linking to allow a reduced immunogen dose or fewer booster vaccinations.

scholarly journals Immunoproteomic Analysis To Identify Shiga Toxin-Producing Escherichia coli Outer Membrane Proteins Expressed during Human Infection

2014 ◽  
Vol 82 (11) ◽  
pp. 4767-4777 ◽  
Author(s):  
David Montero ◽  
Paz Orellana ◽  
Daniela Gutiérrez ◽  
Daniela Araya ◽  
Juan Carlos Salazar ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Membrane Proteins ◽  
Outer Membrane ◽  
Shiga Toxin ◽  
Vaccine Development ◽  
Outer Membrane Proteins ◽  
Antibiotic Use ◽  
Etiologic Agent ◽  
Human Infection ◽  
Content Type

ABSTRACTShiga-toxin producingEscherichia coli(STEC) is the etiologic agent of acute diarrhea, dysentery, and hemolytic-uremic syndrome (HUS). There is no approved vaccine for STEC infection in humans, and antibiotic use is contraindicated, as it promotes Shiga toxin production. In order to identify STEC-associated antigens and immunogenic proteins, outer membrane proteins (OMPs) were extracted from STEC O26:H11, O103, O113:H21, and O157:H7 strains, and commensalE. colistrain HS was used as a control. SDS-PAGE, two-dimensional-PAGE analysis, Western blot assays using sera from pediatric HUS patients and controls, and matrix-assisted laser desorption ionization–tandem time of flight analyses were used to identify 12 immunogenic OMPs, some of which were not reactive with control sera. Importantly, seven of these proteins have not been previously reported to be immunogenic in STEC strains. Among these seven proteins, OmpT and Cah displayed IgG and IgA reactivity with sera from HUS patients. Genes encoding these two proteins were present in a majority of STEC strains. Knowledge of the antigens produced during infection of the host and the immune response to those antigens will be important for future vaccine development.

scholarly journals Structural Analysis of the Interaction between the Bacterial Cell Division Proteins FtsQ and FtsB

mBio ◽  
2018 ◽  
Vol 9 (5) ◽  
Author(s):  
Danguole Kureisaite-Ciziene ◽  
Aravindan Varadajan ◽  
Stephen H. McLaughlin ◽  
Marjolein Glas ◽  
Alejandro Montón Silva ◽  
...  
Keyword(s):  
Cell Division ◽  
Membrane Proteins ◽  
Bacterial Cell ◽  
Outer Membrane Proteins ◽  
Ring Structure ◽  
Globular Domain ◽  
Bacterial Cell Division ◽  
Content Type ◽  
Division Site

ABSTRACT Most bacteria and archaea use the tubulin homologue FtsZ as its central organizer of cell division. In Gram-negative Escherichia coli bacteria, FtsZ recruits cytosolic, transmembrane, periplasmic, and outer membrane proteins, assembling the divisome that facilitates bacterial cell division. One such divisome component, FtsQ, a bitopic membrane protein with a globular domain in the periplasm, has been shown to interact with many other divisome proteins. Despite its otherwise unknown function, it has been shown to be a major divisome interaction hub. Here, we investigated the interactions of FtsQ with FtsB and FtsL, two small bitopic membrane proteins that act immediately downstream of FtsQ. We show in biochemical assays that the periplasmic domains of E. coli FtsB and FtsL interact with FtsQ, but not with each other. Our crystal structure of FtsB bound to the β domain of FtsQ shows that only residues 64 to 87 of FtsB interact with FtsQ. A synthetic peptide comprising those 24 FtsB residues recapitulates the FtsQ-FtsB interactions. Protein deletions and structure-guided mutant analyses validate the structure. Furthermore, the same structure-guided mutants show cell division defects in vivo that are consistent with our structure of the FtsQ-FtsB complex that shows their interactions as they occur during cell division. Our work provides intricate details of the interactions within the divisome and also provides a tantalizing view of a highly conserved protein interaction in the periplasm of bacteria that is an excellent target for cell division inhibitor searches. IMPORTANCE In most bacteria and archaea, filaments of FtsZ protein organize cell division. FtsZ forms a ring structure at the division site and starts the recruitment of 10 to 20 downstream proteins that together form a multiprotein complex termed the divisome. The divisome is thought to facilitate many of the steps required to make two cells out of one. FtsQ and FtsB are part of the divisome, with FtsQ being a central hub, interacting with most of the other divisome components. Here we show for the first time in detail how FtsQ interacts with its downstream partner FtsB and show that mutations that disturb the interface between the two proteins effectively inhibit cell division.

scholarly journals Novel Two-Component System MacRS Is a Pleiotropic Regulator That Controls Multiple Morphogenic Membrane Protein Genes inStreptomyces coelicolor

2018 ◽  
Vol 85 (4) ◽  
Author(s):  
Meng Liu ◽  
Peipei Zhang ◽  
Yanping Zhu ◽  
Ting Lu ◽  
Yemin Wang ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Consensus Sequence ◽  
Aerial Mycelium ◽  
Dnase I ◽  
Inverted Repeats ◽  
Content Type ◽  
Dnase I Footprinting ◽  
Two Component

ABSTRACTAs with most annotated two-component systems (TCSs) ofStreptomyces coelicolor, the function of TCS SCO2120/2121 was unknown. Based on our findings, we have designated this TCS MacRS, formorphogenesis andactinorhodin regulator/sensor. Our study indicated that either single or double mutation of MacRS largely blocked production of actinorhodin but enhanced formation of aerial mycelium. Chromatin immunoprecipitation (ChIP) sequencing, using anS. coelicolorstrain expressing MacR-Flag fusion protein, identifiedin vivotargets of MacR, and DNase I footprinting of these targets revealed a consensus sequence for MacR binding, TGAGTACnnGTACTCA, containing two 7-bp inverted repeats. A genome-wide search revealed sites identical or highly similar to this consensus sequence upstream of six genes encoding putative membrane proteins or lipoproteins. These predicted sites were confirmed as MacR binding sites by DNase I footprinting and electrophoretic mobility shift assaysin vitroand by ChIP-quantitative PCRin vivo, and transcriptional analyses demonstrated that MacR significantly impacts expression of these target genes. Disruption of three of these genes,sco6728,sco4924, andsco4011, markedly accelerated aerial mycelium formation, indicating that their gene products are novel morphogenic factors. Two-hybrid assays indicated that these three proteins, which we have named morphogenic membrane protein A (MmpA; SCO6728), MmpB (SCO4924), and MmpC (SCO4011), interact with one another and with the putative membrane protein and MacR target SCO4225. Notably, SAV6081/82 and SVEN1780/81, homologs of MacRS TCS fromS. avermitilisandS. venezuelae, respectively, can substitute for MacRS, indicating functional conservation. Our findings reveal a role for MacRS in cellular morphogenesis and secondary metabolism inStreptomyces.IMPORTANCETCSs help bacteria adapt to environmental stresses by altering gene expression. However, the roles and corresponding regulatory mechanisms of most TCSs in theStreptomycesmodel strainS. coelicolorare unknown. We investigated the previously uncharacterized MacRS TCS and identified the core DNA recognition sequence, two seven-nucleotide inverted repeats, for the DNA-binding protein MacR. We further found that MacR directly controls a group of membrane proteins, including MmpA-C, which are novel morphogenic factors that delay formation of aerial mycelium. We also discovered that these membrane proteins interact with one another and that otherStreptomycesspecies have conserved MacRS homologs. Our findings suggest a conserved role for MacRS in morphogenesis and/or other membrane-associated activities. Additionally, our study showed that MacRS impacts, albeit indirectly, the production of the signature metabolite actinorhodin, further suggesting that MacRS and its homologs function as novel pleiotropic regulatory systems inStreptomyces.

scholarly journals Biofilm Formation Caused by Clinical Acinetobacter baumannii Isolates Is Associated with Overexpression of the AdeFGH Efflux Pump

2015 ◽  
Vol 59 (8) ◽  
pp. 4817-4825 ◽  
Author(s):  
Xinlong He ◽  
Feng Lu ◽  
Fenglai Yuan ◽  
Donglin Jiang ◽  
Peng Zhao ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Gene Transcription ◽  
Biofilm Formation ◽  
Efflux Pump ◽  
Outer Membrane Proteins ◽  
Content Type ◽  
Potential Association ◽  
Genes Encoding ◽  
Clinical Environments

ABSTRACTChronic wound infections are associated with biofilm formation, which in turn has been correlated with drug resistance. However, the mechanism by which bacteria form biofilms in clinical environments is not clearly understood. This study was designed to investigate the biofilm formation potency ofAcinetobacter baumanniiand the potential association of biofilm formation with genes encoding efflux pumps, quorum-sensing regulators, and outer membrane proteins. A total of 48 clinically isolatedA. baumanniistrains, identified by enterobacterial repetitive intergenic consensus (ERIC)-PCR as types A-II, A-III, and A-IV, were analyzed. Three representative strains, which were designatedA. baumanniiABR2, ABR11, and ABS17, were used to evaluate antimicrobial susceptibility, biofilm inducibility, and gene transcription (abaI,adeB,adeG,adeJ,carO, andompA). A significant increase in the MICs of different classes of antibiotics was observed in the biofilm cells. The formation of a biofilm was significantly induced in all the representative strains exposed to levofloxacin. The levels of gene transcription varied between bacterial genotypes, antibiotics, and antibiotic concentrations. The upregulation ofadeGcorrelated with biofilm induction. The consistent upregulation ofadeGandabaIwas detected in A-III-typeA. baumanniiin response to levofloxacin and meropenem (1/8 to 1/2× the MIC), conditions which resulted in the greatest extent of biofilm induction. This study demonstrates a potential role of the AdeFGH efflux pump in the synthesis and transport of autoinducer molecules during biofilm formation, suggesting a link between low-dose antimicrobial therapy and a high risk of biofilm infections caused byA. baumannii. This study provides useful information for the development of antibiofilm strategies.

scholarly journals Targeted and Random Mutagenesis of Ehrlichia chaffeensis for the Identification of Genes Required for In vivo Infection

2013 ◽  
Vol 9 (2) ◽  
pp. e1003171 ◽  
Author(s):  
Chuanmin Cheng ◽  
Arathy D. S. Nair ◽  
Vijaya V. Indukuri ◽  
Shanzhong Gong ◽  
Roderick F. Felsheim ◽  
...  
Keyword(s):  
Random Mutagenesis ◽  
Ehrlichia Chaffeensis

scholarly journals Assessment of Minocycline and Polymyxin B Combination against Acinetobacter baumannii

2015 ◽  
Vol 59 (5) ◽  
pp. 2720-2725 ◽  
Author(s):  
Dana R. Bowers ◽  
Henry Cao ◽  
Jian Zhou ◽  
Kimberly R. Ledesma ◽  
Dongxu Sun ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Clinical Utility ◽  
Efflux Pump ◽  
Polymyxin B ◽  
Intracellular Concentration ◽  
Bacterial Cells ◽  
Efflux Pump Inhibitor ◽  
Content Type

ABSTRACTAntimicrobial resistance amongAcinetobacter baumanniiis increasing worldwide, often necessitating combination therapy. The clinical utility of using minocycline with polymyxin B is not well established. In this study, we investigated the activity of minocycline and polymyxin B against 1 laboratory isolate and 3 clinical isolates ofA. baumannii. Minocycline susceptibility testing was performed with and without an efflux pump inhibitor, phenylalanine-arginine β-naphthylamide (PAβN). The intracellular minocycline concentration was determined with and without polymyxin B (0.5 μg/ml). Time-kill studies were performed over 24 h using approximately 106CFU/ml of each strain with clinically relevant minocycline concentrations (2 μg/ml and 8 μg/ml), with and without polymyxin B (0.5 μg/ml). Thein vivoefficacy of the combination was assessed in a neutropenic murine pneumonia model. Infected animals were administered minocycline (50 mg/kg), polymyxin B (10 mg/kg), or both to achieve clinically equivalent exposures in humans. A reduction in the minocycline MIC (≥4×) was observed in the presence of PAβN. The intracellular concentration andin vitrobactericidal effect of minocycline were both enhanced by polymyxin B. With 2 minocycline-susceptible strains, the bacterial burden in lung tissue at 24 h was considerably reduced by the combination compared to monotherapy with minocycline or polymyxin B. In addition, the combination prolonged survival of animals infected with a minocycline-susceptible strain. Polymyxin B increased the intracellular concentration of minocycline in bacterial cells and enhanced the bactericidal activity of minocycline, presumably due to efflux pump disruption. The clinical utility of this combination should be further investigated.

BaeR participates in cephalosporins susceptibility by regulating the expression level of outer membrane proteins in Escherichia coli

2020 ◽  
Author(s):  
Shuaiyang Wang ◽  
Chunbo You ◽  
Fareed Qumar Memon ◽  
Geyin Zhang ◽  
Yawei Sun ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Membrane Proteins ◽  
Outer Membrane ◽  
Efflux Pump ◽  
Outer Membrane Proteins ◽  
Expression Level ◽  
Antibiotics Resistance ◽  
Dilution Method ◽  
Expression Levels ◽  
E Coli

Abstract The two-component system BaeSR participates in antibiotics resistance of Escherichia coli. To know whether the outer membrane proteins involve in the antibiotics resistance mediated by BaeSR, deletion of acrB was constructed and the recombined plasmid p-baeR was introduced into E. coli K12 and K12△acrB. Minimum inhibitory concentrations (MICs) of antibacterial agents were determined by 2-fold broth micro-dilution method. Gene expressions related with major outer membrane proteins and multidrug efflux pump-related genes were determined by real-time quantitative reverse transcription polymerase chain reaction. The results revealed that the MICs of K12ΔacrB to the tested drugs except for gentamycin and amikacin decreased 2- to 16.75-folds compared with those of K12. When BaeR was overexpressed, the MICs of K12ΔacrB/p-baeR to ceftiofur and cefotaxime increased 2.5- and 2-fold, respectively, compared with their corresponding that of K12△acrB. At the same time, the expression levels of ompC, ompF, ompW, ompA and ompX showed significant reduction in K12ΔacrB/p-baeR as compared with K12△acrB. Moreover, the expression levels of ompR, marA, rob and tolC also significantly ‘decreased’ in K12ΔacrB/p-baeR. These findings indicated that BaeR overproduction can decrease cephalosporins susceptibility in acrB-free E. coli by decreasing the expression level of outer membrane proteins.

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