Pathogens

861 KiB

Open AccessReview

Parasitic Nematode Immunomodulatory Strategies: Recent Advances and Perspectives

by Dustin Cooper and Ioannis Eleftherianos

Pathogens 2016, 5(3), 58; https://doi.org/10.3390/pathogens5030058 - 14 Sep 2016

Cited by 68 | Viewed by 11754

More than half of the described species of the phylum Nematoda are considered parasitic, making them one of the most successful groups of parasites. Nematodes are capable of inhabiting a wide variety of niches. A vast array of vertebrate animals, insects, and plants [...] Read more.

More than half of the described species of the phylum Nematoda are considered parasitic, making them one of the most successful groups of parasites. Nematodes are capable of inhabiting a wide variety of niches. A vast array of vertebrate animals, insects, and plants are all identified as potential hosts for nematode parasitization. To invade these hosts successfully, parasitic nematodes must be able to protect themselves from the efficiency and potency of the host immune system. Innate immunity comprises the first wave of the host immune response, and in vertebrate animals it leads to the induction of the adaptive immune response. Nematodes have evolved elegant strategies that allow them to evade, suppress, or modulate host immune responses in order to persist and spread in the host. Nematode immunomodulation involves the secretion of molecules that are capable of suppressing various aspects of the host immune response in order to promote nematode invasion. Immunomodulatory mechanisms can be identified in parasitic nematodes infecting insects, plants, and mammals and vary greatly in the specific tactics by which the parasites modify the host immune response. Nematode-derived immunomodulatory effects have also been shown to affect, negatively or positively, the outcome of some concurrent diseases suffered by the host. Understanding nematode immunomodulatory actions will potentially reveal novel targets that will in turn lead to the development of effective means for the control of destructive nematode parasites. Full article

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Figure 1
Insect Pathogenic nematodes modulate the phenoloxidase response via the secretion of a trypsin-like serine protease, Sc-CHYM, and Sc-SRP-6. The identified Trypsin-like serine protease has also been shown to limit spreading of insect hemocytes. Sc-SRP-6 modulates the host immune response by altering the activity of digestive enzymes. Sc-KU-4 acts by modulating the ability of hemocytes to aggregate or encapsulate foreign microbes. Full article ">Figure 2
Plant Pathogenic nematodes use a multitude of strategies for host immunomodulation. The venom allergen-like proteins Gr-VAP1 can disrupt the host’s initial recognition of nematode invasion. Other proteins such as glutathione S-transferase or 10A06 interfere with reactive oxygen species produced in response to recognition of an invading nematode. 10A06 also interrupts salicylic acid signaling. SPRYSEC proteins interact resistance proteins to alter pathogen recognition. Some molecules, such as MiCRT, or groups of molecules, such as the annexins, are implicated in immunomodulation; however, their mechanism of action remains unknown. Full article ">Figure 3
Vertebrate Pathogenic nematodes rely heavily on secreted cystatins for host immunomodulation. Cystatins influence the regulation of host cytokine production, as well as the development of adaptive immune responses by altering antigen and polypeptide processing. SCP/TAPS are believed to assist in inhibiting platelet and neutrophil activity. ES-62 alters a number of immune functions including B and T-Cell activation/proliferation via receptor signaling inhibition and the inhibition of mast cell degranulation through the formation of a complex with TLR-4. Recently, miRNA-containing exosomes secreted by some nematode species have been linked to altered host immune responses. Full article ">

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Open AccessReview

The Influenza NS1 Protein: What Do We Know in Equine Influenza Virus Pathogenesis?

by Marta Barba and Janet M. Daly

Pathogens 2016, 5(3), 57; https://doi.org/10.3390/pathogens5030057 - 31 Aug 2016

Cited by 7 | Viewed by 6386

Abstract

Equine influenza virus remains a serious health and potential economic problem throughout most parts of the world, despite intensive vaccination programs in some horse populations. The influenza non-structural protein 1 (NS1) has multiple functions involved in the regulation of several cellular and viral [...] Read more.

Equine influenza virus remains a serious health and potential economic problem throughout most parts of the world, despite intensive vaccination programs in some horse populations. The influenza non-structural protein 1 (NS1) has multiple functions involved in the regulation of several cellular and viral processes during influenza infection. We review the strategies that NS1 uses to facilitate virus replication and inhibit antiviral responses in the host, including sequestering of double-stranded RNA, direct modulation of protein kinase R activity and inhibition of transcription and translation of host antiviral response genes such as type I interferon. Details are provided regarding what it is known about NS1 in equine influenza, especially concerning C-terminal truncation. Further research is needed to determine the role of NS1 in equine influenza infection, which will help to understand the pathophysiology of complicated cases related to cytokine imbalance and secondary bacterial infection, and to investigate new therapeutic and vaccination strategies. Full article

(This article belongs to the Special Issue Equine Influenza)

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724 KiB

Open AccessArticle

Retrospective Analysis of the Equine Influenza Virus A/Equine/Kirgizia/26/1974 (H7N7) Isolated in Central Asia

by Kobey Karamendin, Aidyn Kydyrmanov, Marat Sayatov, Vitaliy Strochkov, Nurlan Sandybayev and Kulaysan Sultankulova

Pathogens 2016, 5(3), 55; https://doi.org/10.3390/pathogens5030055 - 10 Aug 2016

Cited by 4 | Viewed by 4316

Abstract

A retrospective phylogenetic characterization of the hemagglutinin, neuraminidase and nucleoprotein genes of equine influenza virus A/equine/Kirgizia/26/1974 (H7N7) which caused an outbreak in Kirgizia (a former Soviet Union republic, now Kyrgyzstan) in 1977 was conducted. It was defined that it was closely related to [...] Read more.

A retrospective phylogenetic characterization of the hemagglutinin, neuraminidase and nucleoprotein genes of equine influenza virus A/equine/Kirgizia/26/1974 (H7N7) which caused an outbreak in Kirgizia (a former Soviet Union republic, now Kyrgyzstan) in 1977 was conducted. It was defined that it was closely related to the strain London/1973 isolated in Europe and it shared a maximum nucleotide sequence identity at 99% with it. This Central Asian equine influenza virus isolate did not have any specific genetic signatures and can be considered as an epizootic strain of 1974 that spread in Europe. The absence of antibodies to this subtype EI virus (EIV) in recent research confirms its disappearance as of the 1990s when the antibodies were last found in unvaccinated horses. Full article

(This article belongs to the Special Issue Equine Influenza)

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658 KiB

Open AccessArticle

Clearance of Streptococcus suis in Stomach Contents of Differently Fed Growing Pigs

by Franziska Warneboldt, Saara J. Sander, Andreas Beineke, Peter Valentin-Weigand, Josef Kamphues and Christoph Georg Baums

Pathogens 2016, 5(3), 56; https://doi.org/10.3390/pathogens5030056 - 6 Aug 2016

Cited by 10 | Viewed by 5154

Abstract

Streptococcus (S.) suis translocates across the intestinal barrier of piglets after intraintestinal application. Based on these findings, an oro-gastrointestinal infection route has been proposed. Thus, the objective of this study was to investigate the survival of S. suis in the porcine [...] Read more.

Streptococcus (S.) suis translocates across the intestinal barrier of piglets after intraintestinal application. Based on these findings, an oro-gastrointestinal infection route has been proposed. Thus, the objective of this study was to investigate the survival of S. suis in the porcine stomach. Whereas surviving bacteria of S. suis serotypes 2 and 9 were not detectable after 60 min of incubation in stomach contents with a comparatively high gastric pH of 5 due to feeding of fine pellets, the number of Salmonella Derby bacteria increased under these conditions. Further experiments confirmed the clearance of S. suis serotypes 2 and 9 within 30 min in stomach contents with a pH of 4.7 independently of the bacterial growth phase. Finally, an oral infection experiment was conducted, feeding each of 18 piglets a diet mixed with 10¹⁰ CFU of S. suis serotype 2 or 9. Thorough bacteriological screenings of various mesenteric-intestinal lymph nodes and internal organs after different times of exposure did not lead to any detection of the orally applied challenge strains. In conclusion, the porcine stomach constitutes a very efficient barrier against oro-gastrointenstinal S. suis infections. Conditions leading to the passage of S. suis through the stomach remain to be identified. Full article

(This article belongs to the Special Issue Streptococcus suis)

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Mean survival factors of S. suis ST 2 strain 10, ST 9 strain A3286/94 and Salmonella Derby A147/85 as well as pH values in stomach contents ex vivo of piglets fed either a finely ground and pelleted (A) (n = 5) or coarsely ground meal diet (B) (n = 5). Stomach contents were mixed with bacteria and incubated for the indicated time points in air-tight sealed bags at 37 °C in a water bath. Standard deviations (SDs) are not included for reasons of clarity. At t = 3 min SDs were 0.192, 0.267 and 0.049 for S. suis ST2, ST9 and Salmonella Derby in (A), respectively. All other SDs were below 0.02 except for the values in (A) for Salmonella Derby at 60, 120 and 240 min with SD = 0.135; 0.191 and 1.32, respectively. The survival factor of Salmonella Derby was significantly higher at 240 min in comparison to the values at 120, 60 and 3 min (p < 0.05). Differences between survival factors at 120 and 3 min were also significant. The survival factor was calculated by dividing the specific bacterial content at a specific time point (CFU/g) by the inoculation dose. Full article ">Figure 2
Mean specific bacterial loads of S. suis serotype 2 strain 10 and serotype 9 strain A3286/94 grown either to exponential (exp., OD600 = 0.6) or to stationary phase (stat., OD600 = 1.2) as well as pH values in stomach contents ex vivo of piglets (n = 6) fed a finely ground and pelleted diet. Stomach contents were mixed and incubated for the indicated time points in air-tight sealed bags at 37 °C in a water bath. At t = 3 min SDs were 1.3, 24.6, 4.8 and 19.0 for S. suis serotype (ST) 2 (exp. phase), ST2 (stat. phase), ST9 (exp. Phase) and ST9 (stat. phase), respectively. All other SDs were below 0.01. The differences of the specific bacterial loads at t = 3 min compared to the respective values of any other time point of analysis were significant (p < 0.05). Full article ">Figure 3
Survival factors of Salmonella Derby A147/85, S. suis serotype (ST) 2 strain 10 and S. suis ST 9 strain A3286/94 in compound feed (either in fine pellets without formic acid or as crumb feed including formic acid). Feeds were mixed either with 1.9 × 107 CFU Salmonella Derby A147/85, 7.5 × 108 CFU S. suis ST 2 strain 10 or 6.8 × 108 CFU S. suis ST 9 strain A3286/94 per g feed and incubated for the indicated time points at room temperature (20 to 24 °C). The survival factor was calculated by dividing the specific bacterial content at a specific time point (CFU/g) by the inoculation dose. Full article ">

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Open AccessArticle

Genomic Recombination Leading to Decreased Virulence of Group B Streptococcus in a Mouse Model of Adult Invasive Disease

by Sarah Teatero, Paul Lemire, Ken Dewar, Jessica Wasserscheid, Cynthia Calzas, Gustavo V. Mallo, Aimin Li, Taryn B.T. Athey, Mariela Segura and Nahuel Fittipaldi

Pathogens 2016, 5(3), 54; https://doi.org/10.3390/pathogens5030054 - 5 Aug 2016

Cited by 5 | Viewed by 4831

Abstract

Adult invasive disease caused by Group B Streptococcus (GBS) is increasing worldwide. Whole-genome sequencing (WGS) now permits rapid identification of recombination events, a phenomenon that occurs frequently in GBS. Using WGS, we described that strain NGBS375, a capsular serotype V GBS isolate of [...] Read more.

Adult invasive disease caused by Group B Streptococcus (GBS) is increasing worldwide. Whole-genome sequencing (WGS) now permits rapid identification of recombination events, a phenomenon that occurs frequently in GBS. Using WGS, we described that strain NGBS375, a capsular serotype V GBS isolate of sequence type (ST)297, has an ST1 genomic background but has acquired approximately 300 kbp of genetic material likely from an ST17 strain. Here, we examined the virulence of this strain in an in vivo model of GBS adult invasive infection. The mosaic ST297 strain showed intermediate virulence, causing significantly less systemic infection and reduced mortality than a more virulent, serotype V ST1 isolate. Bacteremia induced by the ST297 strain was similar to that induced by a serotype III ST17 strain, which was the least virulent under the conditions tested. Yet, under normalized bacteremia levels, the in vivo intrinsic capacity to induce the production of pro-inflammatory cytokines was similar between the ST297 strain and the virulent ST1 strain. Thus, the diminished virulence of the mosaic strain may be due to reduced capacity to disseminate or multiply in blood during a systemic infection which could be mediated by regulatory factors contained in the recombined region. Full article

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4168 KiB

Open AccessArticle

Modulation of Human Airway Barrier Functions during Burkholderia thailandensis and Francisella tularensis Infection

by Cornelia Blume, Jonathan David, Rachel E. Bell, Jay R. Laver, Robert C. Read, Graeme C. Clark, Donna E. Davies and Emily J. Swindle

Pathogens 2016, 5(3), 53; https://doi.org/10.3390/pathogens5030053 - 3 Aug 2016

Cited by 6 | Viewed by 5244

Abstract

The bronchial epithelium provides protection against pathogens from the inhaled environment through the formation of a highly-regulated barrier. In order to understand the pulmonary diseases melioidosis and tularemia caused by Burkholderia thailandensis and Fransicella tularensis, respectively, the barrier function of the human [...] Read more.

The bronchial epithelium provides protection against pathogens from the inhaled environment through the formation of a highly-regulated barrier. In order to understand the pulmonary diseases melioidosis and tularemia caused by Burkholderia thailandensis and Fransicella tularensis, respectively, the barrier function of the human bronchial epithelium were analysed. Polarised 16HBE14o- or differentiated primary human bronchial epithelial cells (BECs) were exposed to increasing multiplicities of infection (MOI) of B. thailandensis or F. tularensis Live Vaccine Strain and barrier responses monitored over 24–72 h. Challenge of polarized BECs with either bacterial species caused an MOI- and time-dependent increase in ionic permeability, disruption of tight junctions, and bacterial passage from the apical to the basolateral compartment. B. thailandensis was found to be more invasive than F. tularensis. Both bacterial species induced an MOI-dependent increase in TNF-α release. An increase in ionic permeability and TNF-α release was induced by B. thailandensis in differentiated BECs. Pretreatment of polarised BECs with the corticosteroid fluticasone propionate reduced bacterial-dependent increases in ionic permeability, bacterial passage, and TNF-α release. TNF blocking antibody Enbrel^® reduced bacterial passage only. BEC barrier properties are disrupted during respiratory bacterial infections and targeting with corticosteroids or anti-TNF compounds may represent a therapeutic option. Full article

(This article belongs to the Special Issue Host Defense Against Bacteria)

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Figure 1
B. thailandensis and F. tularensis LVS disrupt the physical barrier properties of bronchial epithelial cells. Polarised 16HBE cells were infected apically with a bacterial suspension of indicated multiplicity of infection (MOI) and the ionic permeability determined by measuring transepithelial resistance (TER). (A) Infection of polarised 16HBE cells with B. thailandensis (MOI from 10−3 to 102). TER was measured after 6 h and 24 h of infection; (B) Polarised 16HBEs were infected with F. tularensis (MOI from 1 to 100). TER was analysed 24 h, 48 h and 72 h after infection. Results are normalised to t = 0 h after subtraction of the background TER of an empty transwell. Means ± SEM, n = 3–14 (A) and n = 3 (B). * p ≤ 0.05 compared to control. Full article ">Figure 2
Disruption of tight junctions in bronchial epithelial cells after F. tularensis infection. Polarised 16HBE cells were apically infected with F. tularensis LVS (MOI of 1) for 72 h and distribution of the tight junction protein occludin (green) analysed by confocal fluorescence microscopy. Nuclei stained with DAPI were shown in pseudo-colouring (red) representative image of three independent experiments. Full article ">Figure 3
Passage of bacteria across the epithelial barrier. Polarised 16HBE cells were apically infected with bacteria and the number of bacteria crossing the epithelial barrier assessed by counting live bacteria in the basolateral compartment. (A) Passage of B. thailandensis across the epithelial barrier after 24 h of infection; and (B) epithelial passage of F. tularensis LVS after 72 h of infection. Results are means ± SEM, n = 3. Full article ">Figure 4
B. thailandensis and F. tularensis infection of airway epithelial cells activated the immunological barrier functions. Polarised 16HBE cells were apically infected with B. thailandensis or F. tularensis and the basolateral release of inflammatory mediators was analysed. Basolateral release of TNF-α after 24 h of infection with B. thailandensis by analysed by ELISA (A) and, after 72 h of F. tularensis LVS infection, by CBA assay (B) normalised to untreated control (A: untreated control: 85.3 ± 45.4 pg/mL; B: 2.4 ± 1.2 pg/mL). Results are means ± SEM, n = 3–6 (A) and n = 3 (B). * p ≤ 0.05 compared to control; and (C) correlation of TNF-α release with ionic permeability determined by measuring transepithelial resistance (TER). Full article ">Figure 5
Differentiated PBECs are more sensitive to infection with B. thailandensis. After apical infection of differentiated PBECs with B. thailandensis, physical and immunological barrier properties were monitored. (A) Ionic barrier permeability was measured by TER after 6 h and 24 h of infection and normalised to t = 0 h after subtraction of the background TER of an empty TW; (B) basolateral release of TNF-α after 24 h of infection was analysed by ELISA; and (C) correlation of TNF-α release with ionic permeability determined by measuring the transepithelial resistance (TER). Results are means ± SEM, n = 3–6 (A) and n = 4–6 (B). * p ≤ 0.05 compared to control. Full article ">Figure 6
Corticosteroids and anti-TNF-α treatment alter epithelial barrier functions during B. thailandensis infection. Polarised 16HBEs were pre-treated with 10 nM fluticasone propionate (FP) or 10 μg/mL anti-TNF-α (Enbrel®) for 1 h before apical infection with B. thailandensis for 24 h. (A) Physical barrier properties measured by TER are normalised to t = 0 h; (B) passage of bacteria across the epithelial barrier are determined by bacterial counts in the basolateral medium; and (C) basolateral release of TNF-α measured by ELISA. Results are means ± SEM, n = 5. * p ≤ 0.05. Full article ">

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Open AccessReview

Deliberate Establishment of Asymptomatic Bacteriuria—A Novel Strategy to Prevent Recurrent UTI

by Björn Wullt and Catharina Svanborg

Pathogens 2016, 5(3), 52; https://doi.org/10.3390/pathogens5030052 - 29 Jul 2016

Cited by 29 | Viewed by 7554

Abstract

We have established a novel strategy to reduce the risk for recurrent urinary tract infection (UTI), where rapidly increasing antibiotic resistance poses a major threat. Epidemiologic studies have demonstrated that asymptomatic bacteriuria (ABU) protects the host against symptomatic infections with more virulent strains. [...] Read more.

We have established a novel strategy to reduce the risk for recurrent urinary tract infection (UTI), where rapidly increasing antibiotic resistance poses a major threat. Epidemiologic studies have demonstrated that asymptomatic bacteriuria (ABU) protects the host against symptomatic infections with more virulent strains. To mimic this protective effect, we deliberately establish ABU in UTI-prone patients, who are refractory to conventional therapy. The patients are inoculated with Escherichia coli (E. coli) 83972, now widely used as a prototype ABU strain. Therapeutic efficacy has been demonstrated in a placebo-controlled trial, supporting the feasibility of using E. coli 83972 as a tool to prevent recurrent UTI and, potentially, to outcompete antibiotic-resistant strains from the human urinary tract. In addition, the human inoculation protocol offers unique opportunities to study host-parasite interaction in vivo in the human urinary tract. Here, we review the clinical evidence for protection using this approach as well as some molecular insights into the pathogenesis of UTI that have been gained during these studies. Full article

(This article belongs to the Special Issue Molecular Aspects of Urinary Tract Infection)

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2459 KiB

Open AccessArticle

FlpS, the FNR-Like Protein of Streptococcus suis Is an Essential, Oxygen-Sensing Activator of the Arginine Deiminase System

by Jörg Willenborg, Anna Koczula, Marcus Fulde, Astrid De Greeff, Andreas Beineke, Wolfgang Eisenreich, Claudia Huber, Maren Seitz, Peter Valentin-Weigand and Ralph Goethe

Pathogens 2016, 5(3), 51; https://doi.org/10.3390/pathogens5030051 - 21 Jul 2016

Cited by 12 | Viewed by 11548

Abstract

Streptococcus (S.) suis is a zoonotic pathogen causing septicemia and meningitis in pigs and humans. During infection S. suis must metabolically adapt to extremely diverse environments of the host. CcpA and the FNR family of bacterial transcriptional regulators are important for metabolic gene [...] Read more.

Streptococcus (S.) suis is a zoonotic pathogen causing septicemia and meningitis in pigs and humans. During infection S. suis must metabolically adapt to extremely diverse environments of the host. CcpA and the FNR family of bacterial transcriptional regulators are important for metabolic gene regulation in various bacteria. The role of CcpA in S. suis is well defined, but the function of the FNR-like protein of S. suis, FlpS, is yet unknown. Transcriptome analyses of wild-type S. suis and a flpS mutant strain suggested that FlpS is involved in the regulation of the central carbon, arginine degradation and nucleotide metabolism. However, isotopologue profiling revealed no substantial changes in the core carbon and amino acid de novo biosynthesis. FlpS was essential for the induction of the arcABC operon of the arginine degrading pathway under aerobic and anaerobic conditions. The arcABC-inducing activity of FlpS could be associated with the level of free oxygen in the culture medium. FlpS was necessary for arcABC-dependent intracellular bacterial survival but redundant in a mice infection model. Based on these results, we propose that the core function of S. suis FlpS is the oxygen-dependent activation of the arginine deiminase system. Full article

(This article belongs to the Special Issue Streptococcus suis)

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Graphical abstract

Graphical abstract
Full article ">Figure 1
Influence of FlpS knock-out on global gene expression during growth of S. suis. (A) Summary of significantly differentially expressed genes during exp and stat growth of S. suis strain 10ΔflpS and classification of clusters of orthologous groups (COG). C, energy production and conversion; D, cell cycle control, cell division; E, amino acid transport and metabolism; F, nucleotide transport and metabolism; G, carbohydrate transport and metabolism; H, coenzyme transport and metabolism; I, lipid transport and metabolism; J, translation; K, transcription; L, replication, recombination and repair; M, cell wall/membrane biogenesis; O, post-translational modification, protein turnover, chaperones; P, inorganic ion transport and metabolism; R, general function prediction only; S, function unknown; T, signal transduction mechanisms; V, defense mechanisms; [−], no prediction; (B) Venn diagram illustration of the number of significant differentially expressed genes during exp and stat growth of S. suis strain 10ΔflpS; (C) Venn diagram illustration of the number of significant differentially expressed genes during exp and stat growth of S. suis strains 10ΔflpS and 10ΔccpA [<a href="#B34-pathogens-05-00051" class="html-bibr">34</a>]. Full article ">Figure 2
Metabolic characterization of S. suis strain 10ΔflpS. (A) The wild-type strain 10 and strain 10ΔflpS were grown in CDM medium containing 40 mM of monosaccharides (left panel) or di-/trisaccharides (right panel) indicated, and OD630 values were recorded at one-hour intervals automatically in a thermostatic 96-well microplate reader. Results and standard deviations are shown for three biological replicates. Carbohydrate substrates that could not be used for streptococcal growth in CDM are marked by asterisks; (B) Color map for the overall 13C excess (mol %) of labeled amino acids after growth of S. suis strains in the presence of [U-13C6]glucose in THB media. Notably, only overall 13C excesses above 0.5 mol % were considered as sufficient labeling rates. The results are shown for exp and stat grown bacteria. Mean values of two biological replicates for which MS measurements were performed in triplicate are given. Full article ">Figure 2 Cont.
Metabolic characterization of S. suis strain 10ΔflpS. (A) The wild-type strain 10 and strain 10ΔflpS were grown in CDM medium containing 40 mM of monosaccharides (left panel) or di-/trisaccharides (right panel) indicated, and OD630 values were recorded at one-hour intervals automatically in a thermostatic 96-well microplate reader. Results and standard deviations are shown for three biological replicates. Carbohydrate substrates that could not be used for streptococcal growth in CDM are marked by asterisks; (B) Color map for the overall 13C excess (mol %) of labeled amino acids after growth of S. suis strains in the presence of [U-13C6]glucose in THB media. Notably, only overall 13C excesses above 0.5 mol % were considered as sufficient labeling rates. The results are shown for exp and stat grown bacteria. Mean values of two biological replicates for which MS measurements were performed in triplicate are given. Full article ">Figure 3
Mice infection with different S. suis regulator mutant strains. (A) Intranasal infection of mice. Specific bacterial loads of tracheonasal lavage (TNL) and indicated inner organs of mice (3 d.p.i.) intranasally infected with indicated S. suis strains. Each symbol represents one individual animal and medians are indicated by horizontal lines. Statistical testing was done for each TNL or organ by a Kruskal-Wallis test with a post hoc Dunn’s multiple comparisons test; (B) Intravenous infection of mice. The upper panel shows specific bacterial loads of blood samples and indicated inner organs of mice intravenously infected with indicated S. suis strains. Statistical testing was done for each blood sample or organ by a Kruskal-Wallis test with a post hoc Dunn’s multiple comparisons test. In the lower panel the respective Kaplan-Meier diagram for mortality of mice is shown. Significant difference is indicated by * with p < 0.05 (log-rank test). Full article ">Figure 4
FlpS is essential for arcABC operon expression and fitness of S. suis. (A) Immunoblot analyses of whole-cell lysates of S. suis strains grown to stat phase in THB medium under anaerobic conditions. Immunoblot for wild-type strain 10, strain 10ΔflpS and complemented mutant strain 10ΔflpScomp probed with polyclonal antisera raised against recombinant ArcB; (B) Real-time qRT-PCR experiments of S. suis strain 10 and strain 10ΔflpS grown under standard batch and anaerobic conditions. Fold changes in relative arcB transcript levels were shown for a time kinetic as described in Materials and Methods. Results for two independent experiments are depicted; (C) Real-time qRT-PCR experiments of S. suis strain 10, strain 10ΔflpS and strain 10ΔflpScomp grown under batch and shaking (shake) conditions. Fold changes in relative arcB transcript levels were shown for a time kinetic as described in Materials and Methods. Data from three biological replicates and are shown as means ± SEM. Statistical analysis was performed using one-way ANOVA followed by a post-Tukey test (**, p < 0.01); (D) GFP reporter assay. Reporter plasmids carrying the GFP under control of the arcABC promoter were transformed in S. suis wild-type strain 10 and strain 10ΔflpS. Bars represent the relative fluorescence units (RFU) after normalization to the values obtained for strain 10 carrying the promoterless gfp construct (10::gfp). Experiments were carried out in triplicate and repeated twice; (E) Intracellular survival of the unencapsulated strain 10∆cpsEF (black bars) and its flpS mutant strain 10∆cpsEF∆flpS (white bars) in HEp-2 cells. HEp-2 cells were either treated with 200 nM bafilomycin (+Baf) for 1 h before infection to inhibit endosomal acidification or left untreated (−Baf). Results are given as percentage of intracellular bacterial survival after 2 h. Data represent means and standard deviation of two independent experiments performed in duplicates. Results were considered statistically significant with p < 0.05 in a two-tailed t-test, as indicated by asterisks. Full article ">

14497 KiB

Open AccessArticle

Bacterial Suppression of RNA Polymerase II-Dependent Host Gene Expression

by Ines Ambite, Nataliya Lutay, Christoph Stork, Ulrich Dobrindt, Björn Wullt and Catharina Svanborg

Pathogens 2016, 5(3), 49; https://doi.org/10.3390/pathogens5030049 - 13 Jul 2016

Cited by 6 | Viewed by 5581

Abstract

Asymptomatic bacteriuria (ABU) is a bacterial carrier state in the urinary tract that resembles commensalism at other mucosal sites. ABU strains often lack the virulence factors that characterize uropathogenic Escherichia coli (E. coli) strains and therefore elicit weak innate immune responses [...] Read more.

Asymptomatic bacteriuria (ABU) is a bacterial carrier state in the urinary tract that resembles commensalism at other mucosal sites. ABU strains often lack the virulence factors that characterize uropathogenic Escherichia coli (E. coli) strains and therefore elicit weak innate immune responses in the urinary tract. In addition, ABU strains are active modifiers of the host environment, which they influence by suppressing RNA polymerase II (Pol II)-dependent host gene expression. In patients inoculated with the ABU strain E. coli 83972, gene expression was markedly reduced after 24 h (>60% of all regulated genes). Specific repressors and activators of Pol II-dependent transcription were modified, and Pol II Serine 2 phosphorylation was significantly inhibited, indicating reduced activity of the polymerase. This active inhibition included disease–associated innate immune response pathways, defined by TLR4, IRF-3 and IRF-7, suggesting that ABU strains persist in human hosts by active suppression of the antibacterial defense. In a search for the mechanism of inhibition, we compared the whole genome sequences of E. coli 83972 and the uropathogenic strain E. coli CFT073. In addition to the known loss of virulence genes, we observed that the ABU strain has acquired several phages and identified the lytic Prophage 3 as a candidate Pol II inhibitor. Intact phage particles were released by ABU during in vitro growth in human urine. To address if Prophage 3 affects Pol II activity, we constructed a Prophage 3 negative deletion mutant in E. coli 83972 and compared the effect on Pol II phosphorylation between the mutant and the E. coli 83972 wild type (WT) strains. No difference was detected, suggesting that the Pol II inhibitor is not encoded by the phage. The review summarizes the evidence that the ABU strain E. coli 83972 modifies host gene expression by inhibition of Pol II phosphorylation, and discusses the ability of ABU strains to actively create an environment that enhances their persistence. Full article

(This article belongs to the Special Issue Molecular Aspects of Urinary Tract Infection)

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Figure 1
Suppression of gene expression in inoculated patients and infected human kidney cells. (A) Schematic of therapeutic inoculation with the ABU strain E. coli 83972, which established ABU in the inoculated patients. RNA was extracted from peripheral blood mononuclear cells (PBMCs) before and 24 h after intravesical inoculation. Significantly regulated genes were identified by transcriptomic analysis. Genes in the Pol II network were suppressed, by the ABU strain; (B) Human kidney epithelial cells (A498) were infected with the ABU strain E. coli 83972 and significantly regulated genes were identified by transcriptomic analysis, compared to uninfected cells. Pol II transcription cycle with gene categories regulated by ABU indicated in red. ABU infection affected different steps in the Pol II cycle, including chromatin opening, escape from pausing and pre-mRNA splicing. The ABU strain failed to activate/inhibit the pathology-associated TLR4 and IFN-β pathways. Adapted from Lutay et al. [<a href="#B14-pathogens-05-00049" class="html-bibr">14</a>]. Full article ">Figure 2
The ABU strain inhibits Pol II Ser 2 phosphorylation in vitro. (A) Immunoperoxidase staining of human kidney epithelial cells (A498), using HRP-conjugated antibodies. Pol II phosphorylation (brown) was reduced by the ABU strain compared to uninfected cells. A scale representing each staining intensity is shown (4 = brown, 0 = colorless). Inhibition of Pol II phosphorylation was expressed in percent of the uninfected controls (0% = control, no inhibition; 100% = complete inhibition). The cells were exposed to 2 × 109 CFU/mL of E. coli CFT073 (APN) or 83972 (ABU), (n = 100 cells per sample, p-value by χ2 test). Scale bar = 50 μm; (B) Phospho-Pol II in human kidney epithelial cells, infected with the ABU or APN strains. Western blots of whole cell extracts. The phospho-specific staining was normalized against total Pol II or GAPDH; (C) Competitive infection. The strain to which the cells were first exposed, was shown to determine Pol II activation. Pol II Ser 2 staining of human kidney epithelial cells after 2 h + 2 h infection with the ABU strain followed by the APN strain or the APN strain followed by the ABU strain (n ≥ 167 cells, p-values by χ2 test). Full article ">Figure 3
Induction of Prophage 3 by E. coli 83972 and construction of the E. coli 83972 Prophage 3 deletion mutant. (A) Electron microscopy of lambda-like Prophage 3 released by E. coli 83972 during in vitro growth in pooled human urine; (B) Quantification of lytic phages by plaque formation (plaque forming units, PFU) on E. coli C600. E. coli 83972 released lytic phages during growth in urine and Luria Bertani (LB) broth. Moderate release of lytic phages by CFT073 was induced by mytomicin C; (C) The Prophage 3 sequence was deleted from the E. coli 83972 chromosome. The ABU—p3 mutant strain is unable to produce and release lytic Prophage 3 particles; (D) The Prophage 3 sequence was replaced by a chloramphenicol acetyltransferase (cat) cassette, using homologous Lambda red recombination technology and chloramphenicol resistance for selection. The bacterial sit gene, involved in iron uptake, was conserved in the mutant strain. In the construct, the chloramphenicol resistance gene is surrounded by two flippase recognition target (FRT) sites. Full article ">Figure 4
Pol II phosphorylation is not altered by the Prophage 3 deletion. (A) The Pol II Ser 2 staining intensity (brown) did not differ between cells infected with the ABU strain or the -Prophage 3 deletion mutant (109 and 2 × 109 CFU/mL, n = 100 cells, p-values by χ2 test). Histograms show the distribution of human kidney epithelial cells according to their Pol II Ser 2 staining intensity (+++ = highly stained, - = no staining); (B) Supernatants of A498 cells infected with the ABU strain or the ABU-Prophage 3 deletion mutant. Similar effects on Pol II phosphorylation (109 CFU/mL, n = 100 cells, p-values by χ2 test). Pol II Ser 2 staining was quantified by immunoperoxidase staining using Pol II Ser2specific antibodies; (C) Pol II Ser 2 phosphorylation was not inhibited by lytic phage particles released by the ABU strain during growth in human urine, in vitro. Full article ">

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Open AccessReview

A Review of Evidence that Equine Influenza Viruses Are Zoonotic

by Tai Xie, Benjamin D. Anderson, Ulziimaa Daramragchaa, Maitsetset Chuluunbaatar and Gregory C. Gray

Pathogens 2016, 5(3), 50; https://doi.org/10.3390/pathogens5030050 - 12 Jul 2016

Cited by 24 | Viewed by 12612

Abstract

Among scientists, there exist mixed opinions whether equine influenza viruses infect man. In this report, we summarize a 2016 systematic and comprehensive review of the English, Chinese, and Mongolian scientific literature regarding evidence for equine influenza virus infections in man. Searches of PubMed, [...] Read more.

Among scientists, there exist mixed opinions whether equine influenza viruses infect man. In this report, we summarize a 2016 systematic and comprehensive review of the English, Chinese, and Mongolian scientific literature regarding evidence for equine influenza virus infections in man. Searches of PubMed, Web of Knowledge, ProQuest, CNKI, Chongqing VIP Database, Wanfang Data and MongolMed yielded 2831 articles, of which 16 met the inclusion criteria for this review. Considering these 16 publications, there was considerable experimental and observational evidence that at least H3N8 equine influenza viruses have occasionally infected man. In this review we summarize the most salient scientific reports. Full article

(This article belongs to the Special Issue Equine Influenza)

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Open AccessArticle

Virulence Studies of Different Sequence Types and Geographical Origins of Streptococcus suis Serotype 2 in a Mouse Model of Infection

by Jean-Philippe Auger, Nahuel Fittipaldi, Marie-Odile Benoit-Biancamano, Mariela Segura and Marcelo Gottschalk

Pathogens 2016, 5(3), 48; https://doi.org/10.3390/pathogens5030048 - 11 Jul 2016

Cited by 45 | Viewed by 6337

Abstract

Multilocus sequence typing previously identified three predominant sequence types (STs) of Streptococcus suis serotype 2: ST1 strains predominate in Eurasia while North American (NA) strains are generally ST25 and ST28. However, ST25/ST28 and ST1 strains have also been isolated in Asia and NA, [...] Read more.

Multilocus sequence typing previously identified three predominant sequence types (STs) of Streptococcus suis serotype 2: ST1 strains predominate in Eurasia while North American (NA) strains are generally ST25 and ST28. However, ST25/ST28 and ST1 strains have also been isolated in Asia and NA, respectively. Using a well-standardized mouse model of infection, the virulence of strains belonging to different STs and different geographical origins was evaluated. Results demonstrated that although a certain tendency may be observed, S. suis serotype 2 virulence is difficult to predict based on ST and geographical origin alone; strains belonging to the same ST presented important differences of virulence and did not always correlate with origin. The only exception appears to be NA ST28 strains, which were generally less virulent in both systemic and central nervous system (CNS) infection models. Persistent and high levels of bacteremia accompanied by elevated CNS inflammation are required to cause meningitis. Although widely used, in vitro tests such as phagocytosis and killing assays require further standardization in order to be used as predictive tests for evaluating virulence of strains. The use of strains other than archetypal strains has increased our knowledge and understanding of the S. suis serotype 2 population dynamics. Full article

(This article belongs to the Special Issue Streptococcus suis)

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Open AccessFeature PaperArticle

Recruitment of Factor H to the Streptococcus suis Cell Surface is Multifactorial

by David Roy, Daniel Grenier, Mariela Segura, Annabelle Mathieu-Denoncourt and Marcelo Gottschalk

Pathogens 2016, 5(3), 47; https://doi.org/10.3390/pathogens5030047 - 7 Jul 2016

Cited by 23 | Viewed by 5063

Abstract

Streptococcus suis is an important bacterial swine pathogen and a zoonotic agent. Recently, two surface proteins of S. suis, Fhb and Fhbp, have been described for their capacity to bind factor H—a soluble complement regulatory protein that protects host cells from complement-mediated [...] Read more.

Streptococcus suis is an important bacterial swine pathogen and a zoonotic agent. Recently, two surface proteins of S. suis, Fhb and Fhbp, have been described for their capacity to bind factor H—a soluble complement regulatory protein that protects host cells from complement-mediated damages. Results obtained in this study showed an important role of host factor H in the adhesion of S. suis to epithelial and endothelial cells. Both Fhb and Fhbp play, to a certain extent, a role in such increased factor H-dependent adhesion. The capsular polysaccharide (CPS) of S. suis, independently of the presence of its sialic acid moiety, was also shown to be involved in the recruitment of factor H. However, a triple mutant lacking Fhb, Fhbp and CPS was still able to recruit factor H resulting in the degradation of C3b in the presence of factor I. In the presence of complement factors, the double mutant lacking Fhb and Fhbp was similarly phagocytosed by human macrophages and killed by pig blood when compared to the wild-type strain. In conclusion, this study suggests that recruitment of factor H to the S. suis cell surface is multifactorial and redundant. Full article

(This article belongs to the Special Issue Streptococcus suis)

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Open AccessArticle

Simultaneous Quantification and Differentiation of Streptococcus suis Serotypes 2 and 9 by Quantitative Real-Time PCR, Evaluated in Tonsillar and Nasal Samples of Pigs

by Niels Dekker, Ineke Daemen, Koen Verstappen, Astrid De Greeff, Hilde Smith and Birgitta Duim

Pathogens 2016, 5(3), 46; https://doi.org/10.3390/pathogens5030046 - 30 Jun 2016

Cited by 12 | Viewed by 12179

Abstract

Invasive Streptococcus suis (S. suis) infections in pigs are often associated with serotypes 2 and 9. Mucosal sites of healthy pigs can be colonized with these serotypes, often multiple serotypes per pig. To unravel the contribution of these serotypes in pathogenesis [...] Read more.

Invasive Streptococcus suis (S. suis) infections in pigs are often associated with serotypes 2 and 9. Mucosal sites of healthy pigs can be colonized with these serotypes, often multiple serotypes per pig. To unravel the contribution of these serotypes in pathogenesis and epidemiology, simultaneous quantification of serotypes is needed. A quantitative real-time PCR (qPCR) targeting cps2J (serotypes 2 and 1/2) and cps9H (serotype 9) was evaluated with nasal and tonsillar samples from S. suis exposed pigs. qPCR specifically detected serotypes in all pig samples. The serotypes loads in pig samples estimated by qPCR showed, except for serotype 9 in tonsillar samples (correlation coefficient = 0.25), moderate to strong correlation with loads detected by culture (correlation coefficient > 0.65), and also in pigs exposed to both serotypes (correlation coefficient > 0.75). This qPCR is suitable for simultaneous differentiation and quantification of important S. suis serotypes. Full article

(This article belongs to the Special Issue Streptococcus suis)

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Figure 1
Standard curves of cps2J-qPCR (panel A) and cps9H-qPCR (panel B) obtained by serial dilutions of bacterial suspensions. The y-intercept indicates the expected crossing point (Cp) for a sample with a quantity equal to 1 eq. CFU/PCR reaction, i.e., 1 × 102 eq. CFU/mL. The slope indicates the number of cycles between samples that differ 1.0 log10 eq. CFU /mL; a value of 3.3 is optimal. The R2 value indicates the close fit between the regression line of the standard curve and the individual Cp data points; a value of 1.00 indicates a perfect fit. The efficiency indicates the increase in copies per cycle; a value of 2 is optimal. Full article ">Figure 2
Correlation between the log10 of the number of colony forming units (CFU) determined by selective bacterial examination (SBE) and log 10 eq. CFU by qPCR in tonsillar samples (panels A,C) and in nasal samples (panels B,D) from pigs exposed to either S. suis serotype 2 (panels A,B) or serotype 9 (panels C,D). Pigs that were inoculated are marked by ○, and contact exposed pigs by ●. On the label of each individual point, its time point of sampling is presented (in days post inoculation). Full article ">Figure 3
Correlation between the S. suis counts (log10 CFU/mL) determined by selective bacterial examination (SBE) and counts predicted by a linear mixed model with the qPCR results (log10 eq. CFU/mL) of tonsillar (panel A) and nasal samples (panel B) taken from pigs exposed to both serotypes 2 and 9 as input. The model was constructed with data of pigs colonized with either serotype 2 or 9. Pigs that were inoculated are marked by ○, and contact pigs by ●. On the label of each individual point, its time point of sampling is presented (in days post inoculation). Full article ">

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Open AccessReview

Current Taxonomical Situation of Streptococcus suis

by Masatoshi Okura, Makoto Osaki, Ryohei Nomoto, Sakura Arai, Ro Osawa, Tsutomu Sekizaki and Daisuke Takamatsu

Pathogens 2016, 5(3), 45; https://doi.org/10.3390/pathogens5030045 - 24 Jun 2016

Cited by 118 | Viewed by 8640

Abstract

Streptococcus suis, a major porcine pathogen and an important zoonotic agent, is considered to be composed of phenotypically and genetically diverse strains. However, recent studies reported several “S. suis-like strains” that were identified as S. suis by commonly used methods [...] Read more.

Streptococcus suis, a major porcine pathogen and an important zoonotic agent, is considered to be composed of phenotypically and genetically diverse strains. However, recent studies reported several “S. suis-like strains” that were identified as S. suis by commonly used methods for the identification of this bacterium, but were regarded as distinct species from S. suis according to the standards of several taxonomic analyses. Furthermore, it has been suggested that some S. suis-like strains can be assigned to several novel species. In this review, we discuss the current taxonomical situation of S. suis with a focus on (1) the classification history of the taxon of S. suis; (2) S. suis-like strains revealed by taxonomic analyses; (3) methods for detecting and identifying this species, including a novel method that can distinguish S. suis isolates from S. suis-like strains; and (4) current topics on the reclassification of S. suis-like strains. Full article

(This article belongs to the Special Issue Streptococcus suis)

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