bluuebunny/crossref_metadata_2025 huggingface dataset:
Influence of theSalmonella typhimuriumPathogenicity Island 2 Type III Secretion System on Bacterial Growth in the Mouse
Authors: Shea Jacqueline E. , Beuzon Carmen R. , Gleeson Colin , Mundy Rosanna , Holden David W.
Published: December 2019

We have investigated the in vivo growth kinetics of aSalmonella typhimuriumstrain (P11D10) carrying a mutation inssaJ, aSalmonellapathogenicity island 2 (SPI2) gene encoding a component of a type III secretion system required for systemic growth in mice. Similar numbers of mutant and wild-type cells were recovered from the spleens and livers of BALB/c mice up to 8 h after inoculation by the intraperitoneal route. Thereafter, the numbers of wild-type cells continued to increase logarithmically in these organs, whereas those of P11D10 remained relatively static for several days before being cleared. Gentamicin protection experiments on spleen cell suspensions recovered from infected mice showed that viable intracellular wild-type bacteria accumulated over time but that intracellular P11D10 cells did not. Infection experiments were also performed with wild-type and P11D10 cells carrying the temperature-sensitive plasmid pHSG422 to distinguish between bacterial growth rates and killing in vivo. At 16 h postinoculation there were 10-fold more wild-type cells than mutant cells in the spleens of infected mice, but the numbers of cells of both strains carrying the nonreplicating plasmid were very similar, showing that there was little difference in the degree of killing sustained by the two strains and that the SPI2 secretion system must be required for bacterial replication, rather than survival, in vivo. The SPI2 mutant phenotype in mice is similar to that of strains carrying mutations in theSalmonellavirulence plasmidspvgenes. To determine if these two sets of genes interact together, a double mutant strain carrying SPI2 andspvmutations was constructed and compared with strains carrying single mutations in terms of virulence attenuation. These experiments failed to provide any evidence showing that the SPI2 andspvgene products interact together as part of the same virulence mechanism.
Role of Gamma Interferon inHelicobacter pylori-Induced Gastric Inflammatory Responses in a Mouse Model
Authors: Sawai Naoki , Kita Masakazu , Kodama Tadashi , Tanahashi Toshihito , Yamaoka Yoshio , Tagawa Yoh-ichi , Iwakura Yoichiro , Imanishi Jiro
Published: December 2019

The immune responses toHelicobacter pyloriinfection play important roles in gastroduodenal diseases. The contribution of gamma interferon (IFN-γ) to the immune responses, especially to the induction of gastric inflammation and to protection fromH. pyloriinfection, was investigated with IFN-γ gene knockout (IFN-γ−/−) mice. We first examined the colonizing abilities of eightH. pyloristrains with a short-term infection test in order to selectH. pyloristrains which could colonize the mouse stomach. Only three strains (ATCC 43504, CPY2052, and HPK127) colonized C57BL/6 wild-type mice, although all of the strains except for ATCC 51110 could colonize IFN-γ−/−mice. The number ofH. pyloriorganisms colonizing the stomach in wild-type mice was lower than that in IFN-γ−/−mice. Oral immunization with the CPY2052 sonicate and cholera toxin protected against infection with strain CPY2052 in both types of mouse. These findings suggested that IFN-γ may play a protective role inH. pyloriinfection, although the degree of its protective ability was estimated to be low. In contrast, in a long-term infection test done to examine the contribution of IFN-γ to gastric inflammation, CPY2052-infected wild-type mice developed a severe infiltration of mononuclear cells in the lamina propria and erosions in the gastric epithelium 15 months after infection, whereas CPY2052-infected IFN-γ−/−mice showed no inflammatory symptoms. This result clearly demonstrated that IFN-γ plays an important role in the induction of gastric inflammation caused byH. pyloriinfection.
Saccharomyces boulardii
Protease Inhibits the Effects of
Clostridium difficile
Toxins A and B in Human Colonic Mucosa
Authors: Castagliuolo Ignazio , Riegler Martin F. , Valenick Leyla , LaMont J. Thomas , Pothoulakis Charalabos
Published: December 2019

Saccharomyces boulardii
is a nonpathogenic yeast used in the treatment of
Clostridium difficile
diarrhea and colitis. We have reported that
S. boulardii
inhibits
C. difficile
toxin A enteritis in rats by releasing a 54-kDa protease which digests the toxin A molecule and its brush border membrane (BBM) receptor (I. Castagliuolo, J. T. LaMont, S. T. Nikulasson, and C. Pothoulakis, Infect. Immun. 64:5225–5232, 1996). The aim of this study was to further evaluate the role of
S. boulardii
protease in preventing
C. difficile
toxin A enteritis in rat ileum and determine whether it protects human colonic mucosa from
C. difficile
toxins. A polyclonal rabbit antiserum raised against purified
S. boulardii
serine protease inhibited by 73% the proteolytic activity present in
S. boulardii
conditioned medium in vitro. The anti-protease immunoglobulin G (IgG) prevented the action of
S. boulardii
on toxin A-induced intestinal secretion and mucosal permeability to [
3
H]mannitol in rat ileal loops, while control rabbit IgG had no effect. The anti-protease IgG also prevented the effects of
S. boulardii
protease on digestion of toxins A and B and on binding of [
3
H]toxin A and [
3
H]toxin B to purified human colonic BBM. Purified
S. boulardii
protease reversed toxin A- and toxin B-induced inhibition of protein synthesis in human colonic (HT-29) cells. Furthermore, toxin A- and B-induced drops in transepithelial resistance in human colonic mucosa mounted in Ussing chambers were reversed by 60 and 68%, respectively, by preexposing the toxins to
S. boulardii
protease. We conclude that the protective effects of
S. boulardii
on
C. difficile
-induced inflammatory diarrhea in humans are due, at least in part, to proteolytic digestion of toxin A and B molecules by a secreted protease.
The Polysaccharide Portion of Lipopolysaccharide Regulates Antigen-Specific T-Cell Activation via Effects on Macrophage-Mediated Antigen Processing
Authors: Zirk Nicole M. , Hashmi Saira F. , Ziegler H. Kirk
Published: December 2019

The lipopolysaccharide (LPS) structure ofSalmonella typhimuriumhas been correlated with the virulence of wild-type strain LT2. Mutants of LT2 with truncated polysaccharide portions of LPS are less virulent than strains with a complete LPS structure. Polyclonal T cells and monoclonal T-cell hybridomas were more reactive to heat-killed rough mutants than to heat-killed smooth strains, as measured by interleukin-2 (IL-2) production. Using a large panel of strains with truncated LPS molecules, we found that T-cell reactivity decreased with certain lengths of polysaccharide. The decreased response was not due to differential phagocytic uptake, IL-12 production, or major histocompatibility complex class II surface expression by macrophages. Also, LT2 did not mediate any global suppression since addition of LT2 did not diminish the response of T cells specific for antigens unrelated toSalmonella. In an experiment in which processing times were varied, we found that antigens from rough strains were processed and presented more quickly than those associated with smooth strains. At longer processing times, epitopes from LT2 were presented well. We hypothesize that the slower antigen processing and presentation of wild-typeSalmonellamay be caused by masking of surface antigens by the longer polysaccharide portion of smooth LPS. This blocking of effective antigen presentation may contribute to the virulence ofSalmonella.
Helicobacter pyloriInfection in Immunized Mice Lacking Major Histocompatibility Complex Class I and Class II Functions
Authors: Pappo Jacques , Torrey Deirdre , Castriotta Lillian , Savinainen Anneli , Kabok Zita , Ibraghimov Alexander
Published: December 2019

The role of major histocompatibility complex (MHC) class I- and class II-restricted functions inHelicobacter pyloriinfection and immunity upon oral immunization was examined in vivo. Experimental challenge withH. pyloriSS1 resulted in significantly greater (P≤ 0.025) colonization of MHC class I and class II mutant mice than C57BL/6 wild-type mice. Oral immunization withH. pyloriwhole-cell lysates and cholera toxin adjuvant significantly reduced the magnitude ofH. pyloriinfection in C57BL/6 wild-type (P= 0.0083) and MHC class I knockout mice (P= 0.0048), but it had no effect on theH. pyloriinfection level in MHC class II-deficient mice. Analysis of the anti-H. pyloriantibody levels in serum showed a dominant serum immunoglobulin G1 (IgG1) response in immunized C57BL/6 wild-type and MHC class I mutant mice but no detectable serum IgG response in MHC class II knockout mice. Populations of T-cell-receptor (TCR) αβ+CD4+CD54+cells localized to gastric tissue of immunized C57BL/6 wild-type and MHC class I knockout mice, but TCRαβ+CD8+cells predominated in the gastric tissue of immunized MHC class II-deficient mice. These observations show that CD4+T cells engaged after mucosal immunization may be important for the generation of a protective anti-H. pyloriimmune response and that CD4+CD8−and CD4−CD8+T cells regulate the extent ofH. pyloriinfection in vivo.
Specificity of Infection-Induced Immunity amongBorrelia burgdorferiSensu Lato Species
Authors: Barthold Stephen W.
Published: December 2019

The specificity of infection-induced immunity in mice infected with cultured or host-adaptedBorrelia burgdorferisensu lato, the agent of Lyme disease, was examined. Sera obtained from mice following infection with high and low doses of culturedB. burgdorferisensu stricto, transplantation of infected tissue (host-adapted spirochetes), or tick-borne inoculation all showed protective activity in passive immunization assays. Infection and disease were similar in mice infected with cultured spirochetes or by transplantation. Thus, the adaptive form of inoculated spirochetes did not influence the immune response during active infection. Mice infected withB. burgdorferisensu stricto and then cured of infection with an antibiotic during early or late stages of infection were resistant to challenge with high doses of homologous cultured spirochetes for up to 1 year. In contrast, actively immune mice infected with differentBorreliaspecies (B. burgdorferisensu lato,B. burgdorferisensu stricto cN40,Borrelia afzeliiPKo, andBorrelia gariniiPBi) and then treated with an antibiotic were resistant to challenge with cultured homologous but not heterologous spirochetes. Similar results were achieved for actively immune mice challenged by transplantation and by passive immunization with sera from mice infected with each of theBorreliaspecies and then challenged with cultured spirochetes. Arthritis and carditis in mice that had immunizing infections withB. afzeliiandB. gariniiand then challenged by transplantation withB. burgdorferisensu stricto were equivalent in prevalence and severity to those in nonimmune recipient mice. These results indicate that protective immunity and disease-modulating immunity that develop during active infection are universal among species related toB. burgdorferisensu lato but are species specific.
Increased Levels of Soluble CD14 in Sera of Periodontitis Patients
Authors: Hayashi Joichiro , Masaka Tamami , Ishikawa Isao
Published: December 2019

Soluble CD14 (sCD14) mediates the response to lipopolysaccharide (LPS) in cells lacking membrane-bound CD14. We determined sCD14 concentrations in the sera of 38 periodontitis patients and 25 healthy controls by enzyme-linked immunosorbent assay. The sCD14 levels in the sera of patients with periodontitis were significantly higher than those of healthy subjects and decreased after treatment. Enhanced levels of sCD14 in serum may contribute to the host response to LPS in periodontitis. Furthermore, we showed in vitro that addition of LPS enhanced the release of sCD14 by monoblastic U937 cells treated with 1α,25-dihydroxyvitamin D3. Thus, increased sCD14 levels in periodontitis patients may be due to chronic exposure to LPS.
Virulence Properties of
Escherichia coli
83972, a Prototype Strain Associated with Asymptomatic Bacteriuria
Authors: Hull Richard A. , Rudy Delbert C. , Donovan William H. , Wieser Inge E. , Stewart Colleen , Darouiche Rabih O.
Published: December 2019

Little is known about bacteria associated with asymptomatic bacteriuria (ABU) with regard to urinary tract colonization mechanisms. In this study, virulence properties of
Escherichia coli
83972, a strain that was isolated from a clinical ABU episode, were examined. The genetic potential for expression of P and type 1 pili was demonstrated, and DNA sequences related to type 1C and G (UCA) pilus genes were also detected. However,
E. coli
83972 did not express
d
-mannose-resistant or
d
-mannose-sensitive hemagglutination after growth under standard conditions in vitro or upon isolation from the urine of colonized test subjects. Limited uroepithelial cell adherence was observed in vivo, and weak
d
-mannose-sensitive hemagglutination was detected after extended growth in urine in vitro.
Nonrestricted Differential Intoxication of Cells by Pertussis Toxin
Authors: el Bayâ Ali , Brückener Kerstin , Schmidt M. Alexander
Published: December 2019

After uptake and retrograde transport pertussis toxin acts by ADP-ribosylating α-G
i
proteins. We show that uptake via many different receptor proteins followed by retrograde transport and intoxication is not restricted to a particular cell type. The efficiency of cellular intoxication, however, was found to be cell type dependent.
γδ T-Cell Function in Pathogenesis of Cerebral Malaria in Mice Infected withPlasmodium bergheiANKA
Authors: Yañez D. M. , Batchelder J. , van der Heyde H. C. , Manning D. D. , Weidanz W. P.
Published: December 2019

Mice depleted of γδ T cells by monoclonal antibody treatment and infected withPlasmodium bergheiANKA did not develop cerebral malaria (CM). In striking contrast, δ0/0mice infected withP. bergheideveloped CM despite their γδ T-cell deficiency. γδ T cells appear to be essential for the pathogenesis of CM in mice having experienced normal ontogeny but not in mice genetically deprived of γδ T cells from the beginning of life.
Binding of
Actinobacillus pleuropneumoniae
Lipopolysaccharides to Glycosphingolipids Evaluated by Thin-Layer Chromatography
Authors: Abul-Milh Maan , Paradis Sonia-Élaine , Dubreuil J. Daniel , Jacques Mario
Published: December 2019

The binding profile of
Actinobacillus pleuropneumoniae
serotypes 1 and 2 to various glycosphingolipids was evaluated by using thin-layer chromatogram overlay.
A. pleuropneumoniae
whole cells recognized glucosylceramide (Glcβ1Cer), galactosylceramide (Galβ1Cer) with hydroxy and nonhydroxy fatty acids, sulfatide (SO
3
-3Galβ1Cer), lactosylceramide (Galβ1-4Glcβ1Cer), gangliotriaosylceramide GgO
3
(GalNAcβ1-4Galβ1-4Glcβ1Cer), and gangliotetraosylceramide GgO
4
(Galβ1-3GalNAcβ1-4Galβ1-4Glcβ1Cer) glycosphingolipids. We observed no binding to globoseries, globotriaosylceramide Gb
3
, globoside Gb
4
, or Forssman Gb
5
glycosphingolipids or to gangliosides GM1, GM2, GM3, GD1a, GD1b, GD3, and GT1b. The
A. pleuropneumoniae
strains tested also failed to detect phosphatidylethanolamine or ceramide. Interestingly, extracted lipopolysaccharide (LPS) of serotype 1 and serotype 2 as well as detoxified LPS of serotype 1 showed binding patterns similar to that of whole bacterial cells. Binding to GlcCer, GalCer, sulfatide, and LacCer, but not to GgO
3
and GgO
4
glycosphingolipids, was inhibited after incubation of the bacteria with monoclonal antibodies against LPS O antigen. These findings indicate the involvement of LPS in recognition of three groups of glycosphingolipids: (i) GlcCer and LacCer, where glucose is probably an important saccharide sequence required for LPS binding; (ii) GalCer and sulfatide glycosphingolipids, where the sulfate group is part of the binding epitope of the isoreceptor; and (iii) GgO
3
and GgO
4
, where GalNacβ1-4Gal disaccharide represents the minimal common binding epitope. Taken together, our results indicate that
A. pleuropneumoniae
LPS recognize various saccharide sequences found in different glycosphingolipids, which probably represents a strong virulence attribute.
Limited Interleukin-18 Response inSalmonella-Infected Murine Macrophages and inSalmonella-Infected Mice
Authors: Elhofy Adam , Bost Kenneth L.
Published: December 2019

Optimal immune responses against an intracellular bacterial pathogen, such asSalmonella, involve the production of gamma interferon (IFN-γ), which activates macrophages. It has recently been suggested that, interleukin-18 (IL-18), in addition to IL-12, contributes to the induction of IFN-γ following infection. Given this hypothesis, an optimal host immune response against intracellular bacterial pathogens would include the induction of IL-18 secretion by macrophages due toSalmonellainfection. We questioned whetherSalmonellacould induce macrophages to upregulate their expression of IL-18 mRNA and secretion of IL-18. With cultures of murine macrophages, we were surprised to find that infection by wild-typeSalmonella dublinresulted in decreased expression of IL-18 mRNA and IL-18 secretion rather than an increase. Reduction of macrophage-derived IL-18 expression by wild-typeSalmonellaoccurred early in the response, suggesting a direct effect. Furthermore, mice orally inoculated with wild-typeSalmonellawere shown to have reduced IL-18 mRNA expression at mucosal sites within hours postinoculation. Together these studies demonstrateSalmonella-induced reductions in IL-18 expression, suggesting that this intracellular pathogen may be capable of limiting a potentially protective immune response.
Modulation of Cytokine Response of Pneumonic Foals by VirulentRhodococcus equi
Authors: Giguère Steeve , Wilkie Bruce N. , Prescott John F.
Published: December 2019

The ability ofRhodococcus equito induce pneumonia in foals depends on the presence of an 85- to 90-kb plasmid. In this study, we evaluated whether plasmid-encoded products mediate virulence by modulating the cytokine response of foals. Foals infected intrabronchially with a virulence plasmid-containing strain ofR. equihad similar gamma interferon (IFN-γ) and interleukin-12 (IL-12) p35 but significantly higher IL-1β, IL-10, IL-12 p40, and tumor necrosis factor alpha (TNF-α) mRNA expression in lung tissue compared to foals infected with the plasmid-cured derivative. IFN-γ mRNA expression levels in CD4+T lymphocytes isolated from bronchial lymph nodes (BLN) were similar for the two groups ofR. equi-infected foals on day 3 postinfection. However, on day 14, in association with pneumonia and marked multiplication of virulentR. equibut with complete clearance of the plasmid-cured derivative, IFN-γ mRNA expression in BLN CD4+T lymphocytes was significantly (P< 0.001) higher in foals infected with the plasmid-cured derivative. These results suggests an immunomodulating role forR. equivirulence plasmid-encoded products in downregulating IFN-γ mRNA expression by CD4+T lymphocytes.
Molecular Cloning and Characterization of the
afa-7
and
afa-8
Gene Clusters Encoding Afimbrial Adhesins in
Escherichia coli
Strains Associated with Diarrhea or Septicemia in Calves
Authors: Lalioui Lila , Jouve Mabel , Gounon Pierre , Le Bouguenec Chantal
Published: December 2019

The
afa
gene clusters, which encode proteins involved in adhesion to epithelial cells, from
Escherichia coli
strains associated with urinary and intestinal infections in humans have been characterized. Pathogenic isolates of bovine and porcine origin that possess
afa
-related sequences have recently been described. We report in this work the cloning and characterization of the
afa-7
and
afa-8
gene clusters from bovine isolates. Hybridization and sequencing experiments revealed that despite similarity in genetic organization, the
afa-7
and
afa-8
genes, and the well-characterized
afa-3
operon expressed by human-pathogenic isolates, correspond to three different members of the
afa
family of gene clusters. However, like the
afa-3
gene cluster, both the
afa-7
and
afa-8
gene clusters were found to encode an afimbrial adhesin (AfaE) and an invasin (AfaD). The AfaD peptides encoded by the three gene clusters were only 45% identical, but functional complementation experiments indicated that they belong to the same family of invasins. Hemagglutination and adhesion assays demonstrated that the AfaE-VII and AfaE-VIII adhesins bind to different receptors and that these receptors are not the human decay-accelerating factor recognized to be the receptor of all previously described AfaE adhesins. The AfaE-VIII adhesin is very similar to the M agglutinin of human-uropathogenic strains. We used PCR assays to screen 25 bovine strains for
afaD
and
afaE
genes of either the
afa-7
or
afa-8
gene cluster. The
afa-8
gene cluster was highly prevalent in bovine isolates previously reported to carry
afa
-related sequences (23 of 24 strains), particularly in strains producing cytotoxic necrotizing factors (16 of 16 strains). The location of the
afa-8
gene cluster on the plasmids or chromosome of these isolates suggests that it could be carried by a mobile element, facilitating its dissemination among bovine-pathogenic
E. coli
strains.
Mycobacterium avium
Infection of Epithelial Cells Results in Inhibition or Delay in the Release of Interleukin-8 and RANTES
Authors: Sangari Felix J. , Petrofsky Mary , Bermudez Luiz E.
Published: December 2019

Mycobacterium avium
is an opportunistic pathogen in AIDS patients, who acquire the infection mainly through the gastrointestinal tract. Previous studies in vitro have shown that
M. avium
invades epithelial cells of both intestinal and laryngeal origin. In addition,
M. avium
enters the intestinal mucosa of healthy mice. Because
M. avium
invasion of the intestinal mucosa in vivo initially is not accompanied by significant influx of inflammatory cells, we sought to determine whether
M. avium
would trigger chemokine release upon entry into epithelial cells by using HT-29 intestinal and HEp-2 laryngeal epithelial cell lines. Chemokine synthesis was measured both by the presence of specific mRNA and protein secretion in the cell culture supernatant as determined by enzyme-linked immunosorbent assay. Infection of HT-29 intestinal cells with
M. avium
did not induce the release of interleukin-8 (IL-8) or RANTES for up to 7 days postinfection. However, infection of HEp-2 cells resulted in the release of IL-8 and RANTES at 72 h. Similar findings were observed with other AIDS
M. avium
isolates belonging to different serovars. Secretion of IL-8 by HEp-2 cells was dependent upon bacterial uptake. In addition, prior infection with
M. avium
suppressed IL-8 production by HT-29 cells infected with
Salmonella typhimurium
. Our results suggest that
M. avium
infection of epithelial cells is associated with a delay in IL-8 and RANTES production which, in the case of HT-29, is prolonged up to 1 week. These findings may explain the weak inflammatory response after intestinal mucosa invasion in mice and are probably related with the ability of the bacterium to evade the host’s immune response.
Differential Transcription of the
tcpPH
Operon Confers Biotype-Specific Control of the
Vibrio cholerae
ToxR Virulence Regulon
Authors: Murley Yvette M. , Carroll Patricia A. , Skorupski Karen , Taylor Ronald K. , Calderwood Stephen B.
Published: December 2019

Epidemic strains of
Vibrio cholerae
O1 are divided into two biotypes, classical and El Tor. In both biotypes, regulation of virulence gene expression depends on a cascade in which ToxR activates expression of ToxT, and ToxT activates expression of cholera toxin and other virulence genes. In the classical biotype, maximal expression of this ToxR regulon in vitro occurs at 30°C at pH 6.5 (ToxR-inducing conditions), whereas in the El Tor biotype, production of these virulence genes only occurs under very limited conditions and not in response to temperature and pH; this difference between biotypes is mediated at the level of
toxT
transcription. In the classical biotype, two other proteins, TcpP and TcpH, are needed for maximal
toxT
transcription. Transcription of
tcpPH
in the classical biotype is regulated by pH and temperature independently of ToxR or ToxT, suggesting that TcpP and TcpH couple environmental signals to transcription of
toxT
. In this study, we show a near absence of
tcpPH
message in the El Tor biotype under ToxR-inducing conditions of temperature and pH. However, once expressed, El Tor TcpP and TcpH appear to be as effective as classical TcpP and TcpH in activating
toxT
transcription. These results suggest that differences in regulation of virulence gene expression between the biotypes of
V. cholerae
primarily result from differences in expression of
tcpPH
message in response to environmental signals. We present an updated model for control of the ToxR virulence regulon in
V. cholerae
.
Serological Investigation of
Chlamydia trachomatis
Heat Shock Protein 10
Authors: Betsou Fotini , Sueur Jean Marie , Orfila Jeanne
Published: December 2019

The humoral immune response to
Chlamydia trachomatis
10-kDa heat shock protein (Chsp10) in populations of Russian and French origin was studied by using a recombinant Chsp10 enzyme-linked immunosorbent assay. A physiological but not a serological correlation of Chsp10 exposure with Chsp60 exposure was observed in the Russian population. In the French population studied, there was a significant association between detection of anti-r-Chsp10 immunoglobulin G (IgG) antibodies and chronic genital tract infections. Chsp10 residues 50 to 67 were found to contain an immunodominant although not universal B epitope. Cross-reactions with
Chlamydia pneumoniae
or
Escherichia coli
GroES protein are limited but may occur. Our study suggests that detection of anti-Chsp10 IgG antibodies is associated with chronicity of
C. trachomatis
genital tract infection and does not parallel that of anti-Chsp60 IgG antibodies.
Differential Regulation of Macrophage Interleukin-1 (IL-1), IL-12, and CD80-CD86 by Two Bacterial Toxins
Authors: Foss Dennis L. , Zilliox Michael J. , Murtaugh Michael P.
Published: December 2019

The ability of innate immune cells to differentially respond to various bacterial components provides a mechanism by which the acquired immune response may be tailored to specific pathogens. The response of innate immune cells to bacterial components provides regulatory signals to cognate immune cells. These signals include secreted cytokines and costimulatory molecules, and to a large extent they determine the quantitative and qualitative nature of the immune response. In order to determine if innate immune cells can differentially respond to bacterial components, we compared the responses of macrophages to two bacterially derived molecules, cholera toxin (CT) and lipopolysaccharide (LPS). We found that CT and LPS differentially regulated the expression of interleukin-12 (IL-12) and CD80-CD86 but not that of IL-1β. LPS and CT each induced IL-1β expression in macrophages, while only LPS induced IL-12 and only CT induced CD80-CD86. These differences were markedly potentiated in gamma interferon (IFN-γ)-treated macrophages, in which LPS potently induced IL-12 and CD80-CD86 expression. In contrast, IFN-γ treatment had no effect on the expression of IL-1β. These results define a molecular basis for the differential pathogenicities of bacterial toxins and are relevant to the design of vaccine adjuvants able to selectively induce desired types of immunity.
Involvement of the Enteroaggregative
Escherichia coli
Plasmid-Encoded Toxin in Causing Human Intestinal Damage
Authors: Henderson Ian R. , Hicks Susan , Navarro-Garcia Fernando , Elias Waldir P. , Philips Alan D. , Nataro James P.
Published: December 2019

Enteroaggregative
Escherichia coli
(EAEC) strains have been shown to adhere to human intestinal tissue in an in vitro organ culture (IVOC) model, and certain strains manifest mucosal toxicity. We have recently described the EAEC plasmid-encoded toxin (Pet), a member of a specific serine protease subclass of the autotransporter proteins. When injected into rat ileal loops, Pet both elicited fluid accumulation and had cytotoxic effects on the mucosa. Furthermore, the Pet protein caused rises in short circuit current from rat jejunal tissue mounted in a Ussing chamber and rounding of intestinal epithelial cells in culture. We therefore hypothesized that the mucosal pathology induced by EAEC strains in the IVOC model was related to expression of the Pet protein. Here, we have examined the effects of EAEC strain 042 and its isogenic
pet
mutant in the IVOC model. 042-infected colonic explants exhibited dilation of crypt openings, increased cell rounding, development of prominent intercrypt crevices, and absence of apical mucus plugs. Colonic tissue incubated with the
pet
mutant exhibited significantly fewer mucosal abnormalities both subjectively and as quantitated morphometrically by measurement of crypt aperture diameter. Mucosal effects were restored upon complementation of the
pet
mutation in
trans
. Interestingly, we found that the ability of 042 to damage T84 cells was not dependent upon Pet. The data suggest that the Pet toxin is active on the human intestinal mucosa but that EAEC may have other mechanisms of eliciting mucosal damage.
Examination of Early Interactions between
Haemophilus ducreyi
and Host Cells by Using Cocultured HaCaT Keratinocytes and Foreskin Fibroblasts
Authors: Zaretzky Franca R. , Kawula Thomas H.
Published: December 2019

Haemophilus ducreyi
is the etiologic agent of chancroid, a sexually transmitted genital ulcer disease. Keratinocytes are likely the first cell type encountered by
H. ducreyi
upon infection of human skin; thus, the interaction between
H. ducreyi
and keratinocytes is probably important for the ability of
H. ducreyi
to establish infection. We have used the HaCaT keratinocyte cell line grown in monolayers and in cocultures with HS27 fibroblasts to investigate
H. ducreyi
interactions with keratinocytes and the host-cell response to
H. ducreyi
infection. Using quantitative adherence and gentamicin protection assays, we determined that approximately 13% of
H. ducreyi
adhered to HaCaT cell monolayers, while only a small proportion (0.0052%) was intracellular. By transmission electron microscopy, we observed numerous
H. ducreyi
organisms adherent to but rarely within HaCaT cells cocultured with fibroblasts. Both live
H. ducreyi
and purified
H. ducreyi
lipooligosaccharide (LOS) induced significant interleukin 8 (IL-8) expression from HaCaT cell-HS27 cell cocultures. However, the level of IL-8 expression in response to LOS alone was not as pronounced.
H. ducreyi
LOS was a more potent inducer of IL-8 from cocultures than
Escherichia coli
lipopolysaccharide (LPS) at the same concentration, suggesting a unique effect of
H. ducreyi
LOS on cocultures. Neither live
H. ducreyi
nor purified
H. ducreyi
LOS or
E. coli
LPS induced tumor necrosis factor alpha expression from cocultures.
H. ducreyi
induced drastically different cytokine profiles from cocultures than from HS27 or HaCaT cells cultured separately. IL-8 expression by skin cells in response to
H. ducreyi
infection in vivo may be responsible for the massive influx of polymorphonuclear leukocytes and other inflammatory cells to the site of infection. This influx of inflammatory cells may be partly responsible for the tissue destruction characteristic of chancroid.
Functional Characterization of a Serine/Threonine Protein Kinase of
Pseudomonas aeruginosa
Authors: Motley S. Timothy , Lory Stephen
Published: December 2019

Protein kinases play a key role in signal transduction pathways in both eukaryotic and prokaryotic cells. Using in vivo expression technology, we have identified several promoters in
Pseudomonas aeruginosa
which are preferentially activated during infection of neutropenic mice. One of these promoters directs the transcription of a gene encoding a putative protein kinase similar to the enzymes found in eukaryotic cells. The full characterization of this protein, termed PpkA, is presented in this communication. The
ppkA
gene encodes a 1,032-amino-acid polypeptide with an N-terminal catalytic domain showing all of the conserved residues of protein kinases with the substrate phosphorylation specificities for serine and threonine residues. The catalytic domain is linked to the rest of the protein by a short proline-rich segment. The enzymes showed anomalous migration behavior when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which could be attributed to autophosphorylation activity. The full-length enzyme was expressed as an oligohistidine fusion protein and was shown to phosphorylate several artificial protein substrates. Both autophosphorylation and phosphorylation of added substrates were strongly reduced by a single-amino-acid substitution in the catalytic domain of PpkA. Although PpkA appears to be differentially phosphorylated by autocatalysis, the levels of phosphorylation have minimal effect on its overall enzymatic activity. Our results, therefore, indicate the operation of a novel protein phosphorylation mechanism during transduction of signals in
P. aeruginosa
, and this pathway may be important in regulating the expression of virulence factors by this pathogen during certain phases of infection.
Conservation and Accessibility of an Inner Core Lipopolysaccharide Epitope ofNeisseria meningitidis
Authors: Plested Joyce S. , Makepeace Katherine , Jennings Michael P. , Gidney Margaret Anne J. , Lacelle Suzanne , Brisson J.-R. , Cox Andrew D. , Martin Adele , Bird A. Graham , Tang Christoph M. , Mackinnon Fiona M. , Richards James C. , Moxon E. Richard
Published: December 2019

We investigated the conservation and antibody accessibility of inner core epitopes ofNeisseria meningitidislipopolysaccharide (LPS) because of their potential as vaccine candidates. An immunoglobulin G3 murine monoclonal antibody (MAb), designated MAb B5, was obtained by immunizing mice with agalEmutant ofN. meningitidisH44/76 (B.15.P1.7,16 immunotype L3). We have shown that MAb B5 can bind to the core LPS of wild-type encapsulated MC58 (B.15.P1.7,16 immunotype L3) organisms in vitro and ex vivo. An inner core structure recognized by MAb B5 is conserved and accessible in 26 of 34 (76%) of group B and 78 of 112 (70%) of groups A, C, W, X, Y, and Z strains.N. meningitidisstrains which possess this epitope are immunotypes in which phosphoethanolamine (PEtn) is linked to the 3-position of the β-chain heptose (HepII) of the inner core. In contrast,N. meningitidisstrains lacking reactivity with MAb B5 have an alternative core structure in which PEtn is linked to an exocyclic position (i.e., position 6 or 7) of HepII (immunotypes L2, L4, and L6) or is absent (immunotype L5). We conclude that MAb B5 defines one or more of the major inner core glycoforms ofN. meningitidisLPS. These findings support the possibility that immunogens capable of eliciting functional antibodies specific to inner core structures could be the basis of a vaccine against invasive infections caused byN. meningitidis.
Complete DNA Sequence and Structural Analysis of the Enteropathogenic
Escherichia coli
Adherence Factor Plasmid
Authors: Tobe Toru , Hayashi Tetsuya , Han Chang-Gyun , Schoolnik Gary K. , Ohtsubo Eiichi , Sasakawa Chihiro
Published: December 2019

The complete nucleotide sequence and organization of the enteropathogenic
Escherichia coli
(EPEC) adherence factor (EAF) plasmid of EPEC strain B171 (O111:NM) were determined. The EAF plasmid encodes two known virulence-related operons, the
bfp
operon, which is composed of genes necessary for biosynthesis of bundle-forming pili, and the
bfpTVW
(
perABC
) operon, composed of regulatory genes required for
bfp
transcription and also for transcriptional activation of the
eae
gene in the LEE pathogenicity island on the EPEC chromosome. The 69-kb EAF plasmid, henceforth designated pB171, contains, besides the
bfp
and
bfpTVW
(
perABC
) operons, potential virulence-associated genes, plasmid replication and maintenance genes, and many insertion sequence elements. Of the newly identified open reading frames (ORFs), two which comprise a single operon had the potential to encode proteins with high similarity to a C-terminal region of ToxB whose coding sequence is located on pO157, a large plasmid harbored by enterohemorrhagic
E. coli
. Another ORF, located between the
bfp
and
bfpTVW
operons, showed high similarity with
trcA
, a
bfpT
-regulated chaperone-like protein gene of EPEC. Two sites were found to be putative replication regions: one similar to RepFIIA of p307 or F, and the other similar to RepFIB of R100 (NR1). In addition, we identified a third region that contains plasmid maintenance genes. Insertion elements were scattered throughout the plasmid, indicating the mosaic nature of the EAF plasmid and suggesting evolutionary events by which virulence genes may have been obtained.
Divergent Roles of Tumor Necrosis Factor and Platelet-Activating Factor in Endotoxin-Induced Release of Monocyte Chemoattractant Protein 1 and Macrophage Inflammatory Protein 1β in Chimpanzees
Authors: Dekkers Pascale E. P. , Levi Marcel , van Deventer Sander J. H. , van der Poll Tom
Published: December 2019

A platelet-activating factor receptor antagonist reduced the release of macrophage inflammatory protein 1β (MIP-1β) during endotoxemia in chimpanzees but did not influence the secretion of monocyte chemoattractant protein 1 (MCP-1). Anti-tumor necrosis factor alpha monoclonal antibody completely prevented MCP-1 release and simultaneously enhanced the secretion of MIP-1β. Levels of MIP-1β and MCP-1 release were differentially regulated during endotoxemia.
Treatment with Succinic Anhydride Improves the Immunogenicity ofShigella flexneriType 2a O-Specific Polysaccharide–Protein Conjugates in Mice
Authors: Pavliakova Danka , Chu Chiayung , Bystricky Slavomir , Tolson Nathaniel W. , Shiloach Joseph , Kaufman Jeanne B. , Bryla Dolores A. , Robbins John B. , Schneerson Rachel
Published: December 2019

Seroepidemiological data and a clinical trial with aShigella sonneiO-specific polysaccharide (O-SP)–Pseudomonas aeruginosarecombinant exoprotein A (rEPA) conjugate provide evidence that a critical level of immunoglobulin G (IgG) lipopolysaccharide (LPS) antibodies in serum confers protection against shigellosis. We evaluated the immunogenicity of conjugates whose carrier proteins and O-SPs were treated with succinic anhydride (SA), which reacts with amino groups at neutral pH to form amide-linked carboxyls (succinylation). Conjugates were synthesized with either of two genetically inactivated medically useful toxins, the diphtheria protein CRM9orrEPA, bound to the O-SP ofShigella flexneritype 2a. Conjugates composed of the succinylated protein, succinylated O-SP, or both succinylated components were administered to mice by a clinically relevant scheme, and their levels of serum IgG anti-LPS and anti-proteins were assayed 7 days after the second and third injections. CRM9served as a more immunogenic carrier thanrEPA. Conjugates composed of succinylated components were more immunogenic than the conjugates composed of the native components. SA treatment of both the carrier protein and the O-SP did not confer an advantage over the succinylated protein alone. Conjugates prepared with native proteins, in general, elicited slightly higher levels of IgG protein antibodies than conjugates composed of the SA-treated proteins.
Pseudomonas aeruginosaInduces Type-III-Secretion-Mediated Apoptosis of Macrophages and Epithelial Cells
Authors: Hauser Alan R. , Engel Joanne N.
Published: December 2019

Pseudomonas aeruginosais a gram-negative opportunistic pathogen that is cytotoxic towards a variety of eukaryotic cells. To investigate the effect of this bacterium on macrophages, we infected J774A.1 cells and primary bone-marrow-derived murine macrophages with theP. aeruginosastrain PA103 in vitro. PA103 caused type-III-secretion-dependent killing of macrophages within 2 h of infection. Only a portion of the killing required the putative cytotoxin ExoU. By three criteria, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling assays, cytoplasmic nucleosome assays, and Hoechst staining, the ExoU-independent but type-III-secretion-dependent killing exhibited features of apoptosis. Extracellular bacteria were capable of inducing apoptosis, and some laboratory and clinical isolates ofP. aeruginosainduced significantly higher levels of this form of cell death than others. Interestingly, HeLa cells but not Madin-Darby canine kidney cells were susceptible to type-III-secretion-mediated apoptosis under the conditions of these assays. These findings are consistent with a model in which theP. aeruginosatype III secretion system transports at least two factors that kill macrophages: ExoU, which causes necrosis, and a second, as yet unidentified, effector protein, which induces apoptosis. Such killing may contribute to the ability of this organism to persist and disseminate within infected patients.
Dichotomy of Cytokine Profiles in Patients and High-Risk Healthy Subjects Exposed to Tuberculosis
Authors: Bhattacharyya S. , Singla R. , Dey A. B. , Prasad H. K.
Published: December 2019

To better understand the role of cytokines in susceptible and resistant subjects exposed toMycobacterium tuberculosisinfection, intracellular gamma interferon (IFN-γ) and interleukin-4 (IL-4) in ex vivo peripheral blood-derived CD4+T cells were examined by flow cytometry. Of the 37 individuals examined, 20 had clinical evidence of pulmonary tuberculosis and showed acid-fast bacilli in the sputum. Other individuals in close contact with these patients showed no evidence of disease. Patients had a higher number of CD4+T cells expressing IFN-γ and IL-4 in unstimulated cultures compared to healthy subjects. Despite this, the ratio of IFN-γ+to IL-4+CD4+T cells was similar in both groups. The Th1 response seen in CD4+T cells in patients was also observed in the overall pattern of IFN-γ and IL-4 detected in control culture supernatants by enzyme-linked immunosorbent assay (ELISA). However, after in vitro stimulation of PBMC with heat-killedM. tuberculosisthere was a significant reduction in the percentage of IFN-γ+CD4+T cells (P< 0.001) in patients. This trend was reflected in the IFN-γ ELISA assay with supernatants derived from stimulated cultures. However, the accumulated levels of IFN-γ were higher than those for IL-4. The reduction of IFN-γ+CD4+T cells resulted in the dominance of IL-4+CD4+T cells in 13 patients (P< 0.05). The elevated levels of IL-4+CD4+T cells seen in patients may contribute to the downregulation of IFN-γ expression and the crucial effector function of CD4 T cells, leading to the persistence of disease and the immunopathology characteristically seen in patients. Preliminary data on the indicators of apoptosis in antigen-stimulated cultures in PBMC derived from patients are presented. Of the 17 high-risk healthy individuals examined, 11 differed in that, after mycobacterial-antigen stimulation, there was an enhancement in IFN-γ+CD4+T cells.
Identification of Promiscuous Epitopes from the Mycobacterial 65-Kilodalton Heat Shock Protein Recognized by Human CD4+T Cells of theMycobacterium lepraeMemory Repertoire
Authors: Mustafa Abu S. , Lundin Knut E. A. , Meloen Robert H. , Shinnick Thomas M. , Oftung Fredrik
Published: December 2019

By using a synthetic peptide approach, we mapped epitopes from the mycobacterial 65-kDa heat shock protein (HSP65) recognized by human T cells belonging to theMycobacterium lepraememory repertoire. A panel of HSP65 reactive CD4+T-cell lines and clones were established from healthy donors 8 years after immunization with heat-killedM. lepraeand then tested for proliferative reactivity against overlapping peptides comprising both theM. lepraeandMycobacterium tuberculosisHSP65 sequences. The results showed that the antigen-specific T-cell lines and clones established responded to 12 mycobacterial HSP65 peptides, of which 9 peptides represented epitopes crossreactive between theM. tuberculosisandM. lepraeHSP65 (amino acids [aa] 61 to 75, 141 to 155, 151 to 165, 331 to 345, 371 to 385, 411 to 425, 431 to 445, 441 to 455, and 501 to 515) and 3 peptides (aa 343 to 355, 417 to 429, and 522 to 534) representedM. lepraeHSP65-specific epitopes. Major histocompatibility complex restriction analysis showed that presentation of 9 of the 12 peptides to T cells were restricted by one of the 2 HLA-DR molecules expressed from selfHLA-DRB1genes, whereas 3 peptides with sequences completely identical between theM. lepraeandM. tuberculosisHSP65 were presented to T cells by multiple HLA-DR molecules: peptide (aa 61 to 75) was presented by HLA-DR1, -DR2, and -DR7, peptide (aa 141 to 155) was presented by HLA-DR2, -DR7, and -DR53, whereas both HLA-DR2 and -DR4 (Dw4 and Dw14) were able to present peptide (aa 501 to 515) to T cells. In addition, the T-cell lines responding to these peptides in proliferation assays showed cytotoxic activity against autologous monocytes/macrophages pulsed with the same HSP65 peptides. In conclusion, we demonstrated that promiscuous peptide epitopes from the mycobacterial HSP65 antigen can serve as targets for cytotoxic CD4+T cells which belong to the human memory T-cell repertoire againstM. leprae. The results suggest that such epitopes might be used in the peptide-based design of subunit vaccines against mycobacterial diseases.
Salmonella typhimurium
Virulence Genes Are Induced upon Bacterial Invasion into Phagocytic and Nonphagocytic Cells
Authors: Pfeifer Cheryl G. , Marcus Sandra L. , Steele-Mortimer Olivia , Knodler Leigh A. , Finlay B. Brett
Published: December 2019

Survival and growth of salmonellae within host cells are important aspects of bacterial virulence. We have developed an assay to identify
Salmonella typhimurium
genes that are induced inside
Salmonella
-containing vacuoles within macrophage and epithelial cells. A promoterless luciferase gene cassette was inserted randomly into the
Salmonella
chromosome, and the resulting mutants were screened for genes upregulated in intracellular bacteria compared to extracellular bacteria. We identified four genes in
S. typhimurium
that were upregulated upon bacterial invasion of both phagocytic and nonphagocytic cells. Expression of these genes was not induced by factors secreted by host cells or media alone. All four genes were induced at early time points (2 to 4 h) postinvasion and continued to be upregulated within host cells at later times (5 to 7 h). One mutant contained an insertion in the
ssaR
gene, within
Salmonella
pathogenicity island 2 (SPI-2), which abolished bacterial virulence in a murine typhoid model. Two other mutants contained insertions within SPI-5, one in the
sopB/sigD
gene and the other in a downstream gene,
pipB
. The insertions within SPI-5 resulted in the attenuation of
S. typhimurium
in the mouse model. The fourth mutant contained an insertion within a previously undescribed region of the
S. typhimurium
chromosome,
iicA
(induced intracellularly A). We detected no effect on virulence as a result of this insertion. In conclusion, all but one of the genes identified in this study were virulence factors within pathogenicity islands, illustrating the requirement for specific gene expression inside mammalian cells and indicating the key role that virulence factor regulation plays in
Salmonella
pathogenesis.