bluuebunny/crossref_metadata_2025 huggingface dataset:
Growth of Virulent and AvirulentMycobacterium tuberculosisStrains in Human Macrophages
Authors: Zhang Ming , Gong Jianhua , Lin Yuanguang , Barnes Peter F.
Published: December 2019

Mycobacterium tuberculosisH37Rv causes progressive disease in animals, whereas the H37Ra strain does not. The relevance of this difference in virulence to human infection is uncertain because these strains have been shown to have similar growth rates in human macrophages. To evaluate the intracellular growth ofM. tuberculosisstrains in macrophages under conditions similar to those encountered in vivo, we infected human monocyte-derived macrophages with H37Ra, H37Rv, or one of four isolates from tuberculosis patients at a low bacillus-to-macrophage ratio. H37Rv and the patient isolates grew significantly faster than H37Ra, based on the numbers of CFU and acid-fast bacilli. These findings did not result from extracellular mycobacterial growth, differential macrophage viability, or bacillary clumping. In contrast to other published results, these findings indicate that the virulence characteristics ofM. tuberculosisstrains in animal models are relevant to human tuberculosis infection.
Granulocyte-Macrophage Colony-Stimulating Factor inStaphylococcus aureus-Induced Arthritis
Authors: Verdrengh Margareta , Tarkowski Andrej
Published: December 2019

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine that is able to increase not only the production of phagocytic cells but also their efficacy with respect to, e.g., bactericidal properties. In this study, we wanted to analyze the impact of GM-CSF on experimentalStaphylococcus aureus-induced arthritis. For that purpose, mice were administered GM-CSF before and after bacterial inoculation. Although there was an increase in the total number of leukocytes as well as in the granulocyte fraction, there was no favorable effect on the severity of arthritis or on survival rates. There were no obvious differences between the GM-CSF-pretreated animals and controls with regard to growth of staphylococci in joints and kidneys 4 days after the bacterial inoculation. In contrast, mice that had been pretreated with GM-CSF prior to bacterial inoculation showed approximately four times lower numbers of bacteria in their blood 24 h later. These results, along with those of our previous studies, suggest that on the one hand the granulocyte is the main protective cell during the course ofS. aureusinfection but that on the other hand, upregulation of granulocyte-macrophage production will not exert any additional protective effects with respect to tissue injury.
M Protein of the Group AStreptococcusBinds to the Seventh Short Consensus Repeat of Human Complement Factor H
Authors: Blackmore Timothy K. , Fischetti Vincent A. , Sadlon Tania A. , Ward Helena M. , Gordon David L.
Published: December 2019

Streptococcus pyogenesevades complement by binding the complement-regulatory protein factor H (fH) via the central conserved C-repeat region of M protein. However, the corresponding binding region within fH has not previously been precisely localized. fH is composed of 20 conserved modules called short consensus repeats (SCRs), each of which contains approximately 60 amino acids. A series of fH truncated and deletion mutants were prepared, and their interaction with M6 protein was examined. The M protein binding site was initially localized to SCRs 6 to 15 as demonstrated by ligand dot blotting, chemical cross-linking, and enzyme-linked immunosorbent assay. SCR 7 was then shown to contain the M protein binding site, as a construct consisting of the first seven SCRs bound M protein but a construct containing the first six SCRs did not bind. In addition, deletion of SCR 7 from full-length fH abolished binding to M protein. SCR 7 is known to contain a heparin binding domain, and binding of fH to M6 protein was almost totally inhibited in the presence of 400 U of heparin per ml. These results localize the M6 protein binding site of fH to SCR 7 and indicate that it is in close proximity to the heparin binding site.
Signal Transduction Pathways Involved in EnterohemorrhagicEscherichia coli-Induced Alterations in T84 Epithelial Permeability
Authors: Philpott Dana J. , McKay Derek M. , Mak Walter , Perdue Mary H. , Sherman Philip M.
Published: December 2019

EnterohemorrhagicEscherichia coli(EHEC) infection is associated with watery diarrhea and can lead to complications, including hemorrhagic colitis and the hemolytic-uremic syndrome. The mechanisms by which these organisms produce diarrheal disease remain to be elucidated. Changes in T84 epithelial cell electrophysiology were examined following EHEC infection. T84 cell monolayers infected with EHEC O157:H7 displayed a time-dependent decrease in transepithelial resistance. Increases in the transepithelial flux of both [3H]mannitol and51Cr-EDTA accompanied the EHEC-induced decreases in T84 resistance. Altered barrier function induced by EHEC occurred at the level of the tight junction since immunofluorescent staining of the tight-junction-associated protein ZO-1 was disrupted when examined by confocal microscopy. Decreased resistance induced by EHEC involved a protein kinase C (PKC)-dependent pathway as the highly specific PKC inhibitor, CGP41251, abrogated the EHEC-induced drop in resistance. PKC activity was also increased in T84 cells infected with EHEC. Calmodulin and myosin light chain kinase played a role in EHEC-induced resistance changes as inhibition of these effector molecules partially reversed the effects of EHEC on barrier function. These studies demonstrate that intracellular signal transduction pathways activated following EHEC infection link the increases in T84 epithelial permeability induced by this pathogen.
Protective Effects of a Human 18-Kilodalton Cationic Antimicrobial Protein (CAP18)-Derived Peptide against Murine Endotoxemia
Authors: Kirikae Teruo , Hirata Michimasa , Yamasu Hiromi , Kirikae Fumiko , Tamura Hiroshi , Kayama Fumio , Nakatsuka Keisuke , Yokochi Takashi , Nakano Masayasu
Published: December 2019

CAP18 (an 18-kDa cationic antimicrobial protein) is a granulocyte-derived protein that can bind lipopolysaccharide (LPS) and inhibit various activities of LPS in vitro. The present study examined the protective effect of a synthetic 27-amino-acid peptide (CAP18109–135) from the LPS-binding domain of CAP18 against antibiotic-induced endotoxin shock, using highly LPS-sensitived-(+)-galactosamine (d-GalN)-sensitized C3H/HeN mice. The antibiotic-induced endotoxin (CAZ-endotoxin) was prepared from the culture filtrate ofPseudomonas aeruginosaPAO1 exposed to ceftazidime (CAZ). Injection of CAP18109–135protected the mice injected with LPS or CAZ-endotoxin from death and lowered their tumor necrosis factor (TNF) levels in serum in a dose-dependent manner. Treatment with CAZ caused death of thed-GalN-sensitizedP. aeruginosaPAO-infected mice within 48 h, while injection with CAP18109–135rescued the mice from death. In the mice rescued from death by injection with CAP18109–135, endotoxin levels in plasma and TNF production by liver tissues were decreased but the numbers of viable infecting bacteria in their blood were not decreased significantly and remained at the levels in CAZ-treated mice. These results indicate that CAP18109–135is capable of preventing antibiotic-induced endotoxic shock in mice with septicemia and that the effect is due to its LPS-neutralizing activity rather than to its antibacterial activity.
Identification of
Salmonella typhimurium
Genes Required for Colonization of the Chicken Alimentary Tract and for Virulence in Newly Hatched Chicks
Authors: Turner Arthur K. , Lovell Margaret A. , Hulme Scott D. , Zhang-Barber Li , Barrow Paul A.
Published: December 2019

From a collection of 2,800 Tn
5
-TC1 transposon mutants of
Salmonella typhimurium
F98, 18 that showed reduced intestinal colonization of 3-week-old chicks were identified. The sites of transposon insertion were determined for most of the mutants and included insertions in the lipopolysaccharide biosynthesis genes
rfaK
,
rfaY
,
rfbK
, and
rfbB
and the genes
dksA
,
clpB
,
hupA
, and
sipC
. In addition, identification was made of an insertion into a novel gene that encodes a protein showing similarity to the IIC component of the mannose class of phosphoenolpyruvate-carbohydrate phosphotransferase systems, which we putatively called
ptsC
. Transduction of most of the transposon mutations to a fresh
S. typhimurium
F98 genetic background and construction of defined mutations in the
rfbK
,
dksA
,
hupA
,
sipC
, and
ptsC
genes of
S. typhimurium
F98 supported the role in colonization of all but the
pts
locus. The virulence of the
rfbK
,
dksA
,
hupA
,
sipC
, and
ptsC
defined mutants and
clpB
and
rfaY
transductants in 1-day-old chicks was tested. All but the
ptsC
and
rfaY
mutants were attenuated for virulence. A number of other phenotypes associated with some of the mutations are described.
Immunohistological Localization of the MrkD Adhesin in the Type 3 Fimbriae of
Klebsiella pneumoniae
Authors: Tarkkanen Ann-Mari , Westerlund-Wikström Benita , Erkkilä Leena , Korhonen Timo K.
Published: December 2019

The adhesive minor protein MrkD of the type 3 fimbria of
Klebsiella pneumoniae
was expressed and purified from
Escherichia coli
as a fusion protein with an N-terminal polyhistidine tail. Polyclonal antibodies raised against MrkD specifically recognized the MrkD peptide in Western blots of fimbrial preparations. Immunoelectron microscopic analyses showed that the anti-MrkD immunoglobulins bound to the tip of the plasmid-encoded variant of the type 3 fimbria of
K. pneumoniae
, whereas no binding to the chromosomally encoded MrkD-deficient type 3 fimbrial variant of
K. pneumoniae
was detected. Immunoglobulins from an antiserum raised against purified type 3 fimbrial filaments bound laterally to both type 3 fimbrial variants. The anti-MrkD antibodies also bound to the tip of a
papG
deletion derivative of the
E. coli
P fimbria complemented with
mrkD
, indicating that MrkD structurally complements a PapG mutation in the P fimbria of
E. coli.
Biophysical Characterization of the Stability of the 150-Kilodalton Botulinum Toxin, the Nontoxic Component, and the 900-Kilodalton Botulinum Toxin Complex Species
Authors: Chen Flora , Kuziemko Geoffrey M. , Stevens Raymond C.
Published: December 2019

Botulinum neurotoxin serotype A is initially released from the bacterium
Clostridium botulinum
as a stable 900-kDa complex. The serotype A 900-kDa complex is one of the forms of the toxin being used as a therapeutic agent for the treatment of various neuromuscular disorders. Previous experiments have demonstrated that the 900-kDa complex form of the toxin protects the toxin from the harsh conditions of the gastrointestinal tract. To provide molecular level details of the stability and equilibrium of the 900-kDa complex, the nontoxic component, and the toxic (botulinum neurotoxin) component, the three species have been investigated with a series of biophysical techniques at the molecular level (dynamic light scattering, proteolysis, circular dichroism, pH incubations, and agglutination assays). These experiments were conducted under harsh conditions which mimic those found along the gastrointestinal tract. Separately, exposure to denaturing and proteolytic conditions degrades both the botulinum neurotoxin and the nontoxic component. In the 900-kDa complex, the botulinum neurotoxin is protected during exposure to the gastrointestinal environment and the nontoxic component is slightly modified. Surprisingly, the toxin protects the ability of the nontoxic component to agglutinate erythrocytes. Contrary to previous reports, the purified 900-kDa complex did not have agglutination ability until after exposure to the proteolytic conditions. These experiments provide new evidence and detail for the theory that the nontoxic component and the toxic component protect one another during exposure to harsh conditions, and a molecular model is presented for the passage of the toxin through the gastrointestinal tract.
Borrelia burgdorferiEscape Mutants That Survive in the Presence of Antiserum to the OspA Vaccine Are Killed When Complement Is Also Present
Authors: Solé Mónica , Bantar Carlos , Indest Karl , Gu Yan , Ramamoorthy Ramesh , Coughlin Richard , Philipp Mario T.
Published: December 2019

As an initial attempt to investigate the possible role of outer surface protein A (OspA) escape mutants ofBorrelia burgdorferiin decreasing the efficacy of the OspA vaccine, mutants of the HB19 strain ofB. burgdorferisensu stricto were selected in vitro from an uncloned, low-passage-number isolate. The antiserum used for selection was obtained from rhesus monkeys that had been given a vaccine of the same formulation and dose, and by the same route of administration, as that given to humans in several trials. All of the mutants selected in liquid medium and subsequently cloned twice in solid medium expressed a single abundant protein of 28 to 34 kDa instead of both OspA and OspB. Depending on the mutant, this protein reacted strongly, weakly, or not detectably with the anti-OspA antibody used for selection. Analysis of theospABlocus of each of four representatives from these three groups of mutants by PCR with oligonucleotide primers that hybridize to flanking regions of theospABoperon, and of the corresponding phenotype with monoclonal antibodies that bind to the amino or carboxyl terminus of the OspA or OspB polypeptide, indicated that in all cases a deletion within the operon had occurred. Spirochetes from the four mutant strains chosen for further analysis could be killed in antibody-dependent, complement-mediated killing assays with the selecting anti-OspA antibody, despite their resistance to killing with this antibody in the absence of complement. Complement-mediated killing occurred at an antibody concentration higher than that required to kill wild-type spirochetes. If anti-OspA antibody acts only within the tick, where complement is probably ineffective due to tick-derived decomplementing factors, then OspA escape mutants, if infectious, could seriously diminish the efficacy of OspA vaccines. On the other hand, if the killing ofB. burgdorferiwith anti-OspA antibody also takes place within the human host, then our results indicate that chimeric/deletion escape mutants will be killed as well.
A Fibrinogen-Binding Protein of
Staphylococcus epidermidis
Authors: Nilsson Martin , Frykberg Lars , Flock Jan-Ingmar , Pei Lei , Lindberg Martin , Guss Bengt
Published: December 2019

The present study reports on fibrinogen (Fg) binding of
Staphylococcus epidermidis
. Adhesion of different
S. epidermidis
strains to immobilized Fg was found to vary significantly between different strains, and the component responsible was found to be proteinaceous in nature. To further characterize the Fg-binding activity, a shotgun phage display library covering the
S. epidermidis
chromosome was constructed. By affinity selection (panning) against immobilized Fg, a phagemid clone, pSEFG1, was isolated, which harbors an insert with an open reading frame of ∼1.7 kilobases. Results from binding and inhibition experiments demonstrated that the insert of pSEFG1 encodes a specific Fg-binding protein. Furthermore, affinity-purified protein encoded by pSEFG1 completely inhibited adhesion of
S. epidermidis
to immobilized Fg. By additional cloning and DNA sequence analyses, the complete gene, termed
fbe
, was found to consist of an open reading frame of 3,276 nucleotides encoding a protein, called Fbe, with a deduced molecular mass of ∼119 kDa. With a second phage display library made from another clinical isolate of
S. epidermidis
, it was possible to localize the Fg-binding region to a 331-amino-acid-long fragment. PCR analysis showed that the
fbe
gene was found in 40 of 43 clinical isolates of
S. epidermidis
. The overall organization of Fbe resembles those of other extracellular surface proteins of staphylococci and streptococci. Sequence comparisons with earlier known proteins revealed that this protein is related to an Fg-binding protein of
Staphylococcus aureus
called clumping factor.
Enhanced Levels of
Staphylococcus aureus
Stress Protein GroEL and DnaK Homologs Early in Infection of Human Epithelial Cells
Authors: Qoronfleh M. Walid , Bortner Carol A. , Schwartzberg Paul , Wilkinson Brian J.
Published: December 2019

Antibodies to
Staphylococcus aureus
heat shock proteins (Hsps) are present in the sera of patients with
S. aureus
endocarditis (M. W. Qoronfleh, W. Weraarchakul, and B. J. Wilkinson, Infect. Immun. 61:1567–1570, 1993). Although these proteins are immunogenic, their role in infection has not been established. We developed a cell culture system as a model to examine the potential involvement of staphylococcal Hsps in the initial events of infection. This study supports a model in which a clinical endocarditis isolate responds to host cell signals by selectively regulating the synthesis of numerous proteins, including the stress proteins Hsp60 (GroEL homolog) and Hsp70 (DnaK homolog) and a unique 58-kDa protein.
Phenotypic Effect of IsogenicuspA1anduspA2Mutations onMoraxella catarrhalis035E
Authors: Aebi Christoph , Lafontaine Eric R. , Cope Leslie D. , Latimer Jo L. , Lumbley Sheryl L. , McCracken George H. , Hansen Eric J.
Published: December 2019

The UspA surface antigen ofMoraxella catarrhaliswas recently shown to be comprised of two different proteins (UspA1 and UspA2) which share an internal region containing 140 amino acids with 93% identity (C. Aebi, I. Maciver, J. L. Latimer, L. D. Cope, M. K. Stevens, S. E. Thomas, G. H. McCracken, Jr., and E. J. Hansen, Infect. Immun. 65:4367–4377, 1997). IsogenicuspA1,uspA2, anduspA1 uspA2mutants were tested in a number of in vitro systems to determine what effect these mutations, either individually or together, might exert on the phenotype ofM. catarrhalis035E. Monoclonal antibodies specific for UspA1 or UspA2 were used in an indirect antibody accessibility assay to prove that both of these proteins were expressed on the surface ofM. catarrhalis. All three mutants grew in vitro at the same rate and did not exhibit autoagglutination or hemagglutination properties that were detectably different from those of the wild-type parent strain. When tested for the ability to adhere to human epithelial cells, the wild-type parent strain and theuspA2mutant readily attached to Chang conjunctival cells. In contrast, theuspA1mutant and theuspA1 uspA2double mutant both attached to these epithelial cells at a level nearly 2 orders of magnitude lower than that obtained with the wild-type parent strain, a result which suggested that expression of UspA1 byM. catarrhalisis essential for attachment to these epithelial cells. Both the wild-type parent strain and theuspA1mutant were resistant to the bactericidal activity of normal human serum, whereas theuspA2mutant and theuspA1 uspA2double mutant were readily killed by this serum. This latter result indicated that the presence of UspA2 is essential for expression of serum resistance byM. catarrhalis.
Immune Responses against Major Outer Membrane Antigens of
Neisseria meningitidis
in Vaccinees and Controls Who Contracted Meningococcal Disease during the Norwegian Serogroup B Protection Trial
Authors: Wedege E. , Høiby E. A. , Rosenqvist E. , Bjune G.
Published: December 2019

Sera from vaccinees and controls who contracted serogroup B meningococcal disease during the blinded and open parts of a two-dose protection trial in Norway were compared for antigen-specific and bactericidal antibodies against vaccine strain 44/76 (B:15:P1.7,16). From 16 of 20 (80%) vaccinees and 26 of 35 (74%) controls, one or more serum samples (
n
= 104) were collected during the acute phase (1 to 4 days), early convalescent phase (5 to 79 days), and late convalescent phase (8 to 31 months) after onset of disease. Binding of immunoglobulin G (IgG) to the major outer membrane antigens (80- and 70-kDa proteins, class 1, 3, and 5 proteins, and lipopolysaccharide [LPS]) on immunoblots was measured by digital image analysis. Specific IgG levels in vaccinees increased from acute to early convalescent phases, followed by a decline, while controls showed a small increase over time. Vaccinees had significantly higher levels than controls against class 1 and 3 porins and LPS in acute sera, against all antigens during early convalescence, and against class 1 and 3 porins in the later sera. Vaccinees who were infected with strains expressing subtype P1.7,16 proteins demonstrated a level of IgG binding to protein P1.7,16 with early-convalescent-phase sera that was fourfold higher than that of those infected with other strains. Bactericidal titers in serum against the vaccine strain were 192-fold higher for vaccinees than those for controls during early convalescence, but similarly low levels were found during late convalescence. A vaccine-induced anamnestic response of specific and functional antibody activities was thus shown, but the decrease in protection over time after vaccination indicated that two vaccine doses did not induce sufficient levels of long-term protective antibodies.
The
inlA
Gene of
Listeria monocytogenes
LO28 Harbors a Nonsense Mutation Resulting in Release of Internalin
Authors: Jonquières Renaud , Bierne Hélène , Mengaud Jérôme , Cossart Pascale
Published: December 2019

Internalin is a surface protein that mediates entry of
Listeria monocytogenes
EGD into epithelial cells expressing the cell adhesion molecule human E-cadherin or its chicken homolog, L-CAM, which act as receptors for internalin. After observing that entry of
L. monocytogenes
LO28 into S180 fibroblasts, in contrast to that of EGD, did not increase after transfection with L-CAM, we examined both the expression and the structure of internalin in strain LO28. We discovered a nonsense mutation in
inlA
which results in a truncated protein released in the culture medium. Mutations leading to release of internalin were also detected in clinical and food isolates. These results question the role of internalin as a virulence factor in murine listeriosis.
Multigene Families Encoding the Major Hemagglutinins in Phylogenetically Distinct Mycoplasmas
Authors: Noormohammadi A. H. , Markham P. F. , Duffy M. F. , Whithear K. G. , Browning G. F.
Published: December 2019

Mycoplasma synoviae
has two major membrane antigens, MSPA and MSPB, both of which are phase variable and which may be coordinately involved in adhesion of the organism to erythrocytes. A single gene (
vlhA
) from
M. synoviae
was characterized, and polypeptides were expressed from nonoverlapping 5′ and 3′ regions in
Escherichia coli
. The expression product of the
vlhA
5′ region reacted with specific reagents against MSPB, while that of the 3′ region reacted with specific reagents against MSPA. Analysis of the predicted amino acid sequence showed a characteristic signal peptidase II cleavage site, and the presence of the acylation site was confirmed by identification of a lipid-associated membrane protein, similar in molecular mass to MSPB, in [
3
H]palmitate-labelled membrane proteins. Further sequence analysis of the
vlhA
gene revealed a high identity with the
Mycoplasma gallisepticum
pMGA1.7 gene, a member of a large translated family. The
vlhA
gene was shown to hybridize to multiple restriction fragments of the
M. synoviae
genome, suggesting that it was also a member of a multigene family. These findings indicate that coordinate phase variation of the two major surface antigens of
M. synoviae
WVU may be due to their expression from the same gene and that homologous gene families encode the major hemagglutinins of two phylogenetically distinct mycoplasmas. The presence of homologous multigene families in such phylogenetically distinct species, but not in the genomes of more closely related species, suggests that the families may have been transferred horizontally.
Interleukin-9 Enhances Resistance to the Intestinal NematodeTrichuris muris
Authors: Faulkner Helen , Renauld J.-C. , Van Snick J. , Grencis R. K.
Published: December 2019

Upon infection with the cecum-dwelling nematodeTrichuris muris, the majority of inbred strains of mice launch a Th2-type immune response and in doing so expel the parasite before patency. In contrast, there are a few mouse strains which develop a nonprotective Th1-type response resulting in a chronic infection and the presence of adult worms. Of the Th2 cytokines known to be associated with the resistant phenotype (interleukin-4 [IL-4], IL-5, IL-9, and IL-13), comparatively little is known about the contribution that IL-9 makes towards the protective immune response. In this study we demonstrate that IL-9 is expressed early during the Th2-type response and that its elevation in vivo results in the enhancement of intestinal mastocytosis and the production of both the immunoglobulin E (IgE) and IgG1 isotypes. In addition, elevated IL-9 levels in vivo facilitated the loss ofT. murisfrom the intestine. That IL-9 is important in promoting worm expulsion was also seen following infection of IL-9-transgenic mice, which constitutively overexpress the cytokine. These animals displayed an extremely rapid, but immune mediated, expulsion of the parasite. Also evident in these animals was a pronounced intestinal mastocytosis, which was previously shown by us to be responsible for the expulsion of the related nematodeTrichinella spiralisfrom these animals. Taken together with observations of IL-9 production following infection with other helminths, the results imply that IL-9 contributes to the general mast cell and IgE response characteristic of these infections and, more specifically, enhances resistance toT. muris.
Immunogenicity of Outer Membrane Proteins in a Lipopolysaccharide-Deficient Mutant ofNeisseria meningitidis: Influence of Adjuvants on the Immune Response
Authors: Steeghs Liana , Kuipers Betsy , Hamstra Hendrik Jan , Kersten Gideon , van Alphen Loek , van der Ley Peter
Published: December 2019

The immunogenicity of outer membrane complexes (OMCs) or heat-inactivated bacteria of a lipopolysaccharide (LPS)-deficient mutant derived from meningococcal strain H44/76 was studied. The immune response in BALB/c mice to the major outer membrane proteins was poor compared to the immune response elicited by wild-type immunogens. However, addition of external H44/76 LPS to mutant OMCs entirely restored the immune response. By using an LPS-deficient mutant, it may be possible to substitute a less toxic compound as adjuvant in meningococcal outer membrane vaccines. Therefore, a broad panel of adjuvants were tested for their potential to enhance the immunogenicity of LPS-deficient OMCs. AlPO4,Rhodobacter sphaeroidesLPS, monophosphoryl lipid A and alkali-hydrolyzed meningococcal LPS showed significantly lower adjuvant activity than did H44/76 LPS. Adjuvant activity similar to H44/76 LPS was found forEscherichia coliLPS, meningococcalicsBandrfaCLPS, QuilA, subfractions of QuilA, and MF59. Good adjuvant activity was also found with meningococcalhtrB1LPS, containing penta-acylated lipid A. Antisera elicited with the less active adjuvants showed relatively high immunoglobulin G1 (IgG1) titers, whereas strong adjuvants also induced high IgG2a and IgG2b responses in addition to IgG1. Antisera with the IgG2a and IgG2b isotypes showed high bactericidal activity, indicating that adjuvants promoting the IgG2a and IgG2b response contribute most to the protective mechanism. Thus, this study demonstrates that the immunogenicity of meningococcal LPS-deficient OMCs can be restored by using less toxic adjuvants, which opens up new avenues for development of vaccines against meningococcal disease.
Identification of theznuA-Encoded Periplasmic Zinc Transport Protein ofHaemophilus ducreyi
Authors: Lewis David A. , Klesney-Tait Julia , Lumbley Sheryl R. , Ward Christine K. , Latimer Jo L. , Ison Catherine A. , Hansen Eric J.
Published: December 2019

TheznuAgene ofHaemophilus ducreyiencodes a 32-kDa (mature) protein that has homology to both the ZnuA protein ofEscherichia coliand the Pzp1 protein ofH. influenzae; both of these latter proteins are members of a growing family of prokaryotic zinc transporters. Inactivation of theH. ducreyi35000znuAgene by insertional mutagenesis resulted in a mutant that grew more slowly than the wild-type parent strain in vitro unless ZnCl2was provided at a final concentration of 100 μM. Other cations tested did not restore growth of thisH. ducreyimutant to wild-type levels. TheH. ducreyiZnuA protein was localized to the periplasm, where it is believed to function as the binding component of a zinc transport system. Complementation of theznuAmutation with the wild-typeH. ducreyi znuAgene provided intransrestored the ability of thisH. ducreyimutant to grow normally in the absence of exogenously added ZnCl2. The wild-typeH. ducreyi znuAgene was also able to complement aH. influenzae pzp1mutation. TheH. ducreyi znuAisogenic mutant exhibited significantly decreased virulence (P= 0.0001) when tested in the temperature-dependent rabbit model for experimental chancroid. This decreased virulence was not observed when theznuAmutant was complemented with the wild-typeH. ducreyi znuAgene provided intrans.
Outer Membrane Proteins as a Carrier for Detoxified Lipooligosaccharide Conjugate Vaccines for NontypeableHaemophilus influenzae
Authors: Wu Ting-Huai , Gu Xin-Xing
Published: December 2019

NontypeableHaemophilus influenzae(NTHi) is a common cause of otitis media and respiratory tract infections. Outer membrane proteins (OMP) and lipooligosaccharide (LOS) are major surface antigens of NTHi and potential vaccine candidates. De-O-acylated LOS (dLOS) or oligosaccharide (OS) was coupled to total OMP to form dLOS-OMP and OS-OMP conjugates, while a dLOS-tetanus toxoid (TT) was synthesized for comparison. These conjugates were evaluated in mice and rabbits for immunogenicity. dLOS-OMP elicited a better boostable antibody response against LOS than did dLOS-TT, while OS-OMP was not immunogenic. Formulation of the conjugates with Ribi adjuvant significantly enhanced the immunogenicity of dLOS-OMP and dLOS-TT but not that of OS-OMP. In addition, rabbit antisera elicited by dLOS-OMP but not dLOS-TT (or OMP alone) demonstrated bactericidal activity against 40% of the NTHi strains tested. These results indicate that dLOS is a better derivative of LOS than OS and that OMP is a good carrier for NTHi LOS-based conjugate vaccines.
Differential T-Cell Recognition of Native and RecombinantMycobacterium tuberculosisGroES
Authors: Rosenkrands Ida , Weldingh Karin , Ravn Pernille , Brandt Lise , Højrup Peter , Rasmussen Peter Birk , Coates Anthony R. , Singh Mahavir , Mascagni Paolo , Andersen Peter
Published: December 2019

Mycobacterium tuberculosisGroES was purified from culture filtrate, and its identity was confirmed by immunoblot analysis and N-terminal sequencing. Comparing the immunological recognition of native and recombinant GroES, we found that whereas native GroES elicited a strong proliferative response and release of gamma interferon-γ by peripheral blood mononuclear cells from healthy tuberculin reactors, the recombinant protein failed to do so. The same difference in immunological recognition was observed in a mouse model of TB infection. Both the native and recombinant preparations were recognized by mice immunized with the recombinant protein. Biochemical characterization including sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two-dimensional electrophoresis, and mass spectrometry analysis of both proteins demonstrated no differences between the native and recombinant forms of GroES except for the eight additional N-terminal amino acids derived from the fusion partner in recombinant GroES. The recombinant fusion protein, still tagged with the maltose binding protein, was recognized by T cells isolated from TB-infected mice if mixed with culture filtrate before affinity purification on an amylose column. The maltose binding protein treated in the same manner as a control preparation was not recognized. Based on the data presented, we suggest that the association of biologically active molecules from culture filtrate with the chaperone GroES may be responsible for the observed T-cell recognition of the native preparation.
Identification of Regions of the Chromosome of
Neisseria meningitidis
and
Neisseria gonorrhoeae
Which Are Specific to the Pathogenic
Neisseria
Species
Authors: Perrin Agnes , Nassif Xavier , Tinsley Colin
Published: December 2019

Neisseria meningitidis
and
Neisseria gonorrhoeae
give rise to dramatically different diseases. Their interactions with the host, however, do share common characteristics: they are both human pathogens which do not survive in the environment and which colonize and invade mucosa at their port of entry. It is therefore likely that they have common properties that might not be found in nonpathogenic bacteria belonging to the same genetically related group, such as
Neisseria lactamica
. Their common properties may be determined by chromosomal regions found only in the pathogenic
Neisseria
species. To address this issue, we used a previously described technique (C. R. Tinsley and X. Nassif, Proc. Natl. Acad. Sci. USA 93:11109–11114, 1996) to identify sequences of DNA specific for pathogenic neisseriae and not found in
N. lactamica
. Sequences present in
N. lactamica
were physically subtracted from the
N. meningitidis
Z2491 sequence and also from the
N. gonorrhoeae
FA1090 sequence. The clones obtained from each subtraction were tested by Southern blotting for their reactivity with the three species, and only those which reacted with both
N. meningitidis
and
N. gonorrhoeae
(i.e., not specific to either one of the pathogens) were further investigated. In a first step, these clones were mapped onto the chromosomes of both
N. meningitidis
and
N. gonorrhoeae
. The majority of the clones were arranged in clusters extending up to 10 kb, suggesting the presence of chromosomal regions common to
N. meningitidis
and
N. gonorrhoeae
which distinguish these pathogens from the commensal
N. lactamica
. The sequences surrounding these clones were determined from the
N. meningitidis
genome-sequencing project. Several clones corresponded to previously described factors required for colonization and survival at the port of entry, such as immunoglobulin A protease and PilC. Others were homologous to virulence-associated proteins in other bacteria, demonstrating that the subtractive clones are capable of pinpointing chromosomal regions shared by
N. meningitidis
and
N. gonorrhoeae
which are involved in common aspects of the host interaction of both pathogens.
Inhibition of Neutrophil Apoptosis by Verotoxin 2 Derived from
Escherichia coli
O157:H7
Authors: Liu Jiajia , Akahoshi Tohru , Sasahana Takeshi , Kitasato Hidero , Namai Rie , Sasaki Takeji , Inoue Matsuhisa , Kondo Hirobumi
Published: December 2019

In order to evaluate the pathological role of verotoxin 2 (VT2), we investigated the effects of VT2 on neutrophil apoptosis in vitro. The results showed that VT2 caused a significant delay in spontaneous neutrophil apoptosis and that the effect was abrogated by a protein kinase C inhibitor. These data indicate that longer survival of neutrophils may aggravate neutrophil-mediated tissue damage in VT2-associated diseases.
Induction of Protective T Cells againstListeria monocytogenesin Mice by Immunization with a Listeriolysin O-Negative Avirulent Strain of Bacteria and Liposome-Encapsulated Listeriolysin O
Authors: Tanabe Yoshinari , Xiong Huabao , Nomura Takamasa , Arakawa Masaaki , Mitsuyama Masao
Published: December 2019

Only listeriolysin O (LLO)-producing strains ofListeria monocytogenesgenerate protective immunity in mice. Based on the findings that endogenous gamma interferon (IFN-γ) production was induced only by such strains and that purified LLO could induce IFN-γ from NK cells, we have postulated that LLO may play a pivotal role in the induction of Th1-type protective T cells, which are highly dependent on IFN-γ. In this study, mice were immunized withL. monocytogenesATCC 15313, an LLO-nonproducing avirulent strain, along with LLO encapsulated in liposome (LLO-liposome). LLO-liposome was highly potent in the induction of various cytokines, including IFN-γ. Immunization of mice with either LLO-liposome or the viable strain ATCC 15313 alone did not induce protection against challenge infection. In contrast, the combination of LLO-nonproducing bacteria plus LLO-liposome induced a significant level of protective immunity mediated mainly by Th1-type cells capable of producing a large amount of IFN-γ in an antigen-specific manner. The protection afforded by the combination was not dependent on LLO-specific cytotoxic T cells. These results support the idea that the inability of an LLO-nonproducing avirulent strain or killed bacteria to induce the generation of protective T cells is due not to the lack of a central T-cell epitope(s) but to the lack of ability to induce the production of endogenous cytokine during the early stage of immunization; the results also suggest that an appropriate use of LLO at least in an animal model may be effective in the induction of antigen-specific Th1-dependent protective immunity to various kinds of intracellular parasitic bacteria.
Urokinase-Type Plasminogen Activator in Inflammatory Cell Recruitment and Host Defense againstPneumocystis cariniiin Mice
Authors: Beck James M. , Preston Angela M. , Gyetko Margaret R.
Published: December 2019

Effective host defense againstPneumocystis cariniidepends upon the integrated actions of inflammatory cells and mediators in the lungs. Using immunocompetent and immunosuppressed mice, our laboratory has defined inflammatory changes in the lungs in response toP. carinii. However, the essential molecules and mechanisms required for cellular recruitment and for host defense againstP. cariniiare undefined. We hypothesized that urokinase-type plasminogen activator (uPA), a protease intimately involved in inflammatory cell migration and activation, is required for clearance ofP. carinii. To test this hypothesis in vivo, we compared the intensity ofP. cariniiinfection and inflammation in the lungs of mice lacking the uPA gene (uPA knockout mice) and in the lungs of wild-type mice. After intratracheal inoculation withP. cariniiorganisms, uPA knockout mice developed uniformly heavyP. cariniipneumonia while wild-type mice cleared theP. cariniiinoculum. Bronchoalveolar lavage fluid from uPA knockout mice contained significantly smaller numbers of cells than did lavage fluid from wild-type mice. We conclude that deletion of the uPA gene prevents the clearance ofP. cariniiand reduces inflammatory cell recruitment. Therefore, uPA is an important participant in the network of inflammatory events required for the clearance ofP. carinii, confirming an important role for this molecule in pulmonary host defense against opportunistic pathogens.
Alpha-Toxin and Gamma-Toxin Jointly PromoteStaphylococcus aureusVirulence in Murine Septic Arthritis
Authors: Nilsson Ing-Marie , Hartford Orla , Foster Timothy , Tarkowski Andrzej
Published: December 2019

Septic arthritis is a common and feared complication of staphylococcal infections.Staphylococcus aureusproduces a number of potential virulence factors including certain adhesins and enterotoxins. In this study we have assessed the roles of cytolytic toxins in the development of septic arthritis by inoculating mice withS. aureuswild-type strain 8325-4 or isogenic mutants differing in the expression of alpha-, beta-, and gamma-toxin production patterns. Mice inoculated with either an alpha- or beta-toxin mutant showed degrees of inflammation, joint damage, and weight decrease similar to wild-type-inoculated mice. In contrast, mice inoculated with either double (alpha- and gamma-toxin-deficient)- or triple (alpha-, beta-, and gamma-toxin-deficient)-mutantS. aureusstrains showed lower frequency and severity of arthritis, measured both clinically and histologically, than mice inoculated with the wild-type strain. We conclude that simultaneous production of alpha- and gamma-toxin is a virulence factor inS. aureusarthritis.
Protective Efficacy of Recombinant
Yersinia
Outer Proteins against Bubonic Plague Caused by Encapsulated and Nonencapsulated
Yersinia pestis
Authors: Andrews G. P. , Strachan S. T. , Benner G. E. , Sample A. K. , Anderson G. W. , Adamovicz J. J. , Welkos S. L. , Pullen J. K. , Friedlander A. M.
Published: December 2019

To evaluate the role of
Yersinia
outer proteins (Yops) in conferring protective immunity against plague, six
yop
loci from
Yersinia pestis
were individually amplified by PCR, cloned, and expressed in
Escherichia coli
. The recombinant proteins were purified and injected into mice. Most Yop-vaccinated animals succumbed to infection with either wild-type encapsulated
Y. pestis
or a virulent, nonencapsulated isogenic variant. Vaccination with YpkA significantly prolonged mean survival time but did not increase overall survival of mice infected with the nonencapsulated strain. The only significant protection against death was observed in YopD-vaccinated mice challenged with the nonencapsulated strain.
Epitope Mapping of Monoclonal Antibodies againstBordetella pertussisAdenylate Cyclase Toxin
Authors: Lee S.-J. , Gray M. C. , Guo L. , Sebo P. , Hewlett E. L.
Published: December 2019

Adenylate cyclase (AC) toxin fromBordetella pertussisis a 177-kDa repeats-in-toxin (RTX) family protein that consists of four principal domains; the catalytic domain, the hydrophobic domain, the glycine/aspartate-rich repeat domain, and the secretion signal domain. Epitope mapping of 12 monoclonal antibodies (MAbs) directed against AC toxin was conducted to identify regions important for the functional activities of this toxin. A previously developed panel of in-frame deletion mutants of AC toxin was used to localize MAb-specific epitopes on the toxin. The epitopes of these 12 MAbs were located throughout the toxin molecule, recognizing all major domains. Two MAbs recognized a single epitope on the distal portion of the catalytic domain, two reacted with the C-terminal 217 amino acids, one bound to the hydrophobic domain, and one bound to either the hydrophobic domain or the functionally unidentified region adjacent to it. The remaining six MAbs recognized the glycine/aspartate-rich repeat region. To localize these six MAbs, different peptides derived from the repeat region were constructed. Two of the six MAbs appeared to react with the repetitive motif and exhibited cross-reactivity withEscherichia colihemolysin. The remaining four MAbs appeared to interact with unique epitopes within the repeat region. To evaluate the roles of these epitopes on toxin function, each MAb was screened for its effect on intoxication (cyclic AMP accumulation) and hemolytic activity. The two MAbs recognizing the distal portion of the catalytic domain blocked intoxication of Jurkat cells by AC toxin but had no effect on hemolysis. On the other hand, a MAb directed against a portion of the repeat region caused partial inhibition of AC toxin-induced hemolysis without affecting intoxication. In addition, the MAb recognizing either the hydrophobic domain or the unidentified region adjacent to it inhibited both intoxication and hemolytic activity of AC toxin. These findings extend our understanding of the regions necessary for the complex events required for the biological activities of AC toxin and provide a set of reagents for further study of this novel virulence factor.
Yersinia pseudotuberculosis-Induced Calcium Signaling in Neutrophils Is Blocked by the Virulence Effector YopH
Authors: Andersson Kerstin , Magnusson Karl-Eric , Majeed Meytham , Stendahl Olle , Fällman Maria
Published: December 2019

Pathogenic species of the genusYersiniaevade the bactericidal functions of phagocytes. This evasion is mediated through their virulence effectors, Yops, which act within target cells. In this study we investigated the effect ofYersinia pseudotuberculosison Ca2+signaling in polymorphonuclear neutrophils. The intracellular free calcium concentration in single adherent human neutrophils was monitored during bacterial infection and, in parallel, the encounter between the bacteria and cells was observed. When a plasmid-cured strain was used for infection, adherence of a single bacterium to the cellular surface induced a β1integrin-dependent transient increase in the intracellular concentration of free calcium. This was, however, not seen with Yop-expressing wild-type bacteria, which adhered to the cell surface without generating any Ca2+signal. Importantly, the overall Ca2+homeostasis was not affected by the wild-type strain; the Ca2+signal mediated by the G-protein-coupled formyl-methionyl-leucyl-phenylalanine receptor was still functioning. Hence, the blocking effect was restricted to certain receptors and their signaling pathways. The use of different Yop mutant strains revealed that the protein tyrosine phosphatase YopH was responsible for the inhibition. This virulence determinant has previously been implicated in very rapidYersinia-mediated effects on target cells as the key effector in the blockage of phagocytic uptake. The present finding, thatY. pseudotuberculosis, via YopH, specifically inhibits a self-induced immediate-early Ca2+signal in neutrophils, offers more-detailed information concerning the effectiveness of this virulence effector and implies an effect on Ca2+-dependent, downstream signals.
Molecular Characterization of a
Brucella
Species Large DNA Fragment Deleted in
Brucella abortus
Strains: Evidence for a Locus Involved in the Synthesis of a Polysaccharide
Authors: Vizcaíno Nieves , Cloeckaert Axel , Zygmunt Michel S. , Fernández-Lago Luis
Published: December 2019

A
Brucella melitensis
16M DNA fragment of 17,119 bp, which contains a large region deleted in
B. abortus
strains and DNA flanking one side of the deletion, has been characterized. In addition to the previously identified
omp31
gene, 14 hypothetical genes have been identified in the
B. melitensis
fragment, most of them showing homology to genes involved in the synthesis of a polysaccharide. Considering that 10 of the 15 genes are missing in
B. abortus
and that all the polysaccharides described in the
Brucella
genus (lipopolysaccharide, native hapten, and polysaccharide B) have been detected in all the species, it seems likely that the genes described here might be part of a cluster for the synthesis of a novel
Brucella
polysaccharide. Several polysaccharides have been identified as important virulence factors, and the discovery of a novel polysaccharide in the brucellae which is probably not synthesized in
B. abortus
might be interesting for a better understanding of the pathogenicity and host preference differences observed between the
Brucella
species. However, the possibility that the genes described in this paper no longer encode the synthesis of a polysaccharide cannot be excluded. Brucellae belong to the alpha-2 subdivision of the class
Proteobacteria
, which includes other microorganisms living in association with eucaryotic cells, some of them synthesizing extracellular polysaccharides involved in the interaction with the host cell. The genes described in this paper might be a remnant of the common ancestor of the alpha-2 subdivision of the class
Proteobacteria
, and the brucellae might have lost such extracellular polysaccharide during evolution if it was not necessary for survival or for establishment of the infectious process. Nevertheless, further studies are necessary to identify the entire DNA fragment missing in
B. abortus
strains and to elucidate the mechanism responsible for such deletion, since only 9,948 bp of the deletion was present in the sequenced
B. melitensis
DNA fragment.