bluuebunny/crossref_metadata_2025 huggingface dataset:
Bordetella avium
Virulence Measured In Vivo and In Vitro
Authors: Temple Louise M. , Weiss Alison A. , Walker Kimberly E. , Barnes H. John , Christensen Vern L. , Miyamoto David M. , Shelton Celia B. , Orndorff Paul E.
Published: December 2019

Bordetella avium
causes an upper-respiratory-tract disease called bordetellosis in birds. Bordetellosis shares many of the clinical and histopathological features of disease caused in mammals by
Bordetella pertussis
and
Bordetella bronchiseptica
. In this study we determined several parameters of infection in the domestic turkey,
Meleagris galapavo
, and compared these in vivo findings with an in vitro measure of adherence using turkey tracheal rings. In the in vivo experiments, we determined the effects of age, group size, infection duration, and interindividual spread of
B. avium
. Also, the effect of host genetic background on susceptibility was tested in the five major commercial turkey lines by infecting each with the parental
B. avium
strain and three
B. avium
insertion mutants. The mutant strains lacked either motility, the ability to agglutinate guinea pig erythrocytes, or the ability to produce dermonecrotic toxin. The susceptibilities of 1-day-old and 1-week-old turkeys to
B. avium
were the same, and challenge group size (5, 8, or 10 birds) had no effect upon the 50% infectious dose. Two weeks between inoculation and tracheal culture was optimal, since an avirulent mutant (unable to produce dermonecrotic toxin) persisted for a shorter time. Communicability of the
B. avium
parental strain between confined birds was modest, but a nonmotile mutant was less able to spread between birds. There were no host-associated differences in susceptibility to the parental strain and the three
B. avium
mutant strains just mentioned: in all turkey lines tested, the dermonecrotic toxin- and hemagglutination-negative mutants were avirulent whereas the nonmotile mutants showed no loss of virulence. Interestingly, the ability of a strain to cause disease in vivo correlated completely with its ability to adhere to ciliated tracheal cells in vitro.
Interaction ofListeria monocytogeneswith Human Brain Microvascular Endothelial Cells: InlB-Dependent Invasion, Long-Term Intracellular Growth, and Spread from Macrophages to Endothelial Cells
Authors: Greiffenberg Lars , Goebel Werner , Kim Kwang Sik , Weiglein Inge , Bubert Andreas , Engelbrecht Fredi , Stins Monique , Kuhn Michael
Published: December 2019

Invasion of endothelial tissues may be crucial in aListeria monocytogenesinfection leading to meningitis and/or encephalitis. Internalization ofL. monocytogenesinto endothelial cells has been previously demonstrated by using human umbilical vein endothelial cells as a model system. However, during the crossing of the blood-brain barrier,L. monocytogenesmost likely encounters brain microvascular endothelial cells which are strikingly different from macrovascular or umbilical vein endothelial cells. In the present study human brain microvascular endothelial cells (HBMEC) were used to study the interaction ofL. monocytogeneswith endothelial cells, which closely resemble native microvascular endothelial cells of the brain. We show thatL. monocytogenesinvades HBMEC in an InlB-dependent and wortmannin-insensitive manner. Once within the HBMEC,L. monocytogenesreplicates efficiently over a period of at least 18 h, moves intracellularly by inducing actin tail formation, and spreads from cell to cell. Using a green fluorescent protein-expressingL. monocytogenesstrain, we present direct evidence that HBMEC are highly resistant to damage by intracellularly growingL. monocytogenes. Infection of HBMEC withL. monocytogenesresults in foci of heavily infected, but largely undamaged endothelial cells. Heterologous plaque assays withL. monocytogenes-infected P388D1macrophages as vectors demonstrate efficient spreading ofL. monocytogenesinto HBMEC, fibroblasts, hepatocytes, and epithelial cells, and this phenomenon is independent of theinlCgene product.
Inhibition ofSalmonella typhimuriumInvasion by Host Cell Expression of Secreted Bacterial Invasion Proteins
Authors: Carlson Steve A. , Jones Bradley D.
Published: December 2019

PathogenicSalmonellaspecies initiate infection of a host by inducing their own uptake into intestinal epithelial cells. An invasive phenotype is conferred to this pathogen by a number of proteins that are components of a type III secretion system. During the invasion process, the bacteria utilize this secretion system to release proteins that enter the host cell and apparently interact with unknown host cell components that induce alterations in the actin cytoskeleton. To investigate the role of secreted proteins as direct modulators of invasion, we have evaluated the ability ofSalmonella typhimuriumto enter mammalian cells that express portions of theSalmonellainvasion proteins SipB and SipC. Plasma membrane localization of SipB and SipC was achieved by fusing carboxyl- and amino-terminal portions of each invasion protein to the intracellular carboxyl-terminal tail of a membrane-bound eukaryotic receptor. Expression of receptor chimeras possessing the carboxyl terminus of SipB or the amino terminus of SipC blockedSalmonellainvasion, whereas expression of their chimeric counterparts had no effect on invasion. The effect on invasion was specific forSalmonellasince the perturbation of uptake was not extended to other invasive bacterial species. These results suggest thatSalmonellainvasion can be competitively inhibited by preventing the intracellular effects of SipB or SipC. In addition, these experiments provide a model for examining interactions between bacterial invasion proteins and their host cell targets.
CD4+T-Lymphocyte and Immunoglobulin G2 Responses in Calves Immunized withAnaplasma marginaleOuter Membranes and Protected against Homologous Challenge
Authors: Brown Wendy C. , Shkap Varda , Zhu Daming , McGuire Travis C. , Tuo Wenbin , McElwain Terry F. , Palmer Guy H.
Published: December 2019

Protective immunity against the ehrlichial pathogenAnaplasma marginalehas been hypothesized to require induction of immunoglobulin G2 (IgG2) antibody against outer membrane protein epitopes and coordinated activation of macrophages for phagocytosis and killing. In the present study, cell-mediated immune responses, including induction of IgG isotype switching, were characterized in calves immunized with purified outer membranes of the Florida strain ofA. marginale. Importantly, these calves were subsequently shown to be protected upon experimental challenge with the Florida strain, and calves which developed the highest IgG2 titers were completely protected against infection. Peripheral blood mononuclear cells (PBMC) obtained after immunization proliferated strongly in response to both wholeA. marginalehomogenates and purified outer membranes, and this responsiveness persisted until the time of challenge. Responding cells were shown to be CD4+T cells, and CD4+T-cell lines cultured for 2 to 4 weeks also proliferated specifically in response toA. marginaleand produced high titers of gamma interferon. The helper T-cell response included recognition of conserved epitopes, as PBMC proliferation was stimulated by the homologous Florida strain, four genetically distinctA. marginalestrains, andAnaplasma ovis. The outer membrane proteins stimulating the PBMC responses in protected calves included major surface proteins (MSPs) MSP-1, MSP-2, and MSP-3, which were previously shown to induce partial protection against infection. These studies demonstrate, for the first time, potent helper T-cell responses in cattle protectively immunized with outer membranes againstA. marginalechallenge and identify three MSPs that are recognized by immune T cells. These experiments provide the basis for subsequent identification of the helper T-cell epitopes on MSP-1, MSP-2, and MSP-3 that are needed to evoke anamnestic antibody and effector T-cell responses elicited by protein or nucleic acid immunization.
High-Level Expression ofChlamydia psittaciMajor Outer Membrane Protein in COS Cells and in Skeletal Muscles of Turkeys
Authors: Vanrompay D. , Cox E. , Mast J. , Goddeeris B. , Volckaert G.
Published: December 2019

Theomp1genes encoding the major outer membrane proteins (MOMPs) of avianChlamydia psittaciserovar A and D strains were cloned and sequenced. The nucleotide sequences of the avianC. psittaciserovar A and D MOMP genes were found to be 98.9 and 87.8% identical, respectively, to that of the avianC. psittaciserovar A strain 6BC, 84.6 and 99.8% identical to that of the avianC. psittaciserovar D strain NJ1, 79.1 and 81.1% identical to that of theC. psittaciguinea pig inclusion conjunctivitis strain, 60.9 and 62.5% identical to that of theChlamydia trachomatisL2 strain, and 57.5 and 60.4% identical to that of theChlamydia pneumoniaeIOL-207 strain. The serovar A or D MOMPs were cloned in the mammalian expression plasmid pcDNA1. When pcDNA1/MOMP A or pcDNA1/MOMP D was introduced into COS7 cells, a 40-kDa protein that was identical in size, antigenicity, and electrophoretic mobility to native MOMP was produced. Recombinant MOMP (rMOMP) was located in the cytoplasm of transfected COS7 cells as well as in the plasma membrane and was immunoaccessible. Intramuscular administration of pcDNA1/MOMP in specific-pathogen-free turkeys resulted in local expression of rMOMP in its native conformation, after which anti-MOMP antibodies appeared in the serum.
Murine Model of
Bartonella henselae
Infection in the Immunocompetent Host
Authors: Regnath T. , Mielke M. E. A. , Arvand M. , Hahn H.
Published: December 2019

Bartonella henselae
is an emerging pathogen causing cat scratch disease, bacillary angiomatosis, and peliosis hepatis. Progress in understanding the pathogenesis of and the immune response to these infections has been limited by the lack of an animal model. Following intraperitoneal infection of C57BL/6 mice with
B. henselae
, organs were cleared of cultivatable bacteria within 6 days. In contrast,
B. henselae
DNA could be detected in liver tissue for at least 3 months. Liver tissue showed granulomatous inflammation reaching its highest degree of intensity during the fourth week of infection and resolving within 12 weeks postinfection. This mouse model is applicable to the study of the pathogenesis of
B. henselae
and the immune response to this pathogen in the immunocompetent host.
Molecular and Functional Characterization of
Salmonella enterica
Serovar Typhimurium
poxA
Gene: Effect on Attenuation of Virulence and Protection
Authors: Kaniga Koné , Compton Melissa S. , Curtiss Roy , Sundaram Preeti
Published: December 2019

Salmonella enterica poxA
mutants exhibit a pleiotropic phenotype, including reduced pyruvate oxidase activity; reduced growth rate; and hypersensitivity to the herbicide sulfometuron methyl, α-ketobutyrate, and amino acid analogs. These mutants also failed to grow in the presence of the host antimicrobial peptide, protamine. In this study, PoxA
−
mutants of
S. enterica
serovar Typhimurium (
S. typhimurium
) were found to be 10,000-fold attenuated in orally inoculated BALB/c mice and 1,000-fold attenuated in intraperitoneally inoculated BALB/c mice, compared to wild-type
S. typhimurium
UK-1. In addition,
poxA
mutants were found to be capable of colonizing the spleen, mesenteric lymph nodes, and Peyer’s patches; to induce strong humoral immune responses; and to protect mice against a lethal wild-type
Salmonella
challenge. A 2-kb DNA fragment was isolated from wild-type
S. typhimurium
UK-1 based on its ability to complement an isogenic
poxA
mutant. The nucleotide sequence of this DNA fragment revealed an open reading frame of 325 amino acids capable of encoding a polypeptide of 36.8 kDa that was confirmed in the bacteriophage T7 expression system. Comparison of the translated sequence to the available databases indicated high homology to a family of lysyl-tRNA synthetases. Our results indicate that a mutation of
poxA
has an attenuating effect on
Salmonella
virulence. Further,
poxA
mutants are immunogenic and could be useful in designing live vaccines with a variety of bacterial species. To our knowledge, this is the first report on the effect of
poxA
mutation on bacterial virulence.
Activity of the Mitogenic
Pasteurella multocida
Toxin Requires an Essential C-Terminal Residue
Authors: Ward Philip N. , Miles Antony J. , Sumner Ian G. , Thomas Lewis H. , Lax Alistair J.
Published: December 2019

Pasteurella multocida
toxin (PMT) is a potent mitogen that also affects bone resorption. PMT acts intracellularly and is therefore postulated to have several domains involved in different aspects of its function. The toxin contains eight cysteine residues. Mutants with individual substitutions for each of these residues were constructed, and the effects of these on the biological activity of the toxin were determined by cultured-cell assays. Only the most C-terminal of the eight cysteines (C1165) was essential for full activity, although mutation of the cysteine residue at position 1159 caused a slight but reproducible loss of potency. In animal challenge experiments, mutant toxin (C1165S) was not toxic to piglets, even at doses exceeding a lethal dose of active PMT 1,000-fold. The mutant and wild-type toxins displayed identical purification characteristics, similar susceptibility to proteolytic digestion, and circular dichroism profiles, which indicated that no gross structural changes had taken place. The function of the essential C1165 residue is not yet known, although its most likely role is an enzymatic one at or near the catalytic center of the toxin.
TetR Is a Positive Regulator of the Tetanus Toxin Gene in
Clostridium tetani
and Is Homologous to BotR
Authors: Marvaud Jean-Christophe , Eisel Ulrich , Binz Thomas , Niemann Heiner , Popoff Michel R.
Published: December 2019

The TetR gene immediately upstream from the tetanus toxin (TeTx) gene was characterized. It encodes a 21,562-Da protein which is related (50 to 65% identity) to the equivalent genes (
botR
) in
Clostridium botulinum
. TetR has the feature of a DNA binding protein with a basic pI (9.53). It contains a helix-turn-helix motif and shows 29% identity with other putative regulatory genes in
Clostridium
, i.e.,
uviA
from
C. perfringens
and
txeR
from
C. difficile
. We report for the first time the transformation of
C. tetani
by electroporation, which permitted us to investigate the function of
tetR
. Overexpression of
tetR
in
C. tetani
induced an increase in TeTx production and in the level of the corresponding mRNA. This indicates that TetR is a transcriptional activator of the TeTx gene. Overexpression of
botR/A
(60% identity with TetR at the amino acid level) in
C. tetani
induced an increase in TeTx production comparable to that for overexpression of
tetR
. However,
botR/C
(50% identity with TetR at the amino acid level) was less efficient. This supports that TetR positively regulates the TeTx gene in
C. tetani
and that a conserved mechanism of regulation of the neurotoxin genes is involved in
C. tetani
and
C. botulinum
.
Identification and Characterization of a Phase-Variable NonfimbrialSalmonella typhimuriumGene That Alters O-Antigen Production
Authors: Kwan Lola Y. , Isaacson Richard E.
Published: December 2019

Salmonella typhimurium798, which was isolated from a pig, is known to phase vary from a nonadhesive to an adhesive phenotype. Cells of the adhesive phenotype adhere to porcine enterocytes, are more readily phagocytized by porcine neutrophils and macrophages, and once phagocytized can survive intracellularly, while cells of the nonadhesive phenotype die rapidly. The effect of phenotypic switching also can be visualized by changes in colony morphologies and the presence of between 10 and 15 proteins in the envelopes of cells in the adhesive phenotype. Mutants previously constructed with cells in the adhesive phenotype and the transposon TnphoAwere screened to identify mutants lacking one or more of the unique proteins. One mutation was cloned and sequenced, and the mutation was shown to be inrfaL(O-antigen ligase). Expression of O antigen was shown to be phase variable. The adhesive strain expressed an O antigen that was at least eightfold longer than that for the nonadhesive strain and by virtue of O-antigen production was resistant to porcine complement. The mutant survived intracellularly in phagocytic cells as well as its wild-type parent.
A Vaccine and Monoclonal Antibodies That Enhance Mouse Resistance toCandida albicansVaginal Infection
Authors: Han Yongmoon , Morrison Richard P. , Cutler Jim E.
Published: December 2019

We previously reported that a vaccine composed of liposome-mannan complexes ofCandida albicans(L-mann) stimulates mice to produce protective antibodies against disseminated candidiasis. An immunoglobulin M (IgM) monoclonal antibody (MAb), B6.1, specific for a β-1,2-mannotriose in the complexes protects against the disease, whereas MAb B6 does not. In the present study, the vaccine and MAbs B6.1 and B6 were tested for the ability to protect againstCandidavaginal infection, established by intravaginal (i.vg.) inoculation of yeast cells in mice maintained in pseudoestrus. Fungal CFU in each vagina was determined to assess the severity of infection. Mice vaccinated before infection developed about 62% fewer vaginal CFU than nonimmunized controls. Naive mice that received polyclonal antiserum (from vaccinated mice) i.vg. before infection had 60% fewer CFU than controls. The serum protective factor was stable at 56°C, butC. albicanscells absorbed this factor. Mice given MAb B6.1 i.vg. after infection was established had fewerCandidaCFU in vaginal tissue than control mice given buffer instead of antibody. MAbs B6.1 and B6 given intraperitoneally before infection protected mice, but MAbs preabsorbed with yeast cells did not. MAb B6.1 also protected againstC. tropicalisvaginal infection, but MAb B6 did not. The protective activities of MAbs B6.1 and B6 appeared to be specific because an irrelevant IgM carbohydrate-specific MAb and an irrelevant IgG protein-specific MAb were not protective; also, MAb B6.1 did not affect development of vaginal chlamydial infection. These studies show that an appropriate antibody response, or administration of protective antibodies, can help the host to resistCandidavaginal infection.
Modulation of Expression of the ToxR Regulon in
Vibrio cholerae
by a Member of the Two-Component Family of Response Regulators
Authors: Wong Sandy M. , Carroll Patricia A. , Rahme Laurence G. , Ausubel Frederick M. , Calderwood Stephen B.
Published: December 2019

The ToxRS system in
Vibrio cholerae
plays a central role in the modulation of virulence gene expression in response to environmental stimuli. An integration of multiple signalling inputs mediated by ToxR, -S, and -T controls virulence gene expression leading to cholera toxin (CT) production. Recently, we identified a new virulence locus,
varA
(virulence associated regulator), in classical
V. cholerae
O1 that positively controls transcription of
tcpA
, the major subunit of the toxin-coregulated pilus (TCP) and the production of CT, two key factors in cholera pathogenesis. The
varA
locus is a homolog of
gacA
(originally described for the soil organism
Pseudomonas fluorescens
), which encodes a conserved global regulator belonging to the family of two-component signal transducing molecules. GacA homologs in a number of diverse gram-negative pathogenic bacterial species have been implicated in controlling the production of diverse virulence factors.
varA
mutants showed reduced levels of
tcpA
message and TcpA protein, lacked visible signs of autoagglutination (a phenotype associated with functional TCP), produced decreased levels of CT, and were attenuated in colonizing infant mice. Transcription of
varA
appears to be independent of ToxR, and overexpression of the regulators
tcpPH
and
toxT
from plasmids in the
varA
mutant restored wild-type levels of CT production and the ability to autoagglutinate.
varA
represents an additional modulating factor in the coordinate expression of virulence factors in
V. cholerae.
Evidence for Multiple Pathologic and Protective Mechanisms of Murine Cerebral Malaria
Authors: Jennings Veronica M. , Lal Altaf A. , Hunter Robert L.
Published: December 2019

Murine cerebral malaria (CM) induced byPlasmodium bergheiANKA kills susceptible mice within 24 to 48 h of onset of symptoms and is characterized by the production of inflammatory cytokines in the brain. C57BL/6J mice are sensitive to lethal CM, while A/J mice are resistant. These strains of mice were immunized with an adjuvant vaccine of killed whole-blood-stage parasites. The immunization protected C57BL/6 mice from lethal CM following virulent challenge. The same immunization increased the incidence of lethal CM in A/J mice challenged similarly. Histopathologic examination of the brains of mice from these studies revealed two distinct types of lesions. Type I CM is acute in onset; usually lethal; and characterized by widespread microglial activation, endothelial cell damage, and microvascular disruption in the brain. Type II CM is characterized by intense, but focal, mononuclear cell inflammation without endothelial cell damage or microvascular destruction. Animals with type II lesions were clinically normal and protected from type I lesions. Available clinical, epidemiological, and biochemical evidence suggests that type I and type II lesions might exist in human CM as well.
Internalization ofStaphylococcus aureusby Endothelial Cells Induces Apoptosis
Authors: Menzies Barbara E. , Kourteva Iordanka
Published: December 2019

The ability ofStaphylococcus aureusto invade and survive within endothelial cells is believed to contribute to its propensity to cause persistent endovascular infection with endothelial destruction. In the present study, we show that following invasion of human umbilical vein endothelial cells, intracellularS. aureusorganisms remain viable over a 72-h period and, as determined by transmission electron microscopic examination, that the bacteria exist within vacuoles and free within the cytoplasm. We also demonstrate that endothelial cell death followingS. aureusinvasion occurs at least in part by apoptosis as shown by DNA fragmentation and changes in nuclear morphology. Apoptotic changes were evident as early as 1 h after infection of endothelial cells. Internalization ofS. aureusrather than adherence appears to be necessary, since use of the phagocytosis inhibitor cytochalasin D prevented apoptosis. UV-killed staphylococci, although retaining the capacity to be internalized, were not capable of inducing apoptosis, suggesting that apoptosis is dependent upon a factor associated with viable organisms. The studies demonstrate that viable intracellularS. aureusinduces apoptosis of endothelial cells and that internalized staphylococci can exist free within the cytoplasm.
Identification of the
Helicobacter pylori
VacA Toxin Domain Active in the Cell Cytosol
Authors: de Bernard Marina , Burroni Daniela , Papini Emanuele , Rappuoli Rino , Telford John , Montecucco Cesare
Published: December 2019

Cells exposed to
Helicobacter pylori
toxin VacA develop large vacuoles which originate from massive swelling of membranous compartments at late stages of the endocytic pathway. When expressed in the cytosol, VacA induces vacuolization as it does when added from outside. This and other evidence indicate that VacA is a toxin capable of entering the cell cytosol, where it displays its activity. In this study, we have used cytosolic expression to identify the portion of the toxin molecule responsible for the vacuolating activity. VacA mutants with deletions at the C and N termini were generated, and their activity was analyzed upon expression in HeLa cells. We found that the vacuolating activity of VacA resides in the amino-terminal region, the whole of which is required for its intracellular activity.
Reciprocal Regulation of Th1- and Th2-Cytokine-Producing T Cells during Clearance of Parasitemia inPlasmodium falciparumMalaria
Authors: Winkler Stefan , Willheim Martin , Baier Karin , Schmid Daniela , Aichelburg Alexander , Graninger Wolfgang , Kremsner Peter G.
Published: December 2019

Flow cytometry for the intracellular detection of T-cell cytokines was performed for 15 Gabonese patients during acute uncomplicatedPlasmodium falciparummalaria. A striking expansion of CD4+and CD8+T cells producing gamma interferon (IFN-γ) was found during drug-induced clearance of parasitemia, paralleled by a decrease of interleukin-2 (IL-2) production. The frequency of IL-4- and IL-13-producing CD4+cells gradually decreased, whereas the frequency of T cells producing IL-2+–IFN-γ+, IL-4−–IL-5+, and IL-4+–IL-5+cytokines as well as IL-4+–IFN-γ+and IL-13+–IFN-γ+cytokines was not significantly altered. The capacity for IL-10 production within the CD4+subset increased due to an expansion of both IL-10+–IFN-γ−and IL-10+–IFN-γ+cytokine-expressing cells. Thus, a more pronounced Th2-driven immune response during acute untreatedP. falciparuminfection with a shift towards Th1 responsiveness induced by parasite clearance is suggested.
Changes in Expression of Signal Transduction Proteins in T Lymphocytes of Patients with Leprosy
Authors: Zea Arnold H. , Ochoa Maria T. , Ghosh Paritosh , Longo Dan L. , Alvord W. Gregory , Valderrama Liliana , Falabella Rafael , Harvey Linda K. , Saravia Nancy , Moreno Luis H. , Ochoa Augusto C.
Published: December 2019

Advanced stages of mycobacterial diseases such as leprosy and tuberculosis are characterized by a loss of T-cell function. The basis of this T-cell dysfunction is not well understood. The present report demonstrates major alterations in the expression of signal transduction molecules in T cells of leprosy patients. These alterations were most frequently observed in lepromatous leprosy (LL) patients. Of 29 LL patients, 69% had decreased T-cell receptor ζ-chain expression, 48% had decreased p56lcktyrosine kinase, and 63% had a loss of nuclear transcription factor NF-κB p65. An electrophoretic mobility shift assay with the gamma interferon core promoter region revealed a loss of the Th1 DNA-binding pattern in LL patients. In contrast, tuberculoid leprosy patients had only minor signal transduction alterations. These novel findings might improve our understanding of the T-cell dysfunction observed in leprosy and other infectious diseases and consequently might lead to better immunologic evaluation of patients.
Intracellular Multiplication and Virulence of
Shigella flexneri
Auxotrophic Mutants
Authors: Cersini Antonella , Salvia Anna Maria , Bernardini Maria Lina
Published: December 2019

We have constructed and analyzed a group of
Shigella flexneri
5 auxotrophic mutants. The wild-type strain M90T was mutagenized in genes encoding enzymes involved in the synthesis of (i) aromatic amino acids, (ii) nucleotides, and (iii) diaminopimelic acid. In this way, strains with single (
aroB
,
aroC
,
aroD
,
purE
,
thyA
, and
dapB
) and double (
purE aroB
,
purE aroC
,
purE aroD
,
purE thyA
) mutations were obtained. Although the Aro mutants had the same nutritional requirements when grown in laboratory media, they showed different degrees of virulence in vitro and in vivo. The
aroB
mutant was not significantly attenuated, whereas both the
aroC
and
aroD
strains were severely attenuated.
p
-Aminobenzoic acid (PABA) appeared to be the main requirement for the Aro mutants’ growth in tissue culture. Concerning nucleotides, thymine reduced the pathogenicity, whereas adenine did not. However, when combined with another virulence-affecting mutation, adenine auxotrophy appeared to potentiate that mutation’s effects. Consequently, the association of either the
purE
and
aroC
or the
purE
and
aroD
mutations had a great effect on virulence as measured by the Sereny test, whereas the
purE aroB
double mutation appeared to have only a small effect. All mutants except the
dapB
strain seemed to move within a Caco-2 cell monolayer after 3 h of infection. Nevertheless, the auxotrophs showing a high intracellular generation time were negative in the plaque assay. Knowledge of each mutation’s role in attenuating
Shigella
strains will provide useful tools in designing vaccine candidates.
Nitric Oxide-Mediated Inhibition of the Ability ofRickettsia prowazekiiTo Infect Mouse Fibroblasts and Mouse Macrophagelike Cells
Authors: Turco Jenifer , Liu Hua , Gottlieb Sheldon F. , Winkler Herbert H.
Published: December 2019

The role of the nitric oxide synthase (NOS) pathway in inhibiting the ability ofRickettsia prowazekiito initially infect (invade) mouse cytokine-treated, fibroblastic L929 cells and macrophagelike RAW264.7 cells and the ability of nitric oxide (NO) to damage isolated rickettsiae were investigated. Substantial amounts of nitrite (a degradation product of NO) were produced and the initial rickettsial infection was suppressed in cultures of L929 cells treated with crude lymphokine preparations (LK) or with gamma interferon (IFN-γ) plus tumor necrosis factor alpha (TNF-α) but not in L929 cell cultures treated with IFN-γ alone or TNF-α alone. The NOS inhibitorsNG-methyl-l-arginine and aminoguanidine both inhibited nitrite production and prevented the suppression of the initial rickettsial infection. Antibody-mediated neutralization of the IFN-γ in the LK also inhibited both nitrite production and suppression of the initial rickettsial infection. Cultures of RAW264.7 cells treated with IFN-γ plus lipopolysaccharide exhibited suppression of the initial rickettsial infection, and the suppression was relieved by aminoguanidine. Addition of oxyhemoglobin (a scavenger of extracellular NO) during the rickettsial infection alleviated the suppression of the initial rickettsial infection observed in appropriately treated L929 cells and RAW264.7 cells. In addition, the oxyhemoglobin restored the rickettsia-mediated, rapid killing of the treated RAW264.7 cells. Incubation of isolated rickettsiae with NO inhibited their ability to infect L929 and IFN-γ-treated RAW264.7 cells and to rapidly kill IFN-γ-treated RAW264.7 cells. In contrast, incubation of L929 cells with a solution that contained NO and/or degradation products of NO did not affect their ability to be infected by rickettsiae. The data are consistent with the hypothesis that NO released from appropriately stimulated potential host cells kills extracellular rickettsiae and thus prevents the rickettsiae from infecting the cells.
A Murine Model in Which Protection Correlates with Pertussis Vaccine Efficacy in Children Reveals Complementary Roles for Humoral and Cell-Mediated Immunity in Protection againstBordetella pertussis
Authors: Mills Kingston H. G. , Ryan Mark , Ryan Elizabeth , Mahon Bernard P.
Published: December 2019

The results of phase 3 efficacy trials have shown that acellular and whole-cell pertussis vaccines can confer protection against whooping cough. However, despite the advances in vaccine development, clinical trials have not provided significant new information on the mechanism of protective immunity againstBordetella pertussis. Classical approaches based on measurement of antibody responses to individual antigens failed to define an immunological correlate of protection. A reliable animal model, predictive of acellular and whole-cell pertussis vaccine potency in children, would facilitate an elucidation of the mechanism of immune protection againstB. pertussisand would assist in the regulatory control and future development of pertussis vaccines. In this study, we have shown that the rate ofB. pertussisclearance following respiratory challenge of immunized mice correlated with vaccine efficacy in children. Using this model together with mice with targeted disruptions of the gamma interferon (IFN-γ) receptor, interleukin-4 or immunoglobulin heavy-chain genes, we have demonstrated an absolute requirement for B cells or their products in bacterial clearance and a role for IFN-γ in immunity generated by previous infection or immunization with the whole-cell pertussis vaccine. The results of passive immunization experiments suggested that protection early after immunization with acellular pertussis vaccines is mediated by antibody against multiple protective antigens. In contrast, more complete protection conferred by previous infection or immunization with whole-cell pertussis vaccines reflected the induction of Th1 cells. Our findings suggest that the mechanism of immunity againstB. pertussisinvolves humoral and cellular immune responses which are not directed against a single protective antigen and thus provide an explanation for previous failures to define an immunological correlate of protection.
Effect of Lipopolysaccharide and Inflammatory Cytokines on Interleukin-6 Production by Healthy Human Gingival Fibroblasts
Authors: Kent Leigh W. , Rahemtulla Firoz , Hockett Richard D. , Gilleland Rebecca C. , Michalek Suzanne M.
Published: December 2019

Cytokines are hormone-like proteins which mediate and regulate inflammatory and immune responses. The purpose of this study was to investigate the effect of lipopolysaccharide (LPS) and inflammatory cytokines on regulation of interleukin-6 (IL-6) production by human gingival fibroblasts (HGF). The HGF cell lines used in this study, H-CL and F-CL, were established by the explant technique from healthy gingival tissue. Cultured cells were grown to confluency and incubated with various concentrations of LPS fromEscherichia coliorPorphyromonas gingivalisor with the recombinant human cytokine tumor necrosis factor alpha (TNF-α), IL-1α, or IL-1β. Culture supernatants were collected at various times and assessed for IL-6 production by enzyme-linked immunosorbent assay. Total RNA was isolated from the harvested cells and used to assess levels of IL-6 mRNA by the RNase protection assay. Both LPS preparations induced IL-6 production (1 to 4 ng of IL-6 per ml) by both HGF cell lines. Although TNF-α stimulated IL-6 production by HGF, >10-fold-larger amounts were induced with IL-1α and IL-1β. Furthermore, the addition of both IL-1α and TNF-α to cultured cells resulted in approximately 600- to 800-fold-higher levels of IL-6 than seen in control cultures, suggesting that these cytokines synergistically induced IL-6 production by HGF. IL-6 message in cultured cells was upregulated 20-fold by TNF-α, 1,000-fold by IL-1α and IL-1β, and 1,400-fold by IL-1α plus TNF-α. IL-1α and TNF-α alone upregulate IL-6 production in a dose- and time-dependent fashion. The addition of IL-1α and TNF-α to cultured HGF cells resulted in a synergistic induction of IL-6 after 8 h of incubation and when greater than 10 pg of this combination per ml was used. Our studies show that inflammatory cytokines are hundreds of times more potent than LPS in stimulating IL-6 production by HGF.
Polymorphism in the
Bordetella pertussis
Virulence Factors P.69/Pertactin and Pertussis Toxin in The Netherlands: Temporal Trends and Evidence for Vaccine-Driven Evolution
Authors: Mooi Frits R. , van Oirschot Hans , Heuvelman Kees , van der Heide Han G. J. , Gaastra Wim , Willems Rob J. L.
Published: December 2019

The
Bordetella pertussis
proteins P.69 (also designated pertactin) and pertussis toxin are important virulence factors and have been shown to confer protective immunity in animals and humans. Both proteins are used in the new generation of acellular pertussis vaccines (ACVs), and it is therefore important to study the degree of antigenic variation in these proteins. Sequence analysis of the genes for P.69 and the pertussis toxin S1 subunit, using strains collected from Dutch patients in the period 1949 to 1996, revealed three P.69 and three S1 variants which show differences in amino acid sequence. Polymorphism in P.69 was confined to a region comprised of repeats and located proximal to the RGD motif involved in adherence to host tissues. Variation in S1 was observed in two regions previously identified as T-cell epitopes. P.69 and S1 variants, identical to those included in the Dutch whole-cell pertussis vaccine (WCV), were found in 100% of the strains from the 1950s, the period when the WCV was introduced in The Netherlands. However, nonvaccine types of P.69 and S1 gradually replaced the vaccine types in later years and were found in ∼90% strains from 1990 to 1996. These results suggest that vaccination has selected for strains which are antigenically distinct from vaccine strains. Analysis of strains from vaccinated and nonvaccinated individuals indicated that the WCV protects better against strains with the vaccine type P.69 than against strains with non-vaccine types (
P
= 0.024). ACVs contain P.69 and S1 types which are found in only 10% of recent Dutch
B. pertussis
isolates, implying that they do not have an optimal composition. Our findings cast a new light on the reemergence of pertussis in highly vaccinated populations and may have major implications for the long-term efficacy of both WCVs and ACVs.
Secretory Immune Response to Membrane Antigens duringGiardia lambliaInfection in Humans
Authors: Rosales-Borjas Disney M. , Díaz-Rivadeneyra Juan , Doña-Leyva Antonio , Zambrano-Villa Sergio A. , Mascaró Carmen , Osuna Antonio , Ortiz-Ortiz Librado
Published: December 2019

The secretory immune response in humans infected withGiardia lambliawas studied by using saliva samples and a membrane-rich protein fraction. The membrane fraction, studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, showed 24 antigen bands, ranging from 170 to 14 kDa. Saliva samples from giardiasis patients showed a heterogeneous response against the membrane fraction when they were assayed by immunoblotting. Among the antigens recognized by patient saliva samples, those of 170, 105, 92, 66, 32, 29, and 14 kDa stood out. These antigens were not recognized by saliva samples from healthy individuals. They may be of importance in future studies of protection from or diagnosis ofG. lambliainfections.
Genetic Inactivation of an Extracellular Cysteine Protease (SpeB) Expressed byStreptococcus pyogenesDecreases Resistance to Phagocytosis and Dissemination to Organs
Authors: Lukomski Slawomir , Burns Eugene H. , Wyde Philip R. , Podbielski Andreas , Rurangirwa Jacqueline , Moore-Poveda Donna K. , Musser James M.
Published: December 2019

Streptococcal pyrogenic exotoxin B (SpeB), a conserved cysteine protease expressed by virtually allStreptococcus pyogenesstrains, has recently been shown to be an important virulence factor (S. Lukomski, S. Sreevatsan, C. Amberg, W. Reichardt, M. Woischnik, A. Podbielski, and J. M. Musser, J. Clin. Invest. 99:2574–2580, 1997). Genetic inactivation of SpeB significantly decreased the lethality of a serotype M49 strain for mice and abolished the lethality of a serotype M3 strain after intraperitoneal (i.p.) injection. In the present study, a wild-type M3 isolate and an M3speBmutant derivative were used to investigate the mechanism responsible for altered virulence. Following i.p. injection, the mutant and wild-type strains induced virtually identical cellular inflammatory responses, characterized largely by an influx of polymorphonuclear leukocytes (PMNs). In addition, the mutant and wild-type strains rapidly entered the blood and were recovered from all organs examined. However, significantly fewer (P< 0.05) CFUs of the isogenic mutant derivative than of the wild-type parent strain were recovered from blood and organs. PMNs effectively cleared the M3speBmutant from the peritoneum by 22 h, thereby sparing the host. In contrast, the wild-type M3 strain continued to replicate intraperitoneally and had the ability to kill phagocytes. This process allowed the wild-type strain to continuously disseminate, resulting in host death. Our results indicate that genetic inactivation of the cysteine protease decreased the resistance of the mutant to phagocytosis and impaired its subsequent dissemination to organs. These results provide insight into the detrimental effect of SpeB inactivation on virulence.
In Vivo Formation of Electron Paramagnetic Resonance-Detectable Nitric Oxide and of Nitrotyrosine Is Not Impaired during Murine Leishmaniasis
Authors: Giorgio Selma , Linares Edlaine , Ischiropoulos Harry , Von Zuben Fernando José , Yamada Aureo , Augusto Ohara
Published: December 2019

Recent studies have provided evidence for a dual role of nitric oxide (NO) during murine leishmaniasis. To explore this problem, we monitored the formation of NO and its derived oxidants during the course ofLeishmania amazonensisinfection in tissues of susceptible (BALB/c) and relatively resistant (C57BL/6) mice. NO production was detected directly by low-temperature electron paramagnetic resonance spectra of animal tissues. Both mouse strains presented detectable levels of hemoglobin nitrosyl (HbNO) complexes and of heme nitrosyl and iron-dithiol-dinitrosyl complexes in the blood and footpad lesions, respectively. Estimation of the nitrosyl complex levels demonstrated that most of the NO is synthesized in the footpad lesions. In agreement, immunohistochemical analysis of the lesions demonstrated the presence of nitrotyrosine in proteins of macrophage vacuoles and parasites. Since macrophages lack myeloperoxidase, peroxynitrite is likely to be the nitrating NO metabolite produced during the infection. The levels of HbNO complexes in the blood reflected changes occurring during the infection such as those in parasite burden and lesion size. The maximum levels of HbNO complexes detected in the blood of susceptible mice were higher than those of C57BL/6 mice but occurred at late stages of infection and were accompanied by the presence of bacteria in the cutaneous lesions. The results indicate that the local production of NO is an important mechanism for the elimination of parasites if it occurs before the parasite burden becomes too high. From then on, elevated production of NO and derived oxidants aggravates the inflammatory process with the occurrence of a hypoxic environment that may favor secondary infections.
Inducible Nitric Oxide Synthase Does Not Affect Resolution of Murine Chlamydial Genital Tract Infections or Eradication of Chlamydiae in Primary Murine Cell Culture
Authors: Ramsey Kyle H. , Miranpuri Gurwattan S. , Poulsen Christoffer E. , Marthakis Nancy B. , Braune Laima M. , Byrne Gerald I.
Published: December 2019

Mice lacking inducible nitric oxide synthase (iNOS) or treated with iNOS inhibitors resolved chlamydial genital tract infections. Additionally, treatment of primary murine cell cultures with gamma interferon restricted chlamydial growth in the absence of nitric oxide. From these results, iNOS activity is unnecessary for the resolution of chlamydial genital tract infections in mice and inhibition of chlamydial growth in culture.
Anti-Lipid A Monoclonal Antibody Centoxin (HA-1A) Binds to a Wide Variety of Hydrophobic Ligands
Authors: Helmerhorst E. J. , Maaskant J. J. , Appelmelk B. J.
Published: December 2019

This note describes the binding specificities of four lipid A monoclonal antibodies (MAbs) including Centoxin (HA-1A); these MAbs display similar binding properties. MAbs reacted with lipid A and heat-killed smooth bacteria, whereas no reactivity was observed with smooth lipopolysaccharide (LPS). Immunoblotting of bacterial extracts separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the MAbs bound to many polypeptide bands including the molecular weight markers. Denaturation of bovine serum albumin (BSA) by boiling or dithiothreitol treatment unmasked antibody epitopes. In addition, binding both to a hydrophobic aliphatic C12 chain covalently coupled to BSA and to single-stranded DNA was observed. The polyreactivity of these clones is most likely mediated by a preferential reactivity with hydrophobic molecular patches.
Transposon-Derived
Brucella abortus
Rough Mutants Are Attenuated and Exhibit Reduced Intracellular Survival
Authors: Allen Chris A. , Adams L. Garry , Ficht Thomas A.
Published: December 2019

The O antigen of
Brucella abortus
has been described as a major virulence determinant based on the attenuated survival of fortuitously isolated rough variants. However, the lack of genetic definition of these mutants and the virulence of naturally occurring rough species,
Brucella ovis
and
Brucella canis
, has confused interpretation. To better characterize the role of O antigen in virulence and survival, transposon mutagenesis was used to generate
B. abortus
rough mutants defective in O-antigen presentation. Sequence analysis of DNA flanking the site of Tn
5
insertion was used to verify insertion in genes encoding lipopolysaccharide (LPS) biosynthetic functions. Not surprisingly, each of the rough mutants was attenuated for survival in mice, but unexpected differences among the mutants were observed. In an effort to define the basis for the observed differences, the structure of the rough LPS and the sensitivity of these mutants to individual killing mechanisms were examined in vitro. All of the
B. abortus
rough mutants exhibited a 4- to 5-log-unit increase, compared to the smooth parental strain, in sensitivity to complement-mediated lysis. Little change was evident in the sensitivity of these organisms to hydrogen peroxide, consistent with an inability of O antigen to exclude relatively small molecules. Sensitivity to polymyxin B, which was employed as a model cationic, amphipathic peptide similar to defensins found in phagocytic cells, revealed survival differences among the rough mutants similar to those observed in the mouse. One mutant in particular exhibited hypersensitivity to polymyxin B and reduced survival in mice. This mutant was characterized by a truncated rough LPS. DNA sequence analysis of this mutant revealed a transposon interruption in the gene encoding phosphomannomutase (
pmm
), suggesting that this activity may be required for the synthesis of a full-length core polysaccharide in addition to O antigen.
B. abortus
O antigen appears to be essential for extra- and intracellular survival in mice.
Evidence for Specific Secretion Rather than Autolysis in the Release of Some
Helicobacter pylori
Proteins
Authors: Vanet Anne , Labigne Agnès
Published: December 2019

We investigated whether
Helicobacter pylori
cells actively secrete proteins such as the urease subunits UreA and UreB and the GroES and GroEL homologs HspA and HspB or whether these proteins were present in the extracellular compartment as a consequence of autolysis. Using a subcellular fractionation approach associated with quantitative Western blot analyses, we showed that the supernatant protein profiles were very different from those of the cell pellets, even for bacteria harvested in the late growth phase; this suggests that the release process is selective. A typical cytoplasmic protein, a β-galactosidase homolog, was found exclusively associated with the pellet of whole-cell extracts, and no traces were found in the supernatant. In contrast, UreA, UreB, HspA, and HspB were mostly found in the pellet but significant amounts were also present in the supernatant. HspA and UreB were released into the supernatant at the same rate throughout the growth phase (3%), whereas large portions of HspB and UreA were released during the stationary phase (over 30 and 20%, respectively) rather than during the early growth phase (20% and 6, respectively). The profiles of protein obtained after water extraction of the bacteria with those of the proteins naturally released within the liquid culture supernatants demonstrated that water extraction led to the release of a large amount of protein due to artifactual lysis. Our data support the conclusion that a specific and selective mechanism(s) is involved in the secretion of some
H. pylori
antigens. A programmed autolysis process does not seem to make a major contribution.