bluuebunny/crossref_metadata_2025 huggingface dataset:
Shiga Toxins Stimulate Secretion of Interleukin-8 from Intestinal Epithelial Cells
Authors: Thorpe C. M. , Hurley Bryan P. , Lincicome Lisa L. , Jacewicz Mary S. , Keusch Gerald T. , Acheson David W. K.
Published: December 2019

In the 1980s, Shiga toxin (Stx)-producing
Escherichia coli
O157:H7 (STEC) was identified as a cause of hemorrhagic colitis in the United States and was found to be associated with hemolytic uremic syndrome (HUS), a microangiopathic hemolytic anemia characterized by thrombocytopenia and renal failure. The precise way that Stxs cause hemorrhagic colitis and HUS is unclear. Stxs have been thought to cause disease by killing or irreversibly harming sensitive cells through a nonspecific blockade of mRNA translation, eventually resulting in cytotoxicity by preventing synthesis of critical molecules needed to maintain cell integrity. Because STEC is noninvasive, we have been exploring the host-toxin response at the level of the gastrointestinal mucosa, where STEC infection begins. We have found that Stx is capable of interleukin-8 (IL-8) superinduction in a human colonic epithelial cell line. Despite a general blockade of mRNA translation, Stx treatment results in increased IL-8 mRNA as well as increased synthesis and secretion of IL-8 protein. Our data suggest that an active Stx A subunit is required for this activity. Ricin, which has the same enzymatic activity and trafficking pathway as Stx, has similar effects. Exploration of the effects of other protein synthesis inhibitors (cycloheximide, anisomycin) suggests a mechanism of gene regulation that is distinct from a general translational blockade. Use of the specific p38/RK inhibitor SB202190 showed that blocking of this pathway results in decreased Stx-mediated IL-8 secretion. Furthermore, Stxs induced mRNA of the primary response gene c-
jun
, which was subsequently partially blocked by SB202190. These data suggest a novel model of how Stxs contribute to disease, namely that Stxs may alter regulation of host cell processes in sensitive cells via activation of at least one member of the mitogen-activated protein kinase family in the p38/RK cascade and induction of c-
jun
mRNA. Stx-induced increases in chemokine synthesis from intestinal epithelial cells could be important in augmenting the host mucosal inflammatory response to STEC infection.
A Genomic Island, Termed High-Pathogenicity Island, Is Present in Certain Non-O157 Shiga Toxin-Producing
Escherichia coli
Clonal Lineages
Authors: Karch H. , Schubert S. , Zhang D. , Zhang W. , Schmidt H. , Ölschläger T. , Hacker J.
Published: December 2019

Shiga toxin-producing
Escherichia coli
(STEC) strains cause a wide spectrum of diseases in humans. In this study, we tested 206 STEC strains isolated from patients for potential virulence genes including
stx
,
eae
, and enterohemorrhagic
E. coli hly
. In addition, all strains were examined for the presence of another genetic element, the high-pathogenicity island (HPI). The HPI was first described in pathogenic
Yersinia
species and encodes the pesticin receptor FyuA and the siderophore yersiniabactin. The HPI was found in the genome of distinct clonal lineages of STEC, including all 31
eae
-positive O26:H11/H
−
strains and 7 of 12
eae
-negative O128:H2/H
−
strains. In total, the HPI was found in 56 (27.2%) of 206 STEC strains. However, it was absent from the genome of all 37 O157:H7/H
−
, 14 O111:H
−
, 13 O103:H2, and 13 O145:H
−
STEC isolates, all of which were positive for
eae
. Polypeptides encoded by the
fyuA
gene located on the HPI could be detected by using immunoblot analysis in most of the HPI-positive STEC strains, suggesting the presence of a functional yersiniabactin system. The HPI in STEC was located next to the tRNA gene
asnT
. In contrast to the HPI of other pathogenic enterobacteria, the HPI of O26 STEC strains shows a deletion at its left junction, leading to a truncated integrase gene
int
. We conclude from this study that the
Yersinia
HPI is disseminated among certain clonal subgroups of STEC strains. The hypothesis that the HPI in STEC contributes to the fitness of the strains in certain ecological niches rather than to their pathogenic potential is discussed.
Identification, Characterization, and Expression of Three New Members of the
Borrelia burgdorferi
Mlp (2.9) Lipoprotein Gene Family
Authors: Yang Xiaofeng , Popova Taissia G. , Hagman Kayla E. , Wikel Stephen K. , Schoeler George B. , Caimano Melissa J. , Radolf Justin D. , Norgard Michael V.
Published: December 2019

We previously reported on the existence of a family of lipoprotein genes, designated 2.9 lipoprotein genes, encoded in at least seven versions on the circular (supercoiled) cp32 and cp18 plasmids of
Borrelia burgdorferi
297. A distinguishing feature of the 2.9 lipoproteins were highly similar signal sequences but variable mature polypeptides that segregated into two antigenic classes. Further screenings of
B. burgdorferi
297 genomic libraries led to the identification of three additional 2.9 lipoprotein genes, renamed herein
mlp
, for multicopy lipoprotein genes. Computer analyses and immunoblotting revealed that Mlp-9 segregated with the antigenic class I lipoproteins, whereas Mlp-8 and Mlp-10 were members of class II. Northern blotting showed that all three of the
mlp
genes were expressed when
B. burgdorferi
was cultivated in vitro at 34°C, although
mlp-9
and
mlp-10
transcripts were expressed at very low levels. Additional combined immunoblotting and comparative reverse transcription-PCR analyses performed on borreliae cultivated in vitro at 23, 34, or 37°C indicated that although Mlp-8 was substantially more abundant than Mlp-9 or Mlp-10, all three of the
mlp
genes were upregulated during
B. burgdorferi
replication at 37°C. Expression of the same three lipoproteins was further enhanced upon growth of the spirochetes within dialysis membrane chambers (DMCs) implanted intraperitoneally in rats (i.e., spirochetes in a mammalian host-adapted state), suggesting that temperature alone did not account for maximal upregulation of the
mlp
genes. That certain
mlp
genes are likely expressed during the growth of
B. burgdorferi
in mammalian tissues was supported by findings of antibodies against all three Mlp lipoproteins in mice after challenge with
Ixodes scapularis
nymphs harboring
B. burgdorferi
297. The combined data suggest that as opposed to being differentially expressed in any reciprocal fashion (e.g., OspA/OspC), at least three
mlp
genes are simultaneously upregulated by temperature (37°C) and some other mammalian host factor(s). The findings have importance not only for understanding alternative modes of differential antigen expression by
B. burgdorferi
but also for assessing whether one or more of the Mlp lipoproteins represent new candidate vaccinogens for Lyme disease.
CD4 T Cells and Major Histocompatibility Complex Class II Expression Influence Worm Expulsion and Increased Intestinal Muscle Contraction duringTrichinella spiralisInfection
Authors: Vallance Bruce A. , Galeazzi Francesca , Collins Stephen M. , Snider Denis P.
Published: December 2019

Expulsion of intestinal nematode parasites and the associated increased contraction by intestinal muscle are T cell dependent, since both are attenuated in athymic rodents. The CD4 T-cell subset has been strongly associated with worm expulsion; however, the relationship between these cells, antigen presentation, and worm expulsion is not definitive and the role of these factors in intestinal muscle hypercontractility has not been defined. We infected C57BL/6, athymic, CD4-deficient, CD8α-deficient, and major histocompatibility complex class II (MHC II)-deficient (C2d) mice withTrichinella spiralislarvae. We examined intestinal worm numbers, longitudinal muscle contraction, and MHC II expression. Numerous MHC II-positive cells were identified within the muscularis externa of infected but not uninfected C57BL/6 mice. C57BL/6 and CD8α-deficient mice developed large increases in muscle contraction, expelling the parasite by day 21. Athymic and C2d mice exhibited much smaller increases in muscle contraction and delayed parasite expulsion. CD4-deficient mice exhibited intermediate levels of muscle contraction and delayed parasite expulsion. To further examine the role of MHC II and CD4 T cells, we irradiated C2d mice and reconstituted them with C57BL/6 bone marrow alone or with C57BL/6 CD4 T cells. C57BL/6 bone marrow alone did not affect muscle function or worm expulsion in recipient C2d mice. Partial CD4 T-cell reconstitution was sufficient to restore increased muscle contraction but not worm expulsion. Thus, hematopoietic MHC II expression alone is insufficient for the development of muscle hypercontractility and worm expulsion, but the addition of even small numbers of CD4 T cells was sufficient to induce intestinal muscle pathophysiology.
Cell Death of Human Polymorphonuclear Neutrophils Induced by a
Pseudomonas aeruginosa
Cystic Fibrosis Isolate Requires a Functional Type III Secretion System
Authors: Dacheux D. , Attree I. , Schneider C. , Toussaint B.
Published: December 2019

With a coincubation model incorporating
Pseudomonas aeruginosa
and human polymorphonuclear neutrophils (PMNs), a cystic fibrosis (CF)
P. aeruginosa
isolate has been shown to resist the bactericidal action of PMNs and to induce their cellular death. An isogenic mutant of this CF isolate in which the type III secretion system was rendered nonfunctional was unable to induce cellular death of PMNs.
Modulation of B-Lymphocyte and NK Cell Activities by Glycoinositolphospholipid Purified fromTrypanosoma cruzi
Authors: de Arruda Hinds Luciana B. , Previato Lucia Mendonça , Previato José Osvaldo , Vos Quirijn , Mond James J. , Peçanha Ligia M. T.
Published: December 2019

Glycoinositolphospholipids (GIPLs) are some of the major glycolipids of theTrypanosoma cruzisurface that were previously shown to activate B cells. In the present study, we investigated whether (i)T. cruziGIPLs could induce immunoglobulin secretion from B cells in the absence of T cells and NK cells and whether (ii) NK cells are also stimulated by the GIPLs. B cells purified from mice deficient in both T and NK cells (CD3ɛ transgenic mice) secreted immunoglobulin in response to the GIPL. This response was increased by coculture with a murine NK cell line. TheT. cruziGIPL also increased the NK cell (interleukin-2 induced) proliferative response. Our data indicate that theT. cruziGIPL has a direct stimulatory effect on NK cells and induces immunoglobulin secretion in the absence of T lymphocytes and NK cells. These findings suggest that thisT. cruzi-derived molecule may be one of the stimulators that lead to NK cell activation duringT. cruziinfection.
Identification of Genes Coding for Exported Proteins of
Actinobacillus actinomycetemcomitans
Authors: Mintz Keith P. , Fives-Taylor Paula M.
Published: December 2019

Random fusions of genomic DNA fragments to a partial gene encoding a signal sequence-deficient bacterial alkaline phosphatase were utilized to screen for exported proteins of
Actinobacillus actinomycetemcomitans
in
Escherichia coli
. Twenty-four PhoA
+
clones were isolated and sequenced. Membrane localization signals in the form of signal sequences were deduced from most of these sequences. Several of the deduced amino acid sequences were found to be homologous to known exported or membrane-associated proteins. The complete genes corresponding to two of these sequences were isolated from an
A. actinomycetemcomitans
lambda phage library. One gene was found to be homologous to the outer membrane lipoprotein LolB. The second gene product had homology with a
Haemophilus influenzae
protein and was localized to the inner membrane of
A. actinomycetemcomitans
.
Differential Expression of Caprine β-Defensins in Digestive and Respiratory Tissues
Authors: Zhao Chengquan , Nguyen Tung , Liu Lide , Shamova Olga , Brogden Kim , Lehrer Robert I.
Published: December 2019

We identified two novel β-defensin precursors, preproGBD-1 and preproGBD-2, in the tissues of a goat. Although the precursors were identical in 96.8% of their bases and 88.2% (60 of 68) of their amino acids, preproGBD-1 was expressed principally in the tongue and respiratory tract, whereas preproGBD-2 expression predominated throughout the intestine. These findings exemplify the phenomenon of tissue-specific expression in a family of host defense peptides that arose before the avian and mammalian lineages diverged.
The Mannose Receptor Mediates Uptake of Pathogenic and Nonpathogenic Mycobacteria and Bypasses Bactericidal Responses in Human Macrophages
Authors: Astarie-Dequeker Catherine , N’Diaye Elsa-Noah , Le Cabec Veronique , Rittig Michael G. , Prandi Jacques , Maridonneau-Parini Isabelle
Published: December 2019

The mannose receptor (MR) is involved in the phagocytosis of pathogenic microorganisms. Here we investigated its role in the bactericidal functions of human monocyte-derived macrophages (MDMs), using (i) trimannoside-bovine serum albumin (BSA)-coated latex beads and zymosan as particulate ligands of the MR, and (ii) mannan and mannose-BSA as soluble ligands. We show that phagocytosis of mannosylated latex beads did not elicit the production of O2−. Zymosan, which is composed of α-mannan and β-glucan, was internalized by the MR and a β-glucan receptor, but the production of O2−was triggered only by phagocytosis through the β-glucan receptor. Activation and translocation of Hck, a Src family tyrosine kinase located on lysosomes, has previously been used as a marker of fusion between lysosomes and phagosomes in human neutrophils. In MDMs, Hck was activated and recruited to phagosomes containing zymosan later than LAMP-1 and CD63. Phagosomes containing mannosylated latex beads fused with LAMP-1 and CD63 vesicles but not with the Hck compartment, and the kinase was not activated. We also demonstrate that the MR was unable to distinguish between nonpathogenic and pathogenic mycobacteria, as they were internalized at similar rates by this receptor, indicating that this route of entry cannot be considered as a differential determinant of the intracellular fate of mycobacteria. In conclusion, MR-dependent phagocytosis is coupled neither to the activation of NADPH oxidase nor to the maturation of phagosomes until fusion with the Hck compartment and therefore constitutes a safe portal of entry for microorganisms.
Inhibition ofStaphylococcus aureusAdherence to Collagen under Dynamic Conditions
Authors: Mohamed Nehal , Teeters Mark A. , Patti Joseph M. , Höök Magnus , Ross Julia M.
Published: December 2019

Staphylococcus aureusis the most common etiological agent of bacterial arthritis and acute osteomyelitis and has been shown to bind to type II collagen under static and dynamic conditions. We have previously reported the effect of shear on the adhesion ofS. aureusPhillips to collagen and found that this process is shear dependent (Z. Li, M. Höök, J. M. Patti, and J. M. Ross, Ann. Biomed. Eng. 24[Suppl. 1]:S–55). In this study, we used recombinant collagen adhesin fragments as well as polyclonal antibodies generated against adhesin fragments in attempts to inhibit bacterial adhesion. A parallel-plate flow chamber was used in a dynamic adhesion assay, and quantification of adhesion was accomplished by phase contrast video microscopy coupled with digital image processing. We report that both recombinant fragments studied, M19 and M55, and both polyclonal antibodies studied, α-M17 and α-M55, inhibit adhesion to varying degrees and that these processes are shear dependent. The M55 peptide and α-M55 cause much higher levels of inhibition than M19 and α-M17, respectively, at all wall shear rates studied. Our results demonstrate the importance of using a dynamic system in the assessment of inhibitory strategies and suggest the possible use of M55 and α-M55 in clinical applications to prevent infections caused byS. aureusadhesion to collagen.
Neisseria gonorrhoeae
Mutants Altered in Toxicity to Human Fallopian Tubes and Molecular Characterization of the Genetic Locus Involved
Authors: Arvidson Cindy Grove , Kirkpatrick Risa , Witkamp Manon T. , Larson Jason A. , Schipper Christel A. , Waldbeser Lillian S. , O’Gaora Peadar , Cooper Morris , So Magdalene
Published: December 2019

In an effort to identify potential cytotoxins expressed by
Neisseria gonorrhoeae
, we have identified a locus that, when mutated in the gonococcus, results in a significant increase in toxicity of the strain to human fallopian tube organ cultures (HFTOC). This locus,
gly1
, contains two open reading frames (ORFs) which are likely cotranscribed. ORF1 encodes a polypeptide of 17.8 kDa with a signal sequence that is recognized and processed in
Escherichia coli
and
N. gonorrhoeae
. The 15.6-kDa processed polypeptide has been observed in membrane fractions and filtered spent media from cultures of
E. coli
expressing
gly1
and in outer membrane preparations of wild-type
N. gonorrhoeae
. The
gly1
locus is not essential for bacterial survival, and it does not play a detectable role in epithelial cell adhesion, invasion, or intracellular survival. However, a
gly1
null mutant causes much more damage to fallopian tube tissues than its isogenic wild-type parent. A strain complemented in
trans
for the
gly1
mutation showed a level of toxicity to HFTOC similar to the level elicited by the wild-type parent. Taken together, these results indicate an involvement of the
gly1
locus in the toxicity of
N. gonorrhoeae
to human fallopian tubes.
Use of an Isogenic Mutant Constructed in
Moraxella catarrhalis
To Identify a Protective Epitope of Outer Membrane Protein B1 Defined by Monoclonal Antibody 11C6
Authors: Luke Nicole R. , Russo Thomas A. , Luther Neal , Campagnari Anthony A.
Published: December 2019

Moraxella catarrhalis
-induced otitis media continues to be a significant cause of infection in young children, prompting increased efforts at identifying effective vaccine antigens. We have previously demonstrated that
M. catarrhalis
expresses specific outer membrane proteins (OMPs) in response to iron limitation and that this organism can utilize transferrin and lactoferrin for in vitro growth. One of these proteins, which binds human transferrin, is OMP B1. As the human host presents a naturally iron-limited environment, proteins, like OMP B1, which are expressed in response to this nutritional stress are potential vaccine antigens. In this study, we have developed monoclonal antibody (MAb) 11C6, which reacts to a surface-exposed epitope of OMP B1 expressed by
M. catarrhalis
7169. This antibody was used to clone
ompB1
, and sequence analysis suggested that OMP B1 is the
M. catarrhalis
homologue to the transferrin binding protein B described for pathogenic
Neisseriaceae
,
Haemophilus influenzae
,
Actinobacillus pleuropneumoniae
, and
M. catarrhalis
. Expression of recombinant OMP B1 on the surface of
Escherichia coli
confers transferrin binding activity, confirming that this protein is likely involved in iron acquisition. In addition,
ompB1
was used to construct an isogenic mutant in
M. catarrhalis
7169. This mutant, termed 7169b12, was used as the control in bactericidal assays designed to determine if OMP B1 elicits protective antibodies. In the presence of MAb 11C6 and human complement, wild-type 7169 demonstrated a 99% decline in viability, whereas the
ompB1
isogenic mutant was resistant to this bactericidal activity. Further analysis with MAb 11C6 revealed the presence of this OMP B1 epitope on 31% of the clinical isolates tested. These data suggest that OMP B1 is a potential vaccine antigen against
M. catarrhalis
infections.
The R-Type Pyocin of
Pseudomonas aeruginosa
C Is a Bacteriophage Tail-Like Particle That Contains Single-Stranded DNA
Authors: Lee Frank K. N. , Dudas Kathleen C. , Hanson Julie A. , Nelson M. Bud , LoVerde Philip T. , Apicella Michael A.
Published: December 2019

Pseudomonas aeruginosa
R-type pyocin particles have been described as bacteriocins that resemble bacteriophage tail-like structures. Because of their unusual structure, we reexamined whether they contained nucleic acids. Our data indicated that pyocin particles isolated from
P. aeruginosa
C (pyocin C) contain DNA. Probes generated from this DNA by the random-primer extension method hybridized to distinct bands in restriction endonuclease-digested
P. aeruginosa
C genomic DNA. These probes also hybridized to genomic DNA from 6 of 18
P. aeruginosa
strains that produced R-type pyocins. Asymmetric PCR, complementary oligonucleotide hybridization, and electron microscopy indicated that pyocin C particles contained closed circular single-stranded DNA, approximately 4.0 kb in length. Examination of total intracellular DNA from mitomycin C-induced cultures revealed the presence of two extrachromosomal DNA molecules, a double-stranded molecule and a single-stranded molecule, which hybridized to pyocin DNA. Sequence analysis of 7,480 nucleotides of
P. aeruginosa
C chromosomal DNA containing the pyocin DNA indicated the presence of pyocin open reading frames with similarities to open reading frames from filamentous phages and cryptic phage elements. We did not observe any similarities to known phage structural proteins or previously characterized pseudomonal
prt
genes expressing R-type pyocin structural proteins. These studies demonstrate that pyocin particles from
P. aeruginosa
C are defective phages that contain a novel closed circular single-stranded DNA and that this DNA was derived from the chromosome of
P. aeruginosa
C.
Monocytes That Have IngestedYersinia enterocoliticaSerotype O:3 Acquire Enhanced Capacity To Bind to Nonstimulated Vascular Endothelial Cells via P-Selectin
Authors: Wuorela Maarit , Tohka Sami , Granfors Kaisa , Jalkanen Sirpa
Published: December 2019

Reactive arthritis is usually a self-limiting polyarthritis which develops after certain gastrointestinal or urogenital infections. Microbial antigens found in the inflamed joints are thought to play a key role in the development of this disease. It is not known how antigens of the pathogenic organisms migrate from the mucosal tissues into the joints. The data presented here show that mononuclear phagocytes which mediate the dissemination of several intracellular pathogens acquire an enhanced capacity to bind to nonstimulated vascular endothelial cells after phagocytosis ofYersinia enterocoliticaO:3, one of the causative organisms of reactive arthritis. The increased binding to previously nonstimulated endothelial cells was mediated by P-selectin, whose translocation to the endothelial cell surface was induced by monocytes with intracellularYersiniabacteria. These results suggest that mononuclear phagocytes may be responsible for the dissemination of bacterial antigens and the initiation of the joint inflammation in reactive arthritis.
Impact of the High-Affinity Proline Permease Gene (
putP
) on the Virulence of
Staphylococcus aureus
in Experimental Endocarditis
Authors: Bayer Arnold S. , Coulter Silvija N. , Stover C. Kendall , Schwan William R.
Published: December 2019

Staphylococcus aureus
causes a wide variety of invasive human infections. However, delineation of the genes which are essential for the in vivo survival of this pathogen has not been accomplished to date. Using signature tag mutagenesis techniques and large mutant pool screens, previous investigators identified several major gene classes as candidate essential gene loci for in vivo survival; these include genes for amino acid transporters, oligopeptide transporters, and lantibiotic synthesis (W. R. Schwan, S. N. Coulter, E. Y. W. Ng, M. H. Langhorne, H. D. Ritchie, L. L. Brody, S. Westbrock-Wadman, A. S. Bayer, K. R. Folger, and C. K. Stover, Infect. Immun. 66:567–572, 1998). In this study, we directly compared the virulence of four such isogenic signature tag mutants with that of the parental strain (RN6390) by using a prototypical model of invasive
S. aureus
infection, experimental endocarditis (IE). The oligonucleotide signature tag (OST) mutant with insertional inactivation of the gene (
putP
) which encodes the high-affinity transporter for proline uptake exhibited significantly reduced virulence in the IE model across three challenge inocula (10
4
to 10
6
CFU) in terms of achievable intravegetation densities (
P
, <0.05). The negative impact of
putP
inactivation on in vivo survival in the IE model was confirmed by simultaneous challenge with the original
putP
mutant and the parental strain as well as by challenge with a
putP
mutant in which this genetic inactivation was transduced into a distinct parental strain (S6C). In contrast, inactivation of loci encoding an oligopeptide transporter, a purine repressor, and lantibiotic biosynthesis had no substantial impact on the capacity of OST mutants to survive within IE vegetations. Thus, genes encoding the uptake of essential amino acids may well represent novel targets for new drug development. These data also confirm the utility of the OST technique as an important screening methodology for identifying candidate genes as requisite loci for the in vivo survival of
S. aureus.
Intranasal Immunization Confers Protection against MurinePneumocystis cariniiLung Infection
Authors: Pascale Juan M. , Shaw Margaret M. , Durant Pamela J. , Amador Aytza A. , Bartlett Marilyn S. , Smith James W. , Gregory Richard L. , McLaughlin Gerald L.
Published: December 2019

To evaluate the feasibility of mucosal immunization againstPneumocystis carinii(Pc) experimental infection, female BALB/c mice were intranasally immunized three times with soluble Pc antigens plus cholera toxin fraction B (Pc-CTB); control groups received either Pc antigen, CTB, or phosphate-buffered saline (PBS) alone. Two weeks after the last immunization, five animals from each group were sacrificed, and cellular and humoral immune responses were evaluated. The remaining five mice were CD4 depleted using a monoclonal antibody against mouse CD4 and inoculated with viable Pc. Significantly higher specific lymphoproliferative responses from tracheobronchial lymph node cells, immunoglobulin M (IgM) and IgG antibody levels in serum, and bronchoalveolar lavage (BAL)-derived IgA antibody concentrations were observed in the Pc-CTB group of mice relative to control groups (P< 0.01). Five weeks after challenge, no Pc organisms were observed in the lung smears of the Pc-CTB group, while the animals receiving antigen, adjuvant, or PBS had progressively higher numbers of Pc microorganisms. By Western blot analysis, a strongly reactive 55- to 60-kDa antigen was recognized by BAL IgA and by serum IgG. In summary, mucosal immunization elicited specific cellular and humoral immune responses and protected against Pc lung infection after immunosuppression.
Characterization of a WaaF (RfaF) Homolog Expressed by
Haemophilus ducreyi
Authors: Bauer Beth A. , Lumbley Sheryl R. , Hansen Eric J.
Published: December 2019

Haemophilus ducreyi
lipooligosaccharide (LOS) is capable of inducing an inflammatory response in skin (A. A. Campagnari, L. M. Wild, G. Griffiths, R. J. Karalus, M. A. Wirth, and S. M. Spinola, Infect. Immun. 59:2601–2608, 1991) and likely contributes to the virulence of this sexually transmitted pathogen (B. A. Bauer, M. K. Stevens, and E. J. Hansen, Infect. Immun. 68:4290–4298, 1998). An open reading frame in
H. ducreyi
35000 was found to encode a predicted protein that was 59% identical to the protein product of the
rfaF
(
waaF
) gene of
Salmonella typhimurium
. The
H. ducreyi waaF
gene was able to complement an
S. typhimurium rfaF
(
waaF
) mutant, a result which confirmed the identity of this gene. In contrast to the
rfaF
(
waaF
) gene of enteric bacteria, the
H. ducreyi waaF
gene was not located adjacent to other genes involved in lipopolysaccharide expression. Inactivation of the
H. ducreyi waaF
gene by insertion mutagenesis resulted in expression of a LOS that migrated much faster than wild-type LOS in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The LOS of this mutant also did not bind a monoclonal antibody directed against a cell surface-exposed epitope of wild-type
H. ducreyi
LOS. Testing of the wild-type
H. ducreyi
strain and its isogenic
waaF
mutant in the temperature-dependent rabbit model for dermal lesion production by
H. ducreyi
revealed that this
waaF
mutant was less virulent than the wild-type parent strain. Complementation of the
H. ducreyi waaF
mutant with the wild-type
H. ducreyi waaF
gene resulted in expression of both wild-type LOS and wild-type virulence by this mutant.
Antigen-Specific T-Cell Responses in Humans after Intranasal Immunization with a Meningococcal Serogroup B Outer Membrane Vesicle Vaccine
Authors: Oftung Fredrik , Næss Lisbeth Meyer , Wetzler Lee M. , Korsvold Gro Ellen , Aase Audun , Høiby E. Arne , Dalseg Rolf , Holst Johan , Michaelsen Terje E. , Haneberg Bjørn
Published: December 2019

We have studied the ability of the Norwegian group B meningococcal outer membrane vesicle (OMV) vaccine, when administered intranasally without adjuvant, to induce T-cell responses in humans. A group of 12 vaccinees was immunized with four doses of OMVs (250 μg of protein/dose) at weekly intervals, and a single booster dose was given 5 months later. In vitro T-cell proliferation in response to the OMV vaccine, purified PorA (class 1) protein, PorB (class 3) protein, and one unrelated control antigen (Mycobacterium bovisBCG) was measured by [3H]thymidine incorporation into peripheral blood mononuclear cells obtained from the vaccinees before and after the immunizations. The nasal OMV immunizations induced antigen-specific T-cell responses in the majority of the vaccinees when tested against OMVs (7 of 12) and the PorA antigen (11 of 12). None of the vaccinees showed a vaccine-induced T-cell response to the PorB antigen after the initial four doses. Although some individuals responded to all the vaccine antigens after the booster dose, this response was not significant when the vaccinees were analyzed as a group. We have also demonstrated that the PorA antigen-specific T-cell responses correlated with anti-OMV immunoglobulin A (IgA) levels in nasal secretions, with anti-OMV IgG levels in serum, and with serum bactericidal activity. In conclusion, we have shown that it is possible to induce antigen-specific T-cell responses in humans by intranasal administration of a meningococcal OMV vaccine without adjuvant.
Cloning of the Gene Encoding the
Actinobacillus actinomycetemcomitans
Serotype b OmpA-Like Outer Membrane Protein
Authors: Komatsuzawa Hitoshi , Kawai Toshihisa , Wilson Mark E. , Taubman Martin A. , Sugai Motoyuki , Suginaka Hidekazu
Published: December 2019

The gene encoding an outer membrane protein A (OmpA)-like, heat-modifiable Omp of
Actinobacillus actinomycetemcomitans
ATCC 43718 (strain Y4, serotype b) was cloned by a PCR cloning procedure. DNA sequence analysis revealed that the gene encodes a protein of 346 amino acid residues with a molecular mass of 36.9 kDa. The protein expressed by the cloned gene reacted with a monoclonal antibody to the previously described 29-kDa Omp (Omp29) of strain Y4. This monoclonal antibody reacted specifically with Omp29 of
A. actinomycetemcomitans
(serotype b), but not with any Omp of
Escherichia coli
, including OmpA. This protein exhibited characteristic heat modifiability on sodium dodecyl sulfate-polyacrylamide gels, showing an apparent molecular mass of 29 kDa when unheated and a mass of 34 kDa when heated. The N-terminal amino acid sequence of the protein expressed in
E. coli
perfectly matched those deduced from the purified Omp29 of strain Y4. The deduced amino acid sequence of the gene coding for Omp29 from serotype b matched completely (except for valine at position 321) that of a recently reported
omp34
gene described for
A. actinomycetemcomitans
serotype c (NCTC 9710). Because of the conserved nature of the gene within these serotypes, we designated the gene described herein from serotype b as
omp34
.
Human Intestinal M Cells Display the Sialyl Lewis A Antigen
Authors: Giannasca Paul J. , Giannasca Karen T. , Leichtner Alan M. , Neutra Marian R.
Published: December 2019

The biochemical features that distinguish human M cells from other intestinal epithelial cell types are important for understanding microbial pathogenesis and for targeting vaccines to the mucosal immune system. We applied a large panel of carbohydrate-specific monoclonal antibodies and lectins to Peyer’s patch and cecum biopsy specimens from three normal individuals and a patient with inflammatory bowel disease. The results show that human M-cell glycosylation patterns are distinct from those of other species examined and that human M cells preferentially display the sialyl Lewis A antigen. This carbohydrate epitope is also present in a small subpopulation of enterocytes in the follicle-associated epithelium and in goblet cell mucins.
Pseudomonas aeruginosaKeratitis in Knockout Mice Deficient in Intercellular Adhesion Molecule 1
Authors: Hobden Jeffery A. , Masinick-McClellan Sharon , Barrett Ronald P. , Bark Kenneth S. , Hazlett Linda D.
Published: December 2019

In this study, the role of intercellular adhesion molecule 1 (ICAM-1) in the pathogenesis ofPseudomonas aeruginosakeratitis was examined by using inbred ICAM-1-deficient knockout mice. These mice had significantly less (P≤ 0.02) ocular disease than wild-type mice, suggesting that ICAM-1 contributes to a more severe disease response followingP. aeruginosainfection.
Salmonella typhimuriumand Lipopolysaccharide Stimulate Extracellularly Regulated Kinase Activation in Macrophages by a Mechanism Involving Phosphatidylinositol 3-Kinase and Phospholipase D as Novel Intermediates
Authors: Procyk Katarzyna J. , Kovarik Pavel , von Gabain Alexander , Baccarini Manuela
Published: December 2019

Activation of the extracellularly regulated kinase (ERK) pathway is part of the early biochemical events that follow lipopolysaccharide (LPS) treatment of macrophages or their infection by virulent and attenuatedSalmonellastrains. Phagocytosis as well as the secretion of invasion-associated proteins is dispensable for ERK activation by the pathogen. Furthermore, the pathways used bySalmonellaand LPS to stimulate ERK are identical, suggesting that kinase activation might be solely mediated by LPS. Both stimuli activate ERK by a mechanism involving herbimycin-dependent tyrosine kinase(s) and phosphatidylinositol 3-kinase. Phospholipase D activation and stimulation of protein kinase C appear to be intermediates in this novel pathway of MEK/ERK activation.
The Staphylococcal Transferrin-Binding Protein Is a Cell Wall Glyceraldehyde-3-Phosphate Dehydrogenase
Authors: Modun Belinda , Williams Paul
Published: December 2019

Staphylococcus aureus
and
Staphylococcus epidermidis
possess a 42-kDa cell wall transferrin-binding protein (Tpn) which is involved in the acquisition of transferrin-bound iron. To characterize this protein further, cell wall fractions were subjected to two-dimensional sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis blotted, and the N-terminus of Tpn was sequenced. Comparison of the first 20 amino acid residues of Tpn with the protein databases revealed a high degree of homology to the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Analysis of staphylococcal cell wall fractions for GAPDH activity confirmed the presence of a functional enzyme which, like Tpn, is regulated by the availability of iron in the growth medium. To determine whether Tpn is responsible for this GAPDH activity, it was affinity purified with NAD
+
agarose. Both
S. epidermidis
and
S. aureus
Tpn catalyzed the conversion of glyceraldehyde-3-phosphate to 1,3-diphosphoglycerate. In contrast,
Staphylococcus saprophyticus
, which lacks a Tpn, has no cell wall-associated GAPDH activity. Native polyacrylamide gel electrophoresis of the affinity-purified Tpn revealed that it was present in the cell wall as a tetramer, consistent with the structures of all known cytoplasmic GAPDHs. Furthermore, the affinity-purified Tpn retained its ability to bind human transferrin both in its native tetrameric and SDS-denatured monomeric forms. Apart from interacting with human transferrin, Tpn, in common with the group A streptococcal cell wall GAPDH, binds human plasmin. Tpn-bound plasmin is enzymatically active and therefore may contribute to the ability of staphylococci to penetrate tissues during infections. These studies demonstrate that the staphylococcal transferrin receptor protein, Tpn, is a multifunctional cell wall GAPDH.
Identification and Characterization of a Novel Ibe10 Binding Protein That Contributes to
Escherichia coli
Invasion of Brain Microvascular Endothelial Cells
Authors: Prasadarao Nemani V. , Wass Carol A. , Huang Sheng-He , Kim Kwang Sik
Published: December 2019

The molecular basis of
Escherichia coli
traversal of the blood-brain barrier in the development of
E. coli
meningitis is not well understood. We have previously shown that a novel Ibe10 protein found in cerebrospinal fluid isolates of
E. coli
is necessary for invasion of the brain microvascular endothelial cells (BMEC) that constitute the blood-brain barrier both in vitro and in a newborn rat model of hematogenous meningitis. Here we identified a novel Ibe10 binding molecule/receptor (Ibe10R) on both bovine BMEC (HBMEC) and human BMEC (HBMEC) that is responsible for invasion by
E. coli
. Ibe10R, an approximately 55-kDa protein, was purified from BBMEC by Ibe10-Ni-Sepharose affinity chromatography. Bovine Ibe10R, as well as polyclonal antibodies to Ibe10R, blocked
E. coli
invasion of BBMEC very effectively. The N-terminal amino acid sequence of Ibe10R showed 75% homology to serum albumin. However, the amino acid sequence of an Ibe10R fragment generated by limited enzymatic digestion did not reveal homology to any other proteins, suggesting that Ibe10R represents a novel albumin-like protein. Immunocytochemical analysis of BBMEC using anti-Ibe10R antibody suggested that only a subset of cultured BBMEC express Ibe10R on their surface. Enrichment of Ibe10R-positive BBMEC by fluorescence-activated cell sorting with anti-Ibe10R antibody resulted in enhanced invasion by
E. coli
. The anti-Ibe10R antibody raised against bovine Ibe10R also blocked
E. coli
invasion of HBMEC very effectively. Interestingly, anti-Ibe10R antibody affinity chromatography of HBMEC membrane proteins revealed a smaller protein with an approximate molecular mass of 45 kDa. These results suggest that the Ibe10 of
E. coli
interacts with a novel BMEC surface protein, Ibe10R, for invasion of both BBMEC and HBMEC.
Cloning and Expression of the
dnaK
Gene of
Campylobacter jejuni
and Antigenicity of Heat Shock Protein 70
Authors: Thies Frank L. , Karch Helge , Hartung Hans-Peter , Giegerich Gerhard
Published: December 2019

Campylobacter jejuni
is a leading cause of infectious diarrhea throughout the world. In addition, there is growing evidence that Guillain-Barré syndrome, an inflammatory demyelinating disease of the peripheral nervous system, is frequently preceded by
C. jejuni
infection. In the present study, the
hrcA-grpE-dnaK
gene cluster of
C. jejuni
was cloned and sequenced. The
dnaK
gene consists of an open reading frame of 1,869 bp and encodes a protein with a high degree of homology to other bacterial 70-kDa heat shock proteins (HSPs). The overall percentages of identity to the HSP70 proteins of
Helicobacter pylori
,
Borrelia burgdorferi
,
Chlamydia trachomatis
, and
Bacillus subtilis
were calculated to be 78.1, 60.5, 57.2, and 53.8%, respectively. Regions similar to the
Escherichia coli
ς
70
promoter consensus sequence and to a
cis
-acting regulatory element (CIRCE) are located upstream of the
hrcA
gene. Following heat shock, a rapid increase of
dnaK
mRNA was detectable, which reached its maximum after 20 to 30 min. A 6-His-tagged recombinant DnaK protein (rCjDnaK-His) was generated in
E. coli
, after cloning of the
dnaK
coding region into pET-22b(+), and purified by affinity and gel filtration chromatography. Antibody responses to rCjDnaK-His were significantly elevated, compared to those of healthy individuals, in about one-third of the serum specimens obtained from
C. jejuni
enteritis patients.
Yops ofYersinia enterocoliticaInhibit Receptor-Dependent Superoxide Anion Production by Human Granulocytes
Authors: Visser L. G. , Seijmonsbergen E. , Nibbering P. H. , van den Broek P. J. , van Furth R.
Published: December 2019

The virulence plasmid-borne genes encodingYersiniaadhesin A (YadA) and severalYersiniasecreted proteins (Yops) are involved in the inhibition of phagocytosis and killing ofYersinia enterocoliticaby human granulocytes. One of these Yops, YopH, dephosphorylates multiple tyrosine-phosphorylated proteins in eukaryotic cells and is involved in the inhibition of phagocytosis ofY. enterocoliticaby human granulocytes. We investigated whether antibody- and complement-opsonized plasmid-bearing (pYV+)Y. enterocoliticainhibits O2−production by human granulocytes in response to various stimuli and whether YopH is involved. Granulocytes were preincubated with mutant strains unable to express YadA or to secrete Yops or YopH. O2−production by granulocytes during stimulation was assessed by measuring the reduction of ferricytochromec. PYV+Y. enterocoliticainhibited O2−production by granulocytes incubated with opsonizedY. enterocoliticaorN-formyl-Met-Leu-Phe (f-MLP). This inhibitory effect mediated by pYV did not affect receptor-independent O2−production by granulocytes in response to phorbol myristate acetate, indicating that NADPH activity remained unaffected after activation of protein kinase C. The inhibition of f-MLP-induced O2−production by granulocytes depends on the secretion of Yops and not on the expression of YadA. Insertional inactivation of theyopHgene abrogated the inhibition of phagocytosis of antibody- and complement-opsonizedY. enterocoliticaby human granulocytes but not of the f-MLP-induced O2−production by granulocytes or tyrosine phosphorylation of granulocyte proteins. These findings suggest that the specific targets for YopH are not present in f-MLP receptor-linked signal transduction and that other Yop-mediated mechanisms are involved.
Role of the Extracellular Signal-Regulated Protein Kinase Cascade in Human Neutrophil Killing ofStaphylococcus aureusandCandida albicansand in Migration
Authors: Hii Charles S. T. , Stacey Kathryn , Moghaddami Nahid , Murray Andrew W. , Ferrante Antonio
Published: December 2019

Killing ofStaphylococcus aureusandCandida albicansby neutrophils involves adherence of the microorganisms, phagocytosis, and a collaborative action of oxygen reactive species and components of the granules. While a number of intracellular signalling pathways have been proposed to regulate neutrophil responses, the extent to which each pathway contributes to the killing ofS. aureusandC. albicanshas not been clearly defined. We have therefore examined the effect of blocking one such pathway, the extracellular signal-regulated protein kinase (ERK) cascade, using the specific inhibitor of the mitogen-activated protein kinase/ERK kinase, PD98059, on the ability of human neutrophils to killS. aureusandC. albicans. Our data demonstrate the presence of ERK2 and a 43-kDa form of ERK but not ERK1 in human neutrophils. Upon stimulation with formyl methionyl leucyl phenylalanine (fMLP), the activities of both ERK2 and the 43-kDa form were stimulated. Despite abrogating the activity of both ERK forms, PD98059 only slightly reduced the ability of neutrophils to killS. aureusorC. albicans. This is consistent with our finding that PD98059 had no effect on neutrophil adherence or degranulation, although pretreatment of neutrophils with PD98059 inhibited fMLP-stimulated superoxide production by 50%, suggesting that a change in superoxide production per se is not strictly correlated with microbicidal activity. However, fMLP-stimulated chemokinesis was markedly inhibited, while random migration and fMLP-stimulated chemotaxis were partially inhibited, by PD98059. These data demonstrate, for the first time, that the ERK cascade plays only a minor role in the microbicidal activity of neutrophils and that the ERK cascade is involved primarily in regulating neutrophil migration in response to fMLP.
Human Granulocytic Ehrlichiosis Agent andEhrlichia chaffeensisReside in Different Cytoplasmic Compartments in HL-60 Cells
Authors: Mott Jason , Barnewall Roy E. , Rikihisa Yasuko
Published: December 2019

The human granulocytic ehrlichiosis (HGE) agent resides and multiplies exclusively in cytoplasmic vacuoles of granulocytes. Double immunofluorescence labeling was used to characterize the nature of the HGE agent replicative inclusions and to compare them with inclusions containing the human monocytic ehrlichia,Ehrlichia chaffeensis, in HL-60 cells. Although bothEhrlichiaspp. can coinfect HL-60 cells, they resided in separate inclusions. Inclusions of bothEhrlichiaspp. were not labeled with either anti-lysosome-associated membrane protein 1 or anti-CD63. Accumulation of myeloperoxidase-positive granules were seen around HGE agent inclusions but not aroundE. chaffeensisinclusions. 3-(2,4-Dinitroanilino)-3′-amino-N-methyldipropylamine and acridine orange were not localized to either inclusion type. Vacuolar-type H+-ATPase was not colocalized with HGE agent inclusions but was weakly colocalized withE. chaffeensisinclusions.E. chaffeensisinclusions were labeled with the transferrin receptor, early endosomal antigen 1, and rab5, but HGE agent inclusions were not. Some HGE agent andE. chaffeensisinclusions colocalized with major histocompatibility complex class I and II antigens. These two inclusions were not labeled for annexins I, II, IV, and VI; α-adaptin; clathrin heavy chain; or β-coatomer protein. Vesicle-associated membrane protein 2 colocalized to both inclusions. The cation-independent mannose 6-phosphate receptor was not colocalized with either inclusion type. Endogenously synthesized sphingomyelin, from C6-NBD-ceramide, was not incorporated into either inclusion type. Brefeldin A did not affect the growth of eitherEhrlichiasp. in HL-60 cells. These results suggest that the HGE agent resides in inclusions which are neither early nor late endosomes and does not fuse with lysosomes or Golgi-derived vesicles, whileE. chaffeensisresides in an early endosomal compartment which accumulates the transferrin receptor.
Characterization of Human Bactericidal Antibodies toBordetella pertussis
Authors: Weiss Alison A. , Mobberley Paula S. , Fernandez Rachel C. , Mink ChrisAnna M.
Published: December 2019

TheBordetella pertussisBrkA protein protects against the bactericidal activity of complement and antibody; however, some individuals mount an immune response that overcomes this bacterial defense. To further characterize this process, the bactericidal activities of sera from 13 adults with different modes of exposure toB. pertussis(infected as adults, occupational exposure, immunized with an acellular vaccine, or no identified exposure) against a wild-type strain and a BrkA complement-sensitive mutant were evaluated. All of the sera killed the BrkA mutant, suggesting past exposure toB. pertussisor cross-reactive organisms. Several samples had no or minimal activity against the wild type. All of the sera collected from the infected and occupationally exposed individuals but not all of the sera from vaccinated individuals had bactericidal activity against the wild-type strain, suggesting that some types of exposure can induce an immune response that can overcome the BrkA resistance mechanism. Adsorbing serum with the wild-type strain removed the bactericidal antibodies; however, adsorbing the serum with a lipopolysaccharide (LPS) mutant or an avirulent (bvgmutant) strain did not always result in loss of bactericidal activity, suggesting that antibodies to either LPS orbvg-regulated proteins could be bactericidal. All the samples, including those that lacked bactericidal activity, contained antibodies that recognized the LPS ofB. pertussis. Bactericidal activity correlated best with the presence of the immunoglobulin G3 (IgG3) antibodies to LPS, the IgG subtype that is most effective at fixing complement.