bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of Fur, Aconitase, and Other Proteins Expressed by
Mycobacterium tuberculosis
under Conditions of Low and High Concentrations of Iron by Combined Two-Dimensional Gel Electrophoresis and Mass Spectrometry
Authors: Wong Diane K. , Lee Bai-Yu , Horwitz Marcus A. , Gibson Bradford W.
Published: December 2019

Iron plays a critical role in the pathophysiology of
Mycobacterium tuberculosis
. To gain a better understanding of iron regulation by this organism, we have used two-dimensional (2-D) gel electrophoresis, mass spectrometry, and database searching to study protein expression in
M. tuberculosis
under conditions of high and low iron concentration. Proteins in cellular extracts from
M. tuberculosis
Erdman strain grown under low-iron (1 μM) and high-iron (70 μM) conditions were separated by 2-D polyacrylamide gel electrophoresis, which allowed high-resolution separation of several hundred proteins, as visualized by Coomassie staining. The expression of at least 15 proteins was induced, and the expression of at least 12 proteins was decreased under low-iron conditions. In-gel trypsin digestion was performed on these differentially expressed proteins, and the digestion mixtures were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry to determine the molecular masses of the resulting tryptic peptides. Partial sequence data on some of the peptides were obtained by using after source decay and/or collision-induced dissociation. The fragmentation data were used to search computerized peptide mass and protein sequence databases for known proteins. Ten iron-regulated proteins were identified, including Fur and aconitase proteins, both of which are known to be regulated by iron in other bacterial systems. Our study shows that, where large protein sequence databases are available from genomic studies, the combined use of 2-D gel electrophoresis, mass spectrometry, and database searching to analyze proteins expressed under defined environmental conditions is a powerful tool for identifying expressed proteins and their physiologic relevance.
Generation of Multinucleated Giant Cells In Vitro by Culture of Human Monocytes withMycobacterium bovisBCG in Combination with Cytokine-Containing Supernatants
Authors: Gasser Annette , Möst Johannes
Published: December 2019

Multinucleated giant cells (MGC), a characteristic feature of tuberculous granulomas, form by fusion of monocytes or macrophages, but little is known about the mechanism of the fusion process itself. Several studies report an indirect effect of mycobacteria, i.e., induction of a soluble lymphocyte-derived fusion factor following stimulation by mycobacteria or mycobacterial products. The aim of our study was to determine whether contact with mycobacteria can induce MGC formation from human monocytes in vitro. Stimulation of monocytes withMycobacterium bovisbacillus Calmette-Guérin (BCG) in combination with cytokine-containing supernatants of herpesvirus saimiri-transformed human T-cell clones (T-SN) led to MGC formation with fusion rates of about 27%. In contrast, stimulation with one component alone induced only low fusion rates of up to 10%. Heat-killed BCG in combination with T-SN induced monocyte fusion to the same extent as live mycobacteria. BCG culture supernatant, BCG lysate, or inert particles in combination with T-SN did not induce MGC formation. Experiments using transwell plates containing a semipermeable membrane revealed that induction of the fusion process is dependent on direct contact of monocytes and mycobacteria. MGC formation induced by BCG plus T-SN could be inhibited by addition of monoclonal antibodies to gamma interferon (but not tumor necrosis factor alpha) as well as to the β chain (CD18) of β2-integrins. These results demonstrate that contact with mycobacteria in combination with cytokine-containing supernatants is able to induce human monocytes to form MGC and that membrane-bound molecules of mycobacteria and monocytes are involved in the fusion process.
Cathelicidin Gene Expression in Porcine Tissues: Roles in Ontogeny and Tissue Specificity
Authors: Wu Hua , Zhang Guolong , Ross Christopher R. , Blecha Frank
Published: December 2019

Cathelicidins constitute a family of mammalian antimicrobial peptides that are synthesized in the bone marrow as prepropeptides, stored in neutrophil granules as propeptides, and released as active, mature peptides upon neutrophil degranulation. We investigated the developmental expression of two porcine cathelicidins, PR-39 and protegrin. Both cathelicidins were expressed constitutively in the bone marrow of all pigs at all of the ages tested. Peripheral blood neutrophils from young pigs expressed PR-39 and protegrin mRNA, which were not detectable at 42 days of age. At earlier ages, expression of PR-39 mRNA was detected in the kidney and liver and several lymphoid organs, including the thymus, spleen, and mesenteric lymph nodes, but disappeared at 4 weeks of age. These data provide the first evidence of cathelicidin gene expression in peripheral leukocytes and may indicate a role for these antimicrobial peptides in the development of host defense mechanisms.
Adhesin Expression in Matched Nasopharyngeal and Middle Ear Isolates of Nontypeable
Haemophilus influenzae
from Children with Acute Otitis Media
Authors: Krasan Graham P. , Cutter David , Block Stan L. , St. Geme Joseph W.
Published: December 2019

The HMW1 and HMW2 proteins, Hia, and hemagglutinating pili are important adherence factors in nontypeable
Haemophilus influenzae
. To gain insight into the relative importance of these adhesins in nasopharyngeal colonization and localized respiratory tract disease, we assessed their expression in matched nasopharyngeal and middle ear isolates of nontypeable
H. influenzae
from 17 children with acute otitis media. In all patients, including 11 with bilateral disease, the matched isolates were isogenic based on total protein profiles and genomic fingerprints. Of the nasopharyngeal isolates, 14 expressed only HMW1/HMW2-like proteins, 1 expressed only Hia, 1 expressed only pili, and 1 expressed both Hia and pili. Further analysis revealed concordance between nasopharyngeal isolates and the matched middle ear isolates for expression of the HMW1/HMW2-like proteins and Hia. In contrast, in the two children whose nasopharynges were colonized by piliated organisms, the corresponding middle ear isolates were nonpiliated and could not be enriched for piliation. Nevertheless, Southern analysis revealed that these two middle ear isolates contained all five
hif
genes required for pilus biogenesis and had no evidence of major genetic rearrangement. In summary, the vast majority of isolates of nontypeable
H. influenzae
associated with acute otitis media express HMW1/HMW2-like proteins, with expression present in both the nasopharynx and the middle ear. A smaller fraction of nasopharyngeal isolates express pili, while isogenic strains recovered from the middle ear are often refractory to enrichment for piliation. We speculate that the HMW adhesins and Hia are important at multiple steps in the pathogenesis of otitis media while pili contribute to early colonization and then become dispensable.
Localization of the Intracellular Activity Domain ofPasteurella multocidaToxin to the N Terminus
Authors: Wilson Brenda A. , Ponferrada Virgilio G. , Vallance Jefferson E. , Ho Mengfei
Published: December 2019

We have shown thatPasteurella multocidatoxin (PMT) directly causes transient activation of Gqα protein that is coupled to phosphatidylinositol-specific phospholipase Cβ1 inXenopusoocytes (B. A. Wilson, X. Zhu, M. Ho, and L. Lu, J. Biol. Chem. 272:1268–1275, 1997). We found that antibodies directed against an N-terminal peptide of PMT inhibited the toxin-induced response inXenopusoocytes, but antibodies against a C-terminal peptide did not. To test whether the intracellular activity domain of PMT is localized to the N terminus, we conducted a deletion mutational analysis of the PMT protein, using theXenopusoocyte system as a means of screening for toxin activity. Using PCR and conventional cloning techniques, we cloned from a toxinogenic strain ofP. multocidathe entiretoxAgene, encoding the 1,285-amino-acid PMT protein, and expressed the recombinant toxin as a His-tagged fusion protein inEscherichia coli. We subsequently generated a series of N-terminal and C-terminal deletion mutants and expressed the His-tagged PMT fragments inE. coli. These proteins were screened for cytotoxic activity on cultured Vero cells and for intracellular activity in theXenopusoocyte system. Only the full-length protein without the His tag exhibited activity on Vero cells. The full-length PMT and N-terminal fragments containing the first 500 residues elicited responses in oocytes, but the C-terminal 780 amino acid fragment did not. Our results confirm that the intracellular activity domain of PMT is localized to the N-terminal 500 amino acids of the protein and that the C terminus is required for entry into cells.
An Opsonizing Monoclonal Antibody That Recognizes a Noncapsular Epitope Expressed on
Cryptococcus neoformans
Authors: Merkel Glenn J. , Scofield Barbara A.
Published: December 2019

A mouse hybridoma secreting a monoclonal antibody (MAb) that bound a noncapsular epitope expressed on
C. neoformans
was developed by immunizing BALB/c mice with formalin-killed serotype A yeasts. The hybridoma, designated CSFi, secreted an immunoglobulin G2b MAb that reacted with all
C. neoformans
serotypes tested, including the acapsular mutant ATCC 52817 (Cap67). Postsectioned immune electron microscopy revealed extensive binding of the MAb to the cell walls of both encapsulated and acapsular yeasts. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis of secreted antigens recovered from concentrated culture supernatants from both encapsulated and acapsular strains was conducted. The results showed that this MAb bound predominantly to antigens with molecular masses of approximately 75 and 100 kDa. A competitive enzyme-linked immunosorbent assay was used to demonstrate that the MAb was not cross-reactive with purified glucuronoxylomannan derived from either serotypes A or D. Experiments conducted with mouse peritoneal phagocytes and the mouse phagocyte-like cell line, J774A.1, demonstrated that the CSFi MAb opsonized the yeasts and increased their adherence to both types of phagocytic cells. We conclude, therefore, that antibodies directed at noncapsular epitopes can serve as opsonins and may have a role in modulating cryptococcal infection.
RecombinantStaphylococcusStrains as Live Vectors for the Induction of Neutralizing Anti-Diphtheria Toxin Antisera
Authors: Fromen-Romano Cécile , Drevet Pascal , Robert Alain , Ménez André , Léonetti Michel
Published: December 2019

We have investigated whether the nonpathogenic gram-positive bacteriaStaphylococcus xylosusandS. carnosuscan display a whole domain of a toxic protein on their surface and if such vectors are suitable for immunization of BALB/c mice. The nucleotide sequence encoding the receptor-binding domain (DTR; amino acids 382 to 535) of diphtheria toxin (DT) was inserted into plasmids pSE′mp18ABPXM and pSPPmABPXM, which were designed to display heterologous proteins onS. xylosusandS. carnosuscell surfaces, respectively. Western blot analysis of the resulting bacterial lysates indicates that DTR is produced by each expression system. However, analysis of rabbit anti-DTR antisera binding to the transformed live bacteria shows that DTR is not displayed on the surface ofS. xylosuscells whereas it is efficiently exposed onS. carnosus. A significant anti-DT antibody response was raised in BALB/c mice immunized intraperitoneally withS. carnosusdisplaying DTR, and the antisera abolished DT cytotoxicity on Vero cells. Thus, onlyS. carnosuscan display a whole domain of a toxic protein and represents a potential vector for humoral vaccination.
Complement-Dependent Accumulation and Degradation of Platelets in the Lung and Liver Induced by Injection of Lipopolysaccharides
Authors: Shibazaki Masahiko , Kawabata Yoshihiro , Yokochi Takashi , Nishida Akira , Takada Haruhiko , Endo Yasuo
Published: December 2019

We found unique behaviors among platelets within a few minutes of the intravenous injection of lipopolysaccharide (LPS) into mice. Platelets accumulated primarily in the liver at lower doses of LPS, but at higher doses they accumulated largely in the lungs. When the platelets accumulated in these organs were degraded, there was a rapid anaphylactoid shock. The platelet response depended on the strain of mouse and on the source of LPS. Of various LPSs tested, the LPS from the smooth type ofKlebsiellaO3 (KO3-S LPS) was the most potent at inducing the platelet response and shock. K-76 monocarboxylic acid, an inhibitor of complement C5, effectively prevented theKO3-S LPS-induced degradation (but not accumulation) of platelets and the ensuing rapid shock in BALB/c mice. Moreover, in DBA/2 mice (which are deficient in complement C5), platelets accumulated in the lungs and liver in response toKO3-S LPS but soon returned to the circulation without degradation, and there was no rapid shock. The LPS from the rough type ofKO3 induced an accumulation of platelets in the liver and lungs but not a degradation of platelets. On the basis of these results and those reported by other investigators, we propose that in the platelet response to LPS, the lectin pathway to form C3 convertase from C4 and C2 is involved in the rapid accumulation of platelets in the liver and lungs and that the pathway from C5 to C9 is involved in the destruction of platelets and the consequent anaphylactoid shock.
Characterization of HumanMycobacterium bovisBacille Calmette-Guérin-Reactive CD8+T Cells
Authors: Smith Steven M. , Malin Adam S. , Pauline T.  , Lukey  , Atkinson Sara E. , Content Jean , Huygen Kris , Dockrell Hazel M.
Published: December 2019

Gamma interferon (IFN-γ)-secreting CD4+T cells have long been established as an essential component of the protective immune response againstMycobacterium tuberculosis. It is now becoming evident from studies with the murine model of tuberculosis that an important role also exists for major histocompatibility complex (MHC) class I-restricted CD8+T cells. These cells are capable of acting as both IFN-γ secretors and cytotoxic T lymphocyte (CTL) effectors; however, their exact role in immunity against tuberculosis remains unclear. This study demonstrates the presence ofMycobacterium bovisBCG-reactive CD8+T cells in healthy BCG-vaccinated donors and that these CD8+T cells are potent cytokine producers as well as cytotoxic effector cells. Using FACScan analysis, we have shown that restimulation with liveM. bovisBCG induced more CD8+-T-cell activation than the soluble antigen purified protein derivative and that these cells are actively producing the type 1 cytokines IFN-γ and tumor necrosis factor alpha (TNF-α). These CD8+T cells also contain the cytolytic granule perforin and are capable of acting as potent CTLs againstM. bovisBCG-infected macrophages. The mycobacterial antigens 85A and B (Ag85A and Ag85B, respectively), and to a lesser extent the 19- and 38-kDa proteins, are major antigenic targets for these mycobacterium-specific CD8+T cells, while whole-M. bovisBCG activated effector cells from these BCG-vaccinated donors, as expected, failed to recognize the 6-kDa ESAT-6 protein. The use of metabolic inhibitors and blocking antibodies revealed that the CD8+T cells recognize antigen processed and presented via the classical MHC class I pathway. These data suggest that CD8+T cells may play a critical role in the human immune response to tuberculosis infection.
Common and Specific Characteristics of the High-Pathogenicity Island of
Yersinia enterocolitica
Authors: Rakin A. , Noelting C. , Schubert S. , Heesemann J.
Published: December 2019

Yersinia pestis
,
Y. pseudotuberculosis
O:1, and
Y. enterocolitica
biogroup 1B strains carry a high-pathogenicity island (HPI), which mediates biosynthesis and uptake of the siderophore yersiniabactin and a mouse-lethal phenotype. The HPI of
Y. pestis
and
Y. pseudotuberculosis
(Yps HPI) are highly conserved in sequence and organization, while the HPI of
Y. enterocolitica
(Yen HPI) differs significantly. The 43,393-bp Yen HPI sequence of
Y. enterocolitica
WA-C, serotype O:8, was completed and compared to that of the Yps HPI of
Y. pseudotuberculosis
PB1, serotype O:1A. A common GC-rich region (G+C content, 57.5 mol%) of 30.5 kb is conserved between yersinia strains. This region carries genes for yersiniabactin biosynthesis, regulation, and uptake and thus can be considered the functional “core” of the HPI. In contrast, the second part of the HPI is AT rich and completely different in two evolutionary lineages of the HPI, being 12.8 kb in the Yen HPI and 5.6 kb in the Yps HPI. The variable part acquired one IS
100
element in the Yps HPI and accumulated four insertion elements, IS
1328
, IS
1329
, IS
1400
, and IS
1222
, in the Yen HPI. The insertion of a 125-bp ERIC sequence modifies the structure of the promoter of the
ybtA
yersiniabactin regulator in the Yen HPI. In contrast to the precise excision of the Yps HPI in
Y. pseudotuberculosis
, the Yen HPI suffers imprecise deletions. The Yen HPI is stably integrated in one of the three
asn
tRNA copies in
Y. enterocolitica
biogroup 1B (serotypes O:8, O:13, O:20, and O:21), probably due to inactivation of the putative integrase. The 17-bp duplications of the 3′ end of the
asnT
RNA are present in both
Yersinia
spp. The HPI attachment site is unoccupied in nonpathogenic
Y. enterocolitica
NF-O, biogroup 1A, serotype O:5. The HPI of
Yersinia
is a composite and widely spread genomic element with a highly conserved yersiniabactin functional “core” and a divergently evolved variable part.
Cryptosporidium parvumApical Complex Glycoprotein CSL Contains a Sporozoite Ligand for Intestinal Epithelial Cells
Authors: Langer Rebecca C. , Riggs Michael W.
Published: December 2019

Cryptosporidiosis, caused by the apicomplexan parasiteCryptosporidium parvum, has become a well-recognized diarrheal disease of humans and other mammals throughout the world. No approved parasite-specific drugs, vaccines, or immunotherapies for control of the disease are currently available, although passive immunization withC. parvum-specific antibodies has some efficacy in immunocompromised and neonatal hosts. We previously reported that CSL, an ∼1,300-kDa conserved apical glycoprotein ofC. parvumsporozoites and merozoites, is the antigenic species mechanistically bound by neutralizing monoclonal antibody 3E2 which elicits the circumsporozoite precipitate (CSP)-like reaction and passively protects againstC. parvuminfection in vivo. These findings indicated that CSL has a functional role in sporozoite infectivity. Here we report that CSL has properties consistent with being a sporozoite ligand for intestinal epithelial cells. For these studies, native CSL was isolated from whole sporozoites by isoelectric focusing (IEF) following observations that the ∼1,300-kDa region containing CSL as seen by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was comprised of approximately 15 molecular species (pI 3 to 10) when examined by two-dimensional (2-D) electrophoresis and silver staining. A subset of six ∼1,300-kDa species (pI 4.0 to 6.5) was specifically recognized by 3E2 in 2-D Western immunoblots of IEF-isolated CSL. Isolated native CSL bound specifically and with high affinity to permissive human intestinal epithelial Caco-2 cells in a dose-dependent, saturable, and self-displaceable manner. Further, CSL specifically bound to the surface of live Caco-2 cells inhibited sporozoite attachment and invasion. In addition, sporozoites having released CSL after incubation with 3E2 and occurrence of the CSP-like reaction did not attach to and invade Caco-2 cells. These findings indicate that CSL contains a sporozoite ligand which facilitates attachment to and invasion of Caco-2 cells and, further, that ligand function may be disrupted by CSL-reactive monoclonal antibody. We conclude that CSL is a rational target for passive or active immunization against cryptosporidiosis.
Identification of Genes in an Extraintestinal Isolate of
Escherichia coli
with Increased Expression after Exposure to Human Urine
Authors: Russo Thomas A. , Carlino Ulrike B. , Mong Andrew , Jodush Stephen T.
Published: December 2019

The identification of genes with increased expression in vivo may lead to the identification of novel or unrecognized virulence traits and/or recognition of environmental signals involved in modulating gene expression. Our laboratory is studying an extraintestinal isolate of
Escherichia coli
as a model pathogen. We had previously used human urine ex vivo to identify the unrecognized urovirulence genes
guaA
and
argC
and to establish that arginine and guanine (or derivatives) were limiting in this body fluid (T. A. Russo et al., Mol. Microbiol. 22:217–229, 1996). In this study, we have continued with this approach and identified three additional genes that have increased expression in human urine relative to Luria-Bertani (LB) medium. Expression of
ure1
(urine-responsive element) is increased a mean of 47.6-fold in urine but completely suppressed by exogenous glucose. This finding suggests that
ure1
is regulated by catabolite repression and that limiting glucose in urine is a regulatory signal.
ure1
is present in the
E. coli
K-12 genome, but its function is unknown. Although disruption of
ure1
results in diminished growth in human urine, limiting concentrations of amino acids, nucleosides, or iron (Fe), or changes in osmolarity or pH do not affect the expression of
ure1
. Therefore, Ure1 appears to have a role independent of the synthesis or uptake of these nutrients and does not appear to be involved in osmoprotection.
iroN

E. coli

is a novel
E. coli
gene with 77% DNA homology to a catecholate siderophore receptor gene recently identified in
Salmonella
. Its expression is increased a mean of 27.2-fold in urine and is repressed by exogenous Fe and a urinary pH of 5.0. This finding supports the contention that Fe is a limiting element in urine and that alteration of pH can affect gene expression. It is linked to the P-pilus (
prs
) and F1C fimbrial (
foc
) gene clusters on a pathogenicity island and appears to have been acquired by IS
1230
-mediated horizontal transmission. The homologous

iroN
E. coli

sequence is significantly more prevalent in urinary tract and blood isolates of
E. coli
compared to fecal isolates. Last, the expression of ArtJ, an arginine periplasmic binding protein, is increased a mean of 16.6-fold in urine. This finding implicates arginine concentrations as limited in urine and, in combination with previous data demonstrating that
argC
is important for urovirulence, suggests that the ability of
E. coli
to synthesize or acquire arginine is important for urovirulence.
ure1
,

iroN
E. coli

, and
artJ
all have increased expression in human blood and ascites relative to LB medium as well. The identification of these genes increases our understanding of regulatory signals present in human urine, blood, and ascites. Ure1, IroN

E. coli

, and ArtJ also warrant further evaluation as virulence traits both within and outside the urinary tract.
Phase Variation in
Helicobacter pylori
Lipopolysaccharide due to Changes in the Lengths of Poly(C) Tracts in α3-Fucosyltransferase Genes
Authors: Appelmelk Ben J. , Martin Steve L. , Monteiro Mario A. , Clayton Chris A. , McColm Andrew A. , Zheng Pengyuan , Verboom Theo , Maaskant Janneke J. , van den Eijnden Dirk H. , Hokke Cornelis H. , Perry Malcolm B. , Vandenbroucke-Grauls Christina M. J. E. , Kusters Johannes G.
Published: December 2019

The lipopolysaccharide (LPS) of
Helicobacter pylori
expresses the Lewis x (Le
x
) and/or Le
y
antigen. We have shown previously that
H. pylori
LPS displays phase variation whereby an Le
x
-positive strain yields variants with different LPS serotypes, for example, Le
x
plus Le
y
or nonfucosylated polylactosamine.
H. pylori
has two α3-fucosyltransferase genes that both contain poly(C) tracts. We now demonstrate that these tracts can shorten or lengthen randomly, which results in reversible frameshifting and inactivation of the gene products. We provide genetic and serological evidence that this mechanism causes
H. pylori
LPS phase variation and demonstrate that the on or off status of α3-fucosyltransferase genes determines the LPS serotypes of phase variants and clinical isolates. The role of the α3-fucosyltransferase gene products in determining the LPS serotype was confirmed by structural-chemical analysis of α3-fucosyltransferase knockout mutants. The data also show that the two α3-fucosyltransferase genes code for enzymes with different fine specificities, and we propose the names
futA
and
futB
to designate the orthologs of the
H. pylori
26695 α3-fucosyltransferase genes HP0379 and HP0651, respectively. The data also show that the α3-fucosylation in
H. pylori
precedes α3-fucosyltransferase, an order of events opposite to that which prevails in mammals. Finally, the data provide an understanding at the molecular level of the mechanisms underlying LPS diversity in
H. pylori
, which may play an important role in adaptation to the host.
Redox Imbalance Differentially Inhibits Lipopolysaccharide-Induced Macrophage Activation in the Mouse Liver
Authors: Wang Fuan , Wang Luke Y. , Wright Douglas , Parmely Michael J.
Published: December 2019

Endotoxemia is accompanied by significant changes in the reductive-oxidative (redox) balance of critical target organs. Redox stress has been shown to regulate the expression of proinflammatory genes that are induced by endotoxic lipopolysaccharide (LPS) in vitro; however, much less is known about the effects of redox imbalance on LPS-induced gene expression in vivo. To assess the effects of redox stress on inflammatory responses in endotoxemia, mice were treated with either diethyl maleate (DEM), a glutathione-depleting agent, or buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis, and challenged with LPS. While serum tumor necrosis alpha (TNF-α) responses and the appearance of TNF-α-positive Kupffer cells in the liver were virtually eliminated by DEM or BSO treatment, the expression of both CD14 and inducible NO synthase (iNOS) by Kupffer cells was unaffected by glutathione depletion. By contrast, LPS-induced hepatocyte and hepatic sinusoidal endothelial cell iNOS expression was significantly inhibited in DEM-treated mice. Hepatocyte iNOS induced by recombinant mouse TNF-α was also inhibited by DEM treatment. These results indicate that the effects of oxidative stress in this organ are cell type specific and suggest that both the production and the action of TNF-α are substantially influenced by the redox state of the liver during endotoxemia.
The Intercellular Adhesion (
ica
) Locus Is Present in
Staphylococcus aureus
and Is Required for Biofilm Formation
Authors: Cramton Sarah E. , Gerke Christiane , Schnell Norbert F. , Nichols Wright W. , Götz Friedrich
Published: December 2019

Nosocomial infections that result in the formation of biofilms on the surfaces of biomedical implants are a leading cause of sepsis and are often associated with colonization of the implants by
Staphylococcus epidermidis
. Biofilm formation is thought to require two sequential steps: adhesion of cells to a solid substrate followed by cell-cell adhesion, creating multiple layers of cells. Intercellular adhesion requires the polysaccharide intercellular adhesin (PIA), which is composed of linear β-1,6-linked glucosaminylglycans and can be synthesized in vitro from UDP-
N
-acetylglucosamine by products of the intercellular adhesion (
ica
) locus. We have investigated a variety of
Staphylococcus aureus
strains and find that all strains tested contain the
ica
locus and that several can form biofilms in vitro. Sequence comparison with the
S. epidermidis ica
genes revealed 59 to 78% amino acid identity. Deletion of the
ica
locus results in a loss of the ability to form biofilms, produce PIA, or mediate
N
-acetylglucosaminyltransferase activity in vitro. Cross-species hybridization experiments revealed the presence of
icaA
in several other
Staphylococcus
species, suggesting that cell-cell adhesion and the potential to form biofilms is conserved within this genus.
Characterization of Pic, a Secreted Protease of
Shigella flexneri
and Enteroaggregative
Escherichia coli
Authors: Henderson Ian R. , Czeczulin John , Eslava Carlos , Noriega Fernando , Nataro James P.
Published: December 2019

We have identified and characterized a secreted protein, designated Pic, which is encoded on the chromosomes of enteroaggregative
Escherichia coli
(EAEC) 042 and
Shigella flexneri
2457T. The product of the
pic
gene is synthesized as a 146.5-kDa precursor molecule which is processed at the N and C termini during secretion, allowing the release of a mature protein (109.8 kDa) into the culture supernatant. The deduced amino acid sequence of Pic shows high homology to autotransporter proteins, particularly a subgroup termed the SPATEs (serine protease autotransporters of the
Enterobacteriaceae
). Present in all members of this subgroup is a motif similar to the active sites of certain serine proteases. Pic catalyzes gelatin degradation, which can be abolished by disruption of the predicted proteolytic active site. Functional analysis of the Pic protein implicates this factor in mucinase activity, serum resistance, and hemagglutination. Our data suggest that Pic may be a multifunctional protein involved in enteric pathogenesis.
Experimental Gonococcal Genital Tract Infection and Opacity Protein Expression in Estradiol-Treated Mice
Authors: Jerse Ann E.
Published: December 2019

The development of effective prophylactic agents against gonorrhea and the study of adaptation by
Neisseria gonorrhoeae
to the urogenital mucosa are hindered by the lack of a well-established animal model of gonococcal genital tract infection. Here, a murine model of long-term gonococcal genital tract infection is described. Female BALB/c mice were treated with 17-β-estradiol and inoculated intravaginally with wild-type gonococcal strain FA1090 or MS11.
N. gonorrhoeae
was recovered from vaginal swabs for an average of 12 to 13 days following inoculation with 10
6
CFU of either strain. Inflammation occurred in over 80% of infected mice, and diplococci were associated with epithelial cells and neutrophils in stained vaginal smears. Ascended infection occurred in 17 to 20% of mice inoculated with strain FA1090. An outbred mouse strain (SLC:ddY) previously reported to be naturally susceptible to
N. gonorrhoeae
was also tested; however, as with BALB/c mice, estradiol was required for prolonged infection. Although piliation was not maintained during experimental murine infection, 46 to 100% of vaginal isolates from four of eight BALB/c mice and three of four SLC:ddY mice expressed one or more opacity (Opa) proteins within 4 days after inoculation with an Opa-negative variant of strain FA1090. The observed selection for and/or induction of gonococcal Opa protein expression during murine infection appears to parallel events that occur during experimental urethritis in volunteers.
Outer Membrane Protein A-Promoted Actin Condensation of Brain Microvascular Endothelial Cells Is Required for
Escherichia coli
Invasion
Authors: Prasadarao Nemani V. , Wass Carol A. , Stins Monique F. , Shimada Hiroyuki , Kim Kwang Sik
Published: December 2019

Escherichia coli
is the most common gram-negative bacterium that causes meningitis during the neonatal period. We have previously shown that the entry of circulating
E. coli
organisms into the central nervous system is due to their ability to invade the blood-brain barrier, which is composed of a layer of brain microvascular endothelial cells (BMEC). In this report, we show by transmission electron microscopy that
E. coli
transmigrates through BMEC in an enclosed vacuole without intracellular multiplication. The microfilament-disrupting agents cytochalasin D and latrunculin A completely blocked
E. coli
invasion of BMEC. Cells treated with the microtubule inhibitors nocodazole, colchicine, vincristin, and vinblastine and the microtubule-stabilizing agent taxol also exhibited 50 to 60% inhibition of
E. coli
invasion. Confocal laser scanning fluorescence microscopy showed F-actin condensation associated with the invasive
E. coli
but no alterations in microtubule distribution. These results suggest that
E. coli
uses a microfilament-dependent phagocytosis-like endocytic mechanism for invasion of BMEC. Previously we showed that OmpA expression significantly enhances the
E. coli
invasion of BMEC. We therefore examined whether OmpA expression is related to the recruitment of F-actin. OmpA
+
E. coli
induced the accumulation of actin in BMEC to a level similar to that induced by the parental strain, whereas OmpA
−
E. coli
did not. Despite the presence of OmpA, a noninvasive
E. coli
isolate, however, did not show F-actin condensation. OmpA
+
-
E. coli
-associated condensation of F-actin was blocked by synthetic peptides corresponding to the N-terminal extracellular domains of OmpA as well as BMEC receptor analogues for OmpA, chitooligomers (GlcNAcβ1-4GlcNAc oligomers). These findings suggest that OmpA interaction is critical for the expression or modulation of other bacterial proteins that will subsequently cause actin accumulation for the uptake of bacteria.
Plasmodium falciparum
Field Isolates Commonly Use Erythrocyte Invasion Pathways That Are Independent of Sialic Acid Residues of Glycophorin A
Authors: Okoyeh Jude Nnaemeka , Pillai C. R. , Chitnis Chetan E.
Published: December 2019

Erythrocyte invasion by malaria parasites is mediated by specific molecular interactions. Sialic acid residues of glycophorin A are used as invasion receptors by
Plasmodium falciparum
. In vitro invasion studies have demonstrated that some cloned
P. falciparum
lines can use alternate receptors independent of sialic acid residues of glycophorin A. It is not known if invasion by alternate pathways occurs commonly in the field. In this study, we used in vitro growth assays and erythrocyte invasion assays to determine the invasion phenotypes of 15
P. falciparum
field isolates. Of the 15 field isolates tested, 5 multiply in both neuraminidase and trypsin-treated erythrocytes, 3 multiply in neuraminidase-treated but not trypsin-treated erythrocytes, and 4 multiply in trypsin-treated but not neuraminidase-treated erythrocytes; 12 of the 15 field isolates tested use alternate invasion pathways that are not dependent on sialic acid residues of glycophorin A. Alternate invasion pathways are thus commonly used by
P. falciparum
field isolates. Typing based on two polymorphic markers, MSP-1 and MSP-2, and two microsatellite markers suggests that only 1 of the 15 field isolates tested contains multiple parasite genotypes. Individual
P. falciparum
lines can thus use multiple invasion pathways in the field. These observations have important implications for malaria vaccine development efforts based on EBA-175, the
P. falciparum
protein that binds sialic acid residues of glycophorin A during invasion. It may be necessary to target parasite ligands responsible for the alternate invasion pathways in addition to EBA-175 to effectively block erythrocyte invasion by
P. falciparum
.
Invasion of Human Coronary Artery Cells by Periodontal Pathogens
Authors: Dorn Brian R. , Dunn William A. , Progulske-Fox Ann
Published: December 2019

There is an emerging paradigm shift from coronary heart disease having a purely hereditary and nutritional causation to possibly having an infectious etiology. Recent epidemiological studies have shown a correlation between periodontal disease and coronary heart disease. However, to date, there is minimal information as to the possible disease mechanisms of this association. It is our hypothesis that invasion of the coronary artery cells by oral bacteria may start and/or exacerbate the inflammatory response in atherosclerosis. Since a few periodontal pathogens have been reported to invade oral epithelial tissues, we tested the ability of three putative periodontal pathogens—
Eikenella corrodens
,
Porphyromonas gingivalis
, and
Prevotella intermedia
—to invade human coronary artery endothelial cells and coronary artery smooth muscle cells. In this study we demonstrate by an antibiotic protection assay and electron microscopy that specific species and strains invade coronary artery cells at a significant level. Actin polymerization and eukaryotic protein synthesis in metabolically active cells were required since the corresponding inhibitors nearly abrogated invasion. Many intracellular
P. gingivalis
organisms were seen to be present in multimembranous vacuoles resembling autophagosomes by morphological analysis. This is the first report of oral microorganisms invading human primary cell cultures of the vasculature.
Consequence of
Nramp1
Deletion to
Mycobacterium tuberculosis
Infection in Mice
Authors: North Robert J. , LaCourse Ronald , Ryan Lynn , Gros Philippe
Published: December 2019

129sv mice functionally deleted of the antimicrobial resistance gene,
Nramp1
, were found to be as resistant as wild-type mice to infection with the virulent H37Rv strain of
Mycobacterium tuberculosis
, as determined by monitoring bacterial growth in major organs and recording host survival times. Death of infected mice of both types was associated with extensive infection-induced pathology in the lungs but not in other major organs. These findings are in keeping with the view that
Nramp1
is of limited importance in resistance to tuberculosis in mice.
Local Production of Chemokines during Experimental Vaginal Candidiasis
Authors: Saavedra Michael , Taylor Brad , Lukacs Nicholas , Fidel Paul L.
Published: December 2019

Recurrent vulvovaginal candidiasis, caused byCandida albicans, is a significant problem in women of childbearing age. Although cell-mediated immunity (CMI) due to T cells and cytokines is the predominant host defense mechanism againstC. albicansat mucosal tissue sites, host defense mechanisms againstC. albicansat the vaginal mucosa are poorly understood. Based on an estrogen-dependent murine model of vaginal candidiasis, our data suggest that systemic CMI is ineffective againstC. albicansvaginal infections. Thus, we have postulated that local immune mechanisms are critical for protection against infection. In the present study, the kinetic production of chemokines normally associated with the chemotaxis of T cells, macrophages (RANTES, MIP-1α, MCP-1), and polymorphonuclear neutrophils (MIP-2) was examined following intravaginal inoculation ofC. albicansin estrogen-treated or untreated mice. Results showed significant increases in MCP-1 protein and mRNA in vaginal tissue of infected mice as early as 2 and 4 days postinoculation, respectively, that continued through a 21-day observation period, irrespective of estrogen status. No significant changes were observed with RANTES, MIP-1α, or MIP-2, although relatively high constitutive levels of RANTES mRNA and MIP-2 protein were observed. Furthermore, intravaginal immunoneutralization of MCP-1 with anti-MCP-1 antibodies resulted in a significant increase in vaginal fungal burden early during infection, suggesting that MCP-1 plays some role in reducing the fungal burden during vaginal infection. However, the lack of changes in leukocyte profiles in vaginal lavage fluids collected from infected versus uninfected mice suggests that MCP-1 functions to control vaginalC. albicanstiters in a manner independent of cellular chemotactic activity.
Contribution of Quorum Sensing to the Virulence of
Pseudomonas aeruginosa
in Burn Wound Infections
Authors: Rumbaugh Kendra P. , Griswold John A. , Iglewski Barbara H. , Hamood Abdul N.
Published: December 2019

The
Pseudomonas aeruginosa
quorum-sensing systems,
las
and
rhl
, control the production of numerous virulence factors. In this study, we have used the burned-mouse model to examine the contribution of quorum-sensing systems to the pathogenesis of
P. aeruginosa
infections in burn wounds. Different quorum-sensing mutants of
P. aeruginosa
PAO1 that were defective in the
lasR
,
lasI
, or
rhlI
gene or both the
lasI
and
rhlI
genes were utilized. The following parameters of the
P. aeruginosa
infection were examined: (i) lethality to the burned mouse, (ii) dissemination of the
P. aeruginosa
strain within the body of the infected mouse (by determining the numbers of CFU of
P. aeruginosa
within the liver and spleen), and (iii) spread of the
P. aeruginosa
strain within the burned skin (by determining the numbers of CFU of
P. aeruginosa
at the inoculation site and at a site about 15 mm from the inoculation site [distant site]). In comparison with that of PAO1, the in vivo virulence of
lasI
,
lasR
, and
rhlI
mutants was significantly reduced. However, the most significant reduction in in vivo virulence was seen with the
lasI rhlI
mutant. The numbers of CFU that were recovered from the livers, spleens, and skin of mice infected with different mutants were significantly lower than those of PAO1. At 8 and 16 h post burn infection, comparable numbers of CFU of PAO1 and
lasI
and
rhlI
mutants were obtained from both the inoculation and distant sites of the burned skin of infected mice. In contrast, CFU of the
lasR
mutant and the
lasI rhlI
double mutant were recovered only from the inoculation site of infected mice at 8 and 16 h post burn infection. The ability of a plasmid carrying either the
lasI
or
rhlI
gene or the
lasI
and
rhlI
genes to complement the defect of the
lasI rhlI
double mutant was also examined. The presence of any of these plasmids within the
lasI rhlI
double mutant significantly enhanced its in vivo virulence, as well as its ability to spread within the burned skin. These results suggest that the quorum-sensing systems play an important role in the horizontal spread of
P. aeruginosa
within burned skin and in the dissemination of
P. aeruginosa
within the bodies of burned-and-infected mice and contributed to the overall virulence of
P. aeruginosa
in this animal model.
Intranasal Immunization with Pneumococcal Polysaccharide Conjugate Vaccines with Nontoxic Mutants ofEscherichia coliHeat-Labile Enterotoxins as Adjuvants Protects Mice against Invasive Pneumococcal Infections
Authors: Jakobsen Håvard , Schulz Dominique , Pizza Mariagrazia , Rappuoli Rino , Jónsdóttir Ingileif
Published: December 2019

Host defenses againstStreptococcus pneumoniaedepend largely on phagocytosis following opsonization by polysaccharide-specific immunoglobulin G (IgG) antibodies and complement. Since colonization of the respiratory mucosa is the first step in pneumococcal pathogenesis, mucosal immune responses may play a significant role. In addition to inducing systemic immune responses, mucosal vaccination with an effective adjuvant has the advantage of inducing mucosal IgA antibodies. The heat-labile enterotoxin (LT) ofEscherichia coliis a well-studied mucosal adjuvant, and adjuvant activity of nontoxic LT mutants has been demonstrated for several protein antigens. We investigated the immunogenicity of pneumococcal polysaccharide conjugate vaccines (PNC) of serotypes 1 and 3 in mice after intranasal (i.n.) immunization by using as an adjuvant the nontoxic LT mutant LT-K63 or LT-R72, which has minimal residual toxicity. Pneumococcal serotype-specific antibodies were measured in serum (IgM, IgG, and IgA) and saliva (IgA), and vaccine-induced protection was evaluated by i.n. challenge with virulent pneumococci of the homologous serotype. When administered with LT mutants, i.n. immunization with both conjugates induced systemic and mucosal immune responses, and serum IgG antibody levels were significantly higher than after subcutaneous immunization. All mice immunized i.n. with PNC-1 and LT mutants were protected against bacteremia and cleared the pneumococci from the lung 24 h after i.n. challenge; pneumococcal density correlated significantly with serum IgG antibody levels. Similarly, the survival of mice immunized i.n. with PNC-3 and LT mutants was significantly prolonged. These results demonstrate that i.n. vaccination with PNC and potent adjuvants can protect mice against invasive and lethal pneumococcal infections, indicating that mucosal vaccination with PNC may be an alternative vaccination strategy for humans.
Molecular Characterization of the Locus Encoding Biosynthesis of the Lipopolysaccharide O Antigen of
Escherichia coli
Serotype O113
Authors: Paton Adrienne W. , Paton James C.
Published: December 2019

Shiga toxigenic
Escherichia coli
(STEC) strains are a diverse group of organisms capable of causing severe gastrointestinal disease in humans. Within the STEC family,
eae
-positive STEC strains, particularly those belonging to serogroups O157 and O111, appear to have greater virulence for humans. However, in spite of being
eae
negative, STEC strains belonging to serogroup O113 have frequently been associated with cases of severe STEC disease, including hemolytic-uremic syndrome (HUS). Western blot analysis with convalescent-phase serum from a patient with HUS caused by an O113:H21 STEC strain indicated that human immune responses were directed principally against lipopolysaccharide O antigen. Accordingly, the serum was used to isolate a clone expressing O113 O antigen from a cosmid library of O113:H21 DNA constructed in
E. coli
K-12. Sequence analysis indicated that the O113 O-antigen biosynthesis (
rfb
) locus contains a cluster of nine genes which may be cotranscribed. Comparison with sequence databases identified candidate genes for four glycosyl transferases, an
O
-acetyl transferase, an O-unit flippase, and an O-antigen polymerase, as well as copies of
galE
and
gnd
. Two additional, separately transcribed genes downstream of the O113
rfb
region were predicted to encode enzymes involved in synthesis of activated sugar precursors, one of which (designated
wbnF
) was essential for O113 O-antigen synthesis, and so is clearly a part of the O113
rfb
locus. Interestingly, expression of O113 O antigen by
E. coli
K-12 significantly increased in vitro adherence to both HEp-2 and Henle 407 cells.
T-Cell Recognition ofMycobacterium tuberculosisCulture Filtrate Fractions in Tuberculosis Patients and Their Household Contacts
Authors: Demissie Abebech , Ravn Pernille , Olobo Joseph , Doherty T. Mark , Eguale Tewodros , Geletu Mulu , Hailu Wondewossen , Andersen Peter , Britton Sven
Published: December 2019

We examined the immune responses of patients with active pulmonary tuberculosis (TB) and their healthy household contacts to short-term culture filtrate (ST-CF) ofMycobacterium tuberculosisor molecular mass fractions derived from it. Our goal was to identify fractions strongly recognized by donors and differences among the donor groups of possible relevance for vaccine development. The study population consisted of 65 human immunodeficiency virus-negative donors from the Hossana Regional Hospital, Hossana, Ethiopia. Peripheral blood leukocytes from the donors were stimulated with different antigens and immune responses were determined. Household contacts produced significantly higher levels of gamma interferon (IFN-γ) than the TB patients in response to antigens present in ST-CF and the 10 narrow-molecular-mass fractions. A similar difference in leukocyte proliferative responses to the antigens between the two groups was also found. In general, while all fractions stimulated immune responses, the highest activity was seen with the low-molecular-mass fractions, which include well-defined TB antigens such as ESAT-6. Leukocytes from contacts of TB patients with severe disease produced higher levels of antigen-specific IFN-γ than those from contacts of patients with minimal disease. Both groups of contacts exhibited higher cell-mediated responses than the patients themselves. The enhanced immune response of healthy contacts, especially those of patients with severe disease, to secreted mycobacterial antigens is suggestive of an early stage of infection byM. tuberculosis, which could in time result in overt disease or containment of the infection. This possibility is currently being investigated by follow-up studies of the household contacts.